Jean A. Thomas

Effect of intermittent fasting on prostate cancer tumor growth in a mouse model

Caloric restriction (CR) has been shown to have anti-cancer properties. However, CR may be difficult to apply in humans secondary to compliance and potentially deleterious effects. An alternative is intermittent CR, or in the extreme case intermittent fasting (IF). In a previous small pilot study, we found 2 days per week of IF with ad libitum feeding on the other days resulted in trends toward prolonged survival of mice bearing prostate cancer xenografts. We sought to confirm these findings in a larger study. A total of 100 (7- to 8-week-old) male severe combined immunodeficiency mice were injected subcutaneously with 1 × 105 LAPC-4 prostate cancer cells. Mice were randomized to either ad libitum Western Diet (44% carbohydrates, 40% fat and 16% protein) or ad libitum Western Diet with twice-weekly 24 h fasts (IF). Tumor volumes and mouse bodyweights were measured twice weekly. Mice were killed when tumor volumes reached 1000 mm3. Serum and tumor were collected for analysis of the insulin/insulin-like growth factor 1 (IGF-1) hormonal axis. Overall, there was no difference in mouse survival (P=0.37) or tumor volumes (P⩾0.10) between groups. Mouse body weights were similar between arms (P=0.84). IF mice had significantly higher serum IGF-1 levels and IGF-1/IGFBP-3 ratios at killing (P<0.001). However, no difference was observed in serum insulin, IGFBP-3 or tumor phospho-Akt levels (P⩾0.39). IF did not improve mouse survival nor did it delay prostate tumor growth. This may be secondary to metabolic adaptations to the 24 h fasting periods. Future studies are required to optimize CR for application in humans.

Low-Carbohydrate Diets and Prostate Cancer: How Low Is "Low Enough"?

Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm3. Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P < 0.04). Among the low-carbohydrate arms, NCKD-fed mice were significantly lighter than the 10% or 20% carbohydrate groups (P < 0.05). Tumors were significantly larger in the 10% carbohydrate group on days 52 and 59 (P < 0.05), but at no other point during the study. Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10–20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal). Cancer Prev Res; 3(9); 1124–31. ©2010 AACR.

The effect of carbohydrate restriction on prostate cancer tumor growth in a castrate mouse xenograft model

No‐ and low‐carbohydrate diets delay tumor growth compared to western diet (WD) in prostate cancer (PCa) xenograft studies. The effect of these diets in concert with androgen deprivation is unknown.

Effect of Isocaloric Low-Fat Diet on Prostate Cancer Xenograft Progression in a Hormone-Deprivation Model

PURPOSE Previous mouse studies suggesting that low fat diets slow prostate cancer growth often used corn oil (omega-6), which enhances prostate cancer growth, as the primary fat. Using a saturated fat based diet we previously found no significant difference in tumor growth between low and high fat fed SCID mice (Taconic Farms, Hudson, New York) xenografted with LAPC-4 cells. Whether similar results would hold in a castration model is unclear. MATERIALS AND METHODS A total of 80 male SCID mice were fed a Western diet (40% fat and 44% carbohydrate) and injected with LAPC-4 human prostate cancer cells. When tumors were 200 mm(3), the mice were castrated and randomized to an isocaloric Western or a low fat diet (12% fat and 72% carbohydrate). Animals were sacrificed when tumors were 1,000 mm(3). Serum was collected and assayed for prostate specific antigen, insulin, insulin-like growth factor 1 and insulin-like growth factor binding protein 3. Tumors were assayed for total and phosphorylated Akt. RESULTS Mouse weight was equivalent in the 2 groups. Overall dietary group was not significantly associated with survival (log rank p = 0.32). There were no statistically significant differences in prostate specific antigen (p = 0.53), insulin-like growth factor axis parameters (each p >0.05) or p-Akt-to-t-Akt ratios (p = 0.22) between the groups at sacrifice. CONCLUSIONS In this xenograft model we found no difference in tumor growth or survival between low fat vs Western fed mice when the fat source was saturated fat. These results conflict with those of other studies in which corn oil was used to show that low fat diets delay prostate cancer growth, suggesting that fat type may be as important as fat amount in the prostate cancer setting.

DOES PATHOLOGICAL GLEASON SCORE RE-REVIEW BY A MODERN PATHOLOGIST IMPROVE RISK STRATIFICATION RELATIVE TO HISTORICAL GLEASON SCORE?

INTRODUCTION AND OBJECTIVE: Early detection, stage migration, and emphasis on health related quality of life has led to increasing interest in focal therapy for prostate cancer (PCa). Advocates attempt to map prostate tumors in order to limit their patient population to those with unifocal disease. Unfortunately, no descriptive studies evaluating single focus prostate cancer have been performed and little is known regarding the unique biology of this disease. The objective of this study is to characterize the clinicopathologic behavior of unifocal PCa in comparison to its multifocal counterpart. METHODS: The Center for Prostate Disease Research (CPDR) database was queried for all prostatectomy specimens obtained from 1994 to 2004. All specimens were analyzed at the Armed Forces Institute of Pathology utilizing whole mount and 2 mm step sectioning. Tumors were comprehensively mapped to include number of tumor foci and calculation of total tumor volume. Patients with single focus PCa were compared to those demonstrating multifocal disease. Primary outcomes were pathologic stage and biochemical recurrence, while secondary outcomes were Gleason sum and total tumor volume. A p-value of <0.05 was considered statistically significant using the chi-square, Fisher exact, and student’s T-test. RESULTS: A total of 1012 prostatectomies were examined with 109 (10.7%) demonstrating a single focus of PCa. Median patient age was 60 in the unifocal and 61 in the multifocal cohorts. Median PSA was 10 for the unifocal and 7.4 for the multifocal cohorts. Median total tumor volume was 5.2 cc for unifocal and 4.2 cc for multifocal disease. The unifocal cohort Gleason sums were 2-6 in 51%, 7 in 31.6%, and 8-10 in 17.3%. The multifocal cohort Gleason sums were 2-6 in 56.8%, 7 in 29.9%, and 8-10 in 10.1%. Extraprostatic extension was noted in 56.0% of cases with unifocal and 36.1% of cases with multifocal disease. In total, 27% of unifocal and 15.5% of multifocal disease had biochemical recurrence. Of the patients with organ confined (pT2) disease, 6.4% of unifocal and 5.2% of multifocal disease had a PSA recurrence. The frequency of single focus CaP stratified by year of surgery was: 19931996 (17%), 1997-2000 (9.4%), 2001-2004 (8.6%). CONCLUSIONS: Unifocal prostate cancer is a more aggressive entity than its multifocal counterpart. PSA, Gleason sum, total tumor volume, extracapsular disease, and biochemical recurrence were significantly higher in the unifocal population. With intensive prostate cancer screening the incidence of single focus prostate cancer has decreased.

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 2877: TRAMP-injected mice fed a no-carbohydrate ketogenic diet demonstrate prolonged survival and decreased tumor volumes compared to mice fed western or low-fat diets

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction and Objective: In multiple previous studies, we have shown that mice injected with subcutaneous prostate cancer cells (LNCaP or LAPC-4) and fed a no-carbohydrate ketogenic diet (NCKD, 84% fat, 0% carbohydrate, 16% protein) have significantly decreased tumor volume and significantly prolonged survival, compared to mice fed a western diet (WD, 40% fat, 44% carbohydrate, 16% protein). All previous studies utilized a human prostate cancer cell line, requiring the use of immunodeficient mice. We sought to replicate these previous studies to determine whether diet affected tumor progression and survival in an immunocompetent model. Methods: In this treatment model, 75 male C57bl/6 mice were initially fed WD ad libitum for a period of 14 days. After this 14 day period, all mice were injected subcutaneously with 2.5×105 TRAMP cells and randomized to one of three diets: WD, NCKD, or low-fat diet (LF, 12% fat, 72% carbohydrate, 16% protein). Mice receiving LF diet were fed ad libitum, and served as the reference group in paired-feeding protocols for mice receiving WD and NCKD. Tumor volumes were assessed twice weekly, and mice were sacrificed when tumor volumes reached 1000mm3. Survival was calculated using a Log-rank test and tumor volumes were compared among dietary groups using Kruskal-Wallis test for significance. Results: Mice fed a NCKD demonstrated a trend towards decreased tumor volumes compared to mice fed WD and LF. Median tumor volume at study day 66 for mice fed WD, LF, and NCKD were 1091.2 mm3, 1082.9 mm3, and 661.5mm3 respectively. While the tumor volumes were smaller in mice fed NCKD versus LF or WD, these differences did not reach statistical significance (WD vs. NCKD, p=0.06; LF vs. NCKD, p=0.12). There was no significant difference between tumor volumes among mice fed WD compared to LF (p=0.95). Additionally, a trend towards improved survival was seen in mice fed NCKD vs. WD (p=0.09). Conclusion: This study in an immunocompetent xenograft model supports the findings of multiple previous studies. Mice fed a NCKD demonstrated a trend towards prolonged survival and decreased tumor volume compared to mice fed WD. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2877.

Abstract 3243: Differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a mouse model

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Caloric restriction (CR) has been shown to be anti_cancer. However, whether these effects are the result of generalized reduction in overall caloric intake or reduced intake of macronutrients (i.e. carbohydrates) are unknown. We sought to investigate the differential effects of caloric and carbohydrate restriction upon prostate cancer tumor growth in a xenograft model. Materials and Methods: A total of 100 male SCID mice were injected subcutaneously with the LAPC-4 cell line. Mice were randomized to 1 of 4 diets: ad libitum Western diet (WD, 49% carbohydrates, 35% fat, and 16% protein), pair-fed No Carbohydrate Ketogenic Diet (NCKD; 1% carbohydrates, 83% fat, 16% protein), Western diet calorically restricted (WD-CR), and NCKD calorically restricted (NCKD-CR). Calorically restricted arms were fed 75% of the calories of their reference diet group. Tumor volumes and body weights were measured twice weekly. Mice were sacrificed when tumor volumes reached 1,000 mm3. Results: Relative to WD, there was a suggestion of smaller body weights in both NCKD-CR and WD-CR arms (rank-sum, p≤0.06). There was trend for heavier mice in the NCKD group (rank-sum, p=0.09). By day 50, median tumor volumes for NCKD, WD-CR, and NCKD-CR were 45, 51, and 62% smaller than WD (rank-sum, all p≤0.001). At any time point, there were no significant differences in tumor volume among the NCKD, NCKD-CR, and WD-CR arms. However, there was a suggestion of smaller tumors in the NCKD-CR arm by day 42 relative to NCKD and WD-CR (p<0.11). Diet was significantly associated with mouse survival (log\_rank, p=0.004). In 2\_way comparisons, NCKD, NCKD-CR, and WD-CR had significantly prolonged survival relative to WD (log_rank, all p≤0.02). However, no difference in survival was seen among any caloric or carbohydrate restricted arms (log rank, p≤0.16). A total of 34 mice died due to infection prior to their tumors reaching 1,000mm3 and therefore were not included in the survival analyse (WD-CR, n=14; NCKD, n=5; NCKD-CR, n =15). Conclusions: Both CR and carbohydrate restriction without weight loss are effective in delaying tumor growth and improving mouse survival. We found no differences between caloric and carbohydrate restricted arms, though this may relate to loss of study power due to mouse infections. CR may further weaken the immune system of immunocompromised mice subjecting them to increased risk of infections. A confirmatory study is underway in a high-level barrier facility. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3243.

Abstract 2816: Low-carbohydrate diets and prostate cancer growth: How low is “low enough”

Introduction: Previous dietary studies indicate carbohydrate intake may influence prostate cancer biology, as LAPC-4 and LNCaP xenograft mice fed a no-carbohydrate ketogenic diet (NCKD; 84% fat-0% carbohydrate-16% protein kcal) had significantly smaller tumors and longer survival times compared to mice fed a Western diet (40% fat-44% carbohydrate-16% protein kcal). The NCKD mice were also found to have higher levels of circulating IGFBP-3 and the lowest levels of insulin, IGF-1, and IGF-1:IGFBP-3 ratio despite consuming more calories than the Western group. As it is nearly impossible for a human to consume and maintain a no-carbohydrate diet similar to that in the previous xenograft studies, we sought to determine whether diets containing 10% or 20% kcal from carbohydrates could slow tumor growth in a similar manner to the NCKD in a xenograft model. METHODS: A total of 150 male SCID mice were injected with LAPC-4 cells and placed on a Western diet (35% fat-49% carbohydrate-16% protein kcal) ad libitum. Two weeks post-injection, all mice were randomized to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate. Ten mice not injected with tumor were fed an ad libitum low-fat diet (12% fat-72% carbohydrate-16% protein kcal) and served as the reference group in a modified-paired feeding protocol for the other three groups. Calorie intake and body weights were measured thrice weekly and tumor volumes twice per week. Mice were sacrificed when tumors reached 1,000mm 3 . RESULTS: Despite consuming 5-10% extra calories on average, all mice receiving low-carbohydrate diets were significantly lighter than the mice consuming the low-fat diet (p CONCLUSIONS: LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival to mice consuming a NCKD (0% carbohydrate kcal). Thus, the survival benefit of a NCKD may be achievable with less restrictive low-carbohydrate diets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2816.

EFFECT OF INTERMITTENT FASTING ON PROSTATE CANCER TUMOR GROWTH IN A MOUSE MODEL

Introduction: Caloric restriction is a well established approach to slow tumor growth in animal models. However, this approach is limited in humans due to poor compliance with weight loss regiments. Therefore, we previously investigated intermittent fasting (i.e. intermittent severe caloric restriction) as a means to slow prostate cancer growth in a xenograft model. In our pilot study, we found trends for mice that were fasted 2 days/week and fed ad libitum the other 5 days to have delayed tumor growth while maintaining normal body weight. Herein we sought to validate these findings using a larger sample size and better powered study. Methods: One Hundred male SCID mice (~8 weeks old) were subcutaneously implanted with 1x105 LAPC-4 cells. All mice were initially fed ad libitum Western diet (40% calories from fat; 45% from simple carbohydrates). When tumor volumes reached 200 mm3, mice were randomized to ad libitum feeding or intermittent fasting 2 days/week with ad libitum feeding on the non-fasting days. To date, 32 mice have been randomized to ad libitum feeding and 31 to intermittent fasting. Mice body weights and tumor volumes were measured twice per week. At the conclusion of each fasting day and prior to refeeding, urinary ketone levels for both ad libitum fed and fasted mice were measured. Comparisons of body weight and tumor volumes between groups were performed using the rank sum test. Results: By day 14 after randomization (longest point of follow-up to date), there was no difference in body weights (p=0.81) or tumor volumes (p=0.24) between ad libitum fed and intermittently fasted mice. Intermittently fasted mice were initially ketotic on day 0 after randomization with moderate (~40ng/ml) levels of post-fasting ketonuria. However, by Day 14 after randomization, post-fasting urine ketone levels had decreased to trace levels (~5ng/ml). Ad libitum fed mice had trace to no detectable urinary ketone levels at any time point ( Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 313.