New regulatory mechanisms of WNT signaling in development, stem cells and cancer

WNT信号在发育、干细胞和癌症中的新调控机制

基本信息

批准号：
10487049
负责人：
Andres Lebensohn
金额：
$ 124.45万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10487049
关键词：
APC gene Address Adult Animal Model Animals Apoptosis Attenuated Binding Biochemical Genetics Biochemical Reaction Biochemistry Biological Biological Process Cardiovascular Diseases Cell Communication Cell Differentiation process Cell Proliferation Regulation Cell physiology Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Complex Cues DNA Repair Defect Dental Development Diabetes Mellitus Disease Embryonic Development Evolution FDA approved Gene Expression Genes Genetic Genetic Screening Goals Haploidy Human Human Cell Line Instruction Laboratories Language Lead Life Ligands Malignant Neoplasms Molecular Morphogenesis Mutate Mutation Natural regeneration Neurodegenerative Disorders Oncogenic Organ Organism Organoids Outcome Pathway interactions Pattern Pharmaceutical Preparations Physiological Process Proteins Proteomics Regulation Reporter Reporting Research Signal Pathway Signal Transduction Testing Therapeutic Tissues Transcription Coactivator Transcriptional Activation WNT Signaling Pathway base beta catenin biological adaptation to stress cancer type casein kinase I comparative fascinate forward genetics genetic analysis genome wide screen interest malformation migration programs protein degradation receptor self-renewal skeletal stem cells therapeutic target tissue regeneration tumor tumorigenesis ubiquitin ligase zygote

项目摘要

Through forward genetics screens in haploid human cells harboring a fluorescent reporter of WNT signaling, we discovered regulators of the intact pathway as well as those selectively required to sustain oncogenic signaling. This study (Lebensohn et al., eLife 2016) comprised seven genome-wide screens systematically interrogating the pathway, including screens for positive, negative, and attenuating regulators of ligand-induced signaling, as well as suppressor screens following disruption of key regulators commonly mutated in WNT-driven tumors, such as the tumor suppressor APC. A comparative analysis of the screens revealed new regulatory mechanisms for ligand reception, signal transduction and transcriptional activation. Our current goal is to elucidate the molecular underpinnings of these new regulatory mechanisms, understand their physiological functions and evaluate their potential as therapeutic targets. Since the inception of my laboratory in October of 2018, our specific objective has been to understand new mechanisms of WNT signal transduction independent of changes in beta-catenin levels. Beta-catenin is the main transcriptional co-activator in the WNT pathway, and regulation of beta-catenin abundance by a multi-protein assembly called the destruction complex is considered the principal control point in the WNT signaling cascade. Binding of WNT ligands to their co-receptors triggers inactivation of the destruction complex, which results in accumulation of beta-catenin and expression of WNT target genes. Mutations in components of the destruction complex that lead to aberrant accumulation of beta-catenin are a major driver of tumorigenesis in many types of cancer. Identifying mechanisms that can revert constitutive WNT signaling in cells with compromised destruction complex function is therefore of great therapeutic interest. Genetic suppressor screens are a powerful way to reveal such mechanisms, as they can illuminate intricate connections in signaling networks. We previously reported a comprehensive, comparative forward genetic analysis of WNT signaling using a haploid human cell line containing a fluorescent reporter of WNT activity (Lebensohn et al., eLife 2016). In a suppressor screen to identify genes required for constitutive WNT signaling induced by loss of the destruction complex component casein kinase 1 alpha (CK1alpha), we identified HUWE1. HUWE1 encodes a HECT domain-containing ubiquitin ligase with diverse cellular functions, including regulation of cell proliferation and differentiation, apoptosis, DNA repair, and stress responses. We found that eliminating HUWE1 in cells lacking CK1alpha leads to an 80-90% reduction of WNT target gene expression, but surprisingly, only to a 20-30% reduction of beta-catenin protein abundance (Lebensohn et al., eLife 2016). These results suggest that HUWE1 regulates WNT signaling through additional mechanisms distinct from the control of beta-catenin protein degradation. We are testing this hypothesis and investigating the relevant molecular mechanisms through biochemical, genetic, proteomic and cell biological approaches.

通过具有Wnt信号传导的荧光记者的单倍体人细胞中的正向遗传学筛选，我们发现了完整途径的调节剂以及维持致癌信号传导所需的选择性的调节剂。 This study (Lebensohn et al., eLife 2016) comprised seven genome-wide screens systematically interrogating the pathway, including screens for positive, negative, and attenuating regulators of ligand-induced signaling, as well as suppressor screens following disruption of key regulators commonly mutated in WNT-driven tumors, such as the tumor suppressor APC.对筛选的比较分析揭示了新的调节机制，用于配体接收，信号转导和转录激活。我们目前的目标是阐明这些新调节机制的分子基础，了解它们的生理功能，并评估其作为治疗靶点的潜力。自2018年10月我的实验室成立以来，我们的具体目标是了解Wnt信号转导的新机制，而不是β-catenin水平的变化。 β-catenin是WNT途径中的主要转录共激活因子，并且通过多蛋白组装对β-catenin的丰度调节称为破坏络合物，被认为是Wnt信号级联中的主要控制点。 Wnt配体与其共受体的结合触发破坏复合物的失活，这导致β-catenin的积累和Wnt靶基因的表达。破坏复合物的成分突变导致β-catenin的异常积累是许多类型的癌症的主要驱动力。因此，识别可以恢复具有破坏性复杂功能损害的细胞中构型Wnt信号传导的机制，因此具有极大的治疗意义。遗传抑制器筛选是揭示此类机制的有力方法，因为它们可以照亮信号网络中的复杂连接。我们先前使用包含Wnt活性的荧光记者的单倍体人细胞系对Wnt信号传导进行了全面的比较前瞻性遗传分析（Lebensohn等，Elife，2016年）。在抑制器屏幕上，以识别由损坏的构型WNT信号传导所需的基因，这是由于破坏复合物酪蛋白激酶1α（CK1Alpha）所引起的，我们确定了HUWE1。 HUWE1编码具有不同细胞功能的含域的泛素连接酶，包括调节细胞增殖和分化，凋亡，DNA修复和应激反应。我们发现，消除缺乏CK1Alpha的细胞中的HUWE1导致Wnt靶基因表达的80-90％降低，但令人惊讶的是，仅降低了β-catenin蛋白丰度的20-30％（Lebensohn等人，Elife，Elife 2016）。这些结果表明，HUWE1通过与β-catenin蛋白降解的控制不同的其他机制来调节Wnt信号传导。我们正在测试这一假设，并通过生化，遗传，蛋白质组学和细胞生物学方法研究相关的分子机制。