Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

EphrinB 与替代信号通路之间的串扰机制

• 批准号: 10262044
• 负责人: Ira Daar
• 金额: $ 80.43万
• 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/10262044
• 关键词: Actins; Adhesions; Affect; Amino Acids; Amphibia; Apical; Axon; Benign; Binding; Biological Models; Body Regions; Breast; Cancer cell line; Cell Adhesion; Cell Cycle Progression; Cell physiology; Cell-Cell Adhesion; Cell-Matrix Junction; Cells; Chimeric Proteins; Colon; Congenital Abnormality; Cytoplasmic Tail; Deletion Mutagenesis; Development; Developmental Process; Docking; Dsh protein; Embryo; Embryonic Development; Eph Family Receptors; EphB2 Receptor; Ephrins; Epithelial Cells; Event; Extracellular Domain; Family; Fc domain; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Frequencies; GTPBP1 gene; Glean; Guanine Nucleotide Dissociation Inhibitors; Guanosine Triphosphate Phosphohydrolases; Human; Laboratories; Ligands; Link; Malignant Neoplasms; Malignant neoplasm of gastrointestinal tract; Mediating; Membrane; Modification; Morphogenesis; Movement; Neoplasm Metastasis; Neural Crest Cell; Neuroblastoma; Oligonucleotides; PTEN gene; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Positioning Attribute; Process; Prostate; Proteins; Receptor Protein-Tyrosine Kinases; Reporting; Research; Retina; Role; SW480; Scaffolding Protein; Signal Pathway; Signal Transduction; Signaling Molecule; Small Interfering RNA; System; Tissues; Tumor Angiogenesis; Tumor Cell Invasion; Tumor Stem Cells; Vertebrates; Visual Fields; WNT Signaling Pathway; Xenopus; angiogenesis; cancer cell; cell behavior; cell motility; ciliopathy; cilium biogenesis; embryonic stem cell; epithelial to mesenchymal transition; experimental study; gain of function; hindbrain; human disease; in vivo; insight; interest; kinetosome; loss of function; lung small cell carcinoma; melanoma; member; migration; mutant; neoplastic cell; overexpression; pluripotency; retinal progenitor cell; rho; scaffold; skeletal; stem cell fate specification; stem cells; therapeutic target; tumor; tumor progression; tumorigenesis

During normal development progenitor cells of many tissues undergo progressive restriction of pluripotency, epithelial-to- mesenchymal transition, proliferation, migration, and differentiation. Most, if not all, of these events involve modifications of cell-cell and cell-matrix adhesion, and abnormal modifications of these adhesion systems are often associated with the formation of tumors. The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are frequently over-expressed in a wide variety of cancers, including breast, small-cell lung and gastrointestinal cancers, melanomas, and neuroblastomas. Using the Xenopus embryonic system, we have demonstrated that signaling mediated by the intracellular domain of ephrinB affects cell-cell adhesion, and that this activity can be modulated by interaction with an activated FGF receptor. The transmembrane ephrinB1 protein is a bi-directional signaling molecule that signals through its cytoplasmic domain to promote cellular movements into the eye field, whereas activation of the fibroblast growth factor receptor (FGFR) represses these movements and retinal fate. In Xenopus embryos, ephrinB plays a role in retinal progenitor cell movement into the eye field through an interaction with the scaffold protein Dishevelled (Dsh). We recently identified Drg1 (Developmentally regulated GTP binding protein 1) as a new but requisite interactor and regulator of Dishevelled (Dvl) for proper ciliogenesis. Dvl is a central scaffold that mediates both canonical and non-canonical Wnt signaling and can decisively orchestrate various developmental and cellular processes. One of these processes is ciliogenesis, and while Dvl is known to be critical for apical docking and planar polarization of basal bodies in ciliated epithelial cells, little mechanistic insight into the players and their roles have been gleaned since this original discovery. Regulators of Dvl degradation and Nkd1, as well as its phosphorylation [ie PTEN have been revealed with regard to Dvl's role in ciliogenesis, but few other key players. Here we report the identification of Drg1, a little studied GTP-binding protein 1, that interacts with Dvl and localizes it to the basal body region. This results in another Dvl-associated protein, Daam1, being properly positioned in this basal body region. Daam1 is a formin homology protein that regulates RhoA and the apical actin meshwork that is necessary for apical basal body docking in muticiliated cells (MCCs). Using loss-of-function (via morpholino oligonucleotides) and gain-of-function or rescue experiments in the amphibian system, we provide critical in vivo evidence that a Drg1/Dvl interaction plays a critical role in ciliogenesis in MCCs by regulating the localization of Daam1 and commensurate RhoA activity, contributing to basal body docking and the formation of the apical actin meshwork.We believe that these findings are of broad interest and relevant to Wnt/PCP signaling pathway, as well as understanding ciliopathies. Deregulation of the Eph/ephrin system is associated with the promotion of aggressive and metastatic tumors in various human cancers. Here, we show that a Rho family GTPase regulator, Rho-specific guanine nucleotide dissociation inhibitor 1 (RhoGDI1), can interact with ephrinB1, and this interaction is enhanced upon binding the cognate EphB2 receptor extracellular domain. Deletion mutagenesis revealed that amino acids 327334 of the ephrinB1 intracellular domain are critical for the interaction with RhoGDI1. Stimulation with an EphB2 extracellular domain-Fc fusion protein (EphB2-Fc), induces RhoA activation, and enhances cancer cell migration and invasion in wildtype-ephrinB1 expressing cells. In contrast, these Eph-Fc-induced effects were markedly diminished in cells expressing the mutant ephrinB1 construct (delta327-334) that is ineffective at interacting with RhoGDI1. Furthermore, EphrinB1 depletion by siRNA suppresses EphB2-Fc-induced RhoA activation, and reduces cancer cell migration and invasion in SW480 and Hs578T human cancer cell lines. Our study reveals that the binding of RhoGDI1 and ephrinB1 promotes cancer cell behavior associated with tumor progression and may be a therapeutic target in cancers that express ephrinB1.

在正常发育过程中，许多组织的祖细胞都经历了多能性、上皮细胞向间质细胞转变、增殖、迁移和分化的进行性限制。大多数，如果不是全部，这些事件涉及细胞-细胞和细胞-基质粘附的修饰，这些粘附系统的异常修饰通常与肿瘤的形成有关。Eph家族的受体酪氨酸激酶及其配体ephrin在多种癌症中经常过度表达，包括乳腺癌、小细胞肺癌和胃肠道癌、黑色素瘤和神经母细胞瘤。利用非洲爪蟾胚胎系统，我们已经证明了由ephrinB细胞内结构域介导的信号传导影响细胞间的粘附，并且这种活性可以通过与激活的FGF受体相互作用来调节。跨膜ephrinB1蛋白是一种双向信号分子，通过其细胞质域发出信号，促进细胞运动进入视野，而成纤维细胞生长因子受体（FGFR）的激活抑制这些运动和视网膜命运。在非洲爪蟾胚胎中，ephrinB通过与支架蛋白disheveled （Dsh）的相互作用，在视网膜祖细胞进入视野的过程中发挥作用。我们最近发现Drg1（发育调节GTP结合蛋白1）是一种新的但必需的相互作用因子和disheveled （Dvl）的调节剂，用于正常的纤毛发生。Dvl是一个中心支架，介导规范和非规范Wnt信号，并能决定性地协调各种发育和细胞过程。其中一个过程是纤毛发生，虽然已知Dvl对纤毛上皮细胞的根尖对接和基底的平面极化至关重要，但自这一最初发现以来，对参与者及其作用的机制了解很少。关于Dvl在纤毛发生中的作用，已经揭示了Dvl降解和Nkd1的调节因子，以及它的磷酸化[即PTEN]，但其他关键角色很少。在这里，我们报道了Drg1的鉴定，这是一种很少被研究的gtp结合蛋白1，它与Dvl相互作用，并将其定位于基底体区域。这导致另一种低密度脂蛋白相关蛋白Daam1被正确地定位在这个基底体区域。Daam1是一种双甲素同源蛋白，调节RhoA和顶端肌动蛋白网络，这是多细胞（mcs）的顶端基体对接所必需的。通过两栖动物系统的功能丧失（通过形态学寡核苷酸）和功能获得或挽救实验，我们提供了关键的体内证据，表明Drg1/Dvl相互作用通过调节Daam1的定位和相应的RhoA活性，促进基底体对接和顶端肌动蛋白网络的形成，在mcc的纤毛发生中起着关键作用。我们相信这些发现具有广泛的意义，与Wnt/PCP信号通路以及理解纤毛病有关。在各种人类癌症中，Eph/ephrin系统的失调与促进侵袭性和转移性肿瘤有关。在这里，我们发现Rho家族GTPase调节剂，Rho特异性鸟嘌呤核苷酸解离抑制剂1 （RhoGDI1）可以与ephrinB1相互作用，并且这种相互作用在结合同源EphB2受体胞外结构域后增强。缺失突变表明，ephrinB1胞内结构域的327334氨基酸是与RhoGDI1相互作用的关键氨基酸。EphB2细胞外结构域fc融合蛋白（EphB2- fc）刺激，诱导RhoA激活，增强野生型ephrinb1表达细胞的癌细胞迁移和侵袭。相反，在表达不能与RhoGDI1相互作用的突变型ephrinB1构建体（delta327-334）的细胞中，这些ephf - fc诱导的效应明显减弱。此外，siRNA缺失EphrinB1抑制ephb2 - fc诱导的RhoA激活，减少癌细胞在SW480和Hs578T人癌细胞系中的迁移和侵袭。我们的研究表明，RhoGDI1和ephrinB1的结合促进了与肿瘤进展相关的癌细胞行为，可能是表达ephrinB1的癌症的治疗靶点。