Vitamin D is an essential steroid hormone that regulates systemic calcium homeostasis and cell fate decisions. The prostate gland is hormonally regulated, requiring steroids for proliferation and differentiation of secretory luminal cells. Vitamin D deficiency is associated with an increased risk of lethal prostate cancer, which exhibits a dedifferentiated pathology, linking vitamin D sufficiency to epithelial differentiation. To determine vitamin D regulation of prostatic epithelial differentiation, patient-derived benign prostate epithelial organoids were grown in vitamin D-deficient or -sufficient conditions. Organoids were assessed by phenotype and single-cell RNA sequencing. Mechanistic validation demonstrated that vitamin D sufficiency promoted organoid growth and accelerated differentiation by inhibiting canonical Wnt activity and suppressing Wnt family member DKK3. Wnt and DKK3 were also reduced by vitamin D in prostate tissue explants by spatial transcriptomics. Wnt dysregulation is a known contributor to aggressive prostate cancer, thus findings further link vitamin D deficiency to lethal disease.

Vitamin D deficiency is associated with an increased risk of prostate cancer mortality and is hypothesized to contribute to prostate cancer aggressiveness and disparities in African American populations. The prostate epithelium was recently shown to express megalin, an endocytic receptor that internalizes circulating globulin-bound hormones, which suggests regulation of intracellular prostate hormone levels. This contrasts with passive diffusion of hormones that is posited by the free hormone hypothesis. Here, we demonstrate that megalin imports testosterone bound to sex hormone-binding globulin into prostate cells. Prostatic loss of Lrp2 (megalin) in a mouse model resulted in reduced prostate testosterone and dihydrotestosterone (DHT) levels. Megalin expression was regulated and suppressed by 25-hydroxyvitamin D (25D) in cell lines, patient-derived prostate epithelial cells, and prostate tissue explants. In patients, the relationships between hormones support this regulatory mechanism, as prostatic DHT levels are higher in African American men and are inversely correlated with serum 25D status. Megalin levels are reduced in localized prostate cancer by Gleason grade. Our findings suggest that the free hormone hypothesis should be revisited for testosterone and highlight the impact of vitamin D deficiency on prostate androgen levels, which is a known driver of prostate cancer. Thus, we revealed a mechanistic link between vitamin D and prostate cancer disparities observed in African Americans.

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<p>S1. Accompanies Figure 1 and 2.</p><p>S2. Accompanies Figure 2. No differences in bitransgenic mouse prostate histology, weights and fertility.</p><p>S3. Relates to Figure 3. Vitamin D receptor and androgen receptor response elements in LRP2 promoter.</p><p>S4. Relates to Figure 3. Prostate slices express hormone response components and respond to 25D and T.</p><p>S5. Relates to Figure 4. Androgen levels by Gleason and Age.</p><p>S6. Relates to Figure 6. Expression of LRP2 by age and BCR in DFKZ and TCGA cohorts.</p><p>Table S1. Cell and tissue characteristics</p><p>Table S2. Primer sequences</p>

Prostate cancer is the second leading cause of malignancy-related deaths among American men; however, hundreds of thousands of men live with indolent disease. Active surveillance is a safe option for men with low-risk disease. Many patients are treated with radical prostatectomy or radiation therapy, both of which have associated morbidity. Herein, we sought to identify prostate-derived microRNAs in patient sera and serum extracellular vesicles, and determine if those microRNAs added to current clinical nomograms for prostate cancer prognosis pre- and post-biopsy. Intracellular and extracellular vesicle-contained microRNAs from prostate cancer patient samples were profiled by next-generation RNA sequencing. Abundant microRNAs were included in a custom microRNA PCR panel that was queried in whole serum and serum extracellular vesicles from a diverse cohort of men diagnosed with prostate cancer. Statistical modeling showed that the levels of these circulating microRNAs significantly differed between indolent and aggressive disease and added prognostic value to pretreatment nomograms for prostate cancer disease risk. The microRNAs within the extracellular vesicles had improved prognostic value compared to the microRNAs in the whole serum. In summary, quantifying microRNAs circulating in extracellular vesicles is a clinically feasible assay that may provide additional information for assessing prostate cancer risk stratification.