Role of p53 Missense Mutations on Tumorigenesis in Vivo

描述(申请人提供)：癌症是一种复杂的疾病，每年导致数百万人死亡。基因和表观遗传程序的改变破坏了使细胞保持内稳态的正常对照。P53抑癌基因在维持体内平衡方面起着至关重要的作用，它的活性在大多数癌症的发生发展过程中都会丧失。P53途径中最常见的改变是P53基因本身的错义突变。大量的体内和体外实验表明，含有突变型P53蛋白的细胞比缺乏P53蛋白的细胞具有额外的生长优势。其他研究表明，肿瘤细胞系对突变的p53表达上瘾，因为突变的p53被击倒会逆转转化的表型，并使细胞对化疗更敏感。对人体组织和异种移植的研究表明，肿瘤细胞的进化与基质不同，两者都对肿瘤表型有贡献。间质含有许多成分，如形成为肿瘤提供血管的内皮细胞，试图对抗癌细胞的免疫细胞，以及铺设基质的间质成纤维细胞，这些成纤维细胞通常会改变肿瘤细胞转移的微环境。目前建立的突变型p53小鼠模型不能真实地概括人类散发性肿瘤，要么是因为存在驱动突变型p53表达的外源启动子，要么是因为一开始就有一个零等位基因(具有深刻的生物学效应)。为了解决这些问题，我们通过敲击突变的p53基因，建立了第一个p53错义突变的体细胞模型。该等位基因表达野生型P53，但Cre介导的重组会删除野生型基因，表达突变型P53。这个模型允许我们在一大片野生型细胞中制造一个表达单个突变型p53的细胞，从而更好地建立人类癌症的模型。我们计划在乳腺易患肿瘤模型的乳腺上皮细胞中产生体细胞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，以了解推动肿瘤发生的肿瘤和间质特异性变化。这一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。因为我们的条件小鼠模型与人类散发性肿瘤共享潜在的分子病理学，它将更好地预测人类对药物的反应，因此在临床前测试中是更有价值的工具。最后，为了评估抑制突变型p53在癌症中的潜在治疗效果，我们将开发一种新的条件性突变型p53等位基因。总之，通过建立更好的小鼠模型，这项工作旨在促进我们对突变型p53如何促进肿瘤发生机制的理解，并扩展这一知识，以改进对携带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.