Dept of Pathology

DEPT OF PATHOLOGY

Researcher : Au LK

List of Research Outputs

Au L.K., Wong C.M. and Ng I.O.L., Enhancer of Zeste Homolog 2 (EZH2) was involved in Hepatocellular Carcinoma (HCC) Progression Through Gene Silencing, 16th Hong Kong International Cancer Congress. Hong Kong, 2009.

Au L.K., Wong C.C.L., Lee M.F., Wong C.M. and Ng I.O.L., Frequent upregulation of Enhancer of Zeste Homolog 2 was implicated in Deleted in Liver Cancer 1 gene silencing and HCC progression , The 20th Conference of the Asian Pacific Association for the Study of the Liver (APASL). Beijing, 2010.

Wong C.C.L., Wong C.M., Au L.K. and Ng I.O.L., RhoGTPases and Rho-effectors in hepatocellular carcinoma metastasis: ROCK N' Rho move it , Liver International. 2010, 30: 642-656.

Researcher : Beh SL

Project Title:	The attitudes of doctors toward rape victims
Investigator(s):	Beh SL
Department:	Pathology
Source(s) of Funding:	Other Funding Scheme
Start Date:	06/1993

Abstract:

To have an objective assessment of what the prevalent attitudes are; to ascertain if there is a gender bias; to compare local data with data from other countries; to compare data with other professional groups.

List of Research Outputs

Beh S.L., Causes Of Death - Contemporary, In: Clifton D. Bryant & Dennis L. Peck, Encyclopedia Of Death & The Human Experience. California, London, India, Singapore, SAGE, 2009, One: 159-163.

Beh S.L., Forensic Investigation Of Death - Time For A A Paradigm Shift, International Academy Of Legal Medicine. 2010.

Beh S.L., Infanticide, In: Clifton D. Bryant & Dennis L. Peck, Encyclopedia Of Death & The Human Experience. California, London, India, Singapores, SAGE, 2009, Two: 596-599.

Beh S.L., International Consultant, In: Prof. Peter Vanezis, Medicine, Science & Law. U.K., Royal Society Of Medicine Press, 2010.

Beh S.L., Medical Evaluation Of Claimants Of Torture, In: Hong Kong Academy of Law, Training Programme For Lawyers On Convention Against Torture Claims And Refugee Law. 2009.

Fan J.K.M., Tong D.K.H., Poon J.T.C., Lo O.S.H., Beh S.L., Patil N.G. and Law W.L., Multimodality minimally invasive autopsy -- a feasible and accurate approach to post-mortem examination, Forensic Science International. 2010, 195(1-3): 93-98.

Luk J.K.H., Liu A.N.C., Ng W.C., Lui B., Beh S.L. and Chan F.H.W., End of Life care: towards a more dignified dying process in residential care homes for the elderly, Hong Kong Medical Journal. 2010, 16: 235-236.

Researcher : Castilho AG

List of Research Outputs

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., CD24 is a functional marker that mediates liver tumor initiation via regulation of Nanog, American Association for Cancer Research, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., Identification of a liver cancer stem marker using a chemoresistant tumor model, "Stem cell and Cancer" Gordon Research Conference, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Epithelial-Mesenchymal Transition in Cancer Stem Cells, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Liver cancer stem cells: Implications for a new therapeutic target, Liver International. 2009, 29: 955-965.

Lee K.W., Yung L.H., Cheung C.H., Castilho A.G. and Ng I.O.L., Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis, The American Association for Cancer Research, 2010.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Researcher : Chan ASY

List of Research Outputs

Kosinski C., Stange D.E., Xu C., Chan A.S.Y., Ho C., Yuen S.T., Mifflin R.C., Powell D.W., Clevers H., Leung S.Y. and Chen X., Indian Hedgehog Regulates Intestinal Stem Cell Fate Through Epithelial-Mesenchymal Interactions During Development, Gastroenterology. 2010, 139(3): 893-903.

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Chan AYW

List of Research Outputs

Chan K.Y., Ching D.C.K., Mak C.M., Lam C.W. and Chan A.Y.W., Hereditary spastic paraplegia: identification of an SPG3A gene mutation in a Chinese family, Hong Kong Medical Journal. 2009, 15(4): 304-7.

Chau S.K.C., Kwok K.L., Ng D.K., Lam C.W., Tong S.F., Chan A.Y.W., Siu C.W.K. and Yuen L.Y.P., Maternally inherited Leigh syndrome: an unusual cause of infantile apnea, Sleep Breath. 2010, 14(2): 161-5.

Lee H.H.C., LEE R.S.Y., LAI C.K., Yuen L.Y.P., SIU T.S., Chan A.Y.W. and Lam C.W., A novel duplication at the putative DNA polymerase alpha arrest site and a founder mutation in Chinese in the IVD gene underlie isovaleric acidaemia, Hong Kong Medical Journal. 2010, 16(3): 219-22.

Researcher : Chan EYT

List of Research Outputs

Mok T.M.Y., Huang F.P., Ip W.K., Wong F.Y., Chan E.Y.T. and Xu D., Serum levels of IL-33 and soluble ST2 and their association with disease activity in systemic lupus erythematosus. , HKMJ. 2010, 16: p46 S76.

Researcher : Chan GSW

List of Research Outputs

Chan G.S.W., Tsoi H.W., Wong S.S.Y., Li C.L., Tse H., Un I K., Yuen K.Y. and Chan K.W., BK virus nephropathy due to KOM-3 strain, Am J Kidney Dis. 2009, 54(1): 122-6.

Kwok J.S.Y., Chan G.S.W., Lam M.F., Yan T., Tang L., Kwong K.M., Chan K.W. and Chan D.T.M., Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes, Clinical transplantation. 2010, [Epub ahead of print 2010 Apr 9].

Wong H., Yau T.C.C., Chan P., Ng I.O.L., Chan G.S.W., Hui P., Law W.L., Lo C.M., Hedley A.J. and Epstein R., PPI-delayed diagnosis of gastrinoma: oncologic victim of pharmacologic success, Pathology and Oncology Research. 2010, 16(1): 87-91.

Researcher : Chan J

List of Research Outputs

Chan J., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Integrin-linked kinase promotes hepatocellular carcinoma oncogenesis, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Chan KK

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Yuen H.F., Chiu Y.T., Chan K.K., Chua C.W., McCrudden C.M., Tang K.H., El-Tanani M., Wong Y.C., Wang X. and Chan K.W., Prostate Cancer Cells Modulate Osteoblast Mineralisation And Osteoclast Differentiation Through Id-1 , British Journal of Cancer. 2010, 102: 332-341.

Researcher : Chan KK

List of Research Outputs

Researcher : Chan KW

Project Title:	Identification, Characterization and Therapeutic Targeting of Tumourigenic Liver Cancer Stem Cells
Investigator(s):	Chan KW, Guan XY
Department:	Pathology
Source(s) of Funding:	Michael and Betty Kadoorie Cancer Genetics Research Programme (MBKCGRP) II
Start Date:	01/2008

Abstract:

To characterize cancer stem cells (CSCs) in hepatocellular carcinoma (HCC), by addressing these 4 key objectives: (1) To identify other markers, along with CD133, that can better characterize liver CSCs. (2) To validate CD133 expression as a marker of CSCs in human HCC. (3) To compare the resistence of CD133+ and CD133- HCC CSCs to a panel of commonly used chemotherapeutic drugs followed by elucidation of specific mechanistic pathways that render HCC CSCs resistant to conventional chemotherapies. (4) To examine the role of dysregulated self-renewal pathways (Notch and Hedgehog) in the initiation and maintenance of CD133+ HCC cells.

Project Title:	The regulatory role of miR-616 in the androgen-independent growth of prostate cancer
Investigator(s):	Chan KW, Ma SKY
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2008

Abstract:

Prostate cancer is the most frequently diagnosed malignant tumor and the second leading cause of cancer deaths in North America. In the past ten years, there is also an evidently growing trend of this disease in our local community. One of the most troubling aspects of prostate cancer is that, after androgen ablation therapy, androgen-dependent prostate cancer inevitably progresses to an androgen-independent state, for which no effective treatment has been developed. Although several molecular pathways have been invoked to explain the pathogenesis of this disease, to date the mechanisms for the development and progression of prostate cancer remain largely unknown. A better understanding of the molecular mechanisms by which hormone-sensitive prostate cancer cells acquire the ability to resist hormone deprivation may aid in the development of more effective therapies against this disease. microRNAs (miRNAs) are small, non-coding RNAs that regulate protein expression at the post-transcriptional level. Through the specific targeting of the 3’-untranslated regions (UTRs) of mRNAs, miRNAs down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation. The functional importance of miRNAs is evidenced by many biological processes in which they are implicated, including development, cell proliferation, apoptosis, metabolism, cell differentiation and morphogenesis (Ref. 1-3, listed at end of Section VI). Further, accumulating evidence suggests that miRNAs can function as tumor suppressors or oncogenes and play a central role in oncogenesis. Although differential expression of miRNAs has been reported in human prostate cancer (Ref. 4-7, listed at end of Section VI), the importance of miRNAs specifically in the development of androgen-independent prostate cancer remains elusive. In light of the increasingly recognized role of miRNA in tumor development, we examined whether differences in miRNA expression might distinguish between androgen-dependent (AD) and androgen-independent (AI) states of prostate cancer. Our preliminary study involved a study on the prostate cancer cell line LNCaP. AD LNCaP and AI LNCaP-LNO were analyzed by quantitative PCR (qPCR) for expression of 419 selected miRNAs known to be involved in all facets of cellular regulation (HT419 microRNA qPCR assay panel, System Biosciences – assay comes in 5x 96-well plates). Preliminary data from miRNA profiling of AD LNCaP and AI LNCaP-LNO prostate cell lines revealed several aberrantly expressed miRNAs – of these includes the overexpression of miR-616 in AI LNCaP-LNO cells (Figure 1, please see under Section VI). The 20 miRNAs that were found to be most aberrantly expressed were further confirmed by a second round of quantitative PCR analyses. Of these, miR-616 was found to be most highly dysregulated, with its expression found to be 8 fold greater in AI LNCaP-LNO as compared to AD LNCaP (data not shown). Subsequent preliminary studies by qPCR in a panel of prostate cell lines found expression of miR-616 to correlate with the androgen-dependence state of each cell line (Figure 2, please see under Section VI). The aim of the proposed project is to elucidate the role of differentially expressed miR-616 in the regulation of androgen-independent prostate cancer growth. Specifically, we plan to (1) examine the expression of miR-616 in a panel of prostate cell lines (LNCaP, LNCaP-R, LNCaP-LNO, C4-2B, PC3, DU145, 22rv1, HPr1 and NPTX) using qPCR analyses, and correlate the data with the androgen-dependence state of the cell line; (2) stably overexpress miR-616 in AD LNCaP and immortalized, normal HPr1 or silence miR-616 in AI LNCaP-LNO and 22rv1 cells using lentiviral transduction; (3) study the role of miR-616 in in vitro studies to examine its ability to grow in various culture conditions either with supplemented or depleted synthetic androgens; (4) investigate the role of miR-616 in an in vivo castration mouse model; (5) elucidate potential downstream interacting mRNA/protein molecules of miR-616 via bioinformatics search in the publicly available Sanger database or by way of the novel 2D-difference gel electrophoresis (2D-DIGE) proteomic approach; and (6) determine the expression of miR-616 in human prostate clinical samples using in situ hybridization and correlate the data with patient’s clinic-pathological parameters.

Project Title:	The roles of Id-1 in prostate cancer to bone metastasis
Investigator(s):	Chan KW
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2009

Abstract:

Background Information and Objectives Id (Inhibitor of DNA binding) proteins are a group of helix-loop-helix (HLH) transcription factor which do not contain the basic DNA binding domain. Basic helix-loop-helix (bHLH) transcription factors contain a helix-loop-helix domain for protein-protein interaction and a basic domain for DNA binding. They bind to E-box containing promoter, with CANNTG consensus sequence, and regulate the transcription of their target genes. The group Id-proteins is able to bind to other bHLH proteins and inhibits their binding to DNA. As many cellular processes are controlled by bHLH transcription factors, the negative effect of Id on the DNA binding of bHLH transcription factors plays important roles on development, and on cancer initiation and progression. Id-1 has been shown to be up-regulated in human prostate cancer and play various roles to promote prostate cancer progression. It stimulates cell proliferation through p16(INK4a)/pRB pathway, MAPK pathway and EGFR. It inhibits apoptosis through activation of NFκB pathway and inactivation of TNF-induced apoptosis. It promotes the development of distant metastasis through VEGF and through promoting epithelial-mesenchymal transition. It also allows the development of drug resistant probably through inactivation of JNK pathway. Id-1 is also negatively regulated by the potent tumour suppressor p53, suggesting its oncogenic role in prostate cancer initiation and progression. The use of Id-1 as a molecular marker for prognosis has been studied and it has been shown that high level of Id-1 correlates with poor prognosis of prostate cancer patients. Recent studies in prostate cancer to bone metastasis suggest that there is a pre-requisite of prostate cancer cell to undergo osteomimicry for them to establish secondary metastasis in bone. Osteomimicry is the acquisition of bone cell-like phenotype by cancer cells through up-regulation of genes that are usually expressed in osteoblast. Because Id-1 is expressed during osteoblast differentiation and is regulated by BMP-2 and Wnt, which promote osteoblast differentiation, it is highly probable that Id-1 may play a role in osteomimicry in prostate cancer cells. My research team and others have shown the importance of bone morphogenetic proteins (BMPs) signalling in prostate cancer to bone metastatic progression. The regulatory relationship between BMPs and Id-1 has also been revealed in other cell types. Id-1 is stimulated by addition of BMP-2 to myoblastic cell line and the same is also found in breast cancer cell line. Furthermore, Id-1 has also been demonstrated to be regulated by several BMPs to mediate the BMP-induced osteoblast proliferation and differentiation. Recently, Id-1 has been shown to co-express with BMP-6, which is a potent factor promoting prostate cancer to bone metastasis. Taken together, all these lines of evidence suggest that Id-1 plays an important role in prostate cancer to bone metastasis. Therefore, a study on how Id-1 promotes prostate cancer to bone metastasis may shed light on the use of Id-1 as a prognostic marker and therapeutic target in prostate cancer. We applied the small interfering RNA technique to study the effects of silencing Id-1 on prostate cancer cell line PC-3. Our preliminary results on PC-3 siCon, which expresses high level of Id-1 and PC-3 siId-1, which expresses low level of Id-1, showed that reduced expression of Id-1 reduced the mRNA expression of IL-6 and that of TNF-beta. IL-6 has been shown to be elevated in metastatic prostate cancer and could be used as a prognostic marker. TNF-beta is expressed in human osteoblast like cells. This protein has been shown to modulate the activity of osteoblast and osteoclast, and the proliferation of osteoblast. Besides, TNF-beta promotes secretion of osteoprotegerin, a member of tumor necrosis factor, which in turn promotes osteoblastic bone metastasis through inhibiting osteoclast differentiation. However, the role of TNF-beta in prostate cancer has not been investigated. In addition, conditioned medium from these two cell lines confer significantly different activity on osteoclast differentiation, suggesting Id-1 modulates prostate cancer-mediated osteoclast differentiation. Taken together, our preliminary data also suggest that Id-1 might be involved in the interaction between prostate cancer cell and bone cell and imply that Id-1 might play a significant role in prostate cancer to bone metastasis. As both IL-6 and TNF-beta are both secretory proteins, we believe that Id-1 might regulate expression of these two proteins and therefore the amount they could be secreted into the medium, which leads to differential regulation on the activity of osteoclast. Taken together, these preliminary data guide us to discover how Id-1 modulates prostate cancer-mediated bone cell activity and prompt us to study how the downstream factors of Id-1, such as IL-6 and TNF-beta, govern Id-1 modulated effect on prostate cancer-mediated bone cell activity. Furthermore, we have shown that TWIST is involved in promoting prostate cancer to bone metastasis both in human prostate cancer specimens and in human prostate cancer cell lines in vitro. TWIST is bHLH transcription factor that has been shown to interact with Id-1 and the functions of TWIST have been shown and suggested to be modulated through Id-1 binding. In addition, TWIST and Id-1 have been shown to co-express to regulate cellular differentiation. We would like to further our investigation on how Id-1 modulates TWIST’s functions during prostate cancer progression is required. The objectives are: (i) To study the effects of Id-1 expression in prostate cancer cells on bone cell differentiation (ii) To study whether IL-6 and TNF-beta production in prostate cancer cells is regulated by Id-1 (iii) To study the effects of differential TWIST and Id-1 expressions in prostate cancer cells (iv) To correlate IL-6, TNF-beta, Id-1 and TWIST expression in human prostate specimens

List of Research Outputs

Chan G.S.W., Tsoi H.W., Wong S.S.Y., Li C.L., Tse H., Un I K., Yuen K.Y. and Chan K.W., BK virus nephropathy due to KOM-3 strain, Am J Kidney Dis. 2009, 54(1): 122-6.

Chan K.L., Fan S.T., Lo C.M., Wei W.I., Ng W.M., Chung H.Y., Ng K.K.C., Chan S.C., Chan K.W., Tso W.K., Tsoi N.S. and Tam P.K.H., Pediatric liver transplantation in Hong Kong - a domain with scarce deceased donors, Journal of Pediatric Surgery. 2009, 44(12): 2316-2321.

Chua C.W., Chiu Y.T., Yuen H.F., Chan K.W., Man K., Wang X., Ling M.T. and Wong Y.C., Suppression of androgen-independent prostate cancer cell aggressiveness by FTY720: validating Runx2 as a potential antimetastatic drug screening platform, Clinical Cancer Research. 2009, 15(13): 4322-4335.

Dai Y., Qiao L., Chan K.W., Yang M., Ye J., Zhang R., Ma J., Zou B., Lam S.C., Wang J., Pang R.W.C., Tan V.P.Y., Lan H.Y. and Wong B.C.Y., Adenovirus-mediated down-regulationof X-linked inhibitor of apoptosis protein inhibits colon cancer, Molecular Cancer Therapeutics. 2009, 8(9): 2762-2770.

Kwok J.S.Y., Chan G.S.W., Lam M.F., Yan T., Tang L., Kwong K.M., Chan K.W. and Chan D.T.M., Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes, Clinical transplantation. 2010, [Epub ahead of print 2010 Apr 9].

Liu S., Chan K.W., Tong J., Wang Y., Wang B.Y. and Qiao L., Fibrolamellar hepatocellular carcinoma detected by PET-CT scan: A case report., Hepato-Gastroenterology . 2009, In press.

Liu S., Chan K.W., Wang B.Y. and Qiao L., Fibrolamellar hepatocellular carcinoma. , Am J Gastroenterol . 2009, In press.

Liu S., Chan K.W., Wang B. and Qiao L., Fibrolamellar hepatocellular carcinoma, Am J Gastroenterol. 2009, 104(10): 2617-24; quiz 2625.

Lo P.H.Y., Lung H.L., Cheung A.K.L., Apte S.S., Chan K.W., Kwong F.M., Ko J.M.Y., Cheng Y., Law S.Y.K., Srivastava G., Zabarovsky E.R., Tsao G.S.W., Tang J.C.O., Stanbridge E.J. and Lung M.L., Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis, Cancer Research. 2010, 70(13): 5567-76.

Ma S.K.Y., Chan K.W. and Guan X.Y., Cancer Stem Cells - Concepts, Methodologies and Therapeutic Implications, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Ma S.K.Y., Tang K.H., Chan K.W. and Guan X.Y., Liver Cancer Stem Cells - A Review of Current Literature and Protocols, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Ma S.K.Y., Chan Y.P., Kwan P.S., Tang K.H., Vielkind J.V., Guan X.Y. and Chan K.W., microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression , American Association for Cancer Research. 2010.

Tang S.C.W., Chan Y.Y., Leung J.C.K., Cheng A.S., Chan K.W., Lan H.Y. and Lai K.N., Bradykinin and high glucose promote renal tubular inflammation, Nephrol Dial Transplant. 2010, 25(3): 698-710.

Tong D.K.H., Law S.Y.K., Kwong D.L.W., Chan K.W., Lam A.K.Y. and Wong K.H., Histopathological regression of the primary tumor indicated by percentage of residual viable cells is an important prognostic factor after neoadjuvant chemoradiation therapy for esophageal cancer (Abstract), GASTRO 2009 UEGW/WCOG, London, United Kingdom, 21-25 November 2009.

Tong D.K.H., Law S.Y.K., Kwong D.L.W., Chan K.W., Lam A.K.Y. and Wong K.H., Histopathological regression of the primary tumor indicated by percentage of residual viable cells is an important prognostic factor after neoadjuvant chemoradiation therapy for esophageal cancer, Gut. 2009, Supplement No II Vol 58 - Endoscopy Supplement No I Vol 41: A74.

Tsui K.K. and Chan K.W., Seasonality of Serum Prostate-Specific Antigen Levels and Its Impact on Prostate Cancer Screening in Hong Kong, Journal of Hong Kong Institute of Medical Laboratory Sciences. 2010, 12: 13-24.

Yuen H.F., Chiu Y.T., Chan K.K., Chua C.W., McCrudden C.M., Tang K.H., El-Tanani M., Wong Y.C., Wang X. and Chan K.W., Prostate Cancer Cells Modulate Osteoblast Mineralisation And Osteoclast Differentiation Through Id-1 , British Journal of Cancer. 2010, 102: 332-341.

Researcher : Chan LC

Project Title:	To develop a pilot humanities program in the first year undergraduate medical curriculum 2007-2008.
Investigator(s):	Chan LC
Department:	Pathology
Source(s) of Funding:	Leung Kau Kui Research and Teaching Endowment Fund - Teaching Grants
Start Date:	08/2007

Abstract:

Background: Medical curricula in many universities are undergoing changes to enable students to be life long learners in view of rapid advances in biomedical knowledge and medical technology. Students are also taught clinical interpersonal skills to help them communicate better with patients and their families. Very little, if any, is taught in the area of the humanities, reflective endeavors which seek to retreive, record and interpret the span of human experience (1). The humanities value reason and explore emotion, focus on meaning and ambiguity - perspectives of which come from multiple disciplines including literature, drama, philosophy, theology, anthropology, and law. Through the study of the humanities, one comes to a deeper understanding of the human condition and the nature of suffering as experienced by the patient. The objective of the project proposal is to develop a pilot humanities program in the first year medical undergraduate curricululum to expose students to the field of humanities and its relevance to medical training and practice.

Project Title:	Leukaemogenic potential of ex vivo expanded cord blood haematopoietic stem cells by epigenetic modifying agents
Investigator(s):	Chan LC, Ng RK
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009
Completion Date:	04/2010

Abstract:

The aim of this project is to investigate the effectiveness of epigenetic modifying agents, 5-aza-deoxycytidine (5-aza-dC), trichostatin A (TSA) and valproic acid (VPA), on the ex vivo expansion of functional cord blood haematopoietic stem cells (CB-HSCs). In addition, this project aims to justify the feasibility of using these ex vivo expanded CB-HSCs for therapeutic purposes by determining the leukaemogenic potential of these cells. Haematopoietic stem cell (HSC) is a multipotent cell type which is able to self-renew and differentiate to all blood lineages. It has been shown that bone marrow and umbilical cord blood are the major sources of HSCs. Nevertheless, the proportion of HSCs is usually less than 1% of the total cell population. Because HSCs are able to give rise to all mature blood cell types, transplantation of HSC provides a promising approach in treating haematological disorders, e.g. leukaemias. CB-HSC transplantation does not necessary require a strong immunological compatibility to the recipient because of the immunity of immune system in the donor newborn, which makes the newborn CB-HSC has a wider range of clinical applications than adult BM-HSC. There are several attempts on the ex vivo expansion of HSCs, for examples, by co-culturing with a combination of cytokines or by overexpression of HoxB4. However, even the total population of CD34+ HSCs is increased by these approaches, it is demonstrated that these ex vivo expanded cells loss their competence in repopulating in the transplanted recipients, and hence differentiated into other committed blood progenitors by losing its self-renewal characteristics. Thus, it largely limits the application of HSC for therapeutic purposes. One of the challenges in the area of HSC clinical research would be developing an effective approach to amplify HSC population without deteriorating its stem cell characteristics/functions after transplantation. Recent studies have provided new insights on the epigenetic regulations in stem cell self-renewal and differentiation. Interestingly, both bone marrow (BM-) and cord blood HSCs (CB-HSCs) were shown to have population expanded after treatment with epigenetic modifying agents, such as 5-aza-dC, TSA and VPA. Engraftment experiments demonstrated that there was an enhancement of repopulation in the transplanted mice by a sub-population (CD34+CD90+) of these ex vivo expanded HSCs. Therefore, it raises a potential usage of these agents on the expansion of HSC through manipulation of its epigenetic status, which includes DNA methylation and histone acetylation. It is noted that 5-aza-dC, TSA and VPA have been used for decades in the treatment of human cancers and other diseases. The most common inhibitor of DNA methylation is 5-aza-dC, which is a cytosine analogue. The incorporation of 5-aza-dC into DNA allows an irreversible binding to DNA methyltransferase Dnmt1, and hence inhibits its function. Consequently, the methylation state of parental DNA cannot be maintained in the daughter cells and passive genome-wide demethylation occurs after every round of cell division. On the other hand, both TSA and VPA are the histone deacetylase (HDAC) inhibitors, which abolish the catalytic function of histone acetyltransferase activity of HDACs. The outcome of treatment with both 5-aza-dC and TSA/VPA is the induction of whole genome open chromatin structure and the activation of global transcriptional machinery in the treated cells. It is shown that the VPA-treated BM-HSCs have an up-regulation of HoxB4 expression as well as an activation of Wnt signaling pathway, which was previously shown to have important roles in the ex vivo expansion of HSC. Thus, it is speculated that treatment with these epigenetic modifying agents can activate key regulators involve in stimulating stem cell proliferation and getting out from a quiescent state. However, these agents have effects on the whole epigenome rather than specific loci. It therefore raises a possibility that genes with undesirable functions are also being randomly activated and expressed, e.g. apoptotic genes and proto-oncogenes, after the treatment. It is therefore necessary to justify the safety of utilizing these expanded cells for transplantation purposes, especially in the concern of the leukaemogenic potential of these cells in the recipients. In this project, we ask two important questions concerning about the feasibility of transplanting CB-HSCs after ex vivo expansion: 1) The effectiveness of 5-aza-dC and TSA/VPA on expanding the recently identified functional sub-population (CD34+CD90+CB38-CD45RA-Lin-) of CB-HSCs; 2) The induction of epigenetically silenced proto-oncogenes and leukaemic genes in ex vivo expanded CB-HSCs after incubation with 5-aza-dC and TSA/VPA. The answers of these questions thus provide valuable information on the safety and feasibility of transplanting CB-HSC from this expansion strategy.

List of Research Outputs

Chan L.C., Chen J.Y. and Salter Menzo D.J., Communicating humanistic values through doctors' and patient stories , Frontiers in Medical and Health Sciences Education "Making Sense in Communication" . 2009.

Chan L.C., Creating a learning environment to develop students' reflection and communication about medical humanities. , Frontiers in Medical and Health Sciences in Education 2009.

Chan L.C., Cultivating the Heart of Mindfulness in Everyday Life, Hong Kong Cancer Fund. The University of Hong Kong Taskforce in Medical Humanities, Li Ka Shing Faculty of Medicine. Centre on Behavioral Health, November . 2009.

Chan L.C., Enhancing PBL through the students’ world of learning, Deaprtment of Nursing Studies, Li Ka Shing Faculty of Medicine, November. 2009.

Chan L.C., Facilitator of Seminar on “Curriculum as Conversation”, Institute of Medical and Health Sciences Education (IMHSE) and Enhancement of Teaching and Learning (CETL), The University of Hong Kong, May. 2010.

Chan L.C., How do we meet the needs of students who experience difficulties in learning, Learning and Community and Peer Support Network, Centre for the Enhancement of Teaching and Learning (CETL), The University of Hong Kong, April . 2010.

Chan L.C., Integrating the Teaching of Medical Humanities in the Undergraduate Medical Curriculum – The Hong Kong Experience, New Approaches to Medical Education: Dialogue between the East and the West on Medical Humanities, Beijing, China, October. 2009.

Chan L.C., Chen J.Y. and Salter Menzo D.J., The development of a medical humanities special study module, 7th Asia Pacific Medical Education Conference. 2010.

Chen J.Y., Chan L.C. and Salter Menzo D.J., Communicating humanistic values: students perspectives through visual art, Frontiers in Medical and Health Sciences Education "Making Sense in Communication" . 2009.

Chen J.Y., Salter Menzo D.J. and Chan L.C., Pen, brush, and camera: exploring doctor and patient stories and why they matter – a medical humanities elective module for medical undergraduates, 7th International Medical Education Conference. 2010.

Chen J.Y., Salter Menzo D.J. and Chan L.C., Pen, brush, and camera: exploring doctor and patient stories and why they matter – a medical humanities elective module for medical undergraduates, In: Ron Harden Innovation in Medical Education Award, 7th International Medical Education Conference, International Medical University, Malaysia. 2010.

Chen J.Y., Chan L.C. and Salter Menzo D.J., Student perception of a medical humanities special study module, 7th Asia Pacific Medical Education Conference . 2010.

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Meyer C., Kowarz E., Hofmann J., Renneville A., Zuna J., Trka J., Ben Abdelali R., Macintyre E., De Braekeleer E., De Braekeleer M., Delabesse E E., de Oliveira M.P., Cavé H., Clappier E., van Dongen J.J., Balgobind B.V., van den Heuvel-Eibrink M.M., Beverloo H.B., Panzer-Grümayer R., Teigler-Schlegel A., Harbott J., Kjeldsen E., Schnittger S., Koehl U., Gruhn B., Heidenreich O., Chan L.C., Yip S.F., Krzywinski M., Eckert C., Mőricke A., Schrappe M., Alonso C.N., Schäfer B.W., Krauter J., Lee D.A., Zur Stadt U., Te Kronnie G., Sutton R., Izraeli S., Trakhtenbrot L., Lo Nigro L., Tsaur G., Fechina L., Szczepanski T., Strehl S., Ilencikova D., Molkentin M., Burmeister T., Dingermann T., Klingebiel T. and Marschalek R., New insights to the MLL recombinome of acute leukemias, Leukemia. 2009, 23(8): 1490-1499.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. ELSEVIER, 2010, 38: 481-488.

Salter Menzo D.J., Chan L.C. and Chen J.Y., Creating a learning environment to develop students' reflection and communication about medical humanities , Frontiers in Medical and Health Sciences Education "Making Sense in Communication" . 2009.

So J.C.C., So A.C., Chan A.Y., Tsang S.T.Y., Ma E.S.K. and Chan L.C., Detection and characterisation of beta-globin gene cluster deletions in Chinese using multiplex ligation-dependent probe amplification, J Clin Pathol. 2009, 62(12): 1107-11.

Researcher : Chan LK

Project Title:	AACR 101st Annual Meeting 2010 Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo
Investigator(s):	Chan LK
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Chan L.K., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo , American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Chan L.K., Tung K.K., Ng I.O.L. and Yam J.W.P., Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma, American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Leung T.H.Y., Yam J.W.P., Chan L.K., Ching Y.P. and Ng I.O.L., DLC2 (Deleted in liver cancer 2) suppresses cell growth via regulation of Raf-1-ERK1/2-p70S6K signaling pathway. (accepted), Liver International. 2010.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Chan SY

List of Research Outputs

Long J., Cai Q., Qu S., Li C., Zheng Y., Gu K., Wang W., Xiang Y.B., Cheng J., Chen K., Zhang L., Zheng H., Shen C.Y., Huang C.S., Hou M.F., Shen H., Hu Z., Wang F., Deming S.L., Kelley M.C., Shrubsole M.J., Khoo U.S., Chan Y.K., Chan S.Y., Haiman C.A., Henderson B.E., Le Marchand L., Iwasaki M., Kasuga Y., Tsugane S., Matsuo K., Tajima K., Iwata H., Huang B., Shi J., Li G., Wen W., Gao Y.T., Lu W., Zheng W. and Shu X.O., Identification of a functional genetic variant at 16q12.1 for breast cancer risk: results from the Asia Breast Cancer Consortium., PLoS Genetics. 2010, 6: e1001002.

Researcher : Chan TL

Project Title:	Molecular characterisation of the serrated neoplasia pathway and its role in the development of colorectal cancer with mismatch repair deficiency
Investigator(s):	Chan TL, Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2005

Abstract:

Colorectal cancer (CRC) is one of the commonest cancers world-wide with 850,000 new cases each year. Majority of CRC are known to develop through an adenoma-carcinoma sequence, with molecular genetic changes that characterize each transition step. Adenoma is considered pre-malignant and early prevention and treatment of CRC is possible through regular endoscopic surveillance and removal of tumour at the adenoma stage. However, despite regular surveillance, some patients still develop CRC. One of the possibilities is that there exists another pathway of tumour development. Recently, there are data to suggest the existence of an alternative route, the serrated neoplasia pathway, for CRC development. This latter pathway includes those serrated polyps (SP) that span a morphological spectrum from hyperplastic polyp (HP) to serrated adenoma (SA). The earliest member in this pathway, HP, is a very common lesion in aged individuals and it is a long held belief that HPs are innocent with little propensity for malignant progression. However, a small subset of HPs may progress but the criteria to distinguish the high risk versus the low risk ones are unclear. Concurrently, it is known that a subset (15%) of sporadic CRC manifest a form of genetic instability called microsatellite instability (MSI). This is due to promoter methylation leading to loss of expression of the DNA mismatch repair protein MLH1. The evolution of this subset of CRC is unclear as a preceding adenoma phase is very rarely seen. Recently, studies by us and others have provided molecular evidences to suggest that there may be a strong link between the serrated neoplasia pathway and the development of sporadic CRC with MSI. These include the identification of a high incidence of BRAF mutation in HPs and SAs, the occurrence of MSI in some SPs and the selective association of BRAF mutation with sporadic late-onset CRC with MSI. Our recent pilot study has shown that MLH1 inactivation can be detected in SPs at its very early phase and these can just involve several crypts within the lesion. With these data, we propose to perform a large scale phenotypic and molecular characterization of serrated polyps at their early phase of evolution to define the link between SPs and MSI CRC, and the temporal sequence of genetic changes in this pathway. Aims: 1. To look for evidence of MLH1 inactivation in various stages of evolution of serrated polyps, and to document its incidence and phenotypic characteristics. 2. To document the occurrence of microsatellite instability and frameshift mutation in growth regulatory genes containing microsatellite encoding region as consequences of MLH1 inactivation in various stages of serrated polyps. 3. To analyse the inter-relationship between MLH1 inactivation, BRAF/KRAS mutations and presence of the CpG Island Methylator Phenotype in SPs and their temporal sequence of occurrence. 4. To look for alteration in major molecular genetic pathways in SPs with MLH1 inactivation. 5. To look for evidence of a field effect of MLH1 inactivation in the colon in patients with sporadic late-onset MSI colorectal cancer with MLH1 promoter methylation. 6. To look for clinical, morphological or molecular markers that can distinguish SPs with high risk of progression to MSI CRC.

Project Title:	Expression of microRNAs in various stages and pathways of colorectal carcinogenesis
Investigator(s):	Chan TL, Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007

Abstract:

To profile the expression of miRNAs in normal mucosae, adenomas of the colon, and to compare the differences in miRNA expression profiles of the progenitor cells residing at the base of the crypt and the mature cells residing at the top; to compare the variation in expression of miRNAs in different molecular subtypes of CRC including those with microsatellite instability, microsatellite stabel or microsatellite and chromosome stable phenotype; to identify specific miRNAs if interest that show altered expression in the course of CRC development, or in specific molecular subgroups of CRC; to investigate the function of these miRNAs by over-expression or gene silencing appraoch in cell culture system; to delineated the putative protein-coding target(s) regulated by these miRNA(s) using bioinformatics approach and their validation by western blotting.

Project Title:	The Role and Mechanism of CpG Island Methylator Phenotype (CIMP) in Early-Onset Colorectal Cancer
Investigator(s):	Chan TL
Department:	Pathology
Source(s) of Funding:	Michael and Betty Kadoorie Cancer Genetics Research Programme (MBKCGRP) II
Start Date:	01/2008

Abstract:

(1) To determine the frequency of the CpG Island Methylator Phenotype (CIMP) in the patients with early-onset CRC (i.e. MSI, CIN and MACS sub-groups) (2) To compare the frequencies of promoter methylation in frequently targeted tumour suppressor genes in the CIMP-positive cases between different sub-groups of CRC (3) To identify underlying mechanisms of the CIMP, with a main focus on assessment of SNPs upstream of frequently methylated tumor suppressor genes

Project Title:	Study of the mechanisms underlying heritable germline epimutation of MSH2 in Hereditary Nonpolyposis Colorectal Cancer
Investigator(s):	Chan TL, Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	08/2008

Abstract:

(1) To identify the minimal chromosomal region that is linked to the inheritance of germline methylation of MSH2 gene promoter based on recombination events in two un-related families; (2) To systematically map for the presence of methylation in the adjacent genes and CpG sites in the minimal chromosomal region so as to map the extend of aberrant methylation and to guide downstream genomic analysis; (3) To systematically look for genetic changes in this minimal chromosomal region using the Solexa ultra-high throughput DNA Sequencing Technology and the ultra-high density and long oligo probe-based microarray comparative genomic hybridisation (NimbleGen Systems Inc); (4) To establish lymphoblastoid cell lines from individuals with heritable MSH2 methylation; (5) To look for changes in chromatin modification pattern by examination of the histone codes in the lymphoblastoid cell lines and rectal mucosa, so as to correlate these with the status of MSH2 methylation.

Project Title:	Outstanding Young Researcher Award 2007-2008
Investigator(s):	Chan TL
Department:	Pathology
Source(s) of Funding:	Outstanding Young Researcher Award
Start Date:	10/2008

Abstract:

The Awards are intended to recognize, reward, and promote exceptional research accomplishments of academic and research staff.

Project Title:	An in vivo Model of Genetically Driven Epigenetic Gene Regulation
Investigator(s):	Chan TL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2009
Completion Date:	05/2010

Abstract:

Our group, recently, identified an allele-specific heritable epimutation of MSH2 in a family affected by hereditary non-polyposis colorectal cancer (HNPCC), in which a disease haplotype co-segregated with the epimutation phenotype was found in three successive generations; adenomas, colorectal and endometrial cancers were found in four affected members (Chan Nat Genet 2006). Interestingly, quantitative analysis indicated that the level of methylation varied from tissues to tissues, with the highest in colonic specimens and the lowest in leukocytes. Further inspection identified the same epimutation in an additional Chinese family and a similar epimutation has been reported in four Dutch families. My team and our Dutch collaborator had simultaneously illustrated the basic mechanism for this novel event, which involved a deletion at the 3’ end of a gene (TACSTD1) upstream of MSH2 , inducing loss of termination signal for the nascent RNA. A phenomenon called transcript read-through, which leads to the generation of a fusion transcript encompassing the upstream TACSTD1 and downstream MSH2 genes, was identified. As demonstrated by our group with pyrosequencing and real-time quantitative PCR methods, the mosaic levels of methylation were associated with the differential expressions of TACSTD1 in different tissues, such that the degree of methylation was proportional to the expression of TACSTD1 (Lightenberg*, Kuiper*, Chan* Nat Genet 2008 in press). We have just opened a novel area, establishing the possible contribution of genetic deletion to epimutation and illustrating the tissue- and gene expression-dependent nature of methylation. Further verification of these mechanisms and phenomena in a suitable model is anticipated. In this study, we propose to verify the mechanism of genetic-driven epigenetic regulation in the cells with intact TACSTD1 and MSH2 by homologous recombination of a construct with deletion. It has been demonstrated that level of methylation in the intracisternal A particle (IAP) element located upstream of the Agouti gene associate with variation of phenotypic expression of coat colour in mice. Maternal exposure of external agent such as bisphenol A, which is responsible for the induction of hypomethylation, lead to reduction of CpG methylation in the IAP element and result in shifting coat colour from light yellow to brown in new born mice, Interestingly, maternal dietary supplements can negate the adverse effect of bisphenol A. The successfulness of the assembly of this construct as well as homologous recombination provides a chance to investigate the epigenetic therapy through in vivo study. If we can reproduce the genetic-driven epigenetic regulation by our proposed model, we can also apply this specific mechanism to silent other genes of interest epigenetically rather than genetically. In this proposal, all in vivo studies will be limit to cell culture of, preferably, embryonic stem cells and cells with epithelial origin. Objectives 1) To create genomic constructs suitable for homologous recombination 2) To perform electroporation and estimate the frequency of homologous recombination in different type of cell lines *Co-first authors in this article

Project Title:	The Genome Wide Epigenetic study of Early-onset Colorectal Cancer Patients with CpG Island Methylator Phenotype (CIMP)
Investigator(s):	Chan TL
Department:	Pathology
Source(s) of Funding:	Germany/Hong Kong Joint Research Scheme
Start Date:	01/2010

Abstract:

Refer to hard copy

List of Research Outputs

Chan T.L., Academic editor, PLoS ONE. PloS One, 2010.

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Chan WY

Project Title:	28th Annual Meeting of American Society for Virology Differential Viral Replication kinetics and host innate immune response by Influenza A (H5N1) virus in human bronchial epithelial cells at different differentiation stages
Investigator(s):	Chan WY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	07/2009
Completion Date:	07/2009

Abstract:

N/A

Project Title:	Establishment of primary human conjunctival epithelial cell in vitro and conjunctiva ex vivo organ culture model to study human infection of influenza virus.
Investigator(s):	Chan WY, Peiris JSM, Nicholls JM
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	10/2009

Abstract:

The objective of this study is to establish primary cell and organ cultures of human conjunctival epithelium, which can act as an in vitro and ex vivo model to study human infection by influenza virus in this tissue region. The ongoing swine origin influenza H1N1 virus pandemic started from mid-March 2009 and has caused more than 36 thousands reported human confirmed cases worldwide. Moreover, the previous outbreaks of avian influenza H5N1 virus in year 1997 and 2003 resulted in abnormally severity in human infection with high mortality rate of 33-80%. Sporadically, influenza H7N7 and H7N3 virus caused human conjunctivitis and a sinlge fatality case was reported. As influenza virus is prone to have mutation and frequent reassortment, more virulent mutants equipped with good human-to-human transmissibility and high virulence may eventually appear and cause major impact to humans. While the replication kinetics and the innate immune response of influenza A virus have been extensively studied in the respiratory epithelial cells by our laboratory and others, the investigation to address the possible route of transmission of these viruses is exceptionally crucial. With the fact that human conjunctiva is an epithelium that lines as a barrier between eye and the external environment which contains sialic acid receptors for influenza virus entry, this epithelium should be an area of interest. Up to now, there has been limited study on the infection of influenza virus on the human eye epithelium. The evaluation of influenza virus receptor on the human eye epithelium has been sparse. This lack hinders our understanding of the host-pathogen interaction and the understanding the pathogenesis of this disease. As a result, the establishment of primary in vitro cell culture of human conjunctival epithelial cell and human conjunctival ex vivo organ culture will provide ideal models to study the virus binding, replication and possible physiological and cellular response of the conjunctiva tissue when the virus first encounters the human body at the eye epithelium. These could be addressed using immunohistochemisty, glycan analysis using mass spectrometry and microarrays.

List of Research Outputs

Chan M.C.W., Chan W.Y., Yu C.L., Ho C.C., Chui W.H., Lo C.K., Yuen K.M., Guan Y., Nicholls J.M. and Peiris J.S.M., Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells, Respiratory Research. 2009, 10: 102.

Chan M.C.W., Chan W.Y., Yu C.L., Ho C.C., Yuen K.M., Fong J.H.M., Tang L.L.S., Lai W.W.K., Lo A.C.Y., Chui W.H., Sihoe A.D.L., Kwong D.L.W., Tsao G.S.W., Poon L.L.M., Guan Y., Nicholls J.M. and Peiris J.S.M., Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract, American Journal of Pathology. 2010, 176(4): 1828-40.

Chan W.Y., Chan M.C.W., Wong C.N., Karamanska R., Dell A., Haslam S.M., Sihoe A.D., Chui W.H., Triana-Baltzer G., Li Q., Peiris J.S.M., Fang F. and Nicholls J.M., DAS181 Inhibits H5N1 Influenza virus Infection of Human Lung Tissues., Antimicrobial Agents and Chemotherapy. 2009, 53(9): 3935-3941.

Chan W.Y., Yuen K.M., Yu C.L., Ho C.C., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Differential Viral Replication Kinetics And Host Innate Immune Responses By Influenza A (H5N1) Virus In Human Bronchial Epithelial Cells At Different Differentiation Stages, The 28th Annual meeting of American Society for Virology. 2009.

Chan W.Y., Yu C.L., Yuen K.M., Fong J.H.M., Lo A.C.Y., Lai W.W.K., Wong D.S.H., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Infection of influenza A (H5N1) virus in human eye epithelium, an in vitro and ex vivo study , The 28th Annual Meeting of the American Society of Virology. 2009.

Chan W.Y., Yuen K.M., Yu C.L., Ho C.C., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Influenza H5n1 And H1n1 Virus Replication And Innate Immune Responses In Bronchial Epithelial Cells Are Influenced By The State Of Differentiation, PLoS One. 2010, 5 (1): e8713.

Chan W.Y., Chan M.C.W., Nicholls J.M. and Peiris J.S.M., Tropism and host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract , XII International Symposium on Respiratory Viral Infections. 2010.

Chan W.Y., Young Investigator Award, The Macrae Group LLC. 2010.

Triana-Baltzer G.B., Babizki M., Chan M.C.W., Wong A.C.N., Aschenbrenner L.M., Campbell E.R., Li Q.X., Chan W.Y., Peiris J.S.M., Nicholls J.M. and Fang F., DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis., J Antimicrob Chemother. 2010, 65: 275-84.

Triana-Baltzer G.B., Gubareva L.V., Nicholls J.M., Pearce M.B., Mishin V.P., Belser J.A., Chen L.M., Chan W.Y., Chan M.C.W., Hedlund M., Larson J.L., Moss R.B., Katz J.M., Tumpey T.M. and Fang F., Novel pandemic influenza A(H1N1) viruses are potently inhibited by DAS181, a sialidase fusion protein, PLoS One. 2009, 4: e7788.

Triana-Baltzer G.B., Gubareva L.V., Nicholls J.M., Pearce M.B., Mishin V.P., Belser J.A., Chen L.M., Chan W.Y., Chan M.C.W., Klimov A.A., Hedlund M., Wurtman D., Moss R.B., Katz J.M., Tumpey T.M., Belshe R.B. and Fang F., Pandemic H1N1 2009 and Drug Resistant Influenza Viruses are Potently Inhibited by DAS181, a Sialidase Fusion Protein, Interscience Conference on Antimicrobial Agents and Chemotherapy. 2009.

Researcher : Chan YK

Project Title:	Identification of transcriptional factors interacting with the BRCA1 promoter
Investigator(s):	Chan YK, Khoo US, Ip YC
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2008
Completion Date:	03/2010

Abstract:

Transcription factors (TFs) can regulate gene expression through the formation of transcription initiation complex and recruitment of RNA-polymerase to drive transcription activity. Depending upon genes and promoter sequences, different TFs will be utilized for transcription initiation or suppression. Differential expressions of TFs have previously been observed to play roles in targeting gene expression regulation in breast cancer, and to have correlation with chemotherapeutic treatments and disease prognosis. Some of them can define tumor progression and regulate breast cancer cell invasion, growth and even sensitivity to anti-estrogen treatment. Some TFs are being developed as therapeutic targets for breast cancer. The breast cancer susceptibility gene 1 (BRCA1) plays a significant role in breast cancer development. A few TFs, such as CREB and E2F, which bind to the proximal end of the BRCA1 promoter, have been previously shown to regulate BRCA1 expression, where their expressions are found to have clinical association and are thought to play functional roles in carcinogenesis. Analyzing 1.7kb of the BRCA1 promoter, we have demonstrated the promoter region located -969bp upstream to the transcription-start-site of the gene contained a binding-site for unique TFs. By electrophoretic-mobility-shift assay (EMSA), we demonstrated a genetic change at this site significantly altered the binding affinity of TFs and promoter activity. Genetic association analysis on two independent Chinese cohorts (>3000 subjects) demonstrated that this genetic change resulted in significant reduction of breast cancer risk. Simple TF search, using TFSEARCH, had suggested a few putative TFs to this site; however, co-transfection assay failed to confirm their interaction with the BRCA1 promoter. The conventional in-silico TF search usually makes use existing sequence matrix/algorithm of TF binding-sites to predict putative TFs. However, frequently these predicted TFs may not be the “true” TFs to the promoter of interest after verification by functional analysis. A novel and powerful computational-analysis using gene expression signature has been developed to deduce the promoter composition of genes within which regulatory TFs can be identified. This is based on the rational assumption that coexpressed genes share similar properties of gene regulation. Similar regulated genes should have a higher probability of being regulated by similar transcription factors and transcriptional pathways. This method can minimize the false-positive TFs and has the advantage over the conventional use of DNA sequences, as promoter sequences are well known to maintain a natural bias of being GC-rich. Thus, GC-rich TF binding-sites will not be inappropriately over-represented. By this novel method of computational analysis for TFs, much fewer but biological important TFs will be generated rather than the multitude of TFs normally predicted by conventional in-silico TF search for the BRCA1 promoter, particularly the -969 region. Recently, BRCA1 was found to interact with estrogen receptor alpha (ESR1) resulting in the inhibition of estradiol (E2)-stimulated ESR1 transcriptional activity in breast cancer cells. The effectiveness of anti-estrogen therapy, such as Tamoxifen, is dependent upon positive expression of ESR1 in cancer cells. It will be worth investigating whether TFs, through controlling the expression of BRCA1 may influence the level of inhibition to ESR1 activity which can influence responsiveness of cancer cells to Tamoxifen treatment. We hypothesize that (1) the 1.7kb BRCA1 promoter, particularly at the -969 region, may contain important TFs aside from those previously reported, and (2) these TFs may provide an alternative mechanism to regulate the expression of BRCA1 which could affect cellular characteristics, such as anti-estrogen drug sensitivity, of breast cancer cells. Objectives: 1. To identify biologically important TFs and to map their binding-sites to the 1.7kb of BRCA1 promoter 2. To investigate the functional role of the identified TFs in relation to responsiveness to Tamoxifen treatment on breast cancer cells

Project Title:	Characterization of BRCA2 Variants of Unknown Significance and Mutations
Investigator(s):	Chan YK, Khoo US
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2009
Completion Date:	01/2011

Abstract:

Breast cancer is the commonest cause of cancer in women. Its incidence has been steadily rising in the last few decades and remains the commonest female cancer with an age-standard rate of 47.5/100,000 in Hong Kong (Hong Kong Cancer Registry 2005). Though the rate is lower than that in Caucasians, it is the highest reported in Asian regions. The breast cancer susceptibility genes BRCA1 and BRCA2 are well-known to be associated with breast and ovarian cancer development in females. Mutations found in either of these two genes account for more than half of all familial hereditary breast and/or ovarian cancer cases. Women harboring BRCA1 and/or BRCA2 mutations have a lifetime risk of developing breast cancer as high as 87%. Familial breast cancer cases carrying deleterious/disease-causing BRCA1 and BRCA2 mutations were found to have significant association with several important histological features, such as triple-negative breast cancers higher mitotic counts, higher continuous pushing margin, higher nuclear pleomorphism, etc. Although earlier studies reported no significant differences in survival between BRCA1/2 mutation carriers and non-carriers, it has recently been observed for Israeli Jewish breast cancer patients who received chemotherapy, that BRCA1 mutation carriers for the deleterious truncating founder mutations 185delAG and 5382insC, tended to have better survival than the non-carriers (hazard ratio=0.48, 95%CI=0.19-1.21; p=0.12)1. This suggests that BRCA deleterious mutations may be able to determine response to chemotherapeutic treatment, and to predict disease progression and outcome. In contrast to deleterious mutations causing truncated BRCA proteins, genetic alterations including missense and silence mutations, in-frame insertion/deletions, and mutations in the intron-exon junction and untranslated regions are of uncertain significance, and are named variants of unknown significance (VUSs). These can cause amino-acid substitution, in-frame shift, or aberrant splicing events, some which may be deleterious, whilst others have uncertain functional effect. Furthermore, truncating variants in the carboxyl-domain of BRCA2 have been shown to be neutral polymorphisms. Evidences to suggest a genetic alteration is a VUS include the following: (i) the variant can be found in a control group, but with equal or greater prevalence in a cancer group, (ii) the variant does not co-segregate with the disease in a family with multiple cancer cases, (iii) the variant co-occurred in-trans (opposite allele) with a deleterious mutation in the same gene, (iv) the resulting amino acid substitution is of a similar class of amino acid as the wild type, (v) the wild-type amino acid is not highly conserved among mammalian species. Difficulty in assessing risk and in making clinical recommendations for breast and/or ovarian cancer patients especially arises when a VUS is detected. Therefore, schematic analyses and functional tests have been developed to examine and classify the effect of the BRCA VUSs. A large scale systematic genetic assessment on 1433 VUSs identified from the Myriad Genetic Laboratories database which contains the results of full BRCA genes sequence analysis of 60,000 test probands revealed that 20% of these VUSs are deleterious. Most of cases in this study were of European ancestry. Such assessments would be even more invaluable if they could be validated by functional and in-silico molecular analyses. Two independent studies have integrated clinical parameters and tumor markers, such as tumor grade, estrogen receptor status, and cytokeratin 5/6 and cytokeratin 14 (to define basal-like tumor subtype) of the tumor of the VUSs carriers into the analysis model, suggested that about 9% and 18% of them were deleterious, respectively. For Chinese breast or ovarian cancer, about one hundred deleterious BRCA1 and BRCA2 mutations have been reported. However most of these mutations appear to be distinct from that found in other ethnic groups. Interestingly, many of these mutations are not associated with strong family history, suggesting the possibility of weaker penetrance. Recently, Dr Sharan and his colleagues, from the National Institute of Health (NIH), USA, have established a mouse embryonic-stem (ES)-cell based functional assay that is simple, versatile and reliable to test for the functional significance of BRCA mutations. This ES-cell approach is more superior than other in-vitro assays reported previously because those assays rely on expression vectors to deliver mutant transcripts or proteins to cells that are inherently prone to genomic instability. With the use of this assay, Dr Sharan and his colleagues confirmed that not all truncating mutations are deleterious, and even deleterious mutations had variable capacity to rescue cell viability of conditional knock-out ES-cells. The assay was also able to classify VUSs as deleterious, neutral or low risk, suggesting that different mutations give rise to clear differences in functionality. We postulate that this may account for possible differences in penetrance depending on the expressivity of the mutation and deleterious VUSs. This may explain why not all BRCA mutations give rise to identical pathological features, biological behavior, and chemotherapeutic response. Through our on-going BRCA genes mutation screening project, we have identified several BRCA2 VUSs and novel germline mutation in our local Chinese breast cancer patients. These VUSs and mutations are unique and have not been reported previously in other ethnic groups or deposited in the Breast Cancer Information Core database. Therefore, we believe that there is a need to examine these BRCA2 VUSs and mutations by functional assays in order to better characterize these genetic changes and to assess their functional effect. In this study, we proposed to use the mouse ES-cell based functional assays developed by Dr Sharan to evaluate the functional effect of the gene products harboring these BRCA2 VUSs and mutations and to correlate the functional assay findings with clinical, pathological.

Project Title:	Validating and examining SNPs identified from genome-wide scanning for disease susceptibility to HBV-related liver cancer in Chinese population
Investigator(s):	Chan YK, Ng IOL, Wong CM
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

Hepatitis B virus (HBV) infection is one of the major infectious diseases, with more than 350 million carriers worldwide. HBV infection is particular common in Sub-Saharan Africa, Southeast Asia, and South China. About 5-10% of patients with acute HBV infection may progress to chronic liver disease including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Cirrhosis is the end-stage liver failure and is a frequent cause of death in chronic HBV carriers. HCC is a dreaded complication of chronic HBV infection as it is rapidly fatal when symptomatic and has a high rate of cancer recurrence even after surgical resection. In fact, HCC is the second commonest fatal cancer in HBV prevalent regions. It is commonly believed that a complex combination of host genetic, viral and environmental factors plays a critical role in determining the course of infection. The outcome of HBV infection can be determined to a major degree by the host genetic background. Previous studies based on well-defined human genes with well-known functions have shown a significant relation between chronic HBV infection and specific gene polymorphisms. Those genetic association studies based on Gambia, European, and Asia cohorts have implicated the HLA allele DRB1*1302 in the clearance of HBV infection (1-3). Several other population studies have also revealed that non-HLA loci are associated with persistent HBV infection or HBV clearance. These loci include interferon gamma, vitamin D receptor (4), and estrogen receptor alpha (5). SNP on the eIF-2 gene has also been reported to be of significance as a marker for interferon response (6). With regard to development of HCC in chronic HBV patients, studies of genetic determinants are emerging. Analyses have shown evidence of genetic susceptibility to HCC in patients with chronic HBV infection (7; 8). Furthermore, reports have revealed that polymorphisms of particular genes are associated with HCC development in chronic HBV patients, including cytokine (IL-1B, TNF-alpha) (9) and DNA repair (hMLH1) genes (10), TGF-beta (11) and UDP-glucuronsytransferase genes (12). Associations with a common haplotype at chromosome 4q and microRNA have also been suggested in the development of HCC in HBV patients (13; 14). Using candidate SNPs and genes approach, recent studies published by Gao’s research group reported that polymorphisms of TGF-beta1, CTLA-4, and CRHR2 were significantly increased susceptibility to HBV-related HCC in Han Chinese (15-17). Identification of the host genetic susceptibility loci for progression of chronic HBV infection by individual-gene approach has been difficult, and the number of genes identified so far is small. Development of complications in HBV patients is a complex outcome and it is likely that several polymorphic genes, instead of one major gene, will exert effects. With recent developed high-throughput genotyping technology, genome-wide association analysis has become feasible. By scanning the genomes of a large number of peoples, markers for DNA sequences with increasing risks of particular diseases are being uncovered (e.g. The Wellcome Trust Case Control Consortium 2007). Our research team is hitherto the first using genome-wide scanning to examine host genetic-susceptibility to HBV-related HCC, especially in Chinese population. Our recent genome-wide analysis (GWAS) using Human-610-Quad BeadChip (Illumina) has examined 100 chronic HBV-carriers (control group) and 100 HCC patients (case group). Both cohorts are ethnically Chinese and HBsAg positive. Subjects in the control and case groups were gender and age matched as described in the “Patients recruitment” in the Section VII. After completion of this Phase I study, a number of SNPs were observed with p-values <0.001, indicating that they were substantially associated with the disease susceptibility. In additional to these SNPs, we have selected total of 53 SNPs using our selection criteria. Therefore, we propose to validate our findings of these 53 SNPs and then replicate the validated SNPs using larger sample size. Objectives: (1) To validate SNPs identified from GWAS (2) To genotype the validated SNPs on larger sample size using Sequenom (3) To perform association analysis on the validated SNPs

List of Research Outputs

Chan Y.K., HPV/Pap triage in routine primary cervical cancer screening, 4th Biennial Meeting of the Asia Oceania research organisation on Genital Infections and Neoplasia (AOGIN), New Deli, India. 2010.

Chen J., Chan Y.K., Wu L.H., Ip Y.C., Lam E.W.F. and Khoo U.S., Forkhead Transcription Factor FOXO3a increases Akt Activity in Breast Cancer Cell Lines through a Feed-back Mechanism, The 16th Hong Kong International Cancer Congress, Hong Kong. 2009.

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Khoo U.S., Chan Y.K., Ching C.Y.J., Chan V.S.F., Ip Y.C., Yam L., Chu C.M., Lai S.T., So K.M., Wong T.Y., Chung P.H., Yip S.P., Sham P.C., Leung G.M., Lin C.L. and Peiris J.S.M., Functional role of ICAM-3 polymorphism in genetic susceptibility to SARS infection, Hong Kong Med Journal. 2009, 26-9.

Lau L.Y., Chan Y.K. and Khoo U.S., Identification of MicroRNAs Associated with Tamoxifen Resistance in Breast Cancer, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Liu S., Chan Y.K., Leung C.Y., Luk H.M., Lo S.T., Fong D.Y.T., Cheung A.N.Y., Lin Z.Q. and Ngan H.Y.S., Human papillomavirus infection in Southern Chinese women – a population-based study , American Association for Cancer Research (AACR) 101th annual meeting, Washington DC, USA, April 17-21, 2010.

Long J., Cai Q., Qu S., Li C., Zheng Y., Gu K., Wang W., Xiang Y.B., Cheng J., Chen K., Zhang L., Zheng H., Shen C.Y., Huang C.S., Hou M.F., Shen H., Hu Z., Wang F., Deming S.L., Kelley M.C., Shrubsole M.J., Khoo U.S., Chan Y.K., Chan S.Y., Haiman C.A., Henderson B.E., Le Marchand L., Iwasaki M., Kasuga Y., Tsugane S., Matsuo K., Tajima K., Iwata H., Huang B., Shi J., Li G., Wen W., Gao Y.T., Lu W., Zheng W. and Shu X.O., Identification of a functional genetic variant at 16q12.1 for breast cancer risk: results from the Asia Breast Cancer Consortium., PLoS Genetics. 2010, 6: e1001002.

Zhang L., Chan Y.K., Ip Y.C., Tsang W.H. and Khoo U.S., Splice variant profiling in relation to tamoxifen resistance in breast cancer, The 21st Meeting of the European Association for Cancer Research, Oslo, Norway. 2010.

Researcher : Chan YP

List of Research Outputs

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Ma S.K.Y., Chan Y.P., Kwan P.S., Tang K.H., Vielkind J.V., Guan X.Y. and Chan K.W., microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression , American Association for Cancer Research. 2010.

Researcher : Chen J

List of Research Outputs

Chen J., Chan Y.K., Wu L.H., Ip Y.C., Lam E.W.F. and Khoo U.S., Forkhead Transcription Factor FOXO3a increases Akt Activity in Breast Cancer Cell Lines through a Feed-back Mechanism, The 16th Hong Kong International Cancer Congress, Hong Kong. 2009.

Researcher : Chen WYW

Project Title:	AACR 101st Annual Meeting 2010 Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm
Investigator(s):	Chen WYW
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Cheung ANY

Project Title:	Selecting most suitable students for production of best medical doctors-which criteria should we use?
Investigator(s):	Cheung ANY, Patil NG, Ip MSM, Chan LC
Department:	Pathology
Source(s) of Funding:	Leung Kau Kui Research and Teaching Endowment Fund - Teaching Grants
Start Date:	02/2005

Abstract:

(a) enhancing the processes for evaluating and improving admission criteria for MBBS curriculum; and potentially; (b) improving curriculum design and (c) improving learning opportunities of students on ethical values and communication skills.

Project Title:	Folate and folate receptor alpha in ovarian cancers
Investigator(s):	Cheung ANY, Siu KY
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2007

Abstract:

The project aims at investigating the role of folate and folate receptor alpha in affecting the growth and invasive behaviors of ovarian cancers. World wide, ovarian cancer is a common cancer in women, contributing to the highest mortality among all gynecological cancers. More importantly, there is a recent significant increase in the incidence, rising from 9.8 per 100,000 (crude incidence rate) in 1999 to 12.5 in 2002 [1]. Recent studies in our laboratories have reported several tumor suppressor genes as potential cancer markers, which play a vital role in gynecological malignancies [2-6]. Several epidemiologic factors for this gynecological cancer have been documented. Age, a family history of ovarian or breast cancer, nulliparity and use of oral contraceptive are associated with the risk of ovarian cancer [7]. Recently, attention has focused on the association between high intakes of micronutrients such as folate, vitamin C, vitamin E and lower risk of various cancers [8]. For instance, a high dietary folate intake is related to lower risk of ovarian cancer, particularly among women who consume alcohol [9-11]. Folate, a water-soluble B vitamin found in most vegetable, is required for DNA synthesis, and the related methionine metabolic pathway is essential for DNA methylation. Folate deficiency has found to induce DNA hypomethylation, alter gene expression and misincorporate uracil into DNA leading to chromosome damage, all of which are key factors for carcinogenesis [12,13]. Pregnancy, lactation and alcohol abuse are some common causes for the increase of folate requirements in the body. It is particularly important to notice that alcohol consumption is considered as a major factor account for the inverse relation between folate intake and ovarian cancer risk [10,14,15]. It is interested to note that the folate receptor alpha (FR alpha), a single chain glycosyl-phosphatidylinositol–anchored membrane protein, was found to be overexpressed in nonmucinous ovarian tumors and its expression was directly associated with the progression of ovarian cancer, implicating that uptake of folate by tumor cells may confer tumor growth [16-18]. Such observation suggests a hypothesis that folate may involve in the growth of both normal and tumor cells. Furthermore, the FR alpha was shown to be up-regulated when cells are exposed to folate-deplete media and down-regulated in folate-replete media [19-21]. While the expression of the FR alpha in ovarian cancer among Oriental, such as in Hong Kong, is still unknown, it is worthy to include local data to investigate the hypothesis stated. REFERENCES 1. Hong Kong Department of Health. (2002) The Hong Kong Cancer Registry: Annual Report 2001. *2. Chan, K.Y.K., et al. (2002) Epigenetic factors controlling the BRCA1 and BRCA2 genes in sporadic ovarian cancer. Cancer Research, 63, 4151-4156. *3. Chan, Q.K.Y., et al. (2005) Promoter Methylation and Differential Expression of pi-class Glutathione-S-Transferase in Endometrial Carcinoma. Journal of Molecular Diagnostics, 7, 8-16. *4. Chan, Q.K.Y., et al. (2005) Single Nucleotide Polymorphism of Pi-class Glutathione-S-Transferase and Susceptibility to Endometrial Carcinoma. Clin. Cancer Res., 11, 2981-2985. *5. Shen, D.H., et al. (2005) Epigenetic and genetic alterations of p33ING1b in ovarian cancer. Carcinogenesis, 26, 855-863. *6. Yang, C.Q., et al. (2006) Single nucleotide polymorphisms of follicle-stimulating hormone receptor are associated with ovarian cancer susceptibility. Carcinogenesis, 27, 1502-6. 7. Lukanova, A. and Kaaks, R. (2005) Endogenous hormones and ovarian cancer: epidemiology and current hypotheses. Cancer Epidemiol. Biomarkers Prev., 14, 98-107. 8. Woodside, J.V., et al. (2005) Micronutrients: dietary intake v. supplement use. Proc. Nutr. Soc., 64, 543-553. 9. Larsson, S.C., et al. (2004) Dietary Folate Intake and Incidence of Ovarian Cancer: The Swedish Mammography Cohort. J. Natl. Cancer Inst., 96, 396-402. 10. Kelemen, L.E., et al. (2004) Association of folate and alcohol with risk of ovarian cancer in a prospective study of postmenopausal women. Cancer Causes Control, 15, 1085–1093. 11. Larsson, S.C. and Wolk, A. (2004) Wine consumption and epithelial ovarian cancer. Cancer Epidemiol. Biomarkers Prev., 13, 1823–1824. 12. Blount, B.C., et al. (1997) Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc. Natl. Acad. Sci., 94, 3290-3295. 13. Duthie, S.J. (1999) Folic acid deficiency and cancer: mechanisms of DNA instability. Br. Med. Bull., 55, 578-592. 14. Kelemen, L.E., et al. (2005) Multivitamin and alcohol intake and folate receptor alpha expression in ovarian cancer. Cancer Epidemiol. Biomarkers Prev., 14, 2168-2172. 15. Pelucchi, C., et al. (2005) Dietary folate, alcohol consumption, and risk of ovarian cancer in an Italian case-control study. Cancer Epidemiol. Biomarkers Prev., 14, 2056-2058. 16. Ross, J.F., et al. (1994) Differential regulation of folate receptor isoforms in normal and malignant tissues in vivo and in established cell lines. Physiologic and clinical implications. Cancer, 73, 2432–2443. 17. Campbell, I.G., et al. (1991) Folate-binding protein is a marker for ovarian cancer. Cancer Res., 51, 5329–5338. 18. Toffoli, G., et al. (1997) Overexpression of folate binding protein in ovarian cancers. Int. J. Cancer, 74, 193–198. 19. Luhrs, C.A., et al. (1992) Transfection of a glycosylated phosphatidylinositol-anchored folate-binding protein complementary DNA provides cells with the ability to survive in low folate medium. J. Clin. Invest., 90, 840-847. 20. Matsue, H., et al. (1992) Folate receptor allows cells to grow in low concentrations of 5-methyltetrahydrofolate. Proc. Natl. Acad. Sci. U. S. A., 89, 6006–6009. 21. Hsueh, C.T. and Dolnick, B.J. (1993) Altered folate binding protein mRNA stability in KB cells grown in folate-deficient medium. Biochem. Pharmacol., 45, 2537–2545.

Project Title:	Akt and p21-activated kinase signaling pathways in gestational trophoblastic disease
Investigator(s):	Cheung ANY, Ngan HYS, Siu KY
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007

Abstract:

To elucidate the roles of Akt and p21-activated serine/threnonine kinases (Paks) and their related genes in the pathogenesis of gestational trophoblastic diseases; to evaluate the potential of these genes as markers for predicting clinical progression and molecular targets for therapy.

Project Title:	Cervical cancer screening by enhanced cervical cytology-application of novel markers
Investigator(s):	Cheung ANY, Ngan HYS, Guan XY
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	03/2007

Abstract:

To explore with application of novel markers, some generated by our local genetic studies, to be detected by immunohistochemistry and ISH as an adjunct to enhance sensitivity and specificity of liquid based cervical cytology.

Project Title:	Centre for Research into Circulating Fetal Nucleic Acids
Investigator(s):	Cheung ANY
Department:	Pathology
Source(s) of Funding:	Areas of Excellence Scheme
Start Date:	01/2008

Abstract:

Prenatal diagnosis is an indispensable component of health care. Definitive diagnostic methods in current use, e.g. amniocentesis, are invasive and pose a risk to the unborn child. In 1997, the project coordinator and his research team discovered, for the first time in the world, the presence of cell-free fetal DNA in the plasma of pregnant women, offering new possibilities for non-invasive prenatal diagnosis. The project team has further pioneered many diagnostic applications, a number of which are now used clinically by many centres globally. To maintain Hong Kong at the forefront in non-invasive prenatal diagnostic research, a Centre consisting of a multidisciplinary conglomerate of local and international researchers is formed under this Area of Excellence project, coordinated by The Chinese University of Hong Kong. The Centre will address a number of high-profile unsolved questions in the field of circulating fetal nucleic acids, including non-invasive molecular methods for the diagnosis of fetal Down syndrome. Our ultimate goal is to make safe prenatal diagnosis available to citizens around the world and to promote the development of expertise in molecular diagnostics in this region.

Project Title:	Tropomyosin-related kinase B and Brain-derived neurotrophic factor in ovarian cancer
Investigator(s):	Cheung ANY, Siu KY, Ngan HYS
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2008

Abstract:

Ovarian epithelial tumors are common tumors in women and contribute to the highest mortality among all gynecological cancers. Studies have been carried out on different signaling pathways involved in progression of cancers especially those activated by the growth factors or hormones. We have found in earlier studies that polymorphisms in the follicular stimulating hormone receptor (FSHR) is related to susceptibility to specific histological subtypes of ovarian cancers (Yang et al., 2006) . We are thus particularly interested in receptors that may react to FSH or alternative ligand that may act on FSHR. The interaction between FSH and FSHR with other growth factors and receptors may be investigated to enhance our understanding of ovarian carcinogenesis. Tropomyosin-related kinase (Trk) is a family of single pass transmembrane receptor kinases that are highly expressed in the nervous system and have major roles in the neural functioning and development (Schramm et al., 2005). This family consists of three members: TrkA, TrkB and TrkC. Typical Trk receptors consist of three components: extracellular, transmembrane and intracellular domains. Tropomyosin-related kinase B (TrkB) is a receptor tyrosine kinase that is preferentially activated by brain-derived neurotrophic factor (BDNF). It has been shown that activation of TrkB by BDNF increases cell survival and invasion in some cancers (Koizumi et al., 1998; Ricci et al., 2001). Moreover, in vitro studies showed that TrkB overexpression may contribute to drug resistance of neuroblastoma cells by inhibition of apoptosis (Jaboin et al., 2002). It has been recently postulated that TrkB may be a drug target in anti-cancer therapy (Desmet and Peeper, 2006). On the other hand, substantial evidence have proved that neurotrophins have important roles in the early development of follicles as well as the ovulation process (Dissen et al., 2002). However, their role in ovarian cancers has not been reported. Aims: To explore the role of TrkB-BDNF pathway in ovarian cancer by studying TrkB expression at different stages of ovarian epithelial tumors and investigating its vitro function in ovarian cancer cell lines.

Project Title:	Integrated human papilloma virus analysis as adjunct for triage of atypical cervical cytology
Investigator(s):	Cheung ANY, Ngan HYS, Chan YK, Siu KY
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	01/2008

Abstract:

To investigate, in a cohort of HPV positive (HCT) women with ASCUS, the adjunct value of HPV genotyping by microarray, HPV transcription status by reverse transcription - PCR, and telomerase acitivity by Telomerase Repeat Amplification Protocol (TRAP) Assay.

Project Title:	American Association for Cancer Research Annual Meeting 2008 p21-activated kinase (PAK) in gestational trophoblastic neoplasia
Investigator(s):	Cheung ANY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2008

Abstract:

N/A

Project Title:	FBI-1 in ovarian carcinogenesis and progression
Investigator(s):	Cheung ANY, Siu KY, Ngan HYS
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2009

Abstract:

Ovarian cancers: There has been an increase in the incidence of ovarian cancer in Hong Kong, increased from 9.8 per 100,000 in 1999 to 11.4 in 20031. Moreover, despite advances in multimodality therapy, the mortality of ovarian cancer remains high. To improve strategies for early detection and management of ovarian cancers, enhanced understanding of its carcinogenesis is essential. Ovarian Cancers and p53: p53 tumour suppressor gene is known to play important roles in regulating cell growth through either cell-cycle arrest or apoptosis 2, 3. P53 is also the most commonly mutated tumour suppressor gene in human malignancies with somatic p53 missense mutations found in around half of human cancers 4. p53 pathway can also be inactivated by indirect mechanisms such as Mdm2 amplification leading to p53 destabilization. p53 mutations are frequently found in ovarian cancers (>50%) that derived de novo from the ovarian surface epithelium. FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) is a recently identified transcription factor that was found to suppress the p14ARF-Mdm2-p53 pathway leading to oncogenesis 5-7. FBI-1, encoded by the Zbtb7A gene, also known as Pokemon, LRF and OCZF, is a member of the POK (POZ and Krüppel) family of transcriptional repressors. FBI-1 can suppress activity of p14ARF which in turn is one of the central regulators of p53 pathway. Since p14ARF can inhibit the activity of Mdm2 which induces p53 degradation, p14ARF indirectly activate p53. By suppression of p14ARF, FBI-1 can inhibit p53 and act as a proto-oncogene. Indeed, overexpression of Pokemon and its possible role in tumorigenesis has been reported in epithelial tumors of the bladder, lung, colon, and breast 6, 8, 9. Moreover, overexpression of FBI-1 was found in both p53 mutant and p53 wild-type lung cancers cases, suggesting either a synergistic effect or that FBI-1 exerts its oncogenic properties independently of the p14ARF -Mdm2-p53 axis 9. The role of FBI-1 in ovarian epithelial tumour has not been reported. FBI-1 and ovarian neoplasms: Our preliminary immunohistochemical studies including 5 epithelial inclusion cysts, 10 benign ovarian cystadenomas, 20 borderline ovarian tumours and 59 ovarian carcinomas show significant increase in FBI-1 expression in ovarian tumours with highest expression in carcinomas. The FBI-1 expression is found in both the cytoplasm and the nucleus. From the preliminary results, FBI-1 expression was significantly associated with clear cell histological subtypes. In addition, significantly higher expression in more advanced (Stages II-IV) cancers was demonstrated. FBI-1 may play a role in early carcinogenesis and progression of ovarian cancers.

Project Title:	HPV detection as an adjunct for triage of atypical cervical cytology in screening for cervical cancer
Investigator(s):	Cheung ANY
Department:	Pathology
Source(s) of Funding:	S.K. Yee Medical Foundation - General Award
Start Date:	11/2009

Abstract:

n/a

Project Title:	XV World Congress on Gestational Trophoblastic Diseases Cell Invasion and Apoptosis Signal Pathways in Gestational Trophoblastic Diseases
Investigator(s):	Cheung ANY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	11/2009

Abstract:

N/A

Project Title:	Hedgehog pathway regulator Kif7 in gestational trophoblastic disease
Investigator(s):	Cheung ANY, Ngan HYS, Siu KY, Wong GW
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

Key Issues: Human placental trophoblast may be considered as a pseudo-malignant tissue sharing the propensity to proliferate and invade. Under normal circumstances, trophoblast proliferation, apoptosis, migration and differentiation is regulated by numerous genes and growth factors in a controlled fashion (1). Dysregulation of these processes will lead to diseases complicating pregnancies. Gestational trophoblastic disease (GTD) is a complex family of disease derived from various types of placental trophoblasts and the most common lesions are hydatidiform moles and choriocarcinoma (2). Choriocarcinoma is an aggressive malignant tumour, while hydatidiform moles can be considered as abnormal placentas that are prone to malignant transformation. About 20% of patients with hydatidiform mole require chemotherapy after primary evacuation due to development of persistent gestational trophoblastic neoplasia. However, few indexes have been found to be useful in predicting such progression. In our previous RGC/GRF funded project, we evaluated the role of Stem cell core transcription factors, Oct-4, Sox-2, Nanog and Stat3, in development and progress of GTD. Significant differential expression of mRNA and protein expression of Nanog and Stat3 was demonstrated in choriocarcinoma and HM, particularly those that developed persistent trophoblastic neoplasia requiring chemotherapy. Stable knock-down of Nanog in choriocarcinoma cells demonstrated an anti-apoptosis effect of Nanog in relation to caspases and PARP cleavage activities (3). There was significant positive and inverse correlation between nuclear p-Stat3 immunoreactivity and apoptotic indices and Bcl2 expression respectively (4). By methylation specific PCR and bisulfite sequencing, hypermethylation and corresponding reduced expression of Oct-4 and Sox-2 was demonstrated in hydatidiform mole and choriocarcinoma, correlating with the clinical outcome of the former (5, 6). Such findings suggested that aberrant expression of these stem cell transcription factors contributes to pathogenesis of GTD (1). The Hedgehog signaling pathway is essential for stem cell maintenance and tissue development (7, 8). Aberrant activation of Hedgehog signaling pathway has recently been reported in various human cancers (9). Activation of the pathway is initiated through binding of any of the three ligands, Sonic, Indian and Desert Hedgehog to transmembrane protein receptor Patched. Through alleviating Patched-mediated suppression of another receptor Smoothened, a signal cascade is activated that leads to the translocation of active form of Gli, a family of zinc finger transcriptional factors, to the nucleus and subsequently activates target genes expression (10, 11). Aberrant activation of Hedgehog pathway is caused through sustained increased endogenous expression of Hedgehog (ligand-dependent) or by ligand-independent mutations of Patched or Smoothened in the pathway (12, 13). Kif7 is the mammalian homolog of Drosophila Hedgehog regulator protein Cos2. It roles in Hedgehog signaling has recently been revealed by genetic studies. Kif7 knockout mice exhibited exencephaly and polydactyly, a phenotype reminiscent of Gli3, a major suppressor Gli, knockout mice (14-16). This suggested that Kif7 plays an important role in the suppression of Hedgehog signals during development. However, Patched Kif7 double mutant showed a milder phenotype than Pattched knockout, which suggested Kif7 can also activate HH pathway (16). We have recently reported the overexpression of Patched and Gli1 protein in ovarian cancers and their correlation with poor survival of the patients. Ectopic Gli1 overexpression in ovarian cancer cells conferred increased cell proliferation, cell mobility, invasiveness (17). In addition, overexpression of Gli11 in endometrial cancers through -catenin nuclear accumulation was also demonstrated (18). The role of Hedgehog signaling pathway in human placenta and GTD has not been reported. We hypothesize that the malignant progression of GTD is associated with dysregulation of HH signaling pathways leading to aberrant control of trophoblast apoptosis, cell growth and invasiveness. We also believe in the existence of close interaction between HH signal molecules and the stem cell core transcription factors in particular Oct-4, Sox2 and Nanog. As a pilot study, we shall start our investigations on the hedgehog signaling regulator Kif7 in GTD. Objectives of the proposed investigation: (1) To study the expression profiles of hedgehog signaling regulator Kif7 in gestational trophoblastic disease and normal placentas (2) To investigate mechanisms and effects of Kif7 expression on trophoblast cell function; (3) To evaluate the interaction between Kif7 and stem cell related transcription factors.

List of Research Outputs

Au C.W., Siu M.K., Liao X., Wong E.S., Ngan H.Y.S., Tam K.F., Chan D.C., Chan Q.K. and Cheung A.N.Y., Tyrosine kinase B receptor and BDNF expression in ovarian cancers - Effect on cell migration, angiogenesis and clinical outcome, Cancer Lett. 2009 Aug 28;281(2):151-61. 2009.

Chan D.W., Liu V.W.S., To M.Y., Chiu P.M., Lee Y.W., Yao K.M., Cheung A.N.Y. and Ngan H.Y.S., Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21(WAF1/CIP1) expression in ovarian cancer, Briitish Journal of Cancer. 2009, 101: 1433-1443.

Cheung A.N.Y., 1. Molecular Markers for Cervical Cancer Detection: Role of p16 and other cell cycle markers in triage & 2. Role of immunohistochemistry in problematic cervical biopsies, Asia Oceania (research organisation on) Genital Infections & Neoplasia (AOGIN), New Dehli, India, 26-28 March 2010 & Asia Pacific Cervical Cancer Prevention Round Table Meeting, 25 March. 2010.

Cheung A.N.Y., Cell invasion and apoptosis signal pathways in Gestational Trophoblastic Diseases, XV World Congress on Gestational Trophoblastic Diseases, Cochin, India, 12-15 November. 2009.

Cheung A.N.Y., Cervical Cancer and Precursors, Recent Advances in Cervical Cancer Screening and Prevention - HPV Vaccines, Certificate Course on Updates in Cervical Cancer Screening, co-organized by The Federation of Medical Societies of Hong Kong and The Hong Kong Society for Colposcopy and Cervical Pathology, 28 January. 2010.

Cheung A.N.Y., Epidemiology and Screening of Cervical Cancer in Hong Kong, 1st Biennial Meeting of Asian Society of Gynecologic Oncology (ASGO), Tokyo, Japan, 22 November. 2009.

Cheung A.N.Y., Genetic and molecular signaling studies of ovarian cancers, Joint Retreat organized by Shantou University Medical College and Centre for Cancer Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 4 November. 2009.

Cheung A.N.Y., International Journal of Clinical and Experimental Pathology, 2010.

Cheung A.N.Y., Molecular Markers for Cervical Cancer Detection, Cancer Centre and Pathology Department of Medical College, Shantou University on 9-10, April. 2010.

Cheung A.N.Y., Molecular markers for cervical cancer detection, seminar by Department of Pathology, The University of Hong Kong, 6 August. 2009.

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Cheung A.N.Y., Pathology of Ovarian Carcinoma: New Insights on Origin and Pathogenesis, The 16th Hong Kong International Cancer Congress, Hong Kong, 5 November. 2009.

Cheung A.N.Y., Pathology of abnormal smears and CIN, Basic Coposcopy Workshop 2009, jointly organized by Hong Kong Society for Colposcopy and Cervical Pathology and Department of Obstetrics & Gynaecology, Kwong Wah Hospital, Hong Kong, 28 November. 2009.

Cheung A.N.Y., Women’s Cancers – new Horizons, Discussion caretaker in World Cancer Research Fund Hong Kong 7th Health Professional Conference, Hong Kong, 10 October. 2009.

Ip P.P.C., Cheung A.N.Y. and Clement P.B., Uterine Smooth Muscle Tumors of Uncertain Malignant Potential (STUMP):: A Clinicopathologic Analysis of 16 Cases, The American Journal of Surgical Pathology. 2009, 33(7): 992-1005.

Khoo U.S., Shen D.H., Wong R. and Cheung A.N.Y., Gynaecological Cancers in Genetically Susceptible Women: New thoughts on tubal pathology, Diagnostic Histopathology. 2009, 545-553.

Liu S., Leung C.Y., Chan K.K.L., Cheung A.N.Y. and Ngan H.Y.S., Evaluation of a newly developed GenoArray human papillomavirus (HPV) genotyping assay by comparison with Roche Linear Array HPV genotyping assay, Journal of Clinical Microbiology. 2010, 48(3): 758-64.

Liu S., Chan Y.K., Leung C.Y., Luk H.M., Lo S.T., Fong D.Y.T., Cheung A.N.Y., Lin Z.Q. and Ngan H.Y.S., Human papillomavirus infection in Southern Chinese women – a population-based study , American Association for Cancer Research (AACR) 101th annual meeting, Washington DC, USA, April 17-21, 2010.

Ngan H.Y.S., Cheung A.N.Y., Tam K.F., Chan K.K.L., Tang H.W., Bi D., Descamps D. and Bock H.L., Human papillomavirus-16/18 AS04-adjuvanted cervical cancer vaccine: immunogenicity and safety in healthy Chinese women from Hong Kong, Hong Kong Med J.. 2010, 16(3): 171-9.

Siu K.Y., Wong E.S.Y., Kong S.H., Wong G.W., Tam K.F., Ngan H.Y.S., Le X.F. and Cheung A.N.Y., Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers. , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A., 17 - 21 April 2010.. 2010.

Siu K.Y., Wong G.W. and Cheung A.N.Y., TrkB as a therapeutic target for ovarian cancer, Expert Opin Ther Targets. 2009, 1169-78.

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Wei N., Liu S., Leung T.H.Y., Tam K.F., Liao X., Cheung A.N.Y., Chan K.K.L. and Ngan H.Y.S., Loss of programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer , Molecular Cancer. 2009, 8: 70.

Wong G.W., Siu K.Y., Ngan H.Y.S. and Cheung A.N.Y., Dysregulated expression and function of Plexin-B1 in ovarian cancers , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Cheung CH

List of Research Outputs

Lee K.W., Yung L.H., Cheung C.H., Castilho A.G. and Ng I.O.L., Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis, The American Association for Cancer Research, 2010.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Researcher : Cheung OF

List of Research Outputs

Yau T.O., Leung T.H.Y., Lam S.G.S., Cheung O.F., Tung K.K., Khong P.L., Lam A.K.M., Chung S.K. and Ng I.O.L., Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis., PLoS One. 2009, 4(8): e6566.

Researcher : Cheung YN

Project Title:	Examination of eIF4E level in human breast cancers and study the role of eIF4E in regulating translation of various splice variants of breast cancer genes
Investigator(s):	Cheung YN, Khoo US, Chan YK
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2010

Abstract:

Translation is the last process in the flow of genetic information, and its precise control provides an immediate and rapid response to cope with physiological change. In general, there are four steps in translation: initiation, elongation, termination and ribosome recycling. In eukaryotes, translation initiation is regulated by an array of eukaryotic initiation factors (eIFs) and this is the step being mostly regulated. It has been shown that aberrant function of components of the translation machinery underlies a variety of human diseases including cancers (1). In eukaryotes, including human, most of the mRNAs are translated by the scanning mechanism, in which the ribosome binds to and scans the 5’ leader of the mRNA for the initiation codon. The 5’end of the mRNA is capped with m7GpppN (m is a methyl group and N is any nucleotide) which is required for interaction between the mRNA and ribosome. The 5’cap is recognized by a trimeric initiation factor complex known as eIF4F, consisting of eIF4E, eIF4G and eIF4A. eIF4E interacts with the cap structure, which in turn interacts with the scaffold protein eIF4G. eIF4G interacts with the RNA helicase eIF4A and the ribosome complex. In other words, the eIF4F complex recruits the mRNA to the ribosome complex and unwinds its secondary structures (2). Since eIF4E is the least abundant initiation factor, it is believed that eIF4E is the rate limiting step for translation initiation (3, 4) and its overexpression could lead to deregulation of translation and cellular homeostasis, leading to tumorigenesis. Previously, it has been shown that overexpression of eIF4E caused malignant transformation of immortalized rodent and human cells and conferred solid tumor formation in xenotransplant nude mice model (5, 6). Anti-sense oligonucleotides of eIF4E partially reversed the malignant phenotypes, suggesting that overexpression of eIF4E is the direct cause of transformation (7). Elevated eIF4E mRNA levels have been detected in a variety of rodent cancer cell lines including hepatoma and neuroblastoma (8) as well as human breast carcinoma cell lines (9). Western blot studies showed that eIF4E was elevated 3- to 10-fold in breast carcinomas biopsies and was correlated to the rate of recurrence and mortality (10). However, only a two- to threefold higher eIF4E levels was observed in a variety of breast cancer types of different stages and histopathological grades using immunohistochemistry (IHC) studies(1). The presence of mixed cell types in the tumor mass homogenate possibly contributed to the observed discrepancy. Additional studies are needed to characterize the state of eIF4E in a variety of breast cancers at different stages and grades. The outcome of eIF4E overexpression have been analyzed, showing that high levels of eIF4E facilitates the translation of mRNAs containing structural repeats at the 5’ untranslated region (5’UTR) in vivo (11). Overexpression of eIF4E in cells leaded to an increase in cyclin D1 protein (12) and produced 30-fold higher of Ornithine Decarboxylase protein levels (13). Moreover, eIF4E overexpression caused a dramatic increase in the translations of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) (14, 15). Recently, it was reported that over 60% of human genes are alternatively spliced. Alternative splicing may result in transcripts with different 5’ or 3’ UTRs, different coding sequence or with premature stop codon. The variation of expression levels of alternative splice isoforms of different genes and within the same locus are known to be related with certain diseases (16). However, the regulating mechanisms of the relative and differential expression have yet to be determined. Since overexpression of eIF4E has been detected in breast cancers and it plays a central role in regulating translation, it is possible that the change of eIF4E levels in breast cancer cells allow the translation of specific variant(s) more effectively, which may in turn contribute to breast cancer development. We hypothesized that the breast cancer cells with high eIF4E level would promote the translation of splice variants with highly structured 5’UTRs of some breast cancer genes. In this proposal, we have the following specific aims: Aim 1: Study the correlation of eIF4 levels with the breast cancer stages Aim 2: Study whether different levels of eIF4E in cancer cell lines lead to differential translation of splice variants of breast cancer genes Aim 3: Study whether the different splice variants contain different degree of secondary structure at the 5’ UTRs After this study, we hope to get insight on how translation is involved in breast tumorigenesis and improve the ways for therapeutic treatment of cancer patients. References: 1. R. a. S. Schneider, N, in translational control in biology and medicine. (cold spring harbor laboratory press, 2007). 2. N. Sonenberg, A. G. Hinnebusch, Cell 136, 731 (Feb 20, 2009). 3. R. Duncan, S. C. Milburn, J. W. Hershey, J Biol Chem 262, 380 (Jan 5, 1987). 4. R. Duncan, J. W. Hershey, J Biol Chem 258, 7228 (Jun 10, 1983). 5. A. Lazaris-Karatzas, K. S. Montine, N. Sonenberg, Nature 345, 544 (Jun 7, 1990). 6. A. De Benedetti, R. E. Rhoads, Proc Natl Acad Sci U S A 87, 8212 (Nov, 1990). 7. A. De Benedetti, S. Joshi-Barve, C. Rinker-Schaeffer, R. E. Rhoads, Mol Cell Biol 11, 5435 (Nov, 1991). 8. Y. Miyagi et al., Cancer Lett 91, 247 (May 8, 1995). 9. B. Anthony, P. Carter, A. De Benedetti, Int J Cancer 65, 858 (Mar 15, 1996). 10. B. D. Li, J. C. McDonald, R. Nassar, A. De Benedetti, Ann Surg 227, 756 (May, 1998). 11. A. E. Koromilas, A. Lazaris-Karatzas, N. Sonenberg, Embo J 11, 4153 (Nov, 1992). 12. I. B. Rosenwald, A. Lazaris-Karatzas, N. Sonenberg, E. V. Schmidt, Mol Cell Biol 13, 7358 (Dec, 1993). 13. L. M. Shantz, A. E. Pegg, Cancer Res 54, 2313 (May 1, 1994). 14. C. G. Kevil et al., Int J Cancer 65, 785 (Mar 15, 1996). 15. C. Kevil, P. Carter, B. Hu, A. DeBenedetti, Oncogene 11, 2339 (Dec 7, 1995). 16. M. Cuperlovic-Culf, N. Belacel, A. S. Culf, R. J. Ouellette, Omics 10, 344 (Fall, 2006).

Project Title:	AACR 101st Annual Meeting eIF4E in human breast cancer and its role in regulating translation of splice variants of breast cancer genes
Investigator(s):	Cheung YN
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010

Abstract:

N/A

List of Research Outputs

Cheung Y.N., Nanda J.S., Takacs J.E., Martin-Marcos P., Saini A.K., Hinnebusch A.G. and Lorsch J.R., eIF1 controls multiple steps in start codon recognition during eukaryotic translation initiation, Journal of Molecular Biology. 2009, 394: 268.

Cheung Y.N., Wong S.Y. and Khoo U.S., eIF4E in human breast cancer and its role in regulating translation of splice variants of breast cancer genes, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Researcher : Ching CYJ

List of Research Outputs

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Khoo U.S., Chan Y.K., Ching C.Y.J., Chan V.S.F., Ip Y.C., Yam L., Chu C.M., Lai S.T., So K.M., Wong T.Y., Chung P.H., Yip S.P., Sham P.C., Leung G.M., Lin C.L. and Peiris J.S.M., Functional role of ICAM-3 polymorphism in genetic susceptibility to SARS infection, Hong Kong Med Journal. 2009, 26-9.

Researcher : Ching DCK

List of Research Outputs

Chan K.Y., Ching D.C.K., Mak C.M., Lam C.W. and Chan A.Y.W., Hereditary spastic paraplegia: identification of an SPG3A gene mutation in a Chinese family, Hong Kong Medical Journal. 2009, 15(4): 304-7.

Researcher : Ching YP

Project Title:	Roles of p21-activated protein kinase (Pak) 1 in the pathogenesis of liver cancer
Investigator(s):	Ching YP, Ng IOL, Jin D, Yau TO
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2006

Abstract:

(1) To characterize the mechanisms leading to Pak1 overexpression in human HCC; (2) to delineate the roles of Pak1 in hepatocarcinogenesis: (i) Phosphorylation of possible downstream targets of Pak1 in human HCCs. (ii) characterization of the tumorigenic activity of Pak1 in HCC cells. (iii) characterization of the anti-apoptotic activity of Pak1 in HCC cells. (3) to investigate the role of Pak1 in cancer metastasis: >(i) regulation of cell motility and cell adhesion by Pak1 in HCC cells. (ii) HGF/Rac1/Cdc42/Pak1 signaling in HCC metastasis. (iii) HGF/Pak1 mediated angiogenic activity. (iii) HGF/Pak1 mediated angiogenic activity.

Project Title:	Molecular neurobiology: Regulation of p21-activated protein kinase 5 in neurodegenerative disease
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	Matching Fund for NSFC Young Researcher Award
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

To study molecular neurobiology: regulation of p21-activated protein kinase 5 in neurodegenerative disease.

Project Title:	The roles of CDK5 activator binding protein, LZAP, in the pathogenesis of neurodegenerative disease
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	Travel Grants for NSFC/RGC JRS
Start Date:	12/2007

Abstract:

Travel grants for NSFC/RGC JRS.

Project Title:	Functional characterization of a putative tumour suppressor, AMP-activated protein kinase, in liver cancer
Investigator(s):	Ching YP, Jin D, Ng IOL
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

(1) Investigation of the dysregulation of AMPK in HCC (12 months), i) Expression of AMPK subunits and its regulators in HCC-(LKB1 and AMPK, ii) Confirmation of the dysregulation of AMPK signaling in HCC; (2) investigation of the genetic basis of loss of AMPK 2 exression in human HCC and HCC cells (9 months), i) Epigenetic silencing, ii) Genetic alterations (only LOH); (3) tumor suppressor activity of AMPKa2 in HCC (18 months), i) Characterization of the tumor suppressor activity of AMPK in HCC cells; (4) investigation of AMPKa2 cell signalling and tumor suppression(18 months), i) Negative regulation of mTOR by AMPK in HCC, ii) AMPK-mediated upregulation of p53 in HCC.

Project Title:	Functional characterisation of a novel p14ARF regulating protein, LZAP, in HCC
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2008
Completion Date:	03/2010

Abstract:

Objective: HCC is the second most common fatal cancer in Hong Kong. Different risk factors, such as hepatitis B and C virus infection, cirrhosis, and dietary aflatoxin, are well established in HCC. However, details of the genetic changes in pathogenesis and tumor progression are poorly defined in HCC. In our previous study, we have identified a novel cyclin dependent protein kinase 5 (Cdk5) activator interacting protein, named C53 (now called LZAP, LXXLL/leucine-zipper-containing ARF binding protein), which is ubiquitously expressed in human tissues. Evidence has shown that LZAP regulates apoptosis induced by genotoxic stress and overexpression of LZAP can promote cell proliferation. More recently, LZAP is found to interact with a tumor suppressor, p14ARF, which is a key regulator of cell senescence. In our preliminary study using real-time quantitative RT-PCR, we found that over 69% (33/48) of human HCC samples had significantly higher expression (>2 folds) of LZAP transcripts as compared to their corresponding nontumorous livers. The overexpression of LZAP was statistically correlated with clinicopathological features, i.e. poorer cellular differentiation and liver invasion. Interestingly, our data also indicated that knockdown of LZAP in HCC cells resulted in an upregulation of p14ARF. Thus, we hypothesize that overexpression of LZAP may contribute to the development of HCC by inhibition of cellular senescence. Aims: In order to better understand the roles of LZAP in the pathogenesis of HCC, we aim to: 1.Characterize the overexpression of LZAP protein in human HCC 2.Investigate the tumorigenic roles of LZAP in human HCC 3.Investigate the roles of LZAP/ARF in HCC Our study will characterize how dysregulation of LZAP can lead to the development of HCC and will provide novel insight for new molecular drug targets and for new therapeutic treatment.

Project Title:	The roles of CDK5 activator binding protein, LZAP, in the pathogenesis of neurodegenerative disease
Investigator(s):	Ching YP, Jin D
Department:	Anatomy
Source(s) of Funding:	NSFC/RGC Joint Research Scheme
Start Date:	01/2009

Abstract:

(1) Characterisation of the roles of LZAP in Nclk signaling i) Identification of the Cdk5 phosphorylation sites on LZAP ii) Roles of LZAP in Nclk-mediated neuronal differentiation; (2) Characterisation of the role of LZAP in NF-kappaB activity in neurons; (3) Characterisation of the role of LZAP in neuronal apoptosis i) regulation of neuronal apoptosis by LZAP ii) LZAP and p53 signaling iii) LZAP and JNK signaling; (4) Defining the roles of LZAP in the pathogenesis of neurodegenerative disease.

Project Title:	Characterisation of the role of p21-activated protein kinase 4 in liver cancer
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2009

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is one of the most common cancers worldwide. Different risk factors, such as hepatitis B and C virus infection, cirrhosis, and dietary aflatoxin, are well established in HCC. However, details of the genetic changes in pathogenesis and tumor progression, particularly in metastasis, are poorly defined in HCC. P21-activated protein kinase (PAK) is the main downstream effector of small Rho GTPases, Rac1 and Cdc42, which regulate diverse cellular processes, including cell migration, polarization, gene transcription and cell survival. Recently, we have shown that PAK1 was overexpressed in HCC and is involved in HCC metastasis. However, little information is available for the roles and regulation of other Pak’s family member in carcinogenesis. Here we obtained preliminary data suggesting that another PAK’s family member, PAK4, was overexpressed in HCC. Using quantitative real-time PCR, we have observed that over 60% of human HCC samples (40/63 cases) have significantly higher expression (>2 folds) of PAK4 transcripts as compared to their corresponding nontumourous livers. In addition, the overexpression of PAK4 significantly correlates with clinicopathological features, including venous invasion, liver invasion and poorer cellular differentiation. The overexpression of PAK4 at protein level in HCCs was also confirmed by Western blotting. These data suggest that PAK4 may play a role in the formation of HCC in general and in cancer metastasis in particular. In this proposal, we will further investigate how the overexpression of PAK4 leads to hepatocarcinogenesis. The potential influence of overexpression of PAK4 on apoptosis, tumorigenicity and metastatic potential of HCC cells will be evaluated. Finally, we will try to elucidate the molecular mechanisms by which PAK4 promotes cancer metastasis. Findings derived from our work will provide novel insight into the development and metastasis of liver cancer and might reveal novel strategies for intervention.

Project Title:	Hepatitis B virus promotes hepatocarcinogenesis by activation of Pak1
Investigator(s):	Ching YP, Jin D
Department:	Anatomy
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	01/2010

Abstract:

To investigate the mechanism by which HBX activates Pak1; to define the role of Pak1 in HBV replication; to examine if inhibition of Pak1 activity prevents HBV-induced HCC.

Project Title:	Functional characterisation of a novel centrosomal protein, TAX1BP2, in liver cancer development
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2010

Abstract:

1) Defining the loss of TAX1BP2 expression in human HCC 1.1 Clinicopathological correlation analysis of TAX1BP2 in HCC; 2) Defining the tumor suppressor activity of TAX1BP2 2.1 Confirmation of the tumor suppressor activity of TAX1BP2; 3) Investigating the roles of TAX1BP2 in cell cycle control 3.1 Characterisation of TAX1BP2 phosphorylation in cell cycle progression 3.2 Characterisation of TAX1BP2 phosphorylation in centrosome duplication.

Project Title:	Characterisation of the roles of LKB1 in liver cancer formation
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is one of the most common cancers worldwide. Different risk factors, such as hepatitis B and C virus infection, cirrhosis, and dietary aflatoxin, are well established in HCC. However, details of the genetic changes in pathogenesis and tumor progression, particularly in metastasis, are poorly defined in HCC. LKB1, which is a serine/theonine kinase, regulates diverse cellular processes, including cell migration, polarization, energy metabolism and cell survival. Mutations in the LKB1 gene are found in Peutz-Jeghers syndrome (PJS) patients, who have a much higher chance to develop cancers of epithelial tissue origin. Although LKB1 has been well documented to suppress cancer formation, the detail molecular mechanism by which LKB1 inhibits carcinogenesis is unclear. Using quantitative real-time RT-PCR, we have observed that over 37% of human HCC samples (12/32 cases) have significantly lower expression (>2 folds) of LKB1 transcripts as compared to their corresponding nontumourous livers. In addition, the underexpression of LKB1 significantly correlates with poorer disease free survival rate, suggesting a poorer prognosis of patient. Also, we have obtained preliminary evidence suggesting that LKB1 may phosphorylate beta-catenin at serine residue at position 45, which is important for the breakdown of the protein. Thus, we hypothesize that LKB1 is an important regulator for beta-catenin and loss of LKB1 may promote the accumulation of beta-catenin in HCC cells, leading to hepatocarcinogenesis. In this proposal, we will investigate how underexpression of LKB1 induces HCC formation. More importantly, we will examine the role of LKB1 in regulating beta-catenin activity. Thus our work will derive novel insight into the role of LKB1 in the development of liver cancer and might reveal novel strategies for intervention.

Project Title:	LKB1-AMPK Meeting Underexpression of LKB1 in hepatcellular carcinoma
Investigator(s):	Ching YP
Department:	Anatomy
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2010
Completion Date:	06/2010

Abstract:

N/A

List of Research Outputs

Hui C.F., Tung K.K., Sze M.F., Ching Y.P. and Ng I.O.L., Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma, In: Samuel S. Lee, Liver International. 2010, 30 Issue 1: 65-75.

Ng M.H., Siu K.L., Kok K.H., Ching Y.P. and Jin D., MIP-T3 is a novel inhibitor in TBK1/IRF3-dependent antiviral response. , 28th Annual meeting of the American Society for Virology. 2009.

Researcher : Choi WL

Project Title:	The Role of FOXP1 and Its Isoforms in the Pathogenesis of Diffuse Large B-cell Lymphoma
Investigator(s):	Choi WL, Srivastava G
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009

Abstract:

1. To investigate the effects of FOXP1 isoforms, especially on proliferation and apoptosis, in DLBCL cell lines. 2. To determine whether FOXP1 over-expression and NF-B activation co-exist in DLBCL cells. 3. To identify whether the various FOXP1 isoforms have direct physical interactions with NF-B. 4. To explore whether FOXP1 and NF-B share target promoters, and form a complex at these common DNA targets as transcriptional co-activators or co-repressors.

List of Research Outputs

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Choi W.L., Weisenburger D.D., Greiner T.C., Piris M.A., Banham A.H., Delabie J., Braziel R.M., Geng H., Iqbal J., Lenz G., Vose J.M., Hans C.P., Fu K., Smith L.M., Li M., Liu Z., Gascoyne R.D., Rosenwald A., Ott G., Rimsza L.M., Campo E., Jaffe E.S., Jaye D.L., Staudt L.M. and Chan W.C., A New Immunostain Algorithm Classifies Diffuse Large B-cell Lymphoma Into Molecular Subtypes With High Accuracy, Clinical Cancer Research. 2009, 15: 5494-5502.

Choi W.L., Monoclonal B-cell Lymphocytosis, Hong Kong Society of Haematology January Meeting. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Jacobs C.L., Jima D.D., Zhang J., Dunphy C.W.H., Richards K.L., Choi W.L., Srivastava G., Evens A.M., Gordon L.I., Czader M., Rizzieri D.A., Lagoo A.S., Mann K.P., Flowers C.R., Naresh K., Luftig M., Friedman D.R., Weinberg J.B., Thompson M.A., Gill J., Kahl B.S., Chadburn A. and Dave S., A Comprehensive Identification Of The Microrna Transcriptome And Its Application In B Cell Malignancies, American Society Of Hematology 51st Annual Meeting. 2009, Blood; 114(22): 948-949.

Young K.H., Patten N., Truong S., Eickhoff J., Rocque G.L., Malik J.T., Li Y., Kanehira K., Twohig M., Rehrauer W.M., Waknitz M.A., Kahl B.S., Moller M.B., Tzankov A., Moreno S.M., Piris M.A., Visco C., Dybkaer K., Chiu A., Attilio O., Chang J.C., Bhagat G., Winter J.N., Wang H.-.Y., Dunphy C., O'Neill S., Hsi E.D., Zhao X., Hunt K., Chen W.L., Go R.S., Choi W.L., Zhou F., Czader M., Li Y., Selvaggi S., Malter J.S. and Wu L., Clinical Impact Of TP53 Gene Mutations In Diffuse Large B-cell Lymphoma (DLBCL): An International DLBCL Rituxan-CHOP Consortium Program Study, American Society Of Hematology 51st Annual Meeting. 2009.

Researcher : Chung LP

List of Research Outputs

Lam C.L.D., Girard L., Sihoe A., Cheng L.C., Lui M.M.S., Wong M.P., Chung L.P., Ip M.S.M., Lam W.K. and Minna J.D., Gene expression profiling in lung adenocarcinomas reflects possible different molecular pathogenesis with respect to gender and smoking status, 14th Congress of the Asian Pacific Society of Respirology & 3rd Joint Congress of the Asian Pacific Society of Respirology/American College of Chest Physicians, Seoul, Korea, Nov 2009, Respirology. 2009, 14(Suppl 3): A128 (OS 03-05).

Lam C.L.D., Girard L., Sihoe A.D.L., Cheng L.C., Lui M.A.C.Y., Wong M.P., Chung L.P., Ip M.S.M., Lam W.K. and Minna J.D., Gene expression signatures associated with combination of female non-smokers in lung adenocarcinomas bearing activating epidermal growth factor receptor (EGFR) gene mutations in Chinese, The American Thoracic Society Annual Meeting 2010.

Tam I.Y., Leung L.H., Tin P.C., Chua D.T.T., Sihoe A.D.L., Cheng L.C., Chung L.P. and Wong M.P., Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared to common activating missense mutations, Molecular Cancer Therapeutics. 2009, 8(8): 2142-51.

Researcher : Guo T

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Ho WKJ

List of Research Outputs

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Researcher : Hu X

List of Research Outputs

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Hui CF

List of Research Outputs

Hui C.F., Tung K.K., Sze M.F., Ching Y.P. and Ng I.O.L., Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma, In: Samuel S. Lee, Liver International. 2010, 30 Issue 1: 65-75.

Researcher : Ip PPC

List of Research Outputs

Ip P.P.C., Cheung A.N.Y. and Clement P.B., Uterine Smooth Muscle Tumors of Uncertain Malignant Potential (STUMP):: A Clinicopathologic Analysis of 16 Cases, The American Journal of Surgical Pathology. 2009, 33(7): 992-1005.

Researcher : Ip WK

List of Research Outputs

Mok T.M.Y., Huang F.P., Ip W.K., Wong F.Y., Chan E.Y.T. and Xu D., Serum levels of IL-33 and soluble ST2 and their association with disease activity in systemic lupus erythematosus. , HKMJ. 2010, 16: p46 S76.

Researcher : Ip YC

Project Title:	Targeting Granulin for Breast Cancer Therapy
Investigator(s):	Ip YC, Khoo US
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2008

Abstract:

Background: Granulin-epthelin precursor (GEP) is an important growth factor in breast cancer. GEP overexpression has been shown to associate with resistance to Herceptin (Clin Cancer Res, 12:4192, 2006) and tamoxifen (Cancer Res64:1737, 2004) in breast cancer cells which are main obstacles to complete cure. A newly established neutralizing monoclonal antibody (A23) was shown to inhibit liver cancer growth and increase cisplatin sensitivity, but remains untested in breast cancer. GEP is overexpressed in > 60% of breast carcinoma, but hardly present in benign breast epithelium. Importantly, GEP is expressed in both estrogen receptor-positive and -negative breast cancer. Thus, targeting GEP offers an attractive alternative treatment for estrogen receptor (ER) -negative patients. The GEP monoclonal antibody provides a novel and essential platform for advancement of targeted therapy in breast cancer. Preliminary study: This newly developed monoclonal antibody was used to analyse breast cancer tissue samples in a tissue array study. GEP was highly expressed in breast tumour samples. A panel of breast cancer cell lines was screened for presence of GEP by A23, including tamoxifen-resistant and sensitive cell lines. GEP was expressed independent of ER status or tamoxifen sensitivity. In vitro cell proliferation assay showed that neutralizing antibody was able to inhibit cell proliferation in breast cancer cell lines, including MCF7, T47D and MDA-MB-468, independent of their ER+ and ER- status. In a mouse model established by orthotopic implantation of MCF7, administration of monoclonal antibody was able to suppress tumour growth effectively. Objectives: With the critical role of GEP in therapy resistance, we therefore propose further functional studies to examine the biological roles of GEP contributing to tamoxifen-resistance, targeting to provide a more complete picture on mechanisms leading to tamoxifen-resistance. Neutralizing GEP may increase tamoxifen sensitivity in breast cancer cells. Tamoxifen sensitivity in tamoxifen-resistant cell lines should be restored and breast cancer growth should be inhibited or even abolished by administration of this newly developed GEP antibody. With the virtual absence of expression in benign breast tissue, adverse side effect on normal cells would also be minimal.

List of Research Outputs

Chen J., Chan Y.K., Wu L.H., Ip Y.C., Lam E.W.F. and Khoo U.S., Forkhead Transcription Factor FOXO3a increases Akt Activity in Breast Cancer Cell Lines through a Feed-back Mechanism, The 16th Hong Kong International Cancer Congress, Hong Kong. 2009.

Khoo U.S., Chan Y.K., Ching C.Y.J., Chan V.S.F., Ip Y.C., Yam L., Chu C.M., Lai S.T., So K.M., Wong T.Y., Chung P.H., Yip S.P., Sham P.C., Leung G.M., Lin C.L. and Peiris J.S.M., Functional role of ICAM-3 polymorphism in genetic susceptibility to SARS infection, Hong Kong Med Journal. 2009, 26-9.

Zhang L., Chan Y.K., Ip Y.C., Tsang W.H. and Khoo U.S., Splice variant profiling in relation to tamoxifen resistance in breast cancer, The 21st Meeting of the European Association for Cancer Research, Oslo, Norway. 2010.

Researcher : Jiang L

List of Research Outputs

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Khoo US

Project Title:	Association of the pro-inflammatory and anti-inflammatory cytokine gene polymorphism to breast cancer susceptibility
Investigator(s):	Khoo US, Cheung ANY, Chan YK
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2003

Abstract:

To determine whether the genetic variants of the pro-inflammatory and anti-inflammatory cytokines may (a) contribute towards breast cancer susceptibility or (b) influence prognosis; to investigate whether the joint effects of several of these alleles and/or in combination of specific environmental factors may contribute towards a stronger association.

Project Title:	Infectious Diseases and Global Health: Genetic approach to the identification of host susceptibility factors and pathogen virulence determinants. Genetics of coronaviruses-associated acute respiratory disease
Investigator(s):	Khoo US
Department:	Pathology
Source(s) of Funding:	CGDN NCE Large Scale Collaborative Research Grant
Start Date:	02/2005

Abstract:

To study association of SARS-susceptibility with MHC and KIR; to carry out genetic and functional analysis of coronavirus-associated respiratory disease.

Project Title:	Promoter polymorphisms of L-SIGN in relation to host genetic susceptibility to SARS.
Investigator(s):	Khoo US, Chan YK
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	02/2006

Abstract:

Objective of study : 1. To identify the possible variants in the promoter region of L-SIGN by direct sequencing of 30 unrelated normal Chinese individuals. 2. To examine the pattern of linkage disequilibrium (LD) between all the variants identified, and hence select haptotype tagging single nucleotide polymorphism (htSNP) for risk association study. 3. To analyze these htSNPs for genetic association to SARS CoV using a large case-control study. 4. To examine for possible LD between the htSNPs and the tandem-neck repeats polymorphisms of L-SIGN previously genotyped. 5. To perform in-vitro functional studies to confirm the effect of L-SIGN promoter SNPs on the transcriptional activity of the promoter. Key issues and problems being addressed: L-SIGN or DC-SIGNR (CD209L) is a homologue of DC-SIGN (for dendritic cell-specific ICAM-3 grabbing non-integrin, CD209) and shares 77% amino acid identity with it (1). Located within 30 kb on chromosome 19p13.2-3 in a head-to-head orientation, they are thought to have arisen through a gene duplication event (2;3). The extra-cellular domain of both DC-SIGN and L-SIGN encoded by exon 4, contain tandem repeats of a highly conserved 23-amino acid sequence, followed by a C-terminal C-type carbohydrate recognition domain (CRD)(4-6). Both L-SIGN and DC-SIGN share the ability to bind to high-mannose oligosaccharides through their CRDs and serve as receptors for many viruses such as HIV, HCV and SARS Co-V. Unlike DC-SIGN, L-SIGN has considerable polymorphism in this tandem-repeat domain which encodes the extra-cellular neck region (2). This tandem-repeat segment of 3 to 9 repeats, 7 being predominant (>50%) in the general population. Homo-oligomerization through this tandem-repeat neck region is what determines its high-affinity interaction as well as ligand specificity (7). L-SIGN has been demonstrated to be a binding receptor for SARS Co-V (8). We have recently shown in a genetic association study, that individuals homozygous for L-SIGN tandem repeats are less susceptible to SARS infection, and in a series of in-vitro experiments, that L-SIGN with homozygous tandem-neck repeats had higher binding capacity for SARS-CoV, lower ability for trans infection and increased cell-association for SARS-CoV, thus playing a protective role in SARS infection (Chan et al, Nature Genetics, in press, 9). Promoter polymorphisms can cause altered binding affinity of transcription factors which may effect regulation of gene expression (10). This can give rise to differences in susceptibility to and severity of disease. A promoter polymorphism of DC-SIGN was shown to be associated with risk for parenteral acquisition of HIV-1 infection (11). This same variant was recently reported to be associated with severity of dengue disease and in-vitro study showed that it affects an Sp1-like binding site and transcriptional activity (12). We hypothesize that promoter polymorphisms may also effect the expression of L-SIGN or its association with disease. Having already shown the importance of L-SIGN as a binding receptor for SARS Co-V, and the effect of tandem-neck repeats genotype in susceptibility to SARS infection (9), it would be important to investigate whether promoter polymorphisms of L-SIGN may affect the expression of L-SIGN and thus also contribute towards disease susceptibility and/or severity for SARS. To exclude the possibility that the association with disease may be due to LD between the promoter polymorphism and the functional tandem-neck repeat homo-/heterozygosity, linkage disequilibrium (LD) between these promoter polymorphisms with tandem-neck repeat L-SIGN polymorphisms previously investigated will also be examined. Finally, in-vitro functional studies will be performed to confirm the effect of L-SIGN promoter SNPs on the promoter activity. References: 1. Soilleux EJ, Barten R, Trowsdale J. DC-SIGN; a related gene, DC-SIGNR; and CD23 form a cluster on 19p13. J Immunol 2000 September 15;165(6):2937-42. 2. Bashirova AA, et al. A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 2001 March 19;193(6):671-8. 3. Pohlmann S, et al. DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans. Proc Natl Acad Sci U S A 2001 February 27;98(5):2670-5. 4. Feinberg H, Mitchell DA, Drickamer K, Weis WI. Structural basis for selective recognition of oligosaccharides by DC-SIGN and DC-SIGNR. Science 2001 December 7;294(5549):2163-6. 5. Guo Y, et al. Structural basis for distinct ligand-binding and targeting properties of the receptors DC-SIGN and DC-SIGNR. Nat Struct Mol Biol 2004 July;11(7):591-8. 6. Mitchell DA, Fadden AJ, Drickamer K. A novel mechanism of carbohydrate recognition by the C-type lectins DC-SIGN and DC-SIGNR. Subunit organization and binding to multivalent ligands. J Biol Chem 2001 August 3;276(31):28939-45. 7. Snyder GA, Colonna M, Sun PD. The structure of DC-SIGNR with a portion of its repeat domain lends insights to modeling of the receptor tetramer. J Mol Biol 2005 April 15;347(5):979-89. 8. Jeffers SA, Tusell SM, Gillim-Ross L, Hemmila EM, Achenbach JE, Babcock GJ et al. CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 2004 November 2;101(44):15748-53. 9. VSF Chan, KYK Chan, YX Chen, ….US Khoo* & CL Lin* Homozygous L-SIGN (CD209L) plays a protective role in SARS coronavirus infection. Nature Genetics (in press) (*co-corresponding authors) 10. Theuns J, et al. (2003) Alzheimer-associated C allele of the promoter polymorphism -22C>T causes a critical neuron-specific decrease of presenilin 1 expression. Hum Mol Genet. 12(8), 869-877. 11. Martin MP, et al. Association of DC-SIGN promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type 1 infection. J Virol. 2004; 78(24):14053-6. 12. Sakuntabhai A, et al. (2005) A variant in the CD209 promoter is associated with severity of dengue disease. Nat Genet. 37(5), 507-513. 13. Lewontin RC. The interaction of selection and linkage. II. Optimum Models. Genetics. 1964;50:757-82. 14. Wei Liu,….Khoo US, et al. The functional -969C/T promoter polymorphism in BRCA1 decreases breast cancer risk in Chinese. (Submitted to JNCI)

Project Title:	Splice variant expression in relation to Estrogen Receptor gene expression in Chinese breast cancer
Investigator(s):	Khoo US, Chan YK, Kwong A
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2007

Abstract:

Breast cancer is the commonest cause of cancer in women. Its incidence in Hong Kong Chinese has been steadily rising in the last few decades, with age-standardised rates now at 42.6 per 100,000 which although lower than Caucasian rates, is the highest rate reported in Asia. Differential gene expression profiling has the potential to substantially refine cancer prognosis beyond what is currently possible with traditional clinical and pathological indicators, thus assisting the selection of patients for the most appropriate adjuvant systemic therapies and in the classification of breast cancers based on a better understanding of cancer biology. Micro-array gene-expression profiling has been done by different research groups, with at least 2 models, namely the intrinsic-subtype model (1) and the 70-gene model (2), which have been recently validated using independent data sets (3,4). Recent data using a single data-set on 295 samples applied to five gene-expression-based models have also demonstrated high concordance rates of up to 81% agreement in their outcome predictions for individual samples (5). In spite of this however, there remains a striking lack of overlap in gene identity between gene lists provided by the different studies. The reasons for this are unknown and calls for further study. Alternative splicing (AS) is a key post-transcriptional mechanism for generating multiple protein products from a single gene, occurring in perhaps 40-60% of human genes. Alternatively spliced isoforms of a given protein can display different and even antagonistic biological functions. Mounting evidence suggests that AS is changed or becomes aberrant during the development, progression and metastasis of breast cancer (6). AS in breast cancer cells has been shown to be dependent on cell-type and culture-conditions (7). Traditional microarrays are designed to measure the total level of expression of a gene, without distinguishing between different isoforms, with probe designs being generally biased towards the 3’ end of the gene. Probes considered by standard expression-data analysis to be ‘noise’, may in fact be indicative of different patterns of regulation of multiple splice forms. Over or reduced expression of some gene isoforms may have different functions from their wild-type, and might account for differences in gene identity between the different gene-expression profile models. A genome-wide survey of human AS with exon junction microarrays, discovered AS in nearly 800 genes not previously known to be such and suggested that at least 74% of human multi-exon genes are alternatively spliced (8). Our collaborators, Merck Research Laboratories, Merck & Co., Inc., previously established a 70-gene-based prognostic classifier for breast cancers diagnosed before age 55 (9). This classifier outperformed clinical predictors in selecting out good outcome patients, thus minimizing over-treatment (2). Whilst a uniform gene expression pattern was observed for good outcome patients, patients predicted to have poor outcome had more heterogenous expression patterns. On further investigation, they were able to identify a subset of patients with high estrogen receptor expression for age which strongly predicted extremely poor outcome with distant metastases and homogenous gene expression pattern mainly of cell cycle genes (10). The reason why tumors in young patients with high ER have unique propensity to depend on proliferation associated genes is unclear which led us to hypothesize that there may be specific isoform expression pattern of genes associated with younger age onset of breast cancer. We plan to apply for RGC funding to investigate the contribution of splice-variants in on 140 Chinese breast cancer samples using a custom-made microarray which will focus on alternative splicing of the significant genes contained in their 70-gene profile previously found to be useful for prognostication of breast cancer. To obtain preliminary data in support of this study, we propose to investigate the splice-variants of some selected genes - namely the estrogen receptor alpha and beta, BRCA1 and BRCA2 and some cell cycle genes. Both quantitative expression analysis of both wild type and splice variants will be measured by Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) using Low-density array TaqMan assay (Applied Biosystems). All breast cancer cases studied were primary invasive carcinomas treated by modified mastectomy or breast-conserving treatment, including axillary node dissection, with at least 5 to 10 year clinical follow-up and documented clinicopathological data. In a separate collaborative study with our co-investigator, we will eventually be performing microarray gene-expression profiling using the 70-gene model on these same breast cancer cases. The wild-type gene expression of these cases will thus be available for comparison with wild-type expression and splice variant data obtained in this pilot project. The objectives of this pilot study are thus as follows: Objectives: (1) To identify splice-variants of the estrogen receptor alpha and beta, BRCA1 and BRCA2 and some cell cycle genes differentially expressed in breast cancer, and investigate whether there is any correlation with age and/or clinical outcome. (2) Using high wild-type estrogen receptor levels for age as reference, to identify if there are splice variants of the same gene may be associated with opposing prognoses. Reference: 1. Perou CM, Sorlie T, Eisen MB, van de RM, Jeffrey SS, Rees CA et al. Molecular portraits of human breast tumours. Nature 2000 August 17;406(6797):747-52. 2. van d, V, He YD, van't Veer LJ, Dai H, Hart AA, Voskuil DW et al. A gene-expression signature as a predictor of survival in breast cancer. N Engl J Med 2002 December 19;347(25):1999-2009. 3. Hu Z, Fan C, Oh DS, Marron JS, He X, Qaqish BF et al. The molecular portraits of breast tumors are conserved across microarray platforms. BMC Genomics 2006;7:96. 4. Buyse M, Loi S, van't VL, Viale G, Delorenzi M, Glas AM et al. Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer. J Natl Cancer Inst 2006 September 6;98(17):1183-92. 5. Fan C, Oh DS, Wessels L, Weigelt B, Nuyten DS, Nobel AB et al. Concordance among gene-expression-based predictors for breast cancer. N Engl J Med 2006 August 10;355(6):560-9. 6. Venables JP. Aberrant and alternative splicing in cancer. Cancer Res 2004 November 1;64(21):7647-54. 7. Li C, Kato M, Shiue L, Shively JE, Ares M, Jr., Lin RJ. Cell type and culture condition-dependent alternative splicing in human breast cancer cells revealed by splicing-sensitive microarrays. Cancer Res 2006 February 15;66(4):1990-9. 8. Johnson JM, Castle J, Garrett-Engele P, Kan Z, Loerch PM, Armour CD et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science 2003 December 19;302(5653):2141-4. 9. van ', V, Dai H, van d, V, He YD, Hart AA, Mao M et al. Gene expression profiling predicts clinical outcome of breast cancer. Nature 2002 January 31;415(6871):530-6. 10. Dai H, van't VL, Lamb J, He YD, Mao M, Fine BM et al. A cell proliferation signature is a marker of extremely poor outcome in a subpopulation of breast cancer patients. Cancer Res 2005 May 15;65(10):4059-66.

Project Title:	Ubiquitination of Estrogen Receptor-alpha (ESR1) isoforms
Investigator(s):	Khoo US, Chan YK
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2007

Abstract:

The aim of this project is to examine whether ESR1 isoforms (delta Exon4, delta Exon5, and deltaExon7) undergo proteasome proteolysis degradation by interacting with Carboxyl-terminus of Hsp70 interacting Protein (CHIP). Estrogen Receptor-alpha (ESR1) is a member of the steroid hormone nuclear receptor superfamily and plays an important role in cellular growth and differentiation in breast cancer. Binding with estrogen, ESR1 subsequently dimerizes and then translocates to nuclear leading to transcription of hormone-responsive genes [1]. ESR1 is encoded by eight exons within a genomic locus of greater than 140 kb [2]. It has been well documented that ESR1 isoform variants generated by alternative splicing mechanism are coexpressed in a number of human normal and neoplastic tissues [3]. Thus, ESR1 gene expression and alternative splicing have been postulated to create a heterogeneous population of ESR1 isoforms with differential transcriptional activity, which may help to potentiate the diverse action of estrogen through a single gene. The ESR1 protein contains several structural domains, each of which has a unique function in ligand binding, gene promoter activation, and association with other members of the general transcriptional apparatus [4]. The Domain A/B encoded by exon 1 has a ligand-independent gene activation function (AF-1), shown to be important for stimulating transcription from certain estrogen-responsive genes [5]. The ESR1 DNA binding domain (DBD), composed of two type II zinc (Zn) finger motifs that have been shown to be responsible for DNA promoter sequence recognition [6], lies within Domain C which is encoded by exons 2 and 3. Domain D, encoded by exon 4, contains a constitutive nuclear localization signal as well as sequences required for dimerization of the ESR1. The carboxyl-terminal Domain E, encoded by exons 5–8, is the most functionally characterized domain which contains protein sequences important for heat-shock protein association in the cytoplasm, nuclear localization, ligand dependent receptor dimerization and estrogen and anti-estrogen ligand binding Studies have identified alternatively spliced ESR1 mRNAs that have deletions in various combinations of exons in breast cancer cell lines [7], in normal as well as breast cancer tissues, the frequency and type of variants differing significantly [8]. Breast cancer cell lines that have differential expression of splice variants respond differently to estrogens [9]. Reports suggest that the alternatively spliced ESR1 exhibit variable binding properties to estrogens/anti-estrogens and transcriptional activation/inactivation of estrogen-responsive genes. The estrogen-ESR1 complex can mediate ESR1 turnover through the ubiquitin-proteasome pathway [10]. Ubiquitination is a cellular mechanism to degrade protein through the proteasome proteolysis pathway. This process involves ubiquitin-activating enzyme (E1), ubiquitin conjugating enzyme (E2), ubiquitin ligase (E3), and the 26S proteasome. E3 ligase is the enzyme that can target specific proteins to undergo proteolysis [11. The Carboxyl-terminus of Hsp70 interacting Protein (CHIP) has been found to be one of the E3 ligases which is specific to ESR1 [12]. Previously Tateishi et al using different ESR1 truncated constructs, demonstrated that the Domain E of ESR1, encoded by exon 4-7, was important for ESR1 degradation [12]. They also demonstrated that the carboxyl-terminus of Hsp70 interacting protein (CHIP) was responsible for ESR1 degradation. Their ESR1 truncated constructs were however based on ESR1 functional domains rather than coding exons. The products expressed from these in-vitro constructs were in that sense artificial since they do not represent the splicing isoforms usually differentially expressed in breast cancer cells [13]. CHIP mediates ubiquintin-proteasome degradation of misfolded, mutated, or abnormal forms of ESR1 [12], playing a critical role in regulating ESR1 level. It is observed that estrogen can disrupt the CHIP-ESR1 interaction abrogating ubiquitin-mediated degradation [14] which most probably prevents ESR1 from degradation. Estrogen can thus in turn modulate ESR1 downstream target expression. Our preliminary analysis of ten paired breast cancer tumor and non-tumor tissues has identified alternative splicing ESR1 transcripts lacking either exon 4, 5, or 7. We hypothesize that different ESR1 isoforms undergo proteasome proteolysis degradation differently by affecting interaction between the ESR1 functional domains and CHIP. We will therefore investigate whether ESR1 splicing isoforms, namely delta Exon4, delta Exon5, and delta Exon7 resulting in shorter ligand-binding domains will affect interaction with CHIP and subsequent ESR1 proteosome-mediated degradation. References: 1. Clemons M, et al. Estrogen and the risk of breast cancer. NEngl J Med 2001;344(4):276-85. 2. Ponglikitmongkol M, et al. Genomic organization of the human oestrogen receptor gene. EMBO journal 1988;7(11):3385-8. 3. Fuqua SA, et al. Molecular aspects of estrogen receptor variants in breast cancer. Breast Cancer Res Treatment 1995;35(3):233-41. 4. Kumar V, et al. Functional domains of the human estrogen receptor. Cell 1987;51(6):941-51. 5. Fan JD, et al. Identification of the sequences within the human complement 3 promoter required for estrogen responsiveness provides insight into the mechanism of tamoxifen mixed agonist activity. Molecular endocrinology (Baltimore, Md 1996;10(12):1605-16. 6. Williams DM, et al. Primer design strategies for the targeted amplification of alternatively spliced molecules. Analytical biochemistry 1999;271(2):194-7. 7. Poola I, et al. Identification of twenty alternatively spliced estrogen receptor alpha mRNAs in breast cancer cell lines and tumors using splice targeted primer approach. Journal of steroid biochemistry and molecular biology 2000;72(5):249-58. 8. Poola I, et al. Expression of alternatively spliced estrogen receptor alpha mRNAs is increased in breast cancer tissues. Journal steroid biochemistry and molecular biology 2001;78(5):459-69. 9. Klotz DM, et al. Differential expression of wild-type and variant ER mRNAs by stocks of MCF-7 breast cancer cells may account for differences in estrogen responsiveness. Biochemical and biophysical research communications 1995;210(2):609-15. 10. Nawaz Z, et al. Proteasome-dependent degradation of the human estrogen receptor. PNAS 1999;96(5):1858-62. 11. Ciechanover A. The ubiquitin-proteasome pathway: on protein death and cell life. The EMBO journal 1998;17(24):7151-60. 12. Tateishi Y, et al. Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor. EMBO journal 2004;23(24):4813-23. 13. Kumar VL, et al. Observations on the presence of E domain variants of estrogen receptor-alpha in the breast tumors. J Surgical Oncol 2006;94(4):332-7. 14. Fan M et al. CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha. Molecular endocrinology (Baltimore, Md 2005;19(12):2901-14.

Project Title:	Splicing variant profiling in relation to Estrogen Receptor gene expression in Chinese breast cancer
Investigator(s):	Khoo US, Chan YK, Kwong A, Sham PC
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

To identify splice-variants differentially expressed in breast cancer, and investigate whether they might cluster with age and/or clinical outcome; to perform supervised hierarchical clustering of AS events following the 70-gene profile procedure to identify the splice variants associated with good and poor prognosis; to perform supervised hierarchical clustering of AS events using high wild-type estrogen receptor levels for age as the classifier on the group of poor prognosis cases to identify splice variants associated with poor prognosis.

Project Title:	The role of FOXO transcription factors in the development of hormone refractory breast cancer
Investigator(s):	Khoo US, Chan YK, Ip YC
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2009

Abstract:

Background Breast cancer is the commonest cause of cancer in women. Estrogen is implicated in the development of breast cancer. Nearly 70% of breast cancers express estrogen receptors (ER) and many ER-α positive breast cancers require estrogen for proliferation, while deprivation of estrogen will induce tumor cell apoptosis. Progression of breast cancer often involves the development of hormone refractory disease in which the cancer no longer depends on hormonal stimulation for survival and proliferation. Clinically, the anti-estrogen Tamoxifen is widely used for treating ER-α positive breast cancer. Although most of these cancers initially respond to tamoxifen therapy, tamoxifen-resistant tumors eventually develop which are a cause for treatment failure and mortality. (Johnston 1997). Nowadays, several strategies have been used to block or attenuate estrogen stimulation of breast cancer cells, but tumor cells have been found to adopt different mechanisms to escape attempts to block their growth. Previously, studies have demonstrated that the Akt kinase pathway plays an important role in the development of Tamoxifen resistance (Frogne et al 2005). Activated Akt phosphorylates and suppresses the activity of many pro-apoptotic proteins, including the Forkhead family of transcription factorys (Myatt and Lam, 2007). As part of the Forkhead family, FOXO3a plays a vital role in a variety of processes such as cellular differentiation, tumor suppression, metabolism, cell-cycle arrest, apoptosis and protection from stress (Myatt and Lam, 2007; Paik et al 2007). FOXO3a, whose activity is repressed by the PI3K/Akt kinase signaling cascade, is an important transcriptional regulator of the gene encoding the steroid hormone receptor ER-α. FOXO3a expression levels have been shown to be correlated with ER-α expression in breast cancer cells (Guo et al 2004). Moreover, it was recently discovered that ER-α and ER-ß will bind to unique domains of FOXO3a, indicating direct interaction and regulation between these important proteins (Zou et al 2008). We hypothesize that FOXO3a may play a role in regulating chemo-responsiveness of breast cancer and may be responsible for acquired resistance in cases receiving Tamoxifen therapy. Objectives 1. To examine the effect of Tamoxifen at varying concentrations and time points on a panel of Tamoxifen sensitive (TamS) and Tamoxifen resistant (TamR) breast cancer cell lines for the expression levels of FOXO3a, ER-α and ER-ß, the subcellular distribution of FOXO3a by confocal microscopy and changes in the cell signalling pathways following tamoxifen treatment. 2. To investigate the effect of tamoxifen on selected TamS and TamR breast cancer cell lines by overexpressing FOXO3a by a. transfection of wild-type FOXO3a b. transfection of a triple-mutant form of FOXO3a in which three important phosphorlyation sites are absent. 3. To see the effect of selectively silencing FOXO3a expression using siRNA to knock down FOXO3a in TamS and TamR breast cancer cell lines. References: Frogne T, JS Jepesen et al. Anti-estrogen-resistant human breast cancer cells require activated protein kinase B/Akt for growth. Endocr Relat Cancer 2005; 12(3): 599-614. Guo S, Sonenshein GE. Forkhead box transcription factor FOXO3a regulates estrogen receptor alpha expression and is repressed by the Her-2/neu/phosphatidylinositol 3-kinase/Akt signaling pathway. Mol Cell Biol 2004;24(19):8681-90. Johnston SR. Acquired tamoxifen resistance in human breast cancer--potential mechanisms and clinical implications. Anticancer Drugs 1997;8(10):911-30 Myatt SS and EW Lam. The emerging roles of forkhead box (Fox) proteins in cancer. Nat Rev Cancer 2007; 7(11): 847-59. Paik JH, R Kollipara et al. FoxOs are lineage-restricted redundant tumor suppressors and regulate endothelial cell homeostasis. Cell 2007; 128(2): 309-23. Zou Y, WB Tsai et al. Forkhead box transcription factor FOXO3a suppresses estrogen-dependent breast cancer cell proliferation and tumorigenesis. Breast Cancer Res 2008; 10(1): R21.

Project Title:	A pre-neoplastic marker for basal-like breast cancer
Investigator(s):	Khoo US, Chan YK
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2010

Abstract:

Breast cancer is the leading female cancer in Hong Kong. Gene expression microarray profiling of breast cancer has identified 5 distinct subtypes that are associated with different clinical outcomes, namely luminal A, luminal B, HER-2, basal-like and normal breast-like. Basal-like breast cancers are a distinct subtype of breast cancer characterized by an expression signature that is similar to basal/myoepithelial cells of the breast and is the subtype observed in BRCA1-related breast cancers. It represents 80-90% of breast cancers arising in BRCA1 mutation carriers and about 15% of sporadic breast cancers, with reduced BRCA1 mRNA expression noted in basal-like sporadic breast cancer cases. Patients with basal-like cancer are usually younger, associated with poorer clinical outcome, more strongly associated with family history, more frequently “interval cancers” (i.e. cancers arising between annual mammograms), with specific mammographic features, and demonstrating rapid progression. Although there is as yet no internationally accepted definition for basal-like cancers, basal cytokeratin markers, singly or in combination, such as CK5/6, CK14, and CK17 by immunohistochemistry (IHC) have been used to identify basal phenotype. These cancers are usually of high histological grade and typically do not express hormone receptors or HER-2. They are associated with an aggressive clinical history with development of metastases within the first 5 years, shorter survival and relatively high mortality rate. They also show a specific pattern of distant metastases to brain and lung. Ductal carcinoma in-situ (DCIS) with basal-like phenotype has been reported, suggested to be the precursor lesion to invasive basal-like cancer. TP53 mutations have been found at high frequency in breast cancers with germ-line BRCA1 mutations (97%) as well as in sporadic basal-like breast carcinomas (92%) independent of BRCA1 status. In contrast, a lower incidence of TP53 mutations was found in BRCA1 luminal tumors (53%) and in sporadic breast cancers of luminal sub-type. A recent publication showed that all BRCA1 related breast cancers contain TP53 mutations. Half of these stained positive by IHC for p53 accumulation, which correlated with TP53 hot-spot mutations, whilst those with protein-truncating TP53 mutations stained negative for p53 accumulation. In a study of 245 cases of pure DCIS, DCIS with basal-like phenotype was found strongly associated with IHC staining for p53 accumulation. Pelvic high-grade serous carcinoma, namely high-grade serous ovarian cancer and peritoneal cancer, usually presents at an advanced stage is the most lethal gynecologic malignancy, killing more than 60% of those affected. The site of origin of pelvic high-grade serous carcinoma has been controversial and identification of its precursor lesion had previously been elusive. The acceptance of prophylactic oophorectomy as the treatment strategy for women who have tested positive for BRCA mutations and at high risk for the development of ovarian carcinoma, led to the recognition of clinically occult tubal carcinomas and serous tubal intraepithelial carcinoma (STIC) originating in the distal fallopian tube, particularly the fimbriae thus making an important contribution to determining the ultimate site of origin of this lethal high-grade serous malignancy. Detailed routine pathological examination of fimbrae has since revealed the presence of tubal carcinomas originating in the distal fallopian tube irrespective of BRCA status. STICs have significant cytological atypia, absence of cilia, are highly proliferative, and in 80% of cases highlighted by nuclear accumulation of mutated p53 protein, with TP53 mutations found in almost all cases. In cases having both STIC and ovarian carcinoma, identical TP53 mutations were found, indicating a common primary source of tumor. Furthermore, some TP53 mutations found in the fallopian tube were not found in the ovary. More importantly, p53 immunostaining has revealed the presence of small linear p53 positive foci in non-neoplastic mucosa of the distal fallopian tube, called “p53 signatures”. These are now shown to be a relatively common finding in the fallopian tube, its prevalence in BRCA mutation carriers similar to that in women with unknown BRCA status. Just as STICs and their associated ovarian carcinomas have been shown to share identical mutations, “p53 signatures” have also been seen in continuity with STIC and share common TP53 mutations. These findings support that “p53 signatures” are thus a precursor of pelvic serous carcinoma, and probably the earliest lesion in a continuum of tubal serous carcinogenic sequence. Such findings have important clinical implications which include recommending salpingetcomy at the time of simple hysterectomy. Molecular studies now suggest classifying epithelial ovarian cancers into 2 groups: type II high-grade tumors for which p53 mutations are commonly found, which are all high-grade carcinomas; whilst type I tumors include all the other histological subtypes, particularly the low-grade and borderline tumors, which are associated with mismatch repair genes BRAF, KRAS, beta-catenin and PTEN mutations. Gene expression profile investigating human breast cancer progression has likewise shown that different tumor grades rather than the pathological stages of atypical ductal hyperplasia, DCIS and invasive ductal carcinoma are associated with distinct gene expression signatures. Tumor grade, especially that of grade III samples, were linked with the DCIS-invasive ductal carcinoma stage transition. This parallelism between basal-like breast cancer and pelvic high-grade serous carcinoma prompts us to hypothesize that “p53 signatures” may also be found in non-neoplastic breast epithelium and might be the precursor of basal-like breast cancer. Although p53 overexpression has been demonstrated in pure DCIS with basal-like phenotype, nothing is yet known of TP53 mutation status in DCIS and its relationship with the associated invasive basal-like carcinoma. The identification of “p53 signatures”in non-neoplastic epithelium adjacent to basal-like breast carcinoma will be novel and may be useful as a pre-neoplastic marker for this aggressive form of cancer. The aim of this study is 1. To investigate whether the “p53 signatures” can be found non-neoplastic breast adjacent to basal-like breast carcinoma by IHC. 2. To demonstrate that the “p53 signature” is a precursor lesion to basal-like breast carcinoma. TP53 mutations will be analyzed by direct sequencing of micro-dissected tissue samples of invasive carcinoma, its DCIS component and the “p53 signatures” found adjacently, in order to demonstrate a possible continuity between these three lesions by the sharing of identical mutations. 3. To establish whether “p53 signatures” in non-neoplastic breast may be more readily found in basal-like than in non-basal-like breast cancers. References: 1. Foulkes WD, et al: Germline BRCA1 mutations and a basal epithelial phenotype in breast cancer. J Natl Cancer Inst 95:1482-5, 2003 2. Nielsen TO, et al: Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res 10:5367-74, 2004 3. Fulford LG, et al: Basal-like grade III invasive ductal carcinoma of the breast: patterns of metastasis and long-term survival. Breast Cancer Res 9:R4, 2007 4. Manie E, et al: High frequency of TP53 mutation in BRCA1 and sporadic basal-like carcinomas but not in BRCA1 luminal breast tumors. Cancer Res 69:663-71, 2009 5. Holstege H, et al: High incidence of protein-truncating TP53 mutations in BRCA1-related breast cancer. Cancer Res 69:3625-33, 2009 6. Livasy CA, et al: Identification of a basal-like subtype of breast ductal carcinoma in situ. Hum Pathol 38:197-204, 2007

Project Title:	BIT's 3rd World Cancer Congress 2010 - Breast Cancer Conference The role of forkhead transcription factor FOXO3a in breast endocrine-sensitivity and resistance
Investigator(s):	Khoo US
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Chen J., Chan Y.K., Wu L.H., Ip Y.C., Lam E.W.F. and Khoo U.S., Forkhead Transcription Factor FOXO3a increases Akt Activity in Breast Cancer Cell Lines through a Feed-back Mechanism, The 16th Hong Kong International Cancer Congress, Hong Kong. 2009.

Cheung Y.N., Wong S.Y. and Khoo U.S., eIF4E in human breast cancer and its role in regulating translation of splice variants of breast cancer genes, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Khoo U.S., Chan Y.K., Ching C.Y.J., Chan V.S.F., Ip Y.C., Yam L., Chu C.M., Lai S.T., So K.M., Wong T.Y., Chung P.H., Yip S.P., Sham P.C., Leung G.M., Lin C.L. and Peiris J.S.M., Functional role of ICAM-3 polymorphism in genetic susceptibility to SARS infection, Hong Kong Med Journal. 2009, 26-9.

Khoo U.S., Shen D.H., Wong R. and Cheung A.N.Y., Gynaecological Cancers in Genetically Susceptible Women: New thoughts on tubal pathology, Diagnostic Histopathology. 2009, 545-553.

Khoo U.S., National and Regional Editors , Acta Cytologica. 2009.

Khoo U.S., The role of forkhead transcription factor FOXO3a in breast endocrine-sensitivity and resistance, The 3rd World Cancer Congress 2010-Breast Cancer Conference, Shanghai, China. 2010.

Khoo U.S., “The Role of Forkhead Transcription Factor FOXO3a in Breast Endocrine-Sensitivity and Resistance”, BIT’s 3rd World Cancer Congress - Breast Cancer Conference, Shanghai. 2010.

Lau L.Y., Chan Y.K. and Khoo U.S., Identification of MicroRNAs Associated with Tamoxifen Resistance in Breast Cancer, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Long J., Cai Q., Qu S., Li C., Zheng Y., Gu K., Wang W., Xiang Y.B., Cheng J., Chen K., Zhang L., Zheng H., Shen C.Y., Huang C.S., Hou M.F., Shen H., Hu Z., Wang F., Deming S.L., Kelley M.C., Shrubsole M.J., Khoo U.S., Chan Y.K., Chan S.Y., Haiman C.A., Henderson B.E., Le Marchand L., Iwasaki M., Kasuga Y., Tsugane S., Matsuo K., Tajima K., Iwata H., Huang B., Shi J., Li G., Wen W., Gao Y.T., Lu W., Zheng W. and Shu X.O., Identification of a functional genetic variant at 16q12.1 for breast cancer risk: results from the Asia Breast Cancer Consortium., PLoS Genetics. 2010, 6: e1001002.

Zhang L., Chan Y.K., Ip Y.C., Tsang W.H. and Khoo U.S., Splice variant profiling in relation to tamoxifen resistance in breast cancer, The 21st Meeting of the European Association for Cancer Research, Oslo, Norway. 2010.

Researcher : Ko FCF

Project Title:	Characterization of PKA pathway in hepatocellular carcinoma (HCC)
Investigator(s):	Ko FCF, Yam JWP
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2010

Abstract:

Key issues and problems being addressed: Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer worldwide and the second commonest fatal cancer in Asia and Hong Kong. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. Evidence from our previous work has shown that Deleted in Liver Cancer 1 (DLC1) is a tumor suppressor gene critically involved in hepatocarcinogenesis. DLC1 is widely expressed in normal human tissues, but it is frequently underexpressed in HCC and other cancers. It was shown that chromosome 8p where the locus of DLC1 is located was frequently deleted in human HCC (1). DLC1 serves as the negative regulator of Rho proteins, which are important in remodeling of actin cytoskeleton, transcription regulation, cell proliferation, tumorigenesis and metastasis (2). Recently, tensin, a focal adhesion family was first identified by us as the novel interacting partner of DLC1 (3, 4, 5) and later confirmed to be also of different DLC family members by other independent research groups. Ectopic expression of DLC1 and tensin colocalized with vinculin at focal adhesions. These independent findings suggest that tensin as the interacting partner of DLC1 is responsible for directing the focal adhesion localization of DLC1. These studies also support the notion that proper focal adhesion localization of DLC1 is required for its tumor suppressive activity. Functionally, DLC1 and its binding partner tensin family have been clearly demonstrated to be the suppressors in tumor growth and metastasis (3). Apart from studying the functional significance of DLC1 in human cancers, the upstream modulators of DLC1 have been identified and gained attention recently. Rat homolog of DLC1, p122RhoGAP was phosphorylated by PKB/Akt (6) despite the functional effect this phosphorylation on rat DLC1 is not known. Akt phosphorylation of DLC1 and the subsequent attenuation of tumor suppressive activity of DLC1 upon Akt phosphorylation have been demonstrated by us (7). Apart from Akt, other kinases, PKC/PKD were also shown to phosphorylate DLC1 and modulate nuclear shuttling of DLC1 via alteration of the interaction between DLC1 and 14-3-3 (8). From our preliminary data, we also demonstrated that cAMP/PKA could phosphorylate DLC1 (unpublished data). Phosphorylation signal of DLC1 could be detected using antibody specifically against phospho-PKA (pPKA) substrate (Cell Signaling). Clinical relevance of cAMP/PKA pathway in human cancers has been provided by the enhanced activation of cAMP/PKA pathway in ovarian cancer and leukemia (9, 10). Purpose of proposed project: Compelling evidence has demonstrated the importance of DLC1 in hepatocarcinogenesis and the involvement of cAMP/PKA pathway in the regulation of critical players in HCC. In this proposal, we aim to characterize the nature and functional implications of cAMP/PKA pathway in liver cancer particularly its effects on downstream targets such as CREB and DLC1. The three main objectives of this proposal are: 1) to document the expression profiles of PKA pathway components in HCC, 2) to characterize functions of PKA components in HCC cell model, and 3) to delineate the functional significance of downstream substrates of PKA using HCC cell model.

Project Title:	AACR 101st Annual Meeting 2010 Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma
Investigator(s):	Ko FCF
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Chan J., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Integrin-linked kinase promotes hepatocellular carcinoma oncogenesis, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Chan L.K., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo , American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Chan L.K., Tung K.K., Ng I.O.L. and Yam J.W.P., Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma, American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Kong SH

List of Research Outputs

Siu K.Y., Wong E.S.Y., Kong S.H., Wong G.W., Tam K.F., Ngan H.Y.S., Le X.F. and Cheung A.N.Y., Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers. , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A., 17 - 21 April 2010.. 2010.

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Researcher : Kwok JSY

List of Research Outputs

Kwok J.S.Y., Chan G.S.W., Lam M.F., Yan T., Tang L., Kwong K.M., Chan K.W. and Chan D.T.M., Determination of mismatched donor HLA in kidney transplant recipients with unknown donor HLA phenotypes, Clinical transplantation. 2010, [Epub ahead of print 2010 Apr 9].

Researcher : Lam AKY

List of Research Outputs

Li H., Lam A.K.Y., Xu A., Lam K.S.L. and Chung S.K., High dosage of Exendin-4 increased early insulin secretion in differentiated beta cells from mouse embryonic stem cells, Acta Pharmacol Sin. 2010, 31(5): 570-7.

Tong D.K.H., Law S.Y.K., Kwong D.L.W., Chan K.W., Lam A.K.Y. and Wong K.H., Histopathological regression of the primary tumor indicated by percentage of residual viable cells is an important prognostic factor after neoadjuvant chemoradiation therapy for esophageal cancer (Abstract), GASTRO 2009 UEGW/WCOG, London, United Kingdom, 21-25 November 2009.

Tong D.K.H., Law S.Y.K., Kwong D.L.W., Chan K.W., Lam A.K.Y. and Wong K.H., Histopathological regression of the primary tumor indicated by percentage of residual viable cells is an important prognostic factor after neoadjuvant chemoradiation therapy for esophageal cancer, Gut. 2009, Supplement No II Vol 58 - Endoscopy Supplement No I Vol 41: A74.

Researcher : Lam CCK

List of Research Outputs

So J.C.C., Hwang Y.Y., Shek T.W.H., Lam C.C.K., Lai C.L. and Kwong Y.L., Transfusion-refractory anaemia in liver cirrhosis, Gut. 2010, 59(1): 5.

Yeung C.K., Trendell-Smith N.J., Mak H.K.F., Lam C.C.K. and Kwong Y.L., ‘Western’ or ‘Asian’ intravascular large B-cell lymphoma?, Clinical and Experimental Dermatology. 2009, 34(7): e482–e483.

Researcher : Lam CW

Project Title:	Whole genome scan of genetic susceptibility for hepatitis B carries
Investigator(s):	Lam CW
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	04/2006

Abstract:

To identify genes and alleles with large genetic effects on hepatits B carrier suscepibility in the genomes of Hong Kong Chinese. By identifying these loci, we will have a tool for better prediction of the risk that an adult individual will develop chronic carrier status after acute hepatitis B infection. Additionally, through further study of genes in these loci, we may discover important leads for drugs that could diminish the risks of developing chronic carrier status after acute hepatitis B infection.

Project Title:	Therapeutic antibodies against basal cell carcinomas of the skin
Investigator(s):	Lam CW
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2009

Abstract:

The objective of this project is to identify pharmacological monoclonal antibodies against a membrane oncogene mutants for the treatment of sporadic basal cell carcinomas (BCCs), the most common human cancer. BCCs are a nonmelanocytic form of skin cancer derived from keratinocytes that characterized by slow growth, local invasion and rare metastasis [Reichrath & Querings 2006]. The incidence of BCCs is increasing worldwide [Almahroos & Kurban 2004]. It is the most common human cancer in the Caucasian population, with approximately 1 million new cases per year in the United States but with approximately 200 new cases per year in Hong Kong [Mak et al. 1995]. Fair skin, excess sun exposure and exposure to other carcinogens such as arsenic are known risk factors for the development of BCCs [Preston & Stern 1992; Wong et al. 1998]. Of these, ultraviolet B (UVB) irradiation from sunlight is the most significant risk factor. For Chinese patients, a more specific mutagenic agent such as chronic arsenicism may lead to a different disease mechanism [Wong et al. 1998; Lam et al. 1999]. A highly conserved loss of heterozygosity (LOH), commonly due to uniparental disomy, in the 9q21-q31 region containing a number of documented tumor suppressor genes including FANCC (Fanconi anemia complementation group C), XPA (Xeroderma pigmentosum group A-complementing protein) and PTCH1 (Patched homolog 1, Drosophila) was identified in BCCs [Teh et al. 2005; Danaee et al. 2006]. Loss-function somatic mutations of PTCH1 are widely distributed along the coding sequence and are the pivotal abnormality in BCCs [Hahn et al. 1996; Savino et al. 2004; Lindström et al. 2006]. Activation of the Sonic hedgehog (SHH) signal transduction pathway, results from PTCH1 gene inactivation and less often from SMO (smoothened homolog, Drosophila) gene activation, is an early event in the development of sporadic BCCs [Lam et al. 1999; Xie et al. 1998; Bale & Yu 2001]. The SHH pathway is important during vertebrate embryonic development but its dysregulation can lead to tumorigenesis [Wicking et al. 1999; Bale & Yu 2001]. PTCH1 functions as the SHH receptor. It represses hedgehog target gene expression through its interaction with SMO. However, this repression is inhibited when SHH binds PTCH1 or after PTCH1 gene has been inactivated by mutation. Since PTCH1 is upstream of SMO and SMO may have a higher intrinsic ability to activate the pathway, inhibition of SMO signalling may serve as an effective way to treat BCCs and other extracutaneous tumors caused by PTCH1 inactivating mutation [Bourne 1997; Xie et al. 1997; Taipale et al. 2000; Athar et al. 2004]. The SMO antagonists such as cyclopamine, a plant-derived steroidal alkaloid, and its derivatives were found useful for regulating the SHH pathway activity in the absence of PTCH function [Taipale et al. 2000; Athar et al. 2004]. In a mouse model, direct injection of cyclopamine into BCCs showed that inhibition of the pathway through down-regulation of HIP (heparin/heparin sulphate interacting protein) and Gli1 (glioma-associated oncogene homolog 1) genes and induces Fas expression and apoptosis [Athar et al. 2004]. Unfortunately, cells expressing activated SMO are resistant to cyclopamine [Taipale et al. 2000]. Activated SMO signalling triggers the SHH pathway through Gli-dependent transcription and results in resistant to ligand-mediated apoptosis [Taipale et al. 2000; Kump et al. 2008] Previous studies showed that Chinese population have higher prevalence of activating SMO mutation than that in Caucasian population [Xie et al. 1998; Lam et al. 1999]. Therefore, an alternative pharmacological inhibition of SMO should be developed for our population.

Project Title:	Molecular basis of autism spectrum disorders in Chinese
Investigator(s):	Lam CW
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

The primary objective of this project is to develop a molecular diagnostic protocol for autism spectrum disorders (ASD) that will be more widely effective than present diagnostic methods. Towards this end, we will characterize the genetic mutations that cause ASD in the Hong Kong Chinese population. The second objective is to establish a molecular basis for exploring the natural history of this disease in the Hong Kong Chinese population. By analyzing the nature of the mutations found and predicting their effects on protein function, we will accomplish a third objective, establishing molecular pathology for the disorder.

List of Research Outputs

Chan K.Y., Ching D.C.K., Mak C.M., Lam C.W. and Chan A.Y.W., Hereditary spastic paraplegia: identification of an SPG3A gene mutation in a Chinese family, Hong Kong Medical Journal. 2009, 15(4): 304-7.

Chau S.K.C., Kwok K.L., Ng D.K., Lam C.W., Tong S.F., Chan A.Y.W., Siu C.W.K. and Yuen L.Y.P., Maternally inherited Leigh syndrome: an unusual cause of infantile apnea, Sleep Breath. 2010, 14(2): 161-5.

Cheng T.S., Ho K.M. and Lam C.W., Heterogeneous mutations of the ATP2C1 gene causing Hailey-Hailey disease in Hong Kong Chinese, Journal of the European Academy of Dermatology and Venereology. 2010, 1202-6.

Lam C.W., Lau K.C., Mak C.M., Leung K.Y., Tsoi T.H., Tang H.Y. and Lee P., A fast modified protocol for random-access ultra-high density whole-genome scan: A tool for personalized genomic medicine, positional mapping, and cytogenetic analysis., Clinica Chimica Acta. 2009, 406(1-2): 31-5.

Lam C.W., Associate Editor, Clinica Chimica Acta. 2009.

Lam C.W., Editorial Board member, Clinica Chimica Acta. 2009.

Lam C.W., Editorial Board member, Journal of Chinese Clinical Medicine. 2009.

Lam C.W., Genetic Testing and Personalized Medicine (Theme: Medical Treatment and Diagnosis with Precision), Explore the World of Medicine: Public Lecture Series 2010, Hong Kong, 20 March, 2010. 2010.

Lam C.W., Genome-based diagnosis of genetic disease, Indian Journal of Medical Research. 2010, 131: 484-5.

Lam C.W., Newborn screening for inborn errors of metabolism & Investigations for the causes of obstetrics cholestasis, Maternal Fetal Medicine Postgraduate Meeting – Obstetrics Cholestasis & New Born Screening for Inborn Errors of Metabolism, Tune Mun Hospital, Queen Elizabeth Hospital, Pamela Youde Nethersole Eastern Hospital, Queen Mary Hospital, The University of Hong Kong, 16 October, 2009. 2009.

Lam C.W., Personalized medicine and molecular diagnostics, 2009 International Conference on Personalized Medicine "Genes and Medicine", 5th Joint Conference on Pharmacogenetics of Central South University - Inje University - The Chinese University of Hong Kong held in cooperation with the Pacific Rim Association for Clinical Pharmacogenetics (PRACP), September 19-20, 2009, Hong Kong. 2009.

Lam C.W., Prenatal Molecular Diagnosis of Genetic Disorders: A review of DNA technology in O&G, The Centre for Reproduction, Development and Growth (CRDG) and the Genetics in Reproduction and Fetal Development: From Science to Clinical Practice, Department of Obstetrics and Gynaecology, The University of Hong Kong, 25 October, 2009. 2009.

Lam C.W., Renal Physiology, RCPA AACB Chemical Pathology Course, Australasian Association of Clinical Biochemists, Hotel Grand Chancellor, Hobart TAS, Australia, 15 February, 2010. 2010.

Lee H.H.C., LEE R.S.Y., LAI C.K., Yuen L.Y.P., SIU T.S., Chan A.Y.W. and Lam C.W., A novel duplication at the putative DNA polymerase alpha arrest site and a founder mutation in Chinese in the IVD gene underlie isovaleric acidaemia, Hong Kong Medical Journal. 2010, 16(3): 219-22.

Mak C.M., Lam C.W., Siu T.S., Chan K.Y., Siu C.W.K., Yeung W.L., Hui J., Wong V.C.N., Low L.C.K., Ko C.H., Tam S. and Chan Y.W., Biochemical and molecular characterization of tyrosine hydroxylase deficiency in Hong Kong Chinese, Molecular Genetics and Metabolism. 2010, 99(4): 431-433.

Mak C.M., Fong B.M., Lam C.W. and Tam S., Genotype-confirmed alanine aminotransferase deficiency in a Chinese patient with acute liver failure: a potential diagnostic pitfall, Pathology. 2010, 42(1): 94-5.

Seto W.K., Mak C.M., BUT D., Hung I.F.N., Lam C.W., Tam S., Yuen R.M.F. and Lai C.L., Mutational analysis for Wilson's disease, Lancet. 2009, 374(9690): 662.

Yueng W.L., Lam C.W. and Ng P.C., Intra-familial variation in clinical manifestations and response to ephedrine in siblings with congenital myasthenic syndrome caused by novel COLQ mutations, Developmental Medicine & Child Neurology. 2010, e243-4.

Researcher : Lam QLK

Project Title:	Role of Leptin in Regulating B Cell Maturation and Function
Investigator(s):	Lam QLK, Lu L
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	09/2008
Completion Date:	02/2010

Abstract:

B cells play a fundamental role by secreting antigen-specific antibodies against foreign pathogens during humoral immune responses. B cells developed from the bone marrow migrate to the secondary lymphoid organs to undergo final maturation. Upon encountering their cognate antigens with coupled costimulatory signals, naïve B cells differentiate into antibody-secreting plasma cells in the periphery. In autoimmune diseases, the production of autoreactive antibodies can mediate life-threatening pathology. However, before the control of autoantibody production can be employed, a detailed knowledge of the plasma cell biology is required. Despite considerable understanding of B cell production and selection in the bone marrow, it remains incompletely clear about the regulation of peripheral B cell maturation and activation. Recent studies have revealed the importance of a supportive microenvironment including cytokines and growth factors for the generation and maintenance of plasma cells. Thus, further identification of microenvironmental factors involved in modulating B cell maturation and plasma cell differentiation will contribute to a fuller understanding of B cell biology and humoral immunity. Leptin, the product of the obese gene, is a cytokine produced by adipocytes to regulate body weight through inhibition of food intake and stimulation of energy expenditure. The structure of leptin is in high similarity to the members of the long-chain helical cytokines such as IL-6, IL-12 and G-CSF. Recent studies have demonstrated that leptin acts as a growth factor by stimulating proliferation and inhibiting apoptosis in human and murine lymphocytes (1, 2). Leptin is involved in modulating immune responses towards the Th1 phenotype and suppressing the Th2 effect by stimulating CD4+ T lymphocyte proliferation (3). Leptin has also been shown to promote the survival of T lymphocytes via the upregulation of antiapoptotic proteins such as Bcl-xL. In addition to its critical role in protective immune responses, leptin has been shown to be involved in the development of autoimmune diseases (4). The immunomodulatory effects of leptin in promoting Th1 cytokine production have been linked to enhanced susceptibility to other experimentally-induced autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE) and insulin-dependent diabetes mellitus (5, 6). Although leptin blockade by neutralization in vivo significantly improves clinical score and delays disease progression by inhibiting pathogenic T cell autoreactivity in EAE (7), it is unclear whether leptin exerts a direct effect on B cell maturation, plasma cell generation and antibody production. Because of the destructive effects being mediated by autoantibodies in autoimmune diseases, it is important to understand whether and how leptin will affect the humoral immunity. Encoded by the diabetes (db) gene, the leptin receptor (Ob-R) is a member of class I cytokine receptors, which has signaling capability of IL-6 type cytokine receptors (8). Early studies on db/db mice, in which the leptin receptor Ob-Rb isoform that transduces signals through leptin binding is truncated, have revealed that the numbers of lymphocytes in the peripheral lymphoid organs were signigicantly reduced (9). To understand how leptin modulate immunity directly, I have previously explored the potential role of leptin in regulating dendritic cell development and function by studying dendritic cell (DC) maturation and survival in the pre-diabetic db/db mice (10). We showed that murine DCs derived from the bone marrow (BM) of db/db mice display an immature phenotype with reduced expression levels of MHC class II and co-stimulatory molecules (10). In our recent study, we further showed the molecular mechanisms involved in leptin-mediated CD40 induction in DCs (11). These findings provide the groundwork for me to further investigate the specific role of leptin signaling in the modulation of B cell maturation and plasma cell generation, an essential part of adaptive immunity. Therefore, this proposed study will enhance the understanding of the regulation of humoral immunity, which will be beneficial to the potential design of therapeutic strategies for autoimmune diseases. Objectives of this proposal: 1. To determine the role of leptin in modulating B cell proliferation. 2. To determine the role of leptin in modulating B cell maturation. 3. To evaluate the role of leptin in regulating humoral immune responses. References 1. Howard, J. K., G. M. Lord, G. Matarese, S. Vendetti, M. A. Ghatei, M. A. Ritter, R. I. Lechler, and S. R. Bloom. 1999. Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J Clin Invest 104:1051-1059. 2. Fujita, Y., M. Murakami, Y. Ogawa, H. Masuzaki, M. Tanaka, S. Ozaki, K. Nakao, and T. Mimori. 2002. Leptin inhibits stress-induced apoptosis of T lymphocytes. Clin Exp Immunol 128:21-26. 3. Lord, G. M., G. Matarese, J. K. Howard, R. J. Baker, S. R. Bloom, and R. I. Lechler. 1998. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394:897-901. 4. Lam, Q.L., and Lu, L. 2007. Role of leptin in immunity. Cell Mol Immunol 4:1-13. 5. Sanna, V., Di Giacomo, A., La Cava, A., Lechler, R.I., Fontana, S., Zappacosta, S., and Matarese, G. 2003. Leptin surge precedes onset of autoimmune encephalomyelitis and correlates with development of pathogenic T cell responses. J Clin Invest 111:241-250. 6. Matarese, G., V. Sanna, R. I. Lechler, N. Sarvetnick, S. Fontana, S. Zappacosta, and A. La Cava. 2002. Leptin accelerates autoimmune diabetes in female NOD mice. Diabetes 51:1356-1361. 7. De Rosa, V., Procaccini, C., La Cava, A., Chieffi, P., Nicoletti, G.F., Fontana, S., Zappacosta, S., and Matarese, G. 2006. Leptin neutralization interferes with pathogenic T cell autoreactivity in autoimmune encephalomyelitis. J Clin Invest 116:447-455. 8. Baumann, H., K. K. Morella, D. W. White, M. Dembski, P. S. Bailon, H. Kim, C. F. Lai, and L. A. Tartaglia. 1996. The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci U S A 9. Bennett, B. D., G. P. Solar, J. Q. Yuan, J. Mathias, G. R. Thomas, and W. Matthews. 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr Biol 6:1170-1180. 10. Lam, Q.L., Liu, S., Cao, X., and Lu, L. 2006. Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells. Eur J Immunol 36:3118-3130. 11. Queenie Lai Kwan Lam, Bo-Jian Zheng, Dong-Yan Jin, Xuetao Cao and Liwei Lu 2007. Leptin induces CD40 expression through the activation of Akt in murine dendritic cells. The Journal of Biological Chemistry 38: 27587-27597.

Project Title:	Mechanistic studies on the role of leptin in promoting B cell survival
Investigator(s):	Lam QLK, Lu L
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2010
Completion Date:	06/2010

Abstract:

Apoptosis plays an essential role in the maintenance of homeostasis of B lymphocytes, a process that is regulated by a fine balance between the continual generation of new B lymphocytes and their elimination through multiple checkpoints in both the bone marrow (BM) and the secondary lymphoid organs. In the BM, the successful surface expression of B-cell receptor (BCR) drives B lymphocytes to become immature B cells, whereas cells that fail to produce a functional BCR are eliminated by apoptosis. Immature B cells migrate from the BM to the spleen to differentiate further into mature B cells. To prevent generation of autoreactive immune response, B cells that interact with self-antigens with high affinity are eliminated by anergy, receptor editing or apoptosis. On the other hand, B cells that are activated with antigens at optimal strength yet without additional cytokine-mediated signaling will also enter the apoptotic pathway. The fine balance of survival and death of B lymphocytes is critical because excessive apoptosis may lead to immunodeficiencies whereas insufficient apoptosis may result in autoimmunity and even lymphoid malignancies. During the past decade, remarkable progress in understanding the process of B development has been achieved but much less is known on the survival niches that are critical to the maintenance of B cell homeostasis. Recent studies have shown that a few cytokines including CD40 ligand, BAFF and APRIL can promote the survival of B cells. While it still remains unclear what other microenvironmental factors might be involved in B cell homeostatic maintenance, further characterization of the microenvironmental requirement is critically important for a fuller understanding of adaptive humoral immunity. Bcl-2 family proteins consist of anti-apoptotic and pro-apoptotic members which function to preserve or disturb mitochondrial integrity, respectively, and are required for the maintenance of immune homeostasis. Targeted loss of the anti-apoptotic member gene Bcl-2 has lead to massive apoptosis of lymphocytes which resulted in a complete failure of immune function, whereas overexpression of bcl-2 increased the accumulation of B cells. The role of Bcl-2 as a new class of oncogenes emerged in the late 1980s with the discovery of a chromosomal translocation t(14:18) that resulted in transcriptional activation of the Bcl-2 gene associated with human follicular B cell lymphoma (FBL). Bcl-2 is a prime target for novel therapeutics because its overexpression is common in many types of cancers including the hematological cancers such as non-Hodgkin's lymphoma (NHL), follicular centre B-cell lymphoma (FCCL), chronic lymphocytic leukemia (CLL) and both acute and chronic lymphocytic leukemia (ALL and CLL). The frequently overexpressed Bcl-2 in malignant B cells has been shown to contribute to aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients. It has been shown that interaction between the various Bcl-2 family members and post-translational phosphorylation can regulate the Bcl-2 activity. In recent years, the expression of miRNAs is increasingly recognized to play a prominent role in immune homeostasis and function under physiological conditions, while the accumulating evidence of aberrant miRNA expression in B cell-derived tumors underscores miRNAs as potential targets for cancer therapeutics. Because of the association of Bcl-2 with B cell homeostasis and malignant B cell pathogenesis, elucidation of the miRNA layer of the regulatory mechanisms for its expression in normal B cells and their malignant counterpart will benefit our understanding of not only B cell biology but also the development of a potential target for cancer therapy. The B cell survival factors BAFF and APRIL can upregulate the anti-apoptotic members and downregulate the pro-apoptotic members of the Bcl-2 family proteins. Because of the critical function of Bcl-2 in immune homeostasis and oncogenesis, further identification of microenvironmental factors that regulate the Bcl-2 family proteins in B cells will benefit our understanding to the maintenance of B cell homeostasis and even malignancies. Leptin is a hormone originally discovered to regulate nutrient intake and metabolism, has recently been recognized to play a crucial role in modulating the immune response. We and others have demonstrated that leptin can promote the proliferation and activation of monocytes, neutrophils, natural killer cells, T cells, macrophages and dendritic cells. Despite an early study on leptin deficient ob/ob and leptin-receptor deficient db/db mice revealing markedly reduced numbers of lymphocytes in the peripheral lymphoid organs with impaired humoral responses, it has remained unclear whether and how leptin signaling is involved in modulating B cell biology. On the other hand, a variety of human cancer cells express leptin receptor and proliferate faster in response to leptin stimulation. Common polymorphisms in the leptin gene or its receptor are linked to the pathogenesis of various hematological malignancies including lymphocyte derived cutaneous T-cell lymphoma (CTCL) and NHL. Serum leptin level was significantly increased in multiple myeloma (MM) and CLL patients with correlation to the poor prognostic marker CD38 level. We have previously reported that db/db leptin receptor deficient mouse-derived dendritic cells express reduced level of Bcl-2 protein, and in our pilot studies we found that leptin can enhance the survival capacity of B cells that is associated with their anti-apoptotic function, induction of Bcl-2 expression as well as inhibition of certain miRNAs that are targeted at the 3’ untranslated region (UTR) of Bcl-2. Therefore, we hypothesize that leptin promotes B cell survival via modulation of miRNAs that target Bcl-2 expression. Our proposed studies will provide insights into the potential role of leptin in B cell malignancies, which will be beneficial to the future design of therapeutic strategies. Objectives of this proposal can be summarized as follows: 1) To study the role of leptin in B cell survival and its mechanistic modulation of Bcl-2 expression via miRNAs. 2) To examine the function of leptin in malignant B cell development and its implication in the therapeutic intervention for B cell malignancies.

Project Title:	97th Annual Meeting of the American Association of Immunologies (Immunology 2010) Mechanistic study of leptin-enhanced B cell survival
Investigator(s):	Lam QLK
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	05/2010
Completion Date:	05/2010

Abstract:

N/A

List of Research Outputs

Lo C.K., Lam Q.L.K., Yang M., Ko K.H., Sun L., Ma R., Wang S., Xu H., Tam S., Wu C.Y., Zheng B.J. and Lu L., Leptin signaling protects NK cells from apoptosis during development in mouse bone marrow, Cell Mol Immunol. 2009, 6(5): 353-60.

Researcher : Lam SGS

List of Research Outputs

Yau T.O., Leung T.H.Y., Lam S.G.S., Cheung O.F., Tung K.K., Khong P.L., Lam A.K.M., Chung S.K. and Ng I.O.L., Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis., PLoS One. 2009, 4(8): e6566.

Researcher : Lau KC

List of Research Outputs

Lam C.W., Lau K.C., Mak C.M., Leung K.Y., Tsoi T.H., Tang H.Y. and Lee P., A fast modified protocol for random-access ultra-high density whole-genome scan: A tool for personalized genomic medicine, positional mapping, and cytogenetic analysis., Clinica Chimica Acta. 2009, 406(1-2): 31-5.

Researcher : Lau LY

List of Research Outputs

Lau L.Y., Chan Y.K. and Khoo U.S., Identification of MicroRNAs Associated with Tamoxifen Resistance in Breast Cancer, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Researcher : Lee HHC

List of Research Outputs

Lee H.H.C., LEE R.S.Y., LAI C.K., Yuen L.Y.P., SIU T.S., Chan A.Y.W. and Lam C.W., A novel duplication at the putative DNA polymerase alpha arrest site and a founder mutation in Chinese in the IVD gene underlie isovaleric acidaemia, Hong Kong Medical Journal. 2010, 16(3): 219-22.

Researcher : Lee HLE

List of Research Outputs

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Researcher : Lee KW

Project Title:	The novel role of a splice variant of carboxypeptidase E (CPE-deltaN) in HCC metastasis
Investigator(s):	Lee KW, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	09/2008
Completion Date:	03/2010

Abstract:

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and is the second leading cause of cancer death in Hong Kong (1,2) When presented at the early stage, HCC can be treated with surgical resection or liver transplantation. Unfortunately, more than 80% of HCC patients are presented at their late stage and thus the above treatment cannot provide a cure. The high recurrence rate is commonly associated with a propensity of HCC for intrahepatic and extrahepatic metastases even after surgical resection, leading to poor prognosis (3). Therefore, identification of molecular mechanism of HCC metastasis might lead us to biomarkers to predict the metastatic potential of primary tumors is essential for improving prognosis, treatment strategies, and providing targets for cancer therapy. Acquisition of genetic changes is believed to be critically involved in progression of primary to metastatic tumors. Despite the advances in molecular technologies including comparative genomic hybridisation (CGH), fluorescence in situ hybridization analysis (FISH) and cDNA microarray, which allow the identification of novel targets involved in HCC metastasis, the precise mechanisms involved remain elusive. Recently, establishment of two metastatic HCC cell lines named MHCC-97L and MHCC-97H by Li et al (4), originated from the same parental cell line, MHCC-97 but different in metastatic potential provides a useful tool for studying the molecular mechanism of HCC metastasis. By comparing the gene expression profiles between these two cell lines by cDNA microarray approach, we found 15 fold higher expression of carboxypeptidase E (CPE) with an accession number of NM_001873 in the highly metastatic (MHCC97H) versus low metastatic (MHCC97L) cells. CPE, also known as carboxypeptidase H (CPH) , a ~55kD protein, first identified as a processing enzyme for prohormones (5) which is found primarily in endocrine tissues and peptidergic neurons. However, CPE has recently been reported to be expressed in neuroendocrine tumors in lung (6) and therefore has been proposed to be a useful marker for identifying such tumor, although its role in cancer progression was unknown. Since CPE was first identified in the neuronal system, Dr. Y.P. Loh (Section on Cellular Neurobiology, NICHD, NIH) stably transfected wild-type CPE cDNA into a clone of Neuro2A (WT) cells (a mouse neuroblastoma-glioma cell line) that does not express CPE, they surprisingly found that Neuro2A clones expressing CPE proliferated faster than WT cells (premininary data). This result prompted an exhaustive non-redundant nucleotide sequence database search which uncovered a novel splice variant isoform of CPE (CPE-deltaN) that lacks the N-terminus (preliminary data). By RT-PCR using primer specific for CPE-deltaN mRNA, there showed 10 fold higher expression in MHCC-97H cells when compared with MHCC97L, which is consistent to the result of cDNA microarray. Moreover, WT CPE mRNA were not expressed in these epithelial-derived MHCC97H cells. Taken together, we hypothesize that CPE-deltaN may play a role in cell proliferation during development and controls growth and metastasis of tumor cells, consistent with the notion that cancer represents development gone awry. CPE-deltaN might be a reliable biomarkers that can predict the metastatic potential of HCC tumors, as well as provide targets for therapy. There are 4 main objectives to this study: 1.To examine CPE-deltaN expression HCC clinical samples. 2.To understand the regulatory mechanism of CPE-deltaN in HCC cells. 3.To examine whether CPE-deltaN over-expression or down-regulation would affect HCC cell growth and invasion. 4.To examine the therapeutic role of CPE-deltaN suppression in HCC tumor growth and metastasis in nude mice animal model.

Project Title:	The significance of nucleophosmin phosphorylation (Threonine 234) in HCC metastasis
Investigator(s):	Lee KW, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	02/2010

Abstract:

HCC is the fifth most common malignancy worldwide and is the second most fatal cancer in Southeast Asia and Hong Kong (1,2). When presented at the early stage, HCC can be treated with surgical resection or liver transplantation. However, HCC is frequently complicated by occurrence of intrahepatic and extrahepatic metastases even after surgical resection, thereby leading to poor prognosis (3). To improve the prognosis of HCC patients, there is an urgent need to understand the molecular mechanism of metastasis in HCC. Metastasis is a complicated process attributed to the accumulation of genetic alternations. Despite the recent advances in biomedical technologies, the molecular mechanism underlying cancer metastasis is unclear. As a continuous pursuit in the molecular mechanism of HCC metastasis, we have recently established a pair of primary and its corresponding metastatic lung counterparts (PLC-PT and PLC-LM) by orthotopic injection of parental PLC cell line into the liver of the SCID mice. These two matched HCC cell lines share the same genetic background but differ in invasive ability which was evidenced by wound healing and cell invasion assays. These two cell lines provide an indispensable tools to study HCC metastasis. Since protein kinases and their phosphorylated substrates play key role in signaling process leading to metastasis, we aim to identify a phosphorylated substrate and its pathway which is critical in HCC metastasis. Using CelluSpot™ Serine/Threonine kinase peptide array analysis, we compare the phosphorylation profiling of these two matched HCC cell lines and found phosphorylated level of nucleophosmin (NPM) at Threonine 234 had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Specific phosphorylation of NPM on Threonine 234 was mediated by Cyclin-dependent kinase 1 (CDK1). Western blot analysis confirmed elevated phosphorylated level of NPM at Threonine 234 (Thr234) in PLC-LM cells using phospho-NPM (T234) antibody. The role of NPM (Thr234) in HCC metastasis was further verified in two metastatic HCC cell lines named MHCC-97L and MHCC-97-H by Li et al (4). Consistently, elevated phosphorylated level of NPM (Thr234) was also found in higher metastatic HCC cell line, MHCC97-H when compared with lower metastatic one, MHCC97-L, further suggesting the role of NPM (Thr234) in HCC metastasis. NPM, also known as B23, and located on chromosome 5q35, is a protein of 35 to 40 KD was originally identified as a non-ribosomal necleolar phosphoprotein found at high levels in the granular regions of the nucleolus (5). NPM was implicated in multiple cellular functions including ribosomal protein assembly and transport (5), centrosome duplication (6), and cell cycle progression (7) by direct or indirect mechanism. Although NPM was found to be over-expressed in certain types of carcinomas including colon, bladder and prostate, its role in cancer is far from fully understood. NPM exists in cells as a phosphoprotein and contains multiple phosphorylation sites . Therefore, phosphorylation of NPM can play major role in the functional regulation of NPM. Phosphorylation of NPM at several sites has been previously reported by several different kinases including casein kinase II (CKII), nuclear kinase II, polo-like kinase (Plk) and cdc type kinase during centrosome duplication (8). However, there are no reports showing phosphorylation events on NPM in pathogenesis of cancer. Our pilot study has shown that phosphorylated level of NPM at Threonine 234 was elevated by CDK1 in HCC metastatic cells, suggesting that it may potentially promote HCC progression and metastasis. Further characterization of phosphorylation of NPM in HCC growth and metastasis is warranted. There are three main objectives to this study: 1. To examine NPM (Thr234) expression in HCC clinical samples. 2. To evaluate the biological characteristics, and invasive ability of NPM (Thr234) in HCC both in vitro and in vivo. 3. To delineate the molecular mechanism of NPM (Thr234) in HCC growth and metastasis.

Project Title:	AACR 101st Annual Meeting 2010 Nucleophosmin (Theronine 234) is a novel mediator of tumor metastasis
Investigator(s):	Lee KW
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., CD24 is a functional marker that mediates liver tumor initiation via regulation of Nanog, American Association for Cancer Research, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., Identification of a liver cancer stem marker using a chemoresistant tumor model, "Stem cell and Cancer" Gordon Research Conference, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Epithelial-Mesenchymal Transition in Cancer Stem Cells, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Liver cancer stem cells: Implications for a new therapeutic target, Liver International. 2009, 29: 955-965.

Lee K.W., Yung L.H., Cheung C.H., Castilho A.G. and Ng I.O.L., Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis, The American Association for Cancer Research, 2010.

Lee K.W., PLOS ONE. 2010.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Luk S.U., Yap W.N., Chiu Y.T., Lee D.T.W., Ma S.K.Y., Lee K.W., Vasireddy R.S., Wong Y.C., Ching Y.P., Nelson C.O.L.L.E.E.N., Yap Y.L. and Ling M.T., Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population, International Journal of Cancer. 2010.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Researcher : Lee MF

List of Research Outputs

Au L.K., Wong C.C.L., Lee M.F., Wong C.M. and Ng I.O.L., Frequent upregulation of Enhancer of Zeste Homolog 2 was implicated in Deleted in Liver Cancer 1 gene silencing and HCC progression , The 20th Conference of the Asian Pacific Association for the Study of the Liver (APASL). Beijing, 2010.

Tung K.K., Mak K.M., Lee M.F., Li J.J., Poon R.T.P., Lai C.L., Luk J.M.C. and Ng I.O.L., Serum level of DKK1 as a marker for predicting tumor recurrence of hepatocellular carcinoma , American Association for Cancer Research 101st Annual Meeting 2010.

Researcher : Leung LH

Project Title:	Identification, isolation, expansion and characterization of cancer stem cells in non-small cell lung cancer
Investigator(s):	Leung LH, Chung LP, Wong MP
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	08/2009

Abstract:

Purpose of Study: This study aims at identifying and characterizing cancer stem cells in non-small cell lung cancer. Specific objective: 1. Identification, isolation and expansion of cancer stem cells (CSC) in established non-small cell lung cancer cell lines. 2. Characterization of the isolated human lung CSC using molecular and functional approaches as well as xenograft model. 3. Expression profiling of the identified human lung cancer stem cells. Problems and key issues: Lung cancer is the leading cause of death worldwide. Prognosis of lung cancer is usually poor and the 5 year survival rate is low due to late presentation, disease relapse and lack of definitive chemotherapy. The cancer stem cell theory, originally based on studies on acute leukemia, proposes a new paradigm in the understanding of cancer development and management, but its applicability to different cancers including lung cancer is still incompletely understood. Cancer stem cells can initiate tumor formation, differentiation along pluripotent pathways, and are capable of self-renewal. Cancer stem cells might use novel regulatory pathways to effect tumor perpetuation and drug-resistance which could be revealed by analysis of their gene expression profiles compared to unselected cancer cells. A large series of cancer stem cell markers have been reported in various types of cancers, including liver, colon, skin, brain, prostate, kidney and lung cancer. The rationale, validity and specificity of using these as cancer stem cell markers are still unclear. Systematic study and verification of their utility is needed to provide a better understanding and basis for further investigation on this topic, and to lend support to the cancer stem cell theory for lung cancers. Background: Lung cancer is the leading cause of death worldwide. The prognosis of lung cancer is usually poor and the 5 year survival rate is low due to late presentation, disease relapse and lack of definitive chemotherapy. Recently, the cancer stem cell (CSC) theory suggests that a cancer is initiated by a subpopulation of cancer cells which possess stem cell characteristics and are relatively quiescent, making them resistant to conventional chemotherapeutic agents. These cells are capable of initiating tumor, control tumor differentiation and self-renewal. The existence of cancer stem cells in solid tumours is still being debated, and some investigators prefer to use the term “tumour-initiating cells” to “cancer stem cells”. In this application, for the convenience of communication, the term “cancer stem cell (CSC)” would be used. CSCs were first found in acute myeloid leukemia (AML) cells by Dick J. et al. (1), who demonstrated that only a small subset of human AML cells, which were phenotypically similar to normal hematopoietic stem cells, had the ability to transfer AML when transplanted into immunodeficient mice. The investigators found that other AML cells were incapable of inducing leukemia, suggesting that only a minority of cells, i.e. those that possess stem cell properties, were tumor-initiating. It suggests that cancer is a kind of stem cell disorder and not a simple result of imbalance of cell proliferation and apoptosis signaling. Rather, the unregulated tumor cell growth is thought to be due to a disruption in the regulatory mechanisms controlling stem cell renewal, resulting in impaired stem cell differentiation and unlimited self-renewal. Later on, CSCs were discovered in other types of cancers, including brain, skin (melanoma), prostate, kidney, breast, liver, colon, ovary and lung cancers (2-14). Subsequently, a large panel of CSC markers have been reported in various types of cancers, such as CD133 in cancers of the liver, brain, colon and lung (4, 8, 11, 13) etc. On the other hand, CD44+/CD24-/low were used as CSC markers in breast and prostate cancers. Aldehyde dehydrogenase (ALDH) was also used as a CSC marker of lung and breast cancers (15, 16). CD34 and Sca-1 were used for the identification of murine lung stem cells but Sca-1 is not expressed in human tissues (17). Several ABC transporters and ALDH (15, 18-21) were used to isolate CSC from breast and lung cancers. Expression of embryonic genes such as Oct-4 and Bmi-1 were found in CSC from different tissues (22, 23). However, results from different research groups varied depending on the markers used and with tissue types, making the validity of the reported CSC markers less definitive. Notwithstanding the rationale of using these as CSC markers, the validity and specificity of these markers in lung cancer is still unknown. Currently, it still remains open for investigation whether lung CSC exists and plays a crucial role in tumor initiation, drug resistance and tumor recurrence. To our knowledge, limited studies involving CSC isolation, expansion and characterization using a large series of potential CSC markers have been reported. Specifically, studies on lung cancer stem cells isolated from cell lines of Chinese patients have not been reported previously. The proposed work would aid the understanding of how CSC regulate tumour growth and survival, and provide information for developing new treatment approaches. Rationale: This study is designed to identify and isolate lung CSCs from 26 non-small cell lung cancer (NSCLC) cell lines including those raised from local patients. A panel of stem cell markers reported in different cancers would be used and compared. The identified cells would be isolated, expanded and characterized for their growth and differentiation properties in culture systems, and for their tumorigenicity in xenograft models. Immunohistochemistry (IHC) using these markers would be performed to evaluate their expression in primitive cells of embryonic lung and in regenerating cells of injured lung tissues. The percentage and distribution of these tumor-initiating cells will also be examined on large-scale tissue microarrays and correlated with clinical parameters especially tumor stage and patient outcome. Paired samples of primary and metastatic tumors will also be included to evaluate the expression levels of CSC markers during tumor progression. Since the lung CSCs might signal through dysregulated pathways to enable them to survive and propagate after chemotherapy, the gene expression profiles of isolated CSC and non-CSC would be examined and compared using microarray chips to identify critical genes and pathways involved.

Project Title:	American Association for Cancer Research (AACR) 101st Annual Meeting 2010 Isolation, expansion and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer
Investigator(s):	Leung LH
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Gai G., Leung L.H., Costantino P.D., Aguila J.R., Nguyen D.M., Fink L.M., Ward D.C. and Ma Y., Generation and Characterization of Functional Cardiomyocytes using Induced Pluripotent Stem Cells derived from Human Fibroblasts. , Cell Biol Int.. 2009, 33(11): 1184-93.

Leung L.H., Ma Y., Fiscus R.R., Tung J.W., Tin P.C. and Wong M.P., Isolation, expansion and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer, American Association for Cancer Research Annual Meeting, 2010 Washington DC the United State . 2010.

Leung L.H., Wong J.C., Johlfs M.G., Tsang B.K. and Fiscus R.R., Protein kinase G type Ia activity in human ovarian cancer cells significantly contributes to enhanced Src activation and DNA synthesis/cell proliferation., Molecular Cancer Research. 2010, 8(4): 578-91.

Tam I.Y., Leung L.H., Tin P.C., Chua D.T.T., Sihoe A.D.L., Cheng L.C., Chung L.P. and Wong M.P., Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared to common activating missense mutations, Molecular Cancer Therapeutics. 2009, 8(8): 2142-51.

Researcher : Leung RYY

List of Research Outputs

Hwang Y.Y., Wong K.Y., Leung R.Y.Y., Wong S.H.M., Chan S.C., Srivastava G. and Au W.Y., Post-rituximab Burkitt transformation of PTLD: loss of CD20 expression accompanied by a switch in light-chain expression, Annals of Hematology. 2010, 89(1): 97-99.

Researcher : Leung SY

Project Title:	Identification of BRAF mutation in various stages of colorectal carcinogenesis and its relationship with KRAS mutation
Investigator(s):	Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2002

Abstract:

To examine the biological relationship of BRAF mutation with KRAS mutation. The association of mutations in these two genes with clinico-pathological features, molecular parameters and prognosis will be sought.

Project Title:	Detail study of relationship of BRAF and KRAS mutation with microstatellite instability in colorectal cancer
Investigator(s):	Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2003

Abstract:

To examine in detail the incidence of BRAF and KRAS mutation in a large series of MSI colorectal cancer with detail characterization of germine MMR gene mutation and MLH1 promoter methylation.

Project Title:	Genomic screening for potential tumour suppressors silenced by promoter hypermethylation in gastric adenocarcinomas using cDNA microarray
Investigator(s):	Leung SY, Yuen ST, Chu KM, Fung TWK, Lin MC, Chen X
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2005

Abstract:

Gastric cancer, being the second most common cancer worldwide, continues to present with poor prognosis and obscure cause. In view of the relatively high regional incidence in Hong Kong and certain parts of China, our group has initiated an extensive study in gastric cancer using genomics approach in order to build a solid foundation for on-going research. In collaboration with Stanford University (California, U.S.A.), we have reported the gene expression profiles of 126 gastric adenocarcinomatous and non-neoplastic mucosal samples using a cDNA microarray that contains more than 30,000 unique genes. Noted from the expression profiles were around one thousand genes that were significantly down-regulated in gastric tumours compared with the non-neoplastic mucosae. Among these are many potential tumour suppressors which may contribute to the development of gastric cancer. Recent focus on epigenetics has revealed DNA methylation at the gene promoter region as a key mechanism in silencing tumour suppressor genes and hence plays an important role in carcinogenesis. The latest development of genomics approach has also allowed the systematic profiling for genes that are silenced by methylation in tumour cell lines. The data generated can then be analysed in comparison to the gene expression profiles of tumour tissue samples. This approach will lead to the identification of a comprehensive list of genes that are down-regulated in gastric tumour tissues and at the same time, re-expressed in tumour cell lines after demethylation treatment. Aims: 1. To identify genes that are induced after demethylation treatment in a large panel of gastric cancer cell lines by cDNA microarray analysis 2. To analyse the data against the expression profile database of gastric tumour tissue samples, so as to prioritise a list of genes that are down-regulated in the largest proportion of gastric tumour tissues and at the same time, induced after demethylation treatment in the largest number of gastric cancer cell lines 3. To verify the association of promoter demethylation with gene re-expression in selected gastric cancer cell lines by bisulphite genomic sequencing/Pyrosequencing and RT-PCR 4. To systematically determine the promoter methylation pattern of these target genes in gastric tumour tissues, non-neoplastic gastric mucosae and peripheral blood leucocytes, and to correlate the extent of methylation with gene expression levels in gastric tumour tissues. 5. To correlate the promoter methylation-induced silencing of specific target genes with clinico-pathological parameters, response to treatment and patient outcome

Project Title:	Systematic characterisation of genes participating in Wnt signaling pathway that regulate colon stem cell renewal and their functional significance in colon cancer pathogenesis
Investigator(s):	Leung SY, Chen X, Yuen ST, Chan TL
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	12/2006

Abstract:

To identify a set of putative Wnt target genes that are highly likely to contribute to colorectal carcinogenesis and stem cell phenotype using existing genomics databases; to validate their regulation by Wnt using quantitative RT-PCR on colon cancer cell lines with inducible blockage of Wnt signaling; to examine for variation in expression level of these validated Wnt targets in large numbers of CRCs, adenomas and normal colon mucosae using tissue microarray; to classify at a molecular level CRC using expression data of multiple validated Wnt target genes and to correlate expression levels of these validated Wnt targets with clinico-pathological parameters patient outcome and molecular genetic changes; to functionally characterize several validated Wnt targets with significant clinicopathological correlation using cell culture systems; to analyse promoters for putative Tcf binding sites in selected validated Wnt target and their confirmation using luciferase reporter assay.

Project Title:	Research Output Prize (Faculty of Medicine)
Investigator(s):	Leung SY
Department:	Pathology
Source(s) of Funding:	Research Output Prize (in Faculty)
Start Date:	11/2007

Abstract:

The Research Output Prize accords recognition to an author (or team of authors) of a single research output published or created in the preceding calendar year. Faculties are free to determine what form of research output best represents their research achievement and how it should be selected.

Project Title:	Outstanding Researcher Award 2006-2007
Investigator(s):	Leung SY
Department:	Pathology
Source(s) of Funding:	Outstanding Researcher Award
Start Date:	11/2007

Abstract:

Nil

Project Title:	Molecular and functional characterisation of novel tumour suppressor genes aberrantly down-regulated in colorectal cancer
Investigator(s):	Leung SY, Chan TL, Chen X, Yuen ST
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2008

Abstract:

1) To identify a set of putative tumour suppressor genes that are highly expressed in colonic basal crypt stem/progenitor cells but are aberrantly down-regulated in CRCs compared with normal colon mucosae based on bioinformatic analysis of gene expression profiling databases; (2) To examine for potential molecular mechanisms for their down-regulated expression and their possible clinico-pathological significance; (3) To study for the functional roles of the candidate genes in colon carcinogenesis, including the RGMA gene that is found to be down-regulated by aberrant promoter methylation.

Project Title:	3rd Biennial Meeting of the International Society for Gastrointestinal Hereditary Tumours (InSiGHT 2009) DELETION OF THE 3’ EXONS OF TACSTD1 CAUSES HEREDITARY NONPOLYPOSIS COLORECTAL CANCER WITH MSH2 GENE PROMOTER METHYLATION
Investigator(s):	Leung SY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2009

Abstract:

N/A

Project Title:	Research Output Prize
Investigator(s):	Leung SY, Chan TL
Department:	Pathology
Source(s) of Funding:	Research Output Prize (in Faculty)
Start Date:	12/2009

Abstract:

To identify and recognize the best research outputs in different faculties.

List of Research Outputs

Kosinski C., Stange D.E., Xu C., Chan A.S.Y., Ho C., Yuen S.T., Mifflin R.C., Powell D.W., Clevers H., Leung S.Y. and Chen X., Indian Hedgehog Regulates Intestinal Stem Cell Fate Through Epithelial-Mesenchymal Interactions During Development, Gastroenterology. 2010, 139(3): 893-903.

Leung S.Y., Deletion of the 3’ Exons of Epcam (TACSTD1) Causes Hereditary Nonpolyposis Colorectal Cancer with MSH2 Gene Promoter Methylation, Familial Aspects of Cancer: First Announcement – 2009 Research & Practice, Mantra on Salt Beach, Kingscliff, Australia . 2009.

Leung S.Y., Genomics Study of Gastrointestinal Cancers – Molecular Classification, Prognostication and Cancer Biomarkers, The 2nd Annual Scientific Meeting of The Singapore Gastric Cancer Consortium, Biopolis, Singapore. 2009.

Leung S.Y., Germline MSH2 methylation and deletions, Colon Cancer Family Registry Steering Committee In-Person Meeting, Hawaii, U.S.A. . 2009.

Leung S.Y., Journal Editorial Board Membership, Journal of Clinical Pathology. 2010.

Leung S.Y., Molecular Pathways of Colorectal Cancer Development and their Implications in Prevention, Genetic Diagnosis and Treatment, Third Joint Scientific Meeting of The Royal Society of Radiologists and Hong Kong College of Radiologists and 17th Annual Scientific Meeting of the Hong Kong College of Radiologists, Hong Kong. 2009.

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Poulogiannis G., Ichimura K., Hamoudi R.A., Luo F., Leung S.Y., Yuen S.T., Harrison D.J., Wyllie A.H. and Arends M.J., Prognostic relevance of DNA copy number changes in colorectal cancer, J Pathol. 2010, 220(3): 338-47.

Schetter A.J., Nguyen G.H., Bowman E.D., Mathe E.A., Yuen S.T., Hawkes J.E., Croce C.M., Leung S.Y. and Harris C.C., Association of inflammation-related and microRNA gene expression with cancer-specific mortality of colon adenocarcinoma, Clin Cancer Res. 2009, 15(18): 5878-87.

Xia H.H.X., Yang Y., Chu K.M., Gu Q., Zhang Y.Y., He H., Wong W.M., Leung S.Y., Yuen S.T., Yuen R.M.F., Chan A.O.O. and Wong B.C.Y., Serum macrophage migration-inhibitory factor as a diagnostic and prognostic biomarker for gastric cancer, Cancer. 2009, 115: 5441-5449.

Researcher : Li SWV

List of Research Outputs

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Liao X

List of Research Outputs

Wei N., Liu S., Leung T.H.Y., Tam K.F., Liao X., Cheung A.N.Y., Chan K.K.L. and Ngan H.Y.S., Loss of programmed cell death 4 (Pdcd4) associates with the progression of ovarian cancer , Molecular Cancer. 2009, 8: 70.

Researcher : Lo JCY

List of Research Outputs

Wang P., Kudelko M., Lo J.C.Y., Siu Y.L., Kwok K.T.H., Sachse M., Nicholls J.M., Bruzzone R., Altmeyer R.M. and Nal-Rogier B.T.M., Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins, PLoS ONE. 2009, 4(12): e8325.

Researcher : Loong F

List of Research Outputs

Au W.Y., Kim S.J., Yiu H.H., Ngan R.K., Loong F., Kim W.S. and Kwong Y.L., Clinicopathological features and outcome of late relapses of natural killer cell lymphomas 10–29 years after initial remission, American Journal of Hematology. 2010, 85(5): 362-363.

Gill H., Trendell-Smith N.J., Loong F., Yeung C.K. and Kwong Y.L., Paraneoplastic pemphigus due to CD8-positive cytotoxic T-cell lymphoma, British Journal of Haematology. 2010, 149(4): 464.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Iqbal J., Weisenburger D.D., Greiner T.C., Vose J.M., McKeithan T., Kucuk C., Geng H., Deffenbacher K., Smith L., Dybkaer K., Nakamura S., Seto M., Delabie J., Berger F., Loong F., Au W.Y., Ko Y.H., Sng I., Armitage J.O. and chan W.C., Molecular signatures to improve diagnosis in peripheral T-cell lymphoma and prognostication in angioimmunoblastic T-cell lymphoma, Blood. 2010, 115(5): 1026-36.

Iqbal J., Weisenburger D.D., Chowdhury A., Srivastava G., Greiner T.C., Deffenbacher K.E., Kucuk C., Vose J.M., Au W.Y., Nakamura S., Seto M., Delabie J., Berger F., Loong F., Ko Y.H., Sng I., Liu X., Loughran T.P., Tsai M., Armitage J.O. and Chan W.C., NK-Cell Lymphoma Shares Strikingly Similar Molecular Features with a Distinct Set of gamma delta T-Cell Lymphoma and Identification of Aurora Kinase A Inhibitor as a Novel Therapeutic Agent, 51st Annual Meeting of the American-Society-of-Hematology, New Orleans, LA, DEC 05-08, 2009. Blood; 114(22): 132-133.

Loong F., Chan A.C.L., Ho B.C.S., Chau Y.P., Lee H.Y., Cheuk W., Yuen W.K., Ng W.S., Cheung H.L. and Chan J.K.C., Diffuse large B-cell lymphoma associated with chronic inflammation as an incidental finding and new clinical scenarios, Modern Pathology. 2010, 23(4): 493-501.

Researcher : Lu L

Project Title:	Plasma cell development and its regulation in autoimmunity
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007

Abstract:

To study PC generation and functional characteristics upon immunization; to examine the regulatory mechanisms underlying PC migration and survival; to characterize the development of PCs during the progression of autoimmune arthritis; to study the molecular mechanism involved in regulating PC development in autoimmune arthritis.

Project Title:	Regulation of dendritic cell function and its therapeutic application in autoimmune diabetes
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	NSFC/RGC Joint Research Scheme
Start Date:	12/2007

Abstract:

To study the role of leptin in regulating DC maturation and function; to elucidate the molecular mechanisms underlying leptin-induced DC maturation; to determine the effect of leptin signaling on modulating DC-mediated Treg differentiation; to explore the DC-based immune therapy for diabetes in NOD mice.

Project Title:	Functional role of leptin signaling in the regulation of NK cell development and activity
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2008
Completion Date:	10/2009

Abstract:

Natural killer (NK) cells play a crucial role in the elimination of virus-infected cells and transformed cells (1). Derived from the common lymphoid progenitors, NK precursors undergo a sequential developmental process to generate mature NK cells in the bone marrow (BM) (2). During their ontogeny, the committed progenitors to the NK cell lineage express CD122 but do not express the pan-NK cell marker NK1.1 (3). Subsequently, NK precursors differentiate into immature NK cells that are featured with the acquisition of NK cell surface receptors such as LY49 and CD94-NKG2, and undergo extensive proliferation before their final maturation into NK cells expressing DX5 and Mac-1 (2). Although considerable progress has been recently made in understanding the process of NK cell development, much less is known about the regulation of NK cell ontogeny. Gene-targeting studies have demonstrated that transcription factors such as Ets-1 and Id2 are critical for NK cell development (4, 5). Emerging evidence suggests that phenotypic differentiation and functional maturation of NK cells require an intact BM microenvironment that provides not only direct interaction between stromal cells and developing NK cells but also soluble factors such as IL-15 (6). Leptin, a 16 kDa non-glycosylated polypeptide product of the obese (ob) gene, is an adipocyte-derived hormone which has long been recognized as a key factor in regulating a wide range of biological responses including nutrient intake, energy homeostasis, neuroendocrine function, angiogenesis, bone formation and reproduction (7-9). Early studies on db/db mice, in which the leptin receptor that transduces signals through leptin binding is truncated, have revealed that B and T cell development are severely affected with reduced numbers of lymphocytes in the peripheral lymphoid organs(9). Moreover, leptin has been shown to modulate the adaptive immunity via enhancing T cell survival and stimulating their production of pro-inflammatory cytokines such as IFN- and IL-2. Recently, we have demonstrated an essential role for leptin signaling in the survival and maturation of BM-derived dendritic cells (DCs) (10). Previous studies by Zhao et al have shown that NK cells express the leptin receptor (11). In addition, the reduced number of NK cells in various peripheral organs such as spleen and liver has been observed in db/db mice (12). However, it remains unclear whether leptin-receptor deficiency affects early NK cell development in the BM. Importantly, our pilot studies have found significantly impaired NK cell development at various differentiation stages in the bone marrow of db/db mice when compared with wild-type littermates, which provides a strong baseline for our planned studies in this proposal. Objectives of this proposal: 1. To examine the role of leptin signaling in regulating NK cell development. 2. To determine the role of leptin signaling in modulating NK cell activity. References 1. Hamerman, J. A., K. Ogasawara, and L. L. Lanier. 2005. NK cells in innate immunity. Curr Opin Immunol 17:29-35. 2. Kim, S., K. Iizuka, H. S. Kang, A. Dokun, A. R. French, S. Greco, and W. M. Yokoyama. 2002. In vivo developmental stages in murine natural killer cell maturation. Nat Immunol 3:523-528. 3. Rosmaraki, E. E., I. Douagi, C. Roth, F. Colucci, A. Cumano, and J. P. Di Santo. 2001. Identification of committed NK cell progenitors in adult murine bone marrow. Eur J Immunol 31:1900-1909. 4. Barton, K., N. Muthusamy, C. Fischer, C. N. Ting, T. L. Walunas, L. L. Lanier, and J. M. Leiden. 1998. The Ets-1 transcription factor is required for the development of natural killer cells in mice. Immunity 9:555-563. 5. Yokota, Y., A. Mansouri, S. Mori, S. Sugawara, S. Adachi, S. Nishikawa, and P. Gruss. 1999. Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2. Nature 397:702-706. 6. Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J. Morrissey, K. Stocking, J. C. Schuh, S. Joyce, and J. J. Peschon. 2000. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. J Exp Med 191:771-780. 7. Lord, G. M., G. Matarese, J. K. Howard, R. J. Baker, S. R. Bloom, and R. I. Lechler. 1998. Leptin modulates the T-cell immune response and reverses starvation-induced immunosuppression. Nature 394:897-901. 8. Howard, J. K., G. M. Lord, G. Matarese, S. Vendetti, M. A. Ghatei, M. A. Ritter, R. I. Lechler, and S. R. Bloom. 1999. Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J Clin Invest 104:1051-1059. 9. Bennett, B. D., G. P. Solar, J. Q. Yuan, J. Mathias, G. R. Thomas, and W. Matthews. 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr Biol 6:1170-1180. 10. Lam, Q. L., S. Liu, X. Cao, and L. Lu. 2006. Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells. Eur J Immunol 36:3118-3130. 11. Zhao, Y., R. Sun, L. You, C. Gao, and Z. Tian. 2003. Expression of leptin receptors and response to leptin stimulation of human natural killer cell lines. Biochem Biophys Res Commun 300:247-252. 12. Tian, Z., R. Sun, H. Wei, and B. Gao. 2002. Impaired natural killer (NK) cell activity in leptin receptor deficient mice: leptin as a critical regulator in NK cell development and activation. Biochem Biophys Res Commun 298:297-302.

Project Title:	Role of leptin in regulating B cell maturation and function
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2009

Abstract:

1) To determine the role of leptin in modulating B cell proliferation and activation; 2) To study the function of leptin in plasma cell differentiation and elucidate the underlying molecular mechanisms; 3) To evaluate the role of leptin in regulating autoimmunity.

Project Title:	The regulatory B cell and its functional implication in autoimmune arthritis
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	12/2009

Abstract:

B cells are well known for their important functions in antibody production and antigen presentation. Recently, B cells have been increasingly recognized as immune regulators through the production of cytokines in immune responses (1). Although B cells are generally considered as positive regulators for their capacities to produce antibodies, serve as antigen presenting cells and secrete cytokines, certain subsets of B cells have been shown to negatively regulate immune responses, especially under autoimmune conditions (2). Upon the completion of early B cell developmental process in the bone marrow, newly formed immature B cells migrate to peripheral lymphoid organs (3). In the spleen, immature B cells can be further subdivided into two subpopulations: transitional T1 and T2 B cells (4). T1 B cells depend upon signals from B cell receptor (BCR) for survival independent of BAFF (B cell-activating factor of the TNF family) (5). T1 B cells may differentiate either into mature follicular (FO) B cells or sequentially into T2-MZP (transitional stage 2- marginal zone B cell precursors) or MZ (marginal zone) B cells. After T1 stage, the survival of B cells will become dependent upon signals from both BAFF and BCR. Activation of B cells via CD40 has been shown to stimulate their production of IL-10. Several studies have also shown that B cells can produce a large amount of IL-10 following activation through TLR pathways. In particular, splenic MZ B cells are found to produce IL-10 in response to CpG stimulation. Recently, certain B cell subsets have been identified to possess suppressive immune functions via IL-10 production. These B cells with a regulatory function have been defined as regulatory B cells (Breg cells) (2). Splenic B cells with a phenotype of CD1dhiCD21hiCD23- similar to “MZ” B cells have been shown to produce IL-10 in response to CpG stimulation in PN mice, a lupus-like mouse model. Moreover, IL-10-producing B cells with a unique phenotype of CD1dhiCD5+ could control T cell-mediated inflammatory responses, which are also found to share phenotypic markers with MZ B cells and T2-MZP cells (6). Despite the rapid progress in functional characterization of IL-10-producing regulatory B cells, microenvironmental factors and/or cytokines involved in inducing Breg cell differentiation remain largely uncharacterized. BAFF, a member of TNF family cytokines produced by myeloid cells such as macrophages and dendritic cells, is a key regulator for B cell maturation and survival (7). Analyses of BAFF-deficient mice reveal a fundamental role of BAFF during the transition from immature T1 to T2 B cells. New evidence indicates that BAFF induces CD4+Foxp3+ T cells to suppress T cell response through an indirect B cell-dependent manner, suggesting that BAFF may play a regulatory role in addition to its proinflammatory functions in autoimmune reactions (8). In our pilot study, we identified a previously unrecognized function for BAFF in inducing IL-10-producing B cell differentiation. Moreover, we demonstrated that BAFF-induced IL-10-producing B cells posses a regulatory function by suppressing T cell proliferation and Th1 cytokine production (Figure 1). In this proposal, we aim to elucidate the mechanisms by which BAFF induces Breg cell differentiation. Further studies will focus on whether and how Breg cells modulate T cell functions in normal and autoimmune conditions. Accumulating evidence has implicated Breg cells in various autoimmune diseases. Several lines of evidence have shown the existence of Breg cells in murine models of collagen-induced arthritis (CIA) and inflammatory bowel disease (IBD), demonstrating a crucial role of distinct B cell subsets in suppressing the exacerbation of autoimmune progression and deleterious inflammation (9, 10). Using B cell depleting antibodies in mice with otherwise intact immune systems, certain B cell subsets such as IL-10-producing B cells were shown to inhibit pathogenic T cell proliferation and T cell-mediated immune responses in vivo, while remaining B cells could promote disease progression (11). Moreover, adoptive transfer of IL-10-producing B cell subsets can prevent or reverse autoimmune disease progression but these corresponding B cell subsets from IL-10-/- mice show no effect. Although IL-10 produced by B cells has been recognized as a key cytokine in mediating the regulatory function of B cells, it remains largely unclear how Breg cells modulate pathogenic T cell activation and functions during the development of autoimmune arthritis (12). Current therapeutic strategies for autoimmune disorders including autoimmune arthritis are mainly non-specific anti-inflammatory treatment and immunosuppression that are associated with frequently-occurred severe side effects. Recently, we have identified a role of BAFF in modulating the survival of developing B cells in mouse bone marrow (13). Moreover, we have characterized both dysregulated BAFF expression and B cell maturation during the development of autoimmune arthritis (14). To establish a therapeutic strategy for treating autoimmune arthritis, we have successfully demonstrated that local BAFF gene silencing in joint tissue can suppress Th17 cell generation and inhibit inflammatory reaction, resulting in significantly reduced joint pathology in CIA mice (15). In this proposal ,we aim to further explore an effective cellular therapy by using Breg cells to suppress autoimmune reactions in vivo, where we will transfer BAFF-induced Breg cells into CIA mice. Our expected findings will provide us a strong baseline for future RGC grant application to investigate the immune mechanisms underlying the suppressive effect of Bregs on autoimmune progression. The specific objectives of this proposal are: (1).To identify a novel function of BAFF in inducing Breg cell differentiation and study its underlying mechanism. (2).To characterize immune dysregulation during the development of autoimmune arthritis and functional modulation by Breg cells. (3).To investigate the therapeutic effect of in vivo transfer of Breg cells on autoimmune arthritis in CIA mice.

Project Title:	2010 Annual European Congress of Rheumatology (EULAR 2010) Functional induction of regulatory B cells and its application in the treatment of autoimmune arthritis
Investigator(s):	Lu L
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2010
Completion Date:	06/2010

Abstract:

N/A

List of Research Outputs

Chen D., Zeng Y., Zhou J., Yang L., Jiang S., Huang J., Lu L. and Zheng B., Association of candidate susceptible loci with chronic infection with hepatitis B virus in a Chinese population. , J Med Virol.. 2010, 82(3):: 371-378.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Li J., Wan Y., Guo Q., Zou L., Zhang J., Fang Y., Fu X., Liu H., Lu L. and Wu Y., Altered microRNA expression profile with miR-146a upregulation in CD4+ T cells from patients with rheumatoid arthritis, Arthritis Res Ther. 2010, 12(3): R81.

Lo C.K., Lam Q.L.K., Yang M., Ko K.H., Sun L., Ma R., Wang S., Xu H., Tam S., Wu C.Y., Zheng B.J. and Lu L., Leptin signaling protects NK cells from apoptosis during development in mouse bone marrow, Cell Mol Immunol. 2009, 6(5): 353-60.

Lu L., Ageing of the immune system and its underlying mechanisms, Practical Geriatrics. 2010, 24: 181-183.

Lu L., Arthritis pathogenesis and its experimental therapy, The 1st International Rheumatoid Arthritis Conference, Chinese Society for Rheumatology, Beijing, China. 2009.

Lu L., Autoimmune arthritis and its therapeutic intervention, Frontiers in Biomedical Research, HKU. 2009.

Lu L., Role of B cell activating facotor in autoimmune arthritis, Annual European Congress of Rheumatology, Rome, Italy, 2010.

Ma F., Liu X., Li D., Wang P., Li N., Lu L. and Cao X., MicroRNA-466l upregulates IL-10 expression in TLR-triggered macrophages by antagonizing RNA-binding protein tristetraprolin-mediated IL-10 mRNA degradation, J Immunol. 2010, 184(11): 6053-9.

Su Z., Kong F., Wang S., Chen J., Yin R., Zhou C., Zhang Y., He Z., Shi Y., Shi X., Lu L., Xue Y., Shao Q. and Xu H., The rag locus of Porphyromonas gingivalis might arise from Bacteroides via horizontal gene transfer, Eur J Clin Microbiol Infect Dis. 2010, 29(4): 429-37.

Xu H., Su Z., Wang S., Dai X., Chen J., Kong F., Li Y., Peng S., Shao Q., Ezaki T. and Lu L., Four novel resistance integron gene-cassette occurrences in bacterial isolates from zhenjiang, china., Curr Microbiol. 2009, 59(2): 113-7.

Yang M., Sun L., Wang S., Xu H., Zheng B., Cao X. and Lu L., Cutting Edge: Novel function of BAFF in the induction of IL-10-producing regulatory B cells, Journal of Immunology. 2010, 184: 3321-3325.

Yang P., Qiu G., Wang S., Su Z., Chen J., Wang S., Kong F., Lu L., Ezaki T. and Xu H., The mutations of Th1 cell-specific T-box transcription factor may be associated with a predominant Th2 phenotype in gastric cancers, Int J Immunogenet. 2010, 37(2): 111-5.

Zhou J., Huang J., Poon K.M., Chen D., Chan C.S., Ng F., Guan X.Y., Watt R.M., Lu L., Yuen K.Y. and Zheng B., Functional dissection of an IFN-alfa/belta receptor 1 promoter variant that confers higher risk to chronic hepatitis B virus infection. , J Hepat.. 2009, 51(2):: 322-332.

Researcher : Ma ESK

List of Research Outputs

Kwan T.K., Ma E.S.K., Chan Y.Y., Wan T.S.K., Liu H.S.Y., Sim J.P.Y., Yeung Y.M., Lie A.K.W. and Yip S.F., BCR-ABL mutational studies for predicting the response of patients with chronic myeloid leukaemia to second-generation tyrosine kinase inhibitors after imatinib fail, Hong Kong Medical Journal. 2009, 15(5): 365-73.

Kwong A., Ng E.K.O., Leung C.P.H., Tsang W.P., Wong L.P., Kwok T.T. and Ma E.S.K., Role of miR-143 regulating DNA methyltransferases 3A in breast cancer, Ejc Supplements. 2010, 8(3): 172-173.

Ng E.K.O., Leung C.P.H., Au S., Chan A., Wong L.P., Ma E.S.K., Pang R.W.C., Chua D.T.T., Chu K.M., Law W.L., Poon R.T.P. and Kwong A., Plasma microRNA as a potential marker for breast cancer detection, The 101st Annual Meeting of the American Association for Cancer Research Annual Meeting, Washington D.C., U.S.A., 17 - 21 April 2010.

Ng E.K.O., Kwong A., Tsang W.P., Leung C.P.H., Wong L.P., Kwok T.T. and Ma E.S.K., Role of miR-143 regulating DNA methyltransferases 3A in breast cancer, Cancer Research. 2009, 69(24): 695S-695S.

So J.C.C., So A.C., Chan A.Y., Tsang S.T.Y., Ma E.S.K. and Chan L.C., Detection and characterisation of beta-globin gene cluster deletions in Chinese using multiplex ligation-dependent probe amplification, J Clin Pathol. 2009, 62(12): 1107-11.

Researcher : Ma SKY

Project Title:	The role of microRNAs in the regulation of CD133+ liver cancer stem/progenitor cells
Investigator(s):	Ma SKY, Chan KW, Guan XY
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2008
Completion Date:	05/2010

Abstract:

Hepatocellular carcinoma (HCC) ranks the fifth most prevalent cancer worldwide and the second most common fatal cancer locally. Surgical resection and liver transplantation is available for early-stage HCC, but since most patients are diagnosed at an advanced stage, only 25% of patients with this cancer are amendable to surgery. Chemotherapy is one of the main treatments given to the remaining patients with inoperable HCC and also administered as a pre-/post-surgical adjuvant therapy, yet the overall response rate to this treatment is low due to the highly chemotherapy-resistant nature of the disease. In the past several years, accumulating evidence has lent support to the concept of “stem cell model of carcinogenesis”; which suggests that tumors are organized in a hierarchy of heterogeneous cell populations and that the capability to sustain tumor formation and growth exclusively resides in a rare fraction of cells called cancer stem cells (CSCs)(Refs. 1,2, listed at the end of section VI). These cells not only possess unique survival mechanisms but also harbor distinctive stem/progenitor cell properties including the ability to self-renew, differentiate, and proliferate following a prolonged period of quiescence. The stem cell-like characteristics of these cells and their limited number within the tumor bulk are believed to account for their capability to escape therapies. If tumor growth and metastasis are really driven by CSCs, this can explain why current chemotherapies, developed largely against the bulk tumor mass, are only transiently, if at all, able to shrink the primary tumor, but are unable to provide a lasting cure for the disease. It is likely that these residual CSCs, due to their resistance ability, are able to survive in a dormant state after remission and result in tumor relapse, with renewed resilience and aggression (Ref. 3, listed at the end of Section VI). Recent efforts in the study of CSCs in HCC in our laboratory have led to the identification of a novel and prominent HCC CSC marker – CD133 (Ref. 3, listed at the end of Section VI). Using a severe partial hepatectomy model in which over 70% of the mouse liver mass was removed, CD133 was identified to be significantly involved in the liver regeneration process. Subsequent studies of sorted CD133+/- cells from HCC cell lines and xenograft models found CD133+ HCC cells to not only possess a greater colony-forming efficiency, higher proliferative output and greater ability to form tumor in vivo but are also endowed with characteristics similar to those of stem/progenitor cells including the preferential expression of “stemness” genes, the ability to self-renew and the ability to differentiate into non-hepatocyte-like, angiomyogenic-like lineages. Interestingly, CD133 expression is also found to represent only a minority of the tumor cell population in human HCC specimens (Ref. 4, listed at the end of Section VI). Further studies found CD133+ cells to be more resistant to conventional chemotherapeutic drugs such as doxorubicin and 5-fluorouracil; the underlying mechanism of which required the preferential activation of the Akt/PKB and Bcl-2 survival pathway (Ref. 5, listed at the end of Section VI). In continuation with our recent findings, we aim to further characterize the specific molecular and biological properties governing CD133+ HCC CSCs. microRNAs (miRNAs) are non-coding, single stranded RNAs of approximately 22 nucleotides. In the past several years, this specific class of small RNA molecules has gained significant interest in the molecular biology field and is now recognized as important regulators of post-transcriptional gene expression (Ref. 6, listed at the end of Section VI). miRNAs was first found to be crucial for proper stem cell maintenance and function. Embryonic stem (ES) cells deficient in miRNA processing cannot be maintained. Further, studies have shown that changes in specific miRNAs are associated with ES cell self-renewal and differentiation (Refs. 7,8, listed at the end of Section VI). In addition, miRNA have also been strongly implicated in the development and pathology of many cancers (Refs. 9,10, listed at the end of Section VI). Specifically in the context of HCC, accumulating evidence suggests that specific miRNAs are instrumental in the establishment and/or progression of the disease (Refs. 11,12, listed at the end of Section VI). Since miRNAs regulate self-renewal and differentiation and can function as either oncogenes or tumor suppressor genes to regulate tumor development and prognosis, we examined whether differences in miRNA expression might distinguish CD133+ HCC CSCs from their more differentiated progeny. Sorted CD133+/- fractions from HCC cell lines Huh7 and PLC8024 were analyzed by quantitative PCR for expression of 95 selected miRNAs known to play roles in stem cell biology and differentiation (Stem cell microRNA qPCR assay panel, System Biosciences). Preliminary data from miRNA profiling of sorted CD133+/- cells from the two HCC cell lines revealed several common differentially expressed miRNAs that are specifically involved in stem cell self-renewal and/or differentiation – of these includes the overexpression of miR-372 in CD133+ cells (Figure 1, please see under Section VI). Preliminary studies found the expression of miR-372 to correlate with expression of CD133 in several liver cell lines. The aim of the proposed project is to elucidate the role of differentially expressed miR-372 in the regulation of CD133+ HCC CSCs. Specifically, we aim to (1) examine the expression of miR-372 in a panel of liver cell lines (Huh7, PLC8024, Hep3B, H2P, H2M, HepG2, MiHA) and correlate its expression with CD133, using quantitative PCR analyses; (2) stably overexpress and silence miR-372 in CD133- and CD133+ cells isolated from HCC cell lines, respectively, using lentiviral transduction; (3) study the role of miR-372 in in vitro and in vivo studies to examine its role in cell proliferation, self-renewal and differentiation; (4) elucidate potential downstream interacting mRNA/protein molecules of mir-372 using 2D-PAGE proteomic approach; and (5) determine the expression of miR-372 in sorted CD133+/- cells isolated from human clinical samples and correlate the data with patient’s clinic-pathological parameters.

Project Title:	The study of the interplay between microRNA-130b and TP53INP1 and their role in hepatocellular carcinoma
Investigator(s):	Ma SKY, Guan XY, Chan KW
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2010

Abstract:

Key issues and problems being addressed: Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in Asia and locally in Hong Kong. Surgical resection and liver transplantation is available for early-stage HCC, but since most patients are diagnosed at an advanced stage, only 25% of patients with this cancer are amendable to surgery. The disease is further complicated by a high post-surgical recurrence rate and is refractory to chemotherapies. The process of hepatocarcinogenesis is complex, involving de-regulation of both oncogenes and tumor suppressor genes. Despite our growing understanding, the molecular mechanism underlying hepatocarcinogenesis is limited. A novel paradigm in tumor biology suggests that cancer growth is driven by stem-like cells within a tumor called tumor-initiating cells (TICs). Previously, we have identified a group of primitive stem cells from freshly resected HCC clinical tissue specimens and cell lines that is marked by their CD133 surface phenotype. These CD133+ TICs share features common to stem cells and cancer, as they are capable of self-renewal, differentiation as well as tumor initiation. MicroRNA (miRNAs), a novel class of small RNA molecules, has recently been implicated in regulation of stem cells as well as in human cancer development. Because miRNAs can regulate cell-fate decisions, as well as function as either oncogenes or tumor-suppressor genes, it is likely that knowledge gained from human stem cell transformation can provide a basis for understanding of tumor development. Differential miRNA expression profiling of CD133+ TICs and CD133- cells from human HCC clinical specimens and cell lines identified a marked over-expression of miR-130b in our CD133+ TICs. As part of our continual study, our preliminary data also found miR-130b to be over-expressed in HCC as compared with matched non-tumorous (NT) liver. This up-regulation, together with our preliminary functional study on the role of miR-130b in HCC, suggests de-regulation of miR-130b in hepatocarcinogenesis. Integrated analysis using both in silico predictions and mRNA expression profiling; together with findings from our luciferase reporter assay, subsequently identified tumor protein 53-induced nuclear protein 1 (TP53INP1) to be a functional target of miR-130b. However, to date, there is no information available on correlation of miR-130b and TP53INP1; nor the expression profile, tumorigenic potential and functional characteristics of TP53INP1 in HCC. Further characterization of TP53INP1; and its interplay with miR-130b is warranted. Objectives: 1. To evaluate the clinicopathological relevance and significance of miR-130b and TP53INP1 expression in matched non-tumorous and HCC clinical tissue specimens. - Examine miR-130b expression - Examine TP53INP1 expression - Analyze if miR-130b and TP53INP1 expression are correlated - Analyze if miR-130b and TP53INP1 are correlated with clinicopathological parameters as well as survival of HCC patients 2. To characterize the functional role of TP53INP1 in regulating HCC tumorigenesis. - Perform in vitro functional assays (cell proliferation, anchorage-independent growth, cell cycle status, apoptotic rate) - Validate in vitro studies in an in vivo orthotopic animal model

Project Title:	AACR 101st Annual Meeting 2010 microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression
Investigator(s):	Ma SKY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., CD24 is a functional marker that mediates liver tumor initiation via regulation of Nanog, American Association for Cancer Research, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., Identification of a liver cancer stem marker using a chemoresistant tumor model, "Stem cell and Cancer" Gordon Research Conference, 2009.

Chen L., Chan H.M., Yuan Y.F., Hu L., Huang J., Ma S.K.Y., Wang J., Dong S., Tang K.H., Xie D., Li Y. and Guan X.Y., CHD1L promotes hepatocellular carcinoma progression and metastasis in mice and is associated with these processes in human patients, Journal of Clinical Investigation. 2010, 120: 1178-1191.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Epithelial-Mesenchymal Transition in Cancer Stem Cells, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Liver cancer stem cells: Implications for a new therapeutic target, Liver International. 2009, 29: 955-965.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Luk S.U., Yap W.N., Chiu Y.T., Lee D.T.W., Ma S.K.Y., Lee K.W., Vasireddy R.S., Wong Y.C., Ching Y.P., Nelson C.O.L.L.E.E.N., Yap Y.L. and Ling M.T., Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population, International Journal of Cancer. 2010.

Ma S.K.Y., CD133 And Tumor-initiating Cells In The Liver, Inaugural Symposium On Stem Cell And Regenerative Medicine Program. 2009.

Ma S.K.Y., Chan K.W. and Guan X.Y., Cancer Stem Cells - Concepts, Methodologies and Therapeutic Implications, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Ma S.K.Y., Tang K.H., Chan K.W. and Guan X.Y., Liver Cancer Stem Cells - A Review of Current Literature and Protocols, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Ma S.K.Y., Tumor-initiating Cells / Cancer Stem Cells, Surgical Research Workshop 2010. 2010.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Ma S.K.Y., Chan Y.P., Kwan P.S., Tang K.H., Vielkind J.V., Guan X.Y. and Chan K.W., microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression , American Association for Cancer Research. 2010.

Tang K.H., Ma S.K.Y. and Guan X.Y., Liver Tumor-initiating Cells / Cancer Stem Cells: Past Studies, Current Status And Future Perspectives, In: R Scatena, A Mordente, B Giardina, Advances In Cancer Stem Cell Biology. Springer, 2010.

Researcher : Mak CM

List of Research Outputs

Chan K.Y., Ching D.C.K., Mak C.M., Lam C.W. and Chan A.Y.W., Hereditary spastic paraplegia: identification of an SPG3A gene mutation in a Chinese family, Hong Kong Medical Journal. 2009, 15(4): 304-7.

Fung C.W., Poon G.W.K., Kwok A.M.K., Cheung P.T., Low L.C.K., Siu S., Mak C.M., Tam S. and Wong V.C.N., A study of cerebrospinal fluid neurotransmitter assay in children with undiagnosed neurological diseases in Hong Kong, International Symposium on Epilepsy in Neurometabolic Diseases (ISENMD)(Diamond Prize). 2010.

Mak C.M., Fong B.M., Lam C.W. and Tam S., Genotype-confirmed alanine aminotransferase deficiency in a Chinese patient with acute liver failure: a potential diagnostic pitfall, Pathology. 2010, 42(1): 94-5.

Seto W.K., Mak C.M., BUT D., Hung I.F.N., Lam C.W., Tam S., Yuen R.M.F. and Lai C.L., Mutational analysis for Wilson's disease, Lancet. 2009, 374(9690): 662.

Researcher : Mak KM

List of Research Outputs

Tung K.K., Mak K.M. and Ng I.O.L., Clinical significance of target genes of Wnt/beta-catenin pathway in hepatocellular carcinoma, EJC Supplements / 21st Meeting of the European Association for Cancer Research . Elsevier, Inc, 2010, 8: 48.

Tung K.K., Mak K.M., Lee M.F., Li J.J., Poon R.T.P., Lai C.L., Luk J.M.C. and Ng I.O.L., Serum level of DKK1 as a marker for predicting tumor recurrence of hepatocellular carcinoma , American Association for Cancer Research 101st Annual Meeting 2010.

Researcher : Nanji AA

Project Title:	10th Congress of the International Society for Biomedical Research on Alcoholism Mechanisms of Apoptosis in Alcoholic Liver Disease
Investigator(s):	Nanji AA
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	07/2000

Abstract:

N/A

Researcher : Ng IOL

Project Title:	Characterization of T-cadherin in hepatocellular carcinoma
Investigator(s):	Ng IOL, Chan DW
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2003

Abstract:

To evaluate the expression of T-cadherin at mRNA and protein levels in human HCCs and in HCC cell lines; to investigate the factors which induce expression of T-cadherin in HCC.

Project Title:	Characterization of the roles of hepatitis B virus X protein in liver cancer
Investigator(s):	Ng IOL, Chan DW
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	09/2006

Abstract:

To investigate the alterations of HBx cell targets in HCC cells with knockdown of HBx by RNA interference (RNAi); to delineate the role of HBx in deregulation of mitotic checkpoint control; to characterize the cellular effects of natural HBx mutants in HCC cells.

Project Title:	Characterization of regulatory molecules of Wnt/beta-catenin signaling in liver cancer
Investigator(s):	Ng IOL
Department:	Pathology
Source(s) of Funding:	NSFC/RGC Joint Research Scheme
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

To analyze the expression patterns and activities of Dishevelled (Dv1) and DICKKOPf-1 (DKK1) in the Wnt/beta;-catenin signaling pathway in HCC tissues and cells; to functionally characterize these proteins of Wnt/β-catenin signaling pathway in HCC cells; to evaluate the clinicopathological and prognostic significance of these genes in human HCC.

Project Title:	Significance of LRP6 coreceptor upregulation in the aberrant activation of Wnt signaling in liver cancer
Investigator(s):	Ng IOL, Yam JWP, Ching YP, Yau TO
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

To evaluate the expression profile, genetic alterations, and clinical significance of LRP6 in HCC; to characterize the tumorigenic activity of LRP6 in HCC; to generate and characterize mouse models with overexpression of LRP6 in HCC cells.

Project Title:	Molecular pathology of liver cancer - a multidisciplinary study
Investigator(s):	Ng IOL, Cheung ST, Poon RTP, Ching YP, Guan XY, Jin D, Fan ST, Huang J, Lai CL
Department:	Pathology
Source(s) of Funding:	Collaborative Research Fund (CRF) - Group Research Project
Start Date:	04/2007
Completion Date:	03/2010

Abstract:

1) To elucidate the genetic and molecular alterations in multistep hepatocarcinogenesis – We will use a comprehensive approach to perform genome-wide analysis with HCC tissues and cells as well as animal models. We will determine microRNA expression profile and also characterize the role of hepatitis B virus (HBV) in different disease stages of HCC. 2) To characterize Rho/ROCK/PTEN/AKT signaling pathway in this multistep hepatocarcinogenesis - We will define the critical events in this major oncogenic pathway that have significant impact on HCC development and progression. Particularly, we will delineate the role of this pathway in angiogenesis.

Project Title:	Dissecting the PI3K/Akt signaling network and its clinicopathological significance in liver cancer
Investigator(s):	Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2008
Completion Date:	03/2010

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide. In Southeast Asia and Hong Kong, HCC is the second commonest fatal cancer, as a result of high prevalence of hepatitis B viral (HBV) infection. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. Knowledge of the molecular and cellular targets underlying hepatocarcinogenesis can help guide clinical management and design new treatment modalities. In the pursuit of identification of important cellular pathways and novel genes in HCC, we have previously reported on the Rho/RhoGAP/Pak pathway, Wnt/beta-catenin signaling, and angiogenesis, and delineated important cell targets, novel genes and their biological and clinical significance. In this proposal, we aim to characterize the PI3K/Akt signaling pathway systematically in HCC. Analysis on the activation of Akt and its related targets in human HCC will help determine their clinicopathological significance in patients with this cancer. Recently, a somatic mutation of Akt1 (E17K) has been identified and demonstrated to have oncogenic potential. However, its biological functions, oncogenicity, and prognostic significance in HCC are unknown. We aim to characterize this mutation in HCC and also find out its clinicopathological significance. Different approaches, including cutting-edge technologies in gene expression and mutation analyses, cell biology assays and mouse models, will be used to study the functional aspects and clinico-pathological and prognostic significance of the genetic and molecular alterations in this important pathway. Aims - To evaluate the expression levels and activation of players in PI3K/Akt pathway in HCC - To perform genotyping of Akt in human HCC - To assess the frequency of E17K-Akt1 and other Akt mutations in HBV- and non-HBV-associated HCC - To define the biological functions and oncogenicity of E17K-Akt1 and other Akt mutations in HCC - To delineate the clinicopathological and prognostic significance of activated Akt pathway, including E17K-Akt1 mutation, in HCC

Project Title:	Outstanding Research Student Supervisor Award 2007-2008
Investigator(s):	Ng IOL
Department:	Pathology
Source(s) of Funding:	Outstanding Research Student Supervisor Award
Start Date:	10/2008

Abstract:

For recognizing, rewarding and encouraging exceptioal research achievements; and for strengthening the research culture of the University.

Project Title:	Role of mitotic checkpoint gene MAD1 (mitotic arrest deficient 1) in liver cancer
Investigator(s):	Ng IOL, Yam JWP
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2009

Abstract:

To asses the clinical significance of MADi-alpha and MAD1-beta in HCC

Project Title:	Study of the role of the tumor suppressor phosphatase and tensin homolog (PTEN) in liver cancer and metastasis
Investigator(s):	Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is the fifth common malignancy worldwide and is the second commonest fatal cancer in Hong Kong and in this region. It is accompanied with poor patient survival rates. The disease is often diagnosed at late stages and is frequently associated with metastasis, when only limited options are then available for effective therapies. The molecular mechanisms underlying the development and progression of HCC are poorly understood. Knowledge of the molecular and cellular targets underlying hepatocarcinogenesis can help guide clinical management and design new treatment modalities. Despite the fact that the Phosphatase and Tensin Homolg (PTEN) is a frequently mutated tumor suppressor implicated in various cancers, there are relatively few reports delineating the role of PTEN in HCC development. This study is aimed to uncover the involvement of PTEN in HCC metastasis and its possible underlying mechanisms. Aims: 1. Evaluation of mRNA and protein expression profiling and genetic alterations of PTEN in HCCs 2. Delineation of the clinicopathological and prognostic significance of PTEN in human HCC. 3. Establishing cell and animal models for functional characterization 4. Evaluating its biological mechanisms in terms of cell proliferation, migration and invasion and tumorigenicity 5. Characterization of molecular pathway involved in PTEN-related cell invasion and migration

Project Title:	3rd International Liver Cancer Association 2009 Annual Conference (ILCA 2009) PTEN underexpression was associated with more aggressive tumor behaviour in hepatocellular carcinoma and PTEN suppressed cell invasion by downregulating NF-κB signaling pathway
Investigator(s):	Ng IOL
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	09/2009
Completion Date:	09/2009

Abstract:

N/A

Project Title:	Molecular Pathology of Liver Cancer - a Multidisciplinary Study
Investigator(s):	Ng IOL, Cheung ST, Ching YP, Guan XY, Jin D, Lee KW, Poon RTP, Wong CM
Department:	Pathology
Source(s) of Funding:	Collaborative Research Fund (CRF) - Group Research Project
Start Date:	05/2010

Abstract:

1) To uncover the genetic and molecular alterations in tumor formation and cancer metastasis in the HBV-associated multistep hepatocarcinogenesis. The deregulation of miRNA in relation to the early hepatocarcinogenesis and cancer metastasis will be delineated. We will also determine the microRNA expression profile characteristic of liver cancer stem cells in HCC; 2) To define the critical events in the wider network of the major Rho/ROCK/PTEN cell signaling pathway in this multistep hepatocarcinogenesis, particularly in HCC progression and metastasis.

Project Title:	Significance of cten dysregulation in hepatocarcinogenesis and cancer metastasis
Investigator(s):	Ng IOL, Yam JWP
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2010

Abstract:

Purpose of proposed project, key issues and problems being addressed - Liver cancer is a major malignancy worldwide and is prevalent in this region, being the second commonest fatal cancer in Southeast Asia including Hong Kong (because of prevalent HBV infection). About 330,000 and 1,500 of HCC cases are diagnosed each year in Mainland China and Hong Kong, respectively. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. New adjuvant treatment modalities for HCC are much awaited. In this regard, knowledge of the molecular and cellular targets underlying the development and progression of HCC is of importance as this can provide novel opportunities for therapeutic interventions for this cancer. - Focal adhesions are structural links between the extracellular matrix (ECM) and actin cytoskeleton and are composed of diverse molecules including receptors, structural proteins, adaptors, GTPases, kinases and phosphatases. These focal adhesion proteins play critical roles in normal physiological events such as cellular adhesion, movement, cytoskeletal structure and intracellular signaling pathways. In cancers, aberrant expression and altered functions of focal adhesion proteins contribute to adverse tumor behavior. Indeed, focal adhesion proteins have been shown to play critical roles in HCC. - We are the first group to identify the deleted in liver cancer 2 (DLC2) gene and have comprehensively and systematically characterized the closely related DLC1 as well as DLC2. Both DLC1 and DLC2 are focal adhesion proteins and represent a novel group of tumor suppressors that are GTPase-activating proteins for Rho family small GTPases (RhoGAPs). Significantly, DLC1 is now regarded a bona fide tumor suppressor gene not only in HCC, but also in other cancers. Our group has also identified that tensin2 is the first binding protein of DLC1 and have shown it to be important for DLC1 functions. - The tensin family comprises four members - tensin-1, -2, -3, and cten. Cten has been reported to have oncogenic functions in some cancers but tumor-suppressive functions in others. The expression profiles of cten in human cancers implicate its functional variations in different cellular context. As part of our continual study on focal adhesion proteins (including deleted in liver cancer gene 1 and 2 [DLC1, DLC2)] and tensin2) in HCC, we have found that cten binds DLC1 through a phospho-tyrosine binding (PTB) domain-independent manner. Our preliminary study showed that cten was frequently (43%) upregulated in human HCCs. It was also upregulated in metastatic human HCC cell lines as compared with their corresponding primary HCC cell lines. Overexpression of cten also substantially enhanced the cell migration ability and invasiveness of HCC cells. This upregulated expression of cten in HCC suggests that its dysregulation is implicated in hepatocarcinogenesis and cancer metastasis. However, there is little or no information available on the expression profile, tumorigenic potential and functional characteristics of cten in HCC. Further investigations are warranted. - The purpose of this study is to characterize cten genetically and functionally in liver cancer. Our previous studies have already documented the importance of focal adhesion proteins and tumor suppressive genes in HCC development. Here we hypothesize that cten, a newly identified tensin family member, is a putative oncogene in HCC and its dysregulation plays a role in hepatocarcinogenesis and cancer metastasis. We will examine the mechanism of cten upregulation in human HCC, evaluate whether it is a putative oncogene and/or metastasis enhancing gene in HCC, and the molecular pathways by which cten exerts its effects. Our work should shed light on how cten overexpression leads to HCC formation and metastasis. Objectives: - To evaluate the expression profile and clinical significance of cten in human HCC samples and cell lines - To characterize the tumorigenicity of cten in HCC both in vitro and in vivo - To characterize the metastasis enhancing ability of cten in HCC both in vitro and in vivo - To delineate the cell signaling pathways involved by cten in HCC

List of Research Outputs

Au L.K., Wong C.M. and Ng I.O.L., Enhancer of Zeste Homolog 2 (EZH2) was involved in Hepatocellular Carcinoma (HCC) Progression Through Gene Silencing, 16th Hong Kong International Cancer Congress. Hong Kong, 2009.

Au L.K., Wong C.C.L., Lee M.F., Wong C.M. and Ng I.O.L., Frequent upregulation of Enhancer of Zeste Homolog 2 was implicated in Deleted in Liver Cancer 1 gene silencing and HCC progression , The 20th Conference of the Asian Pacific Association for the Study of the Liver (APASL). Beijing, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., CD24 is a functional marker that mediates liver tumor initiation via regulation of Nanog, American Association for Cancer Research, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., Identification of a liver cancer stem marker using a chemoresistant tumor model, "Stem cell and Cancer" Gordon Research Conference, 2009.

Chan C.P., Mak T.Y., Chin K.T. and Ng I.O.L., N-linked glycosylation is required for optimal proteolytic activation of membrance-bound transcription factor CREB-H, 2010 Hong Kong Inter-University Biochemistry Postgraduate Symposium, CUHK, Hong Kong, 15 May. 2010.

Chan C.P., Mak T.Y., Chin K.T., Ng I.O.L. and Jin D., N-linked glycosylation is required for optimal proteolytic activation of membrane-bound transcription factor CREB-H., J. Cell Sci.. The Company of Biologists, 2010, 123: 1438-1448.

Chan J., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Integrin-linked kinase promotes hepatocellular carcinoma oncogenesis, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Chan L.K., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo , American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Fatima S., Lee N.P.Y., Ng I.O.L. and Luk J.M.C., The role of Dickkopf 4 (DKK4) on Wnt signaling in hepatocellular carcinoma (Poster Presentation), The 20th Annual Conference of the Asian Pacific Association for the Study of the Liver (APASL), Beijing, China, 25-28 March 2010.

Hao K., Luk J.M.C., Lee P.Y., Mao M., Zhang C., Ferguson M.D., Lamb J., Dai H., Ng I.O.L., Sham P.C. and Poon R.T.P., Predicting prognosis in hepatocellular carcinoma after curative surgery with common clinicopathologic parameters, BMC Cancer. 2009, 9: 389.

Hui C.F., Tung K.K., Sze M.F., Ching Y.P. and Ng I.O.L., Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma, In: Samuel S. Lee, Liver International. 2010, 30 Issue 1: 65-75.

Ji J., Shi J., Budhu A., Yu Z., Forgues M., Roessler S., Ambs S., Chen Y., Meltzer P.S., Croce C.M., Qin L.X., Man K., Lo C.M., Lee J., Ng I.O.L., Fan J., Tang Z.Y., Sun H.C. and Wang X.W., MicroRNA expression, survival, and response to interferon in liver cancer, New England Journal of Medicine. 2009, 361(15): 1437-1447.

Ko F.C.F., Chan L.K., Tung K.K., Ng I.O.L. and Yam J.W.P., Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma, American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Epithelial-Mesenchymal Transition in Cancer Stem Cells, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Lee K.W., Castilho A.G., Ma S.K.Y. and Ng I.O.L., Liver cancer stem cells: Implications for a new therapeutic target, Liver International. 2009, 29: 955-965.

Lee K.W., Yung L.H., Cheung C.H., Castilho A.G. and Ng I.O.L., Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis, The American Association for Cancer Research, 2010.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Lee N.P.Y., Poon R.T.P., Shek H.P., Ng I.O.L. and Luk J.M.C., Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma, Biochimica et Biophysica Acta. 2010, Epub ahead of print (May): 1-8.

Leung T.H.Y., Yam J.W.P., Chan L.K., Ching Y.P. and Ng I.O.L., DLC2 (Deleted in liver cancer 2) suppresses cell growth via regulation of Raf-1-ERK1/2-p70S6K signaling pathway. (accepted), Liver International. 2010.

Liu L., Lee P.Y., Chan V.W.M., Xue W., Zender L., Zhang C., Mao M., Dai H., Wang X.L., Xu Z., Lee K.W., Ng I.O.L., Chen Y., Kung H.F., Lowe S.W., Poon R.T.P., Wang J.H. and Luk J.M.C., Targeting cadherin-17 inactivates Wnt signaling and inhibits tumor growth in liver carcinoma, Hepatology (Erratum in: Hepatology 2010;51(1):358). 2009, 50(5): 1453-1463.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Mak G.W.Y., Ng I.O.L. and Ching Y.P., Characterization of CDK5RAP3 in hepatocellular carcinoma, AACR 101st Annual Meeting. 2010.

Mak G.W.Y., Ng I.O.L. and Ching Y.P., Functional characterization of CDK5RAP3 in Human Hepatocellular Carcinoma Cells, 16th Hong Kong International Cancer Congress. 2009.

Ng I.O.L., Asian Journal of Oral and Maxillofacial Surgery. 2009.

Ng I.O.L., Asian Journal of Surgery. 2009.

Ng I.O.L., Associate Editor, Liver International (Official journal of International Association for the Study of the Liver) . 2009.

Ng I.O.L., Journal of Gastroenterology and Hepatology. 2009.

Sun S., Poon R.T.P., Lee N.P.Y., Yeung C., Chan K.L., Ng I.O.L., Day P.J.R. and Luk J.M.C., Proteomics of hepatocellular carcinoma: serum vimentin as a surrogate marker for small tumors (£2 cm), Journal of Proteome Research. 2010, 9(4): 1923-1930.

Tam H.K.A., Wong C.M. and Ng I.O.L., Epigenetic Dysregulation of microRNA-9 in Hepatocellular Carcinoma (HCC), The 21st Meeting of the European Association for Cancer Research. Oslo, Norway, 2010.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Tung K.K., Mak K.M. and Ng I.O.L., Clinical significance of target genes of Wnt/beta-catenin pathway in hepatocellular carcinoma, EJC Supplements / 21st Meeting of the European Association for Cancer Research . Elsevier, Inc, 2010, 8: 48.

Tung K.K., Mak K.M., Lee M.F., Li J.J., Poon R.T.P., Lai C.L., Luk J.M.C. and Ng I.O.L., Serum level of DKK1 as a marker for predicting tumor recurrence of hepatocellular carcinoma , American Association for Cancer Research 101st Annual Meeting 2010.

Wong C.C.L., Wong C.M., Au L.K. and Ng I.O.L., RhoGTPases and Rho-effectors in hepatocellular carcinoma metastasis: ROCK N' Rho move it , Liver International. 2010, 30: 642-656.

Wong C.M., Wong C.C.L., Tam H.K.A. and Ng I.O.L., Aberrant expression of epigenetically regulated microRNAs in liver cancer, 101st American Association for Cancer Research (AACR) Annual Meeting 2010. Washington, DC.

Wong H., Yau T.C.C., Chan P., Ng I.O.L., Chan G.S.W., Hui P., Law W.L., Lo C.M., Hedley A.J. and Epstein R., PPI-delayed diagnosis of gastrinoma: oncologic victim of pharmacologic success, Pathology and Oncology Research. 2010, 16(1): 87-91.

Wong L.T., Yau T.O., Sze M.F. and Ng I.O.L., PTEN underexpression was associated with more aggressive tumor behaviour in hepatocellular carcinoma and PTEN suppressed cell invasion by downregulating NF-κB signaling pathway, The 3rd International LIver Cancer Association Annual Conference, Milan, Italy. 2009.

Yam J.W.P., Wong C.M. and Ng I.O.L., Molecular and functional genetics of hepatocellular carcinoma, Frontiers in bioscience (Scholar edition). 2010, 2: 117-134.

Yang W., Shen N., Ye D.Q., Liu Q., Qian X.X., Hirankarn N., Pan H.F., Mok C.C., Chan D.T.M., Wong R.W.S., Lee K.W., Wong S.N., Leung A.M.H., Li X.P., Avihingsanon Y., Wong C.M., Lee T.L., Ho M.H.K., Lee P.P.W., Chang Y.K., Li P.H., Li R., Zhang L., Wong W.H.S., Ng I.O.L., Lau W.C.S., Sham P.C., Lau Y.L. and Asian Lupus Genetics Consortium A.L.G.C., Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus. , PLoS Genetics. 2010, 6: e1000841.

Yau T.O., Leung T.H.Y., Lam S.G.S., Cheung O.F., Tung K.K., Khong P.L., Lam A.K.M., Chung S.K. and Ng I.O.L., Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis., PLoS One. 2009, 4(8): e6566.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Ng KM

List of Research Outputs

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Researcher : Ng MH

List of Research Outputs

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. ELSEVIER, 2010, 38: 481-488.

Researcher : Ng RK

Project Title:	The role of Jarid1 histone demethylases in the development of haematopoietic stem cell
Investigator(s):	Ng RK
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2009

Abstract:

The aim of this project is to investigate the role of histone demethylase Jarid1a and 1b in the specification of haematopoietic stem cells and the differentiation to other blood lineages. In addition, this project aims to study the correlation of Jarid1 family dysregulation and leukaemogenesis in haematopoietic progenitor cells. Haematopoiesis in early embryos is accomplished by a specification of distinct population of cells, which is called haematopoietic stem cell (HSC), in several embryonic locations, e.g. yolk sac, aorta, gonad, mesonephros (AGM), liver and bone marrow, during development. Haematopoietic stem cell (HSC) is a multipotent cell type, which can self-renew and differentiate to all different blood cell lineages. The process of haematopoiesis has been largely studied through genetic manipulations of early embryo. However, this approach is laborious and the amount of embryonic tissue is technically challenging for molecular analysis. Strikingly, the discovery and isolation of embryonic stem cells (ESCs) provide a new route to study the process of haematopoietic differentiation. ESC is a pluripotent cell type which can differentiate to all embryo cell types. By co-culturing with stromal cells and with specific cytokines, ESCs are able to differentiate to HSCs and other blood lineages, which represent an excellent model system to understand cell differentiation and lineage commitment. Recent studies have provided new insights on the epigenetic regulations in stem cell self-renewal and differentiation. Epigenetic modifications, such as DNA methylation and histone modifications, influence gene expression patterns and provide a unique signature of a cell differentiation status. It is generally believed that the inheritance of these epigenetic marks, from mother to daughter cells, is crucial for the maintenance of a cell differentiation status. Interestingly, a recent study of global histone H3 lysine 4 (H3K4) methylation patterns in haematopoietic progenitor cells demonstrated that genes enriched with H3K4 di-methylation (H3K4me2) is strongly correlated with haematopoiesis; and becomes demethylated upon differentiation. Besides, it showed that those haematopoietic genes with H3K4me2+/me3- are developmentally poised for transcription, suggesting a complicated regulatory network of haematopoiesis by epigenetic modifications. Jarid1 family is recently characterized as histone demethylase, which catalyzes the removal of the di- and tri-methyl group of H3K4. Jarid1 family proteins contain both the catalytic Jumonji N (JmjN) and C (JmjC) domains which process lysine demethylase activity. In addition, an ARID (A/T rich interaction domain), a zinc finger and PHD domains within this family proteins are involved in protein-protein interaction and methylated lysine recognition. Jarid1a and 1b are two of the four members in mammalian Jarid1 family. Jarid1a (also called RBP2, retinoblastoma-binding protein 2) was found as a repressor of differentiation genes and its repressive function is inhibited by binding with retinoblastoma protein (Rb). The repressive function of Jarid1a can also be mediated through an Rb-independent pathway, in which Jarid1a demethylates H3K4me2/me3 through the recruitment by Polycomb group protein complex. Jarid1a has a potential function in HSC development, for which the RBP2 (Jarid1a)-/- knockout mice demonstrated a decreased apoptosis and an increased progression of cell cycle of the isolated HSCs. Besides, Jarid1a binds to Myc protein and acts as a co-activator to promote cell growth. Recent study also identified a Jarid1a translocation in an acute myeloid leukaemia (AML) patient. On the other hand, Jarid1b (also called PLU-1) is found overexpressed in breast cancer cells. Importantly, Jarid1b mediates transcriptional repression of tumor suppressor gene BRCA1 by H3K4 demethylation, which suggests its role on promoting breast cancer cell proliferation. Thus, both Jarid1a and 1b play important role in regulation of cell differentiation and growth; and its dysregulation could lead to tumorogenesis. Because of the high homology of Jarid1a and 1b and the mild phenotype of the RBP2-/- knockout mice, it suggests that these two proteins might be functionally redundant. It is therefore necessary to perform a double knockdown of both genes to eliminate the redundant demethylase activities and study the mechanistic roles of these demethylases during haematopoiesis and leukaemogenesis. In this project, I ask three important questions concerning about the role of epigenetic regulation in haematopoiesis: 1) The functional role of Jarid1 demethylases in ESC to HSC differentiation and hence during embryo development; 2) The reprogrammibility of haematopoietic cell fate by epigenetic manipulation; 3) The leukaemogenic potential of Jarid1 family through aberrant epigenetic regulation. Consequently, this proposed project can provide novel insights into the understanding of haematopoietic cell differentiation.

Project Title:	Genome-wide analysis of DNA methylation patterns in MLL-rearranged leukaemia stem cells
Investigator(s):	Ng RK, Chan LC
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2009

Abstract:

1) To investigate the genome-wide promoter DNA methylation patterns in MLL-transformed LSCs and study their correlations in global transcriptional dysregulation; 2) To investigate the epigenomic differences of LSCs from different haematopoietic origins and study how this affects their leukaemogenic potentials; 3) To identify novel leukaemogenic candidates which are regulated by DNA methylation and study their functional roles in MLL-mediated leukaemogenesis.

Project Title:	The functional role of Bmi-1 in self-renewal of haematopoietic stem cell
Investigator(s):	Ng RK
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2010

Abstract:

HSC is a multipotent cell type, which can self-renew and differentiate to all blood cell lineages. Self-renewal refers to a process by which daughter cells are able to identically reproduce itself as the mother cell after symmetrical or asymmetrical cell division. This unique feature of stem cells is a vital process of replenishing the stem cell pool within adult tissues. It has been classically using embryonic stem cells as a model to study the process of self-renewal. However, global gene expression studies with different stem cell types suggested that different self-renewal pathways could be employed in different tissue stem cells. Since the transplantation of HSC provides a promising approach in treating haematological disorders, e.g. leukaemias, we therefore propose studying the self-renewal pathway in HSC, which has a strong clinical potential by expanding the HSC population for transplantation purposes. Hox genes are a cluster of highly conserved genes that encode DNA-binding transcription factors which regulate the positional identity of cell along the anterior-posterior body axis. Several Hox genes have been found associated with self-renewal, including Hoxa9, Hoxb4, Hoxb6 and Hoxc4. In particular, overexpression of Hoxb4 has been widely used in promoting the ex vivo expansion of HSC. Nevertheless, the regulation of these self-renewal associated Hox genes as well as their downstream targets in HSC remains largely unknown. Recent studies of epigenetics provide novel insights into the regulation of stem cell self-renewal and differentiation. Epigenetic modifications, such as DNA methylation and histone modifications, influence gene expression patterns and crucial in maintaining cellular properties, such as self-renewal capacity. Polycomb proteins are a group of repressor proteins involved in the maintenance of cellular memory. Polycomb repressive complex 2 (PRC2) can mediate histone H3 lysine 27 tri-methylation (H3K27me3) at its target gene loci. One of the polycomb repressive mechanisms is triggered by the recruitment of polycomb repressive complex 1 (PRC1) by the H3K27me3 mark, and subsequently introducing H2A lysine 119 mono-ubiquitination (H2AK119ub1) for gene silencing. Bmi-1, which is one of the components of PRC1, has been demonstrated its essential role in silencing Hox gene expression, through mediating the H2AK119ub1 at targeted Hox gene loci. Strikingly, Bmi-1 knockout mice (Bmi-1 -/-) demonstrated an early exhaustion of HSC, which is in contrary to promoting HSC self-renewal by overexpression of Bmi-1. It thus strongly suggests that Bmi-1 has a crucial function in regulating the self-renewal capacity of HSC. We hypothesize that Bmi-1 can maintain the self-renewal capacity in HSC by regulating Hox gene expression. Therefore, we aim to investigate the expression profile of Hox genes in HSC by altering the cellular level of Bmi-1 (overexpression or knockdown). As direct binding of Bmi-1 at Hoxc13 promoter region has been demonstrated in mouse embryonic fibroblasts (MEFs), we therefore hypothesize that Bmi-1 can epigenetically regulated Hox gene expression in HSC with the introduction of H2AK119ub1 at its targeted Hox loci. We thus aim to perform chromatin immunoprecipitation (ChIP) of H2AK119ub1 and Bmi-1 at Hox loci and find out their interactions with Hox gene expression in HSC. In addition, the binding of other components of PRC1 complex, e.g. Ring1a, or PRC2 complex, e.g. Eed, at the Bmi-1-associated Hox loci will be performed to test whether the Hox gene regulation is triggered by the formation of Polycomb complexes in HSC. Taken together, understanding the functional role of Bmi-1 at Hox genes can provide us mechanistic insights into the self-renewal capacity of HSC. Another important issue of stem cell self-renewal is the modulation of cell cycle, which directly controls the replication potential of a cell. Many of the adult stem cells are in a quiescent G0 phase, and they become activated in replication (self-renewal) once tissue growth or repair. It thus implies that a stem cell self-renewal capacity is partly regulated by cell cycle modulators which prevent its entry to the G0 phase. Interestingly, Bmi-1 -/- mice showed an increased level of Ink4a and Arf in HSC, which subsequently causes cell cycle arrest or senescence. Other study has demonstrated a direct binding of Bmi-1 to the Ink4a/Arf locus in MEFs, which suggests a repressive function of Bmi-1 to both senescence associated genes Ink4a and Arf. However, such interaction of Bmi-1 and these cell cycle regulators has not been demonstrated in HSCs; hence the mechanism of Bmi-1 mediated Ink4a and Arf gene silencing remains unclear. We therefore propose studying the Bmi-1 binding and also the H2AK119ub1 enrichment at the Ink4a/Arf locus in HSCs. Apart from the Ink4a/Arf locus, Ink4c is also a potential target regulated by Bmi-1, as it is another Ink4 gene that can enhance the self-renewal capacity of HSC. p18INK4C can compensate for the reduction of hematopoietic repopulation effect caused by p21Cip1/Waf1 deficiency in HSCs. As a result, studying the regulatory role of Bmi-1 with the Ink4 family members could give us valuable information in the cell cycle control of HSC self-renewal. Polycomb complexes, which consist of Bmi-1, have been demonstrated an important function by repressing the expression of differentiation genes in pluripotent embryonic stem cells. Although overexpression of Bmi-1 results in an enhancement of the self-renewal capacity in HSC, it has not been addressed whether the expanded HSCs retain their normal differentiation potential to other haematopoietic lineages. In addition, since Bmi-1 modulates global gene expression pattern, it raises a potential risk of re-activating some leukaemia-associated genes, e.g. Flt3, Npm1 and Cebpa, in these HSCs. Therefore, it is necessary to test if the Bmi-1-induced HSCs would have normal differentiation capacity and also non-leukaemogenic before utilizing these cells for transplantation purposes. To conclude, this proposal focuses on four major objectives: (1) To study the function of Bmi-1 in the regulation of Hox gene expression in HSC; (2) To demonstrate the mechanistic role of Bmi-1 in HSC self-renewal through epigenetic regulation; (3) To investigate the involvement of Bmi-1 in cell cycle regulation with Ink4 genes, and their association with HSC self-renewal; (4) To justify the functional normality, such as differentiation and leukaemogenic potential, of Bmi-1-induced HSCs for transplantation purposes.

List of Research Outputs

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. 2010, 38: 481-488.

Ng M.H., Ng R.K., Kong C.T., Jin D. and Chan L.C., Activation of Ras-dependent Elk-1 activity by MLL-AF4 family fusion oncoproteins, Experimental Hematology. ELSEVIER, 2010, 38: 481-488.

Ng R.K., Aberrant DNA Methylation in MLL-rearranged Leukaemia, In: Department of Pathology, The University of Hong Kong, Pathology Departmental Seminar. 2010.

Ng R.K., Epigenetic Hurdle in Reprogramming to Stem Cell Fate, In: The University of Hong Kong, Inaugural Symposium on Stem Cell and Regenerative Medicine Program, H.K.. 2009.

Ng R.K., Epigenetic regulation in the reprogramming of stem cell fate, In: Centre for Reproduction, Development and Growth, The University of Hong Kong , Centre for Reproduction, Development and Growth. 2010.

Ng R.K., Epigenetic reprogramming in stem cell differentiation, In: The University of Hong Kong, International Course Stem Cell and Regenerative Medicine, H.K.. 2009.

Ng R.K., Epigenetics in Human Acute Leukaemias: a Novel Strategy in Diagnosis and Therapy, In: The University of Hong Kong, 16th Hong Kong International Cancer Congress, H.K.. 2009.

Researcher : Ngan HYS

Project Title:	High dose cis-platinum and cyclophosphamide vs taxol in ovarian cancer
Investigator(s):	Ngan HYS, Wong RLC
Department:	Obstetrics & Gynaecology
Source(s) of Funding:	Other Funding Scheme
Start Date:	07/1994

Abstract:

To study high dose cis-platinum and cyclophosphamide vs taxol in ovarian cancer.

Project Title:	Differential expression of DeltaNp73 controlling the radiotherapeutic response in cervical cancers
Investigator(s):	Ngan HYS, Cheung ANY, Leung THY, Liu S
Department:	Obstetrics & Gynaecology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2008

Abstract:

The purpose of this proposed investigation is to study the role of the p73 isoform, DNp73, in irradiation-induced apoptosis and in relation to the radiosensitivity of cervical cancer. Cervical cancer is a common genital tract cancer in women. Its treatment includes mainly radical hysterectomy and/or radiotherapy. Radiotherapy is the mainstay of treatment, especially in advanced cervical cancer. The patients’ survival rate can be determined by their responsiveness to radiotherapeutic treatment. Response of cancers to ionizing radiation varies widely, and this may be explained by differences in cancer cell death-inducing effectors. Multiple genetic and epigenetic changes in the cancer cell may contribute to radioresistance. p73, a homolog of p53 tumor suppressor, has been suggested to be an alternative p53-independent apoptotic pathway, in particular, in cervical cancer. This is because cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types of which the viral oncoprotein E6 has the ability to inactivate the function of p53 by promoting degradation. Our previous study has demonstrated an association between p73 expression and radiosensitivity of cervical cancers and suggested that p73 might play an important role in controlling cellular radiosensitivity (Liu et al., 2004). p73 has been identified as a structural and functional homologue of the tumour suppressor protein p53 (Kaghad et al., 1997; Zhu et al., 1998). Despite their similarities, these two proteins are likely to display distinct functions, particularly in tumour formation and progression (Melino et al., 2002; Moll et al., 2001; Yang et al., 2000). Mutations of p73 in human tumours are extremely rare, and p73-deficient mice lack a spontaneous tumour phenotype (Moll et al., 2001). One possible explanation for the different roles of p53 and p73 in tumorigenesis possibly lies within their different genomic organization. While p53 encodes one protein, p73 gives rise to multiple protein isoforms due to alternative promoter utilization and alternative mRNA splicing (Irwin and Kaelin, 2001; Melino et al., 2002; Yang and McKeon, 2000). The full length wild-type TA isoform of p73 (TAp73) containing an N-terminal transactivation domain (TA) can activate downstream target genes and induce apoptosis. In contrast, the N-terminal truncated form (DNp73) lacking the transactivation domain acts as “dominant inhibitors” of the wild-type TAp73 and p53 and has anti-apoptotic function. This suggested p73 holds dual roles where the TAp73 isoform harbors pro-apoptotic characteristic while DNp73 holds anti-apoptotic property. DNp73, acting as a dominant-negative inhibitor, can interfere with p53 from binding to the p53-responsive elements and also inhibit the transcription of the TAp73 isoforms. Both isoforms could co-express in the cells, and their relative expression levels might be important in tumor formation and cell death. Our recent study has demonstrated that the expression level of these two p73 isoforms was inversely correlated, and increase of DNp73 expression was associated with tumor radioresistance and the adverse outcome of cervical cancer patients (Liu et al., 2006a). We hypothesize that DNp73 expression may contribute to the radioresistance of the cervical cancer by its dominant-negative effect. In this study, the effect of DNp73 expression on radiosensitivity will be investigated by overexpression (by expression vector) and underexpression (by siRNA) of DNp73 expression in the cancer cell lines and its relationship with TAp73 expression. Since the high-risk HPV infection and viral oncogene E6 and E7 expression are responsible for cervical carcinogenesis, HPV E6 and E7 expression will also be assessed in clinical specimens and correlated with DNp73 expression. Objectives 1 To study the response of cervical cancer cells to radiation treatment by in vitro manipulation of DNp73 expression. 2 To study the relationship between DNp73 and HPV E6 and E7 expression in cervical cancer.

Project Title:	Regulation of TP73 functions by BCA3 and clinical significance of BCA3 gene in cervical cancer.
Investigator(s):	Ngan HYS, Liu S, Leung THY
Department:	Obstetrics & Gynaecology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	05/2009

Abstract:

The purpose of this proposed investigation is to study the newly identified protein binding partner of p73 isoform, DNp73, Breast Cancer Associated gene (BCA3), in relation to the p73 function in cervical cancer. Cervical cancer is a common genital tract cancer in women. Its treatment includes mainly radical hysterectomy and/or radiotherapy. Radiotherapy is the mainstay of treatment, especially in advanced cervical cancer. The patients’ survival rate can be determined by their responsiveness to radiotherapeutic treatment. Response of cancers to ionizing radiation varies widely, and this may be explained by differences in cancer cell death-inducing effectors. Multiple genetic and epigenetic changes in the cancer cell may contribute to radioresistance. p73, a homolog of p53 tumor suppressor, has been suggested to be an alternative p53-independent apoptotic pathway, in particular, in cervical cancer. This is because cervical cancer is strongly linked to infection by high-risk human papillomavirus (HPV) types of which the viral oncoprotein E6 has the ability to inactivate the function of p53 by promoting degradation. Our previous study has demonstrated an association between p73 expression and radiosensitivity of cervical cancers and suggested that p73 might play an important role in controlling cellular radiosensitivity (Liu et al., 2004). The candidate tumor suppressor gene, p73 was identified by Kaghad et al in 1997 (Kaghad et al., 1997). It was predicted to encode a protein with significant similarity to p53. It was mapped the minimal region of 1p36 that is recurrently deleted in many types of cancers such as neuroblastoma (Ichimiya et al., 2001), breast cancer (Dominguez et al., 2001), squamous cell carcinoma (Araki et al., 2002) and B-cell lymphoma (Martinez-Delgado et al., 2002). Unlike the p53, somatic mutation of p73 gene is extremely rare. The p73 exists in two forms: the N-terminal transactivation domain containing form (TAp73) and the isoforms lacking the transactivation domain (DNp73) (including N, N’, exon2, and exon2/3). The TAp73 exhibits growth inhibitory, tumor suppressive and pro-apoptotic functions while the DNp73 promotes oncogenic activity and abolishes the functions of TAp73. The absent of the transactivation domain is due to transcription started at an alternative promoter (P2) which is located in intron 3 (resulting Np73 and N’p73) and alternative splicing variants (p73/exon2 and p73/exon2/3) (Ishimoto et al., 2002). In normal tissue, the expression of p73 is very low (Kovalev et al., 1998). The balance between the expression level of TAp73 and DNp73 has been shown to play an important role in the development of cancer. Overexpression of the oncogenic DNp73 was found in various types of cancers including breast, ovarian, liver, prostate, colon, and neuroblastoma cancers (Concin et al., 2004; Dominguez et al., 2006; Douc-Rasy et al., 2002; Guan and Chen, 2005; Putzer et al., 2003; Zaika et al., 2002). Elevated expression of DNp73 is associated with poor prognosis in cancer patients as DNp73 inhibits the suppressive functions exerted by p53 and TAp73 which resulted in decreased apoptosis response and chemoresistance (Casciano et al., 1999; Concin et al., 2005; Dominguez et al., 2006). BCA3, also known as A-kinase-interacting protein 1 (AKIP1), was cloned by PCR subtractive hybridization using library matched breast tumor and normal breast cell lines mRNAs (Kitching et al., 2003). It contains a number of putative functional domains including a nuclear localization signal, a proline-rich sequence, five src homology 2 (SH2) binding motifs and a PZD binding motif. The protein expression of AKIP1 is higher in the breast cancer cell lines but is much lower in normal breast tissue (Kitching et al., 2003). Immunostaining of AKIP1 in breast tumor tissue section revealed that AKIP1 is expressed in breast tumor cell but not in the surrounding stroma cells (Kitching et al., 2003). Study has shown that neddylated modified BCA3 represses the NFB-dependent transcription by recruiting the class III histone deaceylase SIRT1 (Gao et al., 2006). Furthermore, recent study has indicated that BCA3 promotes the nuclear retention and phosphorylation of p65 which resulted in enhancing the NFB-dependent gene expression (Gao et al., 2008). These result suggest that posttranslational modification of BCA3 protein play an important role on its function. We hypothesize that BCA3 regulates p73 activity via the binding to p73 and oligomerization of p73 might influence its binding with BCA3. From our preliminary study, the binding affinity of BCA3-DNp73 was stronger than BCA3-TAp73. Since TAp73 and DNp73 play an opposite role in cancer cell development as well as cancer treatment, it will be interesting to study the effects on BCA3-TAp73 and BCA3-DNp73 binding in carcinogenesis. In this study, we will elucidate the effects of BCA3 expression on p73 tumor suppressor function. Objectives 1 To elucidate the binding activity of BCA3 and p73 isoforms and p53 family protein 2 To delineate the functional significance of the binding between BCA3 and p73 3 To address BCA3, TAp73 and DNp73 expression and clinical significance in cervical cancer Reference List 1. Araki D, et al (2002). Int J Oncol 20: 355-60. 2. Casciano I, et al (1999). . Cell Death Differ 6: 391-3. 3. Concin N, et al (2004). Cancer Res 64: 2449-60. 4. Concin N, et al (2005). . Clin Cancer Res 11: 8372-83. 5. Dominguez G, et al (2006). J Clin Oncol 24: 805-15. 6. Dominguez G, et al (2001). . Breast Cancer Res Treat 66: 183-90. 7. Douc-Rasy S, et al (2002). Am J Pathol 160: 631-9. 8. Gao F, et al (2006). Nat Cell Biol 8: 1171-7. 9. Gao N, et al (2008). J Biol Chem 283: 7834-43. 10. Guan M, Chen Y (2005). J Clin Pathol 58: 1175-9. 11. Ichimiya S, et al (2001). Med Pediatr Oncol 36: 132-4. 12. Ishimoto O, et al (2002). Cancer Res 62: 636-41. 13. Kaghad M, et al (1997). Cell 90: 809-19. 14. Kitching R, et al (2003). Biochim Biophys Acta 1625: 116-21. 15. Kovalev S, et al (1998). Cell Growth Differ 9: 897-903. 16. Liu SS, ... Ngan HY (2004). Clin Cancer Res 10: 3309-16. 17. Martinez-Delgado B, et al (2002). Int J Cancer 102: 15-9. 18. Putzer BM, et al (2003). Cell Death Differ 10: 612-4. 19. Zaika AI, et al (2002). J Exp Med 196: 765-80. 20. Zaika A, et al (2001). J Biol Chem 276: 11310-6

Project Title:	Functional characterization and clinical significance of C35-DNp73 in ovarian cancer.
Investigator(s):	Ngan HYS, Leung THY, Liu S
Department:	Obstetrics & Gynaecology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

The purpose of this proposed study is to investigate the newly identified protein binding partner of p73, C35, in relation to the p73 function in ovarian cancer. Ovarian cancer is one of the commonest causes of cancer deaths in women in western countries as well as Hong Kong. The mainstay of treatment includes aggressive surgical cytoreduction and administration of platinum-based cytotoxic drugs. Despite optimal primary treatment, recurrences are common and the overall prognosis is poor. The development of novel ovarian cancer diagnostic tests as well as treatment is urgently required. Therefore, understanding of the molecular mechanisms that contribute to the ovarian cancer development is crucial. The candidate tumor suppressor gene, p73 was identified by Kaghad et al in 1997 1. It was predicted to encode a protein with significant similarity to p53. It was mapped to the minimal region of 1p36 that is recurrently deleted in many types of cancers such as neuroblastoma 2, breast cancer 3, squamous cell carcinoma 4 and B-cell lymphoma 5. Unlike p53, somatic mutation of p73 gene is extremely rare. In normal tissue, the expression of p73 is very low 6. The balance between the expression level of TAp73 and DNp73 has been shown to play an important role in the development of cancer. Functionally, TAp73 exhibits growth inhibitory, tumor suppressive and pro-apoptotic functions while the DNp73 promotes oncogenic activity and abolishes the functions of TAp73. Overexpression of the oncogenic DNp73 was found in various types of cancers including breast, ovarian, liver, prostate, colon, and neuroblastoma cancers 7-12. Elevated expression of DNp73 is associated with poor prognosis in cancer patients as DNp73 inhibits the suppressive functions exerted by p53 and TAp73 which ultimately resulted in decreased apoptotic response and chemoresistance 9,13-14. C35, also known as C17ORF37, was identified by substructive hybridization using library matched breast tumor and normal breast cell lines mRNAs 15. It encoded a 12kDa protein and was abunduntly expressed in breast cancer with distant metastases. Low level of C35 was found in normal human tissue. It was reported that C35 was highly expressed in prostate cancer cell lines and clinical prostate cancer tissue. Ectopic expression of C35 enhanced the ability of prostate cancer cell migration and invasion by up-regulating the expression of matrix metallopeptidase 9 (MMP-9), urokinase plasminogen activator (uPA) and vascular endothelial growth factor (VEGF). Down-regulation of C35 by siRNA could also reduce the DNA binding activity of NF-KappaB 16. These results suggest that C35 protein may play an oncogenic in cancer development. We hypothesize that C35 cooperates with DNp73 to promote oncogenesis in ovarian cancer. From our preliminary study, the binding affinity of C35-DNp73 was strong. Since DNp73 play an oncogenic role in cancer development, it will be interesting to study the effects on C35-DNp73 binding in carcinogenesis. In this study, we will elucidate the functional significance of C35-DNp73. Objectives 1 To elucidate the binding activity of C35 and p73 isoforms and p53 family protein 2 To delineate the functional significance of the binding between C35 and p73 3 To address C35 expression and clinical significance in ovarian cancer Reference List 1. Kaghad, M., et al. Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell 90, 809-819 (1997). 2. Ichimiya, S., et al. Downregulation of hASH1 is associated with the retinoic acid-induced differentiation of human neuroblastoma cell lines. Med Pediatr Oncol 36, 132-134 (2001). 3. Dominguez, G., et al. Wild type p73 overexpression and high-grade malignancy in breast cancer. Breast Cancer Res Treat 66, 183-190 (2001). 4. Araki, D., et al. Frequent allelic losses on the short arm of chromosome 1 and decreased expression of the p73 gene at 1p36.3 in squamous cell carcinoma of the oral cavity. Int J Oncol 20, 355-360 (2002). 5. Martinez-Delgado, B., et al. Frequent inactivation of the p73 gene by abnormal methylation or LOH in non-Hodgkin's lymphomas. Int J Cancer 102, 15-19 (2002). 6. Kovalev, S., Marchenko, N., Swendeman, S., LaQuaglia, M. & Moll, U.M. Expression level, allelic origin, and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines. Cell Growth Differ 9, 897-903 (1998). 7. Zaika, A.I., et al. DeltaNp73, a dominant-negative inhibitor of wild-type p53 and TAp73, is up-regulated in human tumors. J Exp Med 196, 765-780 (2002). 8. Concin, N., et al. Transdominant DeltaTAp73 isoforms are frequently up-regulated in ovarian cancer. Evidence for their role as epigenetic p53 inhibitors in vivo. Cancer Res 64, 2449-2460 (2004). 9. Dominguez, G., et al. DeltaTAp73 upregulation correlates with poor prognosis in human tumors: putative in vivo network involving p73 isoforms, p53, and E2F-1. J Clin Oncol 24, 805-815 (2006). 10. Douc-Rasy, S., et al. DeltaN-p73alpha accumulates in human neuroblastic tumors. Am J Pathol 160, 631-639 (2002). 11. Guan, M. & Chen, Y. Aberrant expression of DeltaNp73 in benign and malignant tumours of the prostate: correlation with Gleason score. J Clin Pathol 58, 1175-1179 (2005). 12. Putzer, B.M., Tuve, S., Tannapfel, A. & Stiewe, T. Increased DeltaN-p73 expression in tumors by upregulation of the E2F1-regulated, TA-promoter-derived DeltaN'-p73 transcript. Cell Death Differ 10, 612-614 (2003). 13. Casciano, I., Ponzoni, M., Lo Cunsolo, C., Tonini, G.P. & Romani, M. Different p73 splicing variants are expressed in distinct tumour areas of a multifocal neuroblastoma. Cell Death Differ 6, 391-393 (1999). 14. Concin, N., et al. Clinical relevance of dominant-negative p73 isoforms for responsiveness to chemotherapy and survival in ovarian cancer: evidence for a crucial p53-p73 cross-talk in vivo. Clin Cancer Res 11, 8372-8383 (2005). 15. Evans, E.E., et al. C35 (C17orf37) is a novel tumor biomarker abundantly expressed in breast cancer. Mol Cancer Ther 5, 2919-2930 (2006). 16. Dasgupta, S., et al. Novel gene C17orf37 in 17q12 amplicon promotes migration and invasion of prostate cancer cells. Oncogene 28, 2860-2872 (2009).

List of Research Outputs

Wang Y., Chan D.W., Liu V.W.S., Chiu P.M. and Ngan H.Y.S., Differential functions of growth factor receptor-bound protein 7 (GRB7) and its variant GRB7v in ovarian carcinogenesis, Clinical Cancer Research. AACR, 2010, 16: 2529-39.

Researcher : Nicholls JM

Project Title:	Tissue tropism and cytokine production within the upper and lower respiratory tract of humans following influenza virus infection
Investigator(s):	Nicholls JM, Peiris JSM, Poon LLM
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	08/2007
Completion Date:	07/2009

Abstract:

To determine what the binding patterns of sialic acid (SA)α2,3 and SAα2,6 linkages as detected by lectin histochemistry in paediatric versus adult epithelial tissues from the upper respiratory tract; to determine if there is correlation between surface SA expression and ex-vivo infection with human influenza (H1N1, H3N2), avian influenza (H5N1, H7N7), and a duck influenza virus (H9N2), and to assess changes in lectin expression following infections with these viruses; to determine there a change in the major cytokine (TNFα and interferon ß) and macrophage chemokines (MCP1 and IP10) implicatied in influenza infection in the upper and lower respiratory tract following infection by human influenza (H1N1, H3N2), avian influenza (H5N1, H7N7) and a duck influenza virus (H9N2) as detected by PCR, ELISA, in-situ hybridization and immunohistochemistry; to determine if there is evidence of infection by lymphoid or dendritic cells of the nasopharynx or tonsil with avian or human influenza viruses.

Project Title:	Susceptibility of the upper respiratory tract to influenza virus infection following desialyation
Investigator(s):	Nicholls JM, Chan MCW, Kwong DLW
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	01/2009

Abstract:

To determine the changes in lectin binding of upper respiratory tract tissues following treatment with different neuraminidases and βgalactosidases to test hypothesis that lectin binding will occur after treatment; to determine the changes in lectin binding following treatment with a new sialidase fusion protein to test hypothesis that lectin binding will occur after treatment; to determine the changes in ex-vivo upper respiratory infections by influenza virus infection after neuraminidase (NA) and DAS181 treatment to test hypothesis tha tinfluenza infection may use non-SA receptors for viral entry.

Project Title:	Influenza and sialic acid linked glycan interactions using virus like pseudoparticles (VLP) and STD-NMR
Investigator(s):	Nicholls JM, Poon LLM
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	11/2009

Abstract:

1) Determine the binding affinity of circulating H5,H1 and H3 viruses and by modification of known HA changes analyze the changes in affinity for Siaα2-6Gal-GlcNAc and Siaα2-3Gal-GlcNAc; 2) Using a profile of commonly detected glycans in the upper and lower respiratory tract determine which of these glycans are the potential binding moieties for circulating H5N1, H1N1 and H3N2 viruses; 3) Determine among a profile of commonly detected glycans in the upper and lower respiratory tract which of these glycans are the potential binding moieties for circulating H9N2 viruses; 4) Analyze the cleavage specificity of Siaα2-6Gal and Siaα2-3Gal by VLPs containing the neuraminidase from H5N1, H1N1 and H3N2 and determine if the efficiency of cleavage is affected by changes to the sugars linked to the subterminal galactose or if the cleaving by neuraminidase is affected by the presence of a HA.

Project Title:	Prevention and treatment of swine origin influenza virus (S-OIV) though the use of interferon - an in vivo and ex vivo study
Investigator(s):	Nicholls JM
Department:	Pathology
Source(s) of Funding:	Research Fund for the Control of Infectious Diseases - Full Grants
Start Date:	01/2010

Abstract:

(1) Predict that alfacon treatment will reduce viral replication and deleterious cytokine production in experimental infections of human bronchial and lung tissue and mouse tissues with S-OIV compared with control infected tissues and mice; (2) compare the effect on S-OIV with seasonal influenza virus infection and avian (H5N1) infection.

List of Research Outputs

Chan M.C.W., Chan W.Y., Yu C.L., Ho C.C., Chui W.H., Lo C.K., Yuen K.M., Guan Y., Nicholls J.M. and Peiris J.S.M., Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells, Respiratory Research. 2009, 10: 102.

Chan M.C.W., Chan W.Y., Yu C.L., Ho C.C., Yuen K.M., Fong J.H.M., Tang L.L.S., Lai W.W.K., Lo A.C.Y., Chui W.H., Sihoe A.D.L., Kwong D.L.W., Tsao G.S.W., Poon L.L.M., Guan Y., Nicholls J.M. and Peiris J.S.M., Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract, American Journal of Pathology. 2010, 176(4): 1828-40.

Chan W.Y., Chan M.C.W., Wong C.N., Karamanska R., Dell A., Haslam S.M., Sihoe A.D., Chui W.H., Triana-Baltzer G., Li Q., Peiris J.S.M., Fang F. and Nicholls J.M., DAS181 Inhibits H5N1 Influenza virus Infection of Human Lung Tissues., Antimicrobial Agents and Chemotherapy. 2009, 53(9): 3935-3941.

Chan W.Y., Yuen K.M., Yu C.L., Ho C.C., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Differential Viral Replication Kinetics And Host Innate Immune Responses By Influenza A (H5N1) Virus In Human Bronchial Epithelial Cells At Different Differentiation Stages, The 28th Annual meeting of American Society for Virology. 2009.

Chan W.Y., Yu C.L., Yuen K.M., Fong J.H.M., Lo A.C.Y., Lai W.W.K., Wong D.S.H., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Infection of influenza A (H5N1) virus in human eye epithelium, an in vitro and ex vivo study , The 28th Annual Meeting of the American Society of Virology. 2009.

Chan W.Y., Yuen K.M., Yu C.L., Ho C.C., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Influenza H5n1 And H1n1 Virus Replication And Innate Immune Responses In Bronchial Epithelial Cells Are Influenced By The State Of Differentiation, PLoS One. 2010, 5 (1): e8713.

Chan W.Y., Chan M.C.W., Nicholls J.M. and Peiris J.S.M., Tropism and host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract , XII International Symposium on Respiratory Viral Infections. 2010.

Cheung A.K.L., Lung H.L., Ko J.M.Y., Cheng Y., Stanbridge E.J., Zabarovsky E.R., Nicholls J.M., Chua D.T.T., Tsao G.S.W., Guan X.Y. and Lung M.L., Chromosome 14 transfer and functional studies identify a candidate tumor suppressor gene, mirror image polydactyly 1, in nasopharyngeal carcinoma., In: George Klein, Proc Natl Acad Sci. 2009, 106: 14478-14483.

Cheung A.K.L., Lung H.L., Ko J.M.Y., Cheng Y., Stanbridge E.J., Zabarovsky E.R., Nicholls J.M., Chua D.T.T., Tsao G.S.W., Guan X.Y. and Lung M.L., Functional Studies of a Cell Cycle and Angiogenesis-related Candidate Tumor Suppressor Gene, Mirror Image Polydactyly 1 , in Nasopharyngeal Carcinoma, 16th Hong Kong International Cancer Congress. 2009.

Lai J.C.C., Chan W.W.L., Kien F.S., Nicholls J.M., Peiris J.S.M. and Garcia J., Formation of virus-like particles from human cell lines exclusively expressing Influenza neuraminidase, Journal of General Virology . 2010, 2322-30.

Mok K.P., Wong C.H.K., Cheung C.Y., Chan M.C.W., Lee M.Y., Nicholls J.M., Guan Y. and Peiris J.S.M., Viral Genetic Determinants of H5N1 Influenza Viruses That Contribute to Cytokine Dysregulation, Journal of Infectious Diseases. 2009, 200(7): 1104-12.

Peiris J.S.M., Cheung C.Y., Leung C.Y.H. and Nicholls J.M., Innate immune responses to influenza A H5N1: friend or foe?, Trends in Immunology. 2009, 30(12): 574-84.

Triana-Baltzer G.B., Babizki M., Chan M.C.W., Wong A.C.N., Aschenbrenner L.M., Campbell E.R., Li Q.X., Chan W.Y., Peiris J.S.M., Nicholls J.M. and Fang F., DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis., J Antimicrob Chemother. 2010, 65: 275-84.

Triana-Baltzer G.B., Gubareva L.V., Nicholls J.M., Pearce M.B., Mishin V.P., Belser J.A., Chen L.M., Chan W.Y., Chan M.C.W., Hedlund M., Larson J.L., Moss R.B., Katz J.M., Tumpey T.M. and Fang F., Novel pandemic influenza A(H1N1) viruses are potently inhibited by DAS181, a sialidase fusion protein, PLoS One. 2009, 4: e7788.

Triana-Baltzer G.B., Gubareva L.V., Nicholls J.M., Pearce M.B., Mishin V.P., Belser J.A., Chen L.M., Chan W.Y., Chan M.C.W., Klimov A.A., Hedlund M., Wurtman D., Moss R.B., Katz J.M., Tumpey T.M., Belshe R.B. and Fang F., Pandemic H1N1 2009 and Drug Resistant Influenza Viruses are Potently Inhibited by DAS181, a Sialidase Fusion Protein, Interscience Conference on Antimicrobial Agents and Chemotherapy. 2009.

Van Poucke S.G., Nicholls J.M., Nauwynck H.J. and Van Reeth K., Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution, Virology Journal. 2010, 7: 38.

Wang P., Kudelko M., Lo J.C.Y., Siu Y.L., Kwok K.T.H., Sachse M., Nicholls J.M., Bruzzone R., Altmeyer R.M. and Nal-Rogier B.T.M., Efficient assembly and secretion of recombinant subviral particles of the four dengue serotypes using native prM and E proteins, PLoS ONE. 2009, 4(12): e8325.

Researcher : Shek TWH

List of Research Outputs

Au W.Y., Law M.F., Tung Y. and Shek T.W.H., Concomitant EBV encoded RNA positive cutaneous nasal-type natural killer-cell lymphoma and EBV encoded RNA negative nasopharyngeal carcinoma., Leukaemia & Lymphoma. 2009, 50(9): 1543-1544.

So J.C.C., Hwang Y.Y., Shek T.W.H., Lam C.C.K., Lai C.L. and Kwong Y.L., Transfusion-refractory anaemia in liver cirrhosis, Gut. 2010, 59(1): 5.

Researcher : Shen L

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Siu CWK

List of Research Outputs

Chau S.K.C., Kwok K.L., Ng D.K., Lam C.W., Tong S.F., Chan A.Y.W., Siu C.W.K. and Yuen L.Y.P., Maternally inherited Leigh syndrome: an unusual cause of infantile apnea, Sleep Breath. 2010, 14(2): 161-5.

Mak C.M., Lam C.W., Siu T.S., Chan K.Y., Siu C.W.K., Yeung W.L., Hui J., Wong V.C.N., Low L.C.K., Ko C.H., Tam S. and Chan Y.W., Biochemical and molecular characterization of tyrosine hydroxylase deficiency in Hong Kong Chinese, Molecular Genetics and Metabolism. 2010, 99(4): 431-433.

Researcher : Siu KY

Project Title:	Effects of folate and folate receptor on ovarian and endometrial cancer: an in vitro study
Investigator(s):	Siu KY, Cheung ANY
Department:	Pathology
Source(s) of Funding:	American Institute for Cancer Research (AICR) - General Award
Start Date:	07/2006

Abstract:

To elucidate the involvement of folate receptor (FR) in ovarian and endometrial cancers by examining its expression and localization in both clinical samples of ovarian and endometrial cancer and cell lines; to correlate with clinical outcome to enable better understanding of the roles of dietary folate in the prevention and treatment of ovarian and endometrial cancers; to investigate the effects of folate and the FR on the proliferation, migration, invasion, anchorage-independent growth and apoptosis in ovarian and endometrial cancers using both in vitro monolayer and organotypic culture model.

Project Title:	Functional characterization and subcellular localization of Pak4 in ovarian carcinoma
Investigator(s):	Siu KY, Cheung ANY
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	10/2007
Completion Date:	09/2009

Abstract:

Primarily due to the advanced stage of disease at presentation, ovarian carcinoma is the most lethal gynecological malignancy (1). Patients with advanced stage of ovarian carcinoma may have initial response after treatment with surgery and chemotherapy, but eventually most will relapse or develop drug resistance and subsequently die of their disease (2). In view of that, identifying novel therapeutic targets for ovarian carcinoma is valuable in order to improve the long-term survival rate. It is believable that genes governing metastatic process may be potential novel therapeutic targets and/or prognostic biomarkers in ovarian carcinoma (3). Metastasis is a complex process involving increased cell migration and enhanced cell survival. Cell migration in turn requires cytoskeleton reorganization (4-6). p21-activated kinases (Paks) were first identified as effectors of Rac1 and Cdc42, which are members of the Rho family of small GTPases found to be important regulators for cytoskeleton reorganization (7). To date, six members of Paks has been found and divided into group I (Pak1, Pak2, Pak3) and group II (Pak4, Pak5, Pak6) based on structural organization and mode of regulation (4-6). Other than the small GTPases, Paks can be activated by several upstream mediators, including phosphatidylinositol 3-kinase, Akt, PDK1 and PKA (6). Moreover, there is accumulating evidence that Paks are important linkers for numerous oncogenic signalling pathways, playing a vital role in diverse biological processes, ranging from cytoskeletal remodelling and cell motility to apoptosis, cell proliferation and chromosomal abnormalities, all of which are requisite for tumorigenesis and metastasis (4-6). Dysregulation of Paks has been documented in human carcinomas, and in particular in depth study of Pak1 in breast carcinoma (6). The possible roles of Pak4, a more recently identified Pak family member, in carcinogenesis is receiving increased attention (6). For example, Pak4 is localized to a region of chromosome 19, where amplification is frequently observed in ovarian carcinoma (8, 9). However, differential expression, subcellular localization, functional roles and signaling pathways of Pak4 in ovarian carcinoma remain to be examined. Pilot study of Pak4 in ovarian carcinoma: Pak4 expression was correlated with the progression of ovarian tumours and patient’s overall survival: In our pilot study, differential expression of total Pak4 in 120 clinical samples of ovarian tumours including 10 benign cystadenomas, 10 borderline tumours, 80 carcinomas of different histological subtypes as well as 20 metastatic foci of ovarian carcinomas was evaluated by immunohistochemistry, western blotting and real time PCR. Pak4 was predominantly expressed in the cytoplasm of ovarian carcinoma with faint nuclear Pak-4 staining detected. Moderate to strong cytoplasmic staining of Pak4 (Fig. 1) was observed in ovarian cancer cells. In contrast, there was weak or no expression of Pak4 in borderline tumours and benign cystadenomas (Fig. 1) and the difference was statistically significant difference between Pak4 expression in benign, borderline and malignant tumours with highest level in the ovarian carcinomas. Furthermore, Pak4 expression was correlated with the progression of ovarian tumours and patient’s overall survival (Fig. 2). Nuclear localization of phospho (p)-Pak4 (the activated form) in ovarian carcinoma: In addition to total Pak4, p-Pak4 was also preformed in clinical samples of ovarian tumours. Interestingly, p-Pak4 immunoreactivity was expressed predominantly as strong nuclear staining with moderate cytoplasmic staining (Fig. 3), implicating that Pak4 may play roles in the nucleus, such as modulating transcription. Nuclear staining of p-Pak4 was also observed in borderline tumours but was less extensive and intense than that in carcinomas. Pak4 expression in malignant progression and metastasis of ovarian carcinoma: Not only was increased expression observed with progression from benign cystadenoma to carcinoma, there was also significant increase in Pak4 expression in the metastatic foci when compared with the primary ovarian carcinomas. These findings suggest that Pak4 is a possibly important regulator for the malignant progression and metastasis of ovarian carcinoma. In-vitro expression of Pak4: By Western blot analysis, the expression of Pak4 was confirmed in ovarian carcinoma cell lines (SKOV-3, OVCAR-3, OVCA420, OVCA429 and OVCA433) while its expression was absent in normal ovarian epithelium cell lines (HOSE-11-12 and HOSE-17-1) (Fig. 4). Based on the above preliminary results, Pak4 is likely to play a significant role in ovarian carcinogenesis and is also possibly an important regulator for the malignant progression and metastasis of ovarian cancer. The objectives of our study aim to investigate 1/ the functional roles and 2/ downstream effectors related to migration and invasion of Pak4 in ovarian carcinogenesis using in vitro study; and 3/ to examine the subcellular localization and expression of Pak4 and p-Pak4 in ovarian cancer cell-lines and 4/ its possible functional role on gene transcription. References: 1. Jemal, A., Murray, T., Samuels, A., Ghafoor, A., Ward, E., and Thun, M. J. Cancer statistics, 2003. CA Cancer J Clin, 53: 5-26, 2003. 2. Agarwal, R. and Kaye, S. B. Ovarian cancer: strategies for overcoming resistance to chemotherapy. Nat Rev Cancer, 3: 502-516, 2003. 3. Sheehan, K. M., Calvert, V. S., Kay, E. W., Lu, Y., Fishman, D., Espina, V., Aquino, J., Speer, R., Araujo, R., Mills, G. B., Liotta, L. A., Petricoin, E. F., 3rd, and Wulfkuhle, J. D. Use of reverse phase protein microarrays and reference standard development for molecular network analysis of metastatic ovarian carcinoma. Mol Cell Proteomics, 4: 346-355, 2005. 4. Bokoch, G. M. Biology of the p21-activated kinases. Annu. Rev. Biochem., 72: 743-781, 2003. 5. Kumar, R. and Hung, M. C. Signaling intricacies take center stage in cancer cells. Cancer Res., 65: 2511-2515, 2005. 6. Kumar, R., Gururaj, A. E., and Barnes, C. J. p21-activated kinases in cancer. Nat Rev Cancer, 6: 459-471, 2006. 7. Hall, A. Rho GTPases and the actin cytoskeleton. Science, 279: 509-514, 1998. 8. Callow, M. G., Clairvoyant, F., Zhu, S., Schryver, B., Whyte, D. B., Bischoff, J. R., Jallal, B., and Smeal, T. Requirement for PAK4 in the anchorage-independent growth of human cancer cell lines. J Biol Chem, 277: 550-558, 2002. 9. Park, J. T., Li, M., Nakayama, K., Mao, T. L., Davidson, B., Zhang, Z., Kurman, R. J., Eberhart, C. G., Shih Ie, M., and Wang, T. L. Notch3 gene amplification in ovarian cancer. Cancer Res, 66: 6312-6318, 2006.

Project Title:	p21-activated kinase 4: a prognostic and therapeutic target for ovarian carcinoma?
Investigator(s):	Siu KY, Cheung ANY, Ngan HYS
Department:	Pathology
Source(s) of Funding:	The Hong Kong Anti-Cancer Society (HKACS) - General Award
Start Date:	10/2007

Abstract:

In this study, we propose to investigate whether Pak4 can be developed as a novel therapeutic target in ovarian carcinoma patients using in vivo tumour growth bioluminescence imaging. The downsteam effectors of Pak 4 will be investigated by in vitro studies including real-time PCR and western blot analysis so that the role of Pak4 in ovarian carcinogenesis can be better understood and more potential molecular targets can be identified. The potential of Pak4 as a molecular target to sensitize ovarian cancer cells to conventional chemotherapy will also be explored using in vitro chemotherapy study.

Project Title:	Characterization of αPIX in ovarian cancer
Investigator(s):	Siu KY, Cheung ANY
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2009

Abstract:

Worldwide, ovarian cancer is a common cancer in women, contributing to high mortality among all gynecological cancers(1). Furthermore, the incidence and mortality of ovarian cancer in Hong Kong is on the rising trend in recent 10 years(2). Such high mortality rate is primarily due to the advanced stage of disease at presentation with metastasis. Moreover, patients with advanced stage ovarian cancer may have initial response after treatment with surgery and chemotherapy, but eventually a significant portion relapse and develop drug resistance(3). As such, investigating genes related to metastasis and chemosensitivity with the potential of developing novel prognostic and therapeutic targets for ovarian cancer are thus valuable as attempts to improve the long-term survival rate. p21-activated serine/threonine kinases (PAKs) are major effectors of the small Rho GTPases Rac1 and Cdc42, and act as central signal transducers regulating many cellular processes, including cell morphology, cytoskeletal reorganization, motility, survival and angiogenesis, which are all prerequisite steps for metastasis(4). Six mammalian PAKs have been identified and classified into Group I (PAKs1-3) and Group II (PAKs 4-6)(4). Overexpression of PAK1 has been found in several human cancers(4), such as breast and colorectal cancers. Recently, we have demonstrated a significant association between increased expression of PAK1, as well as PAK4 and its activated form, with shorter overall and disease-free survival in ovarian cancer(5-7). We have also reported the functional roles of PAK1 and PAK4 along with a physiological link between PAK4, c-Src, ERK1/2 and MMP2 in ovarian cancer cell migration and invasion(5-7). Our findings shed light on their potential to be prognostic biomarkers in ovarian cancer patients and as molecular targets of therapy. Our collaborator, Prof. Stromblad (Karolinska Institute, Sweden), is developing PAK4 specific inhibitory small organic compounds that may be applied in ovarian cancer treatment based on our studies. PAKs exert their cellular effects via their interactions with PAK interactive exchange factors (PIXs), also known as Cloned-out of library (Cools), which are their putative binding partners(8). PIXs function as guanine nucleotide exchange factors which activate Rac1 and Cdc42. Thus, they act as crucial modulators regulating the interaction of such GTPases with their effectors PAKs(8, 9). Two PIXs namely αPIX (Cool2) and βPIX (Cool1) are known to date(8, 9). αPIX is predominately expressed in hematopoetic cells and muscle, whereas βPIX is ubiquitously expressed(8). They are involved in different physiological and pathophysiological conditions, such as mental retardation, vascular development and homeostasis, and infectious diseases(8). Since there is accumulating evidence that PAKs play important roles in human malignancies, including ovarian cancer as found by our group, PIXs, being important regulators of PAKs, are likely to be involved in cancer progression. However, the study of PIXs in cancer has been very limited. Overexpression of αPIX in malignant glioma(10) and βPIX in breast cancer(11) are documented. Pilot study of αPIX in ovarian cancer: We analyzed the expression of αPIX in 143 paraffin embedded clinical samples of ovarian tumors including four inclusion cysts, 11 benign cystadenomas, 11 borderline tumors, 80 carcinomas of different histological subtypes as well as 37 metastatic foci of ovarian cancers by immunohistochemistry. The specificity of αPIX (Atlas) antibody was verified by Western blot analysis using lysates from ovarian cancer samples, which showed a single immunoreactive band of αPIX at 87kDa. Strong cytoplasmic αPIX immunoreactivity was observed in ovarian cancer samples. In contrast, there was moderate staining of αPIX in borderline tumors and weak or no staining in benign cystadenomas/inclusion cysts (Fig. 1). The difference of αPIX between benign cystadenomas/inclusion cysts, borderline and malignant tumors is significant (p<0.001). Real-time PCR on cDNA prepared from 34 randomly selected clinical samples of ovarian tumors with available frozen blocks showed similar results. When αPIX expression was correlated with the clinicopathologic parameters of the patients, an association between increased expression of αPIX with metastasis of ovarian cancers, shorter overall and disease-free survival, advanced stage and high grade cancers, serous/clear cell histological subtypes and reduced chemosensitivity was demonstrated (Fig. 2 and Table 1). No association between chemoresistance and αPIX expression has been reported before. Consistent with results of immunohistochemistry, an increase in the steady-state αPIX mRNA and protein level was detected in A2780CP, a chemoresistant cell lines, than its parental chemosensitive cell line, A2780S (Fig. 3B). αPIX overexpression was also observed in ovarian cancer cell lines (SKOV-3, OVCAR-3, OVCA420 and OVCA433) when compared with normal ovarian epithelial cell lines (HOSE-11-12 and HOSE-17-1) by real-time PCR and Western blot analysis (Fig. 3A). Based on the above preliminary results, we hypothesize that ovarian cancer progression and metastasis, and the development of chemoresistance may be associated with dysregulation of αPIX and their related pathways. The objectives of our study aim to investigate (1) the in vitro effects and (2) downstream effectors of αPIX on ovarian cancer cell migration and invasion; and (3) the capability and (4) the related pathways of αPIX in reducing chemosensitivity of ovarian cancer cells. References: 1. Jemal,A., et al.(2007)Cancer statistics, 2007. CA Cancer J Clin 57:43-66. 2. The Hong Kong Cancer Registry(2004)Department of Health Annual Report . Hong Kong, Department of Health, Hospital Authority. 3. Agarwal,R. & Kaye,S.B.(2003)Ovarian cancer: strategies for overcoming resistance to chemotherapy.Nat Rev Cancer 3:502-516. 4. Kumar,R., et al.(2006)p21-activated kinases in cancer.Nat Rev Cancer 6:459-471. 5. Siu,M.K.*, et al.(2006)Differential expression of p21-activated kinases (Paks) in ovarian cancer.In AACR's 97th Annual Meeting: in Washington DC,USA.*Recipient of the AACR International Scholar-in-Training Grant. 6. Siu,M.K., et al.(2007)p21-activated kinase 4 in Ovarian Cancer: Its expression, localization and possible functional role.In: 16th Annual Growth Factor and Signal Transduction Symposium Iowa,US. 7. Siu,M.K., et al.(2007)Biological significant of p21-activated kinase 4 in ovarian carcinoma: Prognostic marker and regulator of migration and invasion.In AACR's 98th Annual Meeting: Los Angeles,CA. 8. Frank,S.R. & Hansen,S.H.(2008)The PIX-GIT complex: a G protein signaling cassette in control of cell shape.Semin Cell Dev Biol 19:234-244. 9. Peterson,J.R. & Chernoff,J.(2006)Src transforms in a Cool way.Nat Cell Biol 8:905-907. 10. Yokota,T., et al.(2006)Identification of histological markers for malignant glioma by genome-wide expression analysis: dynein, alpha-PIX and sorcin.Acta Neuropathol 111:29-38. 11. Ahn,S.J., et al.(2003)Overexpression of betaPix-a in human breast cancer tissues.Cancer Lett 193:99-107.

Project Title:	Differential impact of reduced folate carrier and folate receptor alpha in ovarian cancer
Investigator(s):	Siu KY, Cheung ANY
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	03/2010

Abstract:

Folate and Cancers Ovarian cancer is a common gynecological cancer world-wide and contributes to high mortality despite of advances in treatment modalities(1). Furthermore, the incidence and mortality of ovarian cancer in Hong Kong is on the rising trend in recent 10 years(2). Although the reasons for this change are largely unknown and remain controversial, several lifestyle risk factors have been implicated, including diet, obesity, fertility and parity. For instance, there is increasing evidence supporting the association between high intakes of micronutrients such as folate, vitamin C, vitamin E and lower risk of various cancers(3). As such, better understanding of the effects of nutritional elements on ovarian carcinogenesis will potentially improve the strategies for its prevention and management. A high dietary folate intake is related to a lower risk of developing ovarian cancers, particularly among women who consume alcohol(4, 5). Folate, a water-soluble B vitamin found in most vegetable, is required for DNA synthesis, and the related methionine metabolic pathway is essential for DNA methylation (Fig. 1). Folate deficiency has found to induce DNA hypomethylation, alter gene expression and misincorporate uracil into DNA leading to chromosome damage, all of which are key factors for carcinogenesis(6, 7). Cellular Receptors of Folate Uptake Folate intake can process through several transporters, including folate receptors (FRs), reduced folate carrier (RFC), the family of organic anion transporters (OATs), and a proton-coupled folate transporter (PCFT), the former two being the major and better characterized folate transporters(8, 9). FRa, a single chain glycosyl-phosphatidylinositol–anchored membrane protein, was found to be overexpressed in nonmucinous ovarian tumors and its expression was directly associated with the progression of ovarian cancer, implicating that uptake of folate by FRa through endocytosis (unidirectional) by tumor cells may confer tumor growth (Fig. 1)(10-12). Such observation suggests a hypothesis that folate may be involved in the growth of both normal and tumor cells. Nevertheless, the expression status of the FRa in ovarian cancer among Orientals, such as women in Hong Kong, is still unknown. Furthermore, the underlying mechanisms in mediating its functions and its genetic and epigenetic alterationsin ovarian cancer are ill defined. RFC is an ubiquitously expressed transporter for natural folates such as 5-methyl or 5-formyl tetrahydrofolate and classical antifolates such as methotrexate (MTX) or pemetrexed which uptake folate in a bi-directional manner (Fig. 1)(9). Loss of RFC with subsequent effects of folate deficiency leading to cancer progression has been documented in colorectal cancer(9, 13). However, its expression status and regulatory mechanisms in normal ovarian epithelium and its functional roles in ovarian cancer remain largely unknown. FRa, RFC and folate in ovarian cancer The expression and localization of FRa and RFC in clinical samples of ovarian cancer • We analyzed the expression of FRa and RFC in 160 paraffin embedded clinical samples of ovarian tumors by immunohistochemistry. • Strong FRa immunoreactivity was observed in ovarian cancer sample in contrast to moderate staining of FRa in borderline tumors and weak or absence of staining in benign cystadenomas/inclusion cysts (Fig. 2A). Inversely, RFC protein (Fig. 2B) expression was significantly lower in ovarian cancers. • Real-time PCR confirmed the upregulation of FRa and downregulation of RFC at mRNA level. Correlation with clinicopathologic parameters • In ovarian carcinomas, high FRa expression was found to be significantly associated with advanced stages of disease and poor histological differentiation, factors associated with poor survival. • In contrast, a significant association between reduced expression of RFC, and shorter overall and disease-free survival was demonstrated (Fig. 3A). Moreover, among ovarian carcinomas with high FRa expression, the overall and disease-free survival was significantly longer in those with high RFC expression (Fig. 3B). Expression of FRa and RFC in ovarian normal and cancerous cell lines • Relatively higher mRNA and protein expression of FRawas observed in ovarian cancer cell lines (SKOV-3, OVCAR-3, OVCA 420, OVCA 429 and OVCA 433) as compared with normal ovarian epithelial cell lines (HOSE-6-3 and HSOE 11-12) by real-time PCR and Western blot analysis (Fig. 4A). In contrast, reduced RFC mRNA and protein expression was found in cancer cell lines than in normal cell lines (Fig. 4B). Knockdown of FRa reduced folate-mediated cell proliferation in SKOV-3 cells • Stable knockdown of FRa was performed in SKOV-3 cells. After confirming the specific knockdown of FRa (Fig. 5A), the effect of folate and FRa on cell proliferation was investigated. • Control or shFRaSKOV-3 cells were pretreated with folate-free medium for 3 days and then treated with different doses of folic acid, a synthetic folate, including 0nM, 6 nM, 12 nM, 60 nM and 120 nM. - The folic acid concentrations used are based on the physiological range in plasma, which ranges from <7 nM in individuals with a negative folate balance to >50 nM in individuals with >400µg/d of folate consumption14. • By MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay, 12 nM, 60 nM and 120 nM folic acid was found to be essential for cell proliferation whereas cells treated with 6 nM folic acid failed to proliferate (Fig. 5B). • Knockdown of FRa in SKOV-3 cells blocked cell proliferation when cells were treated with 12 nM or 60 nM folic acid. However, no difference of cell proliferation was found in control and shFRa SKOV-3 cells treated with 120 nM folic acid (Fig. 5B). Folate induced ovarian cancer cell migration and invasion and such effects were dependent on FRa expression • Transwell migration and invasion assays showed that 12 nM and 60 nM folic acid induced cell migration and invasion in control cells whereas knockdown of FRa blocked folate/folic acid-mediated cell migration and invasion (Fig. 6). Based on the above preliminary results, we hypothesize that folate metabolism is a complex process that is important in ovarian cancer. Dyregulated expression of FRa and RFC, the two folate transporters with apparent opposite roles, contribute to ovarian carcinogensis. The objectives of our study aim to investigate (1)the paradoxical functional impact of FRa and RFC in relation to folate in ovarian cancer; (2)the underlying mechanisms in mediating such functions; (3)the impact of genetic and epigenetic alterations on FRa and RFC in ovarian cancer.

Project Title:	AACR 101st Annual Meeting 2010 Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers
Investigator(s):	Siu KY
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Siu K.Y., Wong E.S.Y., Kong S.H., Wong G.W., Tam K.F., Ngan H.Y.S., Le X.F. and Cheung A.N.Y., Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers. , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A., 17 - 21 April 2010.. 2010.

Siu K.Y., Wong G.W. and Cheung A.N.Y., TrkB as a therapeutic target for ovarian cancer, Expert Opin Ther Targets. 2009, 1169-78.

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Wong G.W., Siu K.Y., Ngan H.Y.S. and Cheung A.N.Y., Dysregulated expression and function of Plexin-B1 in ovarian cancers , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : So JCC

Project Title:	Role of chromosomal 6q23 region in the modulation of HbF production
Investigator(s):	So JCC, Ma ESK, Song Y
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2007

Abstract:

To undertake fine mapping of the 6q23 region that is associated with enhanced-chain production; to Search for functional changes associated with the SNP(s); to Investigate the possible regulatory mechanism of candidate gene(s) or chromosomal region(s) in relation to HbF production.

List of Research Outputs

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

So J.C.C., So A.C., Chan A.Y., Tsang S.T.Y., Ma E.S.K. and Chan L.C., Detection and characterisation of beta-globin gene cluster deletions in Chinese using multiplex ligation-dependent probe amplification, J Clin Pathol. 2009, 62(12): 1107-11.

So J.C.C., Globin gene imbalance in beta globin disorders, The Fifth Asian Workshop on Hematology, Hong Kong National Research Council of Thailand, Japan Society for the Promotion of Science and Department of Paediatrics and Adolescent Medicine, HKU. 2009.

So J.C.C., Hwang Y.Y., Shek T.W.H., Lam C.C.K., Lai C.L. and Kwong Y.L., Transfusion-refractory anaemia in liver cirrhosis, Gut. 2010, 59(1): 5.

Researcher : Srivastava G

Project Title:	Therapeutic potential and mechanisms of antitumor activity of proteasome inhibitor bortezomib in extranodal NK/T-cell lymphoma, nasal type
Investigator(s):	Srivastava G, Liang RHS
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2006
Completion Date:	08/2009

Abstract:

To examine whether bortezomib promotes apoptosis and inhibits cell growth in vitro in a large series of NK lymphoma/leukemia cell lines and extranodal NK/T-cell lymphoma, nasal type (NL) primary cultures by analyzing cell viability, cell senescence, cell cycle and apoptosis; (2) to investigate whether bortezomib is effective in treating NL in vivo, we will study the effect of bortezomib on the treatment of NL NUDE mouse model HX-NKTL. In addition, two NL cell lines will be implanted subcutaneously in nude mice to provide tumor xenograft animal models, including HANKI1 that is a xenograft-derived NL cell line. The drug will be injected intravenously to determine its effectiveness; (3) bortezomib directly affects two important intracellular signaling pathways-NF-κB and JNK that are shown to be regulated by ubiquitin-proteasome degradation in multiple myeloma cells. We will investigate whether this anticancer drug works by the similar molecular mechanisms in NL; (4) to further understand the mechanisms of antitumor action of bortezomib, we will examine the molecules associated with the G₂/M checkpoint because we detected G₂/M arrest in a drug-treated NL cell line in a pilot study; (5) because the drug induced apoptosis in most of the analyzed cell lines and mitochondria are belived to play a crucial role in the control of apoptosis, apoptotic signaling molecules, especially those located in mitochondria, will be analyzed to uncover their involvement with the action of bortezomib; we will explore the essential molecular changes in the early stages of drug action. Superoxide production and intracellular Ca²⁺ dysregulation may be the critical determinants, so we will identify their roles in the bortezomib-induced apoptosis pathways.

Project Title:	Genetic and epigenetic mechanisms and clinicopathological significance of the silencing of a 6q22.33-q23.2 putative tumor suppressor PTPRK (protein tyrosine phosphatase, receptor type, kappa) gene in extranodal NK/T-cell lymphoma, nasal type
Investigator(s):	Srivastava G, Liang RHS
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2007

Abstract:

To study the mRNA and protein expression of PTPRK by real-time quantitative RT-PCR, Northern blot, Western blot analysis in NL/NK leukemia cell lines and normal NK cells, and immunohistochemistry on paraffin sections of primary NL; to determine the allelic loss of 6q22.33-q23.2 by GeneScan LOH analysis in cell lines and primary tumors; to identify the intragenic deletions, insertions and mutations and splicing errors of PTPRK by DNA sequencing in cell lines and primary tumors; and correlate the results with PTPRK expression levels; to determine the methylation status of the PTPRK promoter by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS) in cell lines and primary tumors; and correlate it with PTPRK expression levels; and determine whether demethylation 5-aza-dC can restore PTPRK expression in silenced cell lines; to determine whether genetic or epigenetic alterations, or PTPRK protein expression can be used as a potential clinical biomarker for the diagnosis and prognostic prediction of N; to characterize the tumor suppressor functions of PTPRK in NL by in vitro (I) cell growth assay, (II) cell cycle analysis and apoptosis induction assay, (III) anchorage-independent colony formation assay in soft agar, (IV) cell migration and invasion assay.

Project Title:	Identification of direct and indirect targets of the del 6q putative tumor suppressor PRDM1 gene in NK cells, and the role of the aberrant expression of these PRDM1 downstream target genes in the pathogenesis of NK-cell malignancies
Investigator(s):	Srivastava G, Liang RHS
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2009

Abstract:

On-going studies from our group since 1990 have reported that nasal lymphomas consist of both NK-cell and T-cell tumors [1], almost all of which are NK-cell and very few of which are true T-cell lymphomas [2, 4, 5]. Nasal lymphomas are closely associated with EBV [1, 3, 5], and both subtypes express cytotoxic granule-associated proteins [6, 7], implying a derivation from EBV-infected cytotoxic lymphocytes of NK- and T-cell lineage. Nasal lymphoma neoplastic cells escape immune surveillance by downregulation of the immunogenic EBV nuclear antigens with alternative promoter usage [5], preferential selection of the deletion genotype of the latent membrane protein 1 (LMP1) [8, 11], expression of the immune suppressive hIL-10 [9, 10, 12], and mutations in pro-apoptotic Fas [13] and BCL10 [14, 15] genes. In the WHO classification, NK-cell malignancies have been divided into two separate entities: (1) Extranodal NK-cell lymphoma, nasal type (E-NKL) and (2) aggressive NK-cell leukemia (A-NKL) [16]. Both of these entities are characterized by an aggressive clinical course and poor survival. Our earlier findings showing constitutively active NFB signaling in E-NKL [13, 14] gave us a promising lead for NK-cell lymphoma/leukemia (NKLL) treatment. We have recently reported that bortezomib, a proteasome inhibitor that targets NFB activation and is used to treat multiple myeloma and mantle-cell lymphoma, also has therapeutic potential against NKLL [17]. Moreover, we have recently shown that in addition to cell death by apoptosis at lower bortezomib concentrations, NKLL cells resistant to bortezomib-induced apoptosis can be killed via mitotic catastrophe, an alternative cell death mechanism, at higher pharmacological concentrations [18]. Hence, activating mitotic catastrophe by using bortezomib may provide a novel therapeutic approach for treating apoptosis-resistant NKLLs and other cancers. Although little is known about the pathogenetic mechanisms of NK-cell lymphoma/leukemia (NKLL), various studies using karyotyping, CGH, LOH and deletion mapping from other authors and our group have identified 6q deletions being the most common [19-30]. Our recent studies of NKLL cell lines and primary tumors, which include (I) a combination of high resolution array-CGH with gene expression profiling (an international collaborative project) [31] and (II) a genome-wide cDNA microarray screening for genes upregulated by demethylation in seven NKLL cell lines performed by our group, have implicated the transcriptional repressor PRDM1 (positive regulatory domain zinc finger protein 1 gene) as the putative tumor suppressor gene (TSG) in the common minimal deleted 6q21-q22.1 region in NKLLs. Furthermore, sequencing of PRDM1 has revealed mutations in a proportion of NKLL cell lines and primary cases, resulting in a truncated protein with loss of function, and has suggested that putative TSG PRDM1 inactivation in NKLL could be due to both genetic (allelic loss, mutations) and epigenetic (tumor-specific promoter CpG hypermethylation) mechanisms. PRDM1, a transcriptional repressor, is known as a “master regulator” of plasma cell differentiation in B cells [32, 33]. PRDM1 attenuates the expression of many transcription factors, including BCL6, Pax5, and c-Myc, thereby downmodulate B cell proliferation and B cell receptor (BCR)–mediated activation and promoting immunoglobulin (Ig) secretion and plasma cell differentiation [34, 35]. PRDM1 exists as 2 isoforms, that is, as α- and β-isoforms. Generated from alternative transcription initiation, the β-isoform differs from the α-isoform in that it lacks the amino-terminal 101 amino acids and has a disrupted PR domain (Figure 1) [36], which is functionally impaired, with a loss of repressive function on multiple target genes [36]. More recent new findings published in 2006-2008 suggested that the influence of the PRDM1 transcription repressor extends beyond the B cell lineage to include regulation of T cell homeostasis and activation (Figure 2) [37-41]. Newly ascribed functions for PRDM1 in T cells include the attenuation of activation, proliferation, production of IL-2 and IFN-γ and expression of Bcl-6. In T cells, PRDM1 enhances IL-4 and IL-10 production and may enhance activation-induced cell death (AICD). Direct repression of IL2 and the IL2 activator Fos by PRDM1 attenuates T cell proliferation [42], and survival, while PRDM1 induced by IL-4 plays a critical role in suppressing IL-2 production in activated CD4+ T cells [43]. Our studies have shown that the tumor suppressor PRDM1 gene is also expressed in normal NK cells, and it is inactivated in NKLLs by genetic and epigenetic mechanisms, suggesting that PRDM1 from the del 6q21-q22.1 region could be a new potential TSG in NK-cell neoplasms. For the better understanding of the role of the inactivation of PRDM1 in the pathogenesis of NKLL, we aim to perform the following more in-depth studies to identify the targets of the del 6q putative TSG PRDM1 repression in NK cells, and determine the role of the aberrant expression of these PRDM1 downstream target genes in the pathogenesis of NK-cell malignancies. Some of these PRDM1 direct and indirect downstream target genes may be common to both B and T cells, while others may be unique to NK cells. The specific objectives are as follows: 1. To identify direct targets of PRDM1α repression in NKLL using a ChIP-on-chip microarray (chromatin immunoprecipitation coupled with Affymetric high-density promoter array) approach. 2. To identify PRDM1α indirect target genes (in addition to confirming the direct targets) by using the (I) forced expression and (II) knockdown approaches, followed by cDNA microarray and gene expression studies using qRT-PCR combined with ChIP (chromatin immunoprecipitation) assays in NKLLs. 3. To study the expression of the selected PRDM1α downstream target genes in NKLLs in which PRDM1 is inactivated. 4. To investigate whether PRDM1 β-isoform (with a disrupted PR domain) is a negative regulator of the tumor suppressor α-isoform by studying the expression of the PRDM1α target genes in NKLL cell lines after PRDM1β forced expression in PRDM1β-silenced NKLL cell lines expressing PRDM1α. 5. To study the role of the aberrant expression of the selected PRDM1α downstream target genes in the pathogenesis of NKLLs.

Project Title:	Inactivation of the transcriptional repressor PRDM1 and imbalance in the expression of PRDM1 ?and ?isoforms in NK-cell malignancies, and their roles in the pathogenesis
Investigator(s):	Srivastava G, Liang RHS
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	10/2009

Abstract:

1) To study the PRDM1α and PRDM1β protein expression by Western blot (and immunohistochemistry) and identify the NKLL cell lines and primary tumors with defects in PRDM1 or an imbalance in the expression of its two isoforms; 2) To determine whether the inactivation of PRDM1α in NKLL cell lines and primary tumors is due to the genetic and epigenetic alterations and whether these genetic/epigenetic alterations or lack of PRDM1α expression can serve as potential prognostic markers in NKLL cases; 3) To characterize the tumor suppressive functions of PRDM1α in NKLL by re-expression of PRDM1α in PRDM1α-silenced NKLL cell lines with and without PRDM1β expression using the an inducible mammalian expression system that has extremely low basal expression levels; 4) To provide further support for the tumor suppressive role of PRDM1α in NKLL by knocking down its expression using lentiviral vector-delivered shRNA in NKLL cell lines expressing PRDM1α only and those expressing both isoforms and determine whether it will make NKLL cells more aggressive; 5) To determine whether the upregulation of PRDM1β mRNA is due to tumor-specific hypomethylation in NKLL cell lines and cases and determine the prognostic significance of the ratio of PRDM1α/PRDM1β expression in NKLL cases; 6) To investigate whether PRDM1β (with a disrupted PR domain) may be a negative regulator of the tumor suppressor PRDM1α by re-expression of PRDM1β in PRDM1β-silenced NKLL cell lines expressing PRDM1α; 7) To identify putative target genes of PRDM1α in NKLL using a ChIP-on-chip microarray approach.

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Cheung P.P.Y., Deng W., Man C.W.Y., Tse W.W., Srivastava G., Law S.Y.K., Tsao G.S.W. and Cheung A., Genetic alterations in a telomerase-immortalized human esophageal epithelial cell line: Implications for carcinogenesis, Cancer Letters. 2010, 293: 41-51.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Hwang Y.Y., Wong K.Y., Leung R.Y.Y., Wong S.H.M., Chan S.C., Srivastava G. and Au W.Y., Post-rituximab Burkitt transformation of PTLD: loss of CD20 expression accompanied by a switch in light-chain expression, Annals of Hematology. 2010, 89(1): 97-99.

Iqbal J., Weisenburger D.D., Chowdhury A., Srivastava G., Greiner T.C., Deffenbacher K.E., Kucuk C., Vose J.M., Au W.Y., Nakamura S., Seto M., Delabie J., Berger F., Loong F., Ko Y.H., Sng I., Liu X., Loughran T.P., Tsai M., Armitage J.O. and Chan W.C., NK-Cell Lymphoma Shares Strikingly Similar Molecular Features with a Distinct Set of gamma delta T-Cell Lymphoma and Identification of Aurora Kinase A Inhibitor as a Novel Therapeutic Agent, 51st Annual Meeting of the American-Society-of-Hematology, New Orleans, LA, DEC 05-08, 2009. Blood; 114(22): 132-133.

Jacobs C.L., Jima D.D., Zhang J., Dunphy C.W.H., Richards K.L., Choi W.L., Srivastava G., Evens A.M., Gordon L.I., Czader M., Rizzieri D.A., Lagoo A.S., Mann K.P., Flowers C.R., Naresh K., Luftig M., Friedman D.R., Weinberg J.B., Thompson M.A., Gill J., Kahl B.S., Chadburn A. and Dave S., A Comprehensive Identification Of The Microrna Transcriptome And Its Application In B Cell Malignancies, American Society Of Hematology 51st Annual Meeting. 2009, Blood; 114(22): 948-949.

Lo P.H.Y., Lung H.L., Cheung A.K.L., Apte S.S., Chan K.W., Kwong F.M., Ko J.M.Y., Cheng Y., Law S.Y.K., Srivastava G., Zabarovsky E.R., Tsao G.S.W., Tang J.C.O., Stanbridge E.J. and Lung M.L., Extracellular Protease ADAMTS9 Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis, Cancer Research. 2010, 70(13): 5567-76.

Srivastava G., Editor & Editorial Board Member of International Journal of Medical Sciences. 2009.

Researcher : Sze MF

Project Title:	The role of Hepatitis B virus X protein in dysfunction of chromosomal segregation machinery via-interaction with BUBR1 in hepatocellular carcinoma
Investigator(s):	Sze MF, Ng IOL
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	12/2007

Abstract:

Hepatocellular carcinoma (HCC) is the fifth leading cause of cancer worldwide and is highly prevalent in Asia and in Hong Kong. More than 80% of HCC patients in Hong Kong are associated with chronic Hepatitis B viral infection. On the other hand, chromosomal instability (CIN) is commonly in cancers including HCC. In our previous finding, aneuploidy is common (46%) in our human HCCs [Ng et al., 1994]. It is suggested that CIN and genetic alterations facilitate the progression in hepatocarcinogenesis. CIN can lead to dysfunction of DNA damage checkpoint, centrosome amplification and loss of intact mitotic spindle checkpoint. In documented studies, halo-insufficiency of mitotic checkpoint proteins, including Mitotic arrest deficient (MAD1, MAD2 and MAD3/BUBR1) and Budding uninhibited by benzimidazoles (BUB3), in heterozygotic knock-out mice could induce DNA aneuploidy and, in turn, cancer formation [Dai et al., 2004; Iwanaga et al., 2007; Kalitsis et al., 2000; Michel et al., 2001]. Furthermore, viral proteins including SV40 large T antigen and Tax in human T-cell leukemia virus type 1 can physically interact with mitotic checkpoint protein BUB1 and MAD1 respectively to induce abnormal chromosome segregation in cell lines [Cotsiki et al., 2004; Jin et al., 1998]. It is possible that other viral proteins may alter the function of mitotic checkpoint proteins when expressed in cells. In our pilot study, the preliminary results have shown that HBx could physically interact with the full-length form of MAD3/BUBR1 in co-immunoprecipitation assay. Although there have been reports that overexpression of hepatitis B viral X protein (HBx) could induce CIN and centrosome amplification in liver cells [Livezey et al., 2002; Yun et al., 2004], the detailed mechanism by which HBx induces chromosomal instability particularly via the interaction with BUBR1 is not fully understood. In this research proposal, we propose to investigate the role of HBx in the dysregulation of mitotic spindle checkpoint particularly via the physical interaction with BUBR1 in HCC. Objectives of this research proposal: 1. To study the role of HBx in alternation of mitotic spindle checkpoint control in hepatoma cell lines. 2. To study the physical interaction of HBx with other mitotic checkpoint proteins including MAD1-2, BUB1 and BUB3. 3. To map the minimal binding site of BUBR1 for HBx interaction. 4. To investigate the effects of HBx on alteration of BUBR1 function in immortalized normal liver cell line. 5. To investigate whether expression of HBx can alter the protein level of BUBR1 in hepatoma cell lines and in HCC patient samples

List of Research Outputs

Hui C.F., Tung K.K., Sze M.F., Ching Y.P. and Ng I.O.L., Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma, In: Samuel S. Lee, Liver International. 2010, 30 Issue 1: 65-75.

Wong L.T., Yau T.O., Sze M.F. and Ng I.O.L., PTEN underexpression was associated with more aggressive tumor behaviour in hepatocellular carcinoma and PTEN suppressed cell invasion by downregulating NF-κB signaling pathway, The 3rd International LIver Cancer Association Annual Conference, Milan, Italy. 2009.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Szeto EF

List of Research Outputs

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Researcher : Tam HKA

List of Research Outputs

Tam H.K.A., Wong C.M. and Ng I.O.L., Epigenetic Dysregulation of microRNA-9 in Hepatocellular Carcinoma (HCC), The 21st Meeting of the European Association for Cancer Research. Oslo, Norway, 2010.

Wong C.M., Wong C.C.L., Tam H.K.A. and Ng I.O.L., Aberrant expression of epigenetically regulated microRNAs in liver cancer, 101st American Association for Cancer Research (AACR) Annual Meeting 2010. Washington, DC.

Researcher : Tam S

List of Research Outputs

Au W.Y., Lam W.W., Chu W.W., Tam S., Wong W.K., Lau J., Yeung Y.M., Liu H.S. and Liang R.H.S., Organ-specific hemosiderosis and functional correlation in Chinese patients with thalassemia intermedia and hemoglobin H disease, Annals of Hematology. 2009, 88(10): 947-950.

Au W.Y., Hon C., Yau K., Lai W.W.K., Tam S. and Kwong Y.L., Two cases of monocular visual loss during oral arsenic trioxide therapy of acute promyelocytic leukemia., American Journal of Hematology. 2009, 84: 699.

Fung C.W., Poon G.W.K., Kwok A.M.K., Cheung P.T., Low L.C.K., Siu S., Mak C.M., Tam S. and Wong V.C.N., A study of cerebrospinal fluid neurotransmitter assay in children with undiagnosed neurological diseases in Hong Kong, International Symposium on Epilepsy in Neurometabolic Diseases (ISENMD)(Diamond Prize). 2010.

Lam C.M., Lam B., Yao T.J., Lai A.Y.K., Ooi C.G.C., Tam S., Lam K.S.L. and Ip M.S.M., A randomized controlled trial of nCPAP on insulin sensitivity in obstructive sleep apnea, European Respiratory Journal. 2010, 35(1): 138-145.

Mak C.M., Fong B.M., Lam C.W. and Tam S., Genotype-confirmed alanine aminotransferase deficiency in a Chinese patient with acute liver failure: a potential diagnostic pitfall, Pathology. 2010, 42(1): 94-5.

Mok T.M.Y., Yao T.J., Lo Y. and Tam S., Southern Chinese patients with systemic lupus erythematosus in Hong Kong have low vitamin D levels. , HKMJ. 2010, 16: p46 S77.

Researcher : Tang KH

List of Research Outputs

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., CD24 is a functional marker that mediates liver tumor initiation via regulation of Nanog, American Association for Cancer Research, 2010.

Castilho A.G., Lee K.W., Ma S.K.Y., Tang K.H. and Ng I.O.L., Identification of a liver cancer stem marker using a chemoresistant tumor model, "Stem cell and Cancer" Gordon Research Conference, 2009.

Lee K.W., Cheung C.H., Castilho A.G., Tang K.H., Ma S.K.Y. and Ng I.O.L., Phosphorylation profiling of liver cancer stem cells using a CelluSpot kinase peptide array, " Stem Cells and Cancer "Gordon Research Conference, 2009.

Ma S.K.Y., Tang K.H., Chan K.W. and Guan X.Y., Liver Cancer Stem Cells - A Review of Current Literature and Protocols, In: XY Guan, Cancer Stem Cells. Research Signpost, 2009.

Ma S.K.Y., Tang K.H., Chan Y.P., Lee K.W., Castilho A.G., Ng I.O.L., Man K., To K.F., Zheng B., Chan K.W. and Guan X.Y., miR-130b is preferentially upregulated in CD133+ liver cancer stem cells and regulates tumor growth and self-renewal via tumor protein 53-induced nuclear protein 1, Gordon Research Conference - Stem Cells and Cancer. 2009.

Ma S.K.Y., Chan Y.P., Kwan P.S., Tang K.H., Vielkind J.V., Guan X.Y. and Chan K.W., microRNA-616 induces androgen-independent growth of prostate cancer cells through suppression of TFPI-2 expression , American Association for Cancer Research. 2010.

Tang K.H., Ma S.K.Y. and Guan X.Y., Liver Tumor-initiating Cells / Cancer Stem Cells: Past Studies, Current Status And Future Perspectives, In: R Scatena, A Mordente, B Giardina, Advances In Cancer Stem Cell Biology. Springer, 2010.

Yuen H.F., Chiu Y.T., Chan K.K., Chua C.W., McCrudden C.M., Tang K.H., El-Tanani M., Wong Y.C., Wang X. and Chan K.W., Prostate Cancer Cells Modulate Osteoblast Mineralisation And Osteoclast Differentiation Through Id-1 , British Journal of Cancer. 2010, 102: 332-341.

Researcher : Tao Q

List of Research Outputs

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Tin PC

List of Research Outputs

Leung L.H., Ma Y., Fiscus R.R., Tung J.W., Tin P.C. and Wong M.P., Isolation, expansion and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer, American Association for Cancer Research Annual Meeting, 2010 Washington DC the United State . 2010.

Tam I.Y., Leung L.H., Tin P.C., Chua D.T.T., Sihoe A.D.L., Cheng L.C., Chung L.P. and Wong M.P., Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared to common activating missense mutations, Molecular Cancer Therapeutics. 2009, 8(8): 2142-51.

Researcher : Ting KP

List of Research Outputs

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Researcher : Tong SF

List of Research Outputs

Chau S.K.C., Kwok K.L., Ng D.K., Lam C.W., Tong S.F., Chan A.Y.W., Siu C.W.K. and Yuen L.Y.P., Maternally inherited Leigh syndrome: an unusual cause of infantile apnea, Sleep Breath. 2010, 14(2): 161-5.

Researcher : Trendell-Smith NJ

List of Research Outputs

Au W.Y., Trendell-Smith N.J., Ko B.H., Tong A.C. and Wong R.W.S., Oral Epstein–Barr virus-related B-cell lymphoma causing persistent paraneoplastic dermatomyositis after nasopharyngeal and cutaneous carcinomas, Leukaemia & Lymphoma. 2010, 51(4): 715-716.

Gill H., Trendell-Smith N.J., Loong F., Yeung C.K. and Kwong Y.L., Paraneoplastic pemphigus due to CD8-positive cytotoxic T-cell lymphoma, British Journal of Haematology. 2010, 149(4): 464.

Gill H., Leung A.Y.H., Trendell-Smith N.J., Yeung C.K. and Liang R.H.S., Sweet Syndrome due to Myelodysplastic Syndrome: Possible Therapeutic Role of Intravenous Immunoglobulin in Addition to Standard Treatment, Advances in Hematology. 2010, 2010:328316: Epub.

Tsang H.H., Wong R.W.S., Trendell-Smith N.J., Wu A.K.P. and Mok T.M.Y., Diffuse large B-cell lymphoma of the central nervous system in mycophenolate mofetil-treated patients with systemic lupus erythematosus., HKMJ. 2010, 16: p54 S92.

Yeung C.K., Trendell-Smith N.J., Mak H.K.F., Lam C.C.K. and Kwong Y.L., ‘Western’ or ‘Asian’ intravascular large B-cell lymphoma?, Clinical and Experimental Dermatology. 2009, 34(7): e482–e483.

Researcher : Tsang STY

List of Research Outputs

So J.C.C., So A.C., Chan A.Y., Tsang S.T.Y., Ma E.S.K. and Chan L.C., Detection and characterisation of beta-globin gene cluster deletions in Chinese using multiplex ligation-dependent probe amplification, J Clin Pathol. 2009, 62(12): 1107-11.

Researcher : Tse YT

List of Research Outputs

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Tsui WY

List of Research Outputs

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Tsun OKL

List of Research Outputs

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Researcher : Tung KK

Project Title:	21st Meeting of the European Association for Cancer Research Clinical significance of target genes of Wnt/beta-catenin pathway in hepatocellular carcinoma
Investigator(s):	Tung KK
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	06/2010
Completion Date:	06/2010

Abstract:

N/A

List of Research Outputs

Hui C.F., Tung K.K., Sze M.F., Ching Y.P. and Ng I.O.L., Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma, In: Samuel S. Lee, Liver International. 2010, 30 Issue 1: 65-75.

Ko F.C.F., Chan L.K., Tung K.K., Ng I.O.L. and Yam J.W.P., Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma, American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Tung K.K., Mak K.M. and Ng I.O.L., Clinical significance of target genes of Wnt/beta-catenin pathway in hepatocellular carcinoma, EJC Supplements / 21st Meeting of the European Association for Cancer Research . Elsevier, Inc, 2010, 8: 48.

Tung K.K., Mak K.M., Lee M.F., Li J.J., Poon R.T.P., Lai C.L., Luk J.M.C. and Ng I.O.L., Serum level of DKK1 as a marker for predicting tumor recurrence of hepatocellular carcinoma , American Association for Cancer Research 101st Annual Meeting 2010.

Yau T.O., Leung T.H.Y., Lam S.G.S., Cheung O.F., Tung K.K., Khong P.L., Lam A.K.M., Chung S.K. and Ng I.O.L., Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis., PLoS One. 2009, 4(8): e6566.

Researcher : Wan TSK

List of Research Outputs

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Kwan T.K., Ma E.S.K., Chan Y.Y., Wan T.S.K., Liu H.S.Y., Sim J.P.Y., Yeung Y.M., Lie A.K.W. and Yip S.F., BCR-ABL mutational studies for predicting the response of patients with chronic myeloid leukaemia to second-generation tyrosine kinase inhibitors after imatinib fail, Hong Kong Medical Journal. 2009, 15(5): 365-73.

Tsang C.M., Lau P.W.E., Di K., Cheung P.P.Y., Hau P.M., Ching Y.P., Wong Y.C., Cheung A., Wan T.S.K., Tong Y., Tsao G.S.W. and Feng Y., Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells, International Journal of Molecular Medicine. 2009, 24: 131-138.

Researcher : Wong ACN

List of Research Outputs

Triana-Baltzer G.B., Babizki M., Chan M.C.W., Wong A.C.N., Aschenbrenner L.M., Campbell E.R., Li Q.X., Chan W.Y., Peiris J.S.M., Nicholls J.M. and Fang F., DAS181, a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis., J Antimicrob Chemother. 2010, 65: 275-84.

Researcher : Wong CCL

List of Research Outputs

Au L.K., Wong C.C.L., Lee M.F., Wong C.M. and Ng I.O.L., Frequent upregulation of Enhancer of Zeste Homolog 2 was implicated in Deleted in Liver Cancer 1 gene silencing and HCC progression , The 20th Conference of the Asian Pacific Association for the Study of the Liver (APASL). Beijing, 2010.

Wong C.C.L., Wong C.M., Au L.K. and Ng I.O.L., RhoGTPases and Rho-effectors in hepatocellular carcinoma metastasis: ROCK N' Rho move it , Liver International. 2010, 30: 642-656.

Wong C.M., Wong C.C.L., Tam H.K.A. and Ng I.O.L., Aberrant expression of epigenetically regulated microRNAs in liver cancer, 101st American Association for Cancer Research (AACR) Annual Meeting 2010. Washington, DC.

Researcher : Wong CM

Project Title:	Roles of histone methyltransferases SUV39H1 and SUV39H2 in liver cancer
Investigator(s):	Wong CM, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	01/2008
Completion Date:	12/2009

Abstract:

Objectives 1. To evaluate the clinical and prognostic significance of SUV39H1/2 deregulation in liver dysplastic nodules and HCC. 2. To characterize the effect of SUV39H1/2 deregulation on hepatocarcinogenesis and the underlying mechanisms 3. To investigate the possible roles of SUV39H1/2 in aberrant epigenetic regulation in HCC Liver cancer (hepatocellular carcinoma, HCC) is one of the major malignancies worldwide and ranks as the second most common fatal cancer in Southeast Asia and Hong Kong. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. Despite significant improvements in the outcome of patients with HCC, so far, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. New adjuvant treatment modalities for HCC are much awaited. In this regard, knowledge of the molecular and cellular targets underlying the development and progression of HCC is of importance as this can provide novel opportunities for therapeutic interventions for this cancer. We and others have shown that epigenetic alterations, particularly DNA hypermethylation, play a crucial role in silencing important tumor suppressor genes. DNA methylation and histone modifications work very closely in regulating chromatin structure and gene expression. Accumulating evidence has revealed that aberrant DNA methylation and histone modifications are important in the initiation and progression of human cancers. However, most previous studies have focused on DNA methylation, and the implication of aberrant histone modifications in cancer development and progression has not been fully investigated so far. As part of a continual search for epigenetic alterations in HCC, we have recently performed a systematic, large-scale expression analysis on a total of 90 epigenetic regulators in paired primary human HCC and their corresponding non-tumorous livers. Our preliminary data have revealed that histone methyltransferases, SUV39H1 and SUV39H2, are frequently overexpressed in human HCC when compared with their corresponding non-tumorous livers. SUV39H1/2 are human orthologs of Drosophila transcriptional repressor Su(Var)3-9 and are the prototypes of mammalian histone methyltransferases. SUV39H1 and SUV39H2 share 59% sequence identity and contain evolutionary conserved chromo domain and SET domain. Functionally, SUV39H1 and SUV39H2 are histone H3-specific methyltransferases that selectively methylate Lys-9 of histone 3 (1,2). The histone methyltransferase activities of SUV39H1/2 depend mainly on the SET domain located at their C-terminal region. Suv39h1/2 are members of HP1β heterochromatic protein complex, which is a well characterized chromatin remodeling complex implicated in both gene silencing and heterochromatin structure (1,3). H3K9 methylation catalyze by Suv39h1/2 creates a binding site for HP1β and regulates heterochromatin formation (4,5). In addition, SUV39H1 is also implicated in the repression of gene-specific transcription in euchromatin. SUV39H1 forms a repressive complex with Rb and HP1β, where it functions as a co-repressor for Rb-targeted genes via its H3K9 methyltransferase activity (6). Besides, SUV39H1 is also associated with DNMTs (7) and MBD1 (8), thus linking SUV39H1/2 activity to DNA methylation machinery. The exact roles of SUV39H1 and SUV39H2 in human cancers have not been clearly addressed. However, the implication of H3K9 in tumor suppressor gene inactivation has received much attention in recent years (9,10). As the major enzyme responsible for H3K9 methylation, SUV39H1 has found to be overexpressed in primary colorectal cancer (11). In animal models, Suv39h1 transgenic liver cells displayed increased potential for spontaneous immortalization and self-renewal capability. These cellular alterations were accompanied with deregulation of important tumor suppressor genes, including p21, Rb and p53 (12). However, Suv39h1 and Suv39h2 double knockout mice exhibited severe chromosomal instabilities and increased tumor formation (13). When Suv39h1/2 double knockout mice were intercrossed with transgenic mice expressing constitutive active Ras mutant, it was found that loss of Suv39h1/2 impaired oncogene-induced senescence (OIS) leading to increased susceptibility to Ras induced transformation (14). These observations suggest that SUV39H1 may possess dual roles in carcinogenesis. On one hand, SUV39H1 suppresses cellular transformation in pre-neoplastic cells through cell senescence. On the other, SUV39H1 can assist in tumor progression by epigenetic silencing of tumor suppressor genes at later stages of tumor development. The critical issue as to whether SUV39H1/2 fulfill differential functions at early and late phases of tumorigenesis remains to be clarified. Thus, comprehensive investigation on the functional and pathological implications of SUV39H1/2 in premalignant liver cells and liver cancer is warranted. Here, we hypothesize that deregulation of SUV39H1/2 might contribute to the development and progression of liver cancer. Specifically, our unifying model is that the expression of SUV39H1/2 plays a role in oncogene-induced senescence in premalignant liver cells but mediates the epigenetic inactivation of tumor suppressor genes in tumor cells. The purpose of this study is to characterize the exact roles of histone methyltransferases SUV39H1 and SUV39H2 in different phases of hepatocarcinogenesis. We aim to study the roles of histone methyltransferases, SUV39H1 and SUV39H2, in the development of liver cancer by 1) evaluating the expression and clinicopathological significance of SUV39H1/2 in dysplastic nodules and liver cancer, 2) characterizing the effect of SUV39H1/2 deregulation on tumorigenesis and their underlying mechanisms, and 3) delineating the roles of SUV39H1/2 deregulation in epigenetic alterations in premalignant liver cells and liver cancer. We believe our proposed work will provide mechanistic insight into the molecular basis of epigenetic alterations in hepatocarcinogenesis. Our proposed study may open up new avenues for developing novel diagnostic tools and therapeutic interventions for human liver cancer. Reference 1. O'Carroll, D. et al. Mol Cell Biol 20, 9423-33 (2000). 2. Rea, S. et al. Nature 406, 593-9 (2000). 3. Aagaard, L. et al. Embo J 18, 1923-38 (1999). 4. Lachner, M. et al. Nature 410, 116-20 (2001). 5. Bannister, A. J. et al. Nature 410, 120-4 (2001). 6. Nielsen, S. J. et al. Nature 412, 561-5 (2001). 7. Fuks, F. et al. Nucleic Acids Res 31, 2305-12 (2003). 8. Fujita, N. et al. J Biol Chem 278, 24132-8 (2003). 9. Kondo, Y. et al. Mol Cell Biol 23, 206-15 (2003). 10. Nguyen, C. T. et al. Cancer Res 62, 6456-61 (2002). 11. Kang, M. Y. et al. Int J Cancer 121, 2192-2197 (2007). 12. Czvitkovich, S. et al. Mech Dev 107, 141-53 (2001). 13. Peters, A. H. et al. Cell 107, 323-37 (2001). 14. Braig, M. et al. Nature 436, 660-5 (2005).

Project Title:	Roles of histone methyltransferases SUV39H1 and SUV39H2 in liver cancer
Investigator(s):	Wong CM, Ng IOL
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	09/2008

Abstract:

(1) To evaluate the clinical and prognostic significance of SUV39H1/2 deregulation in liver dysplastic nodules and HCC; (2) To characterize the effect of SUV39H1/2 deregulation on hepatocarcinogenesis and the underlying mechanisms; (3) To investigate the possible roles of SUV39H1/2 in aberrant epigenetic regulation in HCC.

Project Title:	Functional and pathological implications of EZH2 in human HCC
Investigator(s):	Wong CM, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	02/2009

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is one of the major malignancies worldwide and ranks as the second most common fatal cancer in Southeast Asia and Hong Kong. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. We and others have shown that epigenetic alterations, particularly DNA hypermethylation, play a crucial role in silencing important tumor suppressor genes. DNA methylation and histone modifications work very closely in regulating chromatin structure and gene expression. Accumulating evidence has revealed that aberrant DNA methylation and histone modifications are important in the initiation and progression of human cancers. However, most previous studies have focused on DNA methylation, and the implication of aberrant histone modifications in cancer development and progression has not been fully investigated. As part of a continual search for epigenetic alterations in HCC, we have recently performed a systematic, large-scale expression analysis on a total of 90 epigenetic regulators in primary human HCC. Our preliminary data have revealed Polycomb group (PcG) protein enhancer of zeste homolog (EZH2) was significantly overexpressed in human HCC when compared with their corresponding non-tumorous livers. Polycomb group proteins were first identified in the 1940s, and named because when mutated in Drosophila, males had additional sex combs (Polycomb) on the second and third thoracic segments whereas wild-type males only carry sex combs on their first leg (1). Polycomb (PcG) and its closely related trithorax (TrxG) group proteins are the key regulators of developmental genes. More specifically while TrxG proteins maintain the activated state of Hox genes in the appropriate developmental segments, PcG genes generate heritable states of gene silencing over extended regions of chromatin to prevent inappropriate Hox gene expression (1). In mammalian, PcG genes mutation led to developmental defeats such as skeletal malformations (2). In addition, PcG also plays a crucial role in the control of cell proliferation, stem cell maintenance, and more recently implicated in human carcinogenesis (3). The PcG family proteins form two distinct complexes, known as Polycomb repressive complex 1 and 2 (PRC1 and PRC2). The two complexes functionally cooperate in maintaining transcriptional repression of target genes. PRC2 contains four core components namely, EZH2, EED, SUZ12 and RBBP4/7. PRC2 has histone-modifying activity and specifically catalyzes tri-methylation on lysine 27 of histone 3 (H3K27). H3K27 tri-methylation was then recognized by PRC1, which composed of other PcG proteins, including BMI-1, MEL18, CBX2, CBX4, PH1, PH2, RING1 and RNF2. The binding of PRC1 will then induce succeeding chromatin changes that eventually will lead to stable gene silencing (3). The exact roles of PcG proteins in human cancers have not been clearly addressed. However, recent studies have indicated that deregulation of PcG proteins is common in human malignancies. As the major enzyme responsible for H3K27 tri-methylation and PcG mediated gene silencing, EZH2 has found to be up-regulated in prostate (4) and breast (5) cancers. Overexpression of EZH2 was closely associated with cancer metastasis and showed a poor prognosis (4,5). Ectopic expression of EZH2 in immortalized human mammary epithelial cell lines promotes anchorage-independent growth and cell invasion (5). In addition, EZH2 was shown to interact with DNA methyltransferases (DNMTs) and promotes DNA methylation of the target genes, this finding link PcG mediated gene silencing to DNA hypermethylation and suggesting an addition mechanism for epigenetic inactivation of tumor suppressor inactivation in human cancers (6). Nevertheless, the roles of EZH2 and its downsteam targets in human HCC remain to be clarified. A comprehensive investigation on the functional and pathological implications of EZH2 in human HCC is therefore much warranted for this regard. Here, we hypothesize that deregulation of EZH2 may contribute to the development and progression of HCC. Specifically, our working model is that the deregulation of EZH2 plays a role in inactivating important tumor suppressor genes via PcG mediated gene silencing and consequently facilitates HCC development and progression. The purpose of this study is to characterize the functional implications of EZH2 and identify the tumor specific PcG targets in HCC. We aim to study the roles of EZH2 in the development of liver cancer by 1) evaluating the expression and clinicopathological significance of EZH2 in human HCC, 2) characterizing the effect of EZH2 overexpression in tumorigenesis and metastasis of HCC, and 3) delineating the tumor specific targets of EZH2 mediated gene silencing in HCC. We believe our proposed work will provide mechanistic insight into the molecular basis of PcG mediated epigenetic alterations in hepatocarcinogenesis. Our proposed study may open up new avenues for developing novel diagnostic tools and therapeutic interventions for human liver cancer. Objectives 1. To evaluate the expression, clinicopathological significance and prognostic value of EZH2 in human HCC. 2. To characterize the effect of EZH2 deregulation on hepatocarcinogenesis and the underlying mechanisms 3. To identify the tumor specific targets of EZH2 mediated epigenetic gene silencing in HCC. Reference: 1 Ringrose, L. & Paro, R., Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38, 413-443 (2004). 2 Akasaka, T. et al., A role for mel-18, a Polycomb group-related vertebrate gene, during theanteroposterior specification of the axial skeleton. Development 122 (5), 1513-1522 (1996). 3 Sparmann, A. & van Lohuizen, M., Polycomb silencers control cell fate, development and cancer. Nat Rev Cancer 6 (11), 846-856 (2006). 4 Varambally, S. et al., The polycomb group protein EZH2 is involved in progression of prostate cancer. Nature 419 (6907), 624-629 (2002). 5 Kleer, C.G. et al., EZH2 is a marker of aggressive breast cancer and promotes neoplastic transformation of breast epithelial cells. Proc Natl Acad Sci U S A 100 (20), 11606-11611 (2003). 6 Vire, E. et al., The Polycomb group protein EZH2 directly controls DNA methylation. Nature 439 (7078), 871-874 (2006).

Project Title:	Epigenetic alteration and miRNA deregulation in liver cancer
Investigator(s):	Wong CM, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	02/2010

Abstract:

Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide and is the second most common fatal cancer in Southeast Asia and Hong Kong, due to the high prevalence of hepatitis B viral infection. Despite definite improvements in the outcome of patients with HCC, the overall prognosis of this cancer is still unsatisfactory because of late presentation and frequent tumor recurrence after surgical resection. New diagnostic and adjuvant treatment modalities for HCC are much awaited. In this regard, knowledge of the molecular mechanism underlying the development and progression of liver cancer is of importance as this can provide novel opportunities for early diagnosis and therapeutic interventions for this cancer. MicroRNAs (miRNA) are a class of small (~22 nt) single-stranded noncoding RNAs widely expressed in eukaryotic cells. Since the discovery of the first miRNA, lin-4 in C. elegans in 1993, thousands of miRNAs have been identified in organisms ranging from plants to humans. miRNAs play an important role in regulating gene expression at the post-transcriptional level, which can bind to their complementary sequences located on 3’UTR of mRNA targets, resulting in mRNA degradation, or more often, translational repression. Although the biological functions of individual miRNAs remain elusive, previous studies on C. elegans and Drosophila models have revealed that miRNAs are involved in almost all major physiological processes, including cell proliferation, differentiation, apoptosis, hormonal secretion and viral infections. There is mounting evidence that miRNAs are implicated in the initiation, development and progression of human malignancies. Some miRNAs have been shown to promote or suppress cancer cell proliferation and survival, in which case they can function as oncogenes or tumor suppressor genes. Following the first report demonstrating the frequent deletion and down-regulation of miR-15 and miR-16 in B-cell chronic lymphocytic leukemia, several studies have shown that miRNA expression profiles are useful in distinguishing tumors from normal tissues as well as classifying human cancer types. Moreover, changes in miRNA expressions have been found to be associated with various clinicopathological features, including hormonal status, metastasis, and proliferation index. Recently, in addition to genetic alterations, many lines of evidence have indicated that epigenetic alterations also play a very important role in human carcinogenesis. The term ‘epigenetic’ generally refers to heritable changes in DNA methylation and histone modifications that stably modify gene transcription but do not involve changes of the DNA sequences. DNA methylation, a covalent addition of methyl group (-CH3) to the 5-position of cytosine, is the most well characterized epigenetic event. In mammalian cells, DNA methylation is mainly found in the cytosine residues of CpG dinucleotides within the repetitive elements or promoter-related CpG islands. DNA hypermethylation on promoter is associated with transcriptional silencing. Histone proteins are the structural component of chromatin. Recent evidence has suggested that methylation on histone 3 Lys-4 (H3K4) is linked to transcriptional active euchromatin. On the other hand, histone 3 Lys-9 (H3K9) and Lys-27 (H3K27) methylation are associated with transcriptional repressive heterchromatin. These post-translational modifications on histone proteins govern the chromatin structure, which is also known as histone code. DNA methylation and histone modification are essential for development and differentiation, but research over the past decade has linked epigenetic alterations to human carcinogenesis. We and others have previously shown that epigenetic alterations, particularly DNA hypermethylation, play a crucial role in silencing tumor suppressor genes in human cancers. As part of a continual search for epigenetic alterations in HCC, we have recently investigated the role of epigenetic alterations and miRNA deregulation in human HCC. To this end, HCC cell lines SMMC-7721 and BEL7402 were treated with DNA demethylating agent (5-Aza-dC) and histone deacetylase inhibitor (Trichostatin A, TSA) and miRNA expression profiles changes upon treatment were compared by stem-loop RT-qPCR based TaqMan low density array. Totally, 98 miRNAs were found to be remarkably up-regulated upon 5-Aza-dC/TSA treatment. Among these, 35 miRNA were commonly upregulated by the epigenetic inhibitors treatment in both cell lines. In this proposed study, we aim at characterize the epigenetic status, expression and functional implications of these epigenetically regulated miRNA on the development and progression of human HCC. Objectives 1. To determine the expression pattern of epigenetically regulated miRNAs in human HCCs 2. To delineate the role of epigenetic alterations on miRNA deregulation in HCC cell lines and primary HCCs. 3. To investigate the functional implication of epigenetically deregulated miRNA in HCC.

Project Title:	American Association for Cancer Resarch (AACR) 101st Annual meeting 2010 Aberrant expression of epigenetically regulated microRNAs in liver cancer
Investigator(s):	Wong CM
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Au L.K., Wong C.M. and Ng I.O.L., Enhancer of Zeste Homolog 2 (EZH2) was involved in Hepatocellular Carcinoma (HCC) Progression Through Gene Silencing, 16th Hong Kong International Cancer Congress. Hong Kong, 2009.

Au L.K., Wong C.C.L., Lee M.F., Wong C.M. and Ng I.O.L., Frequent upregulation of Enhancer of Zeste Homolog 2 was implicated in Deleted in Liver Cancer 1 gene silencing and HCC progression , The 20th Conference of the Asian Pacific Association for the Study of the Liver (APASL). Beijing, 2010.

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Mak K... .Y..., Wong C.M., Leung C... .W..., Huang H... .T..., Chan S... .B..., Shi J..., Mak S.K., Chan M... .M... and Pong P.W.T., Study of cellular behaviour on micro- and nano- structures, The Institution of Engineering and Technology Hong Kong Younger Members Exhibition/ Conference 2009.

Tam H.K.A., Wong C.M. and Ng I.O.L., Epigenetic Dysregulation of microRNA-9 in Hepatocellular Carcinoma (HCC), The 21st Meeting of the European Association for Cancer Research. Oslo, Norway, 2010.

Wong C.C.L., Wong C.M., Au L.K. and Ng I.O.L., RhoGTPases and Rho-effectors in hepatocellular carcinoma metastasis: ROCK N' Rho move it , Liver International. 2010, 30: 642-656.

Wong C.M., Wong C.C.L., Tam H.K.A. and Ng I.O.L., Aberrant expression of epigenetically regulated microRNAs in liver cancer, 101st American Association for Cancer Research (AACR) Annual Meeting 2010. Washington, DC.

Wong C.M., Academic Editor, PLoS ONE. 2009.

Wong C.M., Deregulation of microRNA expression in liver cancer development, A Croucher Foundation‐sponsored Conference RNA Silencing 2009: Mechanisms and Applications. 2009.

Yam J.W.P., Wong C.M. and Ng I.O.L., Molecular and functional genetics of hepatocellular carcinoma, Frontiers in bioscience (Scholar edition). 2010, 2: 117-134.

Yang W., Shen N., Ye D.Q., Liu Q., Qian X.X., Hirankarn N., Pan H.F., Mok C.C., Chan D.T.M., Wong R.W.S., Lee K.W., Wong S.N., Leung A.M.H., Li X.P., Avihingsanon Y., Wong C.M., Lee T.L., Ho M.H.K., Lee P.P.W., Chang Y.K., Li P.H., Li R., Zhang L., Wong W.H.S., Ng I.O.L., Lau W.C.S., Sham P.C., Lau Y.L. and Asian Lupus Genetics Consortium A.L.G.C., Genome-wide association study in Asian populations identifies variants in ETS1 and WDFY4 associated with systemic lupus erythematosus. , PLoS Genetics. 2010, 6: e1000841.

Researcher : Wong CN

List of Research Outputs

Chan W.Y., Chan M.C.W., Wong C.N., Karamanska R., Dell A., Haslam S.M., Sihoe A.D., Chui W.H., Triana-Baltzer G., Li Q., Peiris J.S.M., Fang F. and Nicholls J.M., DAS181 Inhibits H5N1 Influenza virus Infection of Human Lung Tissues., Antimicrobial Agents and Chemotherapy. 2009, 53(9): 3935-3941.

Researcher : Wong ESY

List of Research Outputs

Cheung A.N.Y., Tsun O.K.L., Ng K.M., Szeto E.F., Siu K.Y., Wong E.S.Y. and Ngan H.Y.S., P634A4 and TAp73 immunocytochemistry in liquid-based cervical cytology—potential biomarkers for diagnosis and progress prediction of cervical neoplasia, Modern Pathology. 2010, 23: 559-66.

Siu K.Y., Wong E.S.Y., Kong S.H., Wong G.W., Tam K.F., Ngan H.Y.S., Le X.F. and Cheung A.N.Y., Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers. , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A., 17 - 21 April 2010.. 2010.

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Wong GW

Project Title:	Role of Plexin-B1 signaling in ovarian cancer
Investigator(s):	Wong GW, Siu KY
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2010

Abstract:

Ovarian cancer is the most deadly gynaecological malignancies. While generally responsive to chemotherapy, relapsed cancers often become resistant to the original chemotherapy. Another factor contributing to the poor outcome of ovarian cancer is the lack of specific symptoms early in the development of the disease. For this reason, most ovarian cancer patients are diagnosed at late stages of the disease when therapies are less efficient. Moreover, ovarian cancer has a tendency to spread within the peritoneal cavity early in the disease course. Understanding the metastatic pathways of ovarian cancer is thus of high importance for devising ovarian cancer treatment. Semaphorins are signalling molecules serving diverse functions. They were first recognized as guiding clues for the wiring of the nervous system, but in the last decade their roles in immune function, angiogenesis, organ development, and cancer metastasis have been rapidly unearthed (Basile et al, 2006; Chedotal, 2007; Ji & Ivashkiv, 2009). Most of the physiological effects of Semaphorins are mediated by Plexins, a group of single pass transmembrane receptors with no kinase activity. Plexins can be grouped into 4 subfamilies (Plexin-A, B, C, and D) according to their domain structure. Plexin-B1 is the prototypic member of class B Plexins and the cognate receptor of Sema4D (Tamagnone et al, 1999). Sema4D and Plexin-B1 might take important roles in the development of ovarian follicles since they are expressed in the granulosa cells and their expression increase in response to pregnant mare serum gonadotropin and hCG (Regev et al, 2005; Regev et al, 2007). In a previous Plexins mutation screening analysis in prostate cancer, I have identified a number of missense mutations within the open reading frame of the gene encoding Plexin-B1 (PLXNB1). These Plexin-B1 mutants were overexpressed in prostate cancers and contributed to faster migration of prostate cancer cells (Wong et al, 2007). The role of Plexin-B1 in ovarian carcinogenesis has not been reported. Physiologically, Sema4D binding to Plexin-B1 may trigger different intracellular signalling pathways in different cell types. Sema4D collapses axonal growth cones in neurons. This effect is achieved through the action of Plexin-B1 on several small GTPases of the Ras and Rho families. Plexin-B1 acts as a R-Ras GAP (GTPase activating protein) and hence downregulates R-Ras activity upon Sema4D stimulation (Oinuma et al, 2004). Inactivation of R-Ras leads to decreased adhesiveness of the growth cone through downregulation of integrins. Activated Plexin-B1 also binds to GTP-Rac and sequesters it from activating other downstream proteins such as Pak (Vikis et al, 2002). Inhibition of Rac may cause less efficient formation of lamellipodia on the growth cone. Plexin-B1 may also activate RhoA through PDZ-RhoGEF and LARG which are RhoA specific GEF (Guanine exchange factor) stably interacting with the C-terminal of Plexin-B1 (Swiercz et al, 2002). RhoA activation inhibits Rac action and also increases the traction force against the movement of growth cones. The collective effect of these signalling events is collapse of axonal growth cones. Interestingly, these signalling events are highly relevant in cell migration of epithelial cells and when activated likely decrease cell migration. In fact, we and others have already demonstrated the effect of Plexin-B1 signalling on epithelial cell migration (Basile et al, 2005; Oinuma et al, 2006; Wong et al, 2007). In addition to small GTPases, receptor tyrosine kinases are very important components of Plexin signalling. Plexin-B1 stably interacts with Met and Ron, the two members of the scatter factor receptor family (Conrotto et al, 2004). The scatter factor receptors are the primary transducers of signals for invasive growth, a biological program which instructs cells to dissociate, migrate, degrade the surrounding matrix, proliferate and survive. Both receptors have tyrosine kinase activity and have been implicated in the invasive growth of many cancer cell types. Plexin-B1 interacts with Met constitutively in a ligand independent manner. Stimulation of Plexin-B1 by Sema4D in epithelial cells leads to Met/Ron activation and initiates classical scatter responses, including loss of cell-cell contact and increased migration. Moreover, Plexin-B1 and ErbB-2 form a stable complex in certain epithelial cell type too (Swiercz et al, 2004). The ErbBs are typical receptor tyrosine kinases that are associated with cancer development. Within the ErbB family, ErbB-2 is the most potent oncoprotein although it has no known ligand. ErbB-2 preferentially forms heterodimer with other ErbBs and acts as an active component in the resultant receptor complex. Binding of Sema4D to plexin-B1 stimulates the intrinsic tyrosine kinase activity of ErbB-2, resulting in the phosphorylation of both plexin-B1 and ErbB-2. This activation of both receptors is required for RhoA activation mediated by Plexin-B1. Notably, both Met and ErbB-2 have been demonstrated to be overexpressed in ovarian cancer. The objectives of this study are: 1. To determine the expression profile of Plexin-B1 and Sema4D in normal ovarian surface epithelium and different categories of ovarian epithelial tumour including aggressive ovarian cancers. 2. To investigate the effect of modulating Plexin-B1 level on proliferation, cell migration, invastion and apoptosis of ovarian cancer cells using cell line models 3. To investigate the mutation status of Plexin-B1 in ovarian cancer

Project Title:	AACR 101st Annual Meeting 2010 Dysregulated expression and function of Plexin-B1 in ovarian cancers
Investigator(s):	Wong GW
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Siu K.Y., Wong E.S.Y., Kong S.H., Wong G.W., Tam K.F., Ngan H.Y.S., Le X.F. and Cheung A.N.Y., Dysregulated expression of stem cell transcription factor Nanog in development and progress of ovarian cancers. , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A., 17 - 21 April 2010.. 2010.

Siu K.Y., Wong G.W. and Cheung A.N.Y., TrkB as a therapeutic target for ovarian cancer, Expert Opin Ther Targets. 2009, 1169-78.

Tang V.H.M., Kong K.Y., Wong G.W., Qiu H.F., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p is required for transcription elongation and termination, Mechanisms of Eukaryotic Transcription, Cold Spring Harbor Laboratory. 2009.

Wong C.M., Tang V.H.M., Kong K.Y., Wong G.W., Qiu H., Jin D. and Hinnebusch A.G., Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p, Nucleic Acids Research. 2010, 38(7): 2217-2228.

Wong G.W., Siu K.Y., Ngan H.Y.S. and Cheung A.N.Y., Dysregulated expression and function of Plexin-B1 in ovarian cancers , The 101st Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Wong KY

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Hwang Y.Y., Wong K.Y., Leung R.Y.Y., Wong S.H.M., Chan S.C., Srivastava G. and Au W.Y., Post-rituximab Burkitt transformation of PTLD: loss of CD20 expression accompanied by a switch in light-chain expression, Annals of Hematology. 2010, 89(1): 97-99.

Researcher : Wong LT

List of Research Outputs

Wong L.T., Yau T.O., Sze M.F. and Ng I.O.L., PTEN underexpression was associated with more aggressive tumor behaviour in hepatocellular carcinoma and PTEN suppressed cell invasion by downregulating NF-κB signaling pathway, The 3rd International LIver Cancer Association Annual Conference, Milan, Italy. 2009.

Researcher : Wong MLY

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Chen W.Y.W., Hu X., Liang C.T., Wong M.L.Y., Au W.Y., Wong K.Y., Choi W.L., Wan T.S.K., Chu K.M., Chim J.C.S., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., Molecular features and functional consequence of CD44 activation by a novel recurrent IGH translocation t(11;14) (p13;q32) in mature B-cell lymphoid neoplasm., 101st Annual Meeting of American Association for Cancer Research (AACR), Washington D.C., USA, April 2010.. 2010.

Hu X., Chen W.Y.W., Liang A.C.T., Au W.Y., Wong K.Y., Wan T.S.K., Wong M.L.Y., Shen L., Chan K.K., Guo T., Chu K.M., Tao Q., Chim J.C.S., Loong F., Choi W.L., Lu L., So J.C.C., Chan L.C., Kwong Y.L., Liang R.H.S. and Srivastava G., CD44 activation in mature B-cell malignancies by a novel recurrent IGH translocation, Blood . 2010, 115: 2458-2461.

Researcher : Wong MP

Project Title:	Mutational Analysis of Epidermal Growth Factor Receptor in Non-Small Cell Lung Cancer
Investigator(s):	Wong MP, Chua DTT
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2005

Abstract:

A. Purpose of study: To evaluate the incidence and patterns of EGFR mutation in NSCLC, and to correlate the findings with clinicopathological characteristics as well as clinical responses of the patients to gefitinib therapy. B. Problem being addressed and Key Issues: • Non-small cell lung cancer (NSCLC) is a common cancer in Hong Kong for both men and women. While >90% of lung cancers in men are related to tobacco, <30% of those in women are associated with cigarette smoking, but the underlying carcinogenic mechanism is still unclear. • Patients with NSCLC show poor overall survival due to late presentation defying complete surgical resection. Responses to current protocols of chemotherapy also demonstrate limited success. • Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a crucial role in cell proliferation, survival and differentiation. In recent years, various forms of therapeutic agents targeting EGFR have been developed, including monoclonal antibodies or inhibitors of tyrosine kinase (TKI). Gefitinib (Iressa) is an orally administrated TKI that has been tried for NSCLC treatment. • It has recently been observed that patients with NSCLC containing EGFR mutations show better clinical responses to gefitinib, particularly women who have never smoked, or whose lung cancers show bronchioloalveolar features. However, a statistically significant benefit in survival has not been consistently demonstrated in published clinical series. The reported findings cannot be extrapolated to apply to our population, as the profile of mutation may be different and the response correlation may be population-dependent. It is thus important to evaluate the pattern of EGFR mutations and response to gefitinib in our patients with NSCLC.

Project Title:	Genetic Analysis of Candidate Cancer Genes in Non-small Cell Lung Cancer in Hong Kong
Investigator(s):	Wong MP, Chung LP, Leung LH
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	12/2007

Abstract:

Purpose 1. To analyze the spectrum and patterns of genomic alterations of selective candidate cancer genes in non-small cell lung cancer. 2. To compare and correlate the mutation profiles with clinical-pathological parameters of the tumours including smoking history, gender, age, tumour size, TNM stages and patient outcome. Background Lung cancer is the leading cancer in incidence and mortality worldwide, with chronic tobacco smoking as the major etiology. In Hong Kong, the incidence rate in male patients is high and averages around 62.3/100,000, with more than 90% of chronic smokers. For women, the incidence is around 34.5/100,000 but the majority involves never smokers. This figure falls in the same range as those in many developed countries but in sharp contrast, the latter also involves mainly smokers. The etiology is unclear but suspected mechanisms in non-smokers may include exposure to environmental tobacco smoke or other carcinogenic metabolites of inhaled or ingested materials such as incompletely combusted animal fat, etc. Carcinogenesis in the lung is believed to result from the accumulation of multiple genetic mutations which lead to enhanced tumour cell ability to proliferate, invade, metastasize and escape from programmed cell death and other regulatory processes. Our previous studies of non-small cell lung cancer (NSCLC) of the local population show frequent genomic alterations with DNA gains of 16p (59%), 20q (44%), 5p (34%), 7p (41%), 8q (31%), 17q (34%), and 19q (34%). High-level DNA amplifications were detected at 1q, 7p, 12q, 19q, and 20q. DNA under-representation was observed less commonly and included 8p (28%), 9p (22%), 13q (28%), and 18q (38%) (1). Microsatellite analysis showed frequent allelic imbalances throughout the genome but statistically significant patterns of allelic imbalances between smokers and non-smokers were not apparent (2). On the gene level, common involvement of p53 mutations was also observed (3), but the EGFR and RAS showed distinctive mutation profiles in tobacco and non-tobacco related lung cancers. Chronic smokers showed more K-ras (19.0%) but infrequent EGFR (16%) gene mutations. For non-smokers, there are distinctly frequent EGFR (71%) mutations with significant prevalence over smokers, and less RAS (3.6%) mutations (4). The overall alteration profile in smokers does not significantly differ from those reported on tobacco-associated lung cancers in the literature. On the other hand, the patterns of genetic alterations could reflect differences in the carcinogenic processes, where in smokers, the wide-spread mutagenic effects of tobacco carcinogens select for multiple different loci in the genome that confer growth advantages, while in non-smokers, there is a predominant selection of the EGFR pathway which is known to be important for lung morphogenesis and maturation during embryonic development. Specific tyrosine kinase inhibitor (TKI) therapy targeting EGFR has been found to produce tumour response with better tolerability and reduced toxicity compared to conventional chemotherapy. Response to TKI is associated with EGFR activation mutations, non-smokers and adenocarcinomas. In the West, TKI is effective in only around 10% of patients due to the predominance of tobacco-induced lung cancers. Together with its high cost, TKI treatment initiation is justified only if an EGFR mutation is found, illustrating the concept of ‘personalized medicine’ where a treatment approach is based on individual cancer characteristics. In Hong Kong, official data on TKI response is limited because the drugs are self-funded and mostly used in non-institutional private patients, but our limited study shows >90% response rate in patients with EGFR mutations. The Issues and Problems Most lung cancer patients present at a late stage and curative surgery cannot be rendered. Chemotherapy is the chief treatment but the currently used agents effect in short term remission but produce severe toxic side-effects. Molecular targeted therapy offers a potential for improving on these limitations. With continuous pharmacologic advances, an increasing number of therapeutic agents targeting EGFR and/or other cancer genes or pathways such as src kinase, VEGF, etc, have been trial-tested against lung cancer. A better understanding and prediction of their response profiles would depend on detailed analyses of the changes in candidate gene targets and biologic characteristics of the lung cancers in different cancer subgroups and ethnic populations.

Project Title:	ALK fusion genes in non-small cell lung cancer
Investigator(s):	Wong MP, Leung LH
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	11/2008

Abstract:

Purpose To investigate the frequency, structure and functional role of fusion genes involving ALK in non-small cell lung cancer Key issues • Lung cancer is common with a high mortality worldwide. For all patients, the tumours are usually detected at a late stage and curative surgery is not feasible. Chemotherapy is the chief treatment but the currently used agents result in short term remission and severe toxic side-effects. Hence, the overall mortality is high and survival has not improved significantly in the past decades. • Molecular targeted therapy offers an alternative treatment and some agents, such as the tyrosine kinase inhibitor (TKI) gefitinib has been shown to result in tumour reduction and improved patient survival in lung cancers, mainly in Chinese and Asians, that harbour various EGFR activating mutations. • In order to better understand the molecular carcinogenic pathways of lung cancer development and to identify new targets for drug development, it is essential to identify all possible gene mutations or alterations in lung cancers that develop in different patient groups, especially the smokers and non-smokers. Problems being addressed • Receptor tyrosine kinases (RTK) are involved in many signalling pathways that lead to cancer cell proliferation, resistance to apoptosis, cell survival and other cancer-related cell processes. • Anaplastic lymphoma kinase (ALK) is a RTK known to mediate the anaplastic large cell lymphomas by its activation through chromosomal translocation resulting in the fusion of ALK to another constitutively expressed molecule at its 5’ end. • Although chromosomal rearrangement is a recognized oncogenic mechanism for leukaemias and sarcomas, it is not usually recognized as an important factor for epithelial cancers except prostatic adenocarcinoma. • A new cancer candidate, EML4-ALK fusion gene, has recently been identified from functional screening of cDNA library established from lung cancer of a smoker. It results from a small chromosomal inversion in 2p where a segment of the echinoderm microtubule-associated protein-like 4 (EML4) gene is fused to the ALK gene. The fusion gene has been detected in 6.7% of clinical tumours initially. Functional study in cell systems has shown that the fusion gene transforms NIH3T3. • Our screening for the EML4-ALK fusion transcript in 266 clinical lung cancers of the local population shows that it is present in 13/266 (4.9%) of cases. Eleven tumours are adenocarcinomas and 2 of the 13 tumours are mucoepidermoid carcinomas. The fusion gene is found in mutual exclusion to RAS and EGFR mutations. Overall, there is statistically significant association with non-smokers, suggesting that besides EGFR, this could be one of the molecular pathways that mediate lung carcinogenesis in non-tobacco related cases, particularly those that develop in airways proximal to the terminal bronchiolar-alveolar units. • ALK can be activated by fusion with multiple partners as demonstrated in anaplastic large cell lymphomas. It is postulated that the same phenomenon could occur in lung cancers. Thus, besides EML4, it is important to identify, and study the frequencies and profiles of other fusion partners to ALK in lung cancers. The cellular effects of the fusion genes can also be studied by transfection of the fusion gene into cancer cell models. • Since the EGFR pathway is the predominant oncogenic pathway in non-tobacco related lung cancers, signalling of the ALK fusion genes including EML4-ALK might converge with the downstream effectors of EGFR. It is thus worthwhile to investigate the interaction between EML4-ALK and other ALK fusion genes with the EGFR mutant pathway.

Project Title:	Cancer genes alterations and their biological effects in lung cancer
Investigator(s):	Wong MP, Leung LH
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	12/2008

Abstract:

To analyze the spectrum and patterns of genomic alterations of selective candidate cancer genes in non-small cell lung cancer; To compare and correlate the mutation profiles with clinical-pathological parameters of the tumours including smoking history, gender, age, tumour size, pathological stages and patient outcome; To study the cellular effects of the mutant cancer genes and their interaction with the EGFR pathway in lung cancer cell lines.

Project Title:	Generation of a versatile and inducible EGFRL858R mouse lung cancer model and its application for studying gene collaboration in lung carcinogenesis
Investigator(s):	Wong MP, Leung LH, Cai K
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	04/2010

Abstract:

Purpose of Study: To generate an inducible EGFRL858R mouse lung cancer model using a novel and versatile tissue-specific lentiviral gene delivery system for studying of multiple gene collaboration in lung carcinogenesis. Key Issues and Problems Lung cancer is common worldwide and mostly caused by tobacco carcinogenesis. The incidence is also high in non-smoking women in East Asia and Hong Kong due to frequent activating EGFR mutations such as the EGFRL858R missense or exon 19 deletional mutations (1). Many lung cancers present late and treatment is palliative with chemotherapy and radiotherapy. The prognosis is poor with overall 5 year survival of around 15%. Little improvement in cancer cure has been achieved over decades even with molecular targeted therapy using anti-EGFR tyrosine kinase inhibitors (TKI) because of frequent relapses after initial tumor reduction. New approaches of lung cancer treatment are needed. Cancers result from cumulative changes in the genome with aberrant signaling along genetic pathways that control cellular growth, death, differentiation, invasion and metastasis. Complex profiles of genetic, epigenetic and expression abnormalities have been identified in clinical human lung cancers using high through-put, genome-wide studies (1-5). “Signatures” of these aberrations have also been found to be associated with certain clinicopathological parameters using biostatistical or analytical approaches. However, the biological role and pathophysiological mechanism of the changes cannot be predicted from these global analyses. Investigation of individual genes or genetic pathways in experimental systems is necessary before the information can be translated into therapeutic applications. Recently, Mapk14/p38has been shown to antagonize the EGFR pathway in normal lung homeostasis. Deletion of p38 in adult mice resulted in increased proliferation and defective differentiation of isolated normal lung stem and progenitor cells (6). It inhibits EGFR proliferative signaling, induces differentiation and apoptosis of stem cells (6-8). p38has been suggested as a tumor suppressor candidate, but whether it takes part in lung tumorigenesis is not known. In particular, the recently advocated cancer stem cell theory proposes that cancers are maintained by a subpopulation of cancer stem cells which can self-renew and give rise to progenies that differentiate but lose the capacity of continuous cell division. Studies have shown that analogous subpopulations of tumorigenic cancer stem cells are present in human lung cancers. Our preliminary data on lung cancer cell lines also show that p38 expression level is lower in the stem cell than non-stem cell subpopulations, suggesting that inactivation of p38 may be involved in lung cancer development through its role as a regulator of lung cancer stem cells. To study this hypothesis, and to investigate the interaction of p38with mutant EGFR in the lung cancer stem cell compartment, it is necessary to generate a suitable mouse model that allows genetic manipulation. The transgenic mouse model of human lung cancer driven by a genetically engineered gene of interest offers an important approach of studying lung cancer biology. For example, using conditional bitransgenic technologies and coupling with the alveolar pneumocyte-specific surfactant protein C (SPC) promoter, the FGF7 and KRasG12D mouse models are able to achieve expression of the transgenes in lung epithelial cells, resulting in development of epithelial cell hyperplasia, adenomatous hyperplasia and lung cancer formation in the adult mice. However, development of fully malignant lung cancers with metastatic potentials could only be achieved by the crossing of two separate transgenic mice to achieve conditional activation of Kras2 along with conditional cre/loxP inactivation of Rb or p53 in the compound conditional model (9). Reported models of EGFRL858R transgenic mice were generated by direct cloning of the mutant to the SPC promoter (10), or crossing of Tet-operator (Tet-O-EGFRL858R) (11-12) mice with CCSP-rtTA mice to achieve lung-specific expression of Tet-O EGFRL858R. Tumor formation was found in both models, suggesting that EGFRL858R mutation is potent enough for tumor-initiation and maintenance in mouse. The Varmus laboratory has recently cloned the receptor of the avian leucosis-sarcoma virus (ALSV-A), designated as TVA (13-14). Coupling of TVA with tissue-specific promoters has been utilized to develop a range of transgenic mice with tissue-specific TVA expression (15-23). Subsequently, an HIV-1-based lentiviral vector pseudotyped with the envelope glycoprotein of ALSV-A, named as HIV(ALSV-A), was generated. This vector is useful for efficient delivery of transgenes to mammalian cells that express the TVA and can be used for generation of transgenic mouse models with various advantages. Lentiviral infection is useful for gene delivery to slowly-dividing cells and can accommodate large insert size. Multiple vectors carrying different transgenes including loss- and gain-of-function genes can be conveniently introduced to the same tissue-specific TVA mouse for studying genetic collaboration, which otherwise needs to be produced by crossing different transgenic mice (21, 24-26). Potentially, the number of tumors that develop can be limited by adjusting the viral dosage, thus achieving a more physiologically relevant model for preclinical drug testing. Currently, this system has been used for transgene delivery in cell culture studies (27) but in vivo use has only been reported in a recent breast cancer model (26). Because of the prevalence of EGFR mutations in lung cancers in our population and the almost universal development of drug resistance after TKI treatment, it is important to develop a transgenic mouse model of lung cancer driven by an activating EGFR mutation for studying various aspects of this lung cancer, such as the role of other genetic or biological factors in the maintenance of the cancer, the underlying mechanism of drug resistance, and for identifying new therapeutic targets for drug development. In this study, we aim to generate an EGFRL858R transgenic mouse model of lung cancer by using the HIV(ALSV-A) system for gene delivery to a SPC-TVA transgenic mouse with site-specific expression of TVA in the lung epithelial cells. Specific objective: 1. To generate an inducible EGFRL858R mouse model of lung cancer using the SPC-TVA transgenic mouse with lung-specific expression of the TVA viral receptor and the ALSV-A-pseudotyped lentiviral gene delivery system. 2. To study the role of Mapk14/p38in the formation and maintenance of lung cancers through cancer stem cell regulation using the EGFRL858R mouse model.

List of Research Outputs

Lam C.L.D., Girard L., Sihoe A., Cheng L.C., Lui M.M.S., Wong M.P., Chung L.P., Ip M.S.M., Lam W.K. and Minna J.D., Gene expression profiling in lung adenocarcinomas reflects possible different molecular pathogenesis with respect to gender and smoking status, 14th Congress of the Asian Pacific Society of Respirology & 3rd Joint Congress of the Asian Pacific Society of Respirology/American College of Chest Physicians, Seoul, Korea, Nov 2009, Respirology. 2009, 14(Suppl 3): A128 (OS 03-05).

Lam C.L.D., Girard L., Sihoe A.D.L., Cheng L.C., Lui M.A.C.Y., Wong M.P., Chung L.P., Ip M.S.M., Lam W.K. and Minna J.D., Gene expression signatures associated with combination of female non-smokers in lung adenocarcinomas bearing activating epidermal growth factor receptor (EGFR) gene mutations in Chinese, The American Thoracic Society Annual Meeting 2010.

Leung L.H., Ma Y., Fiscus R.R., Tung J.W., Tin P.C. and Wong M.P., Isolation, expansion and characterization of CD44 positive cells as tumor-initiating/stem/progenitor cells in non-small cell lung cancer, American Association for Cancer Research Annual Meeting, 2010 Washington DC the United State . 2010.

Tam I.Y., Leung L.H., Tin P.C., Chua D.T.T., Sihoe A.D.L., Cheng L.C., Chung L.P. and Wong M.P., Double EGFR mutants containing rare EGFR mutant types show reduced in vitro response to gefitinib compared to common activating missense mutations, Molecular Cancer Therapeutics. 2009, 8(8): 2142-51.

Wong M.P., EGFR – Mutations & Detection, In: AstraZeneca, New Evidence, New Practices - The Role Of Respiratory Specialists In A New First-line Treatment Paradigm For Advanced Nsclc Patients. 2010.

Wong M.P., Mutations of EGFR and other Oncogenes in Non-small cell lung cancer , Joint Scientific Exchange Session, University of Hong Kong and National Health Research Institute of Taiwan. 2009.

Researcher : Wong SY

List of Research Outputs

Cheung Y.N., Wong S.Y. and Khoo U.S., eIF4E in human breast cancer and its role in regulating translation of splice variants of breast cancer genes, The 101th Annual Meeting of the American Association for Cancer Research, Washington DC, U.S.A.. 2010.

Researcher : Xu MS

List of Research Outputs

Ching C.Y.J., Chan Y.K., Lee H.L.E., Xu M.S., Ting K.P., So T.M., Sham P.C., Leung G.M., Peiris J.S.M. and Khoo U.S., Significance of the myxovirus resistance A (MxA) gene -123C>a single-nucleotide polymorphism in suppressed interferon beta induction of severe acute respiratory syndrome coronavirus infection, J Infect Dis. 2010, 201(12): 1899-908.

Researcher : Yam JWP

Project Title:	Characterization of tensin2, the binding partner of DLC1 tumor suppressor in liver cancer
Investigator(s):	Yam JWP, Ng IOL, Jin D
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2007
Completion Date:	12/2009

Abstract:

To verify the DLC1-tensin2 interaction; to determine the impact of DLC1-tensin2 interaction on their biochemical properties; to delineate the biological functions of tensin2 and its implications in HCC; to evaluate the clinicopathological significance of tensin2 in human HCC.

Project Title:	Nucleocytoplasmic shuttling mechanism of DLC1 tumor suppressor in liver cancer
Investigator(s):	Yam JWP, Ng IOL
Department:	Pathology
Source(s) of Funding:	General Research Fund (GRF)
Start Date:	01/2008

Abstract:

To identify structural elements directing nucleocytoplasmic shuttling; to elucidate the nucleocytoplasmic shuttling mechanism of DLC; to delineate the distinctive functional roles of DLC1 in both cytoplasm and nucleus; to evaluate the clinical significance of the cytoplasmic and nuclear localization of DLC1 in liver cancer. Revised Objectives/Work Programme (Each objective text box is provided for maximum 150 words) (Please list in point form where appropriate) 1 Live fluorescence imaging of nuclear transport of DLC1 proposed for achieving objective #1 will be eliminated. It is because using immunofluorescent staining and cellular fractionation of DLC1 transfected cells or cells with endogenous DLC1 after Leptomycin B treatment can already clearly demonstrate the nuclear transport of DLC1 within the cell.

Project Title:	Functional implications of Akt phosphorylation of DLC1 tumor suppressor in liver cancer
Investigator(s):	Yam JWP, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2008
Completion Date:	05/2010

Abstract:

Purpose of the proposed investigation Evidence from our previous work has shown that DLC1 (Deleted in Liver Cancer 1) is a tumor suppressor gene critically involved in hepatocarcinogenesis. Functionally, DLC1 has been clearly demonstrated to be a suppressor in tumor growth and metastasis. However, the upstream modulators and regulatory mechanism of DLC1 has never been addressed. A recent study demonstrated that rat homolog of DLC1, p122RhoGAP was phosphorylated by Akt. This prompted us to question whether Akt also phosphorylates human DLC1. In this proposal, we aim to characterize the nature and functional implications of Akt phosphorylation of DLC1 in liver cancer. Key issues and problems being addressed Hepatocellular carcinoma (HCC) is a major malignancy worldwide and the second commonest fatal cancer in Asia and Hong Kong. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. DLC1, a RhoGAP (Rho GTPase activating protein) was identified as a tumor suppressor in HCC. Rho proteins function as important regulators in remodeling of actin cytoskeleton, regulation of transcription, cell proliferation, tumorigenesis and metastasis. RhoGAPs serve tumor suppressor function by down-regulating Rho proteins. Thus, the RhoGAP activity of DLC1 has been implicated to contribute to the role of DLC1 in tumor suppression. DLC1 is widely expressed in normal human tissues, but it is frequently underexpressed in HCC and other cancers. Our previous study showed that chromosome 8p where the locus of DLC1 is located was frequently deleted in human HCC (1). We also detected homozygous deletion and underexpression of DLC1 in HCC samples and cell lines (2). Hypermethylation of DLC1 CpG island accounted for the transcriptional silencing of DLC1 gene expression (3). More importantly, we are the first group to demonstrate the growth inhibitory effect of DLC1 on HCC (2). We also demonstrated inhibition of cell migration and invasion by ectopic expression of DLC1 in HCC cell lines (4). Inhibition of GTPase activity of RhoA and Cdc42 in vitro by DLC1 was first demonstrated by us (3). Recently, we have shown that the inhibition of stress fiber formation by the RhoGAP activity of DLC1 was associated with its growth suppressive effect on HCC cell lines (4). Recently, tensin, a focal adhesion family was first identified by us as the novel interacting partner of DLC1 (5) and later confirmed to be also of different DLC family members by other independent research groups. Ectopic expression of DLC1 and tensin colocalized with vinculin at focal adhesions. These independent findings suggest that tensin as the interacting partner of DLC1 is responsible for directing the focal adhesion localization of DLC1. These studies also support the notion that proper focal adhesion localization of DLC1 is required for its tumor suppressive activity. Recent report has shown that the rat homolog of DLC1, rat p122RhoGAP was a novel substrate of Akt (6). This prompted us to question whether Akt also phosphorylates human DLC1. Akt phosphorylated p122RhoGAP on residue Ser-322. Sequence analysis of DLC1 revealed sequence homology of characteristic Akt substrate motif, (R/K)X(R/K)XX(S*/T*) (R: arginine; K: lysine; S* and T*: phosphorylated serine and threonine; X: any amino acid) between rat and human DLC1. The three potential Akt phosphorylated residues (Ser-298, Ser-329 and Ser-567) reside in the central region of DLC1. Akt has been implicated in enhancing cell growth, proliferation and survival. Aberrant expression and activation of Akt have been evident in tumorigenesis of various cancers. The importance of DLC1 and Akt in human cancers prompts us to further investigate the molecular basis by which Akt regulates DLC1. Here, we hypothesize that Akt is a novel regulator of DLC1. Mechanistically, Akt interacts and phosphorylates DLC1, and in consequence regulates the activity of DLC1 by Akt-induced phosphorylation. Our preliminary investigation showed that antibody specifically against phospho-Akt substrate (PAS) could detect phosphorylation signal of ectopically expressed and endogenous DLC1. PAS antibody (Cell Signaling) against the consensus Akt substrate motif has been used for the identification of Akt substrates by various research groups. Our propose work on this novel mechanism underlying the regulation of DLC1 by Akt phosphorylation which potentially has great impacts on the functions of DLC1 warrants further investigation. In this study, we propose to characterize the nature and functional implications of Akt phosphorylation of DLC1 in liver cancer by the following objectives: 1) to identify specific region or critical residue(s) of DLC1 phosphorylated by Akt, 2) to elucidate the binding activity between DLC1 and Akt, and 3) to delineate the functional significance of phosphorylated DLC1

Project Title:	Development of diagnostic use of a novel phospho-antibody in liver malignancy
Investigator(s):	Yam JWP, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Applied Research
Start Date:	01/2009
Completion Date:	12/2009

Abstract:

Key issues and problems being addressed: Hepatocellular carcinoma (HCC) is a major malignancy worldwide and the second commonest fatal cancer in Asia and Hong Kong. However, the molecular mechanisms underlying the development and progression of HCC are poorly understood. The high recurrence rate of HCC due to metastasis has led to poor prognosis of HCC. Better understanding of the molecular players underlying the development of HCC may lead to development of more effective prognostic markers and potential therapies for HCC. Deleted in liver cancer 1 (DLC1), a RhoGAP (Rho GTPase activating protein) was identified as a putative tumor suppressor in HCC in 1998 (1). Rho proteins function as important regulators in remodeling of actin cytoskeleton, regulation of transcription, cell proliferation, tumorigenesis and metastasis. RhoGAPs serve tumor suppressor function by down-regulating Rho proteins. Recent study has provided in vivo evidence that DLC1 is a negative regulator of Rho (2, 3). Since the identification of Deleted in liver cancer 1 (DLC1) in HCC in 1998, accumulating studies have been well characterized DLC1 as a bona fide tumor suppressor not only in HCC, but also in various types of human cancers. The growth inhibitory effect of DLC1 was first demonstrated in HCC (4). Ectopic expression of DLC1 in HCC cell lines also demonstrated inhibition of cell migration and invasion (5). The inhibition of stress fiber formation by the RhoGAP activity of DLC1 was associated with its growth suppressive effect on HCC cell lines (5). Recently, tensin, a focal adhesion family was first identified by us as the novel interacting partner of DLC1 (6) and later confirmed to be also of different DLC family members by other independent research groups. Ectopic expression of DLC1 and tensin colocalized with vinculin at focal adhesions. These independent findings suggest that tensin as the interacting partner of DLC1 is responsible for directing the focal adhesion localization of DLC1. These studies also support the notion that proper focal adhesion localization of DLC1 is required for its tumor suppressive activity. DLC1 is widely expressed in normal human tissues, but it is frequently underexpressed in HCC and other cancers. Heterozygous deletion of DLC1 was commonly found in about 50% of liver, breast, lung and colon cancers (2). The other allele of DLC1 has been found to be epigenetically silenced (7). Apart from down regulation of DLC1 expression either by genomic deletion or epigenetic silencing, our recent finding suggested that enhanced phosphorylation level of DLC1 could also be an indicator for tumor progression. Identification of the rat homolog of DLC1, rat p122RhoGAP as a novel substrate of Akt (8) prompted us to question whether Akt also phosphorylates human DLC1. Sequence analysis of DLC1 revealed the presence of three characteristic Akt substrate motif, (R/K)X(R/K)XX(S*/T*) (R: arginine; K: lysine; S* and T*: phosphorylated serine and threonine; X: any amino acid) in human DLC1. The three potential Akt phosphorylated residues (Ser-298, Ser-329 and Ser-567) reside in the central region of DLC1. Our investigation showed that an antibody specifically against phospho-Akt substrate (PAS) in general could detect phosphorylation signal of ectopically expressed and endogenous DLC1. PAS antibody (Cell Signaling Technology) against the consensus Akt substrate motif has been used for the identification of Akt substrates by various research groups. Site-directed mutagenesis further confirmed that Akt phosphorylates DLC1 only at Ser-567. Functional analysis using colony formation assay demonstrated that the phosphomimetic mutant of DLC1 (in which Ser-567 is converted to aspartic acid) lost its tumor suppressive activity to inhibit growth of HCC cells. Based on our findings, phosphorylation could be another mechanism to functionally deregulate DLC1 at the post-translational level. Therefore, in this study, we propose the phosphorylated form of DLC1 may be used as a novel diagnostic marker for tumor development. Purpose of the proposed project: Oncogenic Akt pathway has been implicated in enhancing cell growth, proliferation and survival. Aberrant expression and activation of Akt have been evident in tumorigenesis of various cancers. Our findings suggested that Akt is a novel regulator of DLC1 and DLC1 is hyper-phosphorylated when Akt is activated. Without a specific phospho-DLC1 (pDLC1) antibody against phosphorylated DLC1 at Ser-567, the endogenous DLC1 cannot be analyzed. Cell Signaling Technology and our research team will work on a collaborative basis to generate and validate the use of the specific pDLC1-S567 antibody. The phospho-antibody will be tested for its specificity and sensitivity to detect the endogenous phosphorylated from of DLC1 both in HCC cell lines and in paired human HCC tumor tissues. The multiple involvements of DLC1 in various cancer types will broaden the potential medical applications of the pDLC1 antibody to other human cancers. The analysis using the phospho-antibody will be extended and performed on other types of cancer tissue samples. In this proposal, we aim to establish the use of pDLC1 antibody not only for basic research applications, but also for future use in clinical diagnosis and prognosis of cancer development. Research objectives: 1) To assess the sensitivity and specificity of pDLC1 antibody. 2) To detect endogenous phosphorylated DLC1 in tumor tissues. 3) To determine the clinicopathological significance of phosphorylated DLC1. References 1. Yuen BZ, et al. Cancer Res 1998; 58:2196-2199. 2. Xue W, et al. Genes and Dev 2008; 22:1439-1444. 3. Lahoz A and Hall A. Genes and Dev 2008; 22:1724-1730. 4. Ng IOL, et al. Cancer Res 2000; 60:6581-6584. 5. Wong CM*, Yam JWP*, et al. Cancer Res 2005; 65:8861-8868. *Both contributed equally. 6. Yam JWP, et al. Cancer Res 2006; 66:8367-8372. 7. Wong CM, et al. Cancer Res 2003; 63:7646-7651. 8. Hers I, et al. J Biol Chem 2006; 281:4762-4770.

Project Title:	Significance and transcriptional upregulation of caveolin-1 by PPARγ in liver cancer metastasis
Investigator(s):	Yam JWP, Ng IOL
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2009

Abstract:

Purpose of the proposed investigation Dynamic changes of proteins in the focal adhesions are involved during cellular movement. Dysregulation of focal adhesion proteins very often contributes to the acquired metastatic behavior of cancer cells. Caveolin-1 (Cav1) was named because of its identification as an essential structural component and functional implications in caveolae. Apart from caveolae, localization of Cav1 at focal adhesions is involved in focal adhesion dynamics and cell migration. Emerging evidence has shown a positive correlation of Cav1 expression with metastatic potential. Our previous study of expression analysis and functional studies strongly supported the role of Cav1 in HCC metastasis. In this study, we are particularly interested to elucidate the mechanism that leads to the upregulation of Cav1 in metastatic cells. Our proposed work will derive novel mechanistic insights into the regulation of Cav1 in cancer metastasis. Key issues and problems being addressed Caveolae are flask-shaped invaginations in the plasma membrane and have been implicated in cellular transport processes and signal transductions (1). The major structural proteins of caveolae are caveolins: Cav1, Cav2 and Cav3. Cav1 and Cav2 are abundant in diverse cell types, whereas Cav3 is muscle-specific (2). Caveolar Cav1 serves as a scaffold allowing key regulators of cell migration to associate with caveolae and interact in the intracellular compartment (3, 4). Apart from localizing in caveolae, Cav1 is also found in the nucleus, cytoplasm, focal adhesions and the extracellular milieu. These non-caveolar Cav1 are involved in cell signaling, interactions with intracellular cytoskeleton complex, which in turn regulate cell adhesion and locomotion. Cav1 was first isolated as a tyrosine phosphorylated substrate of v-Src (5). Cav1 was downregulated in oncogenically transformed cells (6) and its diminution in NIH3T3 cells was sufficient to induce a transformed phenotype (7), thus it was plausible to regard Cav1 as a tumor suppressor gene. However, increasing evidence in recent years demonstrated that Cav1 was suppressed at the early stages of transformation and carcinogenesis but upregulated in advanced-stage metastasis (8). The association of Cav1 overexpression with cancer progression, metastasis and poor prognosis was shown in various carcinomas including esophageal squamous cell carcinoma (9, 10), renal cell carcinoma (11) and gastric cancer (8). A study showed that Cav1 was not detected in non-neoplastic liver while 26% of the HCC cases showed an increased expression (12). In mouse hepatocarcinoma cell lines, strong Cav1 expression was found in those with higher invasive ability (13). PPARγ (Peroxisome proliferator-activated receptor gamma) has been identified as a transcriptional regulator of Cav1 in vitro and in vivo (14-16). PPARγ belongs to the family of ligand-activated transcription factors PPARs. PPARs consist of PPARγ, PPARα and PPARβ/δ, they heterodimerize with the 9-cis-retinoic acid receptor (RXR) and bind to specific PPAR-responsive element (PPRE) of the genes to regulate their transcription. Their target genes are involved in lipid metabolism, inflammation and cell differentiation (17, 18). In HCC cell lines, PPARγ expression has been found to be higher in cells with poor differentiation than those well-differentiated ones (19). In human HCC samples, expression of PPARγ is higher in primary tumor cells and peritumoral hepatocytes than in benign hepatocytes (20). Treatment of PPARγ inhibitors to HCC cells inhibits their proliferation, colony formation, migration and invasion profoundly (19). Furthermore, PPARγ inhibitors also induce the cells to undergo apoptosis or anoikis by preventing the cells from adhering to the extracellular matrix (20, 21). Taken together, PPARγ plays a tumorigenic role in HCC. Interestingly, treatment of PPARγ ligands to HCC cells leads to increased expression of Cav1 (16); such upregulation of Cav1 expression was explained by the discovery of a functional putative PPAR/RXR binding site at the Cav1 promoter (14). In this regard, it is highly tempting to speculate the role of PPARγ-mediated transcriptional activation of Cav1 expression in HCC tumorigenesis. Preliminary study Our unpublished study showed that Cav1 was highly expressed in metastatic cell lines while it could only be detected in a few other non-metastatic cells. Real-time quantitative PCR (qPCR) analysis also revealed that Cav1 was highly expressed in metastatic cells. The Cav1 protein expression level correlates well with the transcript level. Upregulation of Cav1 was also shown to be physiologically relevant. Immunohistochemistry (IHC) revealed that in HCC tissue, Cav1 was not expressed in non-tumorous tissues but strongly expressed at the tumor front where cells tend to have greater potential to acquire aggressive phenotype. In surgical resection cases, Cav1 showed higher expression level in extrahepatic tumor metastasis than in the primary HCC of the same patient. Functionally, we also showed that suppression of Cav1 in HCC cells by lentiviral transduction using shRNA targeting Cav1 strikingly inhibited tumorigenicity in nude mice. Our result strongly supports the notion that Cav1 plays a role in cancer metastasis. As a continual study of Cav1, it is important to unravel the mechanism that leads to the enhanced expression of Cav1 in metastatic cells. Based on the findings that Cav1 was elevated at both transcriptional and translational levels, we further examined the Cav1 promoter activity in HCC cells. Luciferase reporter carrying Cav1 5’region (-737 to -37) which is defined as Cav1 promoter region, is kindly provided by Prof. V. Shah (22). Results of the reporter assay revealed that Cav1 promoter activity correlates with the mRNA level in HCC cells. In addition, Cav1 promoter activity was transactivated by PPARγ in a dose-dependent manner. Collectively, our preliminary findings suggested that PPARγ-mediated transcriptional activation of Cav1 expression in HCC. Objectives In this study, we propose to elucidate the mechanism by which Cav1 is upregulated during HCC metastasis by the following objectives: 1) To elucidate the transcriptional regulation of Cav1 in metastatic cells. Our aim is to examine the effect of PPARγ on the expression and promoter activity of Cav1 and demonstrate the role of PPARγ as the transcription factor of Cav1 promoter in metastatic cells in vivo. 2) To document the expression profiles of Cav1 and PPARγ in HCC. Our aim is to examine Cav1 and PPARγ expressions in a large number of human HCC tissues, evaluate the clinicopathological relevance of Cav1 and PPARγ expressions and determine their correlation in HCC.

Project Title:	Outstanding Young Researcher Award 2008-2009
Investigator(s):	Yam JWP
Department:	Pathology
Source(s) of Funding:	Outstanding Young Researcher Award
Start Date:	12/2009

Abstract:

The Awards are intended to recognize, reward, and promote exceptional research accomplishments of academic and research staff.

Project Title:	20th Conference of the Asian Pacific Association for the Study of the Liver (APASL) Caveolin-1 is overexpressed and promotes human hepatocellular carcinoma tumorigenesis via activation of HGF signaling
Investigator(s):	Yam JWP
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	03/2010
Completion Date:	03/2010

Abstract:

N/A

Project Title:	Role of caveolin-1 in liver cancer metastasis through HGF/Met signaling pathway
Investigator(s):	Yam JWP, Wong AST
Department:	Pathology
Source(s) of Funding:	Seed Funding Programme for Basic Research
Start Date:	06/2010

Abstract:

Purpose of the proposed investigation In this study, we aim to elucidate the molecular mechanism of Caveolin-1 (Cav1)-mediated Met activation in HCC metastasis. Metastasis is a key event at the advanced stage of hepatocarcinogenesis. In light of the emergence of Cav1 as a new master of cancer metastasis in various cancer types and its defined role in HCC metastasis by our previous study, it is important to unveil the molecular mechanisms of Cav1 in HCC. The current understanding about the molecular mechanisms of Cav1 in cancer metastasis is conflicting. Also, the underlying basis of Cav1 in HCC metastasis has never been addressed. In this study, we hypothesize that Cav1 acts as a positive regulator of HCC metastasis through the activation of Met signaling cascades. Functionally, Cav1 overexpression promotes the aggressive behavior of cancer cells. Mechanistically, overexpression of Cav1 activates Met and its downstream signaling cascades leading to the acquisition of metastatic behavior of cells. These hypotheses will be vigorously tested in our study. Key issues and problems being addressed Cav1 expression in human cancers Cav1 was named because of its identification as an essential structural and functional component in caveolae. Non-caveolar Cav1 has been found at focal adhesions and is involved in focal adhesion dynamics and cell migration. Expression of Cav1 has been found to be aberrantly altered in a broad spectrum of human cancers (1). At the molecular level, Cavl is involved in vesicular transport, cholesterol homeostatsis and regulation of signal transduction (1). In recent years, increasing evidence implicates that Cav1 is upregulated in the advanced-stage metastasis (2). Indeed, Cav1 overexpression has been shown to associate with higher tumor stage, metastasis and worse prognosis in various carcinomas (1). However, discrepant findings of Cav1 expression have been reported in HCCs. Cav1 has been shown to be increased in most macroregenerative and dysplastic nodules but only in a few HCCs when compared with the adjacent cirrhotic liver (3). But in recent studies, Cav1 expression has been shown to be overexpressed in 70% of HCCs (4) and its overexpression is significantly correlated with metastasis and worse prognosis (5). In mouse hepatocarcinoma cell lines, Zhou et al. has demonstrated a positive correlation of Cav1 expression level with the metastatic potential of cells (6). Accumulating evidence has pointed to an oncogenic role of Cav1 in hepatocarcinogenesis. Role of Cav1 in cell motility Cell motility is one of the critical components in metastasis. Cav1 expression is essential for cell polarization and migration. Localization of tyrosine phosphorylated Cav1 (pY14Cav1) to focal adhesions has been implicated in cell polarization and migration (7). It is also involved in regulating the Rho/ROCK-dependent focal adhesion dynamics and tumor cell migration and invasion (8). Upregulation of Cav1 by FAK during EMT further demonstrates the importance of Cav1 in cell adhesion and migration (9). Knockdown of Cav1 in Ewing’s sarcoma cells leads to upregulation of Snail and loss of E-cadherin, which represents an increase of cancer cell invasiveness (10). Small GTPases, the key regulators of cytoskeletal rearrangement have been reported to interact with Cav1 (11). As shown in lung adenocarcinoma, increased Cav1 expression enhances the ability of cancer cell to metastasize through the induction of filopodia formation (12). Furthermore, secretory Cav1 of prostate cancer cells functions as proangiogenic factor in metastatic progression (13). On the contrary, Cav1 inhibits spontaneous metastasis of breast cancer (14) and head and neck squamous cell carcinoma (15). Recently, Cav1 has been shown to reduce osteosarcoma metastasis by suppressing c-Src activity and Met signaling pathways (16). These findings implicate that the molecular mechanisms of Cav1 in tumorigenesis are complex and vary in different cellular contexts. Preliminary study Focal adhesions have been documented to play crucial role in HCC (17). In light of the importance of Cav1 as a key player in focal adhesion complex and a new master of cancer metastasis, we prompt to study the functional role and underlying mechanisms of Cav1 in hepatocarcinogenesis. We first examined its expression in HCC cell lines with different tumorigenic and metastatic potentials. Western blot and quantitative PCR (qPCR) analyses showed that Cav1 was highly expressed in metastatic cell lines while it could only be detected in a few non-metastatic cells and was not detected in an immortalized normal liver cell line. Analysis of Cav1 expression in tissue microarray of HCC containing 106 cases of paired non-tumorous and corresponding tumorous tissues using immunohistochemistry (IHC) revealed that Cav1 was upregulated in 37% of HCCs. Cav1 overexpression was significantly correlated with venous invasion (P=0.036). We extended our analysis to 31 sets of clinical samples with each comprising non-tumorous liver tissues, primary HCCs and distant metastatic tissues. Our data showed that Cav1 was upregulated in 26% of primary HCCs and 35% of extrahepatic metastases. Although in vitro functional characterization of Cav1 in HCC has been briefly reported (4, 5), demonstration using in vivo model is deficient. Our functional studies revealed that knockdown of Cav1 expression in HCC cells dramatically reduced anchorage independent growth and in vivo tumorigenicity. On the other hand, stable clones of overexpressing Myc-tagged Cav1 established with non-metastatic PLC cells showed higher proliferation rate and enhanced motility and invasiveness. The in vivo tumorigenicity of PLC/Cav1 cells was greatly enhanced when compared with the vector control clone. In PLC/Cav1 cells, we found that Met expression was elevated. In accordance with this observation, Western blot analysis showed that Met was also particularly expressed in metastatic HCC cells and its expression correlated with that of Cav1. Met is the receptor of HGF, treatment of cells with HGF significantly induced the number of scattered colonies in PLC/Cav1 cells but not the control cells. To explore the role of Cav1 in a relevant metastatic HCC model, we suppressed Cav1 expression in metastatic MHCC97H cells by lentiviral transduction. In metastatic MHCC97H cells, diminution of Cav1 significantly inhibited migratory and invasive activities of cells upon HGF stimulation. Taken together, these findings prompt us to speculate that Cav1 contributes to the execution of HGF signaling by modulating Met expression or activity. Objectives 1) To delineate the molecular interaction of Cav1 and Met signaling pathways in HCC. 2) To characterize the functional effect of Cav1-mediated Met activation in HCC metastasis.

List of Research Outputs

Chan J., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Integrin-linked kinase promotes hepatocellular carcinoma oncogenesis, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Chan L.K., Ko F.C.F., Ng I.O.L. and Yam J.W.P., Nuclear targeted Deleted in liver cancer 1 exhibited reduced tumor suppressive function both in vitro and in vivo , American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Chan L.K., Tung K.K., Ng I.O.L. and Yam J.W.P., Deleted in Liver Cancer 1 (DLC1) is a negative regulator of metastasis and deregulated by kinase phosphorylation in hepatocellular carcinoma, American Association for Cancer Research 101st Annual Meeting, Washington DC, USA. 2010.

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Leung T.H.Y., Yam J.W.P., Chan L.K., Ching Y.P. and Ng I.O.L., DLC2 (Deleted in liver cancer 2) suppresses cell growth via regulation of Raf-1-ERK1/2-p70S6K signaling pathway. (accepted), Liver International. 2010.

Tang K.S., Zhou H., Yam J.W.P. and Wong A.S.T., c-Met overexpression contributes to the acquired apoptosis resistance of non-adherent ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2, 101th Annual Meeting of the American Association for Cancer Research, Washington, DC, (Abstract No. 1722). 2010.

Tang K.S., Zhou H., Yam J.W.P. and Wong A.S.T., c-Met overexpression contributes to the acquired apoptotic resistance of ovarian cancer cells through a cross-talk mediated by phosphatidylinositol 3-kinase and extracellular-signal regulated kinase 1/2, Neoplasia. 2010, 12: 128-138.

Tse Y.T., Ko F.C.F., Tung K.K., Chan L.K., Lee K.W., Wong A.S.T., Ng I.O.L. and Yam J.W.P., Caveolin-1 promotes hepatocellular carcinoma tumourigenesis, migration and invasion via Met-ERK1/2 pathway, Days of Molecular Medicine 2010 Systems Biology Approaches to Cancer and Metabolic Disease, Stockholm, Sweden. 2010.

Yam J.W.P., Wong C.M. and Ng I.O.L., Molecular and functional genetics of hepatocellular carcinoma, Frontiers in bioscience (Scholar edition). 2010, 2: 117-134.

Yam J.W.P., Molecular pathology of hepatocellular carcinoma: insights of caveolin-1 in cancer metastasis, 16th Hong Kong International Cancer Congress. 2009.

Yam J.W.P., Outstanding Young Researcher Award 2008-2009, The University of Hong Kong. 2010.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Yan HHN

Project Title:	Characterization of claudin-1 and its interacting partners in gastric cancer
Investigator(s):	Yan HHN, Leung SY
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	01/2009

Abstract:

Epithelial-mesenchymal transition (EMT) converts epithelial cells into mesenchymal migratory cells, which also plays a key role in tumor progression (1, 2). During tumor metastasis, EMT begins with a loss of cell adhesion and apical-basal polarity in carcinoma cells, to be followed by a shift in cytoskeletal dynamics, acquiring the mesenchymal phenotype. This can facilitate the migration of malignant cells to specific destinations. Disassembly of tight junctions (TJs) and adherens junctions (AJs) is believed to be a pre-requisite for tumor cells to gain motility, mobility and invasiveness. Whilst alterations in AJs, such as E-cadherin and β-catenin, are well-known to play critical roles in EMT, less is known about the role of TJ alteration in this process. In normal epithelia, TJ forms a selective permeability barrier between epithelial cells. TJ regulates the passage of ions and solutes across paracellular space and functions as a fence, which maintains cell polarity by preventing the diffusion of lipids and proteins between the apical and basolateral regions of the cell epithelium (3, 4). Three classes of transmembrane proteins are currently identified in TJ: occludins, claudins, and junctional adhesion molecules (JAMs). They are linked to actin cytoskeleton for scaffolding via the binding of cytoplasmic proteins, such as zonula occludens (ZO) 1, 2 and/or 3 at the C-terminus. Thus, the TJ barrier has to be compromised for a metastatic tumor cell to: (i) ‘escape’ from its primary site, and (ii) ‘transplant’ to the new site during tumorigenesis via changes in the integral membrane proteins at the site. If the biology of these events is understood, it is conceivable that better therapeutic approaches can be developed. Recent studies have demonstrated the aberrant up-regulation of several TJ proteins, especially claudins, in various types of cancers (5, 6). For instance, up-regulation of claudin-1 has been found in colorectal cancer via the β-catenin/Tcf signaling (7, 8). Transfection of wild-type adenomatous polyposis coli (APC) gene, a tumor suppressor gene, in APC-deficient colon cell line resulted in significant down-regulation of claudin-1 (7). Besides, enhanced expression of claudin-1 was found to be inversely correlated with another tumor suppressor gene, smad4 (9). Upon overexpression of claudin-1 in colon cancer cell lines, this led to an increase in tumor invasiveness (8). EMT was also observed, accompanied with a loss of E-cadherin and up-regulation of vimentin (8). On the contrary, ZO proteins have been implicated as tumor suppressors. For instance, ZO proteins share high homology to the tumor suppressor gene of Drosophila called discs-large-A (dlgA) at the src homology region 3 (SH3) and guanylate kinase (GUK) domains (10). ZO-1 was shown to induce EMT in Madin-Darby Canine Kidney (MDCK) cells when mutant ZO-1 lacking the GUK domain was expressed at cytosol instead of at the cell surface (11). Expression of ZO-1 was down regulated in poorly differentiated gastric and colon tumors (12). Besides, expression of ZO-1 in gastric is less frequent in diffuse type as compare with intestinal-type adenocarcinoma (13). When there is an open up of intestinal epithelial barrier upon inflammation, there was down-regulation of ZO-1 and 3, but an increase in expression of claudin-1 as demonstrated in Caco-2 cell line treated with mixture of cytokines (14). The changes closely parallel those observed in carcinogenic process. Additionally, claudin-1 is structurally associated with Pals1-assoicated tight junction protein (PATJ) via the PDZ domain (15). The PATJ, together with Crumbs (CRB) and protein associated with Lin seven1 (Pals1, or Stardust) form a polarity complex at the apical side of an epithelium with TJ (16). This complex was firstly identified in Drosophila and conserved among species. Knock-down of PATJ in polarized Caco-2 cell line resulted in mislocalization of occludin and ZO-3 (17), this suggests the importance of PATJ in maintaining TJ stability. Furthermore, PATJ was found to regulate the migration of MDCK cells by recruiting other polarity complex components to the leading edge of migrating cells, illustrating the role of the polarity complex in regulating cell migratory behavior such as those found in metastatic tumor cells (18). These findings thus illustrate: 1) overexpression of specific TJ proteins (e.g., claudin-1) facilitates tumorigenesis, 2) the opposing effects of claudin-1 and ZOs on tumorigenesis, and 3) TJ proteins are likely involved in tumor metastasis, other than their structural function. The regulation of claudin-1, ZOs as well as the CRB polarity complexes during gastric cancer development is poorly explored except the aberrant up-regulation of claudin-1,3,4 and 7 (13, 19). From our available microarray dataset that contains 90 gastric tumors, 14 tumors that had metastasized to lymph nodes, and 22 normal mucosa (20), we have detected up-regulation of claudin-1, 3 and 4 in gastric tumor, with reciprocal down-regulation of claudin-18. For ZO proteins, there are heterogeneous expressions among tumor samples (Fig 1A). Up-regulation of claudin-1 in gastric tumors versus normal mucosa was further confirmed by real-time quantitative PCR and immunoblotting (see Fig 1B-C). These preliminary findings thus support this application to examine the role of claudin-1 and other TJ proteins in gastric cancer. Aim: To understand the changes in protein levels and interactions between TJ proteins (claudin-1, ZOs and PATJ) in gastric cancer development and their contribution to Epithelial-Mesenchymal-Transition. This will be done by examining for morphological changes, cell mobility, invasive behaviour, alter in steady-state protein levels, subcellular localization, protein-protein interaction between claudin-1, ZOs and polarity complexes via forced overexpression and knockdown of claudin-1 in gastric cancer cell lines.

Project Title:	AACR 101st Annual Meeting 2010 Retinoic acid receptor responder 3 expression suppresses colorectal cancer cell growth in vitro and is a stage-independent prognostic marker in colorectal cancer patients in vivo
Investigator(s):	Yan HHN
Department:	Pathology
Source(s) of Funding:	URC/CRCG - Conference Grants for Teaching Staff
Start Date:	04/2010
Completion Date:	04/2010

Abstract:

N/A

List of Research Outputs

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Yang M

List of Research Outputs

Lo C.K., Lam Q.L.K., Yang M., Ko K.H., Sun L., Ma R., Wang S., Xu H., Tam S., Wu C.Y., Zheng B.J. and Lu L., Leptin signaling protects NK cells from apoptosis during development in mouse bone marrow, Cell Mol Immunol. 2009, 6(5): 353-60.

Yang M., Sun L., Wang S., Xu H., Zheng B., Cao X. and Lu L., Cutting Edge: Novel function of BAFF in the induction of IL-10-producing regulatory B cells, Journal of Immunology. 2010, 184: 3321-3325.

Researcher : Yau TO

Project Title:	The role of Frizzled-7, receptor of the WNT/β-catenin signalling pathway, in hepatocellular carcinoma: Implications in tumorigenesis and metastasis
Investigator(s):	Yau TO, Ng IOL
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	10/2006

Abstract:

Hepatocellular carcinoma (HCC) is second commonest in Asia and in Hong Kong. Hepatocarcinogenesis is closely associated with activation of the WNT/β-catenin signaling pathway (HCC) (Wong et al., 2001; Miyoshi et al., 1998; de La Coste A et al., 1998). In the canonical WNT/β-catenin signaling pathway, WNT ligands bind to the transmembrane receptors Frizzled (FZD) and low density lipoprotein receptor related protein (LRP), and the signal is transduced to cytoplasmic protein FRAT1 and Dishevelled (DVL) respectively. FRAT1 and DVL inhibit the APC complex, and β-catenin therefore accumulates. Subsequently, β-catenin enters the nucleus, forms complex with members of T-cell factor/lymphoid enhancing factor (TCF/LEF) and upregulates TCF/LEF-dependent transcription of target proto-oncogenes such as c-MYC (He et al. 1998) and cyclin D1 (Shtutman et al., 1999; Tetsu et al., 1999), and eventually promotes tumourigenesis. We and others have previously demonstrated overexpression of β-catenin, in membranous, cytoplasmic and/or nuclear compartments, in 41 (68%) of 60 HCCs (Wong et al., 2001). We also shown that downregulation of negative regulator HDPR1 may be one of the mechanisms leading to the accumulation of β-catenin in HCC (Yau et al, 2005). To further delineate the deregulation of this important pathway in HCC, we plan to investigate the role of WNT receptor molecules in this deregulation. One of these receptor molecules, FZD7, was recently demonstrated to be overexpressed in HCC (Merle et al., 2004 and 2005) and implicated to promote motility of HCC cells. However, the role of FZD7 in hepatocarcinogenesis and metastasis has not been fully characterized. In this research proposal, we propose to investigate the roles of FZD7 in hepatocarcinogenesis and metastasis. Objectives of this research proposal: 1.To study the expression of FZD7 at mRNA and protein levels in human HCCs and in HCC cell lines. 2.To investigate whether there are any mutations of FZD7 that will result in activation of the pathway. 3.To investigate whether upregulation of FZD7 in HCC is due to gene amplification. 4.To study the role of FZD7 in hepatocarcinogenesis in HCC cell lines. 5.To study the role of FZD7 in metastasis using orthotopic xenograft implantation in immunodeficient mice.

List of Research Outputs

Wong L.T., Yau T.O., Sze M.F. and Ng I.O.L., PTEN underexpression was associated with more aggressive tumor behaviour in hepatocellular carcinoma and PTEN suppressed cell invasion by downregulating NF-κB signaling pathway, The 3rd International LIver Cancer Association Annual Conference, Milan, Italy. 2009.

Yau T.O., Leung T.H.Y., Lam S.G.S., Cheung O.F., Tung K.K., Khong P.L., Lam A.K.M., Chung S.K. and Ng I.O.L., Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis., PLoS One. 2009, 4(8): e6566.

Researcher : Yeung CW

List of Research Outputs

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Researcher : Yeung HY

Project Title:	Systematic characterisation of Notch signaling targets in colon cancer development
Investigator(s):	Yeung HY, Leung SY, Yuen ST
Department:	Pathology
Source(s) of Funding:	Small Project Funding
Start Date:	05/2008
Completion Date:	10/2009

Abstract:

The homeostatic self-renewal of the intestine depends on the interplay of cell proliferation, differentiation, migration, adhesion and cell death. Perturbation to the pathways that regulate self-renewal process play critical role in neoplastic transformation. Although the current understanding on the stem cell renewal and differentiation is primarily derived from the study of small intestine, both small intestine and colon basically share some major pathways in the regulation such as Wnt, BMP, Notch and hedgehog. Among them, Wnt and Notch together act as a gate-keeper in intestinal epithelium (van Es and Cleavers 2005). The role of Wnt signaling in regulation of stem/progenitor cells self-renewal and malignant transformation in small intestine and colon has been well characterized (Gregorief and Cleavers 2005). However, less is known on role of Notch signaling. Suppression of Notch signaling, by injection of g-secretase inhibitor, a Notch inhibitor, or conditional inactivation of its transcription factor CSL/RBP-J, could effectively increase the number and size of mucosecreting goblet cells in intestine and even caused inhibition of cell division and secretory cell conversion in colon adenomas (van Es JH et al 2005; Artavanis-Tsakonas SR et al 1999). Indeed, reduction in goblet cells is one of the early signs of neoplastic transformation in the colon, although a small proportion of CRCs known as mucinous carcnioma still possess the mucous secreting property (Hanski C et al 1997; Ho SB et al 1993; Audie JP et al 1993; Sylvester PA et al 2001). It is known that Notch functions through induction of HES, which in turn suppresses the expression of Hath1. Hath1 expression induces secretory lineage differentiation but how it mediates this function and its downstream signaling component remains largely unknown. Park et al demonstrated that in CRC, expression of Hath1 is suppressed, being detectable in only 7% of conventional CRCs. However, 100% of mucinous and signet ring cell carcinoma still expressed Hath1 (Park ET et al 2006). Further, recent study demonstrated that Hath1 overexpression in HT-29 human colon cancer cells significantly inhibited cell proliferation and tumorigenesis accompanied with induction of the goblet cell differentiation marker mucin2 (Leow CC et al 2004, 2005). All these data suggested that Notch signaling activation, with Hath1 suppression, is associated with stem/progenitor cell proliferation and malignant transformation in the majority of CRC. Interruption of Notch signaling, either through direct inhibition of Notch, or induction of Hath1 or its downstream signaling component, may constitute an alternative strategy to treat CRCs. To this end, we need to have a better understanding of Notch downstream signaling components, and to what extend these are perturbed in CRCs. Our group has previously generated a cDNA microarray expression database that delineated normal colon crypt development between bottom and top compartments (Kosinski and Li et al, submitted to PNAS, in revision). We identified the Notch signaling activation at bottom crypt (Fig.1) that supports its corresponding role in colon stem/progenitor cell maintenance. In addition, we have generated another gene expression dataset that contains 52 CRCs, 11 adenomas and 9 normal mucosae. In the current study, we propose to characterize the downstream signaling component of Hath1 in vitro using expression array. The data generated, when analyzed together with the colon crypt development and the CRC databases, will give us a comprehensive picture of how Notch regulate secretory lineage differentiation through Hath1, and its role in normal colon development as well as neoplastic transformation. Aim 1. To identify the downstream signaling component of Hath1-mediated Notch signaling using expression microarray through forced expression or knockdown of Hath1 in colon cell lines. 2. To compare the altered component with gene expression databases of colon top versus bottom crypt compartments and CRCs, so as to select candidate signaling components of importance both in vitro and in vivo. 3. To validate the regulation of those candidate genes by Notch signaling through examination for gene or protein expression changes after treatment of colon cell lines with Notch inhibitor (g-secretase inhibitor). 4. To examine gene expression levels and protein localizations of those putative molecules in human normal colon or CRC tissues in vivo. 5. To study the functional roles of those candidate genes in human colon cancer cell lines. 6. To demonstrate the involvement of those candidate genes in cell fate determination in human colon cell lines.

List of Research Outputs

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Researcher : Yeung YS

List of Research Outputs

Ko F.C.F., Yeung Y.S., Wong C.M., Chan L.K., Poon R.T.P., Ng I.O.L. and Yam J.W.P., Deleted in liver cancer 1 isoforms are distinctly expressed in human tissues, functionally different and under differential transcriptional regulation in hepatocellular carcinoma, Liver International. 2010, 30: 139-148.

Yeung Y.S., Tse Y.T., Ko F.C.F., Chan L.K., Sze M.F., Ng I.O.L. and Yam J.W.P., Growth suppression activity of tensin2 in human hepatocellular carcinoma is dependent on PTEN and SH2 domains, The 21st Meeting of the European Association for Cancer Research (EACR), Oslo, Norway. 2010.

Researcher : Yip SF

List of Research Outputs

Kwan T.K., Ma E.S.K., Chan Y.Y., Wan T.S.K., Liu H.S.Y., Sim J.P.Y., Yeung Y.M., Lie A.K.W. and Yip S.F., BCR-ABL mutational studies for predicting the response of patients with chronic myeloid leukaemia to second-generation tyrosine kinase inhibitors after imatinib fail, Hong Kong Medical Journal. 2009, 15(5): 365-73.

Ma C.H., Fan K.P., Ward A.C., Liongue C., Lewis R.S., Cheng S.H., Chan P.K., Yip S.F., Liang R.H.S. and Leung A.Y.H., A novel zebrafish jak2a(V581F) model shared features of human JAK2(V617F) polycythemia vera., Experimental Hematology. 2009, 37: 1379-1386.

Meyer C., Kowarz E., Hofmann J., Renneville A., Zuna J., Trka J., Ben Abdelali R., Macintyre E., De Braekeleer E., De Braekeleer M., Delabesse E E., de Oliveira M.P., Cavé H., Clappier E., van Dongen J.J., Balgobind B.V., van den Heuvel-Eibrink M.M., Beverloo H.B., Panzer-Grümayer R., Teigler-Schlegel A., Harbott J., Kjeldsen E., Schnittger S., Koehl U., Gruhn B., Heidenreich O., Chan L.C., Yip S.F., Krzywinski M., Eckert C., Mőricke A., Schrappe M., Alonso C.N., Schäfer B.W., Krauter J., Lee D.A., Zur Stadt U., Te Kronnie G., Sutton R., Izraeli S., Trakhtenbrot L., Lo Nigro L., Tsaur G., Fechina L., Szczepanski T., Strehl S., Ilencikova D., Molkentin M., Burmeister T., Dingermann T., Klingebiel T. and Marschalek R., New insights to the MLL recombinome of acute leukemias, Leukemia. 2009, 23(8): 1490-1499.

Researcher : Yuen HF

List of Research Outputs

Chan K.K., Shen L., Au W.Y., Yuen H.F., Wong K.Y., Guo T., Wong M.L.Y., Shimizu N., Tsuchiyama J., Kwong Y.L., Liang R.H.S. and Srivastava G., Interleukin-2 induces NF-kappaB activation through BCL10 and affects its subcellular localization in natural killer lymphoma cells, J Pathol. 2010, 221(2): 164-74.

Chua C.W., Chiu Y.T., Yuen H.F., Chan K.W., Man K., Wang X., Ling M.T. and Wong Y.C., Suppression of androgen-independent prostate cancer cell aggressiveness by FTY720: validating Runx2 as a potential antimetastatic drug screening platform, Clinical Cancer Research. 2009, 15(13): 4322-4335.

Yuen H.F., Chiu Y.T., Chan K.K., Chua C.W., McCrudden C.M., Tang K.H., El-Tanani M., Wong Y.C., Wang X. and Chan K.W., Prostate Cancer Cells Modulate Osteoblast Mineralisation And Osteoclast Differentiation Through Id-1 , British Journal of Cancer. 2010, 102: 332-341.

Researcher : Yuen HF

List of Research Outputs

Researcher : Yuen KM

List of Research Outputs

Chan M.C.W., Chan W.Y., Yu C.L., Ho C.C., Yuen K.M., Fong J.H.M., Tang L.L.S., Lai W.W.K., Lo A.C.Y., Chui W.H., Sihoe A.D.L., Kwong D.L.W., Tsao G.S.W., Poon L.L.M., Guan Y., Nicholls J.M. and Peiris J.S.M., Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract, American Journal of Pathology. 2010, 176(4): 1828-40.

Chan W.Y., Yuen K.M., Yu C.L., Ho C.C., Nicholls J.M., Peiris J.S.M. and Chan M.C.W., Influenza H5n1 And H1n1 Virus Replication And Innate Immune Responses In Bronchial Epithelial Cells Are Influenced By The State Of Differentiation, PLoS One. 2010, 5 (1): e8713.

Researcher : Yuen LYP

List of Research Outputs

Chau S.K.C., Kwok K.L., Ng D.K., Lam C.W., Tong S.F., Chan A.Y.W., Siu C.W.K. and Yuen L.Y.P., Maternally inherited Leigh syndrome: an unusual cause of infantile apnea, Sleep Breath. 2010, 14(2): 161-5.

Lee H.H.C., LEE R.S.Y., LAI C.K., Yuen L.Y.P., SIU T.S., Chan A.Y.W. and Lam C.W., A novel duplication at the putative DNA polymerase alpha arrest site and a founder mutation in Chinese in the IVD gene underlie isovaleric acidaemia, Hong Kong Medical Journal. 2010, 16(3): 219-22.

Researcher : Yuen ST

List of Research Outputs

Kosinski C., Stange D.E., Xu C., Chan A.S.Y., Ho C., Yuen S.T., Mifflin R.C., Powell D.W., Clevers H., Leung S.Y. and Chen X., Indian Hedgehog Regulates Intestinal Stem Cell Fate Through Epithelial-Mesenchymal Interactions During Development, Gastroenterology. 2010, 139(3): 893-903.

Li S.W.V., Yuen S.T., Chan T.L., Yan H.H.N., Law W.L., Yeung H.Y., Chan A.S.Y., Tsui W.Y., So S., Chen X. and Leung S.Y., Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic and epigenetic mechanisms, Gastroenterology. 2009, 137(1): 176-87.

Poulogiannis G., Ichimura K., Hamoudi R.A., Luo F., Leung S.Y., Yuen S.T., Harrison D.J., Wyllie A.H. and Arends M.J., Prognostic relevance of DNA copy number changes in colorectal cancer, J Pathol. 2010, 220(3): 338-47.

Schetter A.J., Nguyen G.H., Bowman E.D., Mathe E.A., Yuen S.T., Hawkes J.E., Croce C.M., Leung S.Y. and Harris C.C., Association of inflammation-related and microRNA gene expression with cancer-specific mortality of colon adenocarcinoma, Clin Cancer Res. 2009, 15(18): 5878-87.

Xia H.H.X., Yang Y., Chu K.M., Gu Q., Zhang Y.Y., He H., Wong W.M., Leung S.Y., Yuen S.T., Yuen R.M.F., Chan A.O.O. and Wong B.C.Y., Serum macrophage migration-inhibitory factor as a diagnostic and prognostic biomarker for gastric cancer, Cancer. 2009, 115: 5441-5449.

Researcher : Yung LH

List of Research Outputs

Lee K.W., Yung L.H., Cheung C.H., Castilho A.G. and Ng I.O.L., Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis, The American Association for Cancer Research, 2010.

Researcher : Zhang H

List of Research Outputs

Siu K.Y., Yeung C.W., Zhang H., Kong S.H., Ho W.K.J., Ngan H.Y.S., Chan D.C. and Cheung A.N.Y., p21-activated kinase 1 promotes aggressive phenotype, cell proliferation and invasion in gestational trophoblastic disease. , Am J Pathol.. 2010, 176: 3015-22.

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Zhang H

List of Research Outputs

Wong G.W., Huo Z., Siu K.Y., Zhang H., Jiang L., Wong E.S.Y. and Cheung A.N.Y., Hypermethylation of SOX2 Promoter in Endometrial Carcinogenesis, Obstetrics and Gynecology International. 2010.

Researcher : Zhang L

List of Research Outputs

Zhang L., Chan Y.K., Ip Y.C., Tsang W.H. and Khoo U.S., Splice variant profiling in relation to tamoxifen resistance in breast cancer, The 21st Meeting of the European Association for Cancer Research, Oslo, Norway. 2010.

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