Prevention of mammary tumors by metformin in comparison to calorie restriction

说明(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，例如在临床前肥胖方案中限制卡路里。在啮齿动物中，实施卡路里限制很容易，但在人类中，减肥方案的结果很差，包括不遵守规定和体重恢复，这导致了人们对包括二甲双胍在内的卡路里限制模拟药物的兴趣。在这里，我们将直接比较在临床相关的激素反应型乳腺癌模型中限制热量摄入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生率，延长潜伏期，并减轻肿瘤负担，程度与中等热量限制相似。具体目的1：确定二甲双胍和热量限制对乳腺肿瘤发生的影响，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纵向测量与乳腺肿瘤发生有关的血清因子，即胰岛素、血糖和IGF-I的影响。具体目的3：探讨二甲双胍和热量限制对乳腺上皮细胞增殖的影响及其与乳腺组织增殖相关蛋白表达水平和AMPK信号转导途径的关系。我们将使用第二年发生乳腺肿瘤的雌性MMTV-TGF-1/C57BL6小鼠。小鼠将从10周大(正常体重)开始喂以33%的脂肪饮食或以低脂肪的正常小鼠饮食维持。喂食高脂饮食的小鼠将根据30周龄时的体重增加分为三组，如先前报道的那样(国际肥胖杂志，28：956,2004)。最重的小鼠将被指定为容易肥胖，中间的一组被指定为超重，最轻的一组，肥胖抵抗，是指保持在正常体重范围内的小鼠。这种关系与人类相似，即饮食相似，但体重差异很大。在30周龄时，各体重组小鼠分别给予限食25%卡路里或二甲双胍治疗(100或200 mg/kg体重/天)，并追踪至90周龄。食物摄入量、水消耗量和体重将被确定。将在干预之前和特定时间点获取血清样本，以确定IGF-I、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、潜伏期和肿瘤负担与体重和干预的关系将是主要结果。将在干预前和研究结束时测定体内乳腺细胞的增殖情况。肿瘤和乳腺组织将被检测与AMPK通路相关的蛋白质，该通路被认为在卡路里限制和二甲双胍治疗中都是重要的。所获得的信息将提供证据，证明二甲双胍治疗和限制卡路里对与体重状况有关的乳腺肿瘤的发展的影响，并有望导致对患有乳腺癌高风险的肥胖妇女进行人体试验。

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.