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

Abstract

Abstract 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