The role of ephrins and Eph receptors in cancer

Eph receptors are the largest receptor tyrosine kinase family of transmembrane proteins with an extracellular domain capable of recognizing signals from the cells’ environment and influencing cell–cell interaction and cell migration. Ephrins are the ligands to Eph receptors and stimulate bi-directional signaling of the Eph/ephrin axis. Eph receptor and ephrin overexpression can result in tumorigenesis as related to tumor growth and survival and is associated with angiogenesis and metastasis in many types of human cancer. Recent data suggest that Eph/ephrin signaling could play an important role in the development of novel inhibition strategies and cancer treatments to potentially target this receptor tyrosine kinase and/or its ligand. A deeper understanding of the molecular basis for normal versus defective cell–cell interaction through the Eph/ephrin axis will enable the potential development of novel cancer treatments. This review emphasizes the biology of Eph/ephrin as well as the potential for novel targeted therapy through this pathway.     

Eph-ephrin bidirectional signaling in physiology and disease

Receptor tyrosine kinases of the Eph family bind to cell surface-associated ephrin ligands on neighboring cells. The ensuing bidirectional signals have emerged as a major form of contact-dependent communication between cells. New findings reveal that Eph receptors and ephrins coordinate not only developmental processes but also the normal physiology and homeostasis of many adult organs. Imbalance of Eph/ephrin function may therefore contribute to a variety of diseases. The challenge now is to better understand the complex and seemingly paradoxical signaling mechanisms of Eph receptors and ephrins, which will enable effective strategies to target these proteins in the treatment of diseases such as diabetes and cancer.     

Profiling Eph receptor expression in cells and tissues: A targeted mass spectrometry approach

The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression.     

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.     

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.     

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.     

Eph/ephrin signaling in epidermal differentiation and disease

Eph receptor tyrosine kinases mediate cell-cell communication by interacting with ephrin ligands residing on adjacent cell surfaces. In doing so, these juxtamembrane signaling complexes provide important contextual information about the cellular microenvironment that helps orchestrate tissue morphogenesis and maintain homeostasis. Eph/ephrin signaling has been implicated in various aspects of mammalian skin physiology, with several members of this large family of receptor tyrosine kinases and their ligands present in the epidermis, hair follicles, sebaceous glands, and underlying dermis. This review focuses on the emerging role of Eph receptors and ephrins in epidermal keratinocytes where they can modulate proliferation, migration, differentiation, and death. The activation of Eph receptors by ephrins at sites of cell-cell contact also appears to play a key role in the maturation of intercellular junctional complexes as keratinocytes move out of the basal layer and differentiate in the suprabasal layers of this stratified, squamous epithelium. Furthermore, alterations in the epidermal Eph/ephrin axis have been associated with cutaneous malignancy, wound healing defects and inflammatory skin conditions. These collective observations suggest that the Eph/ephrin cell-cell communication pathway may be amenable to therapeutic intervention for the purpose of restoring epidermal tissue homeostasis and integrity in dermatological disorders.     

An ephrin mimetic peptide that selectively targets the EphA2 receptor

Eph receptor tyrosine kinases represent promising disease targets because they are differentially expressed in pathologic versus normal tissues. The EphA2 receptor is up-regulated in transformed cells and tumor vasculature where it likely contributes to cancer pathogenesis. To exploit EphA2 as a therapeutic target, we used phage display to identify two related peptides that bind selectively to EphA2 with high affinity (submicromolar K(D) values). The peptides target the ligand-binding domain of EphA2 and compete with ephrin ligands for binding. Remarkably, one of the peptides has ephrin-like activity in that it stimulates EphA2 tyrosine phosphorylation and signaling. Furthermore, this peptide can deliver phage particles to endothelial and tumor cells expressing EphA2. In contrast, peptides corresponding to receptor-interacting portions of ephrin ligands bind weakly and promiscuously to many Eph receptors. Bioactive ephrin mimetic peptides could be used to selectively deliver agents to Eph receptor-expressing tissues and modify Eph signaling in therapies for cancer, pathological angiogenesis, and nerve regeneration.     

Eph/ephrin signaling in morphogenesis, neural development and plasticity

Ephrins are cell-surface-tethered ligands for Eph receptors, the largest family of receptor tyrosine kinases. During development, the Eph/ephrin cell communication system appears to influence cell behavior such as attraction/repulsion, adhesion/de-adhesion and migration, thereby influencing cell fate, morphogenesis and organogenesis. During adulthood, the Eph/ephrin system continues to play roles in tissue plasticity, for example in shaping dendritic spines during neuronal plasticity. Mechanistically, Eph-ephrin repulsive behavior appears to require ligand-receptor internalization and signaling to Rho GTPases.     

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 .     

Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation

The Eph receptors and their ephrin ligands play crucial roles in a large number of cell–cell interaction events, including those associated with axon pathfinding, neuronal cell migration and vasculogenesis. They are also involved in the patterning of most tissues and overall cell positioning in the development of the vertebrate body plan. The Eph/ephrin signaling system manifests several unique features that differentiate it from other receptor tyrosine kinases, including initiation of bi-directional signaling cascades and the existence of ligand and receptor subclasses displaying promiscuous intra-subclass interactions, but very rare inter-subclass interactions. In this review we briefly discuss these features and focus on recent studies of the unique and expansive high-affinity Eph/ephrin assemblies that form at the sites of cell–cell contact and are required for Eph signaling initiation. This article is part of a Special Issue entitled: Emerging recognition and activation mechanisms of receptor tyrosine kinases.     

Eph/ephrin signaling in epithelial development and homeostasis

Eph receptors and ephrin ligands are widely expressed during embryonic development with well-defined functions in directing neuronal and vascular network formation. Over the last decade, evidence has mounted that Ephs and ephrins are also actively involved in prenatal and postnatal development of epithelial tissues. Their functions beyond developmental settings are starting to be recognized as well. The diverse functions of Eph/ephrin are largely related to the complementary expression pattern of the Eph receptors and corresponding ephrin ligands that are expressed in adjacent compartments, although overlapping expression pattern also exists in epithelial tissue. The interconnection between Ephs or ephrins and classical cell junctional molecules suggests they may function coordinately in maintaining epithelial structural integrity and homeostasis. This review will highlight cellular and molecular evidence in current literature that support a role of Eph/ephrin systems in regulating epithelial cell development and physiology.     

Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application

The Eph family of receptors, with 14 members in humans, makes up the largest group of receptor tyrosine kinases. These Eph receptors, along with their ligands, the 8 members of the ephrin family of ligands are involved in diverse developmental functions, including hindbrain development in vertebrates, tissue patterning, and angiogenesis. These Eph receptors and ephrin ligands have also been identified as important regulators in the development and progression of cancer. We have presented here a systematic and comprehensive investigation of the Eph/ephrin expression profiles of MCF-10A, MCF-7, and MDA-MB-231 cells representing normal breast, non-invasive breast tumor, and invasive tumor, respectively, based on their characteristic phenotypes in Matrigel matrix. The data have allowed us to correlate the gene expression profile with the cell phenotype that has potential application in tumor diagnostics. We demonstrate here that upregulation of EphA2, A7, A10, and ephrinA2 and B3 is likely involved in tumorigenesis and/or invasiveness, while downregulation of EphA1, A3, A4, A8, B3, B4, B6, and ephrinA1 and B1 may be particularly important in invasiveness. Based on these results we discuss the role of EphA2 and ephrinA1 combination in malignancy. The data have provided clues as to the importance of these molecules in the progression of breast cancer and specifically identified EphB6, a kinase-deficient receptor, which is downregulated in the most aggressive cell line, as reported for several other cancer types including neuroblastoma and melanoma suggesting its potential as a prognostic indicator in breast cancer as well.     

The ephrins and Eph receptors in angiogenesis.

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell-cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.