Role of p53 Missense Mutations on Tumorigenesis in Vivo

描述（由申请人提供）：癌症是一种复杂的疾病，每年夺去数百万人的生命。遗传和表观遗传程序的改变破坏了使细胞保持稳态的正常控制。p53肿瘤抑制因子对维持体内平衡至关重要，其活性在大多数癌症的发展中丧失。p53通路中最常见的改变是p53基因本身的错义突变。大量的体内和体外实验表明，与缺乏p53的细胞相比，p53突变蛋白的细胞具有额外的生长优势。其他研究表明，肿瘤细胞系依赖于p53突变体的表达，因为p53突变体的敲除使转化的表型恢复，使细胞对化疗更敏感。对人类组织和异种移植物的研究表明，肿瘤细胞与基质的进化不同，两者都有助于肿瘤表型的形成。基质含有许多成分，如内皮细胞，内皮细胞形成血管为肿瘤提供营养，免疫细胞试图对抗癌细胞，基质成纤维细胞奠定基质，经常改变肿瘤细胞转移的微环境。目前生成的突变p53小鼠模型并不能忠实地再现人类散发性肿瘤，这要么是因为存在外源性启动子驱动突变p53的表达，要么是因为一开始就有一个空等位基因（具有深远的生物学效应）。为了解决这些问题，我们通过p53突变位点的敲入产生了p53错义突变的第一个体细胞模型。该等位基因表达野生型p53，但通过cre介导的重组会删除野生型cDNA并表达突变型p53。这个模型使我们能够在野生型细胞的海洋中制造一个单一的p53突变表达细胞，从而更好地模拟人类癌症。我们计划在乳腺易发肿瘤模型的乳腺上皮中产生体细胞p53突变。我们还将在ErbB2/neu模型中对间质成纤维细胞进行p53突变。在这两项研究中，我们将监测微环境的变化，并最终进行下一代测序和表达分析，以了解驱动肿瘤发生的肿瘤和基质特异性变化。该模型将为理解肿瘤上皮细胞及其微环境的共同进化提供动力。由于我们的条件小鼠模型与人类散发性肿瘤具有相同的潜在分子病理学，因此它将更能预测人类对药物的反应，因此在临床前测试中更有价值。最后，为了评估抑制突变型p53在癌症中的潜在治疗效果，我们将开发一个新的条件突变型p53等位基因。总之，通过生成更好的小鼠模型，这项工作旨在促进我们对突变p53如何促进肿瘤发生的机制的理解，并扩展这一知识，以推进含有这些突变的肿瘤的治疗选择。

DESCRIPTION (provided by applicant): Cancer is a complex disease that kills millions of people annually. Alterations of genetic and epigenetic programs derail the normal controls that keep cells in homeostasis. The p53 tumor suppressor is crucial in maintaining homeostasis and its activity is lost in the development of most cancers. The most common alteration that occurs in the p53 pathway is a missense mutation of the p53 gene itself. Numerous in vivo and in vitro experiments suggest that cells with mutant p53 proteins haven additional growth advantages over cells that lack p53. Other studies show that tumor cell lines are addicted to mutant p53 expression as knockdown of mutant p53 reverts transformed phenotypes and makes cells more responsive to chemotherapy. Studies with human tissues and xenografts indicate that tumor cells evolve differently from the stroma and both contribute to the tumor phenotype. The stroma contains numerous components such as endothelial cells which form vessels to feed the tumor, immune cells that try to fight the cancer cells, and stromal fibroblasts that lay down the matrix and are often changing the microenvironment for a tumor cell to metastasize. Current mutant p53 mouse models generated do not faithfully recapitulate human sporadic tumors, either due to the presence of exogenous promoters driving expression of mutant p53 or the consequence of having a null allele (which has profound biological effects) to start with. To address these issues, we have generated the first somatic model of a p53 missense mutation via a knockin at the mutant p53 locus. This allele expresses wild type p53 but upon Cre-mediated recombination will delete the wild type cDNA and express mutant p53. This model allows us to make one single mutant p53 expressing cell in a sea of wild type cells and as such better models human cancer. We plan to generate somatic p53 mutations in breast epithelium of mammary prone tumor models. We will also make a p53 mutation in stromal fibroblasts in an ErbB2/neu model. In both, we will monitor changes to the microenvironment and ultimately perform next generation sequencing and expression analyses to understand both tumor and stromal specific changes that drive tumorigenesis. This model will provide an impetus for understanding the co-evolution of tumor epithelial cells and their microenvironment. Because our conditional mouse model shares the underlying molecular pathology with human sporadic tumors, it will be more predictive of human responses to drugs, and thus a more valuable tool in preclinical testing. Finally, to evaluate the potential therapeutic efficacy of inhibiting mutant p53 in cancers, we wil develop a novel conditional mutant p53 allele. In summary, through the generation of better mouse models, this work aims to advance our understanding of the mechanisms of how mutant p53 contributes to tumorigenesis and extend this knowledge to advance therapeutic options for tumors harboring these mutations.