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，这是由于其负调节因子Hdm2和HdmX的异常表达导致野生型P53功能的衰减。小鼠模型和体外转染研究表明，P53稳定性和功能输出的控制对其提出的调节因子浓度的微小变化非常敏感。因此，精确量化细胞内P53及其负调节因子（包括H/Mdm2、H/Mdmx和去泛素化酶HAUSP）的浓度对于建立准确的P53调控模型至关重要。该资助的四个特定目标采用定量生化策略、小鼠分子遗传学和siRNA功能分析和筛选来阐明正常细胞和癌细胞在应激前后调节P53肿瘤抑制通路的分子机制。特异性Aims 1和2量化P53及其在细胞质、细胞核和染色质上的调节因子。这些研究旨在测试P53调控的新模型，其中P53介导的H/Mdm2泛素连接酶的激活在一个正反馈回路中起作用，以消除主要的P53转录拮抗剂H/Mdmx。Specific Aim 3采用小鼠模型验证正反馈回路假说，并分析Mdm2和MdmX的结构-功能关系。Specific Aim 4提出了在表达野生型P53的大部分肿瘤中，激活的癌基因使P53通路失活的机制。它还利用siRNA筛选来鉴定介导P53、Hdm2和HdmX蛋白酶体降解的新调节因子。癌症显然是由致癌基因的激活突变导致的，这些突变会加速生长或增加生存，而灭活突变会使P53等肿瘤抑制因子失效。然而，我们现在知道，P53基因在近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。要实现这一目标，需要了解P53在正常细胞中是如何被调节的，从而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P53基因缺陷的治疗方法。本提案旨在提供这类信息。

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.