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，如临床前肥胖协议中的热量限制。在啮齿类动物中，很容易实施热量限制，但在人类中，减肥方案的结果很差，包括不依从和体重反弹，导致对包括二甲双胍在内的热量限制模拟物的兴趣。在此，我们将在激素反应性乳腺癌的临床相关模型中直接比较热量限制与二甲双胍治疗的影响。我们的假设是二甲双胍将降低乳腺肿瘤的发病率，延长潜伏期，并降低肿瘤负荷，其程度与中度热量限制相似。具体目的1：确定二甲双胍和热量限制对乳腺肿瘤发展的影响，即，发病率、潜伏期、肿瘤负荷和肿瘤病理学与体重的关系。具体目标二：比较二甲双胍和热量限制对参与乳腺肿瘤发生的血清因子纵向测量的影响，即，胰岛素、葡萄糖和IGF-I。具体目标3：评估二甲双胍和热量限制对体内乳腺上皮细胞增殖的影响，以及与增殖和AMPK通路相关的蛋白质的乳腺组织表达水平。我们将使用雌性MMTV-TGF-1/C57 BL 6小鼠，其在第二年发生乳腺肿瘤。从10周龄（正常体重）开始，小鼠将喂食33%脂肪饮食或维持低脂正常小鼠饮食。如先前报道的（Int.J.Obesity28：956，2004），将喂食高脂肪饮食的小鼠在30周龄时根据体重增加分为三组。将最重的小鼠指定为肥胖倾向小鼠，将中间组指定为超重小鼠，将最轻组指定为抗肥胖小鼠，这些小鼠的体重保持在正常体重小鼠的体重范围内。这种关系类似于人类中发生的情况，即，饮食相似，但体重差异很大。在30周龄时，对每个体重组中的小鼠自由进食，限制25%卡路里或用二甲双胍（100或200 mg/kg体重/天）处理，并随访至90周龄。将测定摄食量、饮水量和体重。将在干预前和指定时间点采集血清样本，以确定IGF-I、胰岛素和/或葡萄糖是否在肿瘤发生/预防中发挥作用。与体重和干预相关的乳腺肿瘤发病率、潜伏期和肿瘤负荷将是主要结局。将测定干预前和研究结束时的体内乳腺细胞增殖。将测定肿瘤和乳腺组织中与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的作用中均很重要。所获得的信息将提供二甲双胍治疗和热量限制对乳腺肿瘤发展与体重状态相关的影响的证据，并有望导致乳腺癌高风险肥胖女性的人体试验。

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.