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Functional Analysis of the Beclin-1 Tumor Suppressor Interaction With hVps34 (Type-III P13-Kinase) in Breast Cancer Cells
Annual rept. 7 May 2005-6 May 2006
MEDICAL COLL OF OHIO AT TOLEDO
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14. ABSTRACT Macroautophagy plays a pivotal role in type II programmed cell death. Beclin 1 regulates macroautophagy. Overexpression of Beclin promotes autophagy and inhibits tumorigenesis in breast carcinoma cells, and conversely, heterozygous disruption of the Beclin gene can promote tumorigenesis in mice. In Year-1 we established that Beclin associates with the human type-III phosphatidylinositol 3-kinase PI3K, hVps34, but not with another putative partner, Bcl-2. The lipid product of Vps34, PI3P, is required not only for autophagy, but also for assembly of proteins involved in endocytosis and trafficking of enzymes from the trans-Golgi network to the lysosomes. Our studies indicated that Beclin is required for hVps34 to function in autophagy, but is dispensable for hVps34 to function in endocytosis. In Year-2 we have generated a stable MCF7 breast cancer cell line with expression of FLAG-tagged Beclin under the control of an inducible promoter. Using this cell line, we purified the FLAG-Beclin-Vps34 complex and performed mass spectrometry to identify other protein components present in the complex. We established for the first time that p150, a regulatory subunit of type-III PI3K, associates with Beclin. We generated a Beclin mutant that fails to associate with p150, but remains competent to interact with Vps34. We are currently developing stable MCF7 cells in which p150 and Beclin are individually and jointly suppressed by RNAi. Using these resources, we will determine how Beclin and p150 contribute to the regulation of Vps34 kinase activity and downstream signaling to the mTOR pathway. These studies will provide important insights into how Beclin controls type II cell death in breast cancer.
APPROVED FOR PUBLIC RELEASE