Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.

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Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2-4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, upregulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.

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.

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