Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

在正常发育期间，许多组织的祖细胞经历多能性、上皮-间充质转化、增殖、迁移和分化的进行性限制。这些事件中的大多数（如果不是全部）涉及细胞-细胞和细胞-基质粘附的修饰，并且这些粘附系统的异常修饰通常与肿瘤的形成相关。受体酪氨酸激酶的Eph家族及其配体肝配蛋白在多种癌症中经常过表达，包括乳腺癌、小细胞肺癌和胃肠癌、黑色素瘤和神经母细胞瘤。使用非洲爪蟾胚胎系统，我们已经证明，ephrinB的细胞内结构域介导的信号传导影响细胞-细胞粘附，并且这种活性可以通过与活化的FGF受体的相互作用来调节。跨膜ephrinB 1蛋白是一种双向信号分子，通过其胞质结构域发出信号以促进细胞运动进入眼区，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Dishevelled（Dsh）的相互作用在视网膜祖细胞移动到眼区中发挥作用。我们最近确定Drg 1（发育调节GTP结合蛋白1）作为一个新的，但必要的相互作用和调节器的Dishevelled（Dvl）适当的纤毛发生。Dvl是介导经典和非经典Wnt信号传导的中心支架，并且可以决定性地协调各种发育和细胞过程。这些过程之一是纤毛发生，虽然已知Dvl对于纤毛上皮细胞中的基底体的顶端对接和平面极化至关重要，但自从这一最初的发现以来，对参与者及其作用的机制见解很少。Dvl降解和Nkd 1的调节剂以及其磷酸化[即PTEN]已经揭示了Dvl在纤毛发生中的作用，但很少有其他关键参与者。在这里，我们报告的鉴定Drg 1，一个研究很少的GTP结合蛋白1，与Dvl相互作用，并将其定位到基体区域。这导致另一种Dvl相关蛋白Daam 1正确定位在该基体区域中。Daam 1是一种调节RhoA和顶端肌动蛋白网络的同源蛋白，该顶端肌动蛋白网络是多纤毛细胞（MCC）中顶端基底体对接所必需的。使用功能丧失（通过吗啉代寡核苷酸）和两栖动物系统中的功能获得或拯救实验，我们提供了关键的体内证据，即Drg 1/Dvl相互作用通过调节Daam 1的定位和相应的RhoA活性在MCC中的纤毛发生中起关键作用，我们相信这些发现具有广泛的意义，并且与Wnt/PCP信号通路，以及了解纤毛病变。Eph/ephrin系统的失调与各种人类癌症中侵袭性和转移性肿瘤的促进相关。在这里，我们表明，Rho家族GTdR调节剂，Rho特异性鸟嘌呤核苷酸解离抑制剂1（RhoGDI 1），可以与ephrinB 1相互作用，这种相互作用增强后，结合同源EphB 2受体胞外结构域。缺失突变显示ephrinB 1胞内结构域的氨基酸327 - 334对于与RhoGDI 1的相互作用至关重要。用EphB 2细胞外结构域-Fc融合蛋白（EphB 2-Fc）刺激，诱导RhoA活化，并增强癌细胞在野生型肝配蛋白B1表达细胞中的迁移和侵袭。相比之下，这些Eph-Fc诱导的作用在表达突变型埃甫蛋白B1构建体（delta 327 -334）的细胞中显著减弱，所述突变型埃甫蛋白B1构建体在与RhoGDI 1相互作用时无效。此外，EphrinB 1通过siRNA的消耗抑制EphB 2-Fc诱导的RhoA活化，并减少SW 480和Hs 578 T人癌细胞系中的癌细胞迁移和侵袭。我们的研究表明，RhoGDI 1和ephrinB 1的结合促进了与肿瘤进展相关的癌细胞行为，并且可能是表达ephrinB 1的癌症的治疗靶点。

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.