A model system to study the tumor suppressor APC and its role in Wnt regulation.

研究抑癌基因 APC 及其在 Wnt 调节中的作用的模型系统。

基本信息

批准号：
8574208
负责人：
David Roberts
金额：
$ 27.85万
依托单位：
FRANKLIN AND MARSHALL COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2017-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8574208
关键词：
26S proteasome APC2 gene Address Adenomatous Polyposis Coli Adult Affinity Amino Acids Animals Binding Sites Biochemical Biological Biological Assay Biological Models Biology Cancer Etiology Cell Maintenance Cell Proliferation Cells Cessation of life Colon Carcinoma Complex Data Development Developmental Process Diagnosis Disease Drosophila genus Drosophila melanogaster Event Evolution Family Gene Targeting Genetic Genetic Models Goals Homeostasis Human Lead Light Malignant Neoplasms Mammalian Cell Mediating Modeling Molecular Mutation Nuclear Oncogenic Pathway interactions Phenotype Phosphorylation Phosphorylation Site Play Protein Dephosphorylation Proteins Proteomics Reagent Recruitment Activity Regulation Research Role Serine Signal Pathway Signal Transduction Signal Transduction Pathway Speed Stem cells Structure Testing Transgenes Tumor Suppressor Proteins Ubiquitin-mediated Proteolysis Pathway Ubiquitination Work base biochemical model c-myc Genes colon cancer cell line human disease in vivo insight loss of function mutation member novel prevent protein complex public health relevance research study transcription factor tumor progression tumorigenesis ubiquitin-protein ligase

26S proteasome APC2 gene Address Adenomatous Polyposis Coli Adult Affinity Amino Acids Animals Binding Sites Biochemical Biological Biological Assay Biological Models Biology Cancer Etiology Cell Maintenance Cell Proliferation Cells Cessation of life Colon Carcinoma Complex Data Development Developmental Process Diagnosis Disease Drosophila genus Drosophila melanogaster Event Evolution Family Gene Targeting Genetic Genetic Models Goals Homeostasis Human Lead Light Malignant Neoplasms Mammalian Cell Mediating Modeling Molecular Mutation Nuclear Oncogenic Pathway interactions Phenotype Phosphorylation Phosphorylation Site Play Protein Dephosphorylation Proteins Proteomics Reagent Recruitment Activity Regulation Research Role Serine Signal Pathway Signal Transduction Signal Transduction Pathway Speed Stem cells Structure Testing Transgenes Tumor Suppressor Proteins Ubiquitin-mediated Proteolysis Pathway Ubiquitination Work base biochemical model c-myc Genes colon cancer cell line human disease in vivo insight loss of function mutation member novel prevent protein complex public health relevance research study transcription factor tumor progression tumorigenesis ubiquitin-protein ligase

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项目摘要

DESCRIPTION (provided by applicant): To properly orchestrate the dramatic events that occur during animal development, it is essential that cells communicate with their neighbors through the use of cell signaling pathways. While signaling pathways play critical roles in normal developmental processes, inappropriate activation of these pathways frequently results in cancer. The Wnt signaling pathway provides a paradigm for how misregulation of cell signaling pathways can contribute to oncogenesis. Loss-of-function mutations in the tumor suppressor protein, Adenomatous Polyposis Coli (APC), inappropriately activate the pathway and frequently initiate cancer progression. Mutations in APC are responsible for over 80% of all colon cancer cases, thus it is clear that understanding APC biology is crucial to our efforts to treat the disease. To truly understand how inactivation of APC initiates the oncogenic phenotype, it is first crucial to understand how APC contributes to normal Wnt regulation. In the current model of Wnt signaling, APC participates in a multi-protein complex (called the destruction complex) that targets the key effector protein ¿-catenin for ubiquitin-mediated proteolysis. In the absence of APC, the destruction complex is inactivated by an unknown mechanism, and ¿-catenin protein levels hyper- accumulate. Together with its nuclear partners (members of the TCF/LEF family), ¿-catenin functions as a bi- partite transcription factor, promoting expression of Wnt target genes such as c-myc and cyclinD1 that drive cell proliferation. While it is clear that APC participates in the ¿-catenin destruction complex, its mechanistic role in this complex has remained elusive. In the past decade, several models have been proposed to explain APC's mechanistic function in the ¿- catenin destruction complex. While these models are based on convincing biochemical data, they remain to be rigorously tested using functional studies. During my postdoctoral research, I began to address some models of APC function by performing an in vivo structure/function analysis of APC. This study uncovered several surprising findings, including the identification of two largely uncharacterized regions of APC essential for its function in Wnt regulation. Overall, this structure/function approach provided important insight into the destruction complex, and suggested an alternative model of its inner workings. Here, I expand upon these initial findings and propose experiments that will directly evaluate three prominent biochemical models of APC function, and are informed by my prior structure/function study. For these experiments, I propose to use Drosophila APC2 as a model for human APC, as members of the Wnt pathway are highly conserved throughout evolution. Moreover, I have previously shown that Drosophila APC2 works as effectively as human APC in functional studies when expressed in human colon cancer cell lines. Thus we can take advantage of the speed of this simpler genetic model system to investigate questions important to the progression and treatment of human colon cancer.

描述（由适用提供）：为了正确地编排动物发育过程中发生的戏剧性事件，必须通过使用细胞信号通路与邻居进行通信。信号通路在正常发育过程中起着关键作用，但这些途径的不适当激活通常会导致癌症。 Wnt信号通路为细胞信号通路的不正调如何导致肿瘤发生提供了一个范式。肿瘤抑制蛋白，腺瘤性息肉大肠（APC）的功能丧失突变，不当激活该途径并经常引发癌症进展。 APC中的突变负责所有结肠癌病例中的80％以上，因此很明显，了解APC生物学对我们治疗该疾病的努力至关重要。为了真正了解APC的失活是如何启动致癌表型的，它首先是了解APC如何贡献正常的WNT调节的关键。在当前的WNT信号传导模型中，APC参与了多蛋白质复合物（称为破坏复合物），该复合物靶向泛素介导的蛋白水解靶向关键效应蛋白�-Catenin。在没有APC的情况下，破坏复合物被未知的机制灭活，并且 - 钙蛋白蛋白水平高蓄积。 - 帕宁蛋白与其核伴侣（TCF/LEF家族的成员）一起起作用，作为双方转录因子，促进了驱动细胞增殖的Wnt靶基因（例如C-Myc和Cyclind1）的表达。虽然很明显，APC参与者是 - 帕宁蛋白破坏复合物，但其在该复合物中的机械作用仍然难以捉摸。在过去的十年中，已经提出了几种模型来解释APC在Catenin破坏复合物中的机械功能。尽管这些模型基于令人信服的生化数据，但使用功能研究仍需要严格测试它们。在我的博士后研究中，我开始通过执行APC的体内结构/功能分析来解决APC功能的一些模型。这项研究发现了一些令人惊讶的发现，包括鉴定了两个在WNT调节中功能所必需的两个很大程度上未表征的APC区域。总体而言，这种结构/功能方法为破坏复合物提供了重要的见解，并提出了其内部工作的替代模型。在这里，我扩展了这些初始发现和建议实验，这些发现将直接评估APC功能的三种突出的生化模型，并通过我先前的结构/功能研究来告知。对于这些实验，我建议将果蝇APC2用作人类APC的模型，因为WNT途径的成员通过进化是高度保守的。此外，我先前已经表明，在人类结肠癌细胞系中表达时，果蝇APC2在功能研究中的有效工作。我们可以利用这种简单的遗传模型系统的速度来研究对人类结肠癌进展和治疗重要的问题。