Maria Notaridou

Common alleles in candidate susceptibility genes associated with risk and development of epithelial ovarian cancer

Common germline genetic variation in the population is associated with susceptibility to epithelial ovarian cancer. Microcell‐mediated chromosome transfer and expression microarray analysis identified nine genes associated with functional suppression of tumorogenicity in ovarian cancer cell lines; AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1 and STAG3. Sixty‐three tagging single nucleotide polymorphisms (tSNPs) in these genes were genotyped in 1,799 invasive ovarian cancer cases and 3,045 controls to look for associations with disease risk. Two SNPs in RUVBL1, rs13063604 and rs7650365, were associated with increased risk of serous ovarian cancer [HetOR = 1.42 (1.15–1.74) and the HomOR = 1.63 (1.10–1.42), p‐trend = 0.0002] and [HetOR = 0.97 (0.80–1.17), HomOR = 0.74 (0.58–0.93), p‐trend = 0.009], respectively. We genotyped rs13063604 and rs7650365 in an additional 4,590 cases and 6,031 controls from ten sites from the United States, Europe and Australia; however, neither SNP was significant in Stage 2. We also evaluated the potential role of tSNPs in these nine genes in ovarian cancer development by testing for allele‐specific loss of heterozygosity (LOH) in 286 primary ovarian tumours. We found frequent LOH for tSNPs in AXIN2, AKTIP and RGC32 (64, 46 and 34%, respectively) and one SNP, rs1637001, in STAG3 showed significant allele‐specific LOH with loss of the common allele in 94% of informative tumours (p = 0.015). Array comparative genomic hybridisation indicated that this nonrandom allelic imbalance was due to amplification of the rare allele. In conclusion, we show evidence for the involvement of a common allele of STAG3 in the development of epithelial ovarian cancer.

In vitro three-dimensional modeling of fallopian tube secretory epithelial cells

BackgroundFallopian tube secretory epithelial cells (FTSECs) have been implicated as a cell-of-origin for high-grade serous epithelial ovarian cancer. However, there are relatively few in vitro models of this tissue type available for use in studies of FTSEC biology and malignant transformation. In vitro three-dimensional (3D) cell culture models aim to recreate the architecture and geometry of tissues in vivo and restore the complex network of cell-cell/cell-matrix interactions that occur throughout the surface of the cell membrane.ResultsWe have established and characterized 3D spheroid culture models of primary FTSECs. FTSEC spheroids contain central cores of hyaline matrix surrounded by mono- or multi-layer epithelial sheets. We found that 3D culturing alters the molecular characteristics of FTSECs compared to 2D cultures of the same cells. Gene expression profiling identified more than a thousand differentially expressed genes between 3D and 2D cultures of the same FTSEC lines. Pathways significantly under-represented in 3D FTSEC cultures were associated with cell cycle progression and DNA replication. This was also reflected in the reduced proliferative indices observed in 3D spheroids stained for the proliferation marker MIB1. Comparisons with gene expression profiles of fresh fallopian tube tissues revealed that 2D FTSEC cultures clustered with follicular phase tubal epithelium, whereas 3D FTSEC cultures clustered with luteal phase samples.ConclusionsThis 3D model of fallopian tube secretory epithelial cells will advance our ability to study the underlying biology and etiology of fallopian tube tissues and the pathogenesis of high-grade serous epithelial ovarian cancer.

Functional complementation studies identify candidate genes and common genetic variants associated with ovarian cancer survival

Common germline genetic variation and/or somatic alterations in tumours may be associated with survival in women diagnosed with ovarian cancer. The successful identification of genetic associations relies on a suitable strategy for identifying and testing candidate genes. We used microcell-mediated chromosome transfer approach and expression microarray analysis to identify genes that were associated with neoplastic suppression in ovarian cancer cell lines. Sixty-five tagging single nucleotide polymorphisms (tSNPs) in nine candidate genes were genotyped in approximately 1700 invasive ovarian cancer cases to look for associations with survival. For two of these genes, loss of heterozygosity (LOH) analysis of tSNPs in 314 ovarian tumours was used to identify associations between somatic gene deletions and survival. We identified significant associations with survival for a tSNP in caspase 5 (CASP5) [hazard ratio (HR) = 1.13 (95% CI: 1.00-1.27), P = 0.042] and two tSNPs in the retinoblastoma binding protein (RBBP8) gene [HR = 0.85 (95% CI: 0.75-0.95), P = 0.007 and HR = 0.83 (95% CI: 0.71-0.95), P = 0.009]. After adjusting for multiple prognostic factors in a multivariate Cox regression analysis, both associations in RBBP8 remained significant (P = 0.028 and 0.036). We then genotyped 314 ovarian tumours for several tSNPs in CASP5 and RBBP8 to identify gene deletions by LOH. For RBBP8, 35% of tumours in 101 informative cases showed somatic allelic deletion; LOH of RBBP8 was associated with a significantly worse prognosis [HR = 2.19 (95% CI: 1.36-3.54), P = 0.001]. In summary, a novel in vitro functional approach in ovarian cancer cells has identified RBBP8 as a gene for which both germline genetic variation and somatic alterations in tumours are associated with survival in ovarian cancer patients.

Abstract 4728: Functional analysis of candidate genes at ovarian cancer susceptibility loci

Genome-wide association studies (GWAS) have identified a plethora of common, low risk susceptibility alleles associated with multiple disease phenotypes. However, few studies have established the functional mechanisms underlying susceptibility at these loci. This is a challenging field. Evaluating the function of genetic variants that confer small relative disease risks (RRs=0.8-1.2) relies on robust models of normal and diseased tissues. We identified a low-penetrance susceptibility locus on chromosome 19p13.11 that confers susceptibility to serous epithelial ovarian cancer (EOC), breast cancer (BC) in BRCA1 carriers, and in triple negative (PGR-, ER-, HER2-) BC cases. There is also evidence that the region is associated with overall survival after a diagnosis of EOC. The SNPs that are most significantly associated with EOC/BC susceptibility lie in and around two candidate genes at this locus; MERIT40 and ANKLE1. ANKLE1 has no function that suggests it might be involved in cancer development; but MERIT40 is a particularly intriguing gene because the protein product stabilizes the Rap80/BRCA1/BRCC45/BRCC36 complex at double-stranded DNA breaks. We looked for evidence that MERIT40 and ANKLE1 were involved in EOC development using real-time PCR on a panel of 80 normal ovarian epithelial and EOC cell lines. We found MERIT40 to be significantly overexpressed in cancer cell lines (P = 5 × 10-9), but no change in ANKLE1 expression between the two (P = 0.54). MERIT40 is also upregulated in ∼70% of ovarian tumor specimens whereas there were no differences in gene expression for ANKLE1. These data suggests that MERIT40 expression increases during tumor development. We stably overexpressed GFP-tagged cDNAs of MERIT40 and ANKLE1 in normal ovarian, fallopian tube epithelia (the two EOC precursor cell types) and MCF10A breast epithelial cells. Neoplastic transformation was scored using migration, invasion and anchorage-independent growth assays. The phenotype of the MERIT40/ANKLE1-overexpressing cells in 3D cultures was evaluated by analysis of 3D cyst and spheroid architecture, and measurement of the proliferative indices of the cultures. Additionally, to investigate the mechanism by which MERIT40 is associated with EOC survival we knocked down MERIT40 expression in EOC cell lines using stable and doxycycline-inducible lentiviral shRNA vectors. Knockdown of MERIT40 in EOC lines did not significantly affect sensitivity to cisplatin or paclitaxel, but did induce cell cycle arrest during G1-phase in cells with MMR defects. This suggests that in the absence of MERIT40 EOC cells accumulate DNA damage and undergo cell cycle arrest. The in vitro modeling data so far suggests that MERIT40 is the likely target gene at the 19p13.11 locus associated with susceptibility to EOC and BC. We propose that MERIT40 overexpression in advanced tumors may enable cancer cells to tolerate DNA damage in the presence of somatic mutations in MMR genes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4728. doi:10.1158/1538-7445.AM2011-4728

Abstract 5021: A 3D model of genetic transformation of normal ovarian epithelial cells identifies candidate genes associated with the initiation and development of epithelial ovarian cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Novel biomarkers associated with early-stage ovarian cancer (OC) development may represent susceptibility makers that initiate ovarian cancers or screening markers for early disease detection. Little is known about the biology underlying OC development and heterogeneity. It has been hyporthesis that OCs can arise from the ovarian surface epithelium (OSE), a monolayer of epithelial cells covering the surface of the ovary. We have shown that immortalised OSE cells (IOSE) bypass replicative senescence but show no other features of neoplastic transformation. Here, we established an in vitro three-dimensional (3D) genetic model of early-stage ovarian carcinogenesis. By first overexpressing the CMYC oncogene, and then expressing mutant alleles of the KRAS or BRAF oncogenes in IOSE cells, we were able to create a stepwise model of neoplastic transformation of OSE. Overexpression of CMYC in IOSE cells (IOSE-CMYC) induces a significant increase in anchorage-dependent and -independent growth, enhanced progression through the cell cycle and decreased apoptosis. Subsequent expression of mutant KRAS-G12V or BRAF-V600E alleles in IOSE-CMYC cells caused differential rates of anchorage-independent growth, invasiveness and proliferation showing that the different genetic elements mimics the phenotypic heterogeneity seen in primary ovarian tumors in vivo. 3D models of oncogene-expressing clones revealed many characteristics of malignant cells in vivo that could not be detected in 2D monolayer cultures. Gene expression microarray profiling of the different stages of this model identified several candidate tumour suppressor genes that are associated with neoplastic transformation. These included: THBS1, an inhibitor of angiogenesis in several cancer types; FEZ1, which is involved in cell growth and shows loss of expression in ∼40% of OCs; and RGS4, (a regulator of G-protein signaling) shown to promote breast cancer migration and invasion. Novel activated genes were also identified including PITX1, previously shown to be overexpressed in a murine model of ovarian granulosa cell tumours; and HIST1H4C, which is a overexpressed in therapy related myloid leukemias. We used the model to evaluate the functional role of genes that have recently been shown to increase susceptibility to ovarian cancer using genome wide association studies. One locus is 8q24, near the CMYC oncogene, highlighting its significance in OC development. Two other loci are BNC2 at 9q21 and TiPARP at 3q25. Both genes were down-regulated in response to CMYC over-expression and showed significant loss of function with further transformation after KRAS-G12V activation (p=7.8 × 10-7 and p=6.3 × 10-4, respectively). This suggests both behave as tumor suppressor genes. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5021.

Abstract 4729: A genome-wide association study of ovarian cancer prognosis identifies a novel locus for aggressive serous cancer on 19p13

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological cancer in the UK and improved understanding of the mechanisms driving prognosis is greatly needed. There is evidence that inherited genetic variation influences EOC survival but no common variants have been definitively identified to date. The aim of our study was to detect polymorphisms that influence EOC prognosis using a genome wide approach. Subjects were from a recently completed genome-wide association study (GWAS) for genetic susceptibility to EOC. We tested 507,094 SNPs genotyped in 1,817 invasive EOC cases from the UK for association with survival using Cox proportional hazards regression (Stage 1). The 4,649 SNPs showing the strongest association with survival were genotyped in an additional 3,709 EOC cases with survival time data and 4,809 controls (Stage 2). Based on the combined analysis of Stage 1 and 2 data, no SNP showed association with survival at genome-wide significance (P −8 ). However, rs8170 on 19p13 showed a strong association with risk of serous EOC (P trend = 1.0×10 −7 ) and evidence of association with overall survival (P trend = 1.8×10 −5 ). This SNP and an additional disease-associated variant in the same region (rs2363956) were genotyped in an additional 4,043 EOC cases and 5,951 controls (Stage 3). The combined analysis confirmed the association with risk of serous cancer (combined data odds ratio = 1.17 95% CI 1.11 - 1.22, P trend = 2.9 × 10 −11 ). There was no evidence of association with non-serous EOC risk (P trend = 0.76). Of the stage 3 cases, 1,845 had survival time data available. The addition of these samples provided some support for the survival association (combined data hazard ratio =1.16 95% CI 1.08 - 1.23, P trend = 1.6 × 10 −5 ). The effect on survival was not attenuated by adjusting for histopathological subtype suggesting that this association is not solely a consequence of predisposition to serous disease. These two SNPs are in two separate genes on 19p13 - MERIT40 and ANKLE2. MERIT40 is a functionally important component of the BRCA1 complex. CGH microarray data on 102 primary EOCs showed the 19p13 region to be amplified in 40% of tumors. We also found MERIT40 to show significantly higher expression levels in EOC cell lines (n=23) compared to normal ovarian epithelial cells (n=48) (P=5.6×10 −9 ), but the genotypes of neither SNP were significantly correlated with MERIT40 expression. We are currently performing chemosensitivity assays to characterize the impact of MERIT40 on response to platinum-based chemotherapy, the first-line agent for EOC. While we have strong evidence of an association between 19p13 and serous EOC risk, the association with survival requires confirmation in additional cases, an effort which is currently underway. Nevertheless, the detection of this biologically compelling locus may lead to the development of novel treatments for this lethal disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4729.