Regulation of beta-catenin degradation during Wnt signal transduction

描述(申请人提供)：WNT/b-catenin信号是一种保守的发育途径，在人类疾病中发挥重要作用。结肠腺瘤性息肉病是Wnt途径的一个组成部分，它的突变与家族性腺瘤性息肉病综合征和80%的非遗传性结直肠癌有关。然而，自20年前APC被发现以来，对其在WNT信号中的作用仍知之甚少。APC是一个多蛋白质复合体的一部分，它促进泛素介导的转录辅助激活因子β-连环蛋白的降解。APC功能的丧失(由于RNAi截断突变或下调APC水平)导致b-catenin水平升高和Wnt途径的配体无关激活。我们已经开发出一种针对LRP6的单抗，LRP6是Wnt的辅助受体，可以抑制Wnt3a介导的Wnt途径在培养的哺乳动物细胞中的激活。目前的Wnt信号模型表明，APC仅在Wnt受体下游发挥作用。令人惊讶的是，我们的抗LRP6抗体(以及LRP6 RNAi构建物)抑制了几个具有APC突变的癌细胞系中的Wnt信号，以及被siRNA耗尽的APC细胞中的Wnt信号。用抗LRP6抗体处理APC突变癌细胞可下调细胞内β-连环蛋白的水平，这与对b-连环蛋白降解的影响一致。在这个提案中，我们试图揭示APC和LRP6之间在调节Wnt途径激活方面的联系。我们将评估抗LRP6抗体治疗或RNAi导致的LRP6功能丧失是否会抑制APC突变的更大一组癌细胞中的Wnt信号。我们将测试当APC功能丧失时，Wnt通路在Wnt辅助受体(FrizzledLRP6)水平上被激活的可能性。我们将测试是否需要β-连环蛋白降解复合体上游的其他蛋白质，以在APC通过RNAi敲除或表达显性负性蛋白质而失去功能时激活Wnt途径。我们预测APC和LRP6竞争与β-连环素降解复合体的结合，我们将在培养细胞、非洲爪哇卵提取物和纯化的蛋白质中验证这一假说。最后，我们建议利用非洲爪哇胚胎提供体内证据，以证实LRP6下调可以阻止APC变形胚胎中Wnt靶基因转录的激活。这些研究有可能深入了解重要的肿瘤抑制因子APC的功能，并直接影响由于APC突变而导致的Wnt驱动疾病的治疗方法的发展。

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.