The Eph/Ephrin family in cancer metastasis: communication at the service of invasion

NON-THEMATIC REVIEW

The Eph/Ephrin family in cancer metastasis:communication at the service of invasion

Mustapha Kandouz

Published online: 2 May 2012# Springer Science+Business Media, LLC 2012

Abstract Cancer cells rely on intercellular communicationthroughout the different stages of their transformation andprogression into metastasis. They do so by co-opting differ-ent processes such as cell-cell junctions, growth factors,receptors, and vesicular release. Initially characterized inneuronal and vascular tissues, Ephs and Ephrins, the largestfamily of receptor tyrosine kinases, comprised of two classes(i.e., A and B types), is increasingly scrutinized by cancerresearchers. These proteins possess the particular features ofboth the receptors and ligands being membrane-bound which,via mandatory direct cell-cell interactions, undergo a bidirec-tional signal transduction initiated from both the receptor andthe ligand. Following cell–cell interactions, Ephs/Ephrins be-have as guidance molecules which trigger both repulsive andattractive signals, so as to direct the movement of cells throughtheir immediate microenvironment. They also direct processeswhich include sorting and positioning and cytoskeleton rear-rangements, thus making them perfect candidates for thecontrol of the metastatic process. In fact, the role of Ephsand Ephrins in cancer progression has been demonstrated formany of the family members and they, surprisingly, have bothtumor promoter and suppressor functions in different cellularcontexts. They are also able to coordinate between multipleprocesses including cell survival, proliferation, differentiation,adhesion, motility, and invasion. This review is an attempt to

summarize the data available on these Ephs/Ephrins’ biolog-ical functions which contribute to the onset of aggressivecancers. I will also provide an overview of the factors whichcould explain the functional differences demonstrated by Ephsand Ephrins at different stages of tumor progression andwhose elucidation is warranted for any future therapeutictargeting of this signaling pathway in cancer metastasis.

Keywords Eph . Ephrin . Receptor tyrosine kinase .

Communication . Metastasis . Invasion . Adhesion .

Tumor suppressor . Oncogene . Cancer

1 Introduction

Intercellular communication is critical for the life cycle ofany cell, either normal or transformed, although the natureof the mediators of this communication can vary from onetype and condition to another. Cells need to transmit orreceive information to or from their microenvironment inorder to respond to developmental imperatives, performspecific functions or adjust to immediate or imminentthreats. Various processes and entities are used to performthis signaling output/input activity, the outcome of whichdecides the cellular fate. Such processes, which includewithout limitation, cell-cell junctions (e.g., gap, tight, adhe-rens), growth factors and receptors, as well as released vesic-ular structures, are involved in the onset and progression ofcancer. In fact, cancer cells can divert these processes to theirown benefit by increasing their survival, proliferation, motility,invasion, and ultimately, metastasis [1–6].

A group of proteins that gained considerable attention inrecent history are the Ephs and Ephrins. The mammalianEph (Erythropoietin-producing hepatoma) family of recep-tor tyrosine kinases (RTKs) is considered the largest family

M. Kandouz (*)Department of Pathology and Bioactive Lipids Research Program,Wayne State University School of Medicine,5101 Cass Ave, Chemistry Bldg, Rm 425,Detroit, MI 48202, USAe-mail: [email protected]

M. KandouzKarmanos Cancer Institute, Wayne State University,Detroit, MI, USA

Cancer Metastasis Rev (2012) 31:353–373DOI 10.1007/s10555-012-9352-1

of RTKs and includes nine A-type Ephs (EphA1–8 andEphA10), five A-type Ephrins (Ephrins-A1–5), five B-typeEphs (EphB1–4 and EphB6), and 3 B-type Ephrins (Ephrin-B1–3). The extracellular domain of both A and B-type Ephreceptors is composed of a globular ligand-binding domain,a cysteine-rich region, and two fibronectin type III repeats;the intracellular cytoplasmic domain is formed by a juxta-membrane region, a tyrosine kinase domain, a sterile αmotif (SAM) protein–protein interaction domain, and aC-terminal PDZ-binding motif (Fig. 1). The A-type Ephrinsare tethered to the cell membrane by a glycosylphosphati-dylinositol (GPI) anchor and the B-type Ephrins possess atransmembrane domain. In addition to this difference, the

type is defined by the binding partnerships. While as ageneral rule EphAs bind to Ephrin-As and EphBs bind toEphrin-Bs, few exceptions exist where inter-type bindingcan occur.

Three fundamental and related features of this family ofproteins are important for its function: (a) both receptors andligands are membrane-bound, (b) unlike other receptorswhere the ligand released by a cell can target cells even inremote areas, Ephs and Ephrins require direct cell–cellinteractions, although there is evidence that soluble formscan exist [7, 8], and (c) both Ephs and Ephrins are able totransduce a signal to their host cells: this bidirectional signaltransduction includes an Eph-initiated forward signaling andan Ephrin-initiated reverse signaling. As a result, thesefeatures render the concept of “receptor” and “ligand” arbi-trary, for an Eph receptor can act as a ligand and vice-versa.

Following cell–cell interactions, Ephs/Ephrins trigger re-pulsive signals that make cells retract and migrate awayfrom each other, although it is known that sometimes theyalso induce cell adhesion. Thus, they are guidance moleculesthat direct the movement of cells through their immediatemicroenvironment. Based on their respective profile of ex-pression and signaling, they control sorting and segregation ofcell subpopulations in different organs and during differentphysiological processes, for which excellent reviews are avail-able. In particular, Ephs and Ephrins play important rolesduring embryonic development-related processes (i.e.,segmentation, axon guidance, neural crest cells migration,angiogenesis) [9–13].

As for all other receptor/ligand-mediated communicationprocesses, Ephs and Ephrins have been shown to playimportant yet complex roles in cancer diseases. Interestingly,they have been assigned both tumor promoter and suppressorfunctions in different cellular contexts [14–19]. As mentionedearlier, many of the functions regulated by Eph/Ephrin signal-ing are cell guidance functions directly reminiscent of themetastatic process. These include cell morphology, move-ments, cytoskeleton rearrangements, and cell positioning.Furthermore, Eph–Ephrins play a role in the metastatic pro-cess not only by affectingmigration and invasiveness of tumorcells, but also via their role in angiogenesis.

2 Role of Ephs and Ephrins in angiogenesis

2.1 Role of Ephs and Ephrins in vascular development

Ephs and Ephrins play a crucial role during the developmentof the cardiovascular system. In particular, they are involvedin the angiogenic remodeling of blood vessels, by control-ling the sorting and segregation of cells in arterial versusvenous systems [20, 21]. Ephrin-B2 has been shown to beinvolved in vascular remodeling in normal and tumor

Ephrin-B

EphB

Ephrin-A

EphA

Fibronectin type III repeats

Globular domain

C-T

erm

inus

Kinase domain

SAM domainPDZ motif

Forward Signaling

Reverse Signaling

GPI anchor

Eph

Cis

Tra

ns

Fig. 1 The Eph/Ephrin family is constituted of A and B types, whichare all membrane-bound. The extracellular domain of Ephs is com-posed of a globular ligand-binding domain, a cysteine-rich region, andtwo fibronectin type III repeats. The intracellular cytoplasmic domainis formed by a juxtamembrane region, a tyrosine kinase domain, asterile α motif (SAM) protein–protein interaction domain, and a C-terminal PDZ-binding motif. The A-type Ephrins are attached to thecell membrane by a glycosylphosphatidylinositol (GPI) anchor and theB-type Ephrins have a transmembrane domain. As a general rule,EphAs bind to Ephrin-As, and EphBs bind to Ephrin-Bs. Upon inter-action, a bi-directional signal transduction is generated involving bothEphs (forward signaling) and Ephrins (Reverse signaling). In additionto these trans interactions, cis-interactions occur, whereby interactionsbetween Ephs and Ephrins within the same cells, result in the interfer-ence with their signaling

354 Cancer Metastasis Rev (2012) 31:353–373

tissues. Ephrin-B2 and EphB4 are essential for angiogenesisin the mouse embryo [22–24].

During the angiogenic process, the proper organization ofendothelial cells necessitates the recruitment of supportingcells, also called mural cells, such as pericytes. These muralcells need Ephrin-B2 to associate with microvessels, asdemonstrated by the phenotype of mural cells-specificEphrin-B2 mutant mice, which display vascular defects inmany organs including the skin, the lung, the intestine, andkidney, characterized by extensive hemorrhage, in additionto abnormal migration of smooth muscle cells to lymphaticcapillaries [25]. These defects illustrate a role for Ephrin-B2in the maintenance of proper vascular architecture, throughensuring adequate spatial organization of the mural cellswhich cover the microvessels. In vitro, Ephrin-B2-deficientcells show poor spreading, defective nonpolarized lamelli-podia and focal adhesion formation and increased non-directional motility. Overall, these data suggest a role forEphrin-B2 in cell migration and vascular architecturethrough direct cell-cell interactions [25]. Another work sup-ports the role of EphB/Ephrin-B interactions during cell–cellcontacts between pericytes and endothelial cells in postnatalvascular development. It shows that this function involvesthe extracellular matrix and Src-dependent phosphorylationof the intracytoplasmic domain of Ephrin-Bs, resultingin localization at contact points between endothelialcells and pericytes [26]. The role of Ephrin-B2 in muralcells’ spreading, motility, lamellipodia formation, and focaladhesion formation also involves Crk-p130 (CAS) signaling[26].

Ephrin-B2 levels are elevated in angiogenic endothelialcells [27, 28], where its stimulation with soluble EphB4induces migration of endothelial cells [29]. ConstitutiveEphrin-B2 expression in human umbilical vein endothelialcells (HUVECs) triggers repeated contraction-expansionepisodes whereby cells become round and lose their lamelli-podia, before undergoing contraction and membrane bleb-bing, followed by extension of lamellae and membraneruffling [30]. These different processes seem to require differ-ent signaling mechanisms. For example, cell re-spreading butnot retraction or membrane blebbing were found to involveGrb4 and Rac while contraction is actomyosin-dependentand cell retraction and membrane blebbing involve ROCKactivation. The Ephrin-B-induced fast motility and mi-gration of HUVECs seems random [30]. In another study,Ephrin-B1 induced migration and proliferation of HUVECs[31].

2.2 Role of Ephrins and Ephs in tumor-associatedangiogenesis

In view of the role of Ephrins/Ephs in the normal vascularfunction, it is normal that many studies addressed this role in

the course of angiogenesis associated with cancer metastaticprogression. The signaling mechanisms at work are knownto some extent and are associated with processes such asmigration, cell motility, and cytoskeleton organization, mostof which are known for their critical role in the biology oftumor-associated endothelial cells.

Many data show an association between Eph/Ephrinlevels and tumor angiogenesis. For instance, Ephrin-B1level is higher in Hepatocellular carcinoma (HCC) tissuesthan in non-tumor tissues, where it significantly increasestumor growth as well as tumor vessel number in vivo [31].High expression levels of Ephrin-B2 correlate with highertumor vascularization and metastasis in malignant melano-ma [32]. In addition to these correlative studies, the gener-ation and characterization of genetic murine models hasbeen instrumental in demonstrating the role of Ephs andEphrins in angiogenesis. In a transgenic C57Bl/6/ASV-Bmurine model of HCC development, increased levels ofEphrin-B2 were found in correlation with HCC progression,in particular in tumor-associated sinusoidal endothelial cells[33]. Also, using transgenic mice where wild type anddominant negative mutant Ephrin-B2 were expressed spe-cifically in mammary epithelial cells, it was shown that notonly the manipulation affected the epithelial cells’ differen-tiation, but it also affected mammary gland vascularization[34]. The authors reported that while wild type Ephrin-B2induced superfluous but organized capillaries, mutantEphrin-B2 overexpression resulted in irregularities in thevasculature (i.e., blind-ending capillaries). Furthermore,crossing mutant Ephrin-B2-expressing transgenic animalswith a NeuT transgenic animal, a model of mammary trans-formation, resulted in a metastatic phenotype [34]. Mammarytumor cells-endothelial cells’ interaction also involves EphB4and Ephrin-B2, respectively. When EphB4 is devoid of itskinase domain, tumors were found to have higher bloodcontent and increased size of blood vessels than control wildtype EphB4-expressing tumors. The extracellular ligand-binding domain of EphB4 has chemotactic properties andstimulates endothelial cells’ invasion, survival, and prolifera-tion [29]. Taken together, these results suggest that bothreverse and forward signaling are important in the mainte-nance of an EphB/Ephrin-B balance that keeps angiogenesisand metastasis in check in the mammary gland. However,there is evidence that Ephrins might affect endothelial cellmigration and angiogenesis in a receptor-independent manner.Ephrin-B2 is able to modulate endothelial cells’ motilityand cellular morphology independently from Eph binding,by stimulating episodes of cycling between actomyosin-dependent cell contraction and spreading [30]. In addition tothe B-type Ephrins and Ephs, the A-type counterparts areequally important in tumor-associated angiogenesis. Ephrin-A1 is overexpressed in vascularized tumors and induces en-dothelial cell sprouting and migration, as shown in mammary

Cancer Metastasis Rev (2012) 31:353–373 355

cells. This function is related to the metastatic process andtumor-induced angiogenesis via increasing endothelial cells’migration and vascular density [35] and necessitates a bidi-rectional signaling through binding with A-type Ephs. Somedata suggest that co-expression of Ephrin-A1 and cognatereceptor EphA2 is involved in colorectal tumor-induced neo-vascularization, although it was noted that this role seems tooccur in the early stages (smaller tumors less than 5 cm instages I and II) rather than in the late stages [36]. The role ofEphA2 in angiogenesis has been shown both in vitro and invivo. Targeted disruption of EphA2 results in impaired vascu-lar assembly in response to Ephrin-A1 stimulation. EphA2-deficient endothelial cells fail to undergo vascular assemblyand migration in response to Ephrin-A1. Murine pulmonarymicrovascular endothelial cells derived from EphA2-deficientanimals were significantly impaired in their ability to assem-ble into capillary-like structures (vascular assembly) in re-sponse to Ephrin-A1. The role of EphA2 in endothelial cellassembly and migration involves phosphoinositide (PI)3-kinase-mediated activation of Rac1 GTPase. In vitro, endo-thelial cells from EphA2-deficient mice show a decreasedability to respond to the pro-angiogenic effect of Ephrin-A1.In vivo, their vascular assembly is impaired when transplantedinto recipient mice [37]. Using a transplantation model where-by 4T1 metastatic mammary adenocarcinoma cells weretransplanted subcutaneously and orthotopically into EphA2-deficient female mice, it was shown that host microenviron-ment EphA2 function is required for tumor angiogenesis andmetastatic progression [38]. The EphA angiogenic role isdependent on activation of a pathway that involves the phos-phatidylinositol 3-kinase, the Vav2 and Vav3 as guanine nu-cleotide exchange factors (GEFs) and the Rho family GTPaseRac1. According to this model, Ephrin-A1 stimulation recruitsthe Vav proteins to the activated EphA2 receptor. The EphA/Vav association therefore modulates the activity of Vav GEFs,leading to activation of Rac1 GTPase, thus affecting the actincytoskeleton and lamellipodia and filopodia formation, andultimately resulting in endothelial cell migration [39]. In sup-port of this model, the same study results also suggested thatthe kinase function of EphA2 is important in tumor angiogen-esis and, using phosphorylated tyrosines mapping and muta-tional analysis and knock in of EphA2-null endothelial cells,the authors showed that mutations that uncouple EphA2 withVav guanine nucleotide exchange factors or p85 are defectivein Rac1 activation and cell migration. Furthermore, EphA2mutations in tyrosine residues in the juxtamembrane re-gion, kinase domain, or SAM domain inhibited Ephrin-A1-induced vascular assembly and prevented incorporation ofmutant EphA2-reconstituted endothelial cells into the tumorvasculature [40].

In other respect, Ephs/Ephrins are likely to work inconcert with other factors involved in angiogenesis, includ-ing the other major families of RTK ligands, namely

vascular endothelial cell growth factors (VEGFs), angio-poietins, and other growth factors and signaling entities.However, most of the data available are obtained usingendothelial cells and the relevance of these findings in atumorigenic context is not known. Ephrin-A1 is stronglyexpressed at angiogenic sites, presumably following induc-tion by proangiogenic factors. It strongly impairs cellspreading in rat vascular smooth muscle cells (VSMCs). Arole for the Rac/PAK pathway in this process has beensuggested [41]. Ephrin-A1 and the lipid mediatorSphingosine-1-phosphate (S1P) seem to synergize in theblood vessel destabilization necessary for angiogenesis[41]. EphA/Ephrin-A signaling is necessary for a maximalpro-angiogenic effect of VEGF. VEGF induces Ephrin-A1expression, which activates EphA2, resulting in the induc-tion of cell survival, migration, sprouting, and corneal an-giogenesis. This effect was blocked by a soluble, forwardsignaling-devoid, EphA2-Fc receptor or by EphA2 anti-sense oligonucleotides [42]. The Eph/Ephrins’ signalingpathways are tightly connected to the VEGF functions. InHUVECS, VEGF induces Ephrin-B2 and decreases EphB4levels via a signaling pathway that involves Notch familyproteins DLL4 and Notch4, thus affecting endothelial cel-lular migration and differentiation [33]. Ephrin-B2 promotesendothelial cells’ sprouting and motility during angiogene-sis, via the VEGF/Rac1/Akt/Erk signaling axis. Ephrin-B2deficiency either in vitro or in vivo, compromises theinternalization of VEGFR3, the VEGF-C receptor, andthe subsequent pro-angiogenic signaling [43]. Ephrin-B2controls VEGFR2 endocytic internalization, activation,and downstream signaling, thus directing VEGF-inducedfilopodial extension of tip cells, which are specialized endo-thelial cells at the tips of the vascular sprouts [44]. Also, it wasfound that VEGF levels were increased by EphrinA/EphAsignaling which could provide an indication of the mecha-nisms at work [35]. It seems that the EphA2/Ephrin-A1 inter-action is necessary for endothelial cell survival, migration, andsprouting and angiogenesis induced by VEGF [42]. EphB2and EphB4 have also been reported to cooperate withSDF-1, the ligand of the G-protein-coupled receptorCXCR4. SDF-1 promotes endothelial cell clustering andcell–cell contacts, which favors signaling through EphB2and EphB4 and results in endothelial cell movementand alignment into cord-like structures [45]. Likewise,Ephrin-A1 cooperates with Slit2, a protein involved in cy-toskeletal remodeling and migration, to regulate angio-genesis. Actually, Ephrin-A1 opposes the pro-angiogenicfunction of Slit2, by inhibiting Slit2-mediated mTORC2-dependent activation of Akt and Rac GTPase [46]. Finally,with relevance to tumor angiogenesis, hypoxia, a regulator ofexpression of proangiogenic factors, also regulates theexpression of Ephs and Ephrins in a HIF-1α-dependentmanner [47].


3 Eph/ephrin signaling in cancer Cells’ migration,invasion, adhesion, and metastasis

The levels of Ephs and Ephrins have been assessed invarious metastatic tissues (Table 1). As an example, elevatedlevels of Ephrin-B2 are correlated with metastatic progres-sion of oesophageal squamous carcinomas [48]. High ex-pression of EphA4 and low expression of EphB2 arecorrelated with colorectal cancer metastases to the liver[49]. EphB3 in NSCLC cell lines induces cell growth andmigration and promotes tumorigenicity and metastasis invivo in a kinase-independent manner [50]. Some studiessuggest that EphB2 mutations may have an essential rolein cell migration and progression and metastasis of prostatecancer [51]. The activation of EphB2 receptor inhibitedcolon cancer cell growth, adhesion, and migration [52].EphA2 is overexpressed in pancreatic cancer. Although clin-ical data suggest loss of EphA2 in co-existent pancreaticcancer metastases, it still has a potential role in organ specificmetastasis [53]. By contrast to these data suggesting a mainlypro-metastatic role, decreased levels of EphB6 are associatedwith an increased risk of metastases development in earlystage non-small cell lung cancer (NSCLC) [54]. Restorationof EphB6 expression increased adhesion and decreased mi-gration and entirely abolished metastasis formation in vivo[55]. In addition to angiogenesis, many biological processesdetermine cancer cells’ metastatic potential, including migra-tion, invasion, adhesion, intravasation, and extravasation. Al-though many aspects of the functions and mechanisms ofaction of some Ephs and Ephrins have been investigatedrelatively well, the large number of family members andmultiplicity of combinations of binding partnerships rendersit difficult to draw a comprehensive functional map. Forinstance, it is yet to determine whether or not different inter-action partners belonging to the same type (A or B), result insimilar signaling pathways, migratory and invasive functions.

Among the most investigated, the EphA2/Ephrin-A1 in-teraction illustrates the role of this family in the coordinationof cancer cells’ motility and invasiveness (Fig. 2). UponEphrin-A1 stimulation, EphA2 forms a complex with Srcand focal-adhesion kinase (FAK), whereby FAK is phos-phorylated at Tyr-576/577, which is followed by activationof a Rho-dependent actin and myosin contraction and cellrepulsion [56]. Ephrin-A1 induces the activation of the Abltyrosine kinase, resulting in the phosphorylation of the CrkIIadaptor and the disruption of its interaction with C3G, aRap1 guanine nucleotide exchange factor. The ensuing de-crease in the Rap1-GTP explains the Ephrin-A1-inducedcell rounding and loss of adhesion [57]. The IGF-1/IGF-1R signaling stimulates EphA3 expression in Jurkat cells.This increases the response to Ephrin-A5 and loss of celladhesion to fibronectin, an effect that seems to involve aninteraction of EphA3 with the adapter protein CrkII [58].

Ephrin-A5-stimulated EphA3 triggers cell rounding, bleb-bing, and detachment due to a CrkII/Rho-dependent actin/myosin cytoskeleton reorganization and cellular protru-sions’ retraction. EphA3 tyrosine kinase activity is neces-sary for this function [59]. Ephrin-A5 has been shown to bepro-invasive, also enhancing anchorage-independent growthand morphological transformation [60]. Ephrin-B3 expres-sion is increased in migrating and invading glioma cells andclinical specimens and its overexpression and subsequenttyrosine phosphorylation increased cell migration and inva-sion. Ephrin-B3’s loss resulted in less response to EphB2/Fc-mediated migration and invasion. These effects ofEphrin-B3 seem to involve the recruitment and activationof Rac1 [61]. Ephrin-B2 plays a role in migration andinvasion of glioma cells [62]. Ephrin-B2-mediated activa-tion of EphB4 enhances the migratory ability of melanomacells and increases RhoA activity, while the inhibition ofEphB4 forward signaling by overexpressing a kinase deadmutant inhibits cell migration, affects the organization ofactin cytoskeleton and decreases RhoA activity [63]. Incontrast to this pro-invasive function, in breast cancer cellsEphrin-B2-stimulated EphB4 activates a pathway thatinvolves Abl family tyrosine kinases and the Crk adapterprotein, resulting in the inhibition of not only viability andproliferation but equally motility and invasion [15]. Simi-larly, EphA3 overexpression or its stimulation by Ephrin-A5inhibits rhabdomyosarcoma cell adhesion and migration, viaRhoGTPases activity [64]. Ephrin-B1 stimulated by EphB3,regulates leukemic cells’ migration and metastasis. Its phos-phorylation and relocalization to lipid rafts along with thecytoskeletal regulators Rac1 and CrkL, seems to be depen-dent upon the kinase Lck [65]. The role of lipid rafts in Eph/Ephrins invasive properties has also been suggested by thefinding that Ephrin-B2 is brought to close proximity withthe β1 integrin within the so-called Lubrol-RAFTs. Thisinteraction seems to be behind the oncogenic function ofEphrin-B2 in melanoma cells [66].

3.1 Connections between Ephs/Ephrins, integrins,and cytoskeleton remodeling

Eph/Ephrin signaling is closely connected to integrin func-tions. It is involved in integrin clustering and the productionof the extracellular matrix [67]. EphA1 activation inhibitscell spreading and motility through a pathway that involvesRhoA/ROCK and the integrin-linked kinase (ILK). TheEphA1/ILK interaction, although independent of the EphA1kinase activation, involves Ephrin-A1 ligand stimulation[68]. There are many data in normal physiological condi-tions linking Ephs and Ephrins to integrins and adhesion.The study of the neural processes such as axon guidance hasprovided essential insight into these downstream processes.However, their relevance to cancer and metastasis can only


Tab

le1

Sum

maryof

stud

ieswith

data

correlatingEph

andEph

rinlevelsandprog

nostic

parameters

Eph

/Eph

rin

Tissue

Metho

dExp

ression

Clin

ical

sign

ificance

Reference

Eph

rin-A1

Eph

A2

Vertical

grow

thph

ase

cutaneou

smelanom

asIH

CStron

gexpression

ofEph

rin-A1

andEph

A2was

associated

with

increasedmelanom

athickn

ess

Eph

rin-A1staining

isrelated

todecreasedpatient

survival

[166

]

Eph

A2

Pancreatic

cancer

IHC

Primaryandmetastatic

carcinom

asmorestrong

lypo

sitiv

ecomparedto

benign

ductsandPanIN

lesion

s.Poo

rlydifferentiatedcarcinom

asmore

strong

lypo

sitiv

ethan

wellandmod

erately

differentiatedtumors.Adv

ancedcarcinom

asshow

edrelativ

elyless

strong

positiv

elabelin

gthan

prim

arycarcinom

as.Decreased

expression

morecommon

lyfoun

din

liver,

lung

,or

peritoneal

metastasesas

compared

todistantlymph

node

metastases.

Potentialrole

forEPHA2in

organspecific

metastasis

[53]

Eph

A4

Melanom

asNorthernblot

Decreased

orlostin

advanced

melanom

as–

[167

]

Eph

B1

Gastric

carcinom

aRealtim

eRT-PCR/IHC

Eph

B1transcript

overexpressedin

68.9

%andun

derexp

ressed

in14

.8%

ofcases.

Exp

ressionof

proteingreatly

different

from

thetranscript

expression

,with

overexpression

andun

derexp

ression

being17

.2%

and44

.8%,respectiv

ely.

Eph

B1proteinun

derexp

ression

issign

ificantly

associated

with

invasion

,stageandmetastasis

[168

]

Eph

A4Eph

B2

Colorectalcancer

Realtim

eRT-PCR

HighEph

A4andlow

Eph

B2correlatewith

liver

metastasis.Nocorrelationbetween

Eph

A4andEph

B2expression

Overexp

ressionof

Eph

A4andredu

ced

expression

ofEph

B2may

beauseful

predictorof

liver

metastasis

[49]

Eph

B2

Colorectalcancer

IHC

Eph

B2expressedin

100%

ofno

rmal

coloncryp

tbase

cells,78

%of

adenom

as,

55.4

%of

prim

aryCRCs,37

.8%

oflymph

node

metastases,and32

.9%

ofliv

ermetastases

Patientswith

CRCsthat

lose

Eph

B2

expression

hadmoreadvanced

tumou

rstage,po

ordifferentiatio

n,po

orov

erall

survival

anddisease-free

survival,with

thelatterbeingindepend

entof

tumou

rstage.

[52]

Eph

B2

Colorectalcancer

IHC/Insitu

hybridization

Eph

B2expression

was

show

nat

allstages

ofcolorectal

tumorigenesis

Highlevelsof

Eph

B2expression

areassociated

with

along

ermean

duratio

nof

survival

incolorectal

cancer

[169

]

Eph

B3

Colorectalcancer

IHC

Eph

B3expression

was

sign

ificantly

redu

cedin

advanced

Duk

es’

stagetumor

specim

ens

–[170

]

Eph

B3

Non

-small-celllung

cancer

(NSCLC)

Realtim

eRT-PCR

Upregulated

expression

Exp

ressionlevelcorrelated

with

thepatient

patholog

iccharacteristics,

includ

ingtumor

size,differentiatio

n,andmetastasis.

[50]

Eph

B4

Headandneck

squamou

scellcarcinom

a(H

NSCC)

Quantitativ

ePCR

Eph

B4am

plification

–[171

]

Eph

B6

Non

-smallcelllung

cancer

(NSCLC)

Realtim

eRT-PCR

EPHB6redu

cedin

tumorscompared

with

matched

norm

allung

tissue,

correlated

with

hyperm

ethy

latio

n.

Decreased

Eph

B6associated

with

anincreasedrisk

formetastasis

developm

ent.

[55]


Tab

le1

(con

tinued)

Eph

/Eph

rin

Tissue

Metho

dExp

ression

Clin

ical

sign

ificance

Reference

Eph

B6

Non

-smallcelllung

cancer

(NSCLC)

Realtim

eRT-PCR

EPHB6high

insquamou

scellcarcinom

asAssociatio

nwith

redu

cedrisk

ofmetastasiswhenexpressedin

the

prim

arytumor

(I/IIdisease).Bettersurvival

forpatientswith

tumorsexpressing

Eph

B6.

[172

]

Eph

B6

Melanocytic

tumors

Realtim

eRT-PCR/IHC

Reductio

nof

Eph

B6with

melanom

aprog

ressionto

metastatic

disease

–[173

]

Eph

B6Eph

rin-B2

Eph

rin-B3

Neuroblastoma

RT-PCR

High-levelEph

B6,

Eph

rin-B2,

and

Eph

rin-B3isassociated

with

low-

stageNB(stages1,

2,and4S

)

High-levelexpression

ofEph

B6,

Eph

rin-B2,

andEph

rin-B3predictedfavo

rable

NBou

tcom

e.

[174

]

Eph

rin-B2Eph

B4

Uterine

cervical

cancers

Realtim

eRT-PCR/IHC

Eph

B4andEph

rin-B2sign

ificantly

increasedwith

clinical

stages

(I<II<III+IV

)

Poo

r36

-mon

thsurvival

ratesof

patients

with

high

Eph

B4andEph

rin-B2

(31%

and19

%,respectiv

ely)

[175

]

Eph

rin-B2Eph

B4

Ovarian

cancers

Realtim

eRT-PCR/IHC

Eph

B4andephrinB2sign

ificantly

increasedwith

clinical

stages

(I<II<III<IV

)

Poo

r24

-mon

thsurvival

ratesof

patients

with

high

Eph

B4andEph

rin-B2

(25%

and27

%,respectiv

ely)

[176

]

Eph

A1

Gastric

carcinom

aRealtim

eRT-PCR/IHC

Dow

nregulationof

Eph

A1in

34%,

upregu

latio

nin

25%,andno

difference

in41

%.

The

survival

analysisshow

edthat

patients

who

setumor

exhibitedEph

A1up

regu

latio

nhadapo

orer

outcom

ethan

thosewho

setumor

exhibiteddo

wnregulation(P

00.00

5)or

nodifference

inexpression

(P00.00

3).

[177

]

Eph

A1Eph

rin

A1.

Eph

A2

Eph

rin-A5.

Ovarian

cancer

real

timeRT-PCR

Overexp

ressionof

Eph

A1correlated

with

Eph

rinA1.

Amoremod

est

overexpression

andcorrelation

ofEph

A2with

Eph

rin-A1.

Astriking

correlationof

both

ephrin

A1and

ephrin

A5expression

with

poor

survival

[178

]

Eph

A7

Glio

blastoma

multiforme

IHC

Increasedexpression

ofEph

A7in

in43

.7%

ofGBM

samples

Overexp

ressionof

Eph

A7proteinwas

predictiv

eof

theadverseou

tcom

ein

GBM

patients,independ

entof

intratum

ormicrovascular

density

(MVD)

expression

.Highdensity

ofMVD

aswellas

high

erEph

A7expression

predictedthediseaseou

tcom

emore

accurately

than

Eph

A7variable

alon

e

[179

]

Eph

A2Eph

rinA

-1Vulvarsquamou

scellcarcinom

asIH

CHighexpression

ofEph

A2(51%)and

Eph

rinA

-1(56%)in

vulvar

carcinom

as.

Highlevelsof

Eph

A2andEph

rinA

-1associated

with

deep

tumou

rinvasion

andhigh

FIG

Ostagestage.

Inun

ivariate

analysis,high

expression

ofEph

rinA

-1associated

with

poor

survival.In

multiv

ariate

analysisneith

erEph

rinA

-1no

rEph

A2sign

ificantly

correlated

tosurvival.

[180

]

Eph

A2

End

ometrioid

endo

metrial

adenocarcino

mas

IHC

Eph

A2ov

erexpression

in47

%of

tumorsissign

ificantly

associated

with

high

VEGFexpression

and

high

MVD

coun

ts

HighEph

A2expression

,high

VEGF

expression

andhigh

MVD

coun

tssign

ificantly

associated

with

shorter

disease-specific

survival.

[181

]

Eph

A2

End

ometrioid

endo

metrial

carcinom

as(EEC)

IHC

Highexpression

ofEph

A2in

48%

ofEECsamples

versus

10%

ofbenign

samples.

Eph

A2ov

erexpression

associated

with

aggressive

phenotyp

e(highdiseasestage,

high

tumor

grade,increaseddepthof

myo

metrial

invasion

)in

EECand

[182

]


be inferred from the nature of the mechanisms involved,while awaiting further investigation. The activation ofEphrin-A2 or Ephrin-A5 by EphA3, results in a β1-integrin-dependent increased adhesion of Ephrin-A-expressing cells to laminin. This effect correlates with thetyrosine phosphorylation of a protein with a molecular massof 120 kDa by Src, whose identity has not been elucidated[69]. The role of Src signaling as a mediator of the effect ofEphs/Ephrins on integrins is also illustrated by anotherstudy that shows that Ephrin-A5 activation affects shortterm cell adhesion and long term morphological changes,dependently on the activation of β1 integrin, using both Srcfamily members-dependent and -independent pathways.Both pathways lead to prolonged activation of ERK1/ERK2 and change of morphology and the Src-independentpathway has been hypothesized to involve FAK [70]. This isactually possible; EphA2 has been shown to be constitutive-ly associated with FAK in resting cells. FAK is a tyrosinekinase and an important integrin intracellular signaling me-diator. Upon Ephrin-A1-mediated activation, the EphA2kinase recruits the protein tyrosine phosphatase SHP2, lead-ing to the dephosphorylation of FAK and paxillin, anddissociation of the FAK-EphA2 complex. The end result isthe induction of an inactive conformation of integrins andinhibition of cell spreading, migration and integrin-mediatedadhesion [71]. EphA2 possesses pro-invasive functions inpancreatic adenocarcinoma cells, by inducing a FAK-dependent increase in MMP-2 expression. This activity iscounteracted by ligand binding [72]. Ligand-activatedEphB1 induces tyrosine phosphorylation of the focal adhe-sion protein paxillin and is recruited to paxillin-FAK com-plexes to induce cell migration in a c-Src-dependent manner[73]. The role of Ephrin-A1 in integrin-mediated adhesioninvolves the regulation of the redox signaling, via inhibitionof Rac1. This change in redox status affects the activity ofthe tyrosine phosphatase LMW-PTP, whose function indephosphorylation of p190RhoGAP, results in RhoA acti-vation, inhibition of cell adhesion, and spreading in prostaticcarcinoma cells [74]. EphA8, a lesser known A-type Eph,when overexpressed in either NIH3T3 or HEK293 cellsforms a stable complex with and localizes p110gamma PI3-kinase to the plasma membrane in a tyrosine kinase-independent manner. This interaction seems to involve thejuxtamembrane domain of EphA8 and enables a signal thatwould ultimately result in enhanced cell adhesion to fibro-nectin via α5β1- or β3 integrins [75]. The connectionbetween integrins and the B-type Ephs/Ephrins in cancerundoubtedly deserves more attention. It has been proposedthat EphB1 functions as a “ligand density sensor,” wherebyit responds to an Ephrin-B1 gradient by attaching to theextracellular matrix via the α5β1 integrin [76]. EphA4/Ephrin-B2 binding, one of the few inter-type interactions,induces signal transduction via suppression of Cdc42, whichT

able

1(con

tinued)

Eph

/Eph

rin

Tissue

Metho

dExp

ression

Clin

ical

sign

ificance

Reference

associated

inverselywith

expression

ofestrog

enreceptor

(ER)andprog

esterone

receptor

(PR),

Eph

A2

Esoph

agealsquamou

scell

carcinom

a(ESCC)

IHC

Eph

A2ov

erexpression

50%.

Correlatio

nbetweenEph

A2expression

andregion

allymph

node

metastasis,

numberof

lymph

node

metastases,

poor

degree

oftumor

differentiatio

nandpo

orer

survival

rates.

[183

]

Eph

rin-B2Eph

B4

Uterine

endo

metrial

cancers

IHCandreal-tim

eRT-PCR

Overexp

ressionof

Eph

rinB

2andEph

B4

with

clinical

stages

(I<II<III),

dedifferentiatio

n(G

(1)<G(2)<G(3))

andmyo

metrial

invasion

(A<B<C)

Poo

rer60

-mon

thsurvival

ratesin

patients

with

high

Eph

rin-B2(59%)andEph

B4

(62%)than

inpatientswith

low

expression

levels.

[184

]


leads to integrin activation and fibronectin assembly [77].EphA4 interacts with and leads to tyrosine phosphorylationof the guanine nucleotide exchange factor (GEF) Ephexin1,thus activating Rho GTPase RhoA, and modulating thecytoskeleton, axon repulsion, and growth cone collapse[78]. From these data, appears that Rho GTPases play animportant role in Eph/Ephrin-mediated adhesion, migrationand invasion (Fig. 2). Furthermore, many of the effectsdescribed so far are mediated by remodeling of the actincytoskeleton and the plasma membrane.

3.2 Ephs/Ephrins and other intercellular communicationsin epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) is a process byvirtue of which epithelial cells are converted into motileand invasive mesenchymal type cells. The role of EMT incancer progression is the subject of intense investigation[79, 80], but the role of the inverse process, mesenchymal-epithelial transition (MET) is not very well characterized. Itinvolves an active sequence of disruption of intercellularadhesion structures, which facilitates cell motility. Intercel-lular communications via tight, gap and adherens junctionsestablish restrictions on the movement of differentiated nor-mal epithelial cells, which are alleviated during cancer pro-gression. Judging from the available information, Ephs andEphrins have an important role in many aspects of thesestructural–functional changes (Fig. 3). Ephrin-B1 signaling

results in the loss of tight junctions, an effect that involves acompetition between Ephrin-B1 and the small GTPase

Ephrin

Eph

ITG

ITG

FG

FR

1

EG

FR

ErbB

2

Fig. 2 A tentative map of major known signaling pathways recruitedby Ephrins and Ephs during different aspects of the metastatic pathway.Eph/Ephrin interactions-mediated signaling via Src/FAK, Ras/MAPK,and PI3K/Akt and the Rho family GTPases, is emphasized. In somecontexts, such as following the interaction with other RTKs, thesesignaling pathways might be recruited in a ligand-independent manner.N.B.: Precise subcellular localization is not respected in this depiction.

Also, this is a compilation of different pathways which have beenidentified in different contexts. It is not known whether these featuresare shared by all different cell types and all Ephs and Ephrins. Finally,although only the forward signaling is illustrated, the data availableand discussed in the text indicate that the reverse signaling implicatessimilar pathways

EphEphrin

Fig. 3 Schematic representation summarizing some known connec-tions between Eph/Ephrin signaling, cell–cell junction components,and the extracellular matrix (ECM). Ephs and Ephrins interact withdifferent membrane-bound proteins such as E-Cadherin, Claudins, andintegrins, thus controlling the formation of cell-cell junctions and thecell adhesion to the extracellular matrix (ECM). They also modulatethe remodeling of this ECM through regulating the release of metal-loproteases. These processes contribute to the function of Ephs andEphrins in cell adhesion and migration


Cdc42 for the association with the scaffold protein Par-6polarity complex protein [81]. Dynamic expression changesin E-cadherin, an epithelial marker, seem to affect EphA2activation and function. While in benign epithelia, E-cadherin keeps EphA2 in cell–cell contact sites, the latter’slocalization is changed to membrane ruffles in E-cadherin-negative breast cancers, only to become activated again inmetastatic tissues where E-cadherin expression is restored[82]. Another study also showed that E-cadherin maintainsEphA2 receptor localization at cell–cell contact sites inepithelial cells and its absence leads to EphA2 localizationto the perinuclear region [83]. The intestinal epithelium is aninteresting example of how multiple processes (i.e., cell-to-cell adhesion, migration and positioning) are coordinated byEphB/EphrinBs [13]. EphBs are transcriptionally regulatedby β-catenin and Tcf4 in the intestinal epithelium. In normaltissues, they control the positioning of cell types along thecrypt-villus axis and restrict intermingling of the prolifera-tive and differentiated cell populations, by using the repul-sion exerted on EphB-positive cells by cells in Ephrin-B-negative areas [84]. EphB’s tumor suppressor function in theintestine is related to its role in the compartmentalization ofthe expansion of colorectal cancer cells through an E-cadherin-mediated mechanism [14]. Eph/ephrin signallingcontrols the compartmentalization of cells in intestinal epi-thelial tissues by regulating the formation of E-cadherin-based adhesions. EphB receptors interact with E-cadherinand with the metalloproteinase ADAM10 at sites of cell–celladhesion. Their activation upon interaction with Ephrin-B1-expressing cells induces shedding of E-cadherin by AD-AM10 and the perturbation of its localization [85]. EphB3promotes MET by inactivating both CrkL and Rac1, re-distributing E-cadherin and β-catenin, and re-establishingepithelial cell–cell junctions, which could contribute to itstumor suppression function [86]. Similarly, there is evidencethat EphB4/Ephrin-B2 and EphA4/Ephrin-A interactionsregulate MET in normal tissue development [84, 87]. E-Cadherin being the major factor in EMT and MET, the linkof Eph/Ephrin signaling with other cadherins is virtually notknown. Highly aggressive melanoma cells have the abilityto form structures that mimic vascular networks. During thisprocess termed vasculogenic mimicry (VM), cells expressendothelium-specific genes such as VE-cadherin. VE-cadherin interacts with and regulates the expression ofEphA2 at the cell membrane by mediating its ability tobecome phosphorylated through interactions with Ephrin-A1. VE-cadherin also modulates EphA2 localization to cell–cell adhesion junctions [88]. Ephrin-B1 affects cell–celladhesion by an Eph receptor-independent mechanism thatinvolves the creation of a complex with Claudin-1 orClaudin-4, two tight junction molecules, via the extracellu-lar domains of these proteins, leading to tyrosine phosphor-ylation of the cytoplasmic domain of Ephrin-B1 [89].

EphA2 associates with and modulates the localization ofClaudin-4, a constituent of tight junctions. Subsequent phos-phorylation of the cytoplasmic carboxyl terminus ofClaudin-4 attenuates its association with ZO-1 and enhancesparacellular permeability [90]. As will be further discussedin the next paragraph, the effects of Ephs and Ephrins oncell–cell interactions could differ when they are involved ina receptor/ligand complex than when they are acting sepa-rately. For instance, EphB2, acting as a ligand, inducesexpression of markers of epidermal differentiation (i.e.,keratins KRT1 and KRT10, SPRRs, desmosomal proteins)and inhibits basal layer markers and integrins [91]. Al-though little information is at our disposal, another type ofjunctions involving Ephs and Ephrins is the gap junction[92, 93]. It is not clear whether and how this connectioncontributes to the role of gap junctions to cancer progressionand vice-versa.

3.3 Ephs/Ephrins and the stem cells’ genesis of metastases

The role of stem/progenitor cells in cancer metastasis is anactive field of research [94, 95]. In light of this concept,Ephs and Ephrins have been shown to regulate the fate ofstem cells, suggesting a possible mode of action of Eph/Ephrin signaling in the pre-metastatic cells. EphB4 forwardsignaling, independently from Ephrin-B2 reverse signalingleads to tumor metastasis in a model of NeuT mouse mam-mary tumors. It appears that this role occurs in stem/pro-genitor cells even before the initiation of tumor formation[96]. Deregulated Ephrin-B2 expression interferes with theregulation of the stem cell niche and leads to a shift in thedifferentiation pathway [97]. EphB/Ephrin-B signaling con-trols the positioning of cells within the stem cell niche in theintestinal epithelium [98]. High EphB2 expression is part ofa gene signature specific for adult intestinal stem cells thatpredicts disease relapse in colorectal cancer patients [99].

4 Determinants of dual functions of Ephs and Ephrins

There is increasing evidence that Ephs and Ephrins playdifferent functions at different stages of tumor progressionand different related biological processes such as cell pro-liferation, survival, and migration. Also, the same moleculesmight show opposite roles, tumor promoter in one contextand tumor suppressor in another. Many examples of suchdual functions are available [100, 101]. This is unlike clas-sical RTKs, which are traditionally viewed as potential oreffective oncogenes. They are involved in cell survival andproliferation as well as in cell death or differentiation. Inter-estingly, the role of Ephs/Ephrins in the metastatic processcan be dissociated from their role in migration, invasion oradhesion. EphB4 has been reported to promote metastatic


dissemination [102]. Echoing this finding, a study showedthat metastasis but not migration of EphB4-expressing tu-mor cells or their adhesion to Ephrin-B2-expressing endo-thelial cells, requires the forward signaling. Indeed, bothcells transfected with a truncated EphB4 variant which lacksthe cytoplasmic catalytic domain (DeltaC-EphB4) and thewild type-transfected counterparts seem to adhere preferen-tially to Ephrin-B2-expressing endothelial cells. However,the DeltaC-EphB4-transfected cells showed much less meta-static dissemination to the lungs, the liver, and the kidneys[103]. EphB receptors control both cell migration and pro-liferation in the intestinal stem cell niche [98]. Although thelarger picture of the dual functions of Ephs and Ephrins andtheir coordination, as illustrated above, is just beginning touncover, some features constitute possible explanatoryelements.

4.1 A large family, a large picture

The Eph and Ephrin family is a large one. This providesfunctional flexibility if viewed from the redundancy aspect.However, although there is no comprehensive overview ofthe differences in the signaling pathways they involve, theavailable data clearly show that these family members aresometimes capable of fundamentally different functions. Ifwe assume then that not all Ephs and Ephrins are function-ally equal and that they actually might sometimes haveopposite roles, an important question is whether and howthe expression of different combinations of Ephs and Ephrinsis coordinated to ensure a proper function in processes such asproliferation, survival, migration, invasion, and tissue pattern-ing. It is possible that specific receptors, cognate ligands, andcomplexes thereof might have different roles. This is exem-plified by a recent study, showing that all Ephrin-As testedinduce epidermal differentiation markers and suppress celladhesion genes, especially integrins, yet their effects are morespecific rather than redundant [104]. The final outcome of thedifferent expression combinations and functional configura-tions of Ephs and Ephrins could be determined by manyfactors, including their respective expression levels, phosphor-ylation, degradation, proteolytic cleavage, and subcellularlocalization, to name just a few.

4.2 Regulation of combinatory Eph/Ephrin expression

An interesting but unaddressed question is how are thereported gradients of Ephrins established in normal tissuessuch as the intestinal and neuronal tissues [84, 105], so as toprovide an elegant way for the guidance and positioning ofEph-expressing cells. Similarly, the regulation of Eph/Ephrinexpression in tumors is very poorly examined. EphB2 andEphB4 show opposite expression patterns during colorectalcancer progression, decreasing and increasing, respectively.

This has been shown to implicate a differential usage oftranscriptional coactivator cyclic AMP-responsive elementbinding protein-binding protein (CBP) over p300 by theWnt/β-catenin pathway, which is known to regulate the ex-pression of both EphB2 and EphB4. The two proteins seem tohave opposite functions in the colon and loss of EphB4inhibits tumor growth and metastases [102]. EphB2 acts bycontributing to the compartmentalization of the intestinal ar-chitecture and by preventing the intermingling betweenEphB2-expressing progenitor cells in the crypt with theEphrin-B2-expressing cells in the villus.

4.3 Phosphorylation and kinase-dependence of Eph/Ephrinsignaling and function

Phosphorylation seems to be an important factor in thefunction of Ephs/Ephrins in invasion. For instance,tyrosine-phosphorylation of Ephrin-B1 is important in thelatter’s role in invasion of scirrhous gastric cancer cells, andits blocking results in the attenuation of Rac1 GTPase acti-vation [106]. EphB3 has two functions in cell adhesion andmigration that seem to follow two different pathways. WhileEphB3 kinase activity is needed for the inhibition ofintegrin-mediated cell adhesion and induction of cell round-ing, its inhibitory role in HGF/SF directional cell migration,following Ephrin-B1 stimulation, is kinase-independent andinvolves a reduction in Rac1/Cdc42 activities [107]. It hasbeen suggested that EphA3, through its phosphotyrosineresidue at position 602 (Y602), binds the adaptor proteinNck1, and this interaction could regulate cell migration andretraction [108]. Ephrin-B2 was found to be more expressedand phosphorylated in human brain glioblastoma (GBM)tumors than in their normal counterparts. Overexpressionof Ephrin-B2 in the GBM cell line U251 resulted in higherlevels of tyrosine phosphorylation and stimulated cellularmigration and invasion [62]. Upon EphB1 interaction,Ephrin-B1 is tyrosine phosphorylated and transduces apro-migratory signal in endothelial cells. It also induces anintegrin αvβ3 and α5β1-mediated attachment. This processinvolves the JNK signaling pathway, but not ERK1/2 or p38pathways [109]. EphB2 expression and phosphorylationwas found to be increased in glioma cells during migrationand invasion. EphB2 was localized in lamellipodia of mi-grating cells [110].

The importance of Eph phosphorylation is further sub-stantiated by its complex interplay with other signalingpathways and other RTKs (Fig. 2). Kinase signaling andmore specifically the PI3K/Akt pathway can contribute tothe understanding of complex and sometimes seeminglyparadoxical effects of Ephs/Ephrins. Akt can phosphorylateEphA2, thus promoting cell migration and invasion. Uponstimulation by Ephrin-A1, EphA2 behaves as an upstreamnegative regulator of Akt, resulting in the inhibition of cell


migration [111]. Interestingly, the PI3K/Akt and mitogen-activated protein kinase (MAPK) pathways are also impor-tant in other functions of Ephs and Ephrins such as celldeath and autophagy [112–115], suggesting a complex butprecise coordination. The analysis of transgenic mice mod-els of mammary carcinomas where EphA2-deficiency andErbB2 overexpression were combined, suggests that EphA2cooperates with ErbB2 to promote tumor and metastaticprogression. EphA2 forms a complex with ErbB2, thusenhancing the activation of Ras-MAPK signaling and RhoAGTPase [116]. Increased expression of EphA2 and Ephrin-A1 was observed in osteosarcomas. Ephrin-A1 ligand bind-ing induces increased EphA2 tyrosine phosphorylation anddegradation, and downstream MAPK activation. In return,the MAPK pathway regulates Ephrin-A1 expression [117].Activation of the fibroblast growth factor receptor (FGFR1)induces the phosphorylation of EphB2 in the absence ofligand stimulation. Following Ephrin-B1 stimulation,FGFR1 signaling inhibits EphB2 phosphorylation throughthe MAPK pathway [118]. EphA4 also forms a complexwith FGFR1 which enhances the latter’s proliferative andmigrative function in glioma cells through the MAPK/Akt/Rac1/Cdc42 signaling pathways [119]. Adhesion can induceEphA2 expression. Ephrin-A1 stimulation of EphA2 inhib-its ERK phosphorylation, conducting to decreased viability.This effect is counteracted by the activation of the EGFR/Ras/MAPK pathway [120]. In fact, activated EGFR and theconstitutively active EGFR type III deletion mutant, bothinduce the expression of and interact with EphA2. Thelatter’s ligand-mediated stimulation or its silencing opposeEGF-induced cell motility [121]. Taken together, these find-ings suggest that the outcome of the functions of Ephs andEphrins might be greatly conditioned by cooperative orconflicting signaling from other pathways relying heavilyon protein phosphorylation mechanisms, such as in tumorswith RTK mutations or amplifications.

A limitation to the requirement of kinase activity of theEph proteins in their role in invasion is illustrated by the fewkinase-defective members of this family. One such protein,happens to be EphB6, which inhibits the invasiveness ofbreast cancer cells by decreasing the levels of the matrixmetalloproteinases MMP7 and MMP19, and increasing thetissue inhibitors of metalloproteinase 2 (TIMP2) [122]. InNSCLC cells, EphB6 can lead to MAPK activation [54].Interestingly, EphB6 was shown to exert biphasic effects inresponse to different concentrations of Ephrin-B2; while itpromoted cell adhesion and migration at low Ephrin-B2concentrations, it induced repulsion and inhibited migrationat high ligand concentrations. Tyrosine phosphorylation ofits cytoplasmic domain by a Src family kinase appears to beimportant in this dual response of EphB6 [123]. Althoughkinase-defective, EphB6 is able to trigger cytoplasmic sig-naling by interacting with other Ephs; for instance EphB6

undergoes transphosphorylation by EphB1 upon ligandstimulation [124, 125].

4.4 Modulation of Ephs/Ephrins’ functions by proteindegradation, endocytosis, and cleavage

Eph/Ephrin interactions trigger the phosphorylation of bothpartners, which could result in the signal attenuation throughtheir degradation. In glioma cells, unlike EphA2 whichseems to have oncogenic functions, one of its ligandsEphrin-A1 has tumor suppressive and anti-adhesive effects.Ephrin-A1 expression and its interaction with EphA2 resultsin the latter’s phosphorylation and degradation. Consequently,FAK, a downstream effector of EphA2 is also downregulated[126]. EphA2 stimulation by the soluble Ephrin-A1-Fc ligandinduces proteosomal degradation of EphA2, thus reducing itsinvasive activity in pancreatic adenocarcinoma cells [72]. Theattenuation of EphA2 levels has also been shown to involvethe recruitment of SHIP2 which regulates EphA2 endocytosisand signaling through a PI3K/Rac1 pathway [127]. Eph/Ephrin signaling can lead to either cell-cell repulsion or adhe-sion. It has been suggested that the discrimination between thetwo cell fates could involve tyrosine phosphatases, as reportedfor EphA3/Ephrin-A5 signaling in leukemia cells [128]. TheRho family GEF Vav2-dependent endocytosis of the Eph/Ephrin complex was shown to switch the outcome of thisinteraction, from cell–cell adhesion to repulsion [129].

Endocytosis, a process used by cells to recycle or degradeRTKs, thus modulating their signaling, is important in re-pulsive functions of Ephs and Ephrins. The complex gener-ated by interaction between the full-length protein partnersoccurs in a bidirectional manner and its endocytosis pro-motes cell detachment and repulsion [130]. Following cell–cell interaction, membrane ruffles are formed at the EphB4/EphrinB2 contact sites, allowing the endocytic internaliza-tion of the receptor-ligand complexes, which ultimatelyresults in cell repulsion according to a mechanism involvingRac-dependent actin polymerization [131]. EphB1 bindinginduces Ephrin-B1 clustering on the cell surface and subse-quent clathrin-mediated endocytic internalization [132].There is evidence that Rho family GTPases are importantmechanistic intermediates. Following Eph/Ephrin interac-tion, Rho family GEF Vav2-dependent endocytosis of theEph/Ephrin complex leads to the conversion of an adhesiveinteraction into cell repulsion [129].

The role of Ephs/Ephrins interactions and invasion tookan interesting turn with the finding that at least some ofthese proteins can be subject to proteolytic cleavage bymatrix metalloproteases and other proteases. Ephrin-B1’spro-invasive role requires the integrity of its C-terminusend.When following interaction with the EphB receptors, thiscytoplasmic C-terminal end is cleaved by MMPs, it activatesexocytosis of MMP-8 in an Arf1 GTPase-dependent manner.


This MMP-8 release was put forward to explain the role ofEphrin-B1 in invasion [133]. MMP2 and MMP9 mediateEphB2 cleavage upon stimulation with Ephrin-B2 at the cellsurface, thus privileging neural cell repulsion over adhesion[134]. Although the role of these proteolytic processes in cellinvasiveness is somehow addressed, the importance of thereleased C-ter fragment, if any, and its mechanisms of actionare not known.

4.5 Different configurations of ligand-receptor interactionsin the functions of Ephs and Ephrins

In many instances, increased expression of Ephs is notaccompanied with high tyrosine phosphorylation levels.This might be due not only to a reduced expression levelof cognate Ephrins, but also to a diminished availability andproximity due to spatio-temporal limitations. Although bynature, their functions involve direct cell-cell interactions,Ephs and Ephrins have been shown to be expressed in“exposed” cell areas, such as cell protrusions, where theirrole seems “ligand”-independent or “receptor”-independent.Ephrin-B2 is overexpressed in advanced malignant melano-mas, mostly in the invasive front of the tumors, indicating arole for Ephrin-B2 in cell migration and interaction with theextracellular matrix. This function involves a role in lamel-lipodia formation, actin fibers polymerization, activation ofthe focal adhesion kinase, and an increase in β1-integrin-mediated adhesion to laminin and fibronectin [135]. Inbreast cancer, where it is overexpressed, EphA2 has a rolein EGF-triggered cell migration and invasion that is ligand-independent. EphA2 is engaged in a complex comprised ofactivated RhoG, ELMO2 and DOCK4, in addition to theguanine nucleotide exchange factor Ephexin4. Upon EGFstimulation, EphA2 binds to Ephexin4 and activates RhoG,which then recruits ELMO2 and DOCK4. The complex thusformed activates Rac in the cortactin-rich protrusions, whichpromotes cell migration [136]. An additional example isEphB4, which inhibits integrin-mediated cell substrate ad-hesion, spreading and migration, and reduces β1-integrinprotein levels, in a manner independent of Ephrin-B stimu-lation but involving EphB4 kinase activity [137]. EphB4regulates murine melanoma cells migration and actin cyto-skeleton reorganization in a kinase and Rho-A-dependentmanner.

Another level of functional discrimination is the possibilityof cis-interactions between different Ephs/Ephrins within thesame cell, generating different heterodimers (Fig. 1). Forinstance, Ephrin-As co-expressed with EphAs in retinal gan-glion cell axons, abolish the latter’s sensitivity for repellentaxon guidance cues [138]. A cis-interaction has been identi-fied between EphA3 and Ephrin-A5 that results in a loss ofsensitivity of retinal axons to trans-interactions with Ephrin-As by inhibiting the induction of tyrosine phosphorylation of

EphA3 [139]. The kinase-deficient EphB6 can interact withEphB2 and EphA2 and the expression of the three receptors iscorrelated with cell invasiveness. Furthermore, the anti-invasive function of EphB6 seems to be related to its abilityto partner with the kinase-able Eph receptors. The same situ-ation appears to be the case for another kinase-deficient re-ceptor, EphA10 [140]. In fact, there are two aspects to Eph/Ephrin cis co-expression: either direct physical interaction thatwould modify their ability to interact with trans partners, ordifferent subcellular localizations with each one of them sig-naling separately. These aspects are poorly addressed andmore so their relevance to cancer is not known. CoexpressedEphAs and Ephrin-As segregate into different membranedomains from which they signal opposing effects on thegrowth cone, regulating growth cone collapse/repulsion andmotor axon growth/attraction respectively [141]. Similarly,different membrane localizations of Ephrin-A5 and Ephrin-B1 are associated with different signaling patterns and effecton actin reorganization in murine fibroblasts [142]. Ephrinexpression levels and their subcellular distribution to mem-brane patches containing Eph receptors, modulate ephrincis-attenuation of Eph function during spinal motor axonguidance signaling responses [143].

4.6 Heterotypic cell-cell interactions and Eph/Ephrinsignaling

The differential interaction of cancer cells with the tumormicroenvironment constituents is essential. On the otherhand, there are a large number of combinations of Ephs’and Ephrins’ interactions contributed by tumor cells andneighboring non tumor cells alike. When combined, thesetwo concepts reflect the complexity of the ballet of inter-actions at work and the mechanistic compromises beingnegotiated by the tumor cells and the microenvironment,to ultimately lead to different outcomes. In prostate cancercells, the interaction between tumor-embedded EphA2 andEphA4 receptors and Ephrin-As mediate homotypic contactinhibition of locomotion. Meanwhile, the heterotypic inter-action between tumor embedded EphB3 and EphB4 andstromal cells-embedded Ephrin-B2 enhances the tumorcells’ migratory potential. The result of such double inter-action pattern is an increased migration and invasionthrough the stroma, as a prelude possibly to metastasis[144]. Thus, while most of these receptors have beenassigned a tumor suppressor function, they actually workin concert to favor cell invasion. In another study, whenmetastatic prostate cancer cells PC3 are co-cultured withfibroblasts, the cancer cells’ failure to undergo contact inhi-bition and to stop migrating is decided by the combinationof expressed Ephs and Ephrins. This is dependent on acti-vation of EphB3 and EphB4 by Ephrin-B2, through activa-tion of Cdc42. With regard to homotypic contacts, involving


PC3 cancer cells, contact inhibition of locomotion occursand is mediated by EphA-Rho-Rho kinase (ROCK) signal-ing [145].

Eph/Ephrin interactions might involve multiple celltypes, either during angiogenesis or within the metastaticsites, such as the bone marrow microenvironment. Ephrin-B2/EphB4 signaling controls bone homeostasis by regulat-ing the differentiation of osteoblasts and osteoclasts [146].Other studies show the importance of Ephs and Ephrins inthe bone [147]. Decreased levels of Ephrin-A1 were foundto be associated with prostate cancer metastases to the bonein comparison with metastases to other organs [148]. Itwould be interesting to determine the potential impact ofthese findings on our understanding of bone-specific metas-tasis vis-à-vis the interference of metastasizing cancer cellswith osteoclastic and osteoblastic function. It was also sug-gested that Eph/ephrin signaling might be involved in bonecancer pain (BCP) [149]. In other respect, EphB2 is activat-ed in Waldenstrom’s Macroglobulinemia (WM) cells, WMbeing a rare type of non-Hodgkin lymphoma, while Ephrin-B2 was found to be highly expressed on endothelial cellsand bone marrow stromal cells isolated from WM patients.As suggested by the authors of this study, cell–cell interac-tion and the ensuing EphB2/Ephrin-B2 signaling in theendothelial cells results in the latter’s adhesion and angio-genesis [150]. Ephrin-A4 is involved in regulating the ad-hesive and transendothelial migration capacity in chroniclymphocytic leukemia (CLL) cells circulating betweenblood and tissues. Ephrin-A4 binds to EphA2, which ishighly expressed in tumor necrosis factor-alpha-activatedHUVECs and in the CD31(+) endothelial cells of humanlymph nodes. The forward signaling reduced CLL cells’adhesion to intercellular adhesion molecule 1, vascularcell adhesion molecule 1, and other extracellular matrixmolecules [151].

4.7 Other determinents

As research on Ephs and Ephrins progresses, will certainlyemerge novel elements that could potentially explain someaspects of their multiple functions. For example, it wassuggested that heparan sulfate modulates Ephrin-A3/EphAsignaling and might affect their functions [152]. Ephrin-B3’s binding to an unidentified alternative receptor whichis a heparan sulfate proteoglycan, might be involved incellular signaling and Ephrin-Bs’ role in cell rounding andcell spreading [153].

5 Therapeutic targeting of the Eph/Ephrin signaling

The possible therapeutic targeting of Ephs/Ephrins signalinghas recently become a reality although still in its experimental

stage [154]. The therapeutic potential of Ephs and Ephrins hasbeen explored mainly by reducing their expression. For in-stance, silencing EphA2 expression with small-interferingRNA inhibits the growth and migration and inducescaspase-9-mediated apoptosis in malignant mesotheliomacells [155]. The different other approaches take advantage ofone or other feature of the Eph/Ephrin family functions.

Anti-EphB4 monoclonal antibodies have been generatedand shown to inhibit tumor angiogenesis, tumor growth andmetastasis. A combined anti-tumor effect with bevacizumabwas also used using this antibody approach. Interestingly,while one of these antibodies induces EphB4 degradation,the other does not, yet it is actually effective in suppressingthe growth of both EphB4-negative and -positive cells[156]. The intracellular introduction of a synthetic peptideconsisting of a fusion of HIV-TAT and C-terminal aminoacids 331–346 of Ephrin-B1 (PTD-EFNB1-C) blocksEphrin-B1 mediated signaling in scirrhous gastric cancercells and peritoneal dissemination. The peptide acts via theperturbation of the association of Ephrin-B1 with an adaptorprotein Dishevelled, thus blocking RhoA activation. It wasalso found to inhibit extracellular secretion of metallopro-teinases [157]. These effects are reminiscent of the reportedimportance of the C-terminal end of Ephrin-B1 and the roleof its proteolytic cleavage in cell invasion and MMP release.Using the extracellular domain of EphB4 fused with humanserum albumin (sEphB4-HSA) to block Ephrin-B2, resultedin the inhibition of growth factors-induced migration andinvasion of Kaposi sarcoma cells in vitro. In vivo, it reducedblood vessel density, pericyte recruitment, vessel perfusion,and it increased hypoxia [158].

Thanks to their role in vascular development, Ephs andEphrins also provide opportunities to target cancer-associated angiogenesis. Interaction between EphAs andEphrin-As is necessary for VEGF-mediated neovasculariza-tion. VEGF induces Ephrin-A1 expression, thus activatingEphA2. A soluble form of EphA2 (EphA2-Fc), inhibitsVEGF-, but not bFGF-induced endothelial cell survival,migration, sprouting, and corneal angiogenesis. EphA2 an-tisense oligonucleotides inhibited endothelial expression ofEphA2 receptor and suppressed Ephrin-A1- and VEGF-induced cell migration [42]. Nevertheless, the importanceof these EphA2/Ephrin-A1-targeting approaches in cancertherapy is not known. Blocking the activation of EphAsinhibited angiogenesis in two models, the RIP-Tag transgen-ic model of angiogenesis-dependent pancreatic islet cellcarcinoma and the 4T1 model of metastatic mammary ade-nocarcinoma. The use of soluble EphA2-Fc or EphA3-Fcreceptors inhibited tumor angiogenesis by decreasing tumorvascular density, and inhibited tumor growth by decreasingcell proliferation and increasing cell apoptosis. It alsoinhibited migration of endothelial cells in response to tumorcells [159]. Expression of soluble monomeric EphB4


(sEphB4) by melanoma cells reduced their tumor growthand intratumoral microvessel density [160]. Ephrin-A1 co-valently immobilized on the surface of biomimetic poly(ethylene glycol)-diacrylate hydrogels retains its pro-angiogenic properties [161]. Used for anti-angiogenic Ephsor Ephrins targeting, this approach could be eventually usedin cancer therapeutics. A soluble extracellular EphB4 fusedto albumin (sEphB4-Alb) reduced vessel density and tumorgrowth [162]. A specific small molecular weight kinaseinhibitor of the EphB4 kinase, NVP-BHG712, which inhib-its EphB4 kinase activity inhibited VEGF driven vesselformation. Inhibition of EphB4 forward signaling using asmall kinase inhibitor, allowed the inhibition of VEGFinduced angiogenesis, supporting a role for EphB4 forwardsignaling in VEGF induced angiogenesis [163]. Two high-affinity and highly specific monoclonal antibodies (mAbs)directed to the EphA2 receptor, were generated by differen-tial screening of phage-Ab libraries by oligonucleotidemicroarray technology, and block EphA2 using two differ-ent modes. One acts as an agonist that promotes receptorendocytosis and subsequent degradation, and the secondacts as a ligand antagonist, as it blocks the binding to andactivation by the Ephrin-A1 ligand. This antibody-basedapproach proved efficient in inhibiting tumor growth, an-giogenesis and metastasis [164]. A similar approach used anEphA2-agonistic monoclonal antibody, alone or in combi-nation with paclitaxel, in targeting ovarian cancer cells andwas successful at inhibiting tumor growth in vivo, reducingmicrovascular density, proliferation, and VEGF levels, andincreasing endothelial cell apoptosis. This antibody seems totarget EphA2 signaling by inducing rapid dephosphoryla-tion of Src. In combination with paclitaxel, the antibodyreduced tumor growth and angiogenesis and increased me-dian survival in comparison with paclitaxel alone [165].

It is noteworthy that targeting Eph/Ephrin interactionmight have limitations pertinent to the effects proper toindividual proteins independent of their interacting partnersor even their membrane localizations. For instance, EphB4’seffect on cell substrate adhesion, spreading, and migrationwas found to be Ephrin-B-independent. Inhibiting EphB4/Ephrin-B2 binding failed to inhibit this effect on cell sub-strate adhesion [137].

6 Future perspectives

The opportunities allowed by research on the role of Ephsand Ephrins in the understanding of cancer progression ingeneral and in the metastatic process in particular, are juststarting to unravel. They are at the image of the numerousaspects presiding at the fate of this family of proteins. Ephsand Ephrins use their role in intercellular communication toregulate different and sometimes opposing functions in

cancer. Because this is a large family, it also comes withmultiple challenges. It is not known whether and howtheir levels and respective signaling pathways are coor-dinated in specific tissues in specific contexts. For example,how do cancer cells coordinate ligand-dependent versus -independent signaling and functions of an Eph or Ephrinwhen, on their way to the metastatic sites, they migrate fromcontexts where specific ligands/receptors are present, to con-texts where these ligands/receptors are absent? The ability ofdifferent receptor/ligand partners to signal through differentpathways and impact different processes still needs a largeinvestigative effort and most probably warrants a systemsbiology approach, as the amount of expected information islikely to be very large. Because theirs is a bidirectional one,Ephs/Ephrins’ signaling illustrates an instance where not onlythe cancer cell is influenced, but the fate of cells in theimmediate microenvironment cells is also at stake. However,unlike communication through long range acting growth fac-tors and cytokines, Ephs and Ephrins involve a close proxim-ity action. One can speculate that this could provide anadvantage for the cancer cells in their attempt to escape anti-tumor vigilance. In conclusion, although the therapeutic po-tential of the Ephs and Ephrins is increasingly acknowledged,elucidating all of the above-mentioned aspects that conditiontheir function is a pre-requisite for the future of this family as aviable cancer therapeutic target.

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The Eph/Ephrin family in cancer metastasis: communication at the service of invasion