Androgen receptor (AR)-mediated oncogenic pathways have not been fully elucidated. In this study, we used highthroughput microarray analysis on two AR-positive prostate cancer (CaP) cell lines to identify 16 AR-responsive microRNAs (miRNA). We focused on miR-21 because of its previously reported oncogenic activity in other cancers. We show androgen-induced AR binding to the defined miR-21 promoter, miPPR-21, suggesting direct transcriptional regulation. Inhibition of miR-21 diminished androgen-induced CaP cell proliferation, providing new evidence that miRNAs can contribute to androgen-driven cell growth. Elevated expression of miR-21 enhanced CaP tumor growth in vivo and, surprisingly, was sufficient for androgen-dependent tumors to overcome castration-mediated growth arrest. Thus, elevated miR-21 expression alone is sufficient to impart castration resistance. Moreover, quantitative reverse transcription-PCR analysis revealed elevated miR-21 expression in CaP when compared with adjacent normal tissue. These results suggest that miR-21 may contribute to CaP pathogenesis. [Cancer Res 2009;69(18):7165-9]

Dose-escalated radiation therapy for localized prostate cancer (PCa) has a clear therapeutic benefit; however, escalated doses may also increase injury to noncancerous tissues. Radiosensitizing agents can improve ionizing radiation (IR) potency, but without targeted delivery, these agents will also sensitize surrounding normal tissues. Here we describe the development of prostate-targeted RNAi agents that selectively sensitized prostate-specific membrane antigen-positive (PSMA-positive) cells to IR. siRNA library screens identified DNA-activated protein kinase, catalytic polypeptide (DNAPK) as an ideal radiosensitization target. DNAPK shRNAs, delivered by PSMA-targeting RNA aptamers, selectively reduced DNAPK in PCa cells, xenografts, and human prostate tissues. Aptamer-targeted DNAPK shRNAs, combined with IR, dramatically and specifically enhanced PSMA-positive tumor response to IR. These findings support aptamer-shRNA chimeras as selective sensitizing agents for the improved treatment of high-risk localized PCa.

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MicroRNAs (miRNAs) are a natural part of the most recently discovered and global regulatory pathway known as RNA interference. Functional studies have shown how specific miR-NAs can function as tumor suppressors or oncogenes and, correspondingly, deregulated miRNA profiles have been observed in prostate and other cancers. However, the upstream pathways which regulate miRNA expression are only currently being uncovered. The Androgen Receptor (AR) is a nuclear hormone receptor and transcription factor which plays a paramount role in prostate cancer (PCa) pathobiology. We performed high throughput miRNA microarray analysis on two AR-responsive cell lines to identified 16 candidate AR-regulated miRNAs.1 One of the most androgen-induced candidates was a known oncogenic miRNA, miR-21. In a small study of early grade PCa samples we found that miR-21 levels were frequently elevated in comparison to adjacent normal tissue. This observation was supported in the literature2,3 and suggests clinical relevance. We found that the activated AR directly interacts with miR-21 regulatory regions, indicating direct transcriptional induction. Furthermore, we provide new reporter studies supporting AR-regulation. Importantly, in functional studies, we found that a modest overexpression of miR-21 enhanced tumor xenograft growth and was sufficient to support androgen-independent proliferation following surgical castration. Thus, our studies suggest a model where miR-21 contributes to androgen-dependent and androgen-independent PCa growth. However, the AR is only one of many reported transcriptional regulators of miR-21. Here we review our recent discoveries and further analyze the reported miR-21 regulatory regions, inhibitory and stimulatory signaling pathways, and primary transcripts.