Mechanisms of Genetic Instability and Tumor Suppression

描述(申请人提供)：涉及P53转录调节因子的肿瘤抑制通路在几乎所有人类癌症中都是失活的。在许多癌症中，这是由于野生型p53功能因其负调控因子Hdm2和HdmX的异常表达而减弱。小鼠模型和体外转染研究表明，控制P53的稳定性和功能输出对其建议的调节因子浓度的微小变化非常敏感。因此，准确定量P53及其负调控因子H/MDM2、H/MDMX和去泛素化酶Hausp的细胞内浓度对于建立准确的P53调控模型至关重要。这项拨款的四个具体目标是利用定量生化策略、小鼠分子遗传学和siRNA功能分析和筛选来阐明在应激前后调节正常细胞和癌细胞中P53肿瘤抑制途径的分子机制。特异性目标1和2对细胞质、细胞核和染色质上的P53及其调节因子进行定量。这些研究旨在测试一种新的P53调控模型，在该模型中，P53介导的H/MDM2泛素连接酶的激活在正反馈环中作用，以消除主要的P53转录拮抗剂H/MDMX。《特定目的3》使用小鼠模型检验正反馈环路假说，并分析MDM2和MDMX的结构-功能关系。特定目标4建议检查激活的癌基因可以在表达野生型p53的大部分肿瘤中使P53途径失活的机制。它还利用siRNA筛选来识别介导P53、Hdm2和HdmX蛋白酶体降解的新调节因子。癌症显然是由于激活了加速生长或提高存活率的癌基因突变，以及使肿瘤抑制基因失活(如p53)的突变造成的。然而，我们现在了解到，在几乎50%的癌症中，P53基因是正常的，癌基因突变使P53功能丧失。因此，在这类癌症中激活p53可能会为相当数量的癌症患者提供改善癌症治疗的巨大机会。要实现这一目标，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。只有到那时，我们才能开发针对特定肿瘤中使p53失效的遗传缺陷的治疗方法。这项建议旨在提供这样的信息。

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.