Abstract 74: E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer
Authors: Cheng Frank Hsueh-Che , Lin Hon-Yi , Chen Yin-Chen , Hwang Tzy-Wei , Huang Rui-Lan , Chang Chia-Bin , Lin Ru-Inn , Lin Ching-Wen , Chen Gary C.W. , Lin Jora M. J. , Chuang Yu-Ming , Chou Jian-Liang , Li Chin , Cheng Alfred S.L. , Lai Hung-Cheng , Wu Shu-Fen , Tsai Je-Chiang , Chan Michael W.Y. 
Published: August 2018

Abstract
Ovarian cancer is one of the most lethal cancers in the female reproductive system. Previous study 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 decipher this complicated process, we generated a mathematical model and found that estrogen-mediated up-regulation of E2F6 could upregulate the ovarian cancer stem/initiating marker, c-kit by two means one through epigenetic silencing of their co-targeted miR193a by binding of E2F6 which subsequently recruit EZH2 to miR-193a promoter; and second, by competing endogenous (ceRNA) mechanism. To confirm this model, treatment of E2 or environmental hormone, BPA resulted in upregulation of both E2F6 and c-kit but down-regulation of miR-193a in immortalized ovarian surface epithelial cells. Further bisulfite pyrosequencing, ChIP-qPCR and epigenetic treatment found that miR193a was epigenetically silenced by DNA methylation and H3K27me3 in CP70 but not HeyC2 ovarian cancer cells. Overexpression of miR193a inhibited tumor growth in vitro and in vivo. Depletion of EZH2 or E2F6 in CP70 restored miR-193a expression and decreased the number of “ovo” spheroid by reversing the repressive chromatin status of miR-193a promoter. To further explore the biological significance of this E2F6 ceRNA network, integrative RNA-Seq and computational analysis found that PBX1, a miR-193a target and transcriptional activator of the immunosuppressive cytokine IL-10, was down-regulated in E2F6 and EZH2 knockdown CP70 cells. Overexpression of E2F6 3'UTR containing miR-193a MRE but not MRE mutant increased the expression of PBX1 and IL10 in ovarian cancer cells. Importantly, co-culture of conditional media from E2F6 3'UTR overexpressing CP70 cells inhibited the differentiation of THP-1 monocytes into dendritic cell and the T-cell activating function of this THP-1 derived DC. This phenomenon can be rescued by incubation of anti-IL-10 antibody or pretreatment of CP70 cells with EZH2 inhibitor. Finally, clinical studies demonstrated that patients with higher promoter methylation of miR193a were associated with poor survival. Serum IL10 level was found to be higher in high staged ovarian cancer patients and patients with higher E2F6 mRNA level. Additional analysis from TCGA ovarian cancer expression microarray dataset demonstrated that ovarian cancer patients with low expression of EZH2, showed a positive correlation between E2F6, c-KIT and PBX1 resembling the ceRNA phenomenon between these mRNAs. Taken together, our results showed that estrogen-mediated E2F6 ceRNA network can regulate cancer stemness and anti-tumor immunity of DC through epigenetic silencing of miR-193a. Anti-estrogen therapy together with the EZH2 inhibitor may be a novel strategy against this deadly cancer.
Citation Format: Frank Hsueh-Che Cheng, Hon-Yi Lin, Yin-Chen Chen, Tzy-Wei Hwang, Rui-Lan Huang, Chia-Bin Chang, Ru-Inn Lin, Ching-Wen Lin, Gary C.W. Chen, Jora M. J. Lin, Yu-Ming Chuang, Jian-Liang Chou, Chin Li, Alfred S.L. Cheng, Hung-Cheng Lai, Shu-Fen Wu, Je-Chiang Tsai, Michael W.Y. Chan. E2F6-mediated ceRNA and epigenetic silencing of miR193a lead to cancer stemness and anticancer immunity in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 74.