Mechanisms of Genetic Instability and Tumor Suppression

描述（由申请人提供）：涉及P53转录调节因子的肿瘤抑制途径在几乎所有人类癌症中失活。在许多癌症中，这是由于野生型P53功能通过其负调节因子Hdm 2和HdmX的异常表达而减弱。小鼠模型和体外转染研究表明，P53的稳定性和功能输出的控制是非常敏感的，其提出的调节因子的浓度的微小变化。因此，精确定量细胞内P53及其负调控因子（包括H/Mdm 2、H/Mdmx和去泛素化酶HAUSP）的浓度对于开发精确的P53调控模型至关重要。这项资助的四个具体目标采用定量生化策略，小鼠分子遗传学和siRNA功能分析和筛选，以阐明在压力前后调节正常和癌细胞中P53肿瘤抑制途径的分子机制。具体目标1和2量化P53和其在细胞质，细胞核和染色质上的调节剂。这些研究旨在测试P53调控的新模型，其中P53介导的H/Mdm 2泛素连接酶的激活在正反馈环中起作用以消除主要的P53转录拮抗剂H/Mdmx。具体目标3使用小鼠模型来测试正反馈环假设，并分析Mdm 2和MdmX中的结构-功能关系。具体目标4提出研究激活的癌基因在表达野生型P53的肿瘤的实质部分中阻断P53通路的机制。它还利用siRNA筛选来鉴定介导P53、Hdm 2和HdmX的蛋白酶体降解的新调节剂。癌症显然是由癌基因中加速生长或增加存活率的激活突变和使肿瘤抑制因子如P53失活的失活突变引起的。然而，我们现在了解到，P53基因在几乎50%的癌症中是正常的，致癌突变使P53功能丧失。因此，在这些癌症中激活P53可以为大量癌症患者提供改善癌症治疗的巨大机会。实现这一目标需要了解P53在正常细胞中是如何调节的，以推断它在肿瘤中是如何失活的。只有这样，我们才能开发出针对特定肿瘤中使P53丧失功能的遗传缺陷的疗法。本提案旨在提供此类信息。

DESCRIPTION (provided by applicant): The tumor suppressor pathway involving the P53 transcriptional regulator is inactivated in almost all human cancers. In many cancers this is due to attenuation of wild type P53 function by aberrant expression of its negative regulators Hdm2 and HdmX. Mouse models and in vitro transfection studies show that the control of P53 stability and functional output are very sensitive to small changes in the concentrations of its proposed regulatory factors. Therefore, precise quantification of the intracellular concentrations of P53 and its negative regulators including H/Mdm2, H/Mdmx and the deubiquitylating enzyme HAUSP are critical for developing accurate models of P53 regulation. The four Specific Aims of this grant employ quantitative biochemical strategies, mouse molecular genetics, and siRNA functional analyses and screens to elucidate the molecular mechanisms that regulate the P53 tumor suppressor pathway in normal and cancer cells prior to and following stress. Specific Aims 1 and 2 quantify P53 and its regulators in the cytoplasm, nucleus, and on chromatin. The studies are designed to test a new model for P53 regulation in which P53-mediated activation of the H/Mdm2 ubiquitin ligase acts in a positive feedback loop to eliminate H/Mdmx, the primary P53 transcriptional antagonist. Specific Aim 3 uses mouse models to test the positive feedback loop hypothesis, and to analyze structure-function relationships in Mdm2 and MdmX. Specific Aim 4 proposes to examine the mechanisms by which activated oncogenes can inactivate the P53 pathway in the substantial fraction of tumors that express wild type P53. It also utilizes an siRNA screen to identify new regulators that mediate proteasomal degradation of P53, Hdm2 and HdmX. Cancer clearly results from activating mutations in oncogenes that accelerate growth or increase survival, and inactivating mutations that disable tumor suppressors such as P53. However, we now understand that the P53 gene is normal in almost 50% of cancers, and that oncogenic mutations disable P53 function. Thus, activating P53 in such cancers could provide a huge opportunity to improve cancer treatment for a substantial number of cancer patients. Achieving this goal requires understanding how P53 is regulated in normal cells to deduce how it is inactivated in tumors. Only then can we develop therapies to target the genetic defects that disable P53 in a particular tumor. This proposal is designed to provide such information.