Tricia Moore

Dietary Energy Balance Modulates Signaling through the Akt/Mammalian Target of Rapamycin Pathways in Multiple Epithelial Tissues

Abstract The prevalence of obesity, an established risk factor for several types of cancer, has increased steadily over the past several decades in the United States. New targets and strategies for offsetting the effect of obesity on cancer risk are urgently needed. In the present study, we examined the effect of dietary energy balance manipulation on steady-state signaling in multiple epithelial tissues, with a focus on the Akt and mammalian target of rapamycin (mTOR) pathways. For these experiments, male FVB/N and C57BL/6 and female ICR mice were maintained on a control (10 kcal% fat) diet, a diet-induced obesity (DIO; 60 kcal% fat) regimen, or a 30% calorie restriction (CR) regimen for 15 to 17 weeks. Relative to the control group, the DIO regimen increased, whereas CR decreased, circulating insulin-like growth factor-I (IGF-I) as has previously been reported. Western blot analyses showed that the DIO regimen enhanced, whereas CR inhibited, activation of Akt and mTOR, regardless of epithelial tissue or genetic background. In contrast, activation of AMP-activated protein kinase was modulated by dietary energy balance manipulation in the liver but not in the epidermis or dorsolateral prostate. Western blot analyses of epidermal extracts taken from ICR mice also revealed reduced activation of both the IGF-I receptor and epidermal growth factor receptor in CR mice, compared with control mice or mice maintained on the DIO regimen. Taken together, these novel findings suggest that dietary energy balance modulates signaling through cell-surface receptors (i.e., IGF-I receptor and epidermal growth factor receptor), affecting activation of multiple downstream pathways including Akt and mTOR, thus providing important dietary and pharmacologic targets for disrupting the obesity-cancer link.

Reduced Susceptibility to Two-Stage Skin Carcinogenesis in Mice with Low Circulating Insulin-Like Growth Factor I Levels

Calorie restriction has been shown to inhibit epithelial carcinogenesis and this method of dietary restriction reduces many circulating proteins, including insulin-like growth factor I (IGF-I). Previously, we identified a relationship between elevated tissue IGF-I levels and enhanced susceptibility to chemically induced skin tumorigenesis. In this study, liver IGF-I-deficient (LID) mice, which have a 75% reduction in serum IGF-I, were subjected to the standard two-stage skin carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene as the initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as the promoter. We observed a significant reduction in epidermal thickness and labeling index in LID mice treated with either vehicle or TPA. A significant decrease in both tumor incidence and tumor multiplicity was observed in LID mice undergoing two-stage skin carcinogenesis relative to wild-type littermates. Western blot analyses of epidermal extracts revealed reduced activation of both the epidermal growth factor and IGF-I receptors in response to TPA treatment in LID mice. In addition, reduced activation of both Akt and the mammalian target of rapamycin (mTOR) was observed in LID mice following TPA treatment relative to wild-type controls. Signaling downstream of mTOR was also reduced. These data suggest a possible mechanism whereby reduced circulating IGF-I leads to attenuated activation of the Akt and mTOR signaling pathways, and thus, diminished epidermal response to tumor promotion, and ultimately, two-stage skin carcinogenesis. The current data also suggest that reduced circulating IGF-I levels which occur as a result of calorie restriction may lead to the inhibition of skin tumorigenesis, at least in part, by a similar mechanism.

Dietary energy balance modulates prostate cancer progression in Hi-Myc mice

Male Hi-Myc mice were placed on three dietary regimens [30% calorie restriction (CR), overweight control (modified AIN76A with 10 kcal% fat), and a diet-induced obesity regimen (DIO) 60 kcal% fat]. All diet groups had approximately similar incidence of hyperplasia and low-grade prostatic intraepithelial neoplasia in the ventral prostate at 3 and 6 months of age. However, 30% CR significantly reduced the incidence of in situ adenocarcinomas at 3 months compared with the DIO group and at 6 months compared with both the overweight control and DIO groups. Furthermore, the DIO regimen significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared with the overweight control group (96% vs. 65%, respectively; P = 0.02) at the 6-month time point. In addition, at both 3 and 6 months, only in situ carcinomas were observed in mice maintained on the 30% CR diet. Relative to overweight control, DIO increased whereas 30% CR reduced activation of Akt, mTORC1, STAT3, and NFκB (p65) in ventral prostate. DIO also significantly increased (and 30% CR decreased) numbers of T-lymphocytes and macrophages in the ventral prostate compared with overweight control. The mRNA levels for interleukin (IL) 1α, IL1β, IL6, IL7, IL23, IL27, NFκB1 (p50), TNFα, and VEGF family members were significantly increased in the ventral prostate of the DIO group compared with both the overweight control and 30% CR diet groups. Collectively, these findings suggest that enhanced growth factor (Akt/mTORC1 and STAT3) and inflammatory (NFκB and cytokines) signaling may play a role in dietary energy balance effects on prostate cancer progression in Hi-Myc mice. Cancer Prev Res; 4(12); 2002–14. ©2011 AACR.

Rapamycin Is a Potent Inhibitor of Skin Tumor Promotion by 12-O-Tetradecanoylphorbol-13-Acetate

Aberrant activation of phosphoinositide-3-kinase (PI3K)/Akt signaling has been implicated in the development and progression of multiple human cancers. During the process of skin tumor promotion induced by treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), activation of epidermal Akt occurs as well as several downstream effectors of Akt, including the activation of mTORC1. Rapamycin, an established mTORC1 inhibitor, was used to further explore the role of mTORC1 signaling in epithelial carcinogenesis, specifically during the tumor promotion stage. Rapamycin blocked TPA-induced activation of mTORC1 as well as several downstream targets. In addition, TPA-induced epidermal hyperproliferation and hyperplasia were inhibited in a dose-dependent manner with topical rapamycin treatments. Immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin also significantly decreased the number of infiltrating macrophages, T cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Using a two-stage skin carcinogenesis protocol with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as the promoter, rapamycin (5–200 nmol per mouse given topically 30 minutes prior to TPA) exerted a powerful antipromoting effect, reducing both tumor incidence and tumor multiplicity. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited further growth. Overall, the data indicate that rapamycin is a potent inhibitor of skin tumor promotion and suggest that signaling through mTORC1 contributes significantly to the process of skin tumor promotion. The data also suggest that blocking this pathway either alone or in combination with other agents targeting additional pathways may be an effective strategy for prevention of epithelial carcinogenesis. Cancer Prev Res; 4(7); 1011–20. ©2011 AACR.

Energy Balance Modulates Mouse Skin Tumor Promotion through Altered IGF-1R and EGFR Crosstalk

Obesity, an established risk factor for epithelial cancers, remains prevalent in the United States and many other countries. In contrast to positive energy balance states (overweight, obesity), calorie restriction (CR) has been shown to act as a universal inhibitor of tumorigenesis in multiple animal models of human cancer. Unfortunately, the mechanisms underlying the enhancing effects of obesity or the inhibitory effects of CR on cancer etiology remain elusive. Here, we evaluated the impact of dietary energy balance manipulation on epithelial carcinogenesis and identified several potential mechanisms that may account for the differential effects of obesity and CR on cancer. Obesity enhanced tumor promotion during epithelial carcinogenesis, in part, due to altered insulin-like growth factor-1 receptor (IGF-1R)/EGF receptor (EGFR) crosstalk and downstream signaling to effectors such as Akt/mTOR. Obesity-induced changes in cellular signaling subsequently led to altered levels of cell-cycle proteins that favored enhanced epidermal proliferation during tumor promotion. In contrast, CR reduced susceptibility to tumor promotion, attenuated IGF-1R/EGFR crosstalk and downstream signaling, and altered levels of cell-cycle proteins that favored reduced epidermal proliferation during tumor promotion. Collectively, these findings suggest potential targets for the prevention of epithelial cancers, as well as for reversal of obesity-mediated cancer development and progression. Cancer Prev Res; 5(10); 1236–46. ©2012 AACR.

Dietary energy balance modulation of epithelial carcinogenesis: a role for IGF‐1 receptor signaling and crosstalk

Obesity affects more than one third of the U.S. population and is associated with increased risk and/or disease severity for several chronic diseases, including cancer. In contrast, calorie restriction (CR) consistently inhibits cancer across species and cancer types. Differential effects on globally active circulatory proteins, particularly insulin‐like growth factor‐1 (IGF‐1), provide a plausible mechanistic explanation for the energy balance–cancer link. Diet‐induced changes in circulating IGF‐1 modulate IGF‐1R/EGFR activation and downstream signaling to Akt and mTOR. These dietary energy balance effects on signaling ultimately modulate the levels and/or activity of cell cycle regulatory proteins, regulating proliferation, and modulating susceptibility to tumor development. Selective targeting of mTORC1 potently inhibits tumorigenesis in several model systems producing CR mimetic effects. Targeting this and other pathways modulated by dietary energy balance may lead to the development of strategies for cancer chemoprevention and for reversing the effects of obesity on cancer development and progression.

Abstract 1010: Effect of dietary energy balance modulation on the ability of metformin to inhibit skin tumor promotion

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The prevalence of obesity has drastically increased over the last decade and is associated with increased risk for several cancers. In contrast, calorie restriction (CR) universally inhibits cancer. Plausible mechanisms for the energy balance-cancer link include differential signaling through Akt/mTOR via the IGF-1R as well as other growth factor receptors. Previous data from our lab, using topical administration of rapamycin, support the hypothesis that pharmacological disruption of mTORC1 signaling mimics some of the effects of CR on skin tumor promotion. To further explore the role of mTORC1 signaling in tumor promotion we have conducted studies using metformin. In the present study, metformin was administered in the drinking water at doses of 250 mg/kg and 50 mg/kg body weight per day to female FVB/N mice, to assess its ability to reverse the effects of overweight/obesity on skin tumor promotion during two-stage skin carcinogenesis. Metformin treatment during skin tumor promotion significantly inhibited tumor multiplicity in a dose dependent manner in mice on an overweight control diet (AIN76A). There was a 70% reduction in papillomas per mouse at the 250 mg/kg dose and a 35% reduction at the 50 mg/kg dose. Short term mechanistic experiments with these same doses revealed significant decreases in TPA-induced epidermal hyperproliferation (as measured by epidermal hyperplasia and labeling index) after oral administration of metformin. Metformin also activated AMP Kinase in the epidermis as measured by pAMPKαT172 at both doses used as well as attenuated signaling through mTORC1 at the highest dose as measured by downstream signaling proteins p70S6KT389 and prS6S235/236. Treatment with the higher dose of metformin also prevented TPA-induced mTORC1 mediated degradation of translational repressor PDCD4. Current experiments are assessing the ability of metformin to suppress skin tumor promotion in mice maintained on an obesity inducing diet (60kcal% fat) and these studies will also be presented. Overall, the current data support the hypothesis that selectively targeting mTORC1, either directly (rapamycin) or indirectly via AMPK activation, may be an effective strategy for reversing the effects of overweight and obesity on tumor development during epithelial carcinogenesis. 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 1010. doi:1538-7445.AM2012-1010

Abstract 1847: Effect of rapamycin and metformin on skin tumor promotion in mice overweight and obese

The prevalence of obesity in the US has risen drastically over the past few decades and is an established risk factor for several human cancers. Insights into the mechanisms underlying this link are urgently needed to develop strategies for treatment and prevention. Various studies have shown that calorie restriction (CR) during tumor promotion in the two-stage model of skin carcinogenesis causes a significant reduction in tumorigenesis. In addition, diet induced obesity (DIO) leads to insulin resistance, increased circulating levels of IGF-1 and increased susceptibility to tumor development. Current data indicate that signaling through the IGF-1R and EGFR as well as several downstream signaling pathways plays an important role in dietary energy balance effects on tumor promotion during epithelial carcinogenesis. The current study was designed to evaluate the ability of rapamycin (an established mTORC1 inhibitor) to inhibit or reverse the effects of overweight/obesity on skin tumor promotion in mice. In initial experiments using female FVB mice on an AIN76A diet, rapamycin (5-1000 nmol per mouse) was found to be a potent inhibitor of skin tumor promotion by TPA. In this regard, rapamycin reduced both tumor multiplicity and incidence in a dose dependent manner. A dose of 5 nmol rapamycin given 30 min before each TPA treatment reduced the number of papillomas per mouse by approximately 50%. Rapamycin treatment inhibited TPA-induced mTORC1 activation as well as downstream signaling proteins p70S6KT389, prS6S240/244 and p4EBP1T37/46. Multiple treatment experiments were performed to evaluate the effects of rapamycin (5-200 nmol per mouse) on TPA-induced epidermal hyperproliferation (as assessed by epidermal hyperplasia and epidermal labeling index). Rapamycin significantly inhibited TPA-induced epidermal hyperproliferation in a dose dependent manner. In addition, immunohistochemical analyses of the skin from mice in this multiple treatment experiment revealed that rapamycin significantly decreased the number of infiltrating macrophages, T-cells, neutrophils, and mast cells seen in the dermis following TPA treatment. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited their growth. Two-stage skin carcinogenesis studies are currently underway to further evaluate the impact of rapamycin treatment on mice made overweight (AIN76A) or obese (DIO diet; 60 Kcal % fat) via dietary energy balance manipulation. In addition, groups of mice are also being treated with metformin. These ongoing studies will clarify whether suppression of the mTORC1 signaling pathway is a viable strategy for reversing the effects of obesity on tumor development in this well established model of epithelial carcinogenesis. 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 1847. doi:10.1158/1538-7445.AM2011-1847

Abstract 815: Dietary energy balance modulates skin tumor promotion through altered IGF-1R and EGFR crosstalk

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Negative energy balance (calorie restriction, CR) inhibits, while positive energy balance enhances tumor promotion using the two-stage skin carcinogenesis model. Biochemical studies have demonstrated that CR reduced, while diet-induced obesity (DIO) increased insulin-like growth factor 1 receptor (IGF-1R) and epidermal growth factor receptor (EGFR) activation and downstream signaling (i.e., Akt and mTOR) following TPA treatment. Additional studies performed using the liver IGF-1 deficient (LID) mouse model (i.e., 75% reduction in circulating IGF-1) demonstrated reduced susceptibility to epithelial carcinogenesis and attenuated IGF-1R and EGFR signaling during tumor promotion, similar to CR mice. Taken together, these findings suggest that dietary energy balance, primarily through its effects on levels of circulating IGF-1, modulates epithelial carcinogenesis and tumor promotion through diet-induced changes in IGF-1R and EGFR signaling and receptor crosstalk. To determine the impact of IGF-1 on IGF-1R and EGFR signaling and receptor crosstalk, Western blot analyses, immunoprecipiation and qPCR analyses were performed using C50 cells (a nontumorigenic keratinocyte cell line) stimulated with IGF-1. IGF-1 treatment of serum starved cells induced rapid phosphorylation (within 5 minutes) of the IGF-1R, as well as the EGFR and ErbB2. Additional experiments were conducted to evaluate mechanisms underlying the effects of IGF-1 on EGFR activation. Immunoprecipitation experiments demonstrated that IGF-1 induced an association between the IGF-1R and the EGFR. Furthermore, qPCR analysis demonstrated that IGF-1 induced HB-EGF and amphiregulin mRNA expression. Current experiments are exploring the possibility that IGF-1 treatment also leads to increased ectodomain shedding of membrane bound EGFR ligands. Further studies were conducted to examine the impact of caloric consumption on IGF-1R and EGFR crosstalk in vivo. For these experiments, ICR female mice were maintained on either a 30% CR or 60Kcal% fat (DIO) regimen for 15 weeks, and then treated with a single application of either acetone or 3.4 nmol TPA. CR reduced, while DIO increased the interaction between the IGF-1R and the EGFR in the epidermis of TPA treated mice. Furthermore, CR reduced, while DIO increased mRNA expression of EGFR ligands both in the presence and absence of TPA treatment. Additional experiments are in progress to confirm that IGF-1 levels, per se, are directly responsible for these diet-induced changes in IGF-1R/EGFR receptor crosstalk. Collectively, the current data suggest that IGF-1 levels and the activation status of the IGF-1R modulate IGF-1R and EGFR receptor crosstalk. Furthermore, diet-induced changes in IGF-1R/EGFR receptor crosstalk subsequently modulate downstream signaling to Akt and mTOR, thus contributing, at least in part, to the effect of dietary energy balance on skin tumor promotion. 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 815. doi:10.1158/1538-7445.AM2011-815

Abstract 824: Altered growth factor and inflammatory signaling is associated with obesity related prostate cancer progression in HiMyc mice

Obesity is a long-term consequence of energy imbalance that occurs when energy intake exceeds expenditure and is associated with increased mortality rates for various cancers, including prostate cancer. In contrast, calorie restriction (CR) has been shown to act as a potent inhibitor of tumorigenesis. In addition, inflammation has been described to play an important role in carcinogenesis in many tissues. In this study, we have evaluated the effect of both CR and diet induced obesity (DIO) diets on prostate cancer progression in HiMyc mice. Four week old male HiMyc mice were placed on three dietary regimens [30% CR, 10kcal% fat (AIN76A), 60 kcal% fat], thus generating lean, overweight and obese phenotypes. Mice were then sacrificed at 3 and 6 months of age and urogenital tracts were removed and embedded in paraffin for histological analyses. Histopathologic evaluation of the prostate tissue revealed a variety of prostate lesions, with the severity of the lesion correlating with caloric intake. All diet groups had approximately similar incidence of hyperplasia and low grade PIN at 3 and 6 months of age. However, the different energy diets primarily affected the progression of premalignant lesions to malignant lesions in the ventral prostate. At 6 months DIO significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared to the overweight control group (96% vs. 65% respectively, p=0.0221), while only in situ carcinomas but no invasive adenocarcinomas were observed in mice maintained on the CR diet. Immunohistochemistry was performed on additional sections to evaluate differences in activation or total levels of Akt, mTOR, S6 ribosomal, Stat3, NFkB, angiogenesis markers, cell cycle markers and inflammatory cells. DIO increased, while CR reduced activation of signaling through both Akt and mTOR. Similar effects were observed for levels of cyclin D1 and CD31 proteins. In addition, levels of phospho-NFkB (p65) were increased in the DIO group, coupled with the presence of increased numbers of T-lymphocytes and macrophages. Western blot analyses were also performed on protein lysates from the VP of mice maintained on the same dietary regimens for 6 months and confirmed these differential effects of dietary energy balance on Akt/mTOR signaling and Stat3 activation. The observed changes in signaling appeared to be due, at least in part, to changes in signaling through the insulin-like growth factor 1 receptor (IGF-1R) as a result of altered levels of circulating IGF-1. Taken together, these findings suggest that enhanced growth factor (Akt/mTOR and Stat3) and inflammatory (NFkB) signaling may play a role in the diet-induced obesity effects on prostate cancer progression in HiMyc mice. These pathways may represent key targets for preventing and controlling obesity-related prostate cancer progression. 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 824. doi:10.1158/1538-7445.AM2011-824