Cellular Targets of Polyomavirus Tumor Antigens

描述（由申请方提供）：蛋白磷酸酶2A（PP 2A）调节异常对于多瘤病毒（PY）诱导的肿瘤发生至关重要，并导致人类癌症。亮氨酸羧基甲基转移酶（LCMT-1）和蛋白质甲基酯酶-1（PME-1）对PP 2A催化亚基的可逆甲基化是调节PP 2A的最特异性细胞机制，因此可能有望成为基于机制的治疗靶点。因此，重要的是确定LCMT-1和PME-1是否对PP 2A具有特异性。此外，癌基因PYMT取代了由甲基化调控的PP 2A的主要调控亚基B551调控亚基，并以甲基化非依赖性方式与PP 2A结合，这可能是因为甲基化是哺乳动物细胞中PP 2A细胞周期调控的正常机制，PYMT（可能还有PYST）需要绕过甲基化才能诱导癌症。然而，PP 2A甲基化在哺乳动物细胞周期调控中的作用尚不清楚。在这项研究中，PP 2A甲基化，B55指导的PP 2A和PYMT/ST在调节有丝分裂检查点中的作用将通过shRNA敲除方法进行平行检查，其中PP 2A甲基化测定，细胞周期分期（FACS），延时显微镜，微管靶向药物和其他生化方法将被使用。其次，将使用体内（放射性标记结合shRNA敲除）和体外（使用重组酶的蛋白质甲基化/去甲基化试验）方法的组合研究LCMT-1和PME-1对PP 2A的特异性。第三，目前还不清楚PYMT、PYST或甲基化是否影响B“PP 2A调节亚基家族，其功能是调节视网膜母细胞瘤相关蛋白、p107、WNT通路和DNA复制，或影响B4调节亚基，其调节磷酸化和securin的降解。转染、shRNA、腺病毒载体和人细胞转化方法将用于确定B“家族成员或B4是否受PP 2A催化亚基甲基化调节或以转化相关方式被PYMT和PYST靶向置换。最后，我们将在细胞和小鼠模型中测试LCMT-1敲低是否促进转化和肿瘤发生，以及ST介导的转化是否涉及PP 2A依赖性和独立性活动和/或多个B型亚基的下调。这些实验方法将帮助我们深入了解多瘤病毒介导的转化和肿瘤发生，以及PP 2A甲基化和B型亚基在控制正常细胞周期和癌症中的作用。公共卫生相关性：对多瘤病毒诱发癌症的研究已经确定了细胞中导致人类癌症的许多途径和机制。这项拨款提案中的实验将帮助我们更好地了解通过我们的多瘤病毒研究发现的正常细胞生长调节的新机制，这可能代表癌症化疗的新靶点。在这个新靶点水平发挥作用的药物可能毒性较小，因此对抗癌治疗更有用。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.