Mechanisms linking ASPP2 to the Rb/E2F and p53 pathways

描述(由申请人提供)：阐明肿瘤形成和治疗反应的潜在机制对于开发更有效的癌症患者治疗方法是必要的。定义促进癌细胞凋亡的细胞背景是肿瘤学中最基本但尚未完全理解的问题之一。ASPP2(P53 2的凋亡刺激蛋白)是P53结合蛋白家族中受高度调控的成员，通过刺激P53反式激活促凋亡靶基因来促进细胞凋亡。然而，控制ASPP2的重要上游通路的潜在机制仍不清楚。初步数据表明，ASPP2是E2F靶基因，这意味着ASPP2是连接P53和Rb/E2F通路的纽带。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抵抗。这项建议的目的是描述调控ASPP2的上游途径以及这些途径如何影响ASPP2的生物功能。这一建议的中心假设是，E2F调节ASPP2的表达，ASPP2可能在E2F下游发挥作用，促进P53介导的细胞凋亡。这项研究的基本原理是，通过了解控制ASPP2表达的上游途径，将揭示ASPP2促进细胞凋亡的细胞背景。这一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。将询问ASPP2启动子-荧光素酶系统，并操纵内源性ASPP2启动子，以揭示E2F诱导表达的机制-有无细胞损伤。(2)。使用ASPP2功能丧失模型确定ASPP2在多大程度上调节E2F活动。已经构建了一只ASPP2小鼠，并将用于产生ASPP2-/-细胞。在具有特定基因类型的人类肿瘤细胞中，ASPP2也将被siRNA沉默。然后将询问这些系统对E2F(和其他)诱导的细胞凋亡的抵抗力。(3)。在体内确定ASPP2与P53抑癌通路的协同程度。ASPP2小鼠被培育成肿瘤易感的P53背景，并将确定肿瘤形成潜伏期和光谱的变化。我们期望证明ASPP2是E2F凋亡功能的下游介导者，深入了解E2F-1-ASPP2通路介导损伤诱导的细胞凋亡，并获得体内证据表明ASPP2与P53依赖的肿瘤抑制协同作用。这些发现将具有重要意义，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。

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.