Abstract
<title>Abstract</title> <p>BACKGROUND Bipolar androgen therapy (BAT) exploits the paradoxical vulnerability of castration-resistant prostate cancer (CRPC) to supraphysiologic androgen. However, testosterone (T) is used clinically for BAT, which broadly activates wild-type androgen receptor (AR) in normal androgen-responsive tissues and can cause substantial systemic androgenic effects. 5β-Dihydrotestosterone (5β-DHT), a naturally occurring T metabolite, has been considered androgenically inactive because of weak wild-type AR activity. Here, we examined whether 5β-DHT and related 5β-reduced metabolites activate mutant AR signaling and reproduce BAT-like growth suppression. METHODS Six 5β-reduced T metabolites were evaluated in C4-2 and LNCaP prostate cancer cells under androgen-depleted and BAT-like conditions. AR dependence and mutant-specific activity were assessed using enzalutamide, AR-null PC-3 cells, and PC-3 cells expressing AR-W742C or AR-H875Y. Cell proliferation, AR-responsive reporter activity, RT-qPCR, RNA sequencing with gene set enrichment analysis, immunoblotting, and senescence-associated β-galactosidase staining were used to characterize ligand responses. RESULTS At nanomolar concentrations, 5β-DHT and 3β-etiocholanediol (3β-ecdiol) induced AR target genes and promoted AR-dependent C4-2 and LNCaP growth, although less potently than T. RNA sequencing showed that both metabolites activated the T-regulated AR transcriptional program with smaller differentially expressed gene footprints. At high concentrations, 5β-DHT, but not the weaker agonist 3β-ecdiol or progesterone-class steroids, suppressed C4-2 and LNCaP proliferation. High-dose 5β-DHT also activated AR and suppressed growth in PC-3 cells expressing AR-W742C or AR-H875Y, whereas AR-null PC-3 cells were unaffected. High-dose 5β-DHT enriched androgen-response and senescence programs, suppressed G2M, E2F, and MYC-associated pathways, increased p21, p27, and p57, reduced RB phosphorylation and SKP2, and induced senescence-associated β-galactosidase activity. CONCLUSIONS 5β-DHT is an overlooked mutant-AR agonist that promotes growth at low concentrations but induces BAT-like senescence and growth suppression at high concentrations. These findings support an AR-potency threshold that separates mitogenic from antiproliferative signaling and warrant further evaluation of 5β-DHT as a potentially lower-androgenicity T substitute for BAT.</p>