Prevention of mammary tumors by metformin in comparison to calorie restriction

描述（由申请人提供）：肥胖是绝经后乳腺癌的危险因素。然而，很少有研究评估预防策略的影响，如卡路里限制在临床前肥胖方案。在啮齿类动物中，实施卡路里限制很容易，但在人类减肥方案中，结果很差，包括不遵守和体重反弹，导致对卡路里限制模拟物的兴趣，包括二甲双胍。在这里，我们将在激素反应性乳腺癌的临床相关模型中直接比较卡路里限制和二甲双胍治疗的影响。我们的假设是，二甲双胍将减少乳腺肿瘤的发病率，延长潜伏期，减少肿瘤负担，其程度与适度限制卡路里相似。具体目标1：确定二甲双胍和卡路里限制对乳腺肿瘤发展的影响，即发病率、潜伏期、肿瘤负担和肿瘤病理与体重的关系。具体目标2：比较二甲双胍和卡路里限制对乳腺肿瘤发展相关血清因子（即胰岛素、葡萄糖和IGF-I）纵向测量的影响。特异性目标3：评估二甲双胍和卡路里限制对乳腺上皮细胞体内增殖的影响，以及与增殖相关的乳腺组织蛋白表达水平和AMPK途径。我们将使用在第二年出现乳腺肿瘤的雌性MMTV-TGF-1/C57BL6小鼠。小鼠将从10周龄（正常体重）开始饲喂33%脂肪饮食或维持低脂肪正常小鼠饮食。根据先前的报道，在30周龄时根据体重增加将高脂肪饮食的小鼠分为三组(intj。肥胖28:956,2004)。体重最重的小鼠将被指定为肥胖倾向，中间组为超重，体重最轻的组为肥胖抵抗组，其体重保持在正常体重小鼠的范围内。这种关系与人类的情况类似，即饮食相似，但体重差异很大。30周龄时，各体重组小鼠分别饲喂自由食量、25%限热量或二甲双胍（100或200 mg/kg体重/天），随访至90周龄。食物摄取量、饮水量和体重将被确定。在干预前和特定时间点采集血清样本，以确定igf - 1、胰岛素和/或葡萄糖是否在肿瘤发展/预防中发挥作用。乳腺肿瘤发病率、潜伏期和肿瘤负荷与体重和干预措施的关系将是主要结果。在干预前和研究结束时将确定体内乳腺细胞增殖情况。肿瘤和乳腺组织将检测与AMPK通路相关的蛋白质，AMPK通路被认为在热量限制和二甲双胍治疗中都很重要。所获得的信息将为二甲双胍治疗和卡路里限制对乳腺肿瘤发展与体重状况的影响提供证据，并有望导致对乳腺癌高风险肥胖妇女的人体试验。

DESCRIPTION (provided by applicant): Obesity is a risk factor for postmenopausal breast cancer. However, there are few studies that have evaluated the impact of prevention strategies such as calorie restriction in preclinical obesity protocols. In rodents it is easy to implement calorie restriction but in humans weight loss regimens have poor outcomes including noncompliance and weight regain leading to interest in calorie restriction mimetics including metformin. Here, we will directly compare the impact of calorie restriction to metformin treatment in a clinically relevant model of hormone responsive breast cancer. Our hypothesis is that metformin will reduce mammary tumor incidence, extend latency and reduce tumor burden to a similar degree as moderate calorie restriction. Specific Aim 1: To determine effects of metformin and calorie restriction on the development of mammary tumors, i.e., incidence, latency, tumor burden and tumor pathology in relationship to body weight. Specific Aim 2: To compare the impact of metformin and calorie restriction on longitudinal measurement of serum factors involved in mammary tumor development, i.e., insulin, glucose and IGF-I. Specific Aim 3: To assess the impact of metformin and calorie restriction on mammary epithelial cell proliferation in vivo in relationship to mammary tissue expression levels of proteins associated with proliferation and the AMPK pathway. We will use female MMTV-TGF-1/C57BL6 mice which develop mammary tumors in their second year. Mice will be fed a 33% fat diet or maintained on a low-fat normal mouse diet from 10 weeks of age (Normal Weight). Mice fed the high-fat diet will be stratified by body weight gain at 30 weeks of age into three groups as previously reported (Int.J.Obesity 28:956,2004). The heaviest mice will be designated as Obesity-Prone, the middle group as Overweight and the lightest group, Obesity-Resistant, are mice which remain in the weight range of Normal-Weight mice. This relationship is similar to what occurs in humans, i.e., diets are similar but body weights vary considerably. At 30 weeks of age mice in each body weight group will be fed Ad libitum, 25% calorie restricted or treated with metformin (100 or 200 mg/kg body weight/day) and followed until 90 weeks of age. Food intake, water consumption and body weights will be determined. Serum samples will be obtained prior to interventions and at specified time points to determine if IGF-I, insulin and/or glucose play a role in tumor development/prevention. Mammary tumor incidence, latency and tumor burden in relation to body weight and intervention will be the primary outcome. In vivo mammary cell proliferation prior to interventions and at the end of the study will be determined. Tumors and mammary tissue will be assayed for proteins associated with the AMPK pathway which is considered to be important in the actions of both calorie restriction and metformin treatment. The information obtained will provide evidence of the impact of metformin treatment and calorie restriction on mammary tumor development in relation to body weight status and will hopefully lead to human trials of obese women at high risk for breast cancer. PUBLIC HEALTH RELEVANCE: Despite the fact that obesity is a risk factor for postmenopausal breast cancer few preclinical studies have been undertaken to assess how interventions may impact this process. Here, we will compare the effects of moderate calorie restriction and metformin on mammary tumor development in a relevant animal model. The outcome(s) of this study will provide valuable information for identifying mechanisms of action for these prevention strategies and for designing intervention studies in high risk obese women.