Regulation of beta-catenin degradation during Wnt signal transduction
Wnt 信号转导过程中 β-catenin 降解的调控
基本信息
- 批准号:8437474
- 负责人:
- 金额:$ 29.65万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2007
- 资助国家:美国
- 起止时间:2007-07-01 至 2017-08-31
- 项目状态:已结题
- 来源:
- 关键词:APC geneAdenomatous Polyposis Coli ProteinAnimal ModelAntibodiesBindingBiochemicalBiochemistryBiological AssayCancer cell lineCell MaintenanceCell NucleusCell membraneCellsColorectal CancerComplexCultured CellsDataDevelopmentDiseaseDominant-Negative MutationDown-RegulationEmbryoEmbryonic DevelopmentEventExcisionFluorescenceGene ExpressionGenetic TranscriptionInvestigationLDL-Receptor Related ProteinsLigandsLinkMalignant NeoplasmsMammalian CellMediatingMembraneModelingMonoclonal AntibodiesMutationOligonucleotidesOrganismPathway interactionsPhosphorylationPlayProtein BindingProtein OverexpressionProteinsRNA InterferenceRecombinantsRegulationRoleSignal TransductionSignal Transduction PathwaySignaling MoleculeSmall Interfering RNASolidSystemTestingTissuesTranscription CoactivatorTumor Suppressor ProteinsUbiquitinXenopusXenopus laevisbeta catenincancer cellcolon cancer cell lineegghuman diseasein vivoinsightlipoprotein receptor-related protein 6loss of functionmutantprogramsprotein complexprotein functionpublic health relevancereceptorstem cellstherapeutic development
项目摘要
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.
描述(由申请人提供):Wnt/b-catenin信号是一个保守的发育通路,在人类疾病中发挥重要作用。大肠腺瘤性息肉病(一种Wnt通路成分)的突变是家族性腺瘤性息肉病综合征和80%的非遗传性结直肠癌的原因。然而,自20年前被发现以来,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,促进泛素介导的转录辅激活因子β -连环蛋白的降解。APC功能的丧失(由于截断突变或通过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。我们开发了一种针对LRP6的单克隆抗体,LRP6是一种抑制wnt3a介导的哺乳动物细胞中Wnt通路激活的Wnt辅助受体。目前的Wnt信号模型表明APC仅在Wnt受体的下游发挥作用。令人惊讶的是,我们的抗LRP6抗体(以及LRP6 RNAi构建物)抑制了几种APC突变的癌细胞系以及siRNA耗尽APC的细胞中的Wnt信号传导。用抗lrp6抗体治疗APC突变癌细胞可下调细胞内β -连环蛋白水平,这与对b-连环蛋白降解的影响一致。在本提案中,我们试图揭示APC和LRP6在调节Wnt通路激活中的联系。我们将评估抗LRP6抗体治疗或RNAi是否会在APC突变的更大范围的癌症系中抑制Wnt信号。我们将测试在APC功能丧失时,Wnt通路在Wnt协受体(frzzled和LRP6)水平上被激活的可能性。我们将测试当APC因RNAi敲低或显性阴性蛋白的表达而丧失功能时,是否需要β -连环蛋白降解复合物上游的其他蛋白来激活Wnt通路。我们预测APC和LRP6会竞争与β -catenin降解复合物的结合,我们将在培养细胞、爪蟾卵提取物和纯化蛋白中验证这一假设。最后,我们提出利用爪蟾胚胎提供体内证据来证实APC- morphant胚胎中Wnt靶基因转录的激活可以通过LRP6下调来阻断。这些研究有可能深入了解重要的肿瘤抑制因子APC的功能,并直接影响治疗APC突变引起的wnt驱动疾病的治疗方法的发展。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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ETHAN LEE其他文献
ETHAN LEE的其他文献
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{{ truncateString('ETHAN LEE', 18)}}的其他基金
Maximizing Investigators' Research Award (R35 - Clinical Trial Optional)
最大化研究者研究奖(R35 - 临床试验可选)
- 批准号:
10402163 - 财政年份:2017
- 资助金额:
$ 29.65万 - 项目类别:
Maximizing Investigators' Research Award (R35 - Clinical Trial Optional)
最大化研究者研究奖(R35 - 临床试验可选)
- 批准号:
10791528 - 财政年份:2017
- 资助金额:
$ 29.65万 - 项目类别:
Maximizing Investigators' Research Award (R35 - Clinical Trial Optional)
最大化研究者研究奖(R35 - 临床试验可选)
- 批准号:
10596608 - 财政年份:2017
- 资助金额:
$ 29.65万 - 项目类别:
Biochemical reconstitution of heterotrimeric G proteins in the Wnt pathway
Wnt 通路中异源三聚体 G 蛋白的生化重建
- 批准号:
7939072 - 财政年份:2009
- 资助金额:
$ 29.65万 - 项目类别:
Biochemical reconstitution of heterotrimeric G proteins in the Wnt pathway
Wnt 通路中异源三聚体 G 蛋白的生化重建
- 批准号:
7455949 - 财政年份:2007
- 资助金额:
$ 29.65万 - 项目类别:
Biochemical reconstitution of heterotrimeric G proteins in the Wnt pathway
Wnt 通路中异源三聚体 G 蛋白的生化重建
- 批准号:
7619172 - 财政年份:2007
- 资助金额:
$ 29.65万 - 项目类别:
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腺瘤性息肉病大肠杆菌蛋白在小鼠耳蜗中的表达。
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