Analysis of beclin 1 in autophagy and tumor suppression

描述(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。在这一奖项的资助下，我们的实验室鉴定出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体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线虫的发育停滞表型是必不可少的，线虫的发育停滞表型受到线虫相当于哺乳动物致癌的Class I PI3激酶途径的负调控(附录项目#3)。总之，这些发现提出了自噬作为一种肿瘤抑制机制发挥作用的强烈可能性。在这次续订申请中，我们将从以下两个具体目标来评估这一概念。在第一个目标中，我们将检验这一假设，即Beclin 1通过涉及诱导自噬的III类PI3激酶依赖机制发挥肿瘤抑制活性。为了实现这一目标，我们将使用以前建立的人MCF7乳腺癌细胞模型来确定Beclin 1结合伙伴III PI3-K/hVps34是否参与自噬小泡成核，以及参与自噬后期阶段(自噬小泡扩张和完成)的自噬基因是否对Beclin 1的肿瘤抑制功能是必需的。在第二个目标中，我们还将通过研究包含Beclin 1的Vps34结合缺陷突变体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。我们将验证这样的假设，即Beclin 1下游的自噬基因对于负向生长控制和肿瘤抑制是必需的。为了实现这一目标，我们将研究(1)atg3、ATG5和ATG7杂合缺失的小鼠的自发肿瘤形成；(2)自噬基因atg3、ATG5和beclin 1纯合缺失的胚胎干细胞的体内致瘤性；以及(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确定，Beclin 1通过其依赖于Class III-PI3K的自噬功能发挥肿瘤抑制作用，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.