Mutant p53 Gain of Function in Tumorigenesis

描述（申请人提供）：超过50%的肿瘤发生p53突变，包括结肠癌、乳腺癌和肺癌。表达突变型p53水平升高的肿瘤可能比无p53的癌症预后更差。这个研究项目的长期目标是继续关注突变p53促进肿瘤发生的机制。突变型p53增强了p53阴性细胞系的致瘤潜能，这种活性长期以来被认为与其选择性反激活人类多药耐药（MDR1）启动子的能力有关。我们之前已经确定突变型p53调节内源性MDR1和c-myc基因的表达，而这种活性需要一个完整的c端。在之前的资助期内，我们确定了该区域的关键赖氨酸，这些赖氨酸通常在细胞应激时在野生型p53中乙酰化，这是突变型p53转激活所必需的。最重要但矛盾的是，我们证明了这个区域对于突变p53促进致瘤性是必不可少的。这些结果从致瘤性中分离了转激活，并代表了考虑突变型p53功能获得模型的范式转变。相反，我们的数据支持突变p53结合并稳定Mdm2原癌基因蛋白的机制，该机制可能导致非整倍体、增生并最终导致肿瘤细胞生长。根据这一推理，我们构建了一个缺少p19 ARF、Mdm2和p53的三重敲除小鼠模型。来源于这些小鼠的小鼠胚胎成纤维细胞（mef）不具有致瘤性，但值得注意的是，缺乏p53和ARF的双敲除mef很容易在裸鼠中形成肿瘤。这些结果表明Mdm2可能是肿瘤细胞生长所必需的。本研究提出的实验旨在通过基于细胞和动物的方法来验证这一假设，解决以下具体问题：1)Mdm2是否与突变型p53在致瘤性中合作？2) Mdm2在体内对突变体p53功能获得的贡献是什么？3)翻译后修饰是否影响突变体p53的功能获得？我们的研究结果表明，野生型和突变型p53功能的结构要求存在显著差异，这可能为开发治疗癌症的治疗方法提供可利用的基础。

DESCRIPTION (provided by applicant): Mutation of p53 occurs in more than 50% of all tumors, including those of colon, breast and lung. Tumors expressing elevated levels of mutant p53 may be associated with a worse prognosis than p53-null cancers. The long-term goal of this research program continues to focus on the mechanisms by which mutant p53 contributes to tumorigenesis. Mutant p53 enhances the tumorigenic potential of p53-negative cell lines and this activity has long been considered to be linked to its ability to selectively transactivate the human multi-drug resistance (MDR1) promoter. We previously established that mutant p53 regulates endogenous MDR1 and c-myc gene expression and that this activity requires an intact C-terminus. In the previous funding period we identified key lysines in this domain, which normally become acetylated in wild-type p53 during cell stress, that are required for mutant p53-transactivation. Most importantly but paradoxically, we demonstrated that this region is dispensable for mutant p53 to promote tumorigenicity. These results uncouple transactivation from tumorigenicity and represent a paradigm shift in considering models for mutant p53 gain of function. Rather, our data support a mechanism by which mutant p53 binds and stabilizes the Mdm2 protooncogene protein, which could lead to aneuploidy, hyperplasia and ultimately tumor cell growth. Consistent with this reasoning, we generated a triple knockout mouse model that lacks p19 ARF,Mdm2 and p53. Mouse embryo fibroblasts (MEFs) derived from these mice are not tumorigenic, but remarkably, double knockout MEFs lacking both p53 and ARF readily form tumors in nude mice. These results suggest that Mdm2 may be required for tumor cell growth. The experiments proposed in this study are designed to test this hypothesis using cell and animal based approaches by addressing the following specific questions: 1) Does Mdm2 cooperate with mutant p53 in tumorigenicity?; 2) What is the in vivo contribution of Mdm2 to mutant p53 gain of function?; and 3) Do post-translational modifications influence mutant p53 gain of function? Our findings demonstrate significant differences in the structural requirements for wild-type and mutant p53 function, which may provide an exploitable basis for developing therapeutic approaches to treating cancer.