Characterization of Udp-Glucuronate Partitioning in Androgen-Dependent and Castration Resistant Prostate Cancer Reveals Trends for Therapeutic Targets

Prostate epithelial cells rely on a tight balance of androgens to regulate male reproduction, development, and function. To maintain ideal androgen levels, prostatespecific UDP-glucuronosyltransferases (UGT) 2B15 and 2B17 use UDP-glucuronate (UDP-glcA) to inactivate and solubilize excess androgens for elimination. UDP-glcA is generated from UDP-glucose by a two-step NAD+-dependent oxidation catalyzed by UDP-glucose dehydrogenase (UGDH). Androgen stimulated UGDH drives UDP-glcA production and UGT-catalyzed glucuronidation. Here we evaluated glucuronidation potential by characterizing UDP-glcA flux towards androgen glucuronidation. First, an isogenic LNCaP model was used to compare the response of an androgen dependent versus castration resistant prostate cancer (CRPC) to available androgens. Despite a significantly lower glucuronide output, LNCaP 81 cells expressed higher levels of UGDH, UGT2B15, and UGT2B17. However, these cells exhibited blunted liganddependent AR gene response, observed in the magnitude of androgen-activated UGDH and PSA and androgen-repressed UGT2B15/17 expression. Furthermore, ligandactivated binding of AR to the PSA promoter was significantly reduced in the CRPC cells. Steady state and androgen-responsive metabolite analysis revealed channeling of UDP-glcA through the proteoglycan and glycosaminoglycan biosynthetic pathways (instead of androgen glucuronidation), leading to increased surface expression of Notch1. Knocking down UGDH in an androgen-dependent line removed Notch1 and increased the glucuronide output. The second part of this study characterized UGDH’s role in androgen glucuronidation. Steady state and androgen-dependent analyses of steroid clearance in a UGDH knockdown context were performed. The steady state knockdown of UGDH resulted in increased androgen-mediated PSA and UGT2B17 expression, androgen-glucuronide production, and decreased UDP-glcA accumulation. UGDH knockdown also showed increased magnitude of ligand-dependent AR gene expression, particularly with a 5-6-fold stimulation of PSA expression, 55% greater repression of UGT2B17 compared to the controls as well as increased ligand-dependent androgen glucuronidation. Surprisingly, prolonged depletion of exogenous androgens increased expression of the steroid clearing enzymes and the androgen-glucuronides. However, subsequent treatment (48 h) with saturating steroids revealed a higher ligand-dependent AR gene response and a more pronounced prioritization to glucuronidation. Finally, UGDH knockdown stunted androgen-dependent cell growth regardless of available androgens. Overall, these results characterize androgen regulated UDP-glucuronate partitioning and glucuronidation potential