Analysis of beclin 1 in autophagy and tumor suppression

描述（由申请人提供）：自噬的溶酶体途径是降解长寿命细胞蛋白和细胞质细胞器的主要调节分解代谢机制。然而，直到最近，人们对自噬的分子机制或生物学功能知之甚少。在该奖项的资助下，我们的实验室鉴定了第一个哺乳动物自噬基因beclin 1（附录项目#1），并证明它是一个单倍不足的肿瘤抑制基因（附录项目#2）。我们还发现，beclin 1和其他自噬基因是C。elegans，其由哺乳动物致癌I类PI 3激酶途径的线虫等价物负调节（附录项目#3）。总之，这些发现提出了自噬作为肿瘤抑制机制的强烈可能性。在这次更新申请中，我们将在以下两个具体目标中评估这一概念。在第一个目标，我们将测试的假设，beclin 1发挥肿瘤抑制活性，通过III类PI 3激酶依赖性机制，涉及诱导自噬。为了实现这一目标，我们将使用先前建立的人MCF 7乳腺癌细胞模型来确定Beclin 1结合伴侣III类PI 3-K/hVps 34（参与自噬囊泡成核）以及参与自噬后期阶段（自噬囊泡扩张和完成）的自噬基因是否是Beclin 1的肿瘤抑制功能所需的。我们还将通过在含有Beclin 1的Vps 34结合缺陷突变体的敲入小鼠中研究肿瘤发生来测试Beclin 1-Vps 34结合在体内肿瘤发生中的重要性。在第二个目标中，我们将测试的假设，自噬基因下游的beclin 1所需的负生长控制和肿瘤抑制。为了实现这一目标，我们将研究（1）atg 3、atg 5和atg 7杂合缺失小鼠的自发性肿瘤发生;（2）自噬基因atg 3、atg 5和beclin 1纯合缺失胚胎干（ES）细胞的体内致瘤性;（3）自噬基因缺失酵母和哺乳动物细胞的细胞生长控制。总之，我们预计这些研究将确定beclin 1通过其III类PI 3 K依赖性自噬功能作为肿瘤抑制因子发挥作用，并且自噬代表了参与肿瘤抑制和负生长控制的基本机制。

DESCRIPTION (provided by applicant): The lysosomal pathway of autophagy is the major regulated catabolic mechanism for degrading long-lived cellular proteins and cytoplasmic organelles. Yet, until recently, very little was known about the molecular mechanisms or biological functions of autophagy. Under funding from this award, our laboratory identified the first mammalian autophagy gene, beclin 1 (appendix item #1), and demonstrated that it is a haploinsufficient tumor suppressor gene (appendix item #2). We have also found that beclin 1 and other autophagy genes are essential for a developmental arrest phenotype in C. elegans that is negatively regulated by the nematode equivalent of the mammalian oncogenic Class I PI3 kinase pathway (appendix item #3). Together, these findings raise the strong possibility that autophagy functions as a tumor suppressor mechanism. In this renewal application, we will evaluate this concept in the following two specific aims. In the first aim, we will test the hypothesis that beclin 1 exerts tumor suppressor activity through a Class III PI3 kinase-dependent mechanism that involves the induction of autophagy. To accomplish this aim, we will use a previously established human MCF7 breast carcinoma cell model to determine whether the Beclin 1-binding partner, Class III PI3-K/hVps34, (involved in autophagic vesicle nucleation) and whether autophagy genes involved in a later stage of autophagy (autophagic vesicle expansion and completion) are required for the tumor suppressor function of beclin 1. We will also test the importance of Beclin 1-Vps34 binding in tumorigenesis in vivo by studying tumorigenesis in a knock-in mouse that contains a Vps34-binding defective mutant of Beclin 1. In the second aim, we will test the hypothesis that autophagy genes downstream of beclin 1 are required for negative growth control and tumor suppression. To accomplish this aim, we will study (1) spontaneous tumorigenesis in mice with heterozygous deletion of atg3, atg5, and atg7; (2) the in vivo tumorigenicity of embryonic stem (ES) cells with homozygous deletions of the autophagy genes, atg3, atg5 and beclin 1, and (3) cell growth control in yeast and mammalian cells with autophagy gene deletions. Together, we anticipate that these studies will establish that beclin 1 functions as a tumor suppressor through its Class III-PI3K-dependent autophagy function, and that autophagy represents a fundamental mechanism involved in tumor suppression and negative growth control.