Human Cancer Biology miRNA-34b Inhibits Prostate Cancer through Demethylation, Active Chromatin Modifications, and AKT Pathways

Purpose:miRNAs can act as oncomirs or tumor-suppressormiRs in cancer. This studywas undertaken to investigate the status and role of miR-34b in prostate cancer. Experimental Design: Profiling of miR-34b was carried out in human prostate cancer cell lines and clinical samples by quantitative real-time PCR and in situ hybridization. Statistical analyses were done to assess diagnostic/prognostic potential. Biological significance was elucidated by carrying out a series of experiments in vitro and in vivo. Results:We report thatmiR-34b is silenced inhumanprostate cancer and themechanism is throughCpG hypermethylation. miR-34b directly targeted methyltransferases and deacetylases resulting in a positive feedback loop inducing partial demethylation and active chromatin modifications. miR-34b expression could predict overall and recurrence-free survival such that patients with high miR-34b levels had longer survival. Functionally, miR-34b inhibited cell proliferation, colony formation, migration/invasion, and triggered G0/G1 cell-cycle arrest and apoptosis by directly targeting the Akt and its downstream proliferative genes.miR-34b caused adecline in themesenchymalmarkers vimentin, ZO1,N-cadherin, and Snail with an increase in E-cadherin expression, thus inhibiting epithelial-to-mesenchymal transition. Finally we showed the antitumor effect of miR-34b in vivo. MiR-34b caused a dramatic decrease in tumor growth in nude mice compared with cont-miR. Conclusion: These findings offer new insight into the role ofmiR-34b in the inhibition of prostate cancer through demethylation, active chromatin modification, and Akt pathways and may provide a rationale for the development of new strategies targeting epigenetic regulation of miRNAs for the treatment of prostate cancer. Clin Cancer Res; 19(1); 73–84. 2012 AACR. Introduction miRNAs are non–protein-coding RNAs thought to regulate the expression of up to 90% of human genes (1). Growing evidence has strongly implicated the involvement of miRNAs in carcinogenesis (2, 3). Dysregulated miRNAs may function as oncogenes (4), such as the miR-17–92 cluster (5), or tumor suppressor genes such as miR-205 (6– 8). A single miRNA can have hundreds of target mRNAs, highlighting the importance of this gene regulation system in cellular functions (9). The study of miRNAs has become the subject of intense interest, especially after the discovery of their fundamental role in a myriad of cellular and biological processes ranging from development to disease (10). From a clinical point of view, miRNAs have great potential as diagnostic and therapeutic agents. Because microarray analysis shows a general downregulation of miRNAs in tumors when compared with normal tissues (11). As a result of the remarkable tissue specificity of miRNAs, they have become a very useful tool for defining the origin of tumors in poorly differentiated cancers (12). The prognosis and survival of patients depend on the stage of the cancer at diagnosis. For this reason, 1 of the most important issues in clinical cancer research is to find early biomarkers of the tumorigenic process. miRNA signatures have been reported to be powerful tools for early diagnosis in renal cancer and have been found to distinguish between metastatic and nonmetastatic tumors. The 5-year survival rate of patients with primary metastasis was reported to be 10% as compared with 70% to 90% in nonmetastatic patients (13). Among various epigenetic mechanisms of cancer-related gene silencing, DNA hypermethylation of CpG sites within CpG islands is known to lead to the inactivation of many tumor suppressor miRNAs (14). Previous reports have Authors' Affiliations: Department of Urology, VA Medical Center and UCSF; and California Pacific Medical Center Research Institute, San Francisco, California Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). Corresponding Author: Rajvir Dahiya, Urology Research Center (112F), VA Medical Center and UCSF, 4150 Clement Street, San Francisco, CA 94121. Phone: 415-750-4810; Fax: 415-750-6639; E-mail: [email protected] doi: 10.1158/1078-0432.CCR-12-2952 2012 American Association for Cancer Research. Clinical Cancer Research www.aacrjournals.org 73 on April 14, 2017. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from Published OnlineFirst November 12, 2012; DOI: 10.1158/1078-0432.CCR-12-2952