ARID1A, a chromatin remodeler, shows one of the highest mutation rates across many cancer types. Notably, ARID1A is mutated in over 50% of ovarian clear cell carcinomas, which currently has no effective therapy. To date, clinically applicable targeted cancer therapy based on ARID1A mutational status has not been described. Here we show that inhibition of the EZH2 methyltransferase acts in a synthetic lethal manner in ARID1A mutated ovarian cancer cells. ARID1A mutational status correlates with response to the EZH2 inhibitor. We identified PIK3IP1 as a direct ARID1A/EZH2 target, which is upregulated by EZH2 inhibition and contributes to the observed synthetic lethality by inhibiting PI3K/AKT signaling. Significantly, EZH2 inhibition causes regression of ARID1A mutated ovarian tumors in vivo. Together, these data demonstrate for the first time a synthetic lethality between ARID1A mutation and EZH2 inhibition. They indicate that pharmacological inhibition of EZH2 represents a novel treatment strategy for ARID1A mutated cancers.

Summary Oncogene-induced senescence is characterized by a stable cell growth arrest, thus providing a tumor suppression mechanism. However, the underlying mechanisms for this phenomenon remain unknown. Here we show that a decrease in deoxyribonucleotide triphosphates (dNTPs) levels underlies oncogene-induced stable senescence-associated cell growth arrest. The decrease in dNTP levels is caused by oncogene-induced repression of RRM2, the rate-limiting protein in dNTP synthesis. This precedes the senescence-associated cell cycle exit and coincides with the DNA damage response. Consistently, RRM2 downregulation is both necessary and sufficient for senescence. Strikingly, suppression of nucleotide metabolism by RRM2 repression is also necessary for maintenance of the stable senescence-associated cell growth arrest. Further, RRM2 repression correlates with senescence status in benign nevi and melanoma, and its knockdown drives senescence of melanoma cells. These data reveal the molecular basis whereby the stable growth arrest of oncogene-induced senescence is established and maintained through suppression of nucleotide metabolism.

Enhancer of zeste homolog 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that includes noncatalytic subunits suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED). When present in PRC2, EZH2 catalyzes trimethylation on lysine 27 residue of histone H3 (H3K27Me3), resulting in epigenetic silencing of gene expression. Here, we investigated the expression and function of EZH2 in epithelial ovarian cancer (EOC). When compared with primary human ovarian surface epithelial (pHOSE) cells, EZH2, SUZ12, and EED were expressed at higher levels in all 8 human EOC cell lines tested. Consistently, H3K27Me3 was also overexpressed in human EOC cell lines compared with pHOSE cells. EZH2 was significantly overexpressed in primary human EOCs (n ¼ 134) when compared with normal ovarian surface epithelium (n ¼ 46; P < 0.001). EZH2 expression positively correlated with expression of Ki67 (P < 0.001; a marker of cell proliferation) and tumor grade (P ¼ 0.034) but not tumor stage (P ¼ 0.908) in EOC. There was no correlation of EZH2 expression with overall (P ¼ 0.3) or disease-free survival (P ¼ 0.2) in high-grade serous histotype EOC patients (n ¼ 98). Knockdown of EZH2 expression reduced the level of H3K27Me3 and suppressed the growth of human EOC cells both in vitro and in vivo in xenograft models. EZH2 knockdown induced apoptosis of human EOC cells. Finally, we showed that EZH2 knockdown suppressed the invasion of human EOC cells. Together, these data demonstrate that EZH2 is frequently overexpressed in human EOC cells and its overexpression promotes the proliferation and invasion of human EOC cells, suggesting that EZH2 is a potential target for developing EOC therapeutics. Mol Cancer Res; 8(12); 1610-8. Ó2010 AACR.

Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy in the US. Thus, there is an urgent need to develop novel therapeutics for this disease. Cellular senescence is an important tumor suppression mechanism that has recently been suggested as a novel mechanism to target for developing cancer therapeutics. Wnt5a is a non-canonical Wnt ligand that plays a context-dependent role in human cancers. Here, we investigate the role of Wnt5a in regulating senescence of EOC cells. We demonstrate that Wnt5a is expressed at significantly lower levels in human EOC cell lines and in primary human EOCs (n = 130) compared with either normal ovarian surface epithelium (n = 31; p = 0.039) or fallopian tube epithelium (n = 28; p < 0.001). Notably, a lower level of Wnt5a expression correlates with tumor stage (p = 0.003) and predicts shorter overall survival in EOC patients (p = 0.003). Significantly, restoration of Wnt5a expression inhibits the proliferation of human EOC cells both in vitro and in vivo in an orthotopic EOC mouse model. Mechanistically, Wnt5a antagonizes canonical Wnt/β-catenin signaling and induces cellular senescence by activating the histone repressor A (HIRA)/promyelocytic leukemia (PML) senescence pathway. In summary, we show that loss of Wnt5a predicts poor outcome in EOC patients and Wnt5a suppresses the growth of EOC cells by triggering cellular senescence. We suggest that strategies to drive senescence in EOC cells by reconstituting Wnt5a signaling may offer an effective new strategy for EOC therapy.

Epithelial ovarian cancer (EOC) ranks first as the cause of death for gynecological cancers in the United States. SUZ12 is a component of the polycomb repressive complex 2 (PRC2) and is essential for PRC2-mediated gene silencing by generating trimethylation on lysine 27 residue of histone H3 (H3K27Me3). The role of SUZ12 in EOC has never been investigated. Here we show that SUZ12 is expressed at significantly higher levels in human EOC (n=117) compared with either normal human ovarian surface epithelium (n=35, p<0.001) or fallopian tube epithelium (n=15, p<0.001). There is a positive correlation between expression of SUZ12 and EZH2 in human EOC (p<0.001). In addition, expression of SUZ12 positively correlates with Ki67, a marker of cell proliferation (p<0.001), and predicts shorter overall survival (p=0.0078). Notably, knockdown of SUZ12 suppresses the growth of human EOC cells in vitro and in vivo in both orthotopic and subcutaneous xenograft EOC models. In addition, SUZ12 knockdown decreases the levels of H3K27Me3 and triggers apoptosis of human EOC cells. Mechanistically, we identified HRK, a pro-apoptotic gene, as a novel SUZ12 target gene, and demonstrated that HRK upregulation mediates apoptosis induced by SUZ12 knockdown in human EOC cells. In summary, we show that SUZ12 promotes the proliferation of human EOC cells by inhibiting apoptosis and HRK is a novel SUZ12 target gene whose upregulation contributes to apoptosis induced by SUZ12 knockdown.

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Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo-and radio-therapy. Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo-and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.