Abstract 950: Bistable switching of c-KIT by estrogen-mediated ceRNA and epigenetic silencing of miR-193a predicts survival in ovarian cancer
Authors: Cheng Frank Hsueh-Che , Aguda Baltazar D. , Lin Hon-Yi , Tsai Je-Chiang , Kochanczyk Marek , Lin Ru-Inn , Lin Jora M. J. , Chen Gary C. W. , Chang Cheng-Chang , Lai Hung-Cheng , Nephew Kenneth P. , Hwang Tzy-Wei , Chan Michael W. Y. 
Published: July 2016

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&gt;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&lt;0.05) between E2F6 and c-KIT which resembles the ceRNA phenomenon between these two mRNAs. Importantly, ovarian cancer patients with low expression of EZH2 tended to have lower expression of c-KIT. In conclusion, our mathematical model and experimental data suggests that miR-193a can be epigenetically regulated by both ceRNA and promoter methylation. Simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.

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.