Serum Lipids prior to Starting Androgen Deprivation Therapy and Risk of Castration Resistant Prostate Cancer and Metastasis: Results from the SEARCH Database.

PURPOSE: We tested the association of serum lipid levels prior to androgen deprivation therapy with the risk of castration resistant prostate cancer and metastasis. MATERIALS AND METHODS: We identified 302 men in the SEARCH (Shared Equal Access Regional Cancer Hospital) database who received androgen deprivation therapy after radical prostatectomy for nonmetastatic disease, had never received statins before androgen deprivation therapy and had available serum lipid data within 2 years prior to androgen deprivation therapy. Cox proportional hazards models were used to test associations between total cholesterol (less than 200 vs 200 mg/dl or greater), low density lipoprotein (less than 130 vs 130 mg/dl or greater), high density lipoprotein (40 or greater vs less than 40 mg/dl) and triglycerides (less than 150 vs 150 mg/dl or greater) and the risk of castration resistant prostate cancer and metastasis after androgen deprivation therapy while adjusting for potential confounders. Subanalyses were restricted to men who remained statin nonusers after androgen deprivation therapy. RESULTS: Median followup after androgen deprivation therapy was 67 months. Castration resistant prostate cancer and metastasis developed in 42 and 44 men, respectively. Men with elevated cholesterol received androgen deprivation therapy in an earlier year and had longer followup and a higher rate of statin use after androgen deprivation therapy. On multivariable analysis total cholesterol and low density lipoprotein were unrelated to castration resistant prostate cancer. Low high density lipoprotein (less than 40 vs 40 mg/dl or greater) was suggestively linked to an increased risk of castration resistant prostate cancer (HR 1.86, 95% CI 0.99-3.48). The association was stronger in men who remained statin nonusers after androgen deprivation therapy (HR 3.64, 95% CI 1.45-9.17). Results for metastasis were similar to those for castration resistant prostate cancer. CONCLUSIONS: Among men with nonmetastatic prostate cancer who started androgen deprivation therapy serum cholesterol was unrelated to castration resistant prostate cancer or metastasis. Low high density lipoprotein was suggestively associated with risks of increased castration resistant prostate cancer and metastasis, particularly in statin never users. Further studies are needed to explore a potential role for lipids in prostate cancer progression after androgen deprivation therapy.

Evidence for Feedback Regulation Following Cholesterol Lowering Therapy in a Prostate Cancer Xenograft Model.

BACKGROUND: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS: Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS: Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS: Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

The microRNA-183 cluster: the family that plays together stays together.

The microRNA (miR)183 cluster, which is comprised of miRs-183, -96 and -182, is also a miR family with sequence homology. Despite the strong similarity in the sequences of these miRs, minute differences in their seed sequences result in both overlapping and distinct messenger RNA targets, which are often within the same pathway. These miRs have tightly synchronized expression during development and are required for maturation of sensory organs. In comparison to their defined role in normal development, the miR-183 family is frequently highly expressed in a variety of non-sensory diseases, including cancer, neurological and auto-immune disorders. Here, we discuss the conservation of the miR-183 cluster and the functional role of this miR family in normal development and diseases. We also describe the regulation of vital cellular pathways by coordinated expression of these miR siblings. This comprehensive review sheds light on the likely reasons why the genomic organization and seeming redundancy of the miR-183 family cluster was conserved through 600 million years of evolution.

27-hydroxycholesterol and DNA damage repair: implication in prostate cancer.

Introduction: We previously reported that cholesterol homeostasis in prostate cancer (PC) is regulated by 27-hydroxycholesterol (27HC) and that CYP27A1, the enzyme that converts cholesterol to 27HC, is frequently lost in PCs. We observed that restoring the CYP27A1/27HC axis inhibited PC growth. In this study, we investigated the mechanism of 27HC-mediated anti-PC effects. Methods: We employed in vitro models and human transcriptomics data to investigate 27HC mechanism of action in PC. LNCaP (AR+) and DU145 (AR-) cells were treated with 27HC or vehicle. Transcriptome profiling was performed using the Affymetrix GeneChip™ microarray system. Differential expression was determined, and gene set enrichment analysis was done using the GSEA software with hallmark gene sets from MSigDB. Key changes were validated at mRNA and protein levels. Human PC transcriptomes from six datasets were analyzed to determine the correlation between CYP27A1 and DNA repair gene expression signatures. DNA damage was assessed via comet assays. Results: Transcriptome analysis revealed 27HC treatment downregulated Hallmark pathways related to DNA damage repair, decreased expression of FEN1 and RAD51, and induced "BRCAness" by downregulating genes involved in homologous recombination regulation in LNCaP cells. Consistently, we found a correlation between higher CYP27A1 expression (i.e., higher intracellular 27HC) and decreased expression of DNA repair gene signatures in castration-sensitive PC (CSPC) in human PC datasets. However, such correlation was less clear in metastatic castration-resistant PC (mCRPC). 27HC increased expression of DNA damage repair markers in PC cells, notably in AR+ cells, but no consistent effects in AR- cells and decreased expression in non-neoplastic prostate epithelial cells. While testing the clinical implications of this, we noted that 27HC treatment increased DNA damage in LNCaP cells via comet assays. Effects were reversible by adding back cholesterol, but not androgens. Finally, in combination with olaparib, a PARP inhibitor, we showed additive DNA damage effects. Discussion: These results suggest 27HC induces "BRCAness", a functional state thought to increase sensitivity to PARP inhibitors, and leads to increased DNA damage, especially in CSPC. Given the emerging appreciation that defective DNA damage repair can drive PC growth, future studies are needed to test whether 27HC creates a synthetic lethality to PARP inhibitors and DNA damaging agents in CSPC.

27-Hydroxycholesterol Impairs Plasma Membrane Lipid Raft Signaling as Evidenced by Inhibition of IL6-JAK-STAT3 Signaling in Prostate Cancer Cells.

We recently reported that restoring the CYP27A1-27hydroxycholesterol axis had antitumor properties. Thus, we sought to determine the mechanism by which 27HC exerts its anti-prostate cancer effects. As cholesterol is a major component of membrane microdomains known as lipid rafts, which localize receptors and facilitate cellular signaling, we hypothesized 27HC would impair lipid rafts, using the IL6-JAK-STAT3 axis as a model given its prominent role in prostate cancer. As revealed by single molecule imaging of DU145 prostate cancer cells, 27HC treatment significantly reduced detected cholesterol density on the plasma membranes. Further, 27HC treatment of constitutively active STAT3 DU145 prostate cancer cells reduced STAT3 activation and slowed tumor growth in vitro and in vivo. 27HC also blocked IL6-mediated STAT3 phosphorylation in nonconstitutively active STAT3 cells. Mechanistically, 27HC reduced STAT3 homodimerization, nuclear translocation, and decreased STAT3 DNA occupancy at target gene promoters. Combined treatment with 27HC and STAT3 targeting molecules had additive and synergistic effects on proliferation and migration, respectively. Hallmark IL6-JAK-STAT gene signatures positively correlated with CYP27A1 gene expression in a large set of human metastatic castrate-resistant prostate cancers and in an aggressive prostate cancer subtype. This suggests STAT3 activation may be a resistance mechanism for aggressive prostate cancers that retain CYP27A1 expression. In summary, our study establishes a key mechanism by which 27HC inhibits prostate cancer by disrupting lipid rafts and blocking STAT3 activation. IMPLICATIONS: Collectively, these data show that modulation of intracellular cholesterol by 27HC can inhibit IL6-JAK-STAT signaling and may synergize with STAT3-targeted compounds.

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.

microRNAs and DICER1 are regulated by 1,25-dihydroxyvitamin D in prostate stroma.

Vitamin D deficiency increases the risk of lethal prostate adenocarcinomas (PCa) and the majority of older men are deficient. Although PCa arises from the epithelium, the surrounding stroma has hormonal regulatory control over the epithelium and contributes to carcinogenesis. Herein, we describe regulation of microRNAs (miRs) by the active hormone dihydroxyvitamin D (1,25(OH)2D) in human prostate stroma. 1,25(OH)2D binds the vitamin D receptor (VDR) transcription factor to regulate gene expression, including miRs, which have emerged as potent regulators of protein expression. 1,25(OH)2D-regulated miRs were identified by profiling in primary human prostatic stromal cells (PrS) and three miRs, miR-126-3p, miR 154-5p and miR-21-5p were subsequently validated in laser-capture micro-dissected prostate stromal tissue from a vitamin D3 clinical trial (N=45). Regulation of these miRs by 1,25(OH)2D was VDR-dependent. Network analysis of known and putative mRNA targets of these miRs was enriched with cancer and inflammation pathways, consistent with known roles of stroma and of vitamin D in carcinogenesis. Expression of the miR processing ribonuclease, DICER1, positively correlated with vitamin D metabolite levels in the clinical trial specimens. High epithelial/stromal ratios of DICER1 were significantly associated biochemical recurrence (OR 3.1, p=0.03) in a tissue microarray of 170 matched PCa patients. In summary, these results underscore the role of the prostate stroma in regulating responses to the hormone 1,25(OH)2D and identified miRs and DICER1 as being regulated in human prostate stroma. Regulation of stromal DICER1 by 1,25(OH)2D may also have clinical relevance in protection against aggressive PCa.

miR-182, of the miR-183 cluster family, is packaged in exosomes and is detected in human exosomes from serum, breast cells and prostate cells.

Members of the microRNA (miR)-183 family are expressed at high levels in the majority of cancer types, including breast and prostate, and are considered 'oncomiRs'. The purpose of the present study was to investigate the role of exosomes in cell-to-cell transfer of the miR-183 family, which includes miRs-96, -182 and -183. Despite highly detectable levels of these three miRs within prostate and breast cells in vitro, only miR-182 was detectable in exosomes isolated from cell culture supernatant. Similar to the in vitro results, miR-182 was the only miR detected in exosomes isolated from fresh human serum. The packaging of miR-182 into exosomes was examined in MDA-MB-231 (MDA-182) breast cancer cells with miR-182 overexpression. Levels of mature miR-182 increased in exosomes in a dose-dependent manner compared to intracellular expression. Furthermore, co-culture of MDA-182 cells with naïve MDA-MB-231 cells resulted in an increase in mature miR-182 in the naïve cells, which was blocked by a chemical inhibitor of microvesicle formation. In summary, the present study demonstrates that of the miR-183 family members, miR-182 is preferentially packaged in exosomes, detectable in exosomes from human sera and may be transferred between cells via a microvesicle-dependent mechanism.

Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro model.

Previous work on vitamin D in the prostate has focused on the prostatic epithelium, from which prostate cancer arises. Prostatic epithelial cells are surrounded by stroma, which has well-established regulatory control over epithelial proliferation, differentiation, and the inflammatory response. Here we examined the regulation of vitamin D-related genes and inflammatory genes by 1α,25-dihydroxyvitamin D3 (1,25(OH)2D) in laser-capture microdissected prostate tissue from a vitamin D3 clinical trial and in an in vitro model that facilitates stromal-epithelial crosstalk. Analysis of the trial tissues showed that VDR was present in both cell types, whereas expression of the hydroxylases was the highest in the epithelium. Examination of gene expression by prostatic (1,25(OH)2D) concentrations showed that VDR was significantly lower in prostate tissues with the highest concentration of 1,25(OH)2D, and down-regulation of VDR by 1,25(OH) 2D was confirmed in the primary cell cultures. Analysis of inflammatory genes in the patient tissues revealed that IL-6 expression was the highest in the prostate stroma while PTGS2 (COX2) levels were lowest in the prostate cancer tissues from men in the highest tertile of prostatic 1,25(OH)2D. In vitro, TNF-α, IL-6 and IL-8 were suppressed by 1,25 (OH)2D in the primary epithelial cells, whereas TNF-α and PTGS2 were suppressed by 1,25(OH) 2D in the stromal cells. Importantly, the ability of 1,25(OH)2D to alter pro-inflammatory-induced changes in epithelial cell growth were dependent on the presence of the stromal cells. In summary, whereas both stromal and epithelial cells of the prostate express VDR and can presumably respond to 1,25(OH)2D, the prostatic epithelium appears to be the main producer of 1,25(OH)2D. Further, while the prostate epithelium was more responsive to the anti-inflammatory activity of 1,25 (OH)2D than stromal cells, stroma-epithelial crosstalk enhanced the phenotypic effects of 1,25(OH)2D and the inflammatory process in the prostate gland.