Alexis R. Gaines

Association of uridine diphosphate-glucuronosyltransferase 2B gene variants with serum glucuronide levels and prostate cancer risk.

AIMS Uridine diphosphate-glucuronosyltransferase 2B (UGT2B) enzymes conjugate testosterone metabolites to enable their excretion in humans. The functional significance of the UGT2B genetic variants has never been described in humans. We evaluated UGT2B variants in relation to plasma androstane-3α,17β-diol-glucuronide (AAG) levels and the prostate cancer risk. RESULTS AAG levels were measured in sera from 150 controls and compared to the polymorphisms of UGT2B17, UGT2B15, and UGT2B7. Genomic DNA from controls (301) and cases (148) was genotyped for the polymorphisms, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated using unconditional logistic regression analyses. Having two copies of UGT2B17 was associated with higher AAG levels in controls among Whites (p=0.02), but not Blacks (p=0.82). Logistic regression models adjusting for age and race revealed that homozygosity for the G allele of the UGT2B15(D85Y) polymorphism was directly associated with the prostate cancer risk (OR=2.70, 95% CI=1.28, 5.55). CONCLUSIONS While the small sample size limits inference, our findings suggest that an association between the UGT2B17 copy number variant (CNV) and serum AAG levels in Whites, but unexpectedly not in Blacks. This novel observation suggests that genetic determinants of AAG levels in Blacks are unrelated to the UGT2B17 CNV. This study replicates the results that show an association of UGT215(D85Y) with an increased prostate cancer risk.

Associations between Intake of Folate, Methionine, and Vitamins B-12, B-6 and Prostate Cancer Risk in American Veterans

Prostate cancer (PC) is the second leading cause of cancer death in men. Recent reports suggest that excess of nutrients involved in the one-carbon metabolism pathway increases PC risk; however, empirical data are lacking. Veteran American men (272 controls and 144 PC cases) who attended the Durham Veteran American Medical Center between 2004–2009 were enrolled into a case-control study. Intake of folate, vitamin B12, B6, and methionine were measured using a food frequency questionnaire. Regression models were used to evaluate the association among one-carbon cycle nutrients, MTHFR genetic variants, and prostate cancer. Higher dietary methionine intake was associated with PC risk (OR = 2.1; 95%CI 1.1–3.9) The risk was most pronounced in men with Gleason sum <7 (OR = 2.75; 95%CI 1.32– 5.73). The association of higher methionine intake and PC risk was only apparent in men who carried at least one MTHFR A1298C allele (OR = 6.7; 95%CI = 1.6–27.8), compared to MTHFR A1298A noncarrier men (OR = 0.9; 95%CI = 0.24–3.92) (p-interaction = 0.045). There was no evidence for associations between B vitamins (folate, B12, and B6) and PC risk. Our results suggest that carrying the MTHFR A1298C variants modifies the association between high methionine intake and PC risk. Larger studies are required to validate these findings.

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

Abstract 3698: Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer.

Introduction: Western countries have 6 fold higher incidence of prostate cancer (PC). Dietary composition, in addition to other factors, may contribute to this geographic disparity. Cholesterol, the precursor for de novo steroid hormone synthesis, has been hypothesized to promote PC growth via multiple pathways including intratumoral steroidogenesis. While lowering serum cholesterol slows xenograft PC growth, this has not been tested in transgenic models, which allow testing of PC prevention as well as modulating PC growth in an orthotopic position. We determined the effect of cholesterol uptake inhibition, using ezetimibe, on PC growth in Hi-Myc and PTEN transgenic mice. Methods: We randomized 50 c-myc/wt (Hi-Myc) mice and 30 PTEN loxP/loxP -Cre + (PTEN) to one of two diets: high fat high cholesterol (HFHC; 40% fat, 17% protein, 43% carbohydrate, 1.25% cholesterol), or HFHC with ezetimibe (30 mg/kg food) (HFHC+Z). Mice were fed ad libitum starting right after weaning and body weights measured twice weekly. Prostate, liver, spleen, adipose tissue and serum were harvested from Hi-Myc and PTEN mice at age 6 and 4 months, respectively. Serum cholesterol and testosterone (T) and tumor T were measured in Hi-Myc mice and are ongoing in PTEN mice. Results: In Hi-Myc mice, body weights were significantly greater in the HFHC+Z group at sacrifice, relative to the HFHC group (p=0.04), but there were no differences in prostate weight between groups with adjustment for body weight (p=0.84). In PTEN mice, there was no difference in body weights between groups, but prostate weight was significantly lower in the HFHC+Z group, relative to the HFHC group (p=0.046). In Hi-Myc mice, while serum T levels did not vary between groups (p=0.93), tumor T, dihydrotestosterone (DHT) and androstenedione concentrations were significantly reduced in the HFHC+Z group, relative to the HFHC group (p=0.01, p=0.01, p=0.006, respectively). Discussion: Reduction of serum cholesterol levels using cholesterol uptake inhibitor, ezetimibe, significantly reduced prostate weight in PTEN mice consuming a HFHC diet. Furthermore, supplementation of the HFHC diet with ezetimibe significantly reduced tumor concentrations of T, DHT and androstenedione in Hi-Myc mice, although there was no effect on prostate weight. These findings highlight a potentially critical role for cholesterol in PC progression by providing evidence that reduction of hypercholesterolemia may reduce intratumoral de novo steroidogenesis and slow PC growth. This study may provide new insight into the use of cholesterol-lowering drugs for PC prevention and treatment. Citation Format: Emma H. Allott, Elizabeth M. Masko, Alexa Choy, Alexis R. Gaines, Keith R. Solomon, Salvatore V. Pizzo, Stephen J. Freedland. Inhibition of cholesterol uptake with ezetimibe reduces intra-tumoral testosterone levels and slows tumor growth in transgenic mouse models of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3698. doi:10.1158/1538-7445.AM2013-3698

Abstract 5631: The effect of pomegranate extract dose on prostate cancer growth in a carbohydrate restricted mouse xenograft model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Preclinical studies have shown that dietary carbohydrate restriction slows prostate tumor growth in multiple xenograft models in part due to a reduction insulin-like growth factor (IGF) axis signaling. Pomegranate extract (PE) has been shown to slow prostate tumor growth in-vitro and in vivo similarly through this pathway, as well as decrease markers of angiogenesis and inflammation. In this study, we determined the dose-response of PE on prostate tumor growth in the setting of dietary carbohydrate restriction in a slow-growing mouse xenograft model. For this study, we chose a standardized PE (POMx, Pom Wonderful, Los Angeles, CA), at doses which are easily translatable to human equivalents. Materials and Methods: Male athymic nude mice housed 5 per cage (n = 130) were subcutaneously injected with 1 × 10^5 LAPC-4 cells after a 10 day acclimation period. 14 days post-injection, all mice were all placed on an ad-libitum no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein) (day 0). Each cage was randomly assigned to 1 of 4 doses of PE (placebo = 0 mg, low dose = 2.62 mg, intermediate dose = 5.25mg, or high dose = 7.87mg) suspended in 0.2 mL of PBS delivered via oral gavage 5 days per week. These doses are equivalent to 0, 1, 2, or 3 POMx capsules/day consumed by a 70 kg human. Mice will be sacrificed when tumors reach ≥1,000 mm^3. Results: At randomization, there were no differences in median body weight among groups (p = 0.623). Currently, at day 32, overall survival is decreased in the placebo group (hazard ratio 2.26, 95% confidence interval, 0.83 - 6.10, p = 0.109) relative to the low dose PE group with no survival differences among PE groups. Median tumor volumes for the placebo group are 83.3mm^3, compared to 74.5mm^3, 76.3mm^3, and 70.1mm^3 for the low, intermediate, and high PE doses respectively, which are not statistically different (p = 0.98). Conclusions: Preliminary data show a trend toward improved survival with the combination of an NCKD and PE at the selected doses. Currently, tumor volumes are not significantly different, however completed results will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5631. doi:1538-7445.AM2012-5631

Abstract 5255: Methionine restriction limits prostate tumor cell proliferation

Introduction: Methionine restriction has been shown to have growth inhibitory effects in a number of tumor cell lines in vitro. Methionine restriction has previously been demonstrated to slow PC-3 prostate tumor cell growth in vivo, however not at dietary levels tolerable to humans. Up to 90% dietary methionine restriction has been demonstrated to be both tolerable and safe in a preliminary human clinical trial. We hypothesized that 80%-90% methionine restriction could limit prostate tumor cell growth in vitro and in vivo. Methods: We reduced methionine levels by 40%, 80%, 90% and 100% in prostate cancer cell lines in vitro, relative to standard RPMI medium. Cell proliferation and viability were measured using MTS and trypan blue assays, respectively. PC-3 cells were grafted into a xenograft nude mouse model and animals were randomized to standard chow, 80% or 90% methionine restricted diets. Plasma methionine levels were measured before and after the dietary intervention. Tumor volume was measured throughout the study and mice were sacrificed when tumors reached 1000 mm 3 . Results: Methionine restriction exerted a dose and time-dependent effect on PC-3 tumor cell proliferation and viability in vitro. Methionine restriction of 80% and 90% for a period of 6 days reduced PC-3 tumor cell line proliferation by 20% and 40%, respectively, as measured by MTS assay (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5255. doi:1538-7445.AM2012-5255

Abstract 3220: The effects of cholesterol treatment drugs alone and in combination on prostate tumor xenograft growth

Background: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer (PC), but not the incidence of the disease. This may occur by lowering levels of low-density lipoproteins (LDL). Statins and cholesterol-uptake inhibitors like ezetimibe lower serum LDL and are commonly prescribed for patients with high cholesterol. In this study, we determined the therapeutic role of simvastatin, ezetimibe, or both in vitro using several PC cell lines and in vivo using a LAPC-4 xenograft model. Methods: We treated 4 PC cell lines (LAPC-4, CWR22rv1, PC-3, and DU145) with varying concentrations of simvastatin (0-1000nM) or ezetimibe (0-100μM) for 0-5 days. MTS assays were performed to determine cell proliferation, and the IC50 was calculated using BioDataFit 1.02. For our in vivo study, a total of 90 athymic male nude mice were fed a high-fat, high-cholesterol diet (HFHC; 40% fat, 17% protein, 43% carbohydrates, 1.25% cholesterol), subcutaneously injected with 1 x 10^5 LAPC-4 cells, and randomized 2 weeks later to 1 of 4 treatments: vehicle control, 11mg/kg/day simvastatin (delivered via osmotic pump), 30mg/kg diet ezetimibe (delivered in the food), or simvastatin + ezetimibe. Mice remained on study for 42 days post-randomization, at which point they were harvested and samples analyzed. Results: In vitro, simvastatin directly reduced PC cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no inhibitory effect. In vivo, treatment of low continuous dosing of simvastatin or ezetimibe for 42 days had no effect on tumor growth compared to control. However, relative to control, the combination of simvastatin and ezetimibe accelerated tumor growth (p=0.01). The simvastatin-ezetimibe mice also showed lower fat infiltration in the liver and lower serum cholesterol, but higher tumor cholesterol levels than control. Conclusions: Our results suggest that low continuous dosing of simvastatin or ezetimibe may have no effect on PC growth in a slow-growing LAPC-4 xenograft model, but the combination of simvastatin and ezetimibe may accelerate PC growth, possibly by stimulating cholesterol uptake within the tumor. This novel finding may be important in implementing new treatments for men with slow growing PC and high cholesterol. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3220. doi:1538-7445.AM2012-3220

Abstract 4611: Effect of pomegranate and diet on prostate cancer tumor growth and survival

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Pomegranate extract (POM) has been shown to slow prostate cancer (PC) growth in xenograft models. However, mice in these studies ate standard mouse chow, which is very low-fat (∼12% kcals) and not representative of the typical diet for men with PC. Whether POM slows PC growth when taken with a Western diet is unknown. As our group showed diet to separately affect PC xenograft growth, with a no-carbohydrate ketogenic diet (NCKD) being protective vs. a Western diet, we tested the effects of POM on PC growth and survival in mice fed a Western or NCK diet. Methods: 120 male athymic nude mice (age 6-8 weeks) were fed an ad libitum Western diet and injected subcutaneously with 1×10⁁6 LAPC-4 cells. Two weeks later, mice were randomized to remain on Western diet alone or switched to Western diet + POM, NCKD alone, or NCKD + POM treatments (n=30 per arm). Western diet mice were fed ad libitum and NCKD mice via a modified pair-feeding protocol. POM arms received gavage with 0.8 mg POMx extract (PomWonderful) Monday-Friday and control mice received PBS (0.2 mL all arms). Mice were sacrificed when tumors exceeded 1,000 mm⁁3. Tumor volumes were compared between arms using Kruskal-Wallis. Survival between arms was tested using Cox proportional hazards. Results: The study is currently on-going and results are preliminary for day 53 post-injection. To date, significant differences in tumor volume have been noted across the study arms (p=0.007) with NCKD + POM mice having significantly smaller tumors than mice fed base Western diet (p=0.03). In mice fed NCKD, the addition of POM resulted in a trend toward smaller tumors (p=0.07). In contrast, in mice on Western diet, the addition of POM resulted in a trend toward larger tumors (p=0.13). Diet type alone did not affect tumor volume (p=1.00). Preliminary survival data suggest that treatment group impacted survival (p=0.055). Specifically, survival for the NCKD + POM arm was better than either Western + POM or NCKD alone arms (all p<0.046). Conclusion: In a xenograft mouse model, the combination of POM and dietary carbohydrate restriction slows PC tumor growth relative to a control Western diet and improves survival vs. either POM or NCKD treatments alone. Interestingly, our data show that POM delays tumor growth in mice fed NCKD but may actually accelerate tumor growth in mice fed a Western diet. Prior pomegranate studies reporting delayed tumor growth utilized very low-fat diets, which we have also shown to slow tumor growth vs. a Western diet, while our findings suggest that dietary background may profoundly influence the effect of pomegranate extract on PC tumor growth and survival. This is especially important given that men with PC are more likely to consume a Western diet. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4611. doi:10.1158/1538-7445.AM2011-4611

Abstract 2767: Genetic variation in obesity-related genes ADBR2, ADBR3, GHRL, HSD11B1, and SHC1 and risk of prostate cancer: A VA case-control study

INTRODUCTION: Single nucleotide polymorphisms (SNPs) are single nucleotide variations that occur within alleles among individuals. It is believed that SNPs may predispose a person to diseases such as obesity or cancer. One study showed that SNPs within genes linked with obesity were also associated with breast cancer risk. As prostate cancer (PC) mortality is linked with obesity, we determined if SNPs within 5 obesity-related genes were also linked to PC risk in a similar manner to the breast cancer study. METHODS: A total of 53 SNPs were tagged in the genes ADBR2, ADBR3, GHRL, HSD11B1, and SHC1 using the program HAPMAP. DNA collected from 216 subjects (108 incident cases, 108 “healthy” controls) from the Durham VA Hospital were then genotyped for these SNPs. Cases and controls were frequency matched on age and BMI. Logistic regression models were used to determine the risk of obesity (BMI >30 kg/m2) and PC (and high-grade defined as Gleason >7 vs. low-grade/control) for each SNP, adjusting for age and race. RESULTS: In our study population, 83 men were Caucasian and 133 African American. There was no significant differences in age (p=0.18), race (p=0.96), and BMI (p=0.12) for cases and controls. Among the 53 SNPs genotyped, 5 SNPs in ADRB2, ADRB3, and SHC1 were associated with an increased risk of PC (OR 1.88-2.21; p=0.01-0.04), particularly high-grade disease (OR 2.50-3.26; p=0.0004-0.05). Another 4 SNPs in ADRB2 and HSD11B1 were associated with a decreased risk of PC (p=0.01-0.05). A total of 7 SNPs were associated with an increased risk of obesity (p=0.004-0.02). CONCLUSION: In this small pilot study, we found 14 of the 53 analyzed single SNPs in genes previously linked with obesity that were associated with PC risk, both positively and negatively at an alpha of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2767. doi:10.1158/1538-7445.AM2011-2767