(PQA1)Molecular mechanisms by which the diabetic drug metformin kills cancer cell

描述（由申请人提供）：本提案的总体目标是了解并利用常见的糖尿病药物二甲双胍诱导的癌细胞死亡所涉及的代谢变化。二甲双胍治疗癌细胞导致功能失调线粒体的积累，这与细胞死亡有关。我们新的初步数据表明，二甲双胍导致己糖激酶II （HKII）从线粒体中分离，并促进细胞ATP和NAD+的消耗。这些事件，以及二甲双胍介导的细胞死亡，被葡萄糖剥夺强烈增强。这在未转化的细胞中没有观察到。二甲双胍治疗后的NAD+消耗似乎也与蛋白质乙酰化的变化有关。最后，添加外源性NAD+或过度表达NAD合成中的限速酶NAMPT，可以保护细胞免受二甲双胍的细胞毒性。基于这些发现，我们假设二甲双胍介导的癌细胞死亡与ATP和NAD+的消耗以及对关键糖酵解酶HKII和NAD依赖性sirtuin蛋白去乙酰化酶途径的特异性影响有关。这一假设将通过两个具体目标进行检验。目的1是解剖葡萄糖和糖酵解在二甲双胍介导的癌细胞死亡中的作用，确定HKII从线粒体解离的意义，并建立小鼠模型来研究葡萄糖水平和二甲双胍治疗癌症之间的相互作用。遗传学方法将用于改变HKII的表达，然后测量其对二甲双胍细胞毒性的影响。HKII与线粒体关联的重要性将通过表达缺乏线粒体结合结构域的缺失构建体或使用已知破坏HKII与线粒体结合的肽和化合物来检验。一个小鼠模型将使用限制碳水化合物的生酮饮食来降低葡萄糖的可用性，以确定这是否能增强二甲双胍在体内的抗肿瘤活性。此外，二甲双胍将与针对己糖激酶活性或定位的药物联合使用，以确定这是否能提高抗肿瘤效果。目的2是确定NAD+和NAMPT如何保护细胞免受二甲双胍的细胞毒性。我们将研究NAMPT过表达或抑制对二甲双胍介导的能量代谢和细胞杀伤变化的影响。我们将确定特异性sirtuin去乙酰化酶的抑制是否参与二甲双胍介导的代谢和细胞存活的变化。我们将确定乙酰化蛋白在二甲双胍治疗癌细胞时大量改变。我们将使用小鼠模型来检测NAMPT表达和NAD+前体对二甲双胍抑制肿瘤生长的影响。我们将确定NAMPT和sirtuin去乙酰化酶抑制剂的潜力，以增强二甲双胍对抗肿瘤生长的作用。随着这项工作的完成，我们将对二甲双胍作用于癌细胞的分子机制有更全面的了解。我们将对二甲双胍重新用于癌症治疗有一个改进的理论基础，我们将对如何提高药物的疗效有新的见解。

DESCRIPTION (provided by applicant): The broad goal of this proposal is to understand, and take advantage of, metabolic changes that are involved in cancer cell death induced by the common diabetic drug metformin. Metformin treatment of cancer cells leads to accumulation of dysfunctional mitochondria which is associated with cells death. Our new preliminary data show that metformin causes hexokinase II (HKII) to dissociate from mitochondria and promotes depletion of cellular ATP and NAD+. These events, as well as metformin-mediated cell death, are strongly enhanced by glucose deprivation. This is not observed in non-transformed cells. NAD+ depletion following metformin treatment also appears to be associated with changes in protein acetylation. Finally, addition of exogenous NAD+ or overexpression of NAMPT, the rate limiting enzyme in NAD synthesis, protects cells against metformin cytotoxicity. Based on these findings, we hypothesize that metformin-mediated cancer cell death is associated with depletion of ATP and NAD+ and specific effects on a key glycolytic enzyme, HKII, and on NAD-dependent sirtuin protein deacetylase pathways. This hypothesis will be tested through two specific aims. Aim 1 is to dissect the role of glucose and glycolysis on metformin-mediated cell death of cancer cells, to determine the significance of HKII dissociation from mitochondria, and to establish a mouse model to examine the interaction between glucose levels and metformin in treating cancer. Genetic approaches will be used to alter expression of HKII and then the affect on metformin cytotoxicity will be measured. The importance of mitochondrial association by HKII will be examined by expressing deletion constructs that lack the mitochondrial binding domain or by using peptides and compounds that are known to disrupt HKII binding to mitochondria. A mouse model will be developed using a carbohydrate-restricted ketogenic diet to reduce glucose availability to determine if this enhances metformin's anti-tumor activity in vivo. Also metformin will be combined with drugs that target hexokinase activity or localization to determine if this improves the anti-tumor effects. Aim 2 is to determine how NAD+ and NAMPT protect cells against metformin cytotoxicity. We will examine the effects of NAMPT overexpression, or inhibition, on metformin-mediated changes in energy metabolism and cell killing. We will determine if the inhibition of specific sirtuin deacetylases is involved in metforin-mediated changes in metabolism and cell survival. We will identify acetylated proteins that change in abundance upon metformin treatment of cancer cells. We will use mouse models to examine the effects of NAMPT expression and NAD+ precursors on metformin inhibition of tumor growth. We will determine the potential for inhibitors of NAMPT and sirtuin deacetylases to potentiate the action of metformin against tumor growth. With the completion of this work we will have a more complete understanding of the molecular mechanism of action of metformin on cancer cells. We will have an improved rationale for re-purposing of metformin for cancer therapy and we will have new insights on how to improve the efficacy of the drug.