Abstract A27: MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1

Abstract Over the past 30 years, there has been a dramatic increase in the incidence of obesity worldwide. As of 2010, approximately one-third of U.S. adults are obese, while another one-third of adults are overweight. Obesity is one of the risk factors for ovarian cancer. Greater than 75% of ovarian cancer patients present with advanced stage disease. Tumors preferentially spread to the omentum, a major site of abdominal fat, where ovarian cancer cells infiltrate omental adipose tissue. Omental adipocytes may interact directly with cancer cells to create a microenvironment niche that supports cancer growth. Recent studies have shown that cells can communicate through the exchange of bioactive molecules via microvesicles (exosomes and ectosomes). Microvesicles fuse with target cell membranes and transfer proteins, lipids, mRNAs and non-coding RNAs from donor to recipient cells. We hypothesize that adipocytes secrete microvesicles containing tumor-supporting factors that promote ovarian cancer progression. Ion Torrent RNA sequencing and subsequent qRT-PCR analyses were used to identify non-coding microvesicle RNAs secreted by primary cultures of cancer associated adipocytes (CAA), cancer associated fibroblasts (CAF), normal adipocytes, normal fibroblasts and ovarian cancer cells. Of the non-coding microvesicle RNAs identified, microRNA 21 (miR21) showed significantly higher expression in microvesicles secreted from CAA when compared to those from CAF and normal omental adipocytes, fibroblasts, and ovarian cancer cells. Using qRT-PCR of RNA isolated from microdissected frozen tissue and In-situ hybridization on FFPE tissue sections, CAA demonstrated significant higher miR21 expression levels compared to the adjacent cancer cells and normal adipocytes. MiR21 was up-regulated in metastatic ovarian cancer cells in the omentum as compared to those in the primary tumor site. Moreover, strong miR21 staining was observed in omental cancer cells particularly at the invasion front, suggesting that miR21 may be transferred from CAA to their neighboring ovarian cancer cells. To test this hypothesis, SKOV3 ovarian cancer cells were co-cultured with adipocytes transfected with miR21-FAM or incubated with fluorescently labeled adipocyte-derived microvesicles. Confocal microscopy confirmed the delivery of miR21 by adipocyte-derived microvesicles to cancer cells. Since miR21 expression in tumor cells has been linked with resistance to a variety of chemotherapeutic agents, we further delineated the direct effect of miR21 on the chemoresistance of ovarian cancer cell. The ovarian cancer cell lines, OVCA432 and SKOV3, transfected with miR21 precursor showed an increase in cell survival and a decrease in apoptosis in the presence of taxol. Furthermore, miR21 was significantly up-regulated in taxol-resistant HeyA8-MDR and SKOV3-TR cells as compared to the parental cell lines, HeyA8 and SKOV3, respectively. These data strongly suggest that miR21 may confer taxol resistance to ovarian cancer cells. Using microarray analysis of RNA isolated from SKOV3 cells transfected with the miR21 precursor, we identified a set of chemoresistance-related genes associated with miR21 expression. One of the most significantly down-regulated genes is Apoptotic Protease Activating Factor 1 (APAF1). By qRT-PCR, we confirmed that miR21 over-expression decreased APAF1 mRNA expression levels in OVCA432 and SKOV3 cells. A strong miR21 binding site is predicted on the 3′-UTR of APAF1, suggesting APAF1 is the direct target of miR21. In summary, up-regulation of miR21 in omental ovarian cancer cells is mediated through transport of miR21 in adipocyte-derived microvesicles from CAA in the omentum. Further, miR21 confers taxol-resistance in ovarian cancer cells by down-regulating APAF1 expression in ovarian cancer cells to decrease taxol-induced apoptosis. Citation Format: Ngai Na Co, Rosemarie Schmandt, Karen Lu, Samuel Mok. MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1 [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A27.