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

描述(由申请人提供)：这项提案的广泛目标是了解并利用常见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会导致功能障碍的线粒体积聚，这与细胞死亡有关。我们的新的初步数据显示，二甲双胍导致己糖激酶II(HKII)从线粒体解离，并促进细胞内ATP和NAD的耗竭。这些事件，以及二甲双胍介导的细胞死亡，都会在葡萄糖剥夺的情况下强烈增强。这在未转化的细胞中没有观察到。二甲双胍治疗后NAD耗竭似乎也与蛋白质乙酰化的变化有关。最后，加入外源性NAD或过表达NAMPT，NAD合成中的限速酶，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的耗竭以及对关键的糖酵解酶HKII和依赖于NAD的sirtuin蛋白去乙酰化酶途径的特异性作用有关。这一假设将通过两个具体目标进行检验。目的1分析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，并建立小鼠模型以研究葡萄糖和二甲双胍在治疗癌症中的相互作用。将使用遗传方法改变HKII的表达，然后测量其对二甲双胍细胞毒性的影响。通过表达缺乏线粒体结合域的缺失结构，或使用已知的破坏HKII与线粒体结合的多肽和化合物，将检验HKII与线粒体结合的重要性。将使用碳水化合物限制的生酮饮食来开发一种小鼠模型，以减少葡萄糖的可获得性，以确定这是否增强了二甲双胍在体内的抗肿瘤活性。此外，二甲双胍还将与靶向己糖激酶活性或定位的药物联合使用，以确定这是否会改善抗肿瘤效果。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的改变的影响。我们将确定特定的sirtuin脱乙酰酶的抑制是否参与了metforin介导的新陈代谢和细胞存活的变化。我们将鉴定在二甲双胍治疗癌细胞时发生丰度变化的乙酰化蛋白。我们将使用小鼠模型来检测NAMPT的表达和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脱乙酰酶抑制剂增强二甲双胍抗肿瘤生长作用的可能性。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一个更完整的了解。我们将有一个更好的将二甲双胍用于癌症治疗的理由，我们将对如何提高该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.