Dihydrotestosterone (DHT) and testosterone (T), which mediate androgen receptor nuclear translocation and target gene transcription, are crucial androgens and essential molecular triggers required for the proliferation and survival of prostate cancer cells. Therefore, androgen metabolism is commonly targeted in the treatment of prostate cancer. Using a high-pressure liquid chromatographic assay with tandem mass spectral detection, we determined the serum levels of metabolites produced during DHT/T biosynthesis in African American (AA) and European American (EA) men with localized, therapy naïve prostate cancer. Serum progesterone and related metabolites were significantly lower in AA men than in EA men, and these differences were associated with rapid disease progression. Multivariate analysis revealed significant differences between a subset of intermediate androgen metabolites between AA and EA men and between men with <=3 + 4 and >=4 + 3 Gleason score disease. AA men have a significantly higher frequency of single nucleotide polymorphisms in CYP11B1 and CYP11B2, enzymes that regulate corticosterone-aldosterone conversion. Finally, higher levels of T and pregnenolone were associated with a lower risk of progression-free survival only in AA men. This work provides new insight into androgen metabolism and racial disparities in prostate cancer by presenting evidence of dysregulated androgen biosynthesis in therapy naïve disease that correlates with clinical variables.

Vitamin D₃ is a steroid hormone that confers anti-tumorigenic properties in prostate cells. Serum vitamin D₃ deficiency has been associated with advanced prostate cancer (PCa), particularly affecting African American (AA) men. Therefore, elucidating the pleiotropic effects of vitamin D on signaling pathways, essential to maintaining non-malignancy, may provide additional drug targets to mitigate disparate outcomes for men with PCa, especially AA men. We conducted RNA sequencing on an AA non-malignant prostate cell line, RC-77N/E, comparing untreated cells to those treated with 10 nM of vitamin D₃ metabolite, 1α,25(OH)₂D₃, at 24 h. Differential gene expression analysis revealed 1601 significant genes affected by 1α,25(OH)₂D₃ treatment. Pathway enrichment analysis predicted 1α,25(OH)₂D_3- mediated repression of prostate cancer, cell proliferation, actin cytoskeletal, and actin-related signaling pathways (p < 0.05). Prioritizing genes with vitamin D response elements and associating expression levels with overall survival (OS) in The Cancer Genome Atlas Prostate Adenocarcinoma (TCGA PRAD) cohort, we identified ANLN (Anillin) and ECT2 (Epithelial Cell Transforming 2) as potential prognostic PCa biomarkers. Both genes were strongly correlated and significantly downregulated by 1α,25(OH)₂D₃ treatment, where low expression was statistically associated with better overall survival outcomes in the TCGA PRAD public cohort. Increased ANLN and ECT2 mRNA gene expression was significantly associated with PCa, and Gleason scores using both the TCGA cohort (p < 0.05) and an AA non-malignant/tumor-matched cohort. Our findings suggest 1α,25(OH)₂D₃ regulation of these biomarkers may be significant for PCa prevention. In addition, 1α,25(OH)₂D₃ could be used as an adjuvant treatment targeting actin cytoskeleton signaling and actin cytoskeleton-related signaling pathways, particularly among AA men.