Eph Signaling Research Articles

Eph signaling controls mitotic spindle orientation and cell proliferation in neuroepithelial cells.

Mitotic spindle orientation must be tightly regulated during development and adult tissue homeostasis. It determines cell-fate specification and tissue architecture during asymmetric and symmetric cell division, respectively. Here, we uncover a novel role for Ephrin-Eph intercellular signaling in controlling mitotic spindle alignment in Drosophila optic lobe neuroepithelial cells through aPKC activity-dependent myosin II regulation. We show that conserved core components of the mitotic spindle orientation machinery, including Discs Large1, Mud/NuMA, and Canoe/Afadin, mislocalize in dividing Eph mutant neuroepithelial cells and produce spindle alignment defects in these cells when they are down-regulated. In addition, the loss of Eph leads to a Rho signaling-dependent activation of the PI3K-Akt1 pathway, enhancing cell proliferation within this neuroepithelium. Hence, Eph signaling is a novel extrinsic mechanism that regulates both spindle orientation and cell proliferation in the Drosophila optic lobe neuroepithelium. Similar mechanisms could operate in other Drosophila and vertebrate epithelia.

Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling.

The ability of cells to adapt their response to growth factors in relation to their environment is an essential aspect of tissue development and homeostasis. We found that signaling mediated by the Eph family of receptor tyrosine kinases from cell-cell contacts changed the cellular response to the growth factor EGF by modulating the vesicular trafficking of its receptor, EGFR. Eph receptor activation trapped EGFR in Rab5-positive early endosomes by inhibiting Akt-dependent vesicular recycling. By altering the spatial distribution of EGFR activity, EGF-promoted Akt signaling from the plasma membrane was suppressed, thereby inhibiting cell migration. In contrast, ERK signaling from endosomal EGFR was preserved to maintain a proliferative response to EGF stimulation. We also found that soluble extracellular signals engaging the G protein-coupled receptor Kiss1 (Kiss1R) similarly suppressed EGFR vesicular recycling to inhibit EGF-promoted migration. Eph or Kiss1R activation also suppressed EGF-promoted migration in Pten-/- mouse embryonic fibroblasts, which exhibit increased constitutive Akt activity, and in MDA-MB-231 triple-negative breast cancer cells, which overexpress EGFR. The cellular environment can thus generate context-dependent responses to EGF stimulation by modulating EGFR vesicular trafficking dynamics.

Eph Receptor Effector Ephexin Mediates Olfactory Dendrite Targeting in Drosophila.

Deciphering the mechanisms of sensory neural map formation is a central aim in neurosciences. Failure to form a correct map frequently leads to defects in sensory processing and perception. The olfactory map develops in subsequent steps initially forming a rough and later a precise map of glomeruli in the antennal lobe (AL), mainly consisting of olfactory receptor neuron (ORN) axons and projection neuron (PN) dendrites. The mechanisms underpinning the later stage of class-specific glomerulus formation are not understood. Recent studies have shown that the important guidance molecule Eph and its ligand ephrin play a role in class-specific PN targeting. Here, we reveal aspects of the mechanism downstream of Eph signaling during olfactory map formation. We show that the Eph-specific RhoGEF Ephexin (Exn) is required to fine tune PN dendrite patterning within specific glomeruli. We provide the first report showing an in vivo neurite guidance defect in an exn mutant. Interestingly, the quality of the phenotypes is different between eph and exn mutants; while loss of Eph leads to strong misprojections of DM3/Or47a neurons along the medial-lateral axis of the antennal lobe (AL), loss of Exn induces ventral ectopic innervation of a neighboring glomerulus. Genetic interaction experiments suggest that differential signaling of the small GTPases Rac1 and Cdc42 mediated by Exn-dependent and -independent Eph signaling fine tunes spatial targeting of PN dendrites within the olfactory map. We propose that their distinct activities on the actin cytoskeleton are required for precise navigation of PN dendrites within the olfactory map. Taken together, our results suggest that the precise connectivity of an individual neuron can depend on different modes of signaling downstream of a single guidance receptor. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 00: 000-000, 2018.

Epigenetic Control of the Notch and Eph Signaling Pathways by the Prion Protein: Implications for Prion Diseases.

Among the ever-growing number of self-replicating proteins involved in neurodegenerative diseases, the prion protein PrP remains the most infamous for its central role in transmissible spongiform encephalopathies (TSEs). In these diseases, pathogenic prions propagate through a seeding mechanism, where normal PrPC molecules are converted into abnormally folded scrapie isoforms termed PrPSc. Since its discovery over 30years ago, much advance has contributed to define the host-encoded cellular prion protein PrPC as a critical relay of prion-induced neuronal cell demise. A current consensual view is that the conversion of PrPC into PrPSc in neuronal cells diverts the former from its normal function with subsequent molecular alterations affecting synaptic plasticity. Here, we report that prion infection is associated with reduced expression of key effectors of the Notch pathway in vitro and in vivo, recapitulating changes fostered by the absence of PrPC. We further show that both prion infection and PrPC depletion promote drastic alterations in the expression of a defined set of Eph receptors and their ephrin ligands, which represent important players in synaptic function. Our data indicate that defects in the Notch and Eph axes can be mitigated in response to histone deacetylase inhibition in PrPC-depleted as well as prion-infected cells. We thus conclude that infectious prions cause a loss-of-function phenotype with respect to Notch and Eph signaling and that these alterations are sustained by epigenetic mechanisms.

Cross Talk between One-Carbon Metabolism, Eph Signaling, and Histone Methylation Promotes Neural Stem Cell Differentiation

Metabolic pathways, once seen as a mere consequence of cell states, have emerged as active players in dictating different cellular events such as proliferation, self-renewal, and differentiation. Several studies have reported a role for folate-dependent one-carbon (1C) metabolism in stem cells; however, its exact mode of action and how it interacts with other cues are largely unknown. Here, we report a link between the Eph:ephrin cell-cell communication pathway and 1C metabolism in controlling neural stem cell differentiation. Transcriptional and functional analyses following ephrin stimulation revealed alterations in folate metabolism-related genes and enzymatic activity. Invitro and invivo data indicate that Eph-B forward signaling alters the methylation state of H3K4 by regulating 1C metabolism and locksneural stem cell in a differentiation-ready state. Ourstudy highlights a functional link between cell-cell communication, metabolism, and epigenomic remodeling in the control of stem cell self-renewal.

Ephexin1 Is Required for Eph-Mediated Limb Trajectory of Spinal Motor Axons.

The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection.SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype, induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

BackgroundEPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.MethodsEPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.ResultsOur study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.ConclusionsTaken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

The Wound Microenvironment Reprograms Schwann Cells to Invasive Mesenchymal-like Cells to Drive Peripheral Nerve Regeneration

SummarySchwann cell dedifferentiation from a myelinating to a progenitor-like cell underlies the remarkable ability of peripheral nerves to regenerate following injury. However, the molecular identity of the differentiated and dedifferentiated states in vivo has been elusive. Here, we profiled Schwann cells acutely purified from intact nerves and from the wound and distal regions of severed nerves. Our analysis reveals novel facets of the dedifferentiation response, including acquisition of mesenchymal traits and a Myc module. Furthermore, wound and distal dedifferentiated Schwann cells constitute different populations, with wound cells displaying increased mesenchymal character induced by localized TGFβ signaling. TGFβ promotes invasion and crosstalks with Eph signaling via N-cadherin to drive collective migration of the Schwann cells across the wound. Consistently, Tgfbr2 deletion in Schwann cells resulted in misdirected and delayed reinnervation. Thus, the wound microenvironment is a key determinant of Schwann cell identity, and it promotes nerve repair through integration of multiple concerted signals.Video

Effect of hydrogel elasticity and ephrinB2-immobilized manner on Runx2 expression of human mesenchymal stem cells.

Differentiation fate of mesenchymal stem cells (MSC) is modified by biochemical and biophysical factors, such as elasticity and signal proteins. However, there are few experiments about combinations of them. In this study, to evaluate the synergistic effect of them on cell properties of MSC, we established to design the manner of Eph signal ligand, ephrinB2, immobilized onto polyacrylamide hydrogels with varied elasticity. The gene expression level of an osteogenic maker, Runx2, was enhanced by the immobilized manner, and significantly enhanced for the hydrogels of around 4kPa elasticity with bio-specific immobilization of ephrinB2. This is the novel report describing to demonstrate that the Runx2 expression of MSC is synergistically influenced by the hydrogels elasticity and their manner of ephrinB2 immobilized.

Investigation of the role of tyrosine kinase receptor EPHA3 in colorectal cancer

EPH signaling deregulation has been shown to be important for colorectal carcinogenesis and genome-wide sequencing efforts have identified EPHA3 as one of the most frequently mutated genes in these tumors. However, the role of EPHA3 in colorectal cancer has not been thoroughly investigated. We show here that ectopic expression of wild type EPHA3 in colon cancer cells did not affect their growth, motility/invasion or metastatic potential in vivo. Moreover, overexpression of mutant EPHA3 or deletion of the endogenous mutant EPHA3 in colon cancer cells did not affect their growth or motility. EPHA3 inactivation in mice did not initiate the tumorigenic process in their intestine, and had no effects on tumor size/multiplicity after tumor initiation either genetically or pharmacologically. In addition, immunohistochemical analysis of EPHA3 tumor levels did not reveal associations with survival or clinicopathological features of colorectal cancer patients. In conclusion, we show that EPHA3 does not play a major role in colorectal tumorigenesis. These results significantly contribute to our understanding of the role of EPH signaling during colorectal carcinogenesis, and highlighting the need for detailed functional studies to confirm the relevance of putative cancer driver genes identified in sequencing efforts of the cancer genome.

P2.03b-084 Profiling of Eph Signaling in Malignant Pleural Effusions- Identification of Therapy Approaches and Associated Biomarkers: Topic: Biomarkers

For late stage lung cancer (LC) patients few treatment options are at hand and the survival time is very limited, hence novel therapies and associated biomarkers are urgently needed. Erythropoietin-Producing Hepatocellular carcinoma receptor (Eph) tyrosine kinase family and their ligands, Ephrins, drive multiple hallmarks of cancer e.g. proliferation/invasion. The Eph signaling pathways are attractive drug targets due to their dual role in oncogenesis and tumor progression. We analyzed Ephs/Ephrins signaling in LC cells from pleural effusions (PE) to reveal altered kinase pathways and putative BMs. We also assessed cytotoxicity and kinome alterations in PE tumor cells exposed to targeted agents and chemotherapy in vitro. Tumor cells purified from PE, assessed for histology, mutation and translocation status (EGFR, KRAS, BRAF and Alk), were grown in vitro. Toxicity of tyrosine kinase inhibitors (TKIs (e.g. erlotinib, crizotinib, AG1024, AZD9291, dasatinib), EGFR blocker (cetuximab) and/or chemotherapy (e.g. cisplatin, gemcitabine, etoposide) were analyzed after 72 h with the Tox8 assay. Ephs/Ephrins signaling components were studied using western blot, immunoprecipitation and by proximity ligation assay. Mutations and signaling heterogeneity were visualized using padlock probe method. For kinome profiling PathScan RTK signalling antibody array was used. PE isolated tumor cells were identified as adenocarcinoma, squamous cell carcinoma and SCLC and their EGFR, KRAS, BRAF and Alk mutational status determined. High levels of Ephrin B3 and phosphorylated EphA2 ser897, previously shown to be instrumental in driving NSCLC proliferation and invasion in vitro, were confirmed and also shown to directly interact, indicating the importance of this signaling event. The G391R mutation in EphA2, which is reported to cause a constitutive activation of EphA2 and to be linked to metastasis, but also mutations in EGFR (G719A, G719S, T790M and L858R) were detected. The PE derived tumor cells were hetereogenous in their survival response to TKIs and chemotherapy. However, cells with ALK translocation were sensitive to crizotinib and EGFR mutated cells showed response to erlotinib, cetuximab, AG1024, AZD9291 and dasatinib. Kinome analysis revealed selective signaling pathways that could also be targeted in combinational drug treatment. Screening of PE samples from LC patients for targeted agents alongside aberrant Ephs and kinome signaling, can be used to identify novel drug candidates. Together with frontline kinome profiling of NSCLC clinical specimens upon treatment it provides an opportunity to explore/identify new therapeutics for LC.

Differential gene expression profiles in foetal skin of Rex rabbits with different wool density

<p>This study investigated the mechanisms controlling hair follicle development in the Rex rabbit. The Agilent rabbit gene expression microarray was used to determine differentially expressed genes in Rex rabbit foetuses with different wool densities. The expression patterns of selected differentially-expressed genes were further investigated by quantitative real-time PCR. Compared to low wool density rabbits, 1342 differentially expressed probes were identified in high wool density rabbits, including 950 upregulated probes and 392 downregulated probes. Gene ontology analysis revealed that the most upregulated differentially expressed probes belonged to receptors and the most downregulated differentially expressed probes belonged to DNA binding molecules. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the differentially expressed probes were mainly involved in the sonic hedgehog (Shh) and Eph signalling pathways. The results also suggest that transforming growth factor-beta 1, growth hormone receptor, and the keratin-associated protein 6.1 genes, as well as the Shh and Eph signalling pathways, may be involved in the regulation of hair follicle developmental in Rex rabbits.</p>

Decreased maternal behavior and anxiety in ephrin-A5-/- mice.

During development of the nervous system, molecular signals mediating cell-cell interactions play critical roles in the guidance of axonal growth and establishment of synaptic functions. The Eph family of tyrosine kinase receptors and their ephrin ligands has been shown to mediate neuronal interactions in the development of topographic axon projection maps in several brain regions, and the loss of Eph activities result in defects in select axonal pathways. However, effects of deficiencies of the Eph signals on animal behavior have not been well documented. In this study, we showed that inactivation of a ligand of the Eph receptors, ephrin-A5, resulted in defects in maternal behavior and alterations in anxiety. Female ephrin-A5 -/- mice show significant defects in nest building and pup retrieval. In addition, lower levels of anxiety were observed in both male and female null mice. These changes were not due to deficiencies in estradiol, progesterone or corticosterone levels. Our observations suggest that ephrin-A5 plays a key role in the development and/or function of neural pathways mediating mouse maternal care and anxiety.

Tiam-Rac signaling mediates trans-endocytosis of ephrin receptor EphB2 and is important for cell repulsion.

Ephrin receptors interact with membrane-bound ephrin ligands to regulate contact-mediated attraction or repulsion between opposing cells, thereby influencing tissue morphogenesis. Cell repulsion requires bidirectional trans-endocytosis of clustered Eph-ephrin complexes at cell interfaces, but the mechanisms underlying this process are poorly understood. Here, we identified an actin-regulating pathway allowing ephrinB(+) cells to trans-endocytose EphB receptors from opposing cells. Live imaging revealed Rac-dependent F-actin enrichment at sites of EphB2 internalization, but not during vesicle trafficking. Systematic depletion of Rho family GTPases and their regulatory proteins identified the Rac subfamily and the Rac-specific guanine nucleotide exchange factor Tiam2 as key components of EphB2 trans-endocytosis, a pathway previously implicated in Eph forward signaling, in which ephrins act as in trans ligands of Eph receptors. However, unlike in Eph signaling, this pathway is not required for uptake of soluble ligands in ephrinB(+) cells. We also show that this pathway is required for EphB2-stimulated contact repulsion. These results support the existence of a conserved pathway for EphB trans-endocytosis that removes the physical tether between cells, thereby enabling cell repulsion.

Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis.

Mechanisms of ephrin-Eph signalling in development, physiology and disease.

Eph receptor Tyr kinases and their membrane-tethered ligands, the ephrins, elicit short-distance cell-cell signalling and thus regulate many developmental processes at the interface between pattern formation and morphogenesis, including cell sorting and positioning, and the formation of segmented structures and ordered neural maps. Their roles extend into adulthood, when ephrin-Eph signalling regulates neuronal plasticity, homeostatic events and disease processes. Recently, new insights have been gained into the mechanisms of ephrin-Eph signalling in different cell types, and into the physiological importance of ephrin-Eph in different organs and in disease, raising questions for future research directions.

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.

α2-chimaerin is required for Eph receptor-class-specific spinal motor axon guidance and coordinate activation of antagonistic muscles.

Axonal guidance involves extrinsic molecular cues that bind growth cone receptors and signal to the cytoskeleton through divergent pathways. Some signaling intermediates are deployed downstream of molecularly distinct axon guidance receptor families, but the scope of this overlap is unclear, as is the impact of embryonic axon guidance fidelity on adult nervous system function. Here, we demonstrate that the Rho-GTPase-activating protein α2-chimaerin is specifically required for EphA and not EphB receptor signaling in mouse and chick spinal motor axons. Reflecting this specificity, the loss of α2-chimaerin function disrupts the limb trajectory of extensor-muscle-innervating motor axons the guidance of which depends on EphA signaling. These embryonic defects affect coordinated contraction of antagonistic flexor-extensor muscles in the adult, indicating that accurate embryonic motor axon guidance is critical for optimal neuromuscular function. Together, our observations provide the first functional evidence of an Eph receptor-class-specific intracellular signaling protein that is required for appropriate neuromuscular connectivity.

PTP-PEST controls EphA3 activation and ephrin-induced cytoskeletal remodelling.

Eph receptors and their corresponding membrane-bound ephrin ligands regulate cell positioning and establish tissue patterns during embryonic and oncogenic development. Emerging evidence suggests that assembly of polymeric Eph signalling clusters relies on cytoskeletal reorganisation and underlies regulation by protein tyrosine phosphatases (PTPs). PTP-PEST (also known as PTPN12) is a central regulator of actin cytoskeletal dynamics. Here, we demonstrate that an N-terminal fragment of PTP-PEST, generated through an ephrinA5-triggered and spatially confined cleavage mediated by caspase-3, attenuates EphA3 receptor activation and its internalisation. Isolation of EphA3 receptor signalling clusters within intact plasma membrane fragments obtained by detergent-free cell fractionation reveals that stimulation of cells with ephrin triggers effective recruitment of this catalytically active truncated form of PTP-PEST together with key cytoskeletal and focal adhesion proteins. Importantly, modulation of actin polymerisation using pharmacological and dominant-negative approaches affects EphA3 phosphorylation in a similar manner to overexpression of PTP-PEST. We conclude that PTP-PEST regulates EphA3 activation both by affecting cytoskeletal remodelling and through its direct action as a PTP controlling EphA3 phosphorylation, indicating its multifaceted regulation of Eph signalling.

Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.