Dietary energy restriction (DER) inhibits mammary carcinogenesis, yet mechanisms accounting for its protective activity have not been fully elucidated. In this study, we tested the hypothesis that DER exerts effects on intracellular energy sensing pathways, resulting in alterations of phosphorylated proteins that play a key role in the regulation of cancer. Experiments were conducted using the 1-methyl-1-nitrosourea-induced mammary cancer model in which rats were 0%, 20%, or 40% energy restricted during the postinitiation stage of carcinogenesis. Parallel experiments were done in non-carcinogen-treated rats in which effects of DER at 0%, 5%, 10%, 20%, or 40% in liver were investigated. In a DER dose-dependent manner, levels of Thr 172 phosphorylated AMP-activated protein kinase (AMPK) increased in mammary carcinomas with a concomitant increase in phosphorylated acetyl-CoA-carboxylase, a direct target of AMPK, the phosphorylation of which is regarded as an indicator of AMPK activity. Levels of phosphorylated mammalian target of rapamycin (mTOR) decreased with increasing DER, and down-regulation of mTOR activity was verified by a decrease in the phosphorylation state of two mTOR targets, 70-kDa ribosomal protein S6 kinase (p70S6K) and eukaryote initiation factor 4E binding protein 1 (4E-BP1). Coincident with changes in mTOR phosphorylation, levels of activated protein kinase B (Akt) were also reduced. Similar patterns were observed in mammary glands and livers of noncarcinogen-treated rats. This work identifies components of intracellular energy sensing pathways, specifically mTOR, its principal upstream regulators, AMPK and Akt, and its downstream targets, p70S6K and 4E-BP1, as candidate molecules on which to center mechanistic studies of DER. [Cancer Res 2008;68(13):5492-9]

Dietary energy restriction (DER) is a potent inhibitor of carcinogenesis, but chronic DER in human populations is difficult to sustain. Consequently, interest exists in identifying energy restriction mimetic agents (ERMAs), agents that provide the health benefits of DER without reducing caloric intake. The selection of a candidate ERMAs for this study was based on evidence that DER inhibits carcinogenesis by limiting glucose availability. The study objective was to determine if 2-deoxyglucose (2-DG), a glucose analogue that blocks its metabolism, would inhibit mammary carcinogenesis. Pilot studies were done to establish a dietary concentration of 2-DG that would not affect growth. For the carcinogenesis study, ninety 21-day-old female Sprague-Dawley rats were injected i.p. with 50 mg of 1-methyl-1-nitrosourea per kilogram of body weight. Following injection, animals were ad libitum fed AIN-93G diet containing 0.00%, 0.02%, or 0.03% (w/w) 2-DG for 5 weeks. 2-DG decreased the incidence and multiplicity of mammary carcinomas and prolonged cancer latency (P < 0.05). The 0.02% dose of 2-DG had no effect on circulating levels of glucose, insulin, insulin-like growth factor-I, IGF binding protein-3, leptin, or body weight gain. Using MCF-7 human breast cancer cells to investigate the signaling pathways perturbed by disruption of glucose metabolism, 2-DG reduced cell growth and intracellular ATP in a dose-and timedependent manner (P < 0.01). Treatment with 2-DG increased levels of phosphorylated AMP-activated protein kinase and Sirt-1 and reduced phosphorylated Akt (P < 0.05). These studies support the hypothesis that DER inhibits carcinogenesis, in part, by limiting glucose availability and that energy metabolism is a target for the development of ERMA for chemoprevention. (Cancer Res 2005; 65(15): 7023-30)

An increased ratio of dietary n-3 relative to n-6 fatty acids has been shown to inhibit the development of mammary cancer in animal models. However, the molecular mechanisms by which n-3 fatty acids affect tumor growth remain unknown. Here, we investigated the effects of varying dietary ratios of n-3:n-6 fatty acids on cell signaling in a rat model of chemically induced mammary carcinoma. Cell proliferation was reduced by 60% in carcinomas from the high n-3:n-6 treatment group compared with the low n-3:n-6 treatment group. These changes were associated with decreased cyclin-D1 and phospho-retinoblastoma protein expression and increased levels of cyclin-dependent kinase inhibitors, CIP1 (p21) and KIP1 (p27). In addition, the apoptotic index was increased in carcinomas from the high n-3:n-6 group and was associated with elevated apoptotic protease-activating factor 1 and a higher ratio of Bax/Bcl-2. Interestingly, changes in protein expression were consistent with reduced inflammation and suppressed mTOR activity, and the molecular signature associated with high n-3:n-6 treatment revealed changes in PPARg activation and suppression of lipid synthesis. Together, our findings indicate that the molecular effects of high dietary n-3 to n-6 ratios are heterogeneous in nature but point to consistent changes in lipid metabolism pathways, which may serve as potential therapeutic targets for cancer prevention and control. This study identifies the pathways modulated by dietary fatty acid ratios in a rat model of breast cancer, with implications for cancer prevention. Cancer Res; 72(15); 3795-806. Ó2012 AACR.

Caloric restriction has documented beneficial effects on numerous diseases including cancer, yet the mechanism(s) that accounts for these wide ranging benefits is unknown. Part of the difficulty in defining mechanisms has been the long-term nature of experimental protocols in which these beneficial effects have been observed and the inherent difficulty of investigating mechanisms in such studies. The experiments reported were designed: (1) to determine if caloric restriction would inhibit mammary carcinogenesis in a model for this disease process that is 35 days in duration; (2) to determine if progression from pre-malignant to malignant stages of mammary carcinogenesis was affected by caloric restriction; and (3) to explore whether the effects of caloric restriction were associated with changes in adrenal function. Mammary carcinogenesis was induced in female Sprague-Dawley rats by the i.p. administration of 1-methyl-1-nitrosourea (50 mg/kg body weight) at 21 days of age. Rats were randomized to one of four dietary treatment groups: ad libitum fed, or restriction of food intake to 90, 80 or 60% of the ad libitum intake. Rats were palpated for detection of mammary tumors and all mammary lesions excised at necropsy were histologically classified. Twenty-four-hour collections of urine were obtained at weekly intervals throughout the 35-day experiment. Urine was assayed for corticosterone by direct radioimmunoassay. Caloric restriction resulted in both a dose dependent prolongation of latency to palpable carcinomas (P < 0.01) and a reduction in final incidence of mammary cancer; the dose response was linear (P < 0.05). The percentage of pre-malignant mammary lesions in a group increased with increasing degree of caloric restriction, whereas the percentage of carcinomas decreased (P < 0.05). The level of cortical steroid increased linearly with increasing caloric restriction (P < 0.01) an effect that was not attenuated over time. Poisson regression analyses with the number of cancers per rat as the dependent variable, level of caloric restriction as the independent variable and urinary cortical steroid excretion as a co-variate were performed. These analyses indicated that the variation in cancers per rat, irrespective of the treatment group to which an animal was assigned, could be accounted for by urinary cortical steroid excretion (P<0.05); i.e. urinary cortical steroid excretion was an independent predictor of an animal's carcinogenic response. The data reported in this study support the use of a short term model to study the mechanism(s) by which caloric restriction inhibits mammary carcinogenesis and point to both a stage in the disease process, the conversion of pre-malignant to malignant cells, and a target tissue (adrenal gland) and chemical species (adrenal cortical steroid) that may be involved in mediating the protective effects of energy restriction. These data indicate the feasibility of identifying a chemical basis for the protective effect of caloric restriction that is independent of energy restricti...