Erratum: Vitamin D sufficiency enhances differentiation of patient-derived prostate epithelial organoids.

[This corrects the article DOI: 10.1016/j.isci.2020.101974.].

Vitamin D sufficiency enhances differentiation of patient-derived prostate epithelial organoids.

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.

Oncogenic and tumor-suppressive microRNAs in prostate cancer.

MicroRNAs are known to be dysregulated in prostate cancer. These small noncoding RNAs can function as biomarkers and are involved in the biology of prostate cancer. The canonical mechanism for microRNAs is post-transcription regulation of gene expression via binding to the 3' untranslated region of mRNAs, resulting in RNA degradation and/or translational repression. Thus, oncogenic microRNAs, also known as oncomiRs, often have high expression in prostate cancer and target the mRNAs of tumor suppressors. Conversely, tumor-suppressive microRNAs have reduced expression in cancer and typically target oncogenes. Some microRNAs function outside the classical mechanism and serve to stabilize their mRNA targets. Herein, we review contemporary studies that demonstrate oncogenic and tumor-suppressive activity of microRNAs in prostate cancer.

Single-cell RNA-Seq analysis identifies a putative epithelial stem cell population in human primary prostate cells in monolayer and organoid culture conditions.

Human primary prostate epithelial (PrE) cells represent patient-derived in vitro models and are traditionally grown as a monolayer in two-dimensional culture. It has been recently demonstrated that expansion of primary cells into three-dimensional prostatic organoids better mimics prostate epithelial glands by recapitulating epithelial differentiation and cell polarity. Here, we sought to identify cell populations present in monolayer PrE cells and organoid culture, grown from the same patient, using single-cell RNA-sequencing. Single-cell RNA-sequencing is a powerful tool to analyze transcriptome profiles of thousands of individual cells simultaneously, creating an in-depth atlas of cell populations within a sample. Organoids consisted of six distinct cell clusters (populations) of intermediate differentiation compared to only three clusters in the monolayer prostate epithelial cells. Integrated analysis of the datasets allowed for direct comparison of the monolayer and organoid samples and identified 10 clusters, including a distinct putative prostate stem cell population that was high in Keratin 13 (KRT13), Lymphocyte Antigen 6D (LY6D), and Prostate Stem Cell Antigen (PSCA). Many of the genes within the clusters were validated through RT-qPCR and immunofluorescence in PrE samples from 5 additional patients. KRT13+ cells were observed in discrete areas of the parent tissue and organoids. Pathway analyses and lack of EdU incorporation corroborated a stem-like phenotype based on the gene expression and quiescent state of the KRT13+ cluster. Other clusters within the samples were similar to epithelial populations reported within patient prostate tissues. In summary, these data show that the epithelial stem population is preserved in PrE cultures, with organoids uniquely expanding intermediate cell types not present in monolayer culture.

High levels of PIWI-interacting RNAs are present in the small RNA landscape of prostate epithelium from vitamin D clinical trial specimens.

BACKGROUND: Vitamin D, a hormone that acts through the nuclear vitamin D receptor (VDR), upregulates antitumorigenic microRNA in prostate epithelium. This may contribute to the lower levels of aggressive prostate cancer (PCa) observed in patients with high serum vitamin D. The small noncoding RNA (ncRNA) landscape includes many other RNA species that remain uncharacterized in prostate epithelium and their potential regulation by vitamin D is unknown. METHODS: Laser capture microdissection (LCM) followed by small-RNA sequencing was used to identify ncRNAs in the prostate epithelium of tissues from a vitamin D-supplementation trial. VDR chromatin immunoprecipitation-sequencing was performed to identify vitamin D genomic targets in primary prostate epithelial cells. RESULTS: Isolation of epithelium by LCM increased sample homogeneity and captured more diversity in ncRNA species compared with publicly available small-RNA sequencing data from benign whole prostate. An abundance of PIWI-interacting RNAs (piRNAs) was detected in normal prostate epithelium. The obligate binding partners of piRNAs, PIWI-like (PIWIL) proteins, were also detected in prostate epithelium. High prostatic vitamin D levels were associated with increased expression of piRNAs. VDR binding sites were located near several ncRNA biogenesis genes and genes regulating translation and differentiation. CONCLUSIONS: Benign prostate epithelium expresses both piRNA and PIWIL proteins, suggesting that these small ncRNA may serve an unknown function in the prostate. Vitamin D may increase the expression of prostatic piRNAs. VDR binding sites in primary prostate epithelial cells are consistent with its reported antitumorigenic functions and a role in ncRNA biogenesis.

Association of High miR-182 Levels with Low-Risk Prostate Cancer.

A subset of men with prostate cancer develops aggressive disease. We sought to determine whether miR-182, an miRNA with reported oncogenic functions in the prostate, is associated with biochemical recurrence and aggressive disease. Prostate epithelial miR-182 expression was quantified via in situ hybridization of two prostate tissue microarrays and by laser-capture microdissection of prostate epithelium. miR-182 was significantly higher in cancer epithelium than adjacent benign epithelium (P < 0.0001). The ratio of cancer to benign miR-182 expression per patient was inversely associated with recurrence in a multivariate logistic regression model (odds ratio = 0.18; 95% CI, 0.03-0.89; P = 0.044). Correlation of miR-182 with mRNA expression in laser-capture microdissected benign prostate epithelium was used to predict prostatic miR-182 targets. Genes that were negatively correlated with miR-182 were enriched for its predicted targets and for genes previously identified as up-regulated in prostate cancer metastases. miR-182 expression was also negatively correlated with genes previously identified as up-regulated in primary prostate tumors from African American patients, who are at an increased risk of developing aggressive prostate cancer. Taken together, these results suggest that although miR-182 is expressed at higher levels in localized prostate cancer, its levels are lower in aggressive cancers, suggesting a biphasic role for this miRNA that may be exploited for prognostic and/or therapeutic purposes to reduce prostate cancer progression.

The miR-183 family cluster alters zinc homeostasis in benign prostate cells, organoids and prostate cancer xenografts.

The miR-183 cluster, which is comprised of paralogous miRs-183, -96 and -182, is overexpressed in many cancers, including prostate adenocarcinoma (PCa). Prior studies showed that overexpression of individual pre-miRs-182, -96 and -183 in prostate cells decreased zinc import, which is a characteristic feature of PCa tumours. Zinc is concentrated in healthy prostate 10-fold higher than any other tissue, and an >80% decrease in zinc is observed in PCa specimens. Here, we studied the effect of overexpression of the entire 4.8 kb miR-183 family cluster, including the intergenic region which contains highly conserved genomic regions, in prostate cells. This resulted in overexpression of mature miR-183 family miRs at levels that mimic cancer-related changes. Overexpression of the miR-183 cluster reduced zinc transporter and intracellular zinc levels in benign prostate cells, PCa xenografts and fresh prostate epithelial organoids. Microarray analysis of miR-183 family cluster overexpression in prostate cells showed an enrichment for cancer-related pathways including adhesion, migration and wound healing. An active secondary transcription start site was identified within the intergenic region of the miR-183 cluster, which may regulate expression of miR-182. Taken together, this study shows that physiologically relevant expression of the miR-183 family regulates zinc levels and carcinogenic pathways in prostate cells.

Hypoxia-inducible factor-1α promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3.

The function of hypoxia-inducible factor-1α (HIF-1α) in chronic kidney disease is disputed. Here we report that interactions of HIF-1α with transforming growth factor-ß (TGF-ß) signaling may promote its fibrotic effects. Knockout of HIF-1α is protective against glomerulosclerosis and glomerular type-I collagen accumulation in a mouse podocyte ablation model. Transcriptional analysis of cultured renal cells showed that α2(I) collagen expression is directly regulated by HIF-1α binding to a functional hypoxia-responsive element in its promoter at -335 relative to the transcription start site. Activation of COL1A2 transcription by HIF-1α occurred in the absence of hypoxia and is strongly enhanced by TGF-ß signaling. TGF-ß, in addition to increasing HIF-1α levels, increased both HIF-1α binding to the COL1A2 promoter and HIF-1α N-terminal transactivation domain activity. These effects of TGF-ß on HIF-1α were inhibited in Smad3-null mouse embryonic fibroblasts, suggesting a requirement for Smad3. Phosphorylated Smad3 also associated with the -335 hypoxia-responsive element of the COL1A2 promoter independent of a Smad DNA binding sequence. Smad3 binding to the -335 hypoxia-responsive element required HIF-1α both in vitro and in kidney lysate from the disease model, suggesting formation of an HIF-1α-Smad3 transcriptional complex. Thus, HIF-1α-Smad3 has a novel interaction in glomerulosclerosis.

Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1ß and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These results are the first to demonstrate that salmon thrombin has unique analgesic, neuroprotective and anti-inflammatory capabilities compared to human thrombin and that PAR1 may contribute to these actions.