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MicroRNAs (miRNAs) have emerged as key players in cancer progression and metastatic initiation yet their importance in regulating prostate cancer (PCa) metastasis to bone has begun to be appreciated. We employed multimodal strategy based on in-house PCa clinical samples, publicly available TCGA cohorts, a panel of cell lines, in silico analyses, and a series of in vitro and in vivo assays to investigate the role of miR-466 in PCa. Expression analyses revealed that miR-466 is under-expressed in PCa compared to normal tissues. Reconstitution of miR-466 in metastatic PCa cell lines impaired their oncogenic functions such as cell proliferation, migration/invasion and induced cell cycle arrest, and apoptosis compared to control miRNA. Conversely, attenuation of miR-466 in normal prostate cells induced tumorigenic characteristics. miR-466 suppressed PCa growth and metastasis through direct targeting of bone-related transcription factor RUNX2. Overexpression of miR-466 caused a marked downregulation of integrated network of RUNX2 target genes such as osteopontin, osteocalcin, ANGPTs, MMP11 including Fyn, pAkt, FAK and vimentin that are known to be involved in migration, invasion, angiogenesis, EMT and metastasis. Xenograft models indicate that miR-466 inhibits primary orthotopic tumor growth and spontaneous metastasis to bone. Receiver operating curve and Kaplan-Meier analyses show that miR-466 expression can discriminate between malignant and normal prostate tissues; and can predict biochemical relapse. In conclusion, our data strongly suggests miR-466-mediated attenuation of RUNX2 as a novel therapeutic approach to regulate PCa growth, particularly metastasis to bone. This study is the first report documenting the anti-bone metastatic role and clinical significance of miR-466 in prostate cancer.

Prostate carcinogenesis involves alterations in several signaling pathways, the most prominent being the PI3K/AKT pathway. This pathway is constitutively active and drives prostate cancer (PCa) progression to advanced metastatic disease. PTEN, a critical tumor and metastasis suppressor gene negatively regulates cell survival, proliferation, migration and angiogenesis via the PI3K/Akt pathway. PTEN is mutated, downregulated/dysfunctional in many cancers and its dysregulation correlates with poor prognosis in PCa. Here, we demonstrate that microRNA-4534 (miR-4534) is overexpressed in PCa and show that miR-4534 is hypermethylated in normal tissues and cell lines compared to PCa tissues/cells. miR-4534 exerts its oncogenic effects partly by downregulating the tumor suppressor PTEN gene. Knockdown of miR-4534 impaired cell proliferation, migration/invasion and induced G0/G1 cell cycle arrest and apoptosis in PCa. Suppression of miR-4534 and its effects on tumor growth was confirmed in a xenograft mouse model. We performed parallel experiments in non-cancer RWPE1 cells by overexpessing miR-4534 followed by functional assays. Overexpression of miR-4534 induced pro-cancerous characteristics in this non-cancer cell line. Statistical analyses revealed that miR-4534 has potential to independently distinguish malignant from normal tissues and positively correlated with poor overall and PSA recurrence free survival. Taken together, our results show that depletion of miR-4534 in PCa induces a tumor suppressor phenotype partly through induction of PTEN. These results have important implications for identifying and defining the role of new PTEN regulators such as microRNAs in prostate tumorigenesis. Understanding aberrantly overexpressed miR-4534 and its downregulation of PTEN will provide mechanistic insight and therapeutic targets for PCa therapy.

Purpose: miRNAs can act as oncomirs or tumor-suppressor miRs in cancer. This study was 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 that miR-34b is silenced in human prostate cancer and the mechanism is through CpG 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 G(0)/G(1) cell-cycle arrest and apoptosis by directly targeting the Akt and its downstream proliferative genes. miR-34b caused a decline in the mesenchymal markers 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 of miR-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.

The miRNAs have great potential as biomarkers and therapeutic agents owing to their ability to control multiple genes and potential to influence cellular behavior. Here, we identified that miR-23b is a methylation-silenced tumor suppressor in prostate cancer. We showed that miR-23b expression is controlled by promoter methylation and has great promise as a diagnostic and prognostic biomarker in prostate cancer. High levels of miR-23b expression are positively correlated with higher overall and recurrence-free survival in patients with prostate cancer. Furthermore, we elucidated the tumor suppressor role of miR-23b using in vitro and in vivo models. We showed that proto-oncogene Src kinase and Akt are direct targets of miR-23b. Increased expression of miR-23b inhibited proliferation, colony formation, migration/invasion, and triggered G(0)-G(1) cell-cycle arrest and apoptosis in prostate cancer. Overexpression of miR-23b inhibited epithelial-to-mesenchymal transition (EMT) causing a decline in mesenchymal markers Vimentin and Snail and increasing the epithelial marker, E-cadherin. Depletion of Src by RNA interference conferred similar functional effects as that of miR-23b reconstitution. miR-23b expression caused a dramatic decrease in tumor growth in nude mice and attenuated Src expression in excised tumors compared with a control miR. These findings suggest that miR-23b is a methylation-silenced tumor suppressor that may be a useful biomarker in prostate cancer. Loss of miR-23b may confer proliferative advantage and promote prostate cancer migration and invasion, and reexpression of miR-23b may contribute to the epigenetic therapy for prostate cancer.

Background: MicroRNAs (miRNAs) are small noncoding RNAs that regulate the expression of approximately 60% of all human genes. They play important roles in numerous cellular processes, including development, proliferation, and apoptosis. Currently, it is believed that miRNAs elicit their effect by silencing the expression of target genes. In this study, the authors demonstrated that miRNA-205 (miR-205) induced the expression the interleukin (IL) tumor suppressor genes IL24 and IL32 by targeting specific sites in their promoters., Methods: The methods used in this study included transfection of small RNAs; quantitative real-time polymerase chain reaction; in situ hybridization; fluorescence-labeled in situ hybridization; cell cycle, apoptosis, cell viability, migratory, clonability, and invasion assays; immunoblotting; and luciferase reporter, nuclear run-on, and chromatin immunoprecipitation assays., Results: The results revealed that miR-205 was silenced in prostate cancer. Its re-expression induced apoptosis and cell cycle arrest. It also impaired cell growth, migration, clonability, and invasiveness of prostate cancer cells. Micro-RNA-205 induced the expression of tumor suppressor genes IL24 and IL32 at both the messenger RNA and protein levels. The induction of in vitro transcription and enrichment of markers for transcriptionally active promoters in the IL24 and IL32 genes was observed in response to miR-205., Conclusions: In this study, a new function for miR-205 was identified that specifically activated tumor suppressor genes by targeting specific sites in their promoters. These results corroborate a newly identified function that miRNAs have in regulating gene expression at the transcriptional level. The specific activation of tumor suppressor genes (eg, IL24, IL32) or other dysregulated genes by miRNA may contribute to a novel therapeutic approach for the treatment of prostate cancer., (Copyright © 2010 American Cancer Society.)

The minichromosome maintenance (MCM) gene family is essential for DNA replication and is frequently upregulated in various cancers. Here, we examined the role of MCM2 in prostate cancer and the effect of microRNA-1296 (miR-1296), genistein, and trichostatin A (TSA) on the MCM complex. Profiling results showed that expression of MCM genes was higher in tumor samples. Genistein and TSA significantly downregulated the expression of all MCM genes. Genistein, TSA, and small interfering RNA duplexes caused a significant decrease in the S phase of the cell cycle. There was also downregulation of CDT1, CDC7, and CDK2 genes, which govern loading of the MCM complex on chromatin. We also found that miR-1296 was significantly downregulated in prostate cancer samples. In PC3 cells, inhibition of miR-1296 upregulated both MCM2 mRNA and protein, whereas overexpression caused a significant decrease in MCM2 mRNA, protein, and the S phase of the cell cycle. MCM genes are excellent anticancer drug targets because they are essential DNA replication factors that are highly expressed in cancer cells. This is the first report showing anti-MCM effect by miR-1296, genistein, and TSA. TSA is undergoing clinical trials as a prostate cancer treatment but has high toxicity. Genistein, a natural, nontoxic dietary isoflavone, may be an advantageous therapeutic agent for treating prostate cancer. The use of RNA interference is currently being implemented as a gene-specific approach for molecular medicine. The specific downregulation of oncogenes by miR may contribute to novel therapeutic approaches in the treatment of prostate cancer.

Background: : B-cell translocation gene 3 (BTG3/ANA/APRO4) is a candidate tumor suppressor gene in some malignancies. We report here that B-cell translocation gene 3 (BTG3) is transcriptionally down-regulated in prostate cancer and the mechanism of inactivation is through promoter hypermethylation., Methods: : Prostate cancer and normal cell lines were treated with different doses of genistein and 5-aza-2'-deoxycytidine (5Aza-C). BTG3 messenger ribonucleic acid (mRNA) expression was determined by quantitative real-time polymerase chain reaction in tissues and cell lines. Bisulfate-modified polymerase chain reaction, cloning and sequencing were used to examine promoter methylation in tumor samples and cell lines. Enzyme activity/inhibition assays were done to check the effect of genistein and 5Aza-C on DNA methyltransferases. ChIP assay was performed to analyze chromatin modifications caused by genistein treatment., Results: : BTG3 mRNA expression was down-regulated in cancer tissues and cells. Genistein and 5Aza-C induced BTG3 mRNA expression in all PC cell lines. Complete methylation of BTG3 promoter in tumor samples and cancer cell lines was observed. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Genistein and 5Aza-C increased levels of acetylated histones 3, 4, histone 3 dimethylated at lysine 4, histone 3 trimethylated at lysine 4, and RNA polymerase II, decreased DNA methyl transferase and methyl-binding domain protein 2 activity, and increased histone acetyl transferase (HAT) activity., Conclusions: : This is the first report to show that BTG3 is silenced in prostate cancer and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein showed similar effects to that of 5Aza-C, which is currently undergoing phase 2 clinical trials as a treatment for prostate cancer. Because genistein is a natural, nontoxic, and dietary isoflavone, these results indicate that genistein is a novel, advantageous therapeutic agent for treating prostate cancer., (Copyright 2010 American Cancer Society.)