Final Report für DFG

Summary Prostate cancer (PCa), with an incidence of more than 1.46 million new cases per year worldwide, is the most common malignant tumor in elderly men. The major cause of morbidi-ty and mortality is its eventual progression towards a hormone refractory or castration re-sistant stage (CRPC) with its metastatic form (mCRPC). However, even in CRPC, the an-drogen receptor (AR) is still active and sensitive for further therapy options. One out of those is the application of inhibitory AR-binding ligands, like enzalutamide. Unfortunately, in the course of treatment a still significant proportion of patients develop resistance against this therapy, with the AR splice variant AR-V7 playing a major role. Altogether, the search for new therapeutic strategies to treat CRPC/mCRPC is still mandatory. The Mediator (MED) complex is involved in transcription regulation, enhancer–promoter in-teractions and in the chromatin architecture. A major function of the MED complex is to me-diate regulatory signals from DNA-bound transcription factors directly to the RNA Polymerase II, which is responsible for transcription. We aimed at analzying the miRNA-mediated regulation of two key components of the Media-tor Complex, MED12 and MED15, and their functional role in CRPC. This also included re-sensitization of CRPC to anti-androgen treatment (enzalutamide or abiraterone), and com-bined treatment regimens with a TGFß antagonist. We explored two different strategies for reducing AR/AR-V7. A knockdown of the MED com-plex component MED12 decreased the protein expression of AR and AR-V7. Similarly, we identified AR and AR-V7 as targets of the micro RNA miR-454-3p. Accordingly, the transfec-tion of synthetic miR-454-3p reduced the protein expression of AR in both EnzR cell lines and that of AR-V7 in the DuCaP EnzR cell line without affecting MED12. In addition, knockdown of MED12, but not miR-454, reduced cell viability in both cell lines. In a parallel approach, miR-454 was inhibited by using circ-RNAs as miR-454 sponge. This led to a significant induction of cell death and reduced proliferation in different 2D and 3D PCa models. In an in vivo therapy study in prostate xenograft-bearing mice, the therapeutic application of miR-454-specific circ-RNAs formulated in polymeric, polyethylenimine (PEI)-based nanoparticles for RNA delivery led to tumor inhibitory effects. In a second approach, we inhibited MED12 expression in LNCaP (AR+, Enz-sensitive), 22Rv1 (AR-V7+, EnzR), and PC3 (AR−, Enz-insensitive) cells. MED12 inhibition reduced cell proliferation in all cell lines and reduced proliferation in the respective 3D spheroids. Further-more, MED12 knockdown in 22Rv1 cells inhibited the AR response, prostate-specific antigen (PSA) secretion, AR target genes, and AR-V7 expression. Combined with enzalutamide, MED12 inhibition additively decreased the AR activity in both LNCaP and 22Rv1 cells. Taken together, the results of our study suggest MED12 as a potential target for future PCa treatment in conjunction with enzalutamide resistance. MED12 can regulate AR activity and that its inhibition may modulate response to enzalutamide in PCa. Furthermore, we showed that miR-454-3p directly targets AR and AR-V7. The dual approach of either replacing or in-hibiting miRNA-454 provides flexibility in treatment design and may offer personalized options based on individual patient profiles.