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: 7115854
• 负责人: CHARLES D LOPEZ
• 金额: $ 31.04万
• 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 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7115854
• 关键词: 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）：阐明肿瘤形成和对治疗反应的潜在机制是为癌症患者开发更有效治疗的必要条件。定义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。ASPP 2（Apoptosis Stimulating Protein of p53 2）是p53结合蛋白家族的一个高度调节的成员，其通过刺激促凋亡靶基因的p53反式激活来增强凋亡。然而，控制ASPP 2的重要上游途径的机制仍然未知。初步数据表明ASPP 2是E2 F靶基因-这意味着ASPP 2是p53和Rb/E2 F通路之间的联系。广泛的长期目标是了解ASPP 2如何促进癌症发展和对治疗的抵抗。该提案的目的是表征调节ASPP 2的上游途径以及这些途径如何影响ASPP 2生物功能。该提议的中心假设是E2 F调节ASPP 2表达，并且ASPP 2可能在E2 F下游起作用以增强p53介导的细胞凋亡。这项研究的基本原理是，通过了解控制ASPP 2表达的上游途径，将揭示ASPP 2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E2 F如何调节ASPP 2表达。ASPP 2启动子-荧光素酶系统将被询问，内源性ASPP 2启动子操作，以揭示E2 F诱导表达的机制-有和没有细胞损伤。（二）、使用ASPP 2功能丧失模型确定ASPP 2介导E2 F活性的程度。已经构建了ASPP 2小鼠，并将用于产生ASPP 2-/-细胞。ASPP 2也将在具有确定基因型的人肿瘤细胞中被siRNA沉默。然后将询问这些系统对E2 F（和其他）诱导的细胞凋亡的抗性。（三）、确定ASPP 2在体内与p53肿瘤抑制通路合作的程度。将ASPP 2小鼠饲养到肿瘤易感性p53背景中，并测定肿瘤形成潜伏期和谱的变化。我们希望证明ASPP 2是E2 F凋亡功能的下游介导者，深入了解E2 F-1-ASPP 2通路介导损伤诱导的凋亡，并获得体内证据表明ASPP 2与p53依赖性肿瘤抑制合作。这些发现将是重要的，因为它们将证明p53和Rb/E2 F肿瘤抑制通路之间的新联系。