Identifying novel p53 target genes that mediate tumor suppression

描述（由申请人提供）：p53是小鼠和人类中的关键肿瘤抑制因子，并且是人类癌症中最常见的突变基因。该提案的主要目标是了解p53介导肿瘤抑制的分子机制。p53能够感知细胞应激，并通过激活靶基因的表达诱导细胞周期停滞、衰老或凋亡来应答。一些p53靶基因在介导效应子功能如停滞或凋亡中具有已知的作用。然而，对于介导肿瘤抑制重要的基因是未知的。为了鉴定参与肿瘤抑制的新的p53靶基因，我们使用了表达p53的细胞的微阵列分析，其中每个反式激活结构域内的残基发生突变。p53的第一反式激活结构域（p5325，26）的突变严重损害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靶点的表达。然而，保留了抑制体内肿瘤形成的能力。这些数据表明，在介导肿瘤抑制中重要的靶基因尚未确定，但p5325，26可用于识别候选基因。在这个项目中，我们将集中在候选基因识别使用微阵列研究比较基因表达在鼠胚胎成纤维细胞（MEFs）表达p53突变体，可以在肿瘤抑制（p5325，26，p5353，54，p53wt）与p53突变体，不能抑制肿瘤形成（p5325，26，53，54和p53-null）。候选基因进一步筛选已知在各种人类和小鼠肿瘤中下调的基因，以获得14个候选基因的列表。在本实验中，我们将评估候选基因介导癌基因诱导的p53效应子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。将使用HrasV12 MEF（最初鉴定候选基因的细胞）和表达致癌Kras并含有纯合诱导型p53等位基因的鼠肺癌细胞系。在这些细胞中，我们将在不存在p53的情况下表达每种候选物，或者在进行增殖、衰老、转化和迁移测定之前，在存在p53的情况下使用shRNA敲低每种候选物的表达。此外，我们将使用shRNA敲低方法来分析候选物在非小细胞肺癌体内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物对于p53介导肿瘤抑制的能力是重要的。如有必要，这些实验可以很容易地扩展到包括其他候选基因，或组合分析候选基因。了解p53肿瘤抑制功能的机制将对肿瘤细胞生物学和更好的癌症治疗方法的发展产生广泛的影响。

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.