A model system to study the tumor suppressor APC

描述（由申请人提供）：几种关键信号转导途径在动物发育中起关键作用，并且在癌症中也被不适当地激活。其中之一是Wnt途径。它在人类癌症中的作用首先是通过肿瘤抑制剂腺瘤性结肠息肉病（APC）确定的，APC在大多数结肠癌中突变。我们现在知道APC是Wnt信号传导的关键负调节因子。APC是多蛋白“破坏复合物”的一部分，靶向关键的Wnt效应子-连环蛋白，用于磷酸化和最终的蛋白酶体破坏。了解APC功能对于理解Wnt信号如何塑造正常发育以及其不适当的激活如何导致癌症至关重要。除了在Wnt调节中发挥作用外，APC还在细胞骨架调节中发挥作用。APC具有多种细胞骨架功能。在这些研究中，提出了通过对染色体分离的影响在APC突变肿瘤中染色体不稳定性中的作用。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。我们使用果蝇Drosophila，利用果蝇中可用的遗传、细胞生物学和生物化学工具的强大组合。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端不同。在过去的供资期间，我们处理了该领域的几个关键问题。首先，我们发现两个果蝇APC家族成员在许多组织中冗余地参与Wnt信号传导，尽管它们的结构不同并且在细胞内定位上存在显著差异。其次，我们产生了APC 2的无效等位基因。使用现有的无效等位基因的APC 1，这使我们能够创建，第一次，组织和整个动物无效的APC家族成员，揭示了空表型的APC功能在Wnt和细胞骨架调节。我们还产生了一系列新的APC 2等位基因;在其他发现中，这些发现揭示了结肠肿瘤特征性的截短APC蛋白对于Wnt调节是减少的，但不是无效的，支持“恰到好处”的假设。他们还发现，这些截短的蛋白质对细胞骨架具有显性负效应，但对Wnt信号转导没有影响。尽管对APC有很大的兴趣，但关于其在破坏复合物和细胞骨架调节中的作用仍存在关键问题。我们提出了3个具体目标，每个解决关键问题：目标1：定义APC蛋白在破坏复合物中的作用机制。目标二：确定APC结构如何影响破坏复合物的组装和活性，并调节连环蛋白转移到E3连接酶目的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，身体的细胞相互沟通，并在成人组织中调节组织维护和修复伤口。改变的细胞通讯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，以更好地了解人类疾病中的问题。

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.