The influence of histone H3 modifications on darolutamide resistance in prostate cancer

Abstract

Prostate cancer is one of the most frequently diagnosed malignancies in men worldwide and accounts for a substantial proportion of cancer-related deaths. With the introduction of darolutamide, a next-generation androgen receptor (AR) antagonist, a novel therapeutic option with promising clinical results became available in 2020. However, with the expanded clinical use of darolutamide, an increasing number of resistant cases has been observed. Previous studies have identified epigenetic alterations as key drivers in the development and progression of prostate cancer. This study investigated the role of histone H3 lysine 27 trimethylation (H3K27me3) and its writer protein, Enhancer of Zeste Homolog 2 (EZH2), in the development of resistance to darolutamide. VCaP cells were used as an in vitro model of castration-resistant prostate cancer (CRPC) and were treated with increasing concentrations of darolutamide to induce resistance. Western blot analyses revealed elevated protein levels of the repressive histone modification H3K27me3 and EZH2 in darolutamide-resistant VCaP cells (VCaP Daro). These changes correlated with reduced transcription of H3K27me3 target genes, including insulin-like growth factor-binding protein 3 (IGFBP3), homeobox D1 (HOXD1), AR splice variant V7 (ARV7), and AR full-length (Arfl). EZH2 knockdown resulted in decreased H3K27me3 levels and increased transcription of target genes. Similar effects were observed after treatment with EPZ-6438, a selective inhibitor of EZH2 methyltransferase activity. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) revealed a global, genome-wide increase in H3K27me3 occupancy in VCaP Daro, accompanied by a redistribution of H3K27me3 peaks toward gene promoter regions, potentially associated with altered AR binding. Subsequent RNA sequencing identified the sonic hedgehog (SHH) signaling pathway as the most strongly upregulated pathway in VCaP Daro, suggesting a central role in the resistance mechanism. Increased transcription of GLI Family Zinc Finger proteins 1 and 2 (GLI1, GLI2) served as readouts for Hedgehog pathway activation. Hedgehog reporter assays demonstrated SHH ligand secretion in VCaP Daro but not in wild-type VCaP cells (VCaP Wt), indicating possible autocrine or paracrine activation. Furthermore, treatment with the Smoothened inhibitor vismodegib demonstrated that Hedgehog signaling contributes to increased EZH2 expression. Both EPZ-6438 and vismodegib significantly reduced cell viability in both cell lines, as determined by MTS assays. Notably, the inhibitory effects were significantly stronger in VCaP Daro than in VCaP Wt, with vismodegib exhibiting greater cytotoxicity than EPZ-6438. In conclusion, these results suggest that EZH2 and SHH represent promising therapeutic targets for the treatment of darolutamide-resistant CRPC. As corresponding inhibitors are already clinically available, there is substantial translational potential. Further studies are required to elucidate the underlying molecular mechanisms.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed