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和生物学功能知之甚少。在该奖项的资助下，我们的实验室发现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和其他自噬基因对于秀丽隐杆线虫的发育停滞表型至关重要，该表型由线虫等效的哺乳动物致癌I类PI3激酶途径负调控（附件项目#3）。总之，这些发现提高了自噬作为肿瘤抑制机制的可能性。在此更新申请中，我们将在以下两个具体目标中评估此概念。在第一个目标中，我们将验证beclin 1通过III类PI3激酶依赖机制发挥肿瘤抑制活性的假设，该机制涉及诱导自噬。为了实现这一目标，我们将使用先前建立的人MCF7乳腺癌细胞模型来确定Beclin 1结合伙伴III类PI3-K/hVps34（参与自噬囊泡成核）以及参与自噬后期（自噬囊泡扩张和完成）的自噬基因是否需要Beclin 1的抑瘤功能。我们还将通过研究含有vs34结合缺陷突变的Beclin 1的敲入小鼠的肿瘤发生，来测试Beclin 1- vps34结合在体内肿瘤发生中的重要性。在第二个目标中，我们将验证beclin 1下游的自噬基因是负生长控制和肿瘤抑制所必需的假设。为了实现这一目标，我们将研究(1)atg3、atg5和atg7杂合缺失小鼠的自发肿瘤发生；(2)自噬基因atg3、atg5和beclin 1纯合缺失的胚胎干（ES）细胞的体内致瘤性；(3)自噬基因缺失的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建立beclin 1通过其iii类pi3k依赖性自噬功能作为肿瘤抑制因子，并且自噬是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.