Loss of GPR133 Promotes Enzalutamide Resistance in Prostate Cancer by Upregulating HSD3B1 and Intratumoral Androgen Synthesis

Background The progression of prostate cancer (PCa) to a castration‐resistant state (CRPC) remains a major clinical challenge. Resistance to second‐generation androgen receptor (AR) antagonists like enzalutamide often involves the reactivation of AR signaling, frequently through intratumoral androgen synthesis. The molecular drivers that regulate this adaptive resistance mechanism are not fully understood. GPR133 (also known as ADGRD1) is an adhesion G protein‐coupled receptor with emerging roles in various cancers, but its function in prostate cancer is unknown. While androgen signaling is classically mediated by the nuclear AR, GPR133 has recently been identified as a novel membrane androgen receptor, though its functional relationship with the AR pathway in prostate cancer is unknown. Methods We analyzed GPR133 expression in patient‐derived PCa tissues and its correlation with clinical outcomes using publicly available datasets and our patients' samples. We employed gain‐ and loss‐of‐function approaches in vitro to test whether GPR133 specifically mediates resistance to enzalutamide. RNA sequencing was used to identify downstream pathways regulated by GPR133. The role of the downstream effector HSD3B1 was assessed using siRNA‐mediated silencing. The therapeutic implications of GPR133 expression were validated in vivo using xenograft mouse models. Results GPR133 expression is significantly downregulated in prostate cancer tissue compared to benign tissue and is further decreased in CRPC. Low GPR133 expression correlates with poorer disease‐free survival. Silencing GPR133 conferred robust resistance to enzalutamide in vitro and in vivo. Conversely, overexpression of GPR133 could further sensitize cancer cells to enzalutamide. Mechanistically, loss of GPR133 transcriptionally upregulated key enzymes in the steroid hormone biosynthesis pathway, most notably HSD3B1. This upregulation led to elevated intracellular testosterone levels and sustained androgen receptor (AR) signaling, characterized by the persistent expression of AR target genes despite enzalutamide treatment. Silencing HSD3B1 reversed the enzalutamide resistance induced by GPR133 knockdown. Conclusions Our findings identify GPR133 as a novel tumor suppressor in prostate cancer. Loss of GPR133 expression is a key event in the progression to CRPC that promotes therapeutic resistance by activating the intratumoral androgen synthesis pathway. GPR133 may serve as a valuable prognostic biomarker and a potential therapeutic target for advanced prostate cancer.