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：
10262535
负责人：
Andres Lebensohn
金额：
$ 102.87万
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/10262535
关键词：
AXIN2 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 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 Tumor Suppressor Proteins WNT Signaling Pathway base beta catenin biological adaptation to stress cancer type casein kinase 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 a new mechanism 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 1alpha (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 a 90% reduction of WNT reporter activity and to an 80-85% reduction in expression of the endogenous WNT target gene AXIN2, but surprisingly, only to a 20-32% reduction of beta-catenin protein abundance (Lebensohn et al., eLife 2016). These results suggest that HUWE1 regulates WNT signaling through an additional mechanism distinct from the control of beta-catenin protein degradation. We are testing this hypothesis through biochemical, genetic, proteomic and cell biological approaches.

通过对带有 WNT 信号荧光报告基因的单倍体人类细胞进行正向遗传学筛选，我们发现了完整途径的调节因子以及维持致癌信号传导选择性所需的调节因子。这项研究（Lebensohn 等人，eLife 2016）包括七个全基因组筛选，系统地询问该通路，包括筛选配体诱导的信号转导的正、负和减弱调节因子，以及在破坏常见突变的关键调节因子后进行抑制筛选在 WNT 驱动的肿瘤中，例如肿瘤抑制因子 APC。筛选的比较分析揭示了配体接收、信号转导和转录激活的新调节机制。我们当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它们作为治疗靶点的潜力。自我的实验室于 2018 年 10 月成立以来，我们的具体目标是了解一种独立于 β-catenin 水平变化的 WNT 信号转导新机制。 β-连环蛋白是 WNT 通路中的主要转录共激活因子，通过称为破坏复合物的多蛋白组装来调节 β-连环蛋白丰度被认为是 WNT 信号级联中的主要控制点。 WNT 配体与其共受体的结合会触发破坏复合物的失活，从而导致 β-连环蛋白的积累和 WNT 靶基因的表达。导致β-连环蛋白异常积累的破坏复合物成分的突变是许多类型癌症中肿瘤发生的主要驱动因素。因此，确定能够恢复破坏复合物功能受损的细胞中的组成型 WNT 信号转导的机制具有重大的治疗意义。基因抑制筛选是揭示此类机制的有效方法，因为它们可以阐明信号网络中复杂的连接。我们之前报道了使用含有 WNT 活性荧光报告基因的单倍体人类细胞系对 WNT 信号进行全面、比较的正向遗传分析（Lebensohn 等人，eLife 2016）。在通过抑制子筛选来鉴定因破坏复合物成分酪蛋白激酶 1α (CK1α) 缺失而诱导的组成性 WNT 信号传导所需的基因时，我们鉴定出了 HUWE1。 HUWE1 编码含有 HECT 结构域的泛素连接酶，具有多种细胞功能，包括调节细胞增殖和分化、细胞凋亡、DNA 修复和应激反应。我们发现，在缺乏 CK1α 的细胞中消除 HUWE1 会导致 WNT 报告基因活性降低 90%，并使内源性 WNT 靶基因 AXIN2 的表达降低 80-85%，但令人惊讶的是，仅导致 β 降低 20-32% -连环蛋白丰度（Lebensohn 等人，eLife 2016）。这些结果表明 HUWE1 通过不同于控制 β-连环蛋白降解的额外机制来调节 WNT 信号传导。我们正在通过生化、遗传学、蛋白质组学和细胞生物学方法来检验这一假设。