UC Davis

Prostate cancer with distant metastasis (mPCa) is associated with poor prognosis. First-line treatment for localized PCa includes radical prostatectomy (RP) for high-risk disease. Many patients experience androgen receptor (AR)-associated increase of prostate specific antigen (PSA) levels in the serum, aka biochemical recurrence (BCR), increasing potential for mPCa progression. PCa cells that develop resistance to AR based therapies, along with chemotherapy, immunotherapy and radiotherapy, are associated with significant side effects and low overall survival. This dissertation assembles information linking the regulation of BCR to AR-associated metabolic pathways involved with metastatic progression. My goal was to identify metabolic pathways that disrupt BCR and determine potential targets of therapy that eliminate prostate cancer cells unaffected by AR inhibitors.In efforts to achieve this goal, I first conducted metabolomic analysis of prostate tissue from the tumors of 74 patients who underwent prostatectomy as treatment for localized PCa and correlated levels of metabolites with clinical and non-clinical factors. In this process, I identified that elevated levels of 2- hydroxyglutarate (2-HG) in the tumors reduced time-to-recurrence. Exogenous addition of D-2-HG, an enantiomer of 2-HG upregulated in PCa tumors from patients who experience BCR, increased the growth rate of LNCaP and C4 prostate tumor cell lines along with phosphorylation of Akt and ERK.I next studied targets of 2-HG that may affect the course of PCa progression, predominantly through use two castration resistant PCa (CRPC) sublines of hormone sensitive prostate cells (HSPCs)– (i) LNCaP-derived C4 cells, sensitive to AR inhibitors such as enzalutamide; (ii) CWR22-derived 22Rv1 cells, insensitive to enzalutamide. Collaborator-developed small molecule compounds were tested on cell lines of varying AR-sensitivity, including a benzimidazole-based molecule LLS139, observed to significantly decrease cell growth when treated intracellularly at its IC50. LLS139 treatment sensitized the more metastatically aggressive 22Rv1 cells to enzalutamide treatment compared to C4 cells while normal prostate-derived RWPE-1 and Normal Dermal Fibroblasts appear unaffected, highlighting potential in LLS139 as a treatment with reduced side-effects.RNA sequencing and qPCR validation highlighted several genes potentially contributing to mechanism(s) involved in the observed inter and intra differences between C4 and 22Rv1, such as upregulated GPNMB (glycoprotein non-metastatic B; aka HGFIN or osteoactivin)- a type 1 transmembrane glycoprotein expressed on the surface of cells or lysosomes. GPNMB was upregulated following LLS139 treatment of both C4 and 22Rv1 cells yet more significantly in 22Rv1 cells than with C4 cells, supporting drug validation.Proteomics assessment of co-immunoprecipitation experiments yielded Akt2 as a potential binding partner to LLS139. CCT12890 AKT2 inhibitor was employed with GPNMB siRNA transfections to help establish potential protein involvement. GPNMB knockdowns decreased GPNMB and reduced cell death, as observed through MTT experiments, significantly in 22RV1 cells but insignificantly in C4 cells. Treatment efficacy was significantly impacted by the Akt2 inhibitor for both C4 and 22RV1’s, appearing to mimic LLS139 treatment but observed to be less impactful on 22Rv1 than C4 cells. Combinations on inhibitor and siRNA proved fatal for both cell populations as no cell growth was observed. The above results indicate that LLS139 is likely a significant therapeutic agent in AR-insensitive PCa cells that target Akt2, operating through upregulation of the putative tumor suppressor GPNMB.