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.

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.

Disparate adjuvant properties among three formulations of "alum".

Aluminum adjuvants, commonly referred to as "alum," are the most widespread immunostimulants in human vaccines. Although the mechanisms that promote humoral responses to alum-adsorbed antigens are still enigmatic, alum is thought to form antigen depots and induce inflammatory signals that, in turn, promote antibody production. It was recently noted that Imject(®) alum, a commercial aluminum-containing adjuvant commonly used in animal studies, is not the physicochemical equivalent of aluminum adjuvant present in human vaccines. This difference raises concerns about the use of Imject(®) alum in animal research as a model for approved aluminum adjuvants. Here, we compared the capacity of Imject(®) alum, Alhydrogel(®), and a traditional alum-antigen precipitate to induce humoral responses in mice to the hapten-carrier antigen, NP-CGG [(4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin]. The magnitude of humoral responses elicited by Alhydrogel(®) and precipitated alum was significantly greater than that induced by Imject(®) alum. The strength of the humoral responses elicited by different alum formulations was correlated with the quantity of pro-inflammatory cytokines induced and the numbers of inflammatory cells at the site of immunization. Moreover, Imject(®) exhibited a severely reduced capacity to adsorb protein antigens compared to Alhydrogel(®) and precipitated alum. These findings reveal substantial differences in the immunostimulatory properties of distinct alum preparations, an important point of consideration for the evaluation of novel adjuvants, the assessment of new alum-based vaccines, and in mechanistic studies of adjuvanticity.