Dysregulation of Protein Phosphatase 2A (PP2A) is critical for polyomavirus (PY)-induced tumorigenesis and contributes to human cancer. Reversible methylation of the PP2A catalytic subunit by Leucine Carboxy Methyltransferase (LCMT-1) and Protein Methylesterase-1 (PME-1) is the most specific cellular mechanism for regulating PP2A, and thus may have promise as a mechanism-based therapeutic target. It is important, therefore, to determine whether LCMT-1 and PME-1 are specific for PP2A. Moreover, the oncogene, PYMT, displaces the primary regulatory subunit of PP2A regulated by methylation, the B551 regulatory subunit, and binds in a methylation-independent manner to PP2A, presumably because methylation is a normal mechanism for cell-cycle regulation of PP2A in mammalian cells that PYMT (and probably PYST) needs to circumvent to induce cancer. However, nothing is known about the role of PP2A methylation in regulation of the mammalian cell cycle. In this study, the role of PP2A methylation, B55-directed PP2A and PYMT/ST in regulating mitotic checkpoints will be examined in parallel through shRNA knockdown approaches in which PP2A methylation assays, cell cycle staging (FACS), time-lapse microscopy, microtubule targeting drugs and other biochemical approaches will be used. Second, the specificity of LCMT-1 and PME-1 for PP2A will be investigated using a combination of in vivo (radiolabeling combined with shRNA knockdowns) and in vitro (protein methylation/demethylation assays using recombinant enzymes) approaches. Third, it is not known whether PYMT, PYST, or methylation affect the B''PP2A regulatory subunit family, which functions to regulate the retinoblastoma related protein, p107, the WNT pathway, and DNA replication, or the B4 regulatory subunit, which regulates phosphorylation and degradation of securin. Transfection, shRNA, adenovirus vector, and human cell transformation approaches will be used to determine if B''family members or B4 are regulated by PP2A catalytic subunit methylation or targeted for displacement by PYMT and PYST in a transformation- relevant manner. Finally, we will test in cellular and mouse models whether LCMT-1 knockdown promotes transformation and tumorigenesis and whether ST-mediated transformation involves PP2A-dependent and independent activities and/or downregulation of multiple B-type subunits. These experimental approaches will help us gain important insight into polyomavirus-mediated transformation and tumorigenesis and the roles PP2A methylation and B-type subunits play in the control of the normal cell cycle and in cancer.
The study of cancer induced by polyomavirus has identified many pathways and mechanisms in cells that contribute to human cancer. The experiments in this grant proposal will help us better understand a new mechanism of regulation of normal cell growth uncovered through our polyomavirus research that may represent a new target for cancer chemotherapy. Drugs that function at the level of this new target would likely be less toxic, and therefore more useful for anti-cancer therapy.
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