Everardo Macias

CYP27A1 Loss Dysregulates Cholesterol Homeostasis in Prostate Cancer

In this study, we used a bioinformatic approach to identify genes whose expression is dysregulated in human prostate cancers. One of the most dramatically downregulated genes identified encodes CYP27A1, an enzyme involved in regulating cellular cholesterol homeostasis. Importantly, lower CYP27A1 transcript levels were associated with shorter disease-free survival and higher tumor grade. Loss of CYP27A1 in prostate cancer was confirmed at the protein level by immunostaining for CYP27A1 in annotated tissue microarrays. Restoration of CYP27A1 expression in cells where its gene was silenced attenuated their growth in vitro and in tumor xenografts. Studies performed in vitro revealed that treatment of prostate cancer cells with 27-hydroxycholesterol (27HC), an enzymatic product of CYP27A1, reduced cellular cholesterol content in prostate cancer cell lines by inhibiting the activation of sterol regulatory-element binding protein 2 and downregulating low-density lipoprotein receptor expression. Our findings suggest that CYP27A1 is a critical cellular cholesterol sensor in prostate cells and that dysregulation of the CYP27A1/27HC axis contributes significantly to prostate cancer pathogenesis. Cancer Res; 77(7); 1662-73. ©2017 AACR.

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

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.

Impact of carbohydrate restriction in the context of obesity on prostate tumor growth in the Hi-Myc transgenic mouse model

Background:Previously, we showed that carbohydrate restriction with calorie restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction without calorie restriction on tumor development within the context of diet-induced obesity in the Hi-Myc transgenic mouse model of prostate cancer.Methods:Mice were randomized at 5 weeks of age to ad libitum western diet (WD; 40% fat, 42% carbohydrate; n=39) or ad libitum no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n=44). At age 3 or 6 months, mice were killed, prostates weighed and prostate histology, proliferation, apoptosis and macrophage infiltration evaluated by hematoxylin and eosin, Ki67, TUNEL and F4/80 staining, respectively. Body composition was assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western.Results:Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age, relative to the WD group (45 g vs 38g; P=0.008). Despite elevated body weights, serum monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-1α levels were lower in NCKD versus WD mice (P=0.046 and P=0.118, respectively), and macrophage infiltration was reduced in prostates of NCKD versus WD mice (P=0.028). Relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD versus WD mice. However, while mice randomized to NCKD had smaller prostates after adjustment for body weight at 3 and 6 months (P=0.004 and P=0.002, respectively), NCKD mice had higher rates of adenocarcinoma at 6 months compared to WD mice (100 vs 80%, P=0.04).Conclusions:Despite higher caloric intake and elevated body weights, carbohydrate restriction lowered serum MCP-1 levels, reduced prostate macrophage infiltration, reduced prostate weight, but failed to slow adenocarcinoma development. Together, these data suggest that although carbohydrate restriction within the context of obesity may reduce obesity-associated systemic inflammation and perhaps slow tumor growth, it is not sufficient to counteract obesity-associated tumor development.

Serum cholesterol levels and tumor growth in a PTEN-null transgenic mouse model of prostate cancer

BackgroundSome, but not all, epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone synthesis, in prostate cancer. Using a PTEN-null transgenic mouse model of prostate cancer, we tested the effect of modifying serum cholesterol levels on prostate tumor development and growth. We hypothesized that serum cholesterol reduction would lower tumor androgens and slow prostate cancer growth.MethodsPTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (30 mg/kg/day), or no intervention, and sacrificed at 2, 3, or 4 months of age. Serum cholesterol and testosterone were measured by ELISA and intraprostatic androgens by mass spectrometry. Prostate histology was graded, and proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL, respectively.ResultsEzetimibe-treated mice had lower serum cholesterol at 4 months (p = 0.031). Serum cholesterol was positively correlated with prostate weight (p = 0.033) and tumor epithelial proliferation (p = 0.069), and negatively correlated with tumor epithelial apoptosis (p = 0.004). Serum cholesterol was unrelated to body weight (p = 0.195). Tumor stromal cell proliferation was reduced in the ezetimibe group (p = 0.010). Increased serum cholesterol at 4 months was associated with elevated intraprostatic DHEA, testosterone, and androstenedione (p = 0.043, p = 0.074, p = 0.031, respectively). However, cholesterol reduction did not significantly affect adenocarcinoma development at 2, 3, or 4 months of age (0, 78, and 100% in ezetimibe-treated vs. 0, 80, and 100% in mice not receiving ezetimibe).ConclusionsThough serum cholesterol reduction did not significantly affect the rate of adenocarcinoma development in the PTEN-null transgenic mouse model of prostate cancer, it lowered intraprostatic androgens and slowed tumor growth. These findings support a role for serum cholesterol in promoting prostate cancer growth, potentially via enhanced tumor androgen signaling, and may provide new insight into cholesterol-lowering interventions for prostate cancer treatment.

MP83-05 27-HYDROXYCHOLESTEROL INHIBITS PROSTATE CANCER GROWTH BY REDUCING STAT3 SIGNALING

INTRODUCTION AND OBJECTIVES: It has previously been shown that tumor-derived TGF-b related potent immunosuppression and lack of tumor killing specificity promote CaP progression. Here we report a new immunotherapeutic approach using adoptive transfer of patient-derived PSMA-specific, TGF-b-insensitive human CD8+ T cells to inhibit solid xenograft CaP. METHODS: Peripheral blood CD8+ T cells were collected from metastatic castration resistant CaP patient by leukapheresis and cultured in the FDA approved Cell Processing Work Station (CPWS, Panasonic) with CD-3 Biotin/CD28/Anti-Biotin Beads (Bead: Cell 1:2) and IL-2 (100units/ml). We developed a TbRIIDN-TK-IRES-PZ1 chimeric T cell receptor retroviral construct using an anti-PSMA IgTCR(z) gene (PZ1) and a dominant negative TGF-b type II receptor (TbRIIDN) that could induce CD8+ T cells to be PSMA reactive and insensitive to TGF-b. PC-3 cells (PSMA negative; ATCC) or PSMA positive PC-3-PSMA cells (Cleveland Clinic) were used for target cells. Subcutaneous injection of PC-3 and PC-3-PSMA cells (2x105 cells/ each, infected with HSV1-tk-GFP-luciferase reporter) into the left and right flank region respectively in each of 48 immunodeficient RAG-1 mice was performed. One week later, the animals were randomly assigned to one of three adoptive transfer groups (16 mice /each group, 2x106 CD8+ T cells/each mice): Group 1: CD8+ T cells infected with TbRIIDN-TK-IRES-PZ1 (71.1% positive); Group 2: Naive CD8+ T cells; Group 3: No treatment group. Tumor growth was monitored by IVIS luciferase imaging. The animals were provided 2 weekly adoptive transfer treatments and sacrificed after 3 weeks. The size and the weight of the tumor were recorded, and the infiltration of CD8+ T cells and apoptosis was evaluated by immunofluorescence staining and TUNEL. RESULTS: In Group 1, the average tumor weight and volume was significantly lower in the PC3-PSMA tumor (0.413g and 514.2mm3) compared to the PC3 tumor (2.75 g and 3165.2 mm3); (~3-6 , P<0.05). There was no difference between the PC3 tumor (2.36g and 2768.5mm3 ) compared to PC3-PSMA tumor (2.45g and 2411.99 mm3) in Group 2 or Group 3. H&E staining showed large amount of nuclear fusion, fragmentation and necrosis were found in PC3-PSMA tumors in Group 1 compared to Group 2 and 3. In Group 1, tumor apoptosis (72.5/1,000 mm2) and CD8+ T cell infiltration (45.5/1,000 mm2 ) in PC3PSMA tumor parenchyma was significantly higher compared to PC3 tumor (6.7/1,000 mm2 and 3.1/1,000 mm2 respectively). There was no significant apoptosis or CD8+ T cells infiltration observed in either PC3 and PC3-PSMA tumor in Group 2 and Group 3. CONCLUSIONS: Our approach combines TGF-b insensitive with PSMA selectivity that significantly enhance the specificity and tumor killing ability of patient CD8+ T cells, and simultaneously suppress the tumor derived TGF-binduced immunosuppression. Therefore, this construct may offer a novel therapeutic intervention for both primary CaP treatment as well as for recurrence after prostatectomy.

MP99-03 RIOK2 IS A MEDIATOR OF OBESITY ENHANCED PROSTATE CANCER GROWTH

INTRODUCTION AND OBJECTIVES: Extracellular vesicles (EVs) are microvesicles secreted from various cell types. EVs contain microRNAs, proteins, and mRNAs and play a role in intercellular communications via the mechanisms of exocytosis and endocytosis. We aimed to discover a new biomarker for high Gleason score (GS) prostate cancer (PCa) in urinary EVs via quantitative proteomics. METHODS: EVs were isolated from urine after massage from 18 men (negative biopsy [n 1⁄4 6], GS 6 PCa [n 1⁄4 6], or GS 8-9 PCa [n 1⁄4 6]). EV proteins were labeled with iTRAQ and analyzed by LC-MS/MS. Candidate proteins were further analyzed by selected reaction monitoring/multiple reaction monitoring (SRM/MRM). RESULTS: Proteins extracted from EVswere enriched with CD9 protein, which is a marker of EVs, compared with unprocessed urinary proteins. EVs labeled with anti-CD9 antibody conjugated with Au colloids were also confirmed by electron microscopy. We identified and quantified 3530 proteins in the urinary EVs by LC-MS/MS. Thirty-six proteins increased in patients with PCa compared to those with negative biopsy (ratio > 2.0, p < 0.1). Four proteins increased in patients with GS 8-9 PCa compared to those with negative biopsy or GS 6 PCa (ratio > 2.0, p < 0.1). Twenty-seven proteins were chosen for further analysis and verified in 29 independent urine samples (negative [n 1⁄4 11], PCa [n 1⁄4 18]) using SRM/MRM. Among these candidate markers, fatty acid binding protein 5 (FABP5) was higher in the cancer group than in the negative group (p 1⁄4 0.009) and was significantly associated with GS (p for trend 1⁄4 0.011). Univariate logistic analysis showed that FABP5 was significantly associated with prostate cancers with GS 7 or more (p< 0.001). Even after adjusting for age, PSA, and PSA density, FABP5 was significantly associated with prostate cancer with GS 7 or more (p 1⁄4 0.003). The receiver-operator characteristics curve analysis showed that the area under the curve (AUC) for the prediction of GS1⁄4 7 by FABP5 was 0.856 (95% CI 0.708e1.00, p 1⁄4 0.002), whereas the AUC value for prediction by serum PSA was 0.511 (95% CI 0.280e0.757, p 1⁄4 0.87). CONCLUSIONS: We applied the proteomic analysis to discover biomarkers in EVs in urine collected after prostate massage. FABP5 in urinary EVs could be a potential biomarker of high GS prostate cancer. Additional large-scale studies are warranted to confirm this finding.

Abstract 2970: Differential effects of caloric and carbohydrate restriction on prostate cancer in a mouse model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Prostate cancer (PC) is the most common non-cutaneous malignancy in men in the United States. Animal studies have shown that dietary interventions, specifically dietary restriction, demonstrate an ability to inhibit prostate tumor growth and progression. Classically, dietary restriction has come in the form of global caloric restriction (CR). CR, without malnutrition, has been consistently shown to be effective in reducing age-related conditions as well as decreasing the spontaneous incidence and progression of multiple cancers. In contrast to the generalized reduction of calories seen in CR, an alternative manner of dietary restriction would be to restrict specific macronutrients. In prior studies, we have tested the theory that specifically restricting carbohydrates could slow tumor growth by providing mice a diet deplete of carbohydrates (i.e. a no-carbohydrate Ketogenic diet or NCKD). As such, we previously found that in a prostate cancer xenograft study, mice fed a NCKD, in the presence or absence of weight loss, had improved mouse survival as well as slowed tumor growth with respect to a diet complete of carbohydrates (i.e. Western diet). Moreover, NCKD, like CR, appeared to inhibit tumor growth via depression of the insulin/IGF-1 axis. These series of studies suggest that CR and NCKD may act via overlapping molecular mechanisms that inhibit tumor growth, however, no study to date has directly compared the effects of carbohydrate versus caloric restriction. Therefore we sought to investigate the role of global CR and NCKD without CR on tumor growth side by side. In addition, we sought to determine whether calorie restriction of NCKD would provide an additional inhibitory effect (i.e. CR + NCDK). This study consisted of four arms: 1) Western Diet (WD); 2) NCKD (pair fed with WD); 3) western diet 25% calorie restricted (WD-CR) and 4) NCKD 25% CR (NCKD-CR). By day 52 after randomization, when median tumor volumes for WD mice were ∼1000 mm3, NCKD, Western CR and NCKD CR arms had 32.2%, 41.6%, and 62.1% smaller tumor volumes, respectively, relative to WD mice (all p<0.01). At sacrifice, both calorically restricted groups had significantly lower serum glucose levels than the non-calorically restricted arms. Moreover, IGF-1:IGFBP-3 levels of all active experimental arms (NCKD, WD-CR, and NCKD-CR) were significantly lower than WD mice. Among the two CR arms, IGF-1: IGFBP-3 ratios were lower in the NCKD-CR mice. These data suggest that CR and NCKD have similar efficacy in reducing tumor growth and both act, at least in part, via reductions in IGF-1. Also, the combination of CR plus NCKD had an additive effect. Global gene expression analysis of xenograft tumors revealed genes altered in a similar fashion across the three diets in comparison to WD, but also genes uniquely altered in individual diets. Further analysis of these genes will allow us to better understand the molecular mechanisms through which these dietary regimens inhibit tumor growth. Citation Format: Everardo Macias, Jean A. Thomas, Elizabeth M. Masko, Alexis R. Gaines, Brian Whitley, Tanisha Coburn, Susan L. Poulton, Tameika E. Phillips, Stephen J. Freedland. Differential effects of caloric and carbohydrate restriction on prostate cancer in a mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2014-2970