Identifying novel p53 target genes that mediate tumor suppression

描述(申请人提供)：P53是一种关键的肿瘤抑制基因，在小鼠和人类中，是人类癌症中最常见的突变基因。这项建议的主要目的是了解P53介导肿瘤抑制的分子机制。P53能够感知细胞的应激反应，通过激活靶基因的表达诱导细胞周期停滞、衰老或凋亡。一些P53靶基因在介导效应功能方面具有已知的作用，如停滞或凋亡。然而，调节肿瘤抑制的重要基因尚不清楚。为了确定与肿瘤抑制相关的新的p53靶基因，我们使用了表达p53的细胞的微阵列分析，在这些细胞中，每个反式激活结构域的残基都发生了突变。P53的第一个反式激活域(p5325，26)的突变严重损害了P53激活基因表达的能力。该突变体不能诱导大多数已知的P53靶标的表达。然而，在体内抑制肿瘤形成的能力是保留的。这些数据表明，在调节肿瘤抑制中起重要作用的靶基因尚未确定，但p5325、26可用于识别候选基因。在这个项目中，我们将专注于利用微阵列研究确定的候选基因，以比较表达能够抑制肿瘤形成的p53突变(p5325，26，p5353，54，p53wt)和无法抑制肿瘤形成的p53突变(p5325，26，53，54和p53缺失)的小鼠胚胎成纤维细胞(MEF)中的基因表达。这些候选基因被进一步筛选出在各种人类和小鼠肿瘤中下调的基因，得到了14个候选基因的列表。在这项提议的实验中，我们将评估候选基因介导癌基因诱导的P53效应功能的必要性和充分性。对于这些实验，我们将使用两种细胞培养系统。HrasV12 MEF，候选基因最初被发现的细胞，以及表达致癌基因Kras并包含纯合子可诱导的p53等位基因的小鼠肺癌细胞株都将被使用。在这些细胞中，我们将在没有p53的情况下表达每个候选基因，或者在进行增殖、衰老、转化和迁移分析之前，使用shRNAs在存在p53的情况下下调每个候选基因的表达。此外，我们将使用shRNA敲除方法来分析候选基因在体内非小细胞肺癌小鼠模型中的重要性。在每个目标中提出的实验将使我们能够确定哪些候选基因对于P53介导肿瘤抑制的能力是重要的。如果有必要，这些实验可以很容易地扩展到包括更多的候选基因，或者分析候选基因的组合。了解P53抑癌功能的机制将在肿瘤细胞生物学和开发更好的癌症治疗方法方面具有广泛的意义。 公共卫生相关性：P53是一种重要的肿瘤抑制蛋白，在超过一半的人类癌症中发生突变，但对预防癌症具有重要作用的P53的活性尚不清楚。在这个项目中，我们将确定P53抑制肿瘤的分子机制。这些研究的结果将在生物学和医学上具有广泛的意义，并可能有助于设计和开发更好的癌症治疗方法。

DESCRIPTION (provided by applicant): p53 is a critical tumor suppressor in mice and humans, and is the most commonly mutated gene in human cancer. The main goal of this proposal is to understand the molecular mechanisms by which p53 mediates tumor suppression. p53 is able to sense cellular stress and respond by inducing cell cycle arrest, senescence, or apoptosis by activating expression of target genes. Some p53 target genes have known roles in mediating effector functions such as arrest or apoptosis. However the genes important for mediating tumor suppression are unknown. To identify novel p53 target genes involved in tumor suppression, we have used microarray analysis of cells expressing p53 in which residues within each transactivation domain are mutated. Mutation of the first transactivation domain of p53 (p5325,26) severely compromises the ability of p53 to activate gene expression. This mutant is unable to induce expression of most known p53 targets. However, the ability to suppress tumor formation in vivo is retained. These data indicate that the target genes important in mediating tumor suppression have not yet been defined, but p5325,26 can be used to identify candidate genes. In this project we will focus on candidate genes identified using microarray studies to compare gene expression in murine embryonic fibroblasts (MEFs) expressing p53 mutants that can function in tumor suppression (p5325,26, p5353,54, p53wt) with p53 mutants that are unable to suppress tumor formation (p5325,26,53,54 and p53-null). The candidates were further filtered for genes known to be downregulated in a variety of human and mouse tumors, to arrive at a list of 14 candidate genes. In the experiments in this proposal, we will assess both the requirement and sufficiency for candidate genes to mediate oncogene- induced p53 effector function. For these experiments we will utilize two cell culture systems. Both HrasV12 MEFs, the cells in which the candidate genes were originally identified, and a murine lung cancer cell line expressing oncogenic Kras and containing homozygous inducible p53 alleles will be used. In these cells we will express each candidate in the absence of p53 or use shRNAs to knockdown expression of each candidate in the presence of p53 before performing proliferation, senescence, transformation, and migration assays. Further, we will use the shRNA knockdown approach to analyze the importance of the candidates in an in vivo mouse model of non-small cell lung cancer. The experiments proposed in each aim will allow us to determine which candidates are important for the ability of p53 to mediate tumor suppression. If necessary, these experiments can easily be expanded to include additional candidates, or to analyze candidate genes in combination. Understanding the mechanisms that underlie the tumor suppressor function of p53 will have broad implications in tumor cell biology and in development of better cancer treatments. PUBLIC HEALTH RELEVANCE: p53 is an important tumor suppressor protein, mutated in over half of all human cancers, yet the activities of p53 that are important in preventing cancer are unknown. In this project we will identify the molecular mechanisms of tumor suppression by p53. The results of these studies will have broad implications in both biology and medicine, and may aid in the design and development of better cancer therapies.