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

• 批准号: 10702742
• 负责人: Andres Lebensohn
• 金额: $ 146.05万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10702742
• 关键词: APC gene; Address; Adult; Animal Model; Animals; Apoptosis; Attenuated; Binding; Biochemical Reaction; Biochemistry; Biological Process; Cardiovascular Diseases; Cell Communication; Cell Compartmentation; Cell Cycle Arrest; Cell Surface Receptors; Cell membrane; Cell physiology; Cell surface; Cells; Cellular biology; Chemosensitization; Clustered Regularly Interspaced Short Palindromic Repeats; Colorectal Cancer; Complex; Cues; Defect; Dental; Development; Diabetes Mellitus; Disease; Down-Regulation; Embryo; Embryonic Development; Epithelial; Evolution; FDA approved; Family; Family member; Gene Amplification; Gene Family; Genetic Screening; Goals; Growth Factor; Haploidy; Helix-Turn-Helix Motifs; Heparan Sulfate Proteoglycan; Homeostasis; Human; Hyperactivity; Instruction; Language; Leucine Zippers; Life; Ligand Binding; Ligands; Link; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Mediating; Mediator of activation protein; Mesenchymal; Modeling; Molecular; Morphogenesis; Mutate; Natural regeneration; Neoplasm Metastasis; Neurodegenerative Disorders; Oncogenic; Organ; Organism; Organoids; Outcome; Pathway interactions; Pattern; Pharmaceutical Preparations; Phenotype; Physiological; Process; Property; Proteins; Regulator Genes; Regulatory Element; Reporter; Research; Research Project Grants; Role; Signal Pathway; Signal Transduction; Site; Therapeutic; Tissues; Transcription Coactivator; Transcriptional Activation; Ubiquitin; Ubiquitination; Vertebrates; WNT Signaling Pathway; Xenopus laevis; base; beta catenin; cancer type; casein kinase; comparative; fascinate; forward genetics; gastric cancer cell; genetic analysis; genome wide screen; malformation; member; migration; prevent; programs; receptor; response; self-renewal; skeletal; stem cells; therapeutic target; tissue regeneration; tissue stem cells; transcription factor; tumor; ubiquitin ligase; zygote

New regulatory mechanisms in WNT signaling: 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 hyperactive, 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, as well as new gene regulatory elements (Patel, Lebensohn et al., PLoS One 2019). 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. The following research projects are aimed at elucidating some of these new regulatory mechanisms and evaluating their therapeutic potential. 1) The transcription factor TFAP4 is a new limiting component of the WNT signaling pathway. Our genetic screens uncovered a basic helix-loop-helix leucine zipper transcription factor called TFAP4 as a potential regulator of WNT signaling (Lebensohn et al., eLife 2016). We have shown that TFAP4 acts downstream of b-catenin, the principal transcriptional co-activator in the WNT pathway, and is a limiting component for signaling activity. Furthermore, excess TFAP4 can promote ectopic activation of WNT signaling during Xenopus laevis development, causing the formation of a secondary body axis. Interestingly, TFAP4 expression is tightly linked to malignancy in gastrointestinal cancers: TFAP4 is upregulated in colorectal cancer, where it mediates epithelial-mesenchymal transition (EMT) and metastasis, and its down-regulation in gastric cancer cells inhibits proliferation, induces cell cycle arrest and promotes apoptosis. We are dissecting the molecular mechanism by which TFAP4 regulates WNT signaling, and evaluating its potential as a therapeutic target. 2) A new function of the b-catenin destruction complex regulating WNT signaling through the ubiquitin ligase HUWE1. HUWE1 is a HECT-domain ubiquitin ligase involved in dozens of cellular processes through the ubiquitination of diverse substrates. Both oncogenic and tumor suppressive functions have been ascribed to HUWE1. HUWE1 has been postulated as a negative regulator of WNT signaling through at least two distinct mechanisms. However, in our unbiased forward genetic screen for mediators of hyperactive WNT signaling induced by loss of the b-catenin destruction complex kinase casein kinase 1a, we identified HUWE1 as a positive regulator of the WNT pathway (Lebensohn et al., eLife 2016). We also demonstrated that HUWE1 potentiates WNT signaling in cells and in Xenopus laevis embryos. We have now found that HUWE1 promotes WNT/b-catenin signaling through a mechanism independent of the control of b-catenin protein stability. Potentiation of WNT signaling by HUWE1 requires its ubiquitin ligase activity and a subset of b-catenin destruction complex components. These results reveal a new role for some destruction complex components in mediating WNT signaling through HUWE1, distinct from their established activity in controlling b-catenin stability. New regulatory mechanisms in R-spondin signaling: Some WNT responses during embryonic development and in stem cell compartments depend on a second signal provided by the R-spondin family of secreted growth factors, only present in vertebrates. R-spondins are key regulators of WNT signaling strength, but the mechanisms by which they transduce signals are not fully understood and how the four different members of the family control distinct physiological functions is unknown. We discovered that R-spondins 2 and 3 can uniquely potentiate WNT signaling in cells lacking their only known cell-surface receptors, LGRs 4-6, through an alternative interaction with heparan sulfate proteoglycans (HSPGs) (Lebensohn & Rohatgi, eLife 2018; Dubey et al., eLife 2020). This finding is transformative because LGRs were thought to be required to transduce all R-spondin signals and hence determine their site of action, but now we know that R-spondins can also signal in tissues where LGRs are not expressed. The following research projects are aimed at elucidating the molecular mechanisms and physiological functions of 'LGR-independent' signaling by R-spondins. 3) Molecular mechanisms of LGR-independent signaling by R-spondins. A central question regarding any signal transduction mechanism is how binding of the ligand to the receptor communicates the signal across the plasma membrane and into the cell. In the prevailing model, simultaneous binding of R-spondins to their LGR receptors and to the transmembrane ubiquitin ligases ZNRF3 or RNF43 triggers their internalization. This prevents ZNRF3 and RNF43 from targeting WNT receptors for ubiquitin-mediated degradation, thus increasing their abundance on the cell surface and enhancing sensitivity to WNT ligands. We are investigating the molecular mechanisms whereby R-spondins transduce signals in the absence of LGRs through their alternative HSPG receptors to potentiate WNT signaling. 4) Physiological roles for LGR-dependent and LGR-independent signaling by R-spondins during embryonic development and in stem cells. Gene amplifications followed by neo- or sub-functionalization of different gene family members underlie much of the functional diversification generated during evolution. Yet, paralogues are often deemed to be redundant, and drastically different phenotypes resulting from their loss are simply attributed to tissue-specific patterns of expression. This is the prevailing view regarding the four members of the R-spondin family, which potentiate WNT signaling during embryonic development and in adult tissue stem cells. However, the markedly distinct phenotypes caused by the loss of different R-spondins or their LGR receptors could also be due to intrinsic differences in the capacity of these R-spondins to signal through LGR-dependent and LGR-independent mechanisms. We are investigating in what contexts these two modes of signaling are used during development, tissue homeostasis and regeneration. We are also trying to understand what unique properties make LGR-dependent and LGR-independent R-spondin signaling specifically suited to their physiological functions.

WNT信号传导的新调控机制：通过对带有WNT信号传导荧光报告基因的单倍体人类细胞进行正向遗传学筛选，我们发现了完整通路的调节因子以及维持过度活跃的致癌信号传导所需的调节因子。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，包括配体诱导信号传导的正向、负向和减弱调节因子的筛选，以及在破坏通常突变的关键调节因子后的抑制筛选。 WNT驱动的肿瘤，例如抑癌基因APC。对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控机制，以及新的基因调控元件（Patel、Lebensohn 等人，PLoS One 2019）。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。以下研究项目旨在阐明其中一些新的调节机制并评估其治疗潜力。 1）转录因子TFAP4是WNT信号通路的新限制元件。我们的基因筛选发现了一种称为 TFAP4 的基本螺旋-环-螺旋亮氨酸拉链转录因子，它是 WNT 信号传导的潜在调节因子（Lebensohn 等人，eLife 2016）。我们已经证明 TFAP4 作用于 b-catenin 下游，b-catenin 是 WNT 通路中的主要转录共激活因子，并且是信号传导活性的限制成分。此外，过量的 TFAP4 可以在非洲爪蟾发育过程中促进 WNT 信号的异位激活，从而导致次级身体轴的形成。有趣的是，TFAP4的表达与胃肠道癌症的恶性程度密切相关：TFAP4在结直肠癌中上调，介导上皮间质转化（EMT）和转移，而在胃癌细胞中下调则抑制增殖，诱导细胞周期停滞和转移。促进细胞凋亡。我们正在剖析 TFAP4 调节 WNT 信号传导的分子机制，并评估其作为治疗靶点的潜力。 2) b-连环蛋白破坏复合物通过泛素连接酶 HUWE1 调节 WNT 信号传导的新功能。 HUWE1 是一种 HECT 域泛素连接酶，通过不同底物的泛素化参与数十种细胞过程。致癌和肿瘤抑制功能都归因于 HUWE1。 HUWE1 被认为通过至少两种不同的机制作为 WNT 信号传导的负调节因子。然而，在我们对由 b-连环蛋白破坏复合物激酶酪蛋白激酶 1a 丢失引起的过度活跃 WNT 信号转导介质进行的无偏正向遗传筛选中，我们确定 HUWE1 是 WNT 通路的正调节因子（Lebensohn 等人，eLife 2016）。我们还证明 HUWE1 增强细胞和非洲爪蟾胚胎中的 WNT 信号传导。我们现在发现 HUWE1 通过一种独立于 b-catenin 蛋白稳定性控制的机制促进 WNT/b-catenin 信号传导。 HUWE1 增强 WNT 信号传导需要其泛素连接酶活性和 β-连环蛋白破坏复合物成分的子集。这些结果揭示了一些破坏复合物成分在通过 HUWE1 介导 WNT 信号传导中的新作用，与它们在控制 β-连环蛋白稳定性方面的既定活性不同。 R-spondin 信号传导的新调节机制：胚胎发育期间和干细胞区室中的一些 WNT 反应依赖于仅存在于脊椎动物中的分泌性生长因子 R-spondin 家族提供的第二种信号。 R-spondins 是 WNT 信号强度的关键调节因子，但它们转导信号的机制尚未完全了解，并且该家族的四个不同成员如何控制不同的生理功能尚不清楚。 我们发现，R-spondins 2 和 3 可以通过与硫酸乙酰肝素蛋白聚糖 (HSPG) 的另一种相互作用，在缺乏唯一已知细胞表面受体 LGR 4-6 的细胞中独特地增强 WNT 信号转导 (Lebensohn & Rohatgi, eLife 2018; Dubey等人，eLife 2020）。这一发现具有变革性，因为人们认为 LGR 需要转导所有 R-spondin 信号，从而确定其作用位点，但现在我们知道 R-spondins 也可以在 LGR 不表达的组织中发出信号。以下研究项目旨在阐明 R-spondins 的“LGR 独立”信号传导的分子机制和生理功能。 3) R-spondins 的 LGR 独立信号传导的分子机制。关于任何信号转导机制的一个核心问题是配体与受体的结合如何将信号传递穿过质膜并进入细胞。在流行的模型中，R-spondins 与其 LGR 受体和跨膜泛素连接酶 ZNRF3 或 RNF43 同时结合会触发其内化。这可以防止 ZNRF3 和 RNF43 靶向 WNT 受体进行泛素介导的降解，从而增加它们在细胞表面的丰度并增强对 WNT 配体的敏感性。我们正在研究 R-spondins 在 LGR 不存在的情况下通过其替代 HSPG 受体转导信号以增强 WNT 信号传导的分子机制。 4）胚胎发育和干细胞中R-spondins对LGR依赖性和LGR非依赖性信号传导的生理作用。基因扩增之后不同基因家族成员的新功能化或亚功能化是进化过程中产生的大部分功能多样化的基础。然而，旁系同源物通常被认为是多余的，并且由于其丢失而产生的截然不同的表型被简单地归因于组织特异性的表达模式。这是关于 R-spondin 家族四个成员的普遍观点，它们在胚胎发育和成体组织干细胞中增强 WNT 信号传导。然而，由不同 R-spondins 或其 LGR 受体丢失引起的明显不同的表型也可能是由于这些 R-spondins 通过 LGR 依赖性和 LGR 独立机制发出信号的能力存在内在差异。我们正在研究这两种信号传导模式在发育、组织稳态和再生过程中的使用情况。我们还试图了解哪些独特的特性使得 LGR 依赖性和 LGR 独立性 R-spondin 信号特别适合其生理功能。