Abstract B34: Secreted microRNAs from prostate cancer cells: Novel therapeutic targets

Abstract

Abstract Introduction: More than 80% of men with aggressive prostate cancer (PCa) develop metastatic disease and skeletal complications leading to a dramatic reduction in quality of life. There is a deficit in effective biomarkers predicting disease progression, response to treatment, or metastasis at early stages when intervention would be possible. A critical need exists for novel therapeutic targets to prevent PCa metastasis to bone. MicroRNAs (miRNA) are informative biomarkers, as they circulate, are easily detected in human serum, and are coupled to targets reflecting deregulated pathways. Secreted miRNAs may be bound to proteins, membrane-encapsulated, or free RNA molecules and can act as intercellular signaling molecules. Here we tested the hypothesis that miRNAs secreted from prostate cancer cells in the primary tumor function locally and at distal sites to alter the normal tissue environment and promote PCa. Objective: Use global miRNA and gene expression analysis to 1) define cohorts of miRNAs secreted from prostate cancer cells representing different disease stages, 2) identify expression levels of their target mRNAs, and 3) provide insight into mechanisms of secreted miRNA activity in promoting prostate cancer tumor growth and metastasis. Methods: RNA was isolated from normal prostate epithelial (RWPE-1), early stage, androgen responsive PCa (LNCaP), and late stage, androgen insensitive PCa (PC-3) cell lines and from their conditioned media. Global expression of both secreted and extracellular miRNAs were interrogated using Affymetrix miRNA v4.0 microarrays (miRBase v20). Stranded RNA-seq was performed in parallel to identify differential expression of miRNA target mRNAs. We utilized the RNA-seq data to limit predicted mRNAs to those having reciprocal expression changes to their targeting miRNA. Enriched biological pathways of candidate miRNAs were identified for these predicted mRNA targets. Results: To identify secreted miRNAs that are candidates for promoting prostatic tumor growth, cancer progression, and metastatic bone disease, we compared differentially expressed miRNAs between PCa cell lines and their conditioned media. While numerous miRNAs are found to be both secreted and expressed robustly in the cell layer, we find unique subsets of miRNAs in each cell line that are either preferentially secreted or retained within the cell. We examined putative mRNA targets of these miRNAs in our gene expression data and established reciprocal mRNA-miRNA relationships. The miRNAs were found to be associated with pathways known to play a role in PCa and metastatic bone disease including ErbB, Wnt, TGF-beta, and MAPK signaling, as well as previously unassociated biological processes. Functional in vitro assays were performed for several candidate miRNAs that were overexpressed or inhibited in normal prostate epithelial RWPE-1 and highly metastatic PC-3 cell lines. Phenotypic changes, including motility, invasion, migration, and proliferation, were monitored. As an example, inhibition of miR-30a-5p, an established tumor suppressor, increases the rate at which RWPE-1 and PC-3 cells migrate to heal a cell layer scratch while overexpression prevents wound closure in RWPE-1 cells. In vivo orthotopic experiments to assay the ability of candidate miRNAs to promote local and metastatic PCa tumor growth are ongoing. Conclusion: Our combined data identify, for the first time, miRNAs secreted from prostate tumor cells that promote metastatic events of prostate cancer. Several of these miRNAs have been reported to be elevated in PCa patient serum and can provide diagnostic/prognostic biomarkers of tumor development, response to treatment, and recurrence. Our findings link these miRNAs to deregulated cellular pathways for potential intervention of prostate cancer progression. Citation Format: Nicholas H. Farina, Cody J. Callahan, Coralee E. Tye, Joseph R. Boyd, Gary S. Stein, Janet L. Stein, Jane B. Lian. Secreted microRNAs from prostate cancer cells: Novel therapeutic targets. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B34.