Eph- and ephrin-dependent mechanisms in tumor and stem cell dynamics

Abstract

The erythropoietin-producing hepatocellular (Eph) receptors comprise the largest family of receptor tyrosine kinases (RTKs). Initially regarded as axon-guidance and tissue-patterning molecules, Eph receptors have now been attributed with various functions during development, tissue homeostasis, and disease pathogenesis. Their ligands, ephrins, are synthesized as membrane-associated molecules. At least two properties make this signaling system unique: (1) the signal can be simultaneously transduced in the receptor- and the ligand-expressing cell, (2) the signaling outcome through the same molecules can be opposite depending on cellular context. Moreover, shedding of Eph and ephrin ectodomains as well as ligand-dependent and -independent receptor crosstalk with other RTKs, proteases, and adhesion molecules broadens the repertoire of Eph/ephrin functions. These integrated pathways provide plasticity to cell–microenvironment communication in varying tissue contexts. The complex molecular networks and dynamic cellular outcomes connected to the Eph/ephrin signaling in tumor–host communication and stem cell niche are the main focus of this review.

Introduction

The human erythropoietin-producing hepatocellular (Eph) receptors include 14 type I transmembrane proteins comprising the largest family of receptor tyrosine kinases (RTKs). The first member of this family, EphA1, was cloned over 20 years ago from an erythropoietin-producing hepatocellular carcinoma cell line in a screen for oncogenic tyrosine kinases homologous to the viral oncogene v-fps [1]. Besides the first identified functions as axon guidance molecules, Eph receptors are now known to regulate a wide range of cell-to-cell communication events involved in cell positioning and tissue patterning during embryonic development and pathological conditions such as cancer and vascular complications [2–6]. In addition, these receptors have emerged as regulators of specialized cell functions including synaptic plasticity, insulin secretion, bone remodeling, epithelial homeostasis, as well as inflammatory and immune responses [2, 3, 7]. As such, they are expressed by a wide variety of cell types such as neurons, vascular cells, epithelial cells, inflammatory cells, immune cells, and tumor cells including cancer stem cells [8–11].

Unlike for other RTKs, whose ligands are soluble or sequestered within the extracellular matrix (ECM), the ligands for Eph receptors, called ephrins (Eph receptor-interacting), are synthesized as membrane-anchored molecules. At least two properties make this signaling system unique and complex of its own kind: (1) the signal induced by receptor binding of the membrane-bound ligand is simultaneously transduced into both the receptor-expressing cell (forward signaling) and the ligand-expressing cell (reverse signaling), termed bidirectional signaling, (2) the signaling outcome through the same molecules can be opposite depending on the cellular context. The release of functional soluble ligands via shedding of Eph and ephrin ectodomains further suggests the existence of additional paracrine and autocrine Eph/ephrin signaling mechanisms [12–14]. In addition to the diverse interactions and clustering amongst different Eph and ephrins, ligand-dependent and -independent crosstalk occur with other RTKs as well as cell–cell and cell–ECM adhesion molecules [15, 16]. Moreover, close association with Rho family of small GTPases intimately links Eph/ephrin signaling to cytoskeletal dynamics [17–19]. Changes in transmembrane protease interactions, proteolytic processing, endocytosis, and degradation of the receptor complexes determine yet another aspect of signal transduction shift and compartmentalization in response to immediate cell microenvironments [12, 13, 20–22]. These functional interactions provide dynamic regulatory mechanisms to cell–microenvironment communication pathways in varying tissue contexts.

Eph receptors and ephrin ligands

Eph receptors are classified into two subgroups, namely EphAs or EphBs based on sequence homology and binding affinity to their ligands [23]. The human EphA subgroup includes nine receptors (EphA1–8 and EphA10), whereas the EphB subgroup includes five members (EphB1–4 and EphB6). EphA and EphB receptors share a conserved multi-domain structure (Fig. 1; [24, 25]). The extracellular domain (ECD) includes an N-terminal ligand-binding domain (LBD), a cysteine-rich domain (CRD) containing an epidermal growth factor (EGF)-like motif, followed by two fibronectin-type-III repeats (FN-III 1 and FN-III 2). A single-pass transmembrane domain is followed by an intracellular region containing a juxtamembrane region, a tyrosine kinase domain, a sterile α motif (SAM), and a postsynaptic density protein PSD95, Drosophila disc large tumor suppressor DlgA, and zonula occludens-1 protein ZO-1 (PDZ)-binding motif [3, 6]. In the cytoplasmic sequence, the location of tyrosine residues is well conserved within the juxtamembrane and tyrosine kinase domain of all Eph receptors across different species [26]. However, one receptor from each class (EphA10 and EphB6) lacks residues essential for the kinase activity, providing an additional regulatory mechanism for sequestration and signaling attenuation of the active Eph RTKs in receptor hetero-complexes [27, 28].

Fig. 1.

Structure of Eph receptors and ephrin ligands. EphA and EphB receptors share a conserved multi-domain structure [24]. The extracellular domain contains an N-terminal ligand-binding domain (LBD), a cysteine-rich domain (CRD) followed by two fibronectin type-III repeats (FN1 and FN2). A single-pass transmembrane domain (TM) is followed by an intracellular region containing a juxtamembrane region, a tyrosine kinase domain (TK), a sterile α motif (SAM), and a postsynaptic density protein PSD95, Drosophila disc large tumor suppressor DlgA, and zonula occludens-1 protein ZO-1 (PDZ)-binding motif [3, 6]. The ephrin ligands contain a conserved extracellular N-terminal receptor binding domain (RBD). EphrinA ligands are attached to the cell membrane through a glycosylphosphatidylinositol (GPI)-anchor, whereas ephrinBs contain a transmembrane domain, and a C-terminal cytoplasmic tail including a PDZ-binding motif [29]

The ephrin ligands contain a conserved extracellular N-terminal receptor binding domain (RBD) (Fig. 1; [29]). Based on their mode of cell membrane attachment, ephrins are also divided into two subgroups. All five ephrinA ligands (ephrinA1–5) are attached to the cell membrane through a glycosylphosphatidylinositol (GPI)-anchor, whereas the three ephrinBs (ephrinB1–3) contain a transmembrane domain, and a C-terminal cytoplasmic tail including a PDZ-binding motif. Ligand–receptor interactions are specific and promiscuous within each class (A or B), with a few exceptions: EphA4 binds ephrinBs and EphB2 binds ephrinA5, whereas ephrinB2 is the only known ligand for EphB4 [3].

Eph/ephrin signaling

Based on genetic mouse studies, Eph/ephrin signaling contributes to e.g., neuronal, vascular, and skeletal development. In these processes, as well as in branching morphogenesis Eph/ephrin pathways typically contribute to cell sorting and tissue patterning through mechanisms linked to cell attraction/repulsion [7, 16]. However, the abundance, redundancy, and often peculiar context-dependency of Eph/ephrin signaling bring considerable complexity to the patterns of signaling interconnections and integration towards defined cellular outcomes [15, 16]. Moreover, differential signaling directionality and intensities contribute to dynamic cellular outcomes upon multiple receptor/ligand and forward/reverse signaling relationships [4, 30]. Such complexity is also signified in cancer, where Eph/ephrin signaling can (1) be tumor promoting or suppressive, (2) lead to cell adhesion or repulsion, and (3) promote cell migration and proliferation or differentiation and quiescence, depending on mutual relationships within the Eph/ephrin system as well as on cell type-specific traits and cues from the ECM or membrane-bound and soluble co-factors [3, 31, 32].

Ligand-induced forward signaling

Upon binding of the ligand, a 1:1 interaction with the receptor on juxtaposed cell membranes occurs with high affinity [33–36]. The crystal structures resolved for several Eph receptors and Eph/ephrin complexes have helped to describe these receptor/ligand binding properties [33–36]. Using soluble monomeric ephrin ligands, this interaction has been shown to occur through the insertion of the conserved hydrophobic loop (G–H loop) of the ephrin RBD into a hydrophobic cavity within the Eph receptor LBD [16, 24, 35–39]. Recently, ephrin glycosylation has been found to be critical for the ligand binding [40]. This ligand/receptor interaction induces conformational rearrangements in the receptor LBD, which further facilitates the formation of complementary Eph/Eph interaction interfaces and clustering [41]. Other domains, such as the CRD, SAM, and a portion of the FN-III domain within the receptor favor additional Eph/Eph interactions thus stabilizing Eph/ephrin tetramers [24, 33, 37, 42–46].

The conformational changes upon the Eph/ephrin interaction are followed by the receptor autophosphorylation at two tyrosine residues within the juxtamembrane domain and one tyrosine residue within the activation segment of the kinase domain [26]. This disrupts the inhibitory interaction with the kinase domain, enhancing the kinase activity and transphosphorylation of additional tyrosine residues [26]. As typical for RTKs, the phosphorylated tyrosine residues serve as docking sites for Src Homology 2 (SH2) and phosphotyrosine binding (PTB) domain-containing adaptor proteins. These interactions can mediate signal transduction with variable duration and kinetics through multiple downstream signaling pathways including phosphatidylinositol 3′-kinase (PI3K)-AKT, Janus kinase/Signal transducer and activator of transcription (JAK/STAT), Ras/mitogen-activated protein kinase (RAS/MAPK), as well as focal adhesion kinase (FAK) and Src kinase-mediated signals (Fig. 2; [2, 3, 47–49]). Through their PDZ-binding motif, Eph receptors associate with PDZ-domain-containing proteins such as AF6, Pick1, syntenin, and Grip1/2 to further regulate clustering, trafficking, and signaling [50–52].

Fig. 2.

Eph/ephrin signaling. Eph forward signaling (bottom). Ligand binding at cell–cell contact (or involving soluble shed ligand) triggers receptor clustering and phosphorylation of tyrosine residues in juxtamembrane and tyrosine kinase domains [12, 26, 33–36]. The ligand-induced Eph activation mediates downstream signaling pathways such as PI3K–Akt, Janus kinase (JNK)-STAT, as well as focal adhesion kinase (FAK) and Src kinase-mediated signals [2, 3, 47–49]. Forward signaling can also induce transient ERK activation, although the overall effect of EphAs on ERK activity appears suppressive, as e.g., loss of EphA2 results in increased overall Ras/ERK pathway activation [12, 61, 65, 154, 214, 215]. Eph forward signaling also regulates Rho GTPase-mediated actin dynamics through interaction with guanine exchange factors (GEFs), e.g., Ephexin and Vav (EphA), as well as Kalirin and Intersectin (EphB) [17, 82–86]. Low molecular weight protein tyrosine phosphatase (LMW-PTP) activated by Src regulates Eph signaling attenuation or termination by receptor dephosphorylation [82]. Ephrin reverse signaling (top). Eph-ephrin interaction triggers intracellular signaling into the ligand-expressing cell. Reverse signals through GPI-anchored ephrinAs rely on lipid raft-mediated clustering with proteins such as Src family kinase (Fyn) to induce e.g., ERK signaling [53, 87]. EphB/ephrinB interaction triggers reverse signals more directly via ephrinB cytoplasmic tail phosphorylation followed by recruitment of SH2-domain-containing proteins such as Src and Grb4, as well as Rac1 activation, which is attenuated by EphB dephosphorylation by PTP-Basophil-like (PTP-BL) phosphatase [90, 93]. EphrinBs also recruit PDZ-domain-containing proteins through their C-terminus [90], whereas non-phosphorylated ephrinB1 can interact with PAR6, promoting tight junctions [97]. Receptor tyrosine kinase crosstalk (middle right). EphA overexpression coupled with low expression of ephrinA ligand is associated with low tyrosine phosphorylation of the ligand-unbound receptor [32]. Akt downstream of growth factor receptors phosphorylates ligand-unbound EphA2 at serine residue (Ser897) [32, 101]. Src activation can regulate ligand-independent EphA signaling [22, 118]. RTK-EphA2 crosstalk promotes Rho-GTPase activities and EGF-induced Ras/ERK pathway activation in proliferating/differentiated cancer cells whereas suppression of the differentiation-promoting ERK activity in tumor-propagating cells (TPCs) in glioblastoma multiforme (GBM) is mediated by serine-phosphorylated EphA2 [10, 83, 86, 101]

On the cell surface, the Ephs and membrane-anchored ephrins have been found to localize in defined membrane microdomains/lipid rafts, which supports some degree of clustering also prior the ligand/receptor interaction, at least upon high expression [53–55]. Therefore, the Eph signaling can be initiated within these Eph/ephrin clusters, forming signaling centers at the cell–cell interface. Further receptor clustering can follow by progressive formation of higher order homo- and hetero-assemblies of Eph receptors independently of additional ligand binding, which further modulates forward signaling [28, 33, 42, 56, 57]. An increasing number of studies elucidate the complexity and fine-tuning of the signaling propagation in relation to overall cell-surface and soluble ligand and receptor densities as well as to receptor crosstalk, in cis (same cell) receptor/ligand interactions, and intracellular feedback mechanisms [32, 34, 58–67].

Another layer of complexity is brought by the varying mechanisms of receptor/ligand mobilization from the cell junctions to regulate trafficking, signaling compartmentalization, and outcomes. The Eph/ephrin complexes between two juxtaposed cells can be released from one cell via metalloprotease cleavage of membrane-bound ephrin ligands, followed by Eph/ephrin internalization into the other cell [21, 68]. Alternatively, these membrane-bound Eph/ephrin complexes are removed from the cell surface by trans-endocytosis into the Eph- or ephrin-expressing cell [16, 34, 69]. Moreover, although a number of studies has stated the membrane-anchorage of endogenous ephrins or pre-clustering of soluble ligands as prerequisite for signaling, the induction of prominent Eph receptor forward signaling responses by soluble monomeric ephrinA1 has been widely documented [4, 12–14, 42, 70–74]. This can be particularly relevant in the presence of increased metalloproteinase activities during pathological tissue remodeling processes such as cancer. Upon all the above signaling initiation mechanisms, the Eph/ephrin complexes are internalized through caveolae- or clathrin-dependent endocytosis [75, 76]. The activation of the intact or cleavage-released, internalized receptor/ligand complexes can persist, thus allowing signaling shift into either cell [20, 69]. Furthermore, interaction with protein tyrosine phosphatases (PTPases) and the ubiquitin ligase Cbl contribute to Eph/ephrin signaling attenuation or termination by receptor dephosphorylation or degradation [77–82].

Important features of Eph signaling also include the close connections to the cytoskeletal dynamics and cell–cell or cell–matrix adhesion, through crosstalk with Rho GTPases, cadherins and integrins. Functional protein interactions, such as those with the GTPase-regulating guanine-nucleotide exchange factors (GEFs), can occur also independently of receptor tyrosine phosphorylation to regulate actin remodeling and cell morphology [17, 19]. EphA receptors activate Rho GTPases through direct interactions with e.g., Ephexin4, Tiam1 and Vav family GEFs, while EphBs use interactions with e.g., Intersectin and Kalirin GEFs for Rho GTPase regulation (Fig. 2; [17, 82–86]).

Ephrin-mediated reverse signaling

Eph/ephrin interactions trigger signaling also in cells expressing the membrane-bound ligand, a mechanism called “reverse signaling”. Intracellular signals transduced by both ephrinAs and ephrinBs have been found to modify cell behavior. However, reverse signals through GPI-anchored ephrinAs rely on lipid raft-mediated interaction with transmembrane protein complexes. Although ephrinAs lack an intracellular domain for phosphorylation-dependent recruitment of signaling molecules, clustered ephrinA5 has been found to recruit the Src family kinase Fyn to the same caveolae-like membrane domains, upon receptor binding (Fig. 2). This promotes activation of β1 integrin and ERK and increases cell–substrate adhesion [53, 87]. In contrast, EphB/ephrinB interaction triggers ephrinB phosphorylation on conserved tyrosine residues by Src family kinases, thus creating docking sites for SH2-domain-containing proteins such as Grb4 (Fig. 2; [88–93]). Like Eph receptors, ephrinBs also recruit PDZ-domain-containing proteins through their C-terminus, such as the protein tyrosine phosphatase PTP-BL, which in turn can dephosphorylate both ephrinBs and Src as a mechanism of reverse signaling attenuation [90]. The reverse ephrinB signaling has been found to regulate cell invasion via, e.g., matrix metalloproteinase 8 (MMP8) secretion and Rac1 activation [94–96]. Interaction of non-phosphorylated ephrinB1 with the PAR6 polarity protein instead regulates epithelial tight junction formation [97]. EphrinB2 can also exert cell–cell contact-independent functions by stimulating actomyosin-dependent cell contraction through its PDZ-motif with a mechanism that does not require Eph receptor binding [98, 99].

Ligand-independent signaling via growth factor receptor crosstalk

Besides the cell-type and context-dependent bidirectional signals, Eph receptors engage in cell signaling pathways via receptor and cytoplasmic protein crosstalk, which notably broadens their range of functions. These mechanisms have been described with some detail in breast and prostate carcinoma as well as glioblastoma (GBM) cells, where overexpression of EphA2 coupled with down-regulation of its preferred ligand ephrinA1, is linked to enhanced oncogenic EphA2 function via growth factor receptor crosstalk (Fig. 2; [32, 61, 100, 101]). Upon the ligand suppression, attenuation of the counteracting, tumor-suppressive, forward signaling is reflected by low tyrosine phosphorylation of the receptor [32]. In this context, Akt downstream of the growth factor receptors phosphorylates ligand-unbound EphA2 on a serine residue (Ser897), thereby promoting cell invasion [32, 101]. Several growth factors including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor/scatter factor (HGF/SF), and platelet-derived growth factor (PDGF) promote EphA2 serine phosphorylation, suggesting a role for EphA2 as a common co-effector of growth factor signaling [32, 61, 100, 102, 103]. Moreover, a related mechanism has been reported, whereby EGF promotes cell migration through Ephexin4 recruitment to the serine phosphorylated EphA2 and subsequent activation of RhoG/Rac1 axis [83, 86]. Physical association at the cell-surface has been reported for EphA2–ErbB2/EGFR and EphA4–FGFR1 [100, 103, 104]. EphA4 also enhances GBM cell migration and proliferation by interacting with the canonical FGFR signaling pathway [104].

The ligand-independent signaling crosstalk can also occur at different levels and via intracellular signaling effectors. Besides the EphA2–ErbB2/EGFR protein interactions, EGF-induced Ras/ERK pathway transcriptionally enhances EphA2 as well as EphB4, thus further favoring the ligand-independent signaling [61, 103, 105]. Although specific cell-surface and intracellular signaling interactions remain elusive, also EphB receptors can modify cell behavior through ligand-independent mechanisms. For instance, EphB2 and EphB3 receptors regulate cell positioning in the intestinal epithelium via PI3K, independently of kinase activity [106]. Although broadly accepted that, at least in the presence of excess ligand, EphA2 forward signaling is tumor migration- and progression-suppressive, evidence also exists to support the context-dependent role of EphA2 RTK activity and tyrosine phosphorylation on cell migration/invasion [22, 32, 107–109].

The non-receptor tyrosine kinase Src is a common mediator of both upstream and downstream RTK signals regulating cell adhesive, mitogenic, and migratory responses, including Rho-mediated cytoskeletal rearrangements [110–115]. Src is involved in signaling from various RTKs, including PDGFR, EGFR, FGFR, HGFR, and others. Moreover, EphA2 has been reported to increase Src activation in both ligand-dependent and -independent manner co-incident with increased or altered invasion of breast, prostate, and lung carcinoma cells as well as melanoma cells [22, 108, 109, 116–118]. EphA2 is also required for Src-induced invasion of colon carcinoma cells coincident with increased EphA2 tyrosine phosphorylation [119]. The involvement of Src in the Eph-RTK crosstalk has not been well established. However, since EGFR/RAS or the other growth factor receptor pathways are aberrantly activated in various cancer cells, Src is well positioned to function as an important intracellular effector of the pro-invasive EphA2 activities via RTK crosstalk. Through its multiple connections, Src could also serve as a hub for synergy between ligand-dependent and -independent signaling and receptor interaction in the presence of different ligand concentrations (Fig. 3; [120]).

Fig. 3.

Growth factor and adhesion receptor interactions with EphA2 in tumor cell invasion. The context-dependent, often tumor-suppressive, EphA/ephrinA forward signaling supports the maintenance of cadherin junctions abundant in noninvasive epithelial tumors, and involved also in collective cell invasion (left) [63, 157]. In invasive cancer cells, the ligand-independent EphA2 signaling involves physical interaction with EGFR and Akt-mediated EphA2 phosphorylation at Ser897 (right) [32, 100, 101, 103]. This RTK crosstalk between the Eph and growth factor RTKs is linked to the integrin-mediated cell–ECM communication through intracellular pathways via non-receptor tyrosine kinases Src and FAK [22, 171]. These kinases are common mediators of both upstream and downstream RTK and integrin signals regulating cell adhesive, mitogenic, and migratory responses, including Rho family GTPase-mediated cytoskeletal rearrangements (through e.g., RhoA, Rac1, and RhoG). The integrin-mediated adhesion and MT1-MMP-mediated degradation of the ECM, both required for efficient mesenchymal or collective tumor cell invasion across collagen-rich tissues, ephrin cleavages by MMPs as well as EphA2-dependent MT1-MMP induction are also depicted [13, 22]. RhoA-mediated repulsive responses upon EphA2 activation, limited ECM proteolysis, or impaired integrin-mediated adhesion can instead lead to more amoeboid-type single cell invasion by increased actomyosin contractility (see also Fig. 4b)

Eph signaling in tumor cell dynamics

During development, Eph receptors are widely expressed, whereas in adult tissues they are typically found at relatively lower levels [9, 121]. In human cancer, both Ephs and ephrins are frequently deregulated, being either re-expressed or down-regulated [122–131]. In different contexts of carcinogenesis and tumor progression, Eph receptors co-operate with multiple other cellular pathways to mediate either oncogenic or tumor-suppressive functions [2, 100, 103, 104, 132, 133]. Collectively, these pathways exert various effects on cell differentiation, survival, proliferation, cell–cell and cell–ECM adhesion, as well as cell segregation and invasion. Therefore, functional alterations in the signaling through Ephs and their crosstalk with oncogenic signaling pathways are bound to influence tumor initiation and progression at different levels [59, 134].

The mechanisms through which Eph signaling is linked to cancer diverge markedly between tumor types and stages [3]. During metastatic tumor progression, cancer cells are exposed to varying microenvironments within primary site, when invading into stroma or vasculature, and after reaching distant organs [135]. Therefore, a key property of cancer cells is plasticity, i.e. the cells modulate their behavior by integrating intracellular signaling with the varying cell-surface receptor interactions and physical confines or cues of the ECM. Depending on such signals, cancer invasion can occur by either collective cell groups or individual cells [136]. Several crucial Eph receptor-mediated signaling cues have been implicated in the dynamic regulation of such signaling integration in cell–microenvironment communications contributing to the cancer cell invasion plasticity and stem-like properties [22, 118, 137–139].

Although context-dependent, the ligand-induced Eph signaling in tumor cells is broadly considered as migration- and growth-suppressive. Accordingly, the overexpression of Ephs coupled with the down-regulation of the preferred ephrin ligands has been observed in several cancers and associated with tumor aggressiveness and higher grades [140–142]. However, a reverse expression pattern has also been observed in some tumors including breast, colorectal cancer, and acute lymphoblastic leukemia, where low Eph receptor expression through epigenetic silencing or mutations can also correlate with poor prognosis [143–145]. Moreover, the Eph/ephrin system functions also within the stromal compartment, including cancer-associated fibroblasts, vascular cells, and immune cells, thereby regulating heterotypic cell interactions, cell segregation, and cell responses within stroma [126, 146–149]. Eph/ephrin-regulated events within the malignant cells as well as between tumor and the host occur during e.g., (1) tumor cell compartmentalization and segregation to different types of invasive or growing cells within the tumor cell mass and at the invasive edges, (2) vascular cell patterning during tumor angiogenesis, and (3) recruitment of immune and inflammatory cells. Recently, Eph/ephrin signals have also been critically linked to the regulation of cancer cell dedifferentiation and stem-like properties [10, 11, 101]. Hence, although the expression patterns of certain Ephs and ephrins can serve as prognostic markers, additional cell/tumor-context-specific information is essential, in order to implement the Eph/ephrin system into potential tumor/patient-specific prognostic or therapeutic strategies.

Eph/ephrin signaling in cell polarity

Loss of apical-basal polarity normally displayed in epithelia, is a common feature of carcinoma progression [150–152]. Many signaling networks and protein complexes regulate this process that involves, among others, redistribution of the Crumbs, PAR, and Scribble polarity complexes, reorganization of the actin cytoskeleton and localized activation of Rho GTPases [152]. Forward signaling through multiple Eph receptors has been reported to support epithelial polarization, thus inhibiting cell transformation [102, 153–156]. EphrinA1-induced activation of EphA kinases in Madin–Darby canine kidney (MDCK) epithelial cells negatively regulates HGF-induced branching morphogenesis [154]. Confluency or stimulation of MDCK cells with pre-clustered ephrinA1-Fc ligand induces EphA2 tyrosine phosphorylation and suppresses Arf6 GTPase activity, thereby promoting the maturation of E-cadherin-based cell junctions and apical-basal polarity, which enhances EphA2/ephrinA1 signaling via a positive feedback loop [63, 154, 157, 158]. In the intestinal epithelium, EphB expression and activation instead directs the positioning of the different cell types along the crypt-villus axis [159]. In colorectal cancer (CRC), EphB–ephrinB interaction confines the expansion of incipient CRC cells through a mechanism dependent on E-cadherin-based cell-to-cell adhesion, via redistribution of E-cadherin to the basolateral cell membrane [155]. During the adenoma-carcinoma transition, however, the expression of EphB receptors is silenced, thereby promoting cell invasion and tumor progression [106]. These interconnections between Ephs, ephrins, and classical cell-junctional molecules have revealed important Eph/ephrin functions in epithelial structural integrity and homeostasis.

During cancer progression, many types of cancer cells undergo epithelial-to-mesenchymal transition (EMT) during which they lose the epithelial cell–cell contacts and adopt a front-rear polarity required for efficient directional migration and tissue invasion [160, 161]. This process involves asymmetric distribution and activation of Rho GTPases. Generally, Cdc42 and Rac1 are activated at the front of the cell, while RhoA is activated at the cell rear [162]. Through regulation of the activity of these and other Rho GTPases, Eph receptors modulate actin dynamics and cell motility. Regulation of RhoA activation by EphA2 and EphB4 receptor signaling has been reported in various cancer cell lines [109, 114, 118, 163]. Such EMT-promoting Eph functions are also supported by the loss of cell–cell adhesion and apical-basal polarity caused concomitantly with changes in the actin cytoskeleton by ectopic EphA4 expression in developing Xenopus laevis embryos [153].

Eph/ephrin signaling in the dynamics of tumor cell invasion

Metastasis, the main cause of cancer-associated mortality, relies on cancer cell dissemination into distant sites by multiple interchangeable modes of invasion instructed by both cell-intrinsic mechanisms and communication with the microenvironment. EMT and collective migration are both important mechanisms for cell invasion across basement membranes and interstitial tissues via coordinated cell and ECM adhesion, cytoskeletal changes, and ECM proteolysis. Pending on e.g., altered physical properties and composition of the ECM or protease/inhibitor balance, cancer cells can also switch between single cell and collective motility modes [164]. Moreover, impaired integrin-mediated adhesion and limited ECM proteolysis can lead to more amoeboid-type single cell invasion by increased actomyosin contractility [164].

The modulation of invasion modes, efficiency, and plasticity represents the Eph/ephrin function frequently implicated in cancer [22, 109, 117, 165, 166]. Ephs and ephrins directly regulate both homotypic (cancer–cancer) and heterotypic (cancer–host) cell interactions [146, 155, 167]. In many in vitro and in vivo tumor models, ligand-dependent Eph signaling inhibits cell migration and invasion by supporting cell junctional integrity and compartmentalization [16, 32, 74, 155, 168, 169]. However, extensive evidence indicates how the Eph crosstalk with other cell-surface co-factors and intracellular cytoskeleton regulators contributes to dynamic Eph-dependent signaling outcomes towards cancer progression and metastasis. Besides growth factor receptor crosstalk, the Eph receptor functions in cell invasion include effects on actin cytoskeleton, as well as cell–cell and cell–ECM communications through functional interactions modulating e.g., integrins, Rho GTPases and metalloproteinases [22, 58, 146, 170–172].

Eph/ephrin function in the regulation of cell–ECM adhesion and integrin function

Integrins are the main transmembrane receptors that mediate cell–ECM adhesion and mechano-transduction, whose deregulation is involved in abnormal cancer cell spreading, migration, survival, and proliferation [173]. In the invasive cells with Eph–RTK crosstalk, Eph-mediated regulation of cell junctions often involves a shift from constant cell and ECM adhesion to more dynamic integrin-mediated interactions with altered ECM microenvironments, rich in collagen type I, III, fibronectin, and laminins. The adhesion to the ECM ligands induces “outside-in” integrin signaling, which involves the recruitment and activation of downstream effectors such as FAK and Src family kinases, leading ultimately also to changes in MAPK and PI3K signaling pathways [174]. The modulation of integrin binding to their ECM ligands is regulated by conformational changes in their extracellular domains, which in turn can be regulated by intracellular signaling pathways, a mechanism referred to as “inside-out” integrin signaling. Since the key intracellular interacting molecules, including FAK and Src, are shared by both integrin and Eph/ephrin complexes, their downstream effects can be integrated into the regulation of each other’s activities, cell cytoskeleton, and cell or ECM interactions (Fig. 3). However, physical interaction has to date been reported only between EphA4 and integrinα_IIbβ3 [175]. Therefore, the functional interaction between these two receptor pathways can occur also at the intracellular signaling level.

In cancer, both suppressive and promoting Eph functions towards integrin-mediated adhesion have been reported [58, 60, 170, 171, 176]. For instance, in the invasive PC3 prostate cancer cells ligand-unbound EphA2 and β1-integrin promote signaling through FAK [171]. In contrast, ephrinA1-dependent activation of EphA2 leads to FAK inactivation via the recruitment of SHP2 phosphatase, thus inhibiting integrin-mediated spreading and migration of the cells [171]. In mouse embryonic fibroblasts and NIH3T3 cells, ligand-induced EphA2 activation instead promotes cell adhesion and spreading in a FAK and p130^CAS phosphorylation-dependent manner [177]. These differences may be ascribed to different RTK pathways activated in normal and cancer cells, different microenvironmental contexts, or different degrees of ligand-clustering [60].

Interestingly, ligand-induced EphB1 activation leads to increased adhesion of HEK293 embryonic kidney cells and endothelial cells, whereas opposite effects have been reported after EphB3 activation in HEK293 cells and LS174T colorectal epithelial cells [58, 178]. Besides the functions of individual receptors, EphA and EphB co-expression patterns can differentially influence cell adhesion and migration through major effects on cytoskeletal contractility depending on the activation status of each Eph receptor [146]. Modulation of integrin-mediated cell adhesion through ligand-induced Eph activation may also be relevant for the initiation of cell repulsive responses in non-transformed cells and cell segregation [159, 179]. The ligand-independent signaling activated in cancer cells can instead engage Eph receptors in other intracellular signaling complexes with Src and FAK to influence integrin functions [180, 181]. The integration of cytoskeletal migration signaling by Ephs towards intracellular responses triggered by integrins in response to changes in the ECM helps to explain the means for context-dependent regulation of the plastic invasion outcomes.

Rho GTPase–Eph RTK signaling in tumor cell plasticity

The Rho GTPases are key mediators of both RTK and integrin signaling towards the actin cytoskeleton, thereby modulating cell migration and invasion [182]. Increased activities of Rho GTPases are typically involved in tumor progression, representing the key intracellular inducers of switches between different invasion modalities, i.e. cell invasion plasticity [162, 183]. Eph receptors regulate actin dynamics and cell morphology through close co-operation with Rho GTPases [17, 19, 146, 184]. The activated EphA2 and EphA4 trigger contact inhibition of locomotion or repulsive cell movement by causing retraction of membrane protrusions and re-direction of migration through RhoA activation in colliding PC3 prostate cancer cells [146]. However, heterotypic contacts between PC3 and fibroblasts trigger EphB–ephrinB signaling that can override the repulsive EphA–ephrinA signals, and induce continued PC3 migration across the fibroblasts by filopodia and lamellipodia formation through Cdc42 activation [146]. Moreover, ligand-dependent EphA2 signaling triggers rapid cell rounding in PC3 and GBM cells through Src/FAK-mediated RhoA activation concomitant with Rac1 inhibition [12, 32, 146, 171, 172]. A transition from a mesenchymally invasive phenotype to an amoeboid phenotype has also been reported upon RhoA activation by aberrant EphA2 overexpression concomitant with constitutive EphA2 tyrosine phosphorylation in PC3 and melanoma cells [109, 117]. Similarly with EphA2, ephrinA5-induced activation of EphA3 in melanoma cells triggers cell rounding, membrane blebbing concomitant with CrkII-mediated RhoA activation [185]. Therefore, a combination of Eph, ephrin, and cofactor expression patterns within tumor microenvironment can control cancer cell invasion plasticity, through integrated actions of integrin-mediated cell–matrix adhesion, Rho GTPases, and the cytoplasmic signaling responses (Fig. 3).

Metalloproteinase–Eph RTK signaling

Proteolytic ectodomain shedding of cell surface proteins has emerged as an important regulatory mechanism of cell signaling that is implicated in cell adhesion, invasion, segregation/differentiation and growth outcomes [186, 187]. Among the proteases involved in pericellular proteolysis, a disintegrin and metalloprotease (ADAM) transmembrane proteases are closely associated with RTK signaling via ectodomain shedding and activity-regulating cleavages of the receptors or cell-surface associated ligands (Fig. 4; [21, 71]). Consistently, multiple reports describe how, upon Eph/ephrin binding, ADAM can associate with the activated EphA receptor and cleave the receptor-bound ephrin ligand in trans to release the Eph/ephrin complexes [4, 21, 68, 188]. The ephrin cleavage is followed by Eph/ephrin endocytosis, ultimately leading to cell rounding and repulsion [12, 13, 21, 68]. The ephrinA-induced EphA endocytosis is generally associated with the migration-suppressive signaling and receptor degradation. However, potential context-dependent roles of ADAM-EphA interactions in cancer cell segregation and invasion remain incompletely understood [32, 34, 71]. Moreover, a conserved ADAM10 substrate recognition motif has been identified in the extracellular domain of all ephrins, which has been linked to ephrin release, apparently in cis [35, 37, 39, 71]. Since this lies within the ligand G-H loop, the region involved in receptor binding, it remains unclear how this will be involved in the ephrin release by the ADAM cleavages (in trans or in cis), apparently within the juxtamembrane region [13, 16, 24, 35–39]. Of note, ephrinAs are also readily cleaved within defined sites of the juxtamembrane region by soluble MMPs typically enriched in cancer [13]. Therefore, such proteolytic events within Eph/ephrin complexes can represent general phenomena that regulate the signaling axes and cell behavior.

Fig. 4.

Metalloproteinase regulation of Eph signaling and cellular responses. a EphrinA-EphA binding upon cell–cell contact induces activation and conformational changes in EphA, leading to the recruitment of ADAM10. The Eph-interacting ADAM10 then cleaves receptor-bound ephrinA ligands in trans from an adjacent cell, followed by endocytosis of the activated receptor-ligand complexes, and cell–cell repulsion or contact inhibition of locomotion [21]. As typical for RTKs, the transiently activated signal can be attenuated by receptor degradation after endocytosis. This Eph forward signaling leads to cell segregation and migration inhibition. b In invasive carcinoma cells, EphA2 overexpression and ephrinA suppression are frequently coupled with the induction of MT1-MMP [22]. In these cells, growth factor receptors such as ErbB2/EGFR are also often induced or mutationally activated to mediate ligand-independent EphA2 crosstalk as well as migration signaling through Src. In this context, Src activities can also promote the transcriptional activation and phosphorylation of MT1-MMP [198]. EphA2 kinase activity-independent cleavage of the adjacent EphA2 receptor in cis by MT1-MMP triggers Src and EphA2 activity-dependent intracellular translocation of the EphA2 signaling complexes and subsequent RhoA GTPase activation [22]. When coupled with the Src-mediated migration signaling, these events and the intracellular signaling compartmentalization lead to cytoskeletal contractility, cell–cell repulsion, and a switch from collective to single-cell invasion [22]

In invasive basal-like breast carcinoma cells, another mechanism was recently demonstrated, whereby the pericellular collagenase membrane-type-1 matrix metalloproteinase (MT1-MMP) interacts with EphA2 to regulate single-cell dissemination (Fig. 4; [22, 118]). Instead of ephrinA ligand, MT1-MMP directly cleaves the receptor at the FN-III 1 domain in cis on the same cell-surface complexes [22]. This cleavage triggers internalization of the receptor signaling complexes in conjunction with increased RhoA activation, cell repulsion, and a switch from collective to single MDA-MB-231 breast cancer cell invasion [22, 118]. Although somewhat analogous to the changes upon the ligand-induced ADAM-Eph interactions, the signaling requirements and cellular outcomes differ markedly from those involving ADAMs. The protease interaction and cleavage occur also in the absence of EphA2 kinase activity, but EphA2 and Src kinase activities are both required for the intracellular translocation of the signaling complexes and single cell invasion [22]. Interestingly, somatic EphA2 mutations affecting this cleavage site have been found in lung cancer [116]. By inhibiting the MT1-MMP cleavage, the same mutation limits the single-cell invasion, thus inducing rapidly growing coherent cell colonies [22]. Therefore, functional interaction between EphA2 signaling and MT1-MMP regulates switches between collective and single-cell invasion.

In different types of invading or proliferating malignant and normal cells, MT1-MMP interactions and direct or indirect activities have also been reported within membrane complexes of other RTKs, such as FGFR2, FGFR4, VEGFR2, and PDGFRb, as well as the collagen-binding RTK discoidin domain receptor tyrosine kinase 1 (DDR1) [189–194]. Moreover, MT1-MMP has been found to physically associate with ADAM9, ADAM10 and ADAM15, and to cleave at least ADAM9 and ADAM15 [192, 195, 196]. In the FGFR2 complexes, the inactivating cleavage of ADAM9 by MT1-MMP sustains FGF signaling by protecting FGFR2 from ectodomain shedding [192]. Since both ADAM10 and MT1-MMP are expressed and have been shown to associate with EphA2 in e.g., MDA-MB-231 cells, their potential competitive or synergistic activities could be relevant also within the EphA2 complexes towards differential signaling responses [22, 197]. Of note, besides being important for the MT1-MMP-dependent intracellular accumulation/re-localization of the EphA2 and FGFR signaling complexes, Src activity induces direct phosphorylation of a single tyrosine residue in the MT1-MMP cytoplasmic tail [190, 198]. This further supports the central role of Src in the dynamics of Eph crosstalk mechanisms. However, the further relations between the metalloproteinases, RTK crosstalk, and the invasion modulating Eph signaling responses in tumors and tumor/stroma interfaces with differential protease and ligand/receptor expression remain to be explored.

In contrast to ADAMs, MMPs, including the membrane-anchored MT-MMPs, are typically considered as downstream effectors of the signaling pathways, enhancing cancer and stromal cell invasion by ECM degradation [199, 200]. Indeed, extensive evidence indicates that the pericellular collagenolytic functions of MT1-MMP are essential for the tissue invasion and growth of different types of malignant and normal cells [201]. While commonly referred to as separate processes, recent findings have, however, highlighted the close co-operation between proteolytic regulation of the ECM degradation and RTK signaling that can result from the complex modulation of both ECM and non-ECM cell-surface substrates [22, 190, 191, 202, 203]. This concept is exemplified e.g., by the reciprocal regulation described in breast carcinoma, whereby MT1-MMP cleaves EphA2, which in turn promotes collagen invasion by increasing MT1-MMP transcription [22]. In a somewhat analogous manner EphB2/ephrinB1 interaction has been found to increases ECM degradation and cell invasion by enhanced MMP-8 exocytosis, whereas MMP-8 cleaves ephrinB1, thereby regulating the EphB2/ephrinB1 interaction in pancreatic cancer cells [94]. Other Eph receptors involved in the regulation of cell invasion by the modulation of MMPs include EphB4, which stimulation in endothelial cells increases the activation of MMP-2 and MMP-9, which in turn have been found to cleave EphB2 [204, 205]. Moreover, both MMP cleavages of the receptor ectodomains and ADAM cleavages of ephrinBs create truncated membrane proteins, which serve as potential substrates for subsequent intramembranous γ-secretase cleavage [205–209]. While such sequential protease cascades have been reported at least for EphB2, EphA4, as well as ephrinB1, ephrinB2, and ephrinB3 the potential signaling and transcription regulating activities of the released intracellular domains remain poorly understood [205–209].

Eph/ephrin signaling in tumor cell proliferation

The Eph/ephrin signaling per se is best known to influence cell shape, movement, and cancer cell invasion dynamics at various levels, whereas different effects on cell proliferation and survival are also emerging. Overexpression of Eph receptors, accompanied by down-regulation of the ephrin ligands, is most often associated with development and progression of various human cancers [12, 61, 210]. However, Eph forward signaling has been reported to both induce and suppress tumor cell proliferation in different cellular contexts, tumor types, and stages.

EphA/ephrinA axis

Ligand-dependent activation of EphA signaling has a tumor-suppressive effect at least in GBM, colorectal, breast, prostate and skin cancer [12, 32, 74, 168, 169, 211, 212]. In GBM, activation of EphA2 kinase by ephrinA1 has been reported to have an anti-proliferative effect, possibly through down-regulation of EphA2 and FAK activities [12, 212, 213]. EphA2 forward signaling negatively regulates ERK activation in fibroblasts, endothelial cells, and epithelial cells as well as in tumor cells [12, 32, 61, 65, 154, 214]. Accordingly, EphA2 knockout mice display increased tumor cell proliferation and ERK phosphorylation [215]. Ligand stimulation of EphA2 also attenuates EGF-mediated ERK phosphorylation, which correlates with reduced cell proliferation and migration [61, 171]. Altogether, these findings support the tumor growth- and invasion-suppressive EphA2/ephrinA1 signaling. On the other hand, EphA2 activation has also been reported to induce ERK phosphorylation, concomitant with effects on proliferation and cell-ECM attachments [216–218]. While upon stimulation with high doses of ephrinA1 the increased ERK activation and proliferation of GBM cells was modest, one mechanism for MAPK pathway activation in breast cancer cells could involve recruitment of SHC and GRB2 to activated EphA2, translocation of activated ERK to nucleus concomitant with decreased ECM attachments. However, in malignant mesothelioma inhibitory responses towards cell proliferation relied on EphA2 activation-dependent ERK phosphorylation. Therefore, the pathways and intracellular networks connected to EphA2 activation-dependent ERK phosphorylation can differ among cell types. Moreover, other factors such as kinetics and duration of ERK activation as well as basal ERK activities are likely to influence the cellular outcomes connected to EphA2 activation.

EphA2 overexpression has been implicated in aggressive progression of breast, prostate, pancreatic, colon, and lung carcinoma as well as GBM and melanoma [3, 140, 210, 219]. In breast cancer and GBM, EphA2 overexpression is often coupled with low ephrinA1 expression and alternative ligand-independent signaling [9, 32, 61, 83, 101, 212, 220]. EphA2 co-operates with EGFR signaling in MMTV-Neu mice to enhance cell proliferation via RAS/ERK signaling in the absence of ligand stimulation, whereas the requirement for EphA2 function is bypassed in MMTV-PyV-mT tumor model with high basal RAS and ERK activities [100]. Moreover, overexpression of EphB4 receptor commonly induced in breast cancer, promotes tumor initiation and metastasis in MMTV/Neu transgenic mouse model [105]. This suggests that Eph signaling can differentially affect tumor proliferation depending also on the crosstalk with other oncogenic/tumor suppressive signaling pathways. While the promoting function of EphA2 towards glioma cell proliferation and GBM progression can be mediated by an ephrin-independent signaling, EphA2 is also expressed in highly vascular GBM areas. This suggests that EphA2 can also exert a pro-proliferative and tumorigenic role through mitogenic effects on the tumor endothelium, where the contribution of ligand-independent signaling has not yet been investigated [10, 32, 221].

EphB/ephrinB axis

Ligand-induced EphB2 signaling decreases GBM cell proliferation by inhibition of the MAPK pathway through R-Ras signaling, whereas the same receptor increases proliferation of malignant mesothelioma cells [139, 222]. In intestinal adenomas EphB2 drives proliferation of crypt progenitor cells through an Abl-mediated increase in cyclinD1 in the presence of low ligand concentrations [106]. During the progression to CRC, the uncoupling of this pathway from the Eph signaling instead allows continued proliferation after the loss of EphB expression [106, 131, 223].

Although the contrasting effects of the Eph signaling reported for different tumor stages can be ascribed to mechanisms of cancer cell plasticity, the same EphB receptor can also drive different responses in different cellular contexts, likely due to receptor signaling towards distinct downstream effectors. For instance, EphB4 activation results in inhibition of Ras/ERK pathway in endothelial cells through a mechanism involving p120RasGAP, while inducing the proliferation of MCF-7 breast cancer cells largely through the same pathway, via protein phosphatase PP2A [224]. These differential responses could represent a challenge for therapeutic approaches, due to conflicting effects on tumor versus host (including vasculature). Therefore, further investigation of these mechanisms, also with regard to their spatio-temporal occurrence will be useful for developing new therapeutic strategies against the evolving tumor landscapes.

Eph signaling/crosstalk in tumor-associated vascular dynamics

Eph receptors have been widely reported to exert functions in many aspects of pathological vascular remodeling, including tumor angiogenesis, the crucial process for tumor growth and metastatic dissemination. Dysregulation of Eph/ephrin signaling is commonly associated with altered tumor vascularization and cancer prognosis both in human patients and mouse models [126, 225–227]. The fundamental importance of EphB/ephrinB axis in angiogenesis is highlighted by the embryonic lethality due to defective angiogenic remodeling and mural cell functions in ephrinB2, EphB2/EphB3, or EphB4 knockout mice [99, 228–230]. EphA2 signaling deficiency instead seems to have more impact on tumor angiogenesis and other pathological vascular processes [100, 137, 231].

EphB/ephrinB axis in tumor vascularization

EphrinB2 and EphB4 have been found to regulate tumor neovascularization via both tumor cell-vascular cell interactions and receptor-independent ephrin functions in vascular cells [98, 225, 226, 232, 233]. EphB4 expressed in tumor cells can stimulate tumor angiogenesis via activating reverse signaling through ephrinB2 in tumor-associated endothelium [226]. Consistently, specific ephrinB2 knock-in experiments have established the reverse signaling through the PDZ domain of ephrinB2 as a major signaling cue regulating the angiogenic remodeling [234, 235]. However, EphrinB2 can regulate tumor growth and vascularization through VEGFR2 and VEGFR3 signaling also via receptor-independent mechanisms [235, 236]. Moreover, the roles identified for EphB/ephrinB signaling in lymphangiogenesis, vessel assembly, specification and stability hint a broader involvement in more general mechanisms of tumor neovascularization that yet remain to be mechanistically studied [234, 236].

EphA/ephrinA axis in tumor vascularization

EphA2 and ephrinA1 have been found in tumor-associated vessels in various tumor xenograft models in mice and human tumor specimens [126, 220, 237–239]. Implantation of 4T1 metastatic mammary adenocarcinoma cells in EphA2-deficient mice results in decreased microvascular density, tumor volume, and lung metastases [137]. Similarly EphA2 deficiency in MMTV-Neu transgenic mouse model results in decreased tumor microvascular density [100]. Moreover, the administration of soluble EphA2-Fc and EphA3-Fc impairs tumor neovascularization in vivo, and EphA2 silencing or inhibition leads to impaired migration, sprouting, and tube formation of cultured endothelial cells in response to soluble ephrinA1 [100, 132, 239–242]. These findings highlight the importance of endothelial EphA2 signaling in tumor neovascularization.

In RIP-Tag transgenic islet cell adenocarcinoma and in 4T1 transplantable mammary epithelial adenocarcinoma mouse models, ephrinA1 ligand is predominantly detected in tumor cells [239]. This suggests that ephrinA1-expressing tumor cells could induce angiogenesis through EphA2 forward signaling in endothelial cells [239]. However, the EphA2 receptor and ephrin ligand expression patterns are reportedly varying in different types of tumor cells and cancer associated host cells [140, 241]. Therefore, it is conceivable that endothelial EphA2 can regulate tumor angiogenesis through bidirectional signaling upon ephrinA1 interactions, and the plausible involvement and significance of ligand-independent RTK crosstalk remains of future interest [32, 126].

EphA/ephrinA axis in vasculogenic mimicry

Angiogenesis is a way to tumor growth and metastasis. However, some tumors can also metastasize without recruiting host blood vessels, via alternative mechanisms such as vasculogenic mimicry and vascular co-option [243]. Vasculogenic mimicry, first characterized in melanoma, is the de novo formation of vascular-like networks by tumor cells, a mechanism relying on cancer cell plasticity [244]. EphA2 activation is one of the earliest events found to drive vasculogenic mimicry [245, 246]. Activation of EphA2 by ephrinA1 triggers PI3K activation, up-regulation of MT1-MMP and MMP2 activation, which leads to ECM remodeling and proteolytic cleavages of laminin 5 γ2-chain, as wells as migration, invasion and induction of a vasculogenic phenotype of melanoma cells [22, 247, 248]. In this context, melanoma cells gain the expression of endothelium-associated genes, such as VE-Cadherin, EphA2 and laminin 5 γ2-chain, and form vasculogenic-like ECM [244, 248]. While this mechanism is responsible of the vasculogenic phenotype of melanoma, the same signaling pathways triggered upon EphA2 activation can occur within endothelial cells.

EphA/ephrinA axis in vascular co-option

Vascular co-option is a mechanism typical of tumors arising from highly vascularized tissues such as brain, lungs and liver, in which cancer cells migrate and receive oxygen and nutrient supply along the pre-existing host vasculature. In this context Eph receptors exert their effects by promoting adhesion of tumor cells to the host endothelium. EphA2 and EphA3 have been found to be particularly overexpressed in highly vascular GBM areas, both in tumor and endothelial cells [10, 11]. While the molecular mechanisms and determinants of vessel co-option are only recently beginning to be explored, EphA2 and EphA3 could exert a function in vessel co-option by tumor-propagating cells (TPCs), which are particularly enriched in highly vascularized GBM areas.

Eph signaling/crosstalk in stem cell dynamics

The cellular events and fate decisions within stem cell niches involve mechanisms of cell–cell and cell–microenvironment communication, making the Eph/ephrin system a plausible regulator of stem cell function. In adult organisms, stem cells are located in specialized microenvironments, or niches, and ensure tissue homeostasis while enabling tissue regeneration. Stem cell niches are defined as the combination of cellular and microenvironmental determinants orchestrating the self-renewal and differentiation of stem cell pools within specialized tissue locations. Stem cell niche maintenance and its role in determining stem cell fate are not well understood. The expression of Eph receptors and ephrin ligands during embryogenesis and tissue homeostasis/regeneration is consistent with their involvement in stem cell functions during development and in adult tissue homeostasis [249–252]. Only in the last decade, Eph receptors and ephrin ligands have been identified as important regulators of stem cell functions with reportedly variable effects depending on the organ and cellular context. However, a consensus across different reports attributes the Eph/ephrin system a spatio-temporal regulatory function in the balance between stem cell quiescence, self-renewal and differentiation.

Lessons from the intestinal stem cell niche—EphB/ephrinB axis

Intestinal stem cells (ISC) reside at the crypt bottom. As these cells differentiate, they migrate out of the crypts towards lumen, allowing daily renewal of the intestinal epithelium. Wnt/β-catenin/Tcf pathway drives EphB2, EphB3 and ephrinB1 gene expression within the intestinal crypt, generating a gene expression gradient that follows Wnt signals [159]. EphB2 expression, which is highest in the proliferating stem cells at bottom of the crypts, gradually decreases towards the lumen, where differentiated cells that lack Wnt activity express ephrinB1 in a countergradient (Fig. 5a; [159]). EphB2/ephrinB1 bidirectional signaling-mediated repulsive responses are required for cell compartmentalization along the crypt-villus axis [159, 253]. Indeed, disruption of this gradient in EphB2- and ephrinB1-null mice leads to cell-intermingling. EphB signaling also participates in the control of ISC proliferation [254]. These dual EphB functions play an important role also in colorectal tumorigenesis. Most CRCs arise from mutational activation in the Wnt pathway, which leads to a constant crypt progenitor phenotype during the early stages. As a result, intestinal adenomas express high levels of EphB2 and EphB3 that exert mitogenic effects through an Abl/cyclinD1 pathway [106, 159, 223, 254]. EphrinB-expressing cells confine the EphB-expressing adenoma cells and, through a PI3K-dependent repulsive mechanism, prevent them from entering the normal villi area [155, 159, 254].

Fig. 5.

Eph signaling in stem cell dynamics. a EphrinB–EphB signaling regulates both proliferation and migration in intestinal stem cell niche [159, 254]. Intestine stem cells (ISC) reside together with Paneth cells at the bottom of the crypt, where they divide and give rise to differentiated progenitor cells. As these cells differentiate, they migrate up the crypt and villus to sustain renewal of the intestinal epithelium. At the bottom, Paneth cells express EphA3, whereas ISC and progenitor cells express EphB2 in a gradient that decreases towards the lumen. EphrinB1 and ephrinB2 are expressed by the differentiated epithelial cells in a counter gradient [159]. EphB2/ephrinB1 bidirectional signaling mediate repulsive responses required for cell compartmentalization along the crypt-villus axis [159, 253]. In early stage colorectal cancer (adenoma), Wnt pathway enhances the expression of EphB2/EphB3, leading to epithelial evaginations and development of adenomatous polyps (right top) [159]. Along progression to colorectal cancer, the EphB expression is lost allowing spread of tumor cells into ephrinB-expressing areas as well as into surrounding stroma (right bottom) [223]. b EphrinB–EphB signaling regulates lineage plasticity of adult neural stem cell niche cells. The subventricular zone (SVZ) of the brain lateral ventricles contains a single layer of multiciliated ependymal cells lined by differentiated niche astrocytes and ventricle contacting self-renewing astrocytes. In physiological conditions, ependymal cells are involved in maintaining the SVZ stem cell niche, while self-renewing astrocytes are able to differentiate into neural stem cells (progenitors) and further to neuroblasts. Ependymal cells express EphB1/EphB2 receptors and ephrinB1/ephrinB2 ligands, while SVZ astrocytes express EphB1 and ephrinB2 [249]. EphB forward signaling downstream of Notch contributes to the maintenance of ependymal cell and astrocyte characteristics [250, 257]. Upon brain injury, the Notch–EphB signaling is disrupted by EphB downregulation, resulting in cell lineage interconversion between ependymal cells and astrocytes [249]. c In bone marrow, Ephrin–Eph signaling regulates osteoclast differentiation and osteoclast-osteoblast communication [267, 268]. Bone remodeling is sustained by a balance between new bone formation by osteoblasts and old mineralized bone resorption by osteoclasts. Within osteoclast precursors EphA4-dependent ephrinB2 reverse signaling limits, whereas ephrinA2–EphA2 signaling promotes osteoclast differentiation. Upon interaction with EphB4 expressing osteoblasts, EphB4 forward signaling mediates osteoblast differentiation and bone formation, whereas ephrinB2 reverse signaling inhibits bone resorption. d EphA2 and EphA3 signaling maintains cancer stem cell characteristics in human glioblastoma multiforme (GBM) [10, 11]. These tumors are composed of heterogeneous populations of differentiated dividing tumor cells and less differentiated tumor-propagating cells (TPCs). Ligand-independent EphA2 signaling coupled with Ser897 phosphorylation, and ligand-independent EphA3 signaling maintains TPCs in an undifferentiated state, further promoting their self-renewal and tumor-propagating abilities

In a similar manner, the expression of ephrin could for example confine stem-cell progenitors within niche, whereby a disruption of the mutually exclusive Eph/ephrin expression pattern, or a misbalance of counteracting signals, could be responsible of the proliferation and release of regenerating cells. In advanced CRC, the expression of EphB receptors is lost, providing permissive conditions for tissue invasion [223]. In this context the regulation of cyclinD1 dissociates from the EphB signaling, allowing continued proliferation (Fig. 5a). The mobilization of stem/progenitor cells towards sites of regeneration could be achieved through a similar mechanism of signaling uncoupling, thereby sustaining proliferation while conferring migratory capabilities. On the other hand, the changes in the EphB2 expression along CRC could drive spatio-temporally regulated, Eph signaling-driven, tissue regeneration. Down-regulation of Eph receptors in a cell sub-population involved in the actual regeneration process could result in cell mobilization, while the unaltered Eph expression of proliferating progenitors within niche ensures maintenance of the stem cell pool [250].

Lessons from the neural stem cell niche

EphB receptors regulate stem cell proliferation, differentiation, and segregation within the neural stem cell niche. In the adult brain, neurogenesis occurs in two restricted zones: the subgranular zone (SGZ), in the hippocampal dentate gyrus, and the subventricular zone (SVZ) of the lateral ventricles [255]. In the SVZ, a single layer of ependymal cells is lined by differentiated niche astrocytes and self-renewing astrocytes responsible of the neurogenesis (Fig. 5b; [256]). The ability of ependymal cells and astrocytes to mutually convert is suppressed by EphB and Notch signaling [250, 257]. Upon brain injury EphB expression is reduced while Notch signaling remains intact, indicating that a Notch-independent mechanism controls EphB down-regulation following injury [249]. The repression of these pathways allows niche maintenance and regeneration through cellular interconversion of niche astrocytes and ependymal cells. While EphB/ephrinB signaling regulates SVZ niche cell maintenance, EphrinB1 functions in a cell autonomous manner in the developing cerebral cortex. In this context EphrinB1 reverse signaling is required for the maintenance of neural progenitor cells by preventing their differentiation [258]. Ligand-dependent EphA signaling has instead been shown to exert a role in neural stem cell differentiation in the developing central nervous system, through positive regulation of the MAPK pathway [259].

Lessons from other stem cell compartments

Hematopoietic stem cell niche

The Eph/ephrin system has been reported to exert key functions in other stem cell compartments. Purified populations of hematopoietic stem/progenitor cells express several Ephs and ephrins. Hematopoietic stem/progenitor CD34+ cell population express EphA1, EphA2, EphB2, EphB4, ephrinA3, and ephrinA4 [260–262]. EphB4 is expressed particularly by erythroid progenitor cells during early stages of erythropoiesis [263, 264]. Complementarily, EphrinB2 expressed by bone marrow stromal cells regulates erythropoiesis via EphB4 forward signaling [263, 265]. Interaction of EphB4-expressing erythroid progenitors with ephrinB2-expressing stromal cells causes EphB4 down-regulation and accelerates erythropoiesis by inducing detachment of the erythroid progenitors from the bone marrow-derived stromal cells, followed by differentiation into mature erythrocytes [265]. Interaction of erythroid progenitors with ephrinB2-negative stromal cells impairs maturation while ectopic expression of ephrin-B2 increases adhesion to the stromal cells [265]. Thus, the EphB4/ephrinB2 system regulates progenitor cell/stromal cell adhesion, thereby contributing to stem cell function within bone marrow. Moreover, together with the EphB2/ephrinB1 axis, this system has been found to suppress the mesenchymal stem cell-mediated expansion of activated T-cells [266]. In particular, ephrinB1 reverse signaling and EphB4 forward signaling in activated T-cells can inhibit T-cell proliferation through Src, PI3K, Abl and JNK pathways, and through down-regulation of stimulatory cytokines.

The osteoblastic niche

During bone remodeling and homeostasis, Eph/ephrin bidirectional signals regulate osteoclast differentiation and osteoclast-osteoblast communication (Fig. 5c; [267, 268]). Bone remodeling is maintained by a balance between old mineralized bone resorption by osteoclasts and new bone formation by osteoblasts. EphA2/ephrinA2 signaling promotes while ephrinB2 reverse signaling inhibits osteoclast differentiation [267, 268]. Upon interaction with EphB4 expressing osteoblasts, EphB4 forward signaling mediates osteoblast differentiation and bone formation whereas ephrinB2 reverse signaling inhibits bone resorption [268].

Eph signaling/crosstalk in cancer stem cell dynamics—EphA/ephrinA axis

Eph receptors and ephrin ligands regulate both self-renewal of stem/progenitor cells and tumor progression. High-degree similarity between untransformed stem/progenitor cells and cancer cells is also acknowledged. In recent years the concept of numerous cancers harboring a “cancer stem cell” compartment, comprising up to 25 % of the cancer cells, has been described. These cells have been more recently defined as TPCs for their ability to induce tumors in animal hosts, self-renew and give rise to more differentiated cells in expanding tumor cell mass.

Recent studies on GBM have shown that tumors that harbor a large sub-population of TPCs, show increased expression of EphA2 and EphA3 (Fig. 5d; [10, 11]). These receptors regulate central nervous system development whereas their deregulated expression and somatic mutations are associated with growth, progression and metastasis of nervous system tumors [212, 269–272]. EphA2 and EphA3 overexpression in TPCs promote cell self-renewal, via the MAPK/ERK signaling pathway [10, 11]. EphrinA1-mediated EphA2 down-regulation hinders self-renewal through down-regulation of the receptor, associated with a transient increase in ERK phosphorylation and astroglial differentiation. Consistently, EphA2 knockdown depletes the stem cell pool, concomitantly with increased ERK activation [10].

The function of EphA2 on tumor cell proliferation is reportedly controversial and may involve tumor cell-specific feedback mechanisms. However, ephrinA1 expression is usually low in GBM. In this scenario, the activities of EphA2, a receptor particularly enriched in TPCs, are likely to involve ephrin-independent mechanisms and crosstalk with other signaling systems [3, 32, 101, 273, 274]. Consistently, TPCs from GBM show increased EphA2 phosphorylation at the Ser897 [10, 101]. While the level of sustained ERK activation alone might predict the decision between differentiation and proliferation, AKT phosphorylation is also a critical step towards fate decision between differentiation and proliferation [275, 276]. pERK and pAkt signaling pathways converge e.g., into the regulation of cyclinD1 stability to regulate cell-fate decisions in PC12 cells stimulated with nerve growth factor (NGF) [276].

A number of studies have also shown ERK phosphorylation kinetics and duration to be strong determinants of cell fate decisions [276]. Transient ERK activity is linked to increased proliferation, whereas its sustained activation triggers differentiation of PC12 cells [275, 277, 278]. In GBM ephrinA1-mediated signaling can occur for example upon heterotypic cell–cell contacts (i.e. endothelium or immune cells). However, in GBM core, with EphA2 overexpression and suppressed ephrinA1, sustained oncogenic signaling and reciprocal interaction with PI3K-AKT and RAS-ERK pathways, govern cellular outcomes including proliferation and differentiation [3, 32, 273, 274]. Knock-down of EphA2 and EphA3 results in sustained ERK activation in vitro and in vivo, bending cell fate towards differentiation [10, 11]. While EphA receptors can drive differentiation through other mechanisms potentially involving ligand-dependent activation, overexpression and constitutive ligand-independent activation of Eph receptor signaling drives self-renewal and attenuates differentiation.

Altogether, these findings are suggestive of more general mechanisms regulating stem cell properties through EphA receptors. Nevertheless, the co-operating oncogenic signaling pathways potentially activated in TPCs still remain to be investigated in GBM as well as in many other tumor contexts. Whether these mechanisms could sustain cancer-stem cell proliferation also in other aggressive and less-differentiated tumors, such as soft tissue sarcomas or breast and prostate cancers for which EphA2 overexpression and the presence of TPCs have been reported, will be of great interest [22, 142, 279]. Therefore, understanding the common and peculiar features of the Eph/ephrin signaling pathway and receptor crosstalk both in normal tissue homeostasis and cancer could be of great influence towards the development of new therapeutic strategies against more advanced and less differentiated tumors that may have developed resistance to conventional therapies.

Ephs as prognostic factors and therapeutic targets

Eph/ephrin signaling constitutes an important molecular mechanism of many diseases, including various cancers. Given the broad tissue expression, and pleiotropic effects in both tumor cells and the microenvironment, Eph/ephrin signaling represents a promising target for anti-cancer therapies aiming to attack tumors on several fronts. A clear challenge is represented by the dichotomous functions of Eph signaling in different tumor types and stages. Eph receptors have been identified to be either oncogenes or tumor suppressors and to attenuate or potentiate multiple other signaling pathways during carcinogenesis and tumor progression. Therefore, sufficient mechanistic understanding on Eph function still needs to be gained in order to design promising therapeutic agents. Alternatively, overexpressed Eph receptor could be used as drug targeting molecules in cancer immunotherapies. More recently, this signaling system has also been exploited and opened new drug discovery avenues in the field of regenerative medicine [280].

Differential expression of Eph receptor in tumors and normal cells supports their use not only as a therapeutic target but also as a useful biomarker. For example, EphA2 is overexpressed in prostate cancer, breast cancer, melanoma and GBM while in normal tissue counterparts it is expressed at very low levels. In addition, in light of recent findings in GBM, EphA2 serine phosphorylation could be used as a cancer biomarker to identify TPC populations. Moreover, its function in both tumor cells and host intra-tumoral vessels makes this molecule an attractive target for therapeutic intervention. Similarly, soluble EphB4 inhibits tumor growth and angiogenesis in a melanoma mouse model, making this receptor a potential therapeutic target. In addition, Eph receptor expression levels could help to identify patients that would benefit from a targeted therapy. It also remains to be investigated whether Eph protein fragments detected in biological fluids could provide an additional diagnostic marker. The emerging function of EphA2 and EphA3 receptors in cancer stemness highlighted by recent findings in GBM supports differentiation-based tumor-targeting as a therapeutic strategy [10, 11, 281]. Soluble Ephs and ephrins, ephrin mimetics, small molecule inhibitors and monoclonal antibodies have been investigated as cancer therapeutic agents [3, 282–284]. Specific antibodies against EphA2, EphA3, and EphB4 are in advanced pre-clinical or early clinical evaluation. One factor to be considered in developing oncology drugs is the assay system used to measure the efficacy of the drug and this is of particular importance since Eph signaling differentially affects cell behavior in two-dimensional versus three-dimensional microenvironment [22, 197].

Conclusions and perspectives

Metastasis, the main cause of cancer-associated mortality, relies on cancer cell dissemination by interchangeable modes of cell invasion and growth. Such cellular plasticity and stem-like properties also contribute to anticancer treatment responses and escape mechanisms, representing a major challenge in current cancer research. Multiple signaling and tumor-stroma communication pathways are known in these processes. Among them, Eph/ephrin system evidently represents the crossway for the integration of intracellular signaling networks and cytoskeletal dynamics with changes in growth factor signaling, cell–cell interactions and cues or physical confines of the ECM. Within the evolving tissue microenvironments in cancer as well as in physiological and oncogenic stem cell niches, the bidirectionality of Eph/ephrin signaling and the emerging concepts of adhesion and growth factor receptor crosstalk place the Eph/ephrin system at the integration point of pleiotropic cellular responses. Observations of Eph and ephrin interactions with extracellular, transmembrane, and intramembranous proteinases have brought further insight into the dynamic nature of these signaling systems.

Like Ephs, ephrinBs, and the interacting receptors, ADAMs and MT1-MMP are type 1 transmembrane proteins with multidomain structures that provide them with capacity to interact with multiple partners on both sides and within cell membranes. Recent studies suggest that these proteinases can modify RTK signaling through competitive or potentially synergistic extra- and intracellular interactions and pericellular receptor/co-factor cleavages. Thereby, they can form a dynamic regulatory pathway that links the varying extracellular microenvironment with intracellular signaling and cytoskeleton to control the key functions of a cell. How these proteolytic events are integrated with each other or with the complex networks of membrane-bound and intracellular signaling molecules and cell cytoskeleton is, however, poorly understood. Increasing evidence indicates that cell cytoskeleton and invasion dynamics are differentially determined in two- and three-dimensional settings via cadherin junctions, integrin-mediated ECM adhesion, and ECM degradation [201, 285, 286]. Therefore, besides mechanistic studies in isolated cells, better understanding of the context-dependent Eph/ephrin systems undoubtedly requires analyses that take into account the relevant ECM and cell interactions in three-dimensional tissue microenvironments. It is also remains to be investigated what dictates the hierarchy of the signaling or adhesion receptor interactions and coincidental proteolytic activities towards pericellular proteinase activation/inactivation cascades, or the modulation of e.g., growth factor availability and release of the ECM constrains or epitopes by the same proteases. Likewise, the fate and potential functions of cleaved extracellular and further γ-secretase released intracellular Eph and ephrin fragments remain poorly understood.

The known expression patterns and functions of Ephs and ephrins suggest promising application for these molecules as useful prognostic and diagnostic tools. Given the pleiotropic effects on both tumor cells and tumor microenvironment, the Eph/ephrin system represents a promising target for improved anti-cancer therapies aiming at attacking tumor on several fronts. Moreover, the recently emerging functions of Ephs in cancer stemness support their application in differentiation-based therapeutic strategies. However, the crosstalk mediated particularly with the molecular pathways linked to ECM adhesion and remodeling in stem cells remains poorly understood. Thus understanding functions of the Eph/ephrin axis in cell–microenvironment communication required for niche maintenance, stem cell self-renewal, cell differentiation as well as cell invasion and growth dynamics could help to design more effective anti-cancer as well as tissue regeneration therapies.

Abbreviations

ADAM

A disintegrin and metalloprotease

CRC

Colorectal cancer

CRD

Cysteine-rich domain

DDR1

Discoidin domain receptor 1

ECD

Extracellular domain

ECM

Extracellular matrix

EGF

Epidermal growth factor

EMT

Epithelial-to-mesenchymal transition

EPH

Erythropoietin-producing hepatocellular

FAK

Focal adhesion kinase

FGF

Fibroblast growth factor

FN

Fibronectin

GBM

Glioblastoma multiforme

GEF

Guanine-nucleotide exchange factor

GPI

Glycosylphosphatidylinositol

HGF/SF

Hepatocyte growth factor/scatter factor

JAK

Janus kinase

LBD

Ligand-binding domain

MAPK

Mitogen-activated protein kinase

MDCK

Madin–Darby canine kidney

MMP

Matrix metalloproteinase

MT-MMP

Membrane-type metalloproteinase

NGF

Nerve growth factor

PDGF

Platelet-derived growth factor

PDZ

Postsynaptic density protein PSD95, Drosophila disc large tumor suppressor DlgA, and zonula occludens-1 protein ZO-1

PI3K

Phosphatidylinositol 3′-kinase

PTB

Phosphotyrosine binding

PTPase

Protein tyrosine phosphatases

RTK

Receptor tyrosine kinase

SAM

Sterile α motif

SH2

Src homology 2

STAT

Signal transducer and activator of transcription

SVZ

Subventricular zone

TPC

Tumor-propagating cell

VEGF

Vascular endothelial growth factor