Biochemical and functional analysis of the novel androgen receptor antagonist C28 in human prostate cancer cells

Castration-resistant prostate cancer (CRPC) is an aggressive form of prostate cancer (PCa) that develops resists to androgen deprivation therapy and androgen receptor (AR)-antagonists. Mutations in the AR-LBD is a well-known resistance mechanism against clinically used AR-antagonists. Compound 28 (C28) had been shown to inhibit both wild-type and mutant ARs, reducing AR nuclear translocation in transiently transfected HeLa cells and suppresses castration-sensitive PCa cells growth. However, its precise molecular mechanisms in CRPC remain unclear. C4-2 cell line as a CRPC model was used to evaluate C28 efficacy compared to other AR-ligands. Immunofluorescence revealed that C28 significantly reduces AR nuclear import in CRPC cells. Co-immunoprecipitation and Western blot analyses showed that, unlike supraphysiological androgen levels (SAL), but similar to darolutamide (Dar) as clinically used AR-antagonist, C28 decreases HSP27 and AR phosphorylation known to be required for AR nuclear translocation. This proposes a mechanism for C28-mediated AR nuclear import inhibition. This study also addressed how AR-ligands induce cellular senescence in CRPC. C28 was found to inhibit the growth of 2D adherent cells, 3D tumor spheroids, and mouse xenograft tumors by induction of cellular senescence. Mechanistically, the data suggests that C28 activates the DREAM complex signaling pathway, to induce cellular senescence. Inhibition or knockdown of DYRK1A, essential for DREAM complex activation, reduced C28-, Dar-, and SAL-induced cellular senescence. C28 also suppresses G2/M cell cycle genes and promotes the interaction between AR and p130. Additionally, C28 induced expression of the atypical cyclin G2 (CCNG2), contributing to senescence. This was confirmed in both spheroids and xenograft tumors in mice. In summary, this study identifies that AR-p130-DYRK1A-DREAM and CCNG2 signaling pathways are common mechanisms activated by C28, Dar or SAL to mediate cellular senescence in CRPC.