Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVps34 (Type-III P13'-kinase) in Breast Cancer Cells
Final rept. 7 May 2004-6 May 2008
TOLEDO UNIV OH
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Macroautophagy plays a pivotal role in type II programmed cell death. Beclin 1 regulates macroautophagy. Over expression of Beclin has been reported to promote autophagy and inhibit 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. 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 generated stable MCF7 breast cancer cells 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 in the complex. We established that p150, a regulatory subunit of type-III PI3K, and associates with Beclin. In Year-3 we generated stable Beclin knockdown cell lines in ZR-75 and MCF7 breast cancer lines. During the extension year we have used these lines to explore the relationship of autophagy to cell death in breast cancer cells treated with tamoxifen and rottlerin. These studies have revealed that Beclin is not required for initiation of autophagic cell death in breast cancer cells and that under some circumstances autophagy can occur independent of Beclin-1. These studies challenge the accepted notion that Beclin is an essential regulator of autophagy in breast cancer.
- Medicine and Medical Research