Mechanisms linking ASPP2 to the Rb/E2F and p53 pathways

ASPP2 与 Rb/E2F 和 p53 通路的连接机制

• 批准号: 6825521
• 负责人: CHARLES D LOPEZ
• 金额: $ 24.36万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6825521
• 关键词: RNA interference; acetylation; apoptosis; binding proteins; biological signal transduction; chromatin immunoprecipitation; gene induction /repression; genetic regulation; genetically modified animals; genotype; laboratory mouse; mutant; neoplasm /cancer genetics; p53 gene /protein; phosphorylation; small interfering RNA; transcription factor; tumor suppressor genes

DESCRIPTION (provided by applicant): Elucidating the mechanisms underlying tumor formation and response to therapy is necessary to develop more effective treatments for cancer patients. Defining the cellular context that promotes apoptosis in cancer cells is one of the most fundamental, yet incompletely understood, issues in oncology. ASPP2 (Apoptosis Stimulating Protein of p53 2) is a highly regulated member of a family of p53-binding proteins that enhance apoptosis through stimulation of p53-transactivation of pro-apoptotic target genes. However, the mechanisms underlying the important upstream pathways controlling ASPP2 remain unknown. Preliminary data suggests ASPP2 is an E2F target gene--implying that ASPP2 is a link between the p53 and Rb/E2F pathways. The broad long-term objectives are to understand how ASPP2 contributes to cancer development and resistance to therapy. The objective of this proposal is to characterize the upstream pathways regulating ASPP2 and how these affect ASPP2 biologic function. The central hypothesis of this proposal is that E2F regulates ASPP2 expression and that ASPP2 may function downstream of E2F to enhance p53- mediated apoptosis. The rationale for this proposed research is that by understanding the upstream pathways controlling ASPP2 expression, the cellular context in which ASPP2 promotes apoptosis will be revealed. The hypothesis will be tested by three SPECIFIC AIMS: (1). Determine how E2F modulates ASPP2 expression. An ASPP2 promoter-luciferase system will be interrogated, and the endogenous ASPP2 promoter manipulated, to reveal the mechanism of E2F-induced expression---with and without cellular damage. (2). Determine the extent to which ASPP2 mediates E2F activity using ASPP2 loss of function models. An ASPP2 mouse has been constructed and will be used to generate ASPP2-/- cells. ASPP2 will also be silenced with siRNA in human tumor cells with defined genotypes. These systems will be then be interrogated for resistance to E2F (and other)-induced apoptosis. (3). Determine the extent to which ASPP2 cooperates with the p53 tumor suppressor pathway in vivo. ASPP2 mice have been bred into the tumor prone p53 background and alterations in tumor formation latency and spectrum will be determined. We expect to demonstrate that ASPP2 is a downstream mediator of E2F apoptotic function, gain insight into the E2F-1-ASPP2 pathway mediating damage-induced apoptosis, and obtain in vivo evidence that ASPP2 cooperates with p53-dependent tumor suppression. These findings would be significant because they would demonstrate a new link between the p53 and Rb/E2F tumor suppressor pathways.

描述（由申请人提供）：阐明肿瘤形成的机制和对治疗的反应是开发更有效的治疗癌症患者的必要条件。确定促进癌细胞凋亡的细胞环境是肿瘤学中最基本但尚未完全理解的问题之一。ASPP2 （Apoptosis Stimulating Protein of p53 2）是p53结合蛋白家族中受到高度调控的成员，通过刺激促凋亡靶基因的p53转激活来促进细胞凋亡。然而，控制ASPP2的重要上游通路的机制尚不清楚。初步数据表明，ASPP2是E2F靶基因，这意味着ASPP2是p53和Rb/E2F通路之间的联系。广泛的长期目标是了解ASPP2如何促进癌症的发展和对治疗的耐药性。本提案的目的是表征调节ASPP2的上游途径以及这些途径如何影响ASPP2的生物学功能。本研究的中心假设是E2F调控ASPP2的表达，而ASPP2可能在E2F的下游发挥作用，从而增强p53介导的细胞凋亡。本研究的基本原理是通过了解控制ASPP2表达的上游途径，揭示ASPP2促进细胞凋亡的细胞背景。该假设将通过三个具体目标进行检验：(1)。确定E2F如何调节ASPP2的表达。ASPP2启动子-荧光素酶系统将被询问，内源性ASPP2启动子将被操纵，以揭示e2f诱导的表达机制-有或没有细胞损伤。（2）. 利用ASPP2功能缺失模型确定ASPP2介导E2F活性的程度。已经构建了ASPP2小鼠，并将用于生成ASPP2-/-细胞。在确定基因型的人类肿瘤细胞中，ASPP2也将被siRNA沉默。然后，这些系统将被询问对E2F（和其他）诱导的细胞凋亡的抗性。（3）. 确定ASPP2在体内与p53肿瘤抑制通路的合作程度。ASPP2小鼠已被培育到肿瘤易感p53背景下，并将确定肿瘤形成潜伏期和频谱的变化。我们希望证明ASPP2是E2F凋亡功能的下游介质，深入了解E2F-1-ASPP2通路介导损伤诱导的细胞凋亡，并获得ASPP2协同p53依赖性肿瘤抑制的体内证据。这些发现意义重大，因为它们将证明p53和Rb/E2F肿瘤抑制通路之间存在新的联系。