Regulation of beta-catenin degradation during Wnt signal transduction

描述（由申请人提供）：Wnt/b-连环蛋白信号传导是一种保守的发育途径，在人类疾病中发挥重要作用。大肠腺瘤性息肉病（Wnt通路的一个组成部分）的突变导致家族性腺瘤性息肉病综合征和80%的非遗传性结直肠癌。然而，自从二十年前发现APC以来，对APC在Wnt信号传导中的功能仍然知之甚少。APC是多蛋白复合物的一部分，其促进转录辅激活因子β-连环蛋白的泛素介导的降解。APC功能的丧失（由于截短突变或通过RNAi下调APC水平）导致b-连环蛋白水平升高和Wnt途径的配体非依赖性激活。我们已经开发了一种针对LRP 6的单克隆抗体，LRP 6是Wnt辅助受体，可抑制培养的哺乳动物细胞中Wnt通路的Wnt 3a介导的激活。目前的Wnt信号转导模型表明APC仅在Wnt受体下游发挥作用。令人惊讶的是，我们的抗LRP 6抗体（以及LRP 6 RNAi构建体）在具有APC突变的几种癌细胞系中以及在通过siRNA耗尽APC的细胞中抑制Wnt信号传导。用抗LRP 6抗体处理APC突变癌细胞下调β-连环蛋白的细胞内水平，与对β-连环蛋白降解的作用一致。 在这个提议中，我们试图揭示APC和LRP 6在调节Wnt通路激活中的联系。 我们将评估抗LRP 6抗体治疗或RNAi导致的LRP 6功能丧失是否会抑制APC突变的更大组癌症细胞系中的Wnt信号传导。我们将测试APC功能丧失后Wnt通路在Wnt辅助受体（Frizzled和LRP 6）水平被激活的可能性。 我们将测试在通过RNAi敲低或显性阴性蛋白表达导致APC功能丧失后，β-连环蛋白降解复合物上游的其他蛋白是否是激活Wnt通路所必需的。我们预测APC和LRP 6竞争结合β-连环蛋白降解复合物，我们将在培养细胞，爪蟾卵提取物和纯化的蛋白质中测试这一假设。最后，我们建议使用爪蟾胚胎提供体内证据，以证实APC- morphant胚胎中Wnt靶基因转录的激活可以通过LRP 6下调来阻断。这些研究有可能深入了解一种重要的肿瘤抑制因子APC的功能，并直接影响治疗APC突变引起的Wnt驱动疾病的治疗方法的开发。

DESCRIPTION (provided by applicant): Wnt/b-catenin signaling is a conserved developmental pathway that plays important roles in human disease. Mutations in adenomatous polyposis coli, a Wnt pathway component, are responsible for familial adenomatous polyposis syndrome and 80% of nonhereditary colorectal cancers. Since its identification two decades ago, however, the function of APC in Wnt signaling remains poorly understood. APC is part of a multi-protein complex that promotes ubiquitin-mediated degradation of the transcriptional coactivator, beta-catenin. Loss of APC function (due to truncating mutations or downregulating APC levels by RNAi) results in elevated b-catenin levels and ligand-independent activation of the Wnt pathway. We have developed a monoclonal antibody against LRP6, the Wnt coreceptor that inhibits Wnt3a-mediated activation of the Wnt pathway in cultured mammalian cells. Current models of Wnt signaling suggest that APC functions exclusively downstream of Wnt receptors. Surprisingly, our anti-LRP6 antibody (as well as LRP6 RNAi constructs) inhibits Wnt signaling in several cancer cell lines with mutation of APC as well as in cells depleted of APC by siRNA. Treatment of APC mutant cancer cells with the anti-LRP6 antibody downregulates intracellular levels of beta-catenin, consistent with effects on b-catenin degradation. In this proposal, we seek to uncover the link between APC and LRP6 in regulating Wnt pathway activation. We will assess whether loss of LRP6 function by anti-LRP6 antibody treatment or RNAi inhibits Wnt signaling in a larger panel of cancer lines with mutations in APC. We will test the possibility tha the Wnt pathway is activated at the level of the Wnt coreceptors (Frizzled and LRP6) upon loss of APC function. We will test whether other proteins upstream of the beta-catenin degradation complex are required for activation of the Wnt pathway upon APC loss of function by RNAi knockdown or expression of dominant-negative proteins. We predict that APC and LRP6 compete for binding to the beta-catenin degradation complex, and we will test this hypothesis in cultured cells, Xenopus egg extract, and with purified proteins. Finally, we propose to provide in vivo evidence using Xenopus embryos to confirm that the activation of Wnt target gene transcription in APC- morphant embryos can be blocked by LRP6 downregulation. These studies have the potential to provide insight into the function of an important tumor suppressor, APC, and to directly impact the development of therapeutics for the treatment of Wnt-driven diseases due to mutations in APC.