HPS MORBID ANATOMY & CYTOLOGY -PATHOLOGY



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


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