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Aberrant TGFβ signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P< 0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFβ/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.

Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.

Ovarian cancer is one of the most lethal cancers in the female reproductive system. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by the surface antigen CD44 and c-KIT (CD117). Previous study also suggested that long term treatment of estrogen such as hormonal replacement therapy (HRT) may increase the risk of ovarian cancer, however the role of estrogen in ovarian carcinogenesis is still controversial. To unravel this complexity, we propose a mathematical model to explore how the ER signaling pathway contribute to c-KIT expression during ovarian carcinogenesis: one through a ceRNA competition of an ER target,E2F6 and c-KIT for their targeted miRNA, miR-193a; second by binding of E2F6 protein, in association with the polycomb complex, to the promoter of miR-193a to downregulate miR-193a transcription by epigenetic modifications. Our model found that epigenetic silencing of miR-193a generates a bistable switching of c-KIT during ovarian carcinogenesis based on the level of EZH2. To confirm our results, we performed ectopic expression of miR-193a and 3’UTR luciferase in ovarian cancer cell lines and confirmed that E2F6 and c-KIT are the targets of miR-193a. Importantly, treatment of E2 or bisphenol A (BPA) resulted in the up-regulation of E2F6 and c-kit mRNA in IOSE cells in which no or low methylation at the promoter CpG island of miR-193a was found. On the contrary, promoter hypermethylation of miR193a could be observed in miR-193a-underexpressed CP70 ovarian cancer cells but not in HeyC2 cells which showed similar expression level of miR-193a as in IOSE cells. Treatment of demethylating agent (5azaDC) or EZH2 inhibitor (GSK343) resulted in a reexpression of miR-193a in CP70 ovarian cancer cells. Overexpression of miR-193a inhibited tumor growth in vitro and in an animal model. Further ChIP-PCR assay also found that open chromatin mark H3K4me3 was enriched in the promoter region of miR-193a in HeyC2 but not in CP70 cells. On the contrary, repressive chromatin marks H3K9me3 and H3K27me3 were only enriched in CP70 cells. Clinically, ovarian cancer patients (n = 109) with higher promoter methylation of miR-193a were associated with poor survival (p>0.05). Additional analysis of the TCGA ovarian cancer dataset demonstrated that ovarian cancer patients with low expression of EZH2, a polycomb-group protein, showed positive correlation (p Citation Format: Frank Hsueh-Che Cheng, Baltazar D. Aguda, Hon-Yi Lin, Je-Chiang Tsai, Marek Kochanczyk, Ru-Inn Lin, Jora M. J. Lin, Gary C. W. Chen, Cheng-Chang Chang, Hung-Cheng Lai, Kenneth P. Nephew, Tzy-Wei Hwang, Michael W. Y. Chan. Bistable switching of c-KIT by estrogen-mediated ceRNA and epigenetic silencing of miR-193a predicts survival in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 950.