Functional Analysis of the Beclin-1 Tumor Suppressor Interaction with hVps34 (Type-III PI3'-kinase) in Breast Cancer Cells
Abstract
Macroautophagy is associated with 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, PI(3)P, 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. In Year-3 we have generated valuable MCF7 breast cancer cell lines that are essentially deficient in expression of both Beclin-1 and p150. We have also determined that increased autophagy precedes apoptosis in MCF7 cells treated with tamoxifen. Using the Beclin and p150 knockdown cells, we will now extend our studies to determine definitively if autophagy is a protective mechanism or a cause of cell death.
Document Details
- Document Type
- Technical Report
- Publication Date
- Jun 01, 2007
- Accession Number
- ADA472188
Entities
People
- William A. Maltese
Organizations
- University of Toledo