Prostate epithelial cells partly control the potency and availability of androgens by inactivating and excreting them through a process termed glucuronidation. Function of the glucuronidation pathway in prostate cancer is a significant component of the response to androgen deprivation therapy (ADT). The inevitable recurrence of prostate cancer on ADT (castration resistant prostate cancer, CRPC) has few treatment options and ultimately results in mortality. UDP-glucose dehydrogenase (UGDH) is required for glucuronidation and its elevated expression predicts ADT recurrence. Because UGDH is the sole enzyme responsible for synthesis of the UDP-glucuronate precursor for glucuronidation, UGDH is a promising target in the battle against CRPC. Though UDP-glucuronate has three fates in the prostate cell, we have shown that the manipulation of UGDH expression can selectively control activity of the glucuronidation pathway and resultant growth potential of tumor cells. In this collaborative proposal, we will advance work we have done to determine how activity of UGDH in the cell is controlled by the AGC kinase RSK3, and capitalize on a novel peptide inhibitor of UGDH we identified. Hypothesis: Selective inhibition of UGDH will delay, prevent, or reverse emergence of castration resistant hallmarks in prostate tumor cells, thereby optimizing response of prostate cancer to androgen deprivation therapy. Aim 1: Determine the efficacy of RSK3-UGDH combined inhibition on cellular androgen response and prostate tumor growth kinetics in vitro, using isogenic AD and CR cells and PDX models. Aim 2: Use a high throughput approach to identify next generation peptides and small molecule lead compounds for selective inhibition of UGDH.

UDP-glucose dehydrogenase (UGDH) provides an essential precursor for elimination of excess androgen hormones from the prostate by glucuronidation. Its expression is elevated in cancerous prostate glands within biopsied tissue, relative to normal glands and to normal appearing adjacent glands, in which expression is depressed. Using tissue microarrays, which consisted of pathologist-graded biopsy specimens collected from patients prior to beginning androgen deprivation therapy, we evaluated whether UGDH levels could predict response to therapy. The patients had been followed for five years and evaluated for response versus recurrence. Each microarray slide consisted of 100 biopsies. One slide contained specimens from patients who responded. The other slide contained those of patients who experienced recurrence. We immunostained the slides and quantified fluorescence intensity of cancerous and normal appearing adjacent glands in each specimen. All cancerous glands overexpressed UGDH relative to normal glands. Importantly, a significant difference between cancerous and normal adjacent glands was found to correlate with response to androgen deprivation therapy. These preliminary data suggest UGDH will be useful as a predictive biomarker for success of androgen deprivation therapy. Additional work by our lab demonstrated that UGDH expression can be manipulated to control intracellular androgen levels, suggesting it may also be a good therapeutic target to sustain strong androgen response during therapy, thereby sensitizing prostate tumor cells to drugs such as abiraterone or other anti-androgens. In this proposal, we will test this hypothesis by implanting androgen sensitive and castration resistant prostate tumor cells with manipulated UGDH expression into intact and castrated mice. We will evaluate tumor growth kinetics, as well as a complete endpoint profile of glucuronidation pathway proteins and metabolites to determine effects of UGDH modulation on hormone dependent tumor growth. Successful completion of these studies will functionally implicate UGDH in prostate cancer progression and highlight it as a new drug target.

UDP-glucose dehydrogenase (UGDH) is a unique, essential enzyme with the central role of providing UDPglucuronate,a rate-limiting precursor for plasma membrane hyaluronan synthesis, Golgi proteoglycan production, and ER-localized modification of hormones for elimination. Our laboratory has shown that insufficiency of UGDH contributes to loss of control of intracellular steroid levels, and dysregulated tumor cell growth rate in prostate cancer. It is not known how the cytosolic UDP-glucuronate is partitioned to itsrespective fates in the high levels needed for specifically timed product formation, nor how UGDH activity is controlled to limit competition with other pathways. Our hypothesis for this proposal is that hexameric and dimeric units of UGDH sense metabolic status of the cell and respond with increased or decreased enzymatic activity. Information for molecular sensing is conveyed partly through differential protein-protein interactions that occur upon exposure of the dimer-dimer interface. We will test this with two aims. Aim 1: Determine the functional outcome of specific UGDH interactions with components of the androgen elimination pathway. We will directly measure interactions of UGDH with hyaluronan synthase, the Golgi UDP-xylose transporter, and the ER UDP-glucuronate transporter, as the three proteins that mediate the demands for UDP-glucuronate flux. We will quantify UDP-glucuronate, steroid-glucuronide, notch glycosylation and hyaluronan production, which will respectively report overall UGDH activity, and functional distribution to the ER for androgen elimination, the Golgi for proteoglycan secretion, or the plasma membrane for hyaluronan synthesis. Aim 2: Characterize and validate the UGDH interactome using an unbiased approach. We will use mass spectrometry to identify proteins that differentially co-fractionate by size exclusion chromatography with our wellcharacterized hexameric and dimeric point mutants. As a complementary approach, we will identify proteins that cross-link with hexameric versus dimeric UGDH point mutants that incorporate a photo-activatable crosslinker as a non-natural amino acid. Validated interactions and/or post-translational modifications will be used to design strategies for selective partitioning of UDP-glucuronate to favor hormone elimination in future preclinical studies of prostate cancer.