Mechanisms of Cross-talk Between EphrinB and Alternate Signaling Pathways

Division Of Basic Sciences - Nci

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Abstract

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.