Eph/ephrin signaling in cancer

The Eph receptor tyrosine kinases family and their membrane bound ligands, the ephrins, represents a complex signaling network of cell communication for cell sorting during tissue patterning in development and in the normal physiology and homeostasis of adult tissues. This molecular family has adapted to evolving tissue complexity in multicellular organisms through the emergence of more members and complex mechanisms of expression and signaling that result in the fine-tuning of cell positioning. Since their initial identification from an erythropoietin producing hepatocellular (Eph) carcinoma cell line in 1987, Eph/ephrin signaling has been a matter of intensive investigation for their plausible role in cancer. Similarly to their context dependent modus operandi in normal tissues, Eph/ephrin signaling in cancer is an intricate and puzzling network of events that tumors “manage” to their benefit in multiple aspects like cell adhesion to substrate, migration, invasion or growth.     

Tissue transglutaminase clusters soluble A-type ephrins into functionally active high molecular weight oligomers

The Eph receptors and their ligands, the ephrins, are thought to act at points of close cell-cell contact to elicit bi-directional signaling in receptor and ligand expressing cells. However, when cultured in vitro, some A-type ephrins are released from the cell surface and it is unclear if these soluble ephrins participate in Eph receptor activation. We show that soluble ephrin A5 is subject to oligomerization. Ephrins A1 and A5 are substrates for a cross-linking enzyme, tissue transglutaminase, which mediates the formation of oligomeric ephrin. Transglutaminase-cross-linked ephrin binds to A-type Eph receptors, stimulates Eph kinase activity, and promotes invasion and migration of HeLa cells. Transglutaminase-mediated oligomerization of soluble ephrin potentially represents a novel mechanism of forward signaling through Eph receptors and may extend the influence of A-type ephrins beyond cell contact mediated signaling.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Eph receptor and ephrin function in breast,gut, and skin epithelia

Epithelial cells are tightly coupled together through specialized intercellular junctions, including adherens junctions, desmosomes, tight junctions, and gap junctions. A growing body of evidence suggests epithelial cells also directly exchange information at cell-cell contacts via the Eph family of receptor tyrosine kinases and their membrane-associated ephrin ligands. Ligand-dependent and -independent signaling via Eph receptors as well as reverse signaling through ephrins impact epithelial tissue homeostasis by organizing stem cell compartments and regulating cell proliferation, migration, adhesion, differentiation, and survival. This review focuses on breast, gut, and skin epithelia as representative examples for how Eph receptors and ephrins modulate diverse epithelial cell responses in a context-dependent manner. Abnormal Eph receptor and ephrin signaling is implicated in a variety of epithelial diseases raising the intriguing possibility that this cell-cell communication pathway can be therapeutically harnessed to normalize epithelial function in pathological settings like cancer or chronic inflammation.     

Membrane‐mediated regulation of vascular identity

Vascular diseases span diverse pathology, but frequently arise from aberrant signaling attributed to specific membrane‐associated molecules, particularly the Eph‐ephrin family. Originally recognized as markers of embryonic vessel identity, Eph receptors and their membrane‐associated ligands, ephrins, are now known to have a range of vital functions in vascular physiology. Interactions of Ephs with ephrins at cell‐to‐cell interfaces promote a variety of cellular responses such as repulsion, adhesion, attraction, and migration, and frequently occur during organ development, including vessel formation. Elaborate coordination of Eph‐ and ephrin‐related signaling among different cell populations is required for proper formation of the embryonic vessel network. There is growing evidence supporting the idea that Eph and ephrin proteins also have postnatal interactions with a number of other membrane‐associated signal transduction pathways, coordinating translation of environmental signals into cells. This article provides an overview of membrane‐bound signaling mechanisms that define vascular identity in both the embryo and the adult, focusing on Eph‐ and ephrin‐related signaling. We also discuss the role and clinical significance of this signaling system in normal organ development, neoplasms, and vascular pathologies. Birth Defects Research (Part C) 108:65–84, 2016. © 2016 Wiley Periodicals, Inc.     

Control of cell behaviour by signalling through Eph receptors and ephrins

Eph receptor tyrosine kinases and ephrins mediate contact-dependent cell interactions that regulate the repulsion and adhesion mechanisms involved in the guidance and assembly of cells. Recent work has revealed a role of overlapping Eph receptor and ephrin expression in modulating neuronal growth cone repulsion, and has shown that bidirectional activation restricts intermingling and communication between cell populations. In addition, progress has been made in understanding how Eph receptors and ephrins control cell adhesion.     

Fibroblast growth factor receptor-mediated rescue of x-ephrin B1-induced cell dissociation in Xenopus embryos

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.     

Morphogenesis of prechordal plate and notochord requires intact Eph/ephrin B signaling

Eph receptors and their ligands, the ephrins, mediate cell-to-cell signals implicated in the regulation of cell migration processes during development. We report the molecular cloning and tissue distribution of zebrafish transmembrane ephrins that represent all known members of the mammalian class B ephrin family. The degree of homology among predicted ephrin B sequences suggests that, similar to their mammalian counterparts, zebrafish B-ephrins can also bind promiscuously to several Eph receptors. The dynamic expression patterns for each zebrafish B-ephrin support the idea that these ligands are confined to interact with their receptors at the borders of their complementary expression domains. Zebrafish B-ephrins are expressed as early as 30% epiboly and during gastrula stages: in the germ ring, shield, prechordal plate, and notochord. Ectopic overexpression of dominant-negative soluble ephrin B constructs yields reproducible defects in the morphology of the notochord and prechordal plate by the end of gastrulation. Notably disruption of Eph/ephrin B signaling does not completely destroy structures examined, suggesting that cell fate specification is not altered. Thus abnormal morphogenesis of the prechordal plate and the notochord is likely a consequence of a cell movement defect. Our observations suggest Eph/ephrin B signaling plays an essential role in regulating cell movements during gastrulation.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

The role of proteases in regulating Eph/ephrin signaling

Proteases regulate a myriad of cell functions, both in normal and disease states. In addition to protein turnover, they regulate a range of signaling processes, including those mediated by Eph receptors and their ephrin ligands. A variety of proteases is reported to directly cleave Ephs and/or ephrins under different conditions, to promote receptor and/or ligand shedding, and regulate receptor/ligand internalisation and signaling. They also cleave other adhesion proteins in response to Eph-ephrin interactions, to indirectly facilitate Eph-mediated functions. Proteases thus contribute to Eph/ephrin mediated changes in cell-cell and cell-matrix interactions, in cell morphology and in cell migration and invasion, in a manner which appears to be tightly regulated by, and co-ordinated with, Eph signaling. This review summarizes the current literature describing the function and regulation of protease activities during Eph/ephrin-mediated cell signaling.     

Concepts and consequences of Eph receptor clustering

Polymeric receptor-ligand complexes between interacting Eph and ephrin-expressing cells are regarded as dynamic intercellular signalling scaffolds that control cell-to-cell contact: the resulting Eph-ephrin signalling clusters function as positional cues that facilitate cell navigation and tissue patterning during normal and oncogenic development. The considerable complexity of this task, coordinating a multitude of cell movements and cellular interactions, is achieved by accurate translation of spatial information from Eph and ephrin expression gradients into fine-tuned changes in cell-cell adhesion and position. Here we review emerging evidence suggesting that the required combinatorial diversity is not only achieved by the large number of possible Eph-ephrin interactions and selective use of Eph forward and ephrin reverse signals, but in particular through the composition and signal capacity of Eph-ephrin clusters, which is adjusted dynamically to reflect overall Eph and ephrin surface densities on interacting cells. Fine-tuning is provided through multi-layered cluster assembly, where homo- and heterotypic Eph and ephrin interactions define the composition - whilst intracellular signalling feedbacks determine the size and lifetime - of signalling clusters.     

EphA2 Signaling Following Endocytosis: Role of Tiam1

Eph receptors and their membrane‐bound ligands, the ephrins, represent a complex subfamily of receptor tyrosine kinases (RTKs). Eph/ephrin binding can lead to various and opposite cellular behaviors such as adhesion versus repulsion, or cell migration versus cell‐adhesion. Recently, Eph endocytosis has been identified as one of the critical steps responsible for such diversity. Eph receptors, as many RTKs, are rapidly endocytosed following ligand‐mediated activation and traffic through endocytic compartments prior to degradation. However, it is becoming obvious that endocytosis controls signaling in many different manners. Here we showed that activated EphA2 are degraded in the lysosomes and that about 35% of internalized receptors are recycled back to the plasma membrane. Our study is also the first to demonstrate that EphA2 retains the capacity to signal in endosomes. In particular, activated EphA2 interacted with the Rho family GEF Tiam1 in endosomes. This association led to Tiam1 activation, which in turn increased Rac1 activity and facilitated Eph/ephrin endocytosis. Disrupting Tiam1 function with RNA interference impaired both ephrinA1‐dependent Rac1 activation and ephrinA1‐induced EphA2 endocytosis. In summary, our findings shed new light on the regulation of EphA2 endocytosis, intracellular trafficking and signal termination and establish Tiam1 as an important modulator of EphA2 signaling .     

Ephrins in reverse,park and drive

Eph receptors and their membrane-anchored ephrin ligands are thought to orchestrate cell movements by transducing bidirectional tyrosine-kinase-mediated signals into both cells expressing the receptors and cells expressing the ligands. Whether the resulting event is repulsion of an axonal growth cone, directing the orderly segmentation of hindbrain rhombomere cells or controlling angiogenic remodelling, such elaborate and diverse cell movements require intricate changes in the actin cytoskeleton, as well as precise regulation of cellular adhesion. Recent work by several groups has begun to link ephrin reverse signals to intracellular pathways that regulate actin dynamics and might help to explain how these ligands function as receptors to direct cell movement, adhesion and de-adhesion events.     

Association of Dishevelled with Eph tyrosine kinase receptor and ephrin mediates cell repulsion

Eph tyrosine kinase receptors and their membrane-bound ligands, ephrins, are presumed to regulate cell-cell interactions. The major consequence of bidirectional activation of Eph receptors and ephrin ligands is cell repulsion. In this study, we discovered that Xenopus Dishevelled (Xdsh) forms a complex with Eph receptors and ephrin-B ligands and mediates the cell repulsion induced by Eph and ephrin. In vitro re-aggregation assays with Xenopus animal cap explants revealed that co-expression of a dominant-negative mutant of Xdsh affected the sorting of cells expressing EphB2 and those expressing ephrin-B1. Co-expression of Xdsh induced the activation of RhoA and Rho kinase in the EphB2-overexpressed cells and in the cells expressing EphB2-stimulated ephrin-B1. Therefore, Xdsh mediates both forward and reverse signaling of EphB2 and ephrin-B1, leading to the activation of RhoA and its effector protein Rho kinase. The inhibition of RhoA activity in animal caps significantly prevents the EphB2- and ephrin-B1-mediated cell sorting. We propose that Xdsh, which is expressed in various tissues, is involved in EphB and ephrin-B signaling related to regulation of cell repulsion via modification of RhoA activity.     

Targeting Eph receptors with peptides and small molecules: progress and challenges

The Eph receptors are a large family of receptor tyrosine kinases. Their kinase activity and downstream signaling ability are stimulated by the binding of cell surface-associated ligands, the ephrins. The ensuing signals are bidirectional because the ephrins can also transduce signals (known as reverse signals) following their interaction with Eph receptors. The ephrin-binding pocket in the extracellular N-terminal domain of the Eph receptors and the ATP-binding pocket in the intracellular kinase domain represent potential binding sites for peptides and small molecules. Indeed, a number of peptides and chemical compounds that target Eph receptors and inhibit ephrin binding or kinase activity have been identified. These molecules show promise as probes to study Eph receptor/ephrin biology, as lead compounds for drug development, and as targeting agents to deliver drugs or imaging agents to tumors. Current challenges are to find (1) small molecules that inhibit Eph receptor-ephrin interactions with high binding affinity and good lead-like properties and (2) selective kinase inhibitors that preferentially target the Eph receptor family or subsets of Eph receptors. Strategies that could also be explored include targeting additional Eph receptor interfaces and the ephrin ligands.     

Eph receptors: two ways to sharpen boundaries

Eph receptors and ephrins can sharpen domains within developing tissues by mediating repulsion at interfaces. An Eph receptor has now been shown also to regulate cell adhesion within tissue subdivisions.     

Ectodomain structures of Eph receptors

Eph receptors, the largest subfamily of receptor tyrosine kinases (RTKs), and their ephrin ligands are important mediators of cell-cell communication that regulate axon guidance, long-term potentiation, and stem cell development, among others. By now, many Eph receptors and ephrins have also been found to play important roles in the progression of cancer. Since both the receptor and the ligand are membrane-bound, their interaction leads to the multimerization of both molecules to distinct clusters within their respective plasma membranes, resulting in the formation of discrete signaling centers. In addition, and unique to Eph receptors and ephrins, their interaction initiates bi-directional signaling cascades where information is transduced in the direction of both the receptor- and the ligand-bearing cells. The Ephs and the ephrins are divided into two subclasses, A and B, based on their affinities for each other and on sequence conservation. Crystal structures and other biophysical studies have indicated that isolated extracellular Eph and ephrin domains initially form high-affinity heterodimers around a hydrophobic loop of the ligand that is buried in a hydrophobic pocket on the surface of the receptor. The dimers can then further arrange by weaker interactions into higher-order Eph/ephrin clusters observed in vivo at the sites of cell-cell contact. Although the hetero-dimerization is a universal way to initiate signaling, other extracellular domains of Ephs are involved in the formation of higher-order clusters. The structures also show important differences defining the unique partner preferences of the two ligand and receptor subclasses, namely, how subclass specificity is determined both by individual interacting residues and by the precise architectural arrangement of ligands and receptors within the complexes.     

Signal transfer by Eph receptors

The Eph receptors are a unique family of receptor tyrosine kinases that enforce cellular position in tissues through mainly repulsive signals generated upon cell-cell contact. Together, Eph receptors and their membrane-anchored ligands. the ephrins, are key molecules for establishing tissue organization through signaling pathways that control axonal projection, cell migration, and the maintenance of cellular boundaries. Through their SH2 (Src Homology 2) and PDZ (postsynaptic density protein, disks large, zona occludens) domains, several signaling molecules have been demonstrated to interact with the activated cytoplasmic domain of Eph receptors by using the yeast two-hybrid system and in vitro biochemical assays. Most proteins found to interact with Eph receptors are well-known regulators of cytoskeletal organization and cell adhesion, and also cell proliferation. Promoting growth, however, does not appear to be a primary role of Eph receptors. Explaining which signaling interactions identified for the Eph receptors have physiological significance, how Eph receptor signaling cascades are propagated, and characterizing the intrinsic signaling properties of the ephrins are all exciting questions currently being investigated.