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.
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