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

突变体p53具有介导肿瘤侵袭的功能获得机制

• 批准号: 8456806
• 负责人: Apple G Long
• 金额: $ 2.88万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456806
• 关键词: Address; Aging; American; Anchorage-Independent Growth; Binding; Biological Assay; Biological Markers; Cancer Etiology; Cancer Prognosis; Cell Adhesion; Cell Line; Cells; Cellular Stress; Cessation of life; ChIP-seq; Codon Nucleotides; DNA; DNA Binding Domain; DNA Sequence; DNA Structure; Data Set; Development; Disease; Epidermal Growth Factor Receptor; Epithelial Cells; Esophageal; Esophageal Squamous Cell Carcinoma; Event; Exhibits; Extracellular Matrix; Family; Gene Targeting; Genes; Human; Immigration; Immunohistochemistry; In Vitro; Invaded; Investigation; Luciferases; Malignant Neoplasms; Malignant neoplasm of esophagus; Matrix Attachment Regions; Mediating; Mesenchymal; Methods; Microarray Analysis; Migration Assay; Missense Mutation; Modeling; Molecular; Molecular Conformation; Morphology; Mus; Mutate; Mutation; Neoplasm Metastasis; Oral; Pathogenesis; Patients; Phenotype; Play; Processed Genes; Prognostic Marker; Property; Protein Isoforms; Proteins; Reporter; Role; Site; Specificity; Staging; Staining method; Stains; Stress; Structure; Survival Rate; System; TP53 gene; Testing; Tissues; Transformed Cell Line; Transgenic Mice; Tumor Cell Invasion; Tumor Suppressor Proteins; Unresectable; Western Blotting; Work; cell transformation; gain of function; insight; keratinocyte; laser capture microdissection; male; member; men; migration; mouse model; mutant; novel; outcome forecast; overexpression; promoter; protein protein interaction; public health relevance; regenerative; response; transcription factor; tumor; tumor initiation; tumor microenvironment; tumor progression

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.

描述（由申请人提供）：食管癌是美国男性癌症相关死亡的第六大原因，每年有近16，000例病例。这种癌症的预后仍然很差，5年生存率为17%，表明需要进一步研究浸润和转移的分子机制。TP 53突变在超过40%的食管癌病例中发现。虽然p53主要被认为是一种肿瘤抑制因子，在其野生型状态下，通过其反式激活基因的作用，响应细胞应激，最近的研究结果表明，突变型p53可能通过neomorphic机制，以促进肿瘤的进展。我们先前的工作利用EGFR和DNA结合结构域（DBD）突变在TP 53中的过表达来转化永生化的原代食管细胞。这些转化的细胞可以在3D器官型（3D OTC）培养物中不同程度地侵入，这取决于TP 53突变的类型。此外，激光捕获显微切割（LCM）与微阵列分析，比较侵袭性与非侵袭性细胞，表现出优先失调的基因与迁移表型的侵袭性细胞。我们假设这些转化的食管上皮细胞的侵袭性基因签名是由突变型p53及其功能获得性特性介导的。这一假设将通过三个相互关联的具体目标进行检验。目的一：研究突变型p53作为一种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，所述p53三重突变体保持其突变体构象但失去其反式激活能力，以确定其是否可以在体外拯救突变体p53表型。我们将在2D中分析增殖、迁移、侵袭和锚定非依赖性生长，并在3D OTC中分析形态学。此外，我们将在具有突变型p53的转化食管细胞系中进行ChIP-seq，以确定突变型p53的新靶点。目的2：研究突变型p53/p63蛋白间的相互作用及其在侵袭性基因中的作用。Np 63的缺失与食管鳞状细胞癌（ESCC）转移的增加和生存率低有关。此外，突变型p53已被证明与deltaNp 63相互作用。我们将通过测定直接相互作用来表征在我们的转化细胞中体外的这种蛋白质-蛋白质相互作用，并通过荧光素酶报告基因测定来确定这种相互作用是否抑制下游deltaNp 63对靶基因的功能。在目标3中，我们将产生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启动子下驱动组成型活性EGFR和突变型p53的过表达，该启动子对口腔鳞状、食管和前胃组织具有特异性。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靶基因表达，如目的1和2所示。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。