A model system to study the tumor suppressor APC

描述（由申请人提供）：几个关键的信号转导通路在动物发育中起着关键作用，也在癌症中被不适当地激活。其中包括Wnt通路。它在人类癌症中的作用最初是通过肿瘤抑制因子大肠腺瘤性息肉病（APC）确定的，APC在大多数结肠癌中发生突变。我们现在知道APC是Wnt信号的关键负调节因子。APC是多蛋白“破坏复合体”的一部分，其目标是Wnt关键效应物catenin的磷酸化和最终的蛋白酶体破坏。了解APC功能对于理解Wnt信号如何影响正常发育以及其不适当的激活如何导致癌症至关重要。APC除了在Wnt调控中发挥作用外，还在细胞骨架调控中发挥作用。APC具有多种细胞骨架功能。其中之一是APC突变体肿瘤中染色体不稳定的作用，通过对染色体分离的影响。我们建立了一个模型系统来研究APC在正常发育过程中Wnt信号传导和细胞骨架调节中的功能，并模拟其失活如何导致癌症。我们以果蝇为研究对象，利用果蝇遗传、细胞生物学和生化工具的强大组合。哺乳动物和果蝇都有两个APC家族成员，它们都共享一组核心蛋白质结构域，但在N端和c端有所不同。在过去的资助期内，我们解决了该领域的几个关键问题。首先，我们发现两种果蝇APC家族成员在许多组织中都冗余地参与Wnt信号传导，尽管它们的结构不同，细胞内定位也存在显著差异。其次，我们生成了一个APC2的空等位基因。利用APC1现有的空等位基因，我们首次在组织和整个动物中创建了APC家族成员的空等位基因，揭示了APC功能在Wnt和细胞骨架调节中的空表型。我们还生成了一系列新的APC2等位基因；在其他发现中，这些发现揭示了结肠肿瘤特征的截断APC蛋白在Wnt调控中减少，但不是零，支持“刚刚好”假说。他们还发现，这些截断的蛋白对细胞骨架有主要的负面影响，但对Wnt信号传导没有影响。尽管对APC有很大的兴趣，关键问题仍然是关于它在破坏复合体和细胞骨架调节中的作用。我们提出3个具体目标，每个目标都解决关键问题：目标1：定义APC蛋白在破坏复合体中的作用机制。目的2：确定APC结构如何影响破坏复合体的组装和活性，并调节_catenin向E3连接酶的转移。目的3：探索APC家族蛋白调节细胞骨架的机制。公共卫生相关性在胚胎的正常发育过程中，以及在成人组织中，人体细胞之间相互交流，以调节组织维护和修复伤口。细胞通讯的改变是包括结肠癌在内的几种常见癌症的基础，而细胞通讯的丧失会导致某些形式的先天性骨畸形。我们已经开发了一个模型系统来探索肿瘤抑制因子APC如何正常调节细胞通讯和细胞行为，以便更好地了解人类疾病中出现的问题。

DESCRIPTION (provided by applicant): Several key signal transduction pathways play critical roles in animal development, and also are inappropriately activated in cancer. Among these is the Wnt pathway. Its role in human cancer was first identified through the tumor suppressor Adenomatous polyposis coli (APC), mutated in most colon cancers. We now know that APC is a critical negative regulator of Wnt signaling. APC is part of a multiprotein "destruction complex" targeting the key Wnt effector _catenin for phosphorylation and eventual proteasomal destruction. Understanding APC function is essential both to understanding how Wnt signaling shapes normal development and how its inappropriate activation contributes to cancer. In addition to its role in Wnt regulation, APC also plays roles in cytoskeletal regulation. Diverse cytoskeletal functions have been ascribed to APC. Among these is a proposed role in chromosome instability in APC mutant tumors, via effects on chromosome segregation. We established a model system to study APC function in Wnt signaling and cytoskeletal regulation during normal development and to model how its inactivation leads to cancer. We use the fruit fly Drosophila, making use of the powerful combination of genetic, cell biological and biochemical tools available in flies. Both mammals and Drosophila have two APC family members, which all share a core set of protein domains but which differ at their N- and C-termini. In the past funding period, we addressed several key questions in the field. First, we discovered that the two fly APC family members act redundantly in Wnt signaling in many tissues, despite their divergent structures and striking differences in intracellular localization. Second, we generated a null allele of APC2. Using existing null alleles of APC1, this allowed us to create, for the first time, both tissues and whole animals null for both APC family members, revealing the null phenotypes of APC function in both Wnt and cytoskeletal regulation. We also generated a series of new APC2 alleles; among other findings these revealed that the truncated APC proteins characteristic of colon tumors are reduced for Wnt regulation but not null, supporting the "just right" hypothesis. They also revealed that these truncated proteins have dominant negative effects on the cytoskeleton but not in Wnt signaling. Despite the great interest in APC, key questions remain regarding its roles in the destruction complex and cytoskeletal regulation. We propose 3 Specific Aims, each addressing key questions: Aim 1: Define the mechanism(s) of action of APC proteins in the destruction complex. Aim 2: Determine how APC structure influences the assembly and activity of the destruction complex and regulates _catenin transfer to the E3 ligase Aim 3: Explore mechanisms by which APC family proteins regulate the cytoskeleton PUBLIC HEALTH RELEVANCE The body's cells communicate with one another during normal development of an embryo, and in adult tissues to regulate tissue maintenance and repair wounds. Altered cell communication underlies several common cancers including colon cancer, while loss of cell communication causes some forms of congenital bone malformation. We have developed a model system to explore how the tumor suppressor APC normally regulates cell communication and cell behavior, to allow better understanding of what goes wrong in human disease.