5β-Dihydrotestosterone reveals a mutant androgen receptor vulnerability in prostate cancer

Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) cells to rapid cycling between castrate and supraphysiologic androgen concentrations, but clinical BAT uses testosterone, which can also activate wild-type androgen receptor (AR) in androgen-responsive tissues, causing systemic side effects. 5β-dihydrotestosterone (5β-DHT) is a naturally occurring testosterone metabolite generally considered androgenically inactive because it binds wild-type AR weakly, yet its activity against clinically relevant AR mutants has not been systematically evaluated. Here, we tested whether 5β-DHT and related 5β-reduced testosterone metabolites activate AR signaling and growth programs in prostate cancer models that carry AR mutations. In C4-2 cells, 5β-DHT and 3β-etiocholanediol (3β-ecdiol) increased canonical AR target genes, including KLK3 and TMPRSS2 , with weaker activity than testosterone, whereas other 5β metabolites showed limited activity. In androgen-responsive LNCaP and C4-2 models, 5β-DHT and 3β-ecdiol promoted cell growth under androgen-depleted conditions, and this effect was suppressed by enzalutamide, supporting AR dependence. RNA-seq confirmed that 5β-DHT and 3β-ecdiol induced androgen-response gene sets substantially overlapping with testosterone, albeit at lower transcriptional magnitude. Further, we found that 5β-DHT, but not 3β-ecdiol, suppresses cell proliferation of LNCaP, C4-2, and PC-3 cells stably expressing the clinically relevant AR gain-of-function mutants W742C and H875Y through activating AR-induced senescence-like features after high-dose exposure, consistent with the therapeutic logic of BAT. These findings identify 5β-DHT as an overlooked mutant-AR agonist capable of BAT-like tumor suppression and propose it as a testosterone surrogate in BAT with potentially reduced systemic androgenic side effects.