Abstract B70: Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.

Background: Epithelial ovarian cancer (EOC) accounts for the majority of deaths from gynecological malignancies, with a 5-year survival rate for women with advanced stage disease of only 46%. In EOC metastasis primarily occurs by a mechanism termed direct seeding, which involves shedding of tumor cells from the primary site into the peritoneal cavity. To allow survival in suspension before attachment at metastatic sites, ovarian cancer cells must acquire resistance to anoikis, a form of apoptosis initiated by detachment from native extracellular matrix. A targeted therapy that could simultaneously affect multiple factors critical for ovarian cancer progression such as anoikis resistance, chemoresistance and attachment to metastatic sites in the peritoneal cavity, would be a revolutionary breakthrough for this aggressive malignancy. Due to their ability to inhibit multiple targets involved in tumor survival, microRNAs (miRNAs) are attractive therapeutic candidates. We previously demonstrated that miR-200c is downregulated in ovarian cancer cell lines and restoration of miR-200c increased sensitivity to taxanes in vitro, by targeting TUBB3, a known mediator of chemoresistance. We tested the effect of miR-200c restoration in an intraperitoneal xenograft model of human ovarian cancer and observed dramatically decreased tumor formation. Further, even in established tumors, inducible restoration of miR-200c alone and in combination with paclitaxel, resulted in significantly reduced tumor burden. Hypothesis: Since restoration of miR-200c to multiple anoikis resistant EOC cell lines increased anoikis sensitivity and reduced adherence to biological substrates in vitro, we hypothesized that the decreased intraperitoneal tumor burden observed upon miR-200c restoration resulted from increased anoikis sensitivity. Methods and results: To elucidate mechanisms of anoikis resistance in ovarian cancer and identify targets involved in the ability of miR-200c to reverse anoikis resistance, we performed gene and miRNA expression profiling of attached Hey ovarian cancer cells as compared to the same cells grown under conditions of forced suspension for 24h. We identified miR-193 as downregulated in anoikis resistant cells in suspension. Among numerous genes predicted as miR-200c and/or miR-193 targets, SCLC7A11 (xCT), which encodes the light chain of the cystine/glutamate antiporter system xc, was increased by 10.7 fold in suspended versus attached cells. The xc- system is involved in glutathione (GSH) synthesis, mediating intracellular defense against oxidative stress. Reactive oxygen species (ROS) increased in anoikis-sensitive (OvCA 433) cells in suspension, but not in anoikis-resistant cells (Hey), suggesting that xCT might protect these cells from ROS-induced cell death. Consistent with this hypothesis, xCT mRNA and protein increased in anoikis resistant EOC cells in suspension, and transient xCT knockdown rendered cells anoikis sensitive. Interestingly, the xCT 39UTR contains three predicted binding sites for miR-200c and two sites for miR-193b, and restoration of either miRNA blocked the suspension induced-increase in cXT protein, suggesting that both miRNAs target this gene. Conclusions: Our results indicate that the ability of miR-200c to reduce tumor burden results, at least in part, from its ability to restore anoikis sensitivity by inhibiting a suspension-induced increase in xCT that protects tumor cells from ROS. Thus, miR-200c could be a powerful candidate to block progression of ovarian cancer by enhancing anoikis sensitivity and restoring chemo-sensitivity to already attached tumor. Citation Format: Diana M. Cittelly, Dawn R. Cochrane, Erin N. Howe, Irina Dimitrova, Miriam D. Post, Broaddus R. Russell, Monique A. Spillman, Jennifer K. Richer. Decreasing ovarian cancer metastases and progression via restoration of microRNAs that target mediators of anoikis- and chemoresistance.. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B70.