Mutant p53 has gain-of-function mechanisms to mediate tumor invasion

描述(申请人提供)：食道癌是美国男性癌症相关死亡的第六大原因，每年有近16,000例。这种癌症的预后仍然很差，五年生存率为17%，这表明有必要进一步研究其侵袭和转移的分子机制。在超过40%的食道癌病例中发现了TP53的突变。虽然P53最初被认为是野生型状态下的肿瘤抑制因子，其作用是通过反式激活响应细胞压力的基因，但最近的发现表明，突变型P53可能通过新的机制促进肿瘤的进展。我们之前的工作利用TP53中EGFR和DNA结合域(DBD)突变的过表达来转化永生化的原代食道细胞。这些转化的细胞可以在3D器官型(3D OTC)培养中不同程度地入侵，这取决于TP53突变的类型。此外，激光捕获显微切割(LCM)结合微阵列分析，比较侵袭性和非侵袭性细胞，显示与侵袭性细胞中的迁移表型相关的基因优先失调。我们假设这些转化的食道上皮细胞的侵袭基因特征是由突变型P53及其功能获得特性介导的。这一假设将通过三个相互关联的具体目标来检验。目的I：确定突变型P53作为异常转录因子的作用。这将通过在2D和3D系统中表达P53三重突变体来实现，该突变体在2D和3D系统中保持其突变构象但失去其反式激活能力，以确定它是否能够在体外挽救突变的P53表型。我们将在2D中检测增殖、迁移、侵袭和锚定非依赖性生长，并在3D OTC中检测形态学。此外，我们将在突变的p53转化的食道细胞系中进行芯片序列分析，以确定突变的p53的新靶点。目的：研究突变型P53/P63蛋白-蛋白相互作用及其对侵袭性基因特征的影响。Np63基因缺失与食管鳞癌(ESCC)转移增加和生存不良有关。此外，突变型p53已被证明与deltaNp63相互作用。我们将通过分析Dirct相互作用来表征转化细胞中的这种蛋白质-蛋白质相互作用，并通过荧光素酶报告基因分析来确定这种相互作用是否抑制了靶基因下游的deltaNp63功能。在目标3中，我们将建立ESCC的小鼠模型，以确认下游靶点在突变型P53介导的侵袭中的作用。我们将在L2启动子下驱动具有结构性活性的EGFR和突变型p53的过度表达，这是口腔鳞状细胞、食道和前胃组织所特有的。在这些转基因小鼠老化后，我们将检查侵袭性和转移性肿瘤的目标基因的表达，这在AIMS 1和2中确定。此外，我们将表征这些小鼠的肿瘤发展和进展。突变型P53在肿瘤侵袭中的功能作用将获得新的见解。

DESCRIPTION (provided by applicant): Esophageal cancer is the 6th leading cause of cancer related deaths in American males with nearly 16,000 cases each year. Prognosis of this cancer remains poor with a five-year survival rate of 17%, indicating a need for further investigation int the molecular mechanisms underlying invasion and metastases. Mutations in TP53 are found in over 40% of esophageal cancer cases. While p53 has primarily been considered to be a tumor suppressor in its wild-type state through its role in trans-activating genes that respond to cellulr stresses, recent findings have suggested that mutant p53 may act through neomorphic mechanisms to promote tumor progression. Our prior work utilized overexpression of EGFR and DNA-binding domain (DBD) mutations in TP53 to transform immortalized primary esophageal cells. These transformed cells can invade in 3D- organotypic (3D OTC) culture to varying degrees, depending upon the type of TP53 mutation. Additionally, laser capture microdissection (LCM) with microarray analysis, comparing invasive versus non-invasive cells, showed preferential dysregulation in genes associated with a migratory phenotype in the invasive cells. We hypothesize that the invasive gene signature of these transformed esophageal epithelial cells is mediated by mutant p53 and its gain-of-function properties. This hypothesis will be tested through the three interrelated Specific Aims. Aim I: To determine the role of mutant p53 as an aberrant transcription factor. This will be achieved by expression of a p53 triple mutant, which maintains its mutant conformation but loses its trans-activation capability, in 2D and 3D systems, to determine if it can rescue the mutant p53 phenotype in vitro. We will assay for proliferation, migration, invasion, and anchorage-independent growth in 2D, and morphology in 3D OTC. Additionally, we will perform a ChIP-seq in the transformed esophageal cell lines with mutant p53 to determine novel targets of mutant p53. Aim 2: To investigate mutant p53/p63 protein-protein interaction and its contribution to the invasive gene signature. Loss of ¿Np63 has been associated with increase in metastases and poor survival in esophageal squamous cell carcinoma (ESCC). Moreover, mutant p53 has been shown to interact with deltaNp63. We will characterize this protein-protein interaction in vitro in our transformed cells by assaying for dirct interaction and determine if this interaction inhibits downstream deltaNp63 functions on target genes through luciferase reporter assays. In Aim 3, we will generate a mouse model of ESCC, to confirm the role of the downstream targets in mutant p53- mediated invasion. We will drive overexpression of a constitutively active EGFR and mutant p53, under an L2-promoter, which is specific for oral squamous, esophageal, and forestomach tissues. After aging these transgenic mice, we will examine the invasive and metastatic tumors for expression of the target genes, determined in Aims 1 and 2. Furthermore, we will characterize these mice for tumor development and progression. New insights will be acquired into the functional roles of mutant p53 in tumor invasion.