Abstract A30: miRNA Regulators of Ovarian Cancer Metastasis that Predict Patient Outcomes

Abstract

Serous ovarian cancer patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastases. We selected 12 matched primary and omental metastatic serous epithelial ovarian tumors to identify the copy number, mRNA and miRNA expression differences. No significant recurring copy number changes were detected. In contrast, significant recurrence with the same expression changes in all or the large majority of patients was observed for many mRNAs and miRNAs. We identify almost 1,000 genes with recurring significant expression differences across the patient cohort suggesting common phenotypic features are selected from heterogeneous mixture of primary tumors. Genes differentially expressed between metastatic and primary tumors identify a poor prognosis subtype in primary tumors that robustly distinguishes high risk patients across multiple platforms in two large independent data sets. Multivariate analysis suggests that the expression signature is independent of residual disease, a common clinical risk factor. To gain insight into the regulation of these mRNAs, we measured the expression of 377 miRNAs using Taqman qPCR in matched primary and metastatic serous ovarian tumors. We identified 17 miRNAs with significant differential expression in primary and metastatic ovarian human tumors including miR-21, miR-31, and novel miRNAs previously not associated with metastasis. Many of these miRNAs have >10 fold expression differences across multiple patients. We confirmed that the expression differences originate from cancer cells for many of the miRNAs by in situ hybridization and laser capture microdissection of cancer cells from tumors followed by qPCR. We identify combinations of metastatic miRNAs with significantly stronger predictions of patient outcomes than random combinations of miRNAs in The Cancer Genome Atlas data. Some of these same miRNAs show the same expression changes in liver metastases compared to colorectal primary tumors. Ovarian cancer cells form multicellular aggregates, or spheroids, as they disseminate throughout the peritoneal cavity and we find that these metastatic miRNAs affect spheroid formation and growth. All 7 of the metastatic miRNAs expressed in two ovarian cancer cell lines are up-regulated in spheroids compared to monolayers, recapitulating the observations in human metastases compared to primary tumors, suggesting that similar adaptations required for 3D culture are needed to establish metastases. Interestingly, we find miR-31 promotes metastasis in ovarian cancer suggesting a context dependent function compared to breast cancer. miR-31 is up-regulated in metastases by qPCR and in situ hybridization, up-regulated in spheroids compared to monolayers and inhibition reduces spheroid size and viability using three different inhibitors without significant effects on monolayer growth. We have tested the function of 6 other metastatic miRNAs, which predict patient survival, using both gain and loss of function experiments. We find that many of the metastatic miRNAs mediate colony formation, mobility, and/or spheroid size, but do not significantly affect monolayer culture. Predicted targets negatively correlate with miRNA expression better than sets of random permutations in the tumors, and some of these targets negatively correlate with miRNAs in spheroids vs. monolayers. These metastatic miRNAs appear to promote metastasis in part by enhancing β-catenin signaling through repression of APC and suppressing apoptosis. Using miRNA expression profiles and functional studies, we have established the utility of spheroid cultures to examine these clinically relevant metastatic miRNAs. In sum, we have identified metastatic miRNAs, from one of the first miRNA profiles of metastases, critical for aggressive disease in ovarian, and perhaps other, cancers, with potential for biomarker and therapeutic development. We find that multiple miRNAs, many expressed in metastases and not in primary tumors in some patients, are likely important to drive metastasis by regulating multiple gene networks. Citation Format: Alexander S. Brodsky, Hsin-Ta Wu, Souriya Vang, Benjamin Raphael, Laurent Brard. miRNA Regulators of Ovarian Cancer Metastasis that Predict Patient Outcomes [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 A30.