Oxidative stress reactivates androgen receptor signaling via USP36 to drive castration resistance in prostate cancer

Castration-resistant prostate cancer (CRPC) often emerges within a few years following androgen deprivation therapy, and therapeutic options remain limited. Androgen deprivation induces oxidative stress in prostate cancer (PCa) cells, leading to aberrant activation of androgen receptor (AR) signaling. This study aims to clarify the molecular mechanism underlying oxidative stress-induced AR activation in CRPC. Transcriptional activity of the prostate-specific antigen (PSA) promoter was evaluated using a dual-luciferase reporter assay under various treatments. To identify AR-interacting proteins under oxidative stress, TurboID-mediated proximity biotin labeling coupled with mass spectrometry was employed following H₂O₂ exposure. Protein-protein interactions between AR and ubiquitin-specific peptidase 36 (USP36) were validated by co-immunoprecipitation (Co-IP). Subcellular AR expression was assessed via Immunofluorescence in PCa cells. Low doses H₂O₂ (10 and 20 μM) enhanced viability and induced oxidative stress in PCa cells, and these concentrations were therefore selected for subsequent experiments. H₂O₂ treatment activated the AR-PSA signaling axis. The deubiquitinating enzyme USP36 was identified among the proteins that interact with AR upon H₂O₂ stimulation. Co-IP confirmed the specific binding between AR and USP36. Functional studies revealed that USP36 deubiquitinates and stabilizes AR. Notably, knockdown of USP36 abolished H₂O_2-induced activation of the AR-PSA pathway. H₂O₂ promotes the interaction between USP36 and AR, resulting in AR stabilization, transcriptional activation of PSA, and conferring androgen resistance. These findings provide mechanistic insights into how oxidative stress reactivates AR signaling in PCa and highlight potential therapeutic strategies for different stages of PCa.