MicroRNA mediated regulation of key components of the Mediator Complex (MED) and its functional role in CRPC

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Prostate cancer (PCa) is the second most frequent malignant tumor in men worldwide. It usually responds well to androgen deprivation therapy, but eventually castration resistant prostate cancer (CRPC) will develop that is still difficult to treat. In CRPC development and progression, transcription and its regulation are key processes. Transcription is performed by RNA polymerase II, functioning in a pre-initiation complex that includes the Mediator (MED) complex. However, the regulation of MED is largely unknown. In our project, we will focus on two key components of MED, MED12 and MED15, which have recently been described as strongly overexpressed in CRPC compared to androgen-sensitive PCa or benign tissue. MicroRNAs (miRNAs) are important post-transcriptional regulators, and in our in silico analyses we have identified candidate miRNAs that are underexpressed in CRPC and predicted to target MED12/MED15. Therefore, we hypothesize that miRNAs regulating the expression of MED12/MED15 contribute to the development of CRPC, castration resistance and resistance against anti-androgen therapy.The aims of our project are to analyze the miRNA-mediated regulation of MED12/MED15, and its functional relevance in CRPC from the side of the tumor epithelial cells and the stromal cells in vitro and in vivo. This also includes pursuing a resensitization of CRPC for anti-androgen treatment by miRNA replacement and combined treatment with an antagonist of TGFß, regulating candidate miRNAs and MED components.Key objectives of the project:i) Validation of in silico predicted miRNAs as regulators of MED12 or MED15, and characterization of their functional relevance for the treatment of androgen-sensitive as well as enzalutamide or abiraterone resistant PCa cell lines in vitro. ii) Therapeutic application of the identified miRNAs, formulated in polymeric nanoparticles, to inhibit tumor xenograft growth and/or to affect sensitivity of CRPC xenografts towards anti-androgen therapy in vivo. This also includes tissue slice cultures derived from the xenografts for detailed molecular ex vivo analyses. iii) Assessing the clinical relevance of the expression of MED12/15 and their regulating miRNAs for disease progression and survival of CRPC patients by simultaneous miRNA in situ hybridization and immunohistochemistry in PCa tissues. iv) Analysis of MED12/15 regulation by tumor adjacent stromal cells through TGFß and miRNAs under anti-androgen therapy.v) Assessment of combined miRNA replacement and TGFß inhibition for enhanced antitumor effects in vitro, and the therapeutic evaluation of optimal combinations in patient-derived xenografts (PDX) in vivo. In summary, we will elucidate the regulation of MED12/15 by miRNAs and TGFß in PCa tumor cells in context of their interaction with stromal cells. We expect tumor suppressive effects of miRNA replacement and additive/synergistic effects upon its combination with anti-androgen therapy or TGFß inhibition in vitro/in vivo.