Eph-ephrin Interaction Research Articles

Potential role of the Eph/ephrin system in colorectal cancer: emerging druggable molecular targets.

The Eph/ephrin system regulates many developmental processes and adult tissue homeostasis. In colorectal cancer (CRC), it is involved in different processes including tumorigenesis, tumor angiogenesis, metastasis development, and cancer stem cell regeneration. However, conflicting data regarding Eph receptors in CRC, especially in its putative role as an oncogene or a suppressor gene, make the precise role of Eph-ephrin interaction confusing in CRC development. In this review, we provide an overview of the literature and highlight evidence that collaborates with these ambiguous roles of the Eph/ephrin system in CRC, as well as the molecular findings that represent promising therapeutic targets.

A Pharmacological Investigation of Eph-Ephrin Antagonism in Prostate Cancer: UniPR1331 Efficacy Evidence.

The Eph kinases are the largest receptor tyrosine kinases (RTKs) family in humans. PC3 human prostate adenocarcinoma cells are a well-established model for studying Eph-ephrin pharmacology as they naturally express a high level of EphA2, a promising target for new cancer therapies. A pharmacological approach with agonists did not show significant efficacy on tumor growth in prostate orthotopic murine models, but reduced distal metastasis formation. In order to improve the comprehension of the pharmacological targeting of Eph receptors in prostate cancer, in the present work, we investigated the efficacy of Eph antagonism both in vitro and in vivo, using UniPR1331, a small orally bioavailable Eph-ephrin interaction inhibitor. UniPR1331 was able to inhibit PC3 cells' growth in vitro in a dose-dependent manner, affecting the cell cycle and inducing apoptosis. Moreover, UniPR1331 promoted the PC3 epithelial phenotype, downregulating epithelial mesenchymal transition (EMT) markers. As a consequence, UniPR1331 reduced in vitro PC3 migration, invasion, and vasculomimicry capabilities. The antitumor activity of UniPR1331 was confirmed in vivo when administered alone or in combination with cytotoxic drugs in PC3-xenograft mice. Our results demonstrated that Eph antagonism is a promising strategy for inhibiting prostate cancer growth, especially in combination with cytotoxic drugs.

Cholenic acid derivative UniPR1331 impairs tumor angiogenesis via blockade of VEGF/VEGFR2 in addition to Eph/ephrin

Angiogenesis, the formation of new blood vessels from preexisting ones, is crucial for tumor growth and metastatization, and is considered a promising therapeutic target. Unfortunately, drugs directed against a specific proangiogenic growth factor or receptor turned out to be of limited benefit for oncology patients, likely due to the high biochemical redundancy of the neovascularization process. In this scenario, multitarget compounds that are able to simultaneously tackle different proangiogenic pathways are eagerly awaited. UniPR1331 is a 3β-hydroxy-Δ5-cholenic acid derivative, which is already known to inhibit Eph–ephrin interaction. Here, we employed an analysis pipeline consisting of molecular modeling and simulation, surface plasmon resonance spectrometry, biochemical assays, and endothelial cell models to demonstrate that UniPR1331 directly interacts with the vascular endothelial growth factor receptor 2 (VEGFR2) too. The binding of UniPR1331 to VEGFR2 prevents its interaction with the natural ligand vascular endothelial growth factor and subsequent autophosphorylation, signal transduction, and in vitro proangiogenic activation of endothelial cells. In vivo, UniPR1331 inhibits tumor cell-driven angiogenesis in zebrafish. Taken together, these data shed light on the pleiotropic pharmacological effect of UniPR1331, and point to Δ5-cholenic acid as a promising molecular scaffold for the development of multitarget antiangiogenic compounds.

Abstract 5035: Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers

Abstract Introduction: Identifying targets in the tumor microenvironment (TME) that act as barriers to an effective anti-tumor immune response has become an area of intense investigation. In the current study, we established EphB4-ephrin-B2 signaling as a key pathway that regulates both innate and adaptive arms of the immune system. Eph receptor tyrosine kinases and their membrane-bound ephrin ligands have been implicated in human malignancies and in immune cell development, migration, and activation in inflammatory models. However, direct evidence that supports the role of Eph-ephrin interaction in cancer-related immune response is lacking. We hypothesized that EphB4-ephrin-B2 interaction regulates TME by sustaining immunosuppressive cells-Tregs and TAMs thus negatively impacting the functional ability of CD8 T cells. Materials and methods: We used orthotopic models of head and neck squamous cell carcinoma to determine the role of EphB4-ephrin-B2 interaction in tumor immune microenvironment. Mice were treated with control agent or an EphB4-ephrin-B2 blocker in the absence or presence of radiation (RT). Tumor immune cell infiltrates were analyzed using mass cytometry and flow cytometry applications. ELISA or multiplex cytokine array were utilized to determine circulating cytokine/chemokine levels in plasma. Results: We observed that inhibition of EphB4-ephrin-B2 signaling in vivo significantly reduced tumor growth and decreased intratumoral Tregs, TAMs, and increased the activation of Teffector cells, without affecting CD4 T cell numbers. This was correlated with decreased Treg proliferation and activation when EphB4-ephrin-B2 signaling is inhibited. Since RT remains the mainstay in treatment of head and neck squamous cell cancer (HNSCC) patients, we combined EphB4-ephrin-B2 inhibitor with RT in our tumor model and observed further increase in CD8 and CD4 T cell infiltrates and activation status, and a significant decline in circulating TGF-β1 levels and an increase in CXCL10 levels compared to the control group. A significant reduction of TAMs, favoring a polarization towards an anti-tumoral M1 phenotype, was also observed in EphB4-ephrin-B2 inhibitor+RT group. We also compared the efficacy of combining EphB4-ephrin-B2 inhibitor with RT to anti-PDL1+RT in an in vivo model known to develop resistance to anti-PDL1+RT therapy. Our data demonstrated that combining EphB4-ephrin-B2 inhibitor with RT was equally effective to that of anti-PDL1+RT in terms of anti-tumor growth response. Conclusions: Our study provides the first insight into a novel role for EphB4-ephrin-B2 interaction in modulating tumor immune microenvironment in HNSCC. Our findings present a potential alternative in the form of EphB4-ephrin-B2 targeted therapeutics that can be tested in clinical trials in combination with RT for HNSCC patients. Citation Format: Shilpa Bhatia, Ayman Oweida, Shelby Lennon, Laurel Darragh, Sanjana Bukkapatnam, Benjamin Van Court, David Raben, Natalie Serkova, Antonio Jimeno, Eric Clambey, Elena Pasquale, Sana Karam. Blockade of EphB4-ephrin-B2 interaction remodels the tumor immune microenvironment in head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5035.

71 - Targeting glioblastoma with UniPR1331, a new and stable bioavailable small molecule inhibiting Eph–ephrin interaction: In vitro and in vivo evidence

71 - Targeting glioblastoma with UniPR1331, a new and stable bioavailable small molecule inhibiting Eph–ephrin interaction: In vitro and in vivo evidence

The ellagitannin colonic metabolite urolithin D selectively inhibits EphA2 phosphorylation in prostate cancer cells.

The Eph-ephrin system comprises emerging proteins involved in many pathophysiological processes. The pharmacological activity of the main metabolites derived from the intake of some classes of (poly)phenolic compounds, such as caffeoylquinic acids, flavan-3-ols, and ellagitannins, on the Eph-ephrin interaction was evaluated at physiological concentrations. Functional studies to elucidate their role in prostate cancer were also performed. Among the 21 phenolics screened by an ELISA-binding assay, just urolithin C, urolithin D, and ellagic acid succeeded to inhibit the EphA2-ephrin-A1 binding. Urolithin D, the most active, was a competitive and reversible antagonist of EphA receptors able to discriminate between EphA and EphB receptors, showing intra-classes selectivity. Molecular modeling and structure-activity relationships shed light on the binding mode and selective activity of urolithin D. This catabolite blocked EphA2 phosphorylation mediated by ephrin-A1, while lacking cytotoxicity and anti-proliferative effects, and was inactive on the EphA2 kinase assay. The mechanisms behind the cancer preventive properties of foods rich in flavan-3-ols and caffeoylquinic acids are not associated with metabolic pathways directly linked to the Eph-ephrin system. However, the ellagitannin-derived colonic metabolite urolithin D was able to exert remarkable and selective EphA-ephrin-A inhibition, which might impact on prostate cancer prevention.

EphB–EphrinB Interaction Controls Odontogenic/Osteogenic Differentiation with Calcium Hydroxide

IntroductionCalcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide–related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide. MethodsPrimary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed. ResultsCalcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1–positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1–EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide–induced mineralization from primary pulp cells. ConclusionsEphrinB1–EphB2 interaction contributes to calcium hydroxide–induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide–induced odontogenic/osteogenic differentiation.

Eph-Ephrin A System Regulates Human Choriocarcinoma–Derived JEG-3 Cell Invasion

The Eph-ephrin system is a unique system that can induce multiple cellular responses such as cell migration, regulation of angiogenesis, and axonal guidance. Previously, the Eph-ephrin system was reported to regulate human extravillous trophoblast invasion. In this study, we examined the possible involvement of the Eph-ephrin system in the invasion of malignant gestational trophoblastic diseases using a human choriocarcinoma-derived cell line, JEG-3. The mRNA expression of class A Ephs and ephrins on JEG-3 cells was examined by reverse transcription-polymerase chain reaction. The effects of recombinant human Eph A1 (r-Eph A1) and r-ephrin A4 on the proliferation and invasion of JEG-3 cells were investigated by cell proliferation and Matrigel invasion assays. The alterations of integrin expression on JEG-3 cells in the presence of r-Eph A1 and r-ephrin A4 were investigated by flow cytometry. The induction of phosphorylation of focal adhesion kinase in JEG-3 cells by r-ephrin A4 was examined by Western blot analysis. By reverse transcription-polymerase chain reaction, mRNAs of Eph A1, A2, and A4 and ephrin A1, A4, and A5 were detected on JEG-3 cells. In Matrigel invasion assay, both r-Eph A1 and r-ephrin A4 promoted the invasion of JEG-3 cells without affecting cell proliferation. During 24-hour culture with r-Eph A1 and r-ephrin A4, the increase in integrin α 5 expression on JEG-3 cells was observed by flow cytometry. Western blotting analysis showed that r-ephrin A4 induced dephosphorylation of focal adhesion kinase in JEG-3 cells. These findings suggest that Eph-ephrin interaction plays some role in the regulation of choriocarcinoma invasion in cooperation with integrins.

Osteoblastogenesis regulation signals in bone remodeling

Bone remodeling is essential for adult bone homeostasis. The failure of this process often leads to the development of osteoporosis, a present major global health concern. The most important factor that affects normal bone remodeling is the tightly controlled and orchestrated regulation of osteoblasts and osteoclasts. The present review summarized the recent discoveries related to osteoblast regulation from several signals, including transforming growth factor-β, bone morphogenetic proteins, Wnt signal, Notch, Eph-Ephrin interaction, parathyroid hormone/parathyroid hormone-related peptide, and the leptin-serotonin-sympathetic nervous systemic pathway. The awareness of these mechanisms will facilitate further research that explores bone remodeling and osteoporosis. Future investigations on the endogenous regulation of osteoblastogenesis will increase the current knowledge required for the development of potential drug targets in the treatment of osteoporosis.

EphrinA1 stimulates cell attachment and inhibits cell aggregation through the EphA receptor pathway in human endometrial carcinoma-derived Ishikawa cells

Recently, the Eph-ephrinA system was proposed to contribute to the initial interaction between the maternal endometrial epithelium and embryonic trophectoderm. Since the Eph-ephrin interaction can induce adhesive and/or repulsive forces into the cells, we examined the possible role of this system in functional changes in endometrial epithelial cells using endometrial carcinoma-derived Ishikawa cells. The expressions of EphA1, A2 and A4 on Ishikawa cells were examined by RT-PCR and western blotting analyses. The effects of recombinant ephrinA1 on Ishikawa cells were also examined by western blot analysis and cell attachment and aggregation assays. EphA1, A2 and A4 were expressed on Ishikawa cells. Recombinant ephrinA1 bound to the surfaces of Ishikawa cells and induced phosphorylation of EphA2 and A4. In bovine serum albumin-blocked nitrocellulose-coated dishes, Ishikawa cells remained floating and aggregated with each other. Under these conditions, immobilized ephrinA1 promoted Ishikawa cell attachment with increased tyrosine phosphorylation in focal adhesion kinase. In addition, immobilized ephrinA1 reversibly inhibited Ishikawa cell aggregation. Gene-reduction of EphA1, A2 and A4 by siRNAs attenuated the inhibitory effects of ephrinA1 on cell aggregation, confirming that ephrinA1 affects Ishikawa cell functions through Eph-ephrinA interaction. This study demonstrated that the Eph-ephrinA system can promote cell attachment along with intercellular dissociation in Ishikawa cells. These findings suggest that this system can induce functional changes in endometrial epithelial cells.

Ephrin A1 induces intercellular dissociation in Ishikawa cells: possible implication of the Eph-ephrin A system in human embryo implantation

During implantation, the human embryo invades endometrial stromal tissues, reducing the intercellular connections among epithelial cell layers. Since Eph-ephrin interaction can induce repulsive forces to control cell position and movement, we examined the possible involvement of this system in intercellular dissociation among endometrial epithelial cells. The expression of Eph A receptor on human endometrial epithelial cells and endometrial carcinoma-derived Ishikawa cells was examined by RT-PCR, immunohistochemistry and western blotting. The effects of recombinant ephrin A1 on Eph A2 phosphorylation in Ishikawa cells were also examined by western blotting. A permeability assay was performed to determine the effects of ephrin A1 on cell-to-cell adhesion. Eph A1, A2 and A4 mRNAs were detected in human endometrial epithelial cells and Ishikawa cells, and ephrin A1 was present in human blastocysts. Immunohistochemical staining showed that Eph A1, A2 and A4 receptors were expressed on the cell surface region of luminal and glandular epithelial cells in human endometrium in both the proliferative and secretory phase. The presence of Eph A2 protein in the human endometrium was confirmed by western blot analysis. Recombinant ephrin A1 was bound to Ishikawa cells and induced phosphorylation of Eph A2 expressed in Ishikawa cells. In addition, stimulation by ephrin A1 for 20 min increased the permeability of monolayer Ishikawa cells versus control cultures (P < 0.01), without affecting cell viability. This study demonstrated that the Eph-ephrin A system can promote intercellular dissociation in Ishikawa cells suggesting an important role in the initial step of embryo implantation by opening the endometrial epithelial cell barrier.

The role of Eph receptors and ephrin ligands in colorectal cancer

Eph receptors and their ephrin ligands constitute the largest subfamily of receptor tyrosine kinases and are components of the cell signaling pathways involved during development. Eph and ephrin overexpression have been documented in a variety of human cancers including gastrointestinal malignancies and in particular colorectal malignancies. EphB and ephrin B proteins have been implicated in the homeostasis of the gastrointestinal tract where EphB2- and EphB3-ephrin B signaling regulates cell sorting in the mature epithelium. These proteins are also reported to be upregulated in colon carcinomas. The EphA/ephrin A system has also been implicated in epithelial tissue structure and function. More recently, EphA receptors and their corresponding ligands have been implicated in numerous malignancies. Of these, EphA2 in particular has been intensively investigated and has been proposed as a therapeutic target. An interesting observation emerging from these studies is the role for Ephs and ephrins in critical aspects of cell adhesion, migration and positioning, and a crucial role in tumor progression and metastasis. However, the underlying role of Ephs and ephrins in these processes has generally been studied on individual Eph or ephrin genes. Given the multiplicity of Eph expression on gut epithelial cells, a more global approach is needed to define the precise role of Eph-ephrin interaction in malignant transformation. Here, we will review the recent advances on the role of Eph-ephrin signaling in colorectal malignancies.

Eph–ephrin A system regulates murine blastocyst attachment and spreading

Although numerous adhesion molecules are expressed on mammalian endometrial epithelial cells, there have not been any studies of a mechanism to prevent premature attachment of the embryo. In this study, we examined the possible involvement of Eph-ephrin interaction, which can induce repulsive forces. In mice, Eph A1, A2, and A4 were expressed on endometrial epithelial cells and ephrin A1-4 on blastocysts. Reverse transcriptase-polymerase chain reaction showed that mRNA expression of ephrin A1-4 on embryos transiently decreased around the implantation period. Immunohistochemistry demonstrated that the expression of Eph A1 on endometrial epithelial cells and ephrin A1 and A3 expression on embryos decreased at implantation sites. Recombinant Eph A1 reacted with cell the surface of ephrin A-bearing trophectoderm cells. Attachment assays using Eph A1-coated dishes showed that blastocyst attachment was reversibly inhibited by Eph A1. These findings suggest an important role of the Eph-ephrin A system in regulating the initial embryo-maternal contact during the cross-talk period that precedes embryo implantation.

Selectivity and Promiscuity in Eph Receptors

In this issue of Structure , Chrencik et al. (2006) report a structural and thermodynamic analysis of EphB4 in complex with an antagonistic peptide; the insight into Eph-ephrin interaction suggests determinants for Eph receptor specificity. These findings will contribute to the development of EphB4 antagonists for therapeutic applications.

Cell adhesion molecules in the CNS

Cell-cell adhesions are important for brain morphology and highly coordinated brain functions such as memory and learning ([Sanes and Yamagata, 1999][1]; [Yamagata et al., 2003][2]; [Washbourne et al., 2004][3]). During early development of the nervous system, neurons elongate their axons towards

Vessels and Nerves: Marching to the Same Tune

The patterning of the nervous system is achieved through coordinated action of a variety of repulsive or attractive neuronal guidance factors that direct the growth of growing axons to specific pathways. Recent studies suggest that the same or similar sets of factors also guide migration of endothelial cells, helping to direct the stereotypical embryonic patterning of the developing vertebrate vascular system.

Control of axonophilic migration of oligodendrocyte precursor cells by Eph–ephrin interaction

The migration of oligodendrocyte precursor cells (OPCs) is modulated by secreted molecules in their environment and by cell-cell and matrix-cell interactions. Here, we ask whether membrane-anchored guidance cues, such as the ephrin ligands and their Eph receptors, participate in the control of OPC migration in the optic nerve. We postulate that EphA and EphB receptors, which are expressed on axons of retinal ganglion cells, interact with ephrins on the surface of OPCs. We show the expression of ephrinA5, ephrinB2 and ephrinB3 in the migrating OPCs of the optic nerve as well as in the diencephalic sites from where they originate. In addition, we demonstrate that coated EphB2-Fc receptors, which are specific for ephrinB2/B3 ligands, induce dramatic changes in the contact and migratory properties of OPCs, indicating that axonal EphB receptors activate ephrinB signaling in OPCs.Based on these findings, we propose that OPCs are characterized by an ephrin code, and that Eph-ephrin interactions between axons and OPCs control the distribution of OPCs in the optic axonal tracts, and the progress and arrest of their migration.

An early developmental role for Eph-ephrin interaction during vertebrate gastrulation

Eph receptor tyrosine kinases (RTK) and their ephrin ligands are involved in the transmission of signals which regulate cytoskeletal organisation and cell migration, and are expressed in spatially restricted patterns at discrete phases during embryogenesis. Loss of function mutants of Eph RTK or ephrin genes result in defects in neuronal pathfinding or cell migration. In this report we show that soluble forms of human EphA3 and ephrin-A5, acting as dominant negative inhibitors, interfere with early events in zebrafish embryogenesis. Exogenous expression of both proteins results in dose-dependent defects in somite development and organisation of the midbrain-hindbrain boundary and hindbrain. The nature of the defects as well as the distribution and timing of expression of endogenous ligands/receptors for both proteins suggest that Eph-ephrin interaction is required for the organisation of embryonic structures by coordinating the cellular movements of convergence during gastrulation.