Seminars in Cancer Biology xxx (xxxx) xxx–xxx

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Seminars in Cancer Biology

journal homepage: www.elsevier.com/locate/semcancer

Review

Targeting receptor tyrosine kinase EphB4 in cancer therapy

⁎ ⁎
Yinnan Chenb, Hongmei Zhangc, , Yanmin Zhanga,
a
School of Pharmacy, Health Science Center, Xi’an Jiaotong University, No. 76, Yanta West Street, #54, Xi’an, Shaanxi Province 710061, PR China
b
School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA
c
Department of Endocrinology, Xinhua Hospital affiliated to Shanghai Jiaotong University School of Medicine, 1665 Kongjiang Road, Shanghai 200092, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: Eph receptors and their Eph receptor-interacting (ephrin) ligands together form an important cell commu-
Receptor tyrosine kinases nication system with diverse roles. Experimental evidence demonstrated Eph receptor bidirectional signaling
EphB4 receptor with both tumor-suppressing and tumor-promoting activities in cancer cells. The tyrosine kinase EphB4, a
Ephrin signaling member of the Eph receptor family, has been associated with tumor angiogenesis, growth and metastasis, thus
Cancer therapy
making it a valuable and attractive target for drug design for therapeutic applications. In the past decade, many
Drug target
studies have focused on elucidating the structure and function of EphB4 in complex with its ligand ephrinB2 for
their role in carcinogenesis. Meanwhile, an array of compounds targeting EphB4 have been studied and several
selective inhibitors have been tested in clinical studies. This review discusses the structure and function of the
EphB4 receptor, analyzes its potential as a target for anticancer therapy, and summarizes the information about
inhibitors of EphB4 kinase activity. Conclusively, EphB4 is a challenging but promising therapeutic target in
cancer.

1. Introduction on their ligand-binding characteristics, Ephs have been subdivided into

Kinases present one of the largest families of enzymes. Protein ki-
nases regulate key biological processes including cell differentiation,
proliferation, and motility [1]. Receptor tyrosine kinases (RTKs) are
transmembrane proteins that undergo a conformational change upon
the binding of signaling molecules to the extracellular (N-terminal)
domain. The conformational change triggers autophosphorylation of
tyrosine residues in the intracellular domain of the RTK. Subsequently,
the activated RTKs can phosphorylate a variety of cytoplasmic signaling
proteins, which transduce a cellular response. Among other processed,
this signaling may induce the transcription of specific genes [1]. The
Eph receptors family in erythropoietin-producing hepatocellular carci-
noma belongs to the largest class of RTKs. Like other RTKs, they
transduce signals from the cell exterior to the cell interior via the li-
gand-induced activation of their kinase domains. Eph receptor tyrosine
kinases constitute a large family of transmembrane proteins, containing
a single cytoplasmic kinase domain that is activated in response to the
binding of ephrin ligands to the extracellular globular domain of the
receptor [2]. Eph receptors, however, have distinctive features: instead
of binding soluble ligands, they generally mediate contact-dependent
cell-cell communication by interacting with surface-associated ligands
(ephrins on neighboring cells). Ephrins can also attenuate signaling
through Eph receptors that are co-expressed on the same cell. [3]. Based
Eph A and Eph B receptors, although there is significant redundancy
and cross talk between subclasses [4].
In general, EphA receptors (EphA1-A10) are promiscuously acti-
vated by ephrinA ligands (ephrinA1-ephrinA6), whereas EphB receptors
(EphB1-EphB6) are activated by ephrinB ligands (ephrinB1-ephrinB3).
Cross interactions have been observed between EphB2 and ephrinA5 as
well as between EphA4 and ephrinB2/B3. Unlike other receptors in the
family, EphB4 display a distinct specificity for its ligand ephrinB2,
while exhibiting a very weak binding affinity for either ephrinB1 or
ephrinB3 [5]. The EphB4-ephrinB2 interaction plays a prominent role
in cardiovascular development and regulates vascularization in malig-
nant tumors [6].
The Eph receptor/ephrin upregulation in cancer cells, the angio-
genic vasculature, and injured or diseased tissues offer opportunities for
Eph/ephrin-targeted drug delivery [7]. In fact, EphB4 overexpression
has been linked to several tumor types such as breast, prostate, colon,
uterus, melanoma, and ovarian cancer among others [8–13]. Tyrosine
kinase EphB4 has been associated with angiogenesis, tumor growth and
metastasis, making it a valuable and attractive target for drug design for
therapeutic applications. However, the role of EphB4 in cancer remains
a matter for debate, since Eph-ephrin interactions have been shown to
either promote or inhibit tumor growth [14]. The primary focus of this
review is to analyze the structure and function of EphB4, briefly discuss

⁎
Corresponding authors.
E-mail addresses: zhanghongmei02@xinhuamed.com.cn (H. Zhang), zhang2008@mail.xjtu.edu.cn (Y. Zhang).

http://dx.doi.org/10.1016/j.semcancer.2017.10.002
Received 15 June 2017; Received in revised form 13 September 2017; Accepted 4 October 2017
1044-579X/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Chen, Y., Seminars in Cancer Biology (2017), http://dx.doi.org/10.1016/j.semcancer.2017.10.002
Y. Chen et al.
its potential as a target for anticancer therapy and summarize updated
research about inhibitors of EphB4 kinase activity.
2. The structure of the EphB4 protein
2.1. The molecular structure of the EphB receptor family
Himanen and colleagues. have reported the crystal structure of the
EphB2 and ephrinB2 complex [15]. Thus. a tetramer is formed by two
Eph-ephrin receptor/ligand dimers bound to each other. The extra-
cellular domains of EphB family members consistently include a glob-
ular N-terminal ligand-binding domain, a cysteine-rich region, com-
posed of an epidermal growth factor (EGF)-like motif, and two
fibronectin type III repeats [15]. The ephrin-binding domains of EphBs
have a high- affinity binding site that mediates the biochemical com-
munication between Ephs and the ephrins in adjacent cells [16], as well
as low affinity ephrin- binding sites that mediate the clustering of Eph-
ephrin complexes [17]. The extracellular ligand-binding domain of the
Eph receptor is connected to its cytoplasmic portion, which contains a
tyrosine kinase domain and protein-protein interacting modules, in-
cluding a sterile-α-motif (SAM) and a PSD95, DLG, ZO1-binding motif
(PDZ)-binding motif [18]. The SAM domain is a protein-interacting
domain that promotes homodimerization and the oligomerization of
receptors. The PDZ-binding motif mediates the organization of protein
complexes at the plasma membrane [19] (Fig. 1).
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Seminars in Cancer Biology xxx (xxxx) xxx–xxx
2.2. Structure of EphB4 binding domain
EphB and ephrinB are both transmembrane proteins. The B ephrins
are composed of an extracellular Eph receptor- binding domain, a
single-pass transmembrane region, and an intracellular domain con-
taing several phosphorylation sites and a PDZ binding motif at the C-
terminus (Fig. 1). Chrencik et al. identified that the ligand- binding
domain of EphB4 forms a complex with the extracellular domain of
ephrinB2 [20]. The overall structure of the EphB4-ephrinB2 complex is
similar to that of the EphB2-ephrinB2 complex, and a second portion of
the high- affinity heterodimerization interface forms immediately ad-
jacent to the ligand- binding cavity. The high-affinity EphB4- ephrinB2
heterodimer is formed through the insertion of the solvent-exposed li-
gand G-H loop into the upper convex, hydrophobic surface of the EphB4
receptor. The G-H loop of ephrin-B2 is buttressed by the G-H and J-K
loops of EphB4, and should form similar main chain hydrogen bonds
and numerous van der Waals interactions with EphB4 (Fig. 2A). This
high-affinity binding interface is highly hydrophobic, and includes re-
sidues Phe-120, Pro-122, Leu-124, Trp-125, and Leu-127 of ephrin-B2.
Phe-120 forms hydrophobic interactions with Leu-95, Leu-100, and
Pro-101, whereas Leu-124 interacts with Thr-147, in the receptor J-K
loop. Meanwhile, Trp-125 extends to the surface of the receptor, in-
between the J-K and G-H loops, participating in hydrophobic interac-
tions with residues Leu-48, Glu-50, Val-159, Leu-188, and Ala-186 of
EphB4. The conserved Cys-61-Cys-184 disulfide bridge of EphB4 is
stabilized by Pro-122, in the conserved FSPN segment of the ephrin-B2
Fig. 1. Schematic domain structure of EphB4 and its
ligand, ephrinB2. Eph receptors have an extracellular
region including an ephrin-binding domain, a cy-
steine-rich region and two fibronectin type III re-
peats, with an intracellular region containing a tyr-
osine kinase domain, a sterile alpha motif domain
and a PDZ-binding motif. EphrinB ligands are
transmembrane proteins with a cytoplasmic tail and
a PDZ-binding domain. Bi-directional signaling re-
sults in forward signaling through Eph receptors and
reverse signaling through ephrin ligands.
Y. Chen et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

Fig. 2. The binding domain of the EphB4 receptor in complex with the ephrinB2 extracellular domain (yellow) (PDB ID: 2HLE and 2BBA) [20,21]. (A) The EphB4 receptor consists of a
jellyroll folding topology with 13 anti-parallel β-sheets (cyan) connected by loops of varying lengths, whereas the ephrin ligand (yellow) resembles the Greek key folding topology. The
interface is formed through the insertion of the ligand G-H loop into the hydrophobic binding cleft of EphB4. Key residues directly interacting with ephrinB2 are colored in purple. (B)
Surface representation of the three-dimensional structure of the EphB4 binding surface with EphrinB2. Key residues interacting with Ephrin B2 colored in purple.

Fig. 3. Crystal structure of the kinase domain of EphB4 (PDB ID: 3zew) [29]. Left: Backbone ribbon representation of the EphB4 kinase domain (alpha-helixes and beta sheets are shown
in red and cyan respectively). Right: ATP- binding site of EphB4, with highlighted residues used as targets for kinase inhibitors. The key side chains that can potentially interact with
kinase inhibitors are shown as colored sticks.

G-H loop [20] (Fig. 2B).
The structure of the EphB4 receptor in complex with the high-affi-
nity (40 nM) antagonistic TNYL-RAW (TNYLFSPNGPIARAW) peptide
has been elucidated. The peptide is inserted into the same cleft occu-
pied by ephrinB2, along the hydrophobic upper convex portion of the
EphB4 receptor, which is situated on top of a β-sheet ‘floor’, formed by
the D and E β strands. In addition, loops D-E, E-F, G-H, and J-K effec-
tively buttress the peptide in the cavity, forming numerous van der
Waals interactions and main chain hydrogen bonds that stabilize
binding. These studies reveal structural features for drug discovery of
EphB4 antagonists [21].
EphB4 is the sole member of the Eph receptor family that
preferentially interacts with only one ephrin ligand, ephrinB2. Several
amino acid residues that make important contacts with the ephrin G-H
loop in the high-affinity dimerization interface of EphB2 are not con-
served in EphB4. For example, Ser-194 of EphB2 is conserved in other
EphB receptors, but not in EphB4, where an alanine is present at the
corresponding position. Moreover, EphB receptors have an aromatic
residue at the position corresponding to Tyr-57 of EphB2. In EphB4, this
position is occupied by Leu 48, which cannot form a hydrogen bond
with the main chain oxygen of Pro-150 of ephrinB2 or an aromatic-
aromatic interaction with Phe-113 of ephrinB2, as observed with Tyr-
57 of EphB2. Rather, Leu-48 can only form weak hydrophobic inter-
actions with ephrinB2. A leucine that replaces the arginine at position

3
Y. Chen et al.
95 of the EphB4 receptor appears to be sufficient to cause a substantial
structural rearrangement of the receptor J-K loop, resulting in the ab-
sence of another salt bridge that is present in the dimerization interface
of EphB2 with both ephrinB2 and ephrinA5 [15,20]. Further studies
from Dai et al. demonstrated that two pairs of dynamic salt bridges are
unique to EphB4, resulting in ephrinB2 recognition and conformational
selection [22]. These specificities for the interaction between EphB4
and ephrinB2 could be applied to target therapy with decreased side
effects [23], which attribute to decrease the non-specific interaction of
inhibitor and its potential targets. The ability to modulate the EphB4-
ephrinB2 binding will be critical to dissect the roles of these molecules
in tumorigenesis and angiogenesis. The high-resolution structure of the
ephrin binding domain of EphB4 in complex with a high-affinity se-
lective antagonist will allow for the design of novel compounds that
recapitulate the critical contacts of the peptide with EphB4, while
presenting good pharmacokinetic properties.
2.3. Design of EphB4 inhibitors for targeting the kinase domain
Previously Tokarski et al. reported a co-structure dasatinib, which is
a multitargeted tyrosine kinase inhibitor, in complex with kinase do-
main [24]. Tyrosine kinase domains are well conserved among Eph
receptors. A series of studies have reported the design of EphB4 in-
hibitors by modifying the leading compound based on the structure of
the kinase domain (Fig. 3), and those of the available co- structures
[25–28]. Several groups of compounds have been designed to bind the
catalytic domain of EphB4. For instance, some inhibitors can form a key
hydrogen bond or present a donor-acceptor pair to Met-696. Moreover,
some can be buried in the selectivity pocket, the hydrophobic region
beyond the Thr-693 gatekeeper residue, to disrupt ATP binding [28].
Furthermore, the hinge and Glu-697, or the glycine-rich loop and Ile-
621, can form sulfonamides that promote hydrogen bonding with some
inhibitors in the active site of the kinase [27]. Some compounds provide
hydrogen donor and acceptor bonds to the carbonyl side chain of
Glu664 and the backbone amide of Asp-758. This helps to explain why
these compounds retain affinity for the kinase despite the presence of a
polar group in the relatively lipophilic environment of the back pocket
of the ATP-binding site [26]. Overman et al. presented a crystallization
analysis of human EphB family kinases, including the structure of
staurosporine-bound EphB4 [29]. Staurosporine-bound EphB4 adopts
the most closed conformation observed, with the Gly-rich loop folding
tightly over the ligand. In contrast, the apoprotein structures of EphB1
and EphB2 are more open, with the Gly-rich loops partly disordered.
The rational design and optimized synthetic gene constructs of the
EphB4 intracellular kinase domain utilized by Overman et al. provided
a useful platform for researchers to study or improve the derivatives of
EphB4 kinase inhibitors [30].
3. The rgulatiory function of EphB4 in cancer
The functions of the Eph/ephrin system in cancer are complex be-
cause many tumor cells express varying degrees of Eph receptors and
ligands. Eph signaling controls cell morphology, adhesion, migration,
invasion, and the epithelial phenotype by modifying the organization of
the actin cytoskeleton and influencing the activities of integrins and
intercellular adhesion molecules [18,31]. Eph-ephrin complexes trans-
duce emanate bidirectional signals: forward signals depend on Eph ki-
nase activity for propagation in receptor-expressing cells, whereas re-
verse signals depend on Src family kinases for propagation in the
ephrin-expressing cells [18]. Eph receptors and ephrins are widely ex-
pressed in cancer cells and tumor stroma, but may be down regulated in
advanced stages of cancer [14]. Moreover, dysregulating mutations
affecting Eph function also play a role in cancer pathogenesis [14].
However, although bidirectional signaling promotes tumor angiogen-
esis, the mechanism underlying its role in cancer progression is poorly
understood.
4
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
3.1. EphB4 as a tumor repressor
3.1.1. Forward signaling in cancer cells
Eph receptor signaling induced by ephrin binding is initiated though
autophosphorylation and Src family kinase- mediated phosphorylation
of intracellular tyrosine residues, resulting in the activation of the tyr-
osine kinase catalytic domain [32].
In many cancer cell lines, EphB4 receptors appear to be highly ex-
pressed but poorly activated by ephrins, as evaluated by the low level of
tyrosine phosphorylation. For example, EphB4 tyrosine phosphoryla-
tion is much lower in breast cancer cell lines, when compared with non-
transformed MCF10A epithelial cells [14,33,34]. This is one of the clues
suggesting that ephrin- dependent Eph forward signaling might be
detrimental to tumor progression. Moreover, forcing EphB4 receptor
activation with soluble Fc fusion proteins of ephrin ligands can inhibit
proliferation, survival, and migration of many types of cancer cells in
culture as well as tumor growth in mouse models [34,35]. EphB4 re-
ceptors that are activated by ephrins acquire the remarkable ability to
inhibit oncogenic signaling pathways, including Abl-Crk, PI3K-Akt and
HRAS-Erk [36]. Xiao et al. reported that the functional coupling of
EphB4 to different downstream effectors determines the roles of EPhB4
in inhibition or promotion of signaling. For instance, activation of
EphB4 resulted in inhibition of Ras/ERK in endothelial cells (HUVEC)
because of the effectors like p120 RasGAP. However, MCF7 cells are
stimulated by activation of EphB4 in due to PP2A is the effector [37]. In
esophageal squamous cell carcinoma (ESCC) compared with the paired
normal tissues, the expression of EphB4 was up regulated while
ephrinB2 was down regulated. Phosphorylation of EphB4 induced by its
ligand, ephrinB2-Fc, inhibited growth, migration, and colony formation
in ESCC cells. Moreover, overexpression of EphB4 or that of an inactive
mutant EphB4 kinase in ESCC cells promoted cell growth and migra-
tion, suggesting EphB4 promoted cell growth and migration in-
dependently of its kinase activity, and that repressed tumorigenesis
[38]. Consistent with this result, Rutkowski et al. found that EphB4
overexpressing cells demonstrated an enhanced anchorage-independent
growth, migration and invasion, all characteristics associated with an
aggressive phenotype. This supports the hypothesis that the over-
expression of EphB4 facilitates tumor progression. However, these ef-
fects were reversed in the presence of ephrinB2, which led to a reduc-
tion in EphB4 protein levels, demonstrating that ligand-dependent
signaling is tumor suppressive in prostate cancer [39].
EphB4 mutations may also contribute to the disruption of forward
signaling by impairing ephrin binding or kinase activity. The over-
expression of EphB4 in breast cancer produced less malignancy in ad-
vanced stages [40]. Furthermore, EphB4 has been shown to reprogram
transcription, leading to anti-proliferative and anti-invasive outcomes.
A dominant negative form of EphB4 has been shown to promote col-
orectal cancer proliferation and invasion [41]. Mutations that impair
Eph receptor signaling or promote the up regulation of tyrosine phos-
phatases that dephosphorylate Eph receptors may also promote tu-
morigenesis [42]. Using dose and time dependent studies in breast
cancer cells, Barneh et al. showed that EphB4 can differentially inhibit
cell growth in a post-confluent state and that the presence of ligands
promotes the growth-inhibitory properties of the EphB4 receptor. In
this case, the inhibition of growth may have occurred due to the ex-
tended treatment with ligand, a process that leads to the down reg-
ulation of the receptor [43].
3.1.2. EphrinB2 reverse signaling in cancer cells
Unlike Eph receptors, B ephrins do not possess intrinsic catalytic
activity, and thus rely on the recruitment of signaling molecules. Ephrin
B signaling is initiated by the recruitment and activation of Src family
kinases, which phosphorylate specific tyrosine residues in the in-
tracytoplasmic domain of B ephrins, resulting in receptor engagement
and clustering [32]. Ephrin reverse signaling may also contribute to
tumor suppression in some cancers. Eph receptors may further decrease
Y. Chen et al.
tumor invasiveness by promoting the formation of tight junctions in
neighboring epithelial cells through the stimulation of ephrinB. Similar
to its role in neurons, ephrinB reverse signaling may also inhibit the
migratory and invasive effects of the CXCR4G protein-coupled chemo-
kine receptor in cancer cells [44,45]. Loss of ephrinB2 has also been
reported leading to carcinogenesis in a mouse mammary tumor model.
The Wnt/β- catenin/Tcf4 pathway in colorectal cancer cells are down
regulated with over-expression of ephrinB2 [46,47].
3.2. EphB4 as a tumor promotor
3.2.1. Forward signaling in cancer cells
It has been reported that EphB4 is over expressed in various types of
cancer, including colon, prostate, breast, esophageal, pancreatic, lung,
mesothelioma and colorectal cancers [33,38,48–52]. The forward and
reverse Eph-ephrin signaling has been shown to enhance malignant
transformation in some cases. Mounting evidence supports the notion
that Eph receptors are also capable of unconventional signaling activ-
ities independent of ephrin ligand activation. Meanwhile, it is well es-
tablished that the Eph-ephrin system promotes cancer −associated
angiogenesis. Yang et al. reported ephrinB2 dependent EphB4 signaling
that enhances the migratory and invasive ability of melanoma cells,
which also requires the activation of RhoA GTPase [53]. EphB4 sti-
mulated by ephrinB2 can promote proliferation of breast cancer in
MCF7 by the activation of Ras/ERK signaling [37]. Moreover, Becerikli
et al. found that EphB4 was overexpressed in soft tissue sarcomas.
Therefore, either decreasing the expression of EphB4 or inhibiting its
kinase activity can reduce the proliferation of sarcoma cells [54].
3.2.2. Reverse signaling
EphrinB reverse signaling involves Src family kinases, that phos-
phorylate the ephrinB cytoplasmic domain and regulate its interaction
with signaling molecules [45]. EphrinB2 is up regulated in the invading
cells of glioma and melanoma biopsy samples. The overexpression of
EphrinB2 in cultured cancer cells enhances integrin mediated attach-
ment, migration and invasion [55,56]. Tumor cells expressing domi-
nant negative EphB4 are incapable of forward signaling, but remain
able to stimulate ephrinB2 reverse signaling, that in turn promotes cell
invasion and proliferation. The overexpression of EphB4 correlates with
tumors with larger blood vessels and a higher blood content [57]. Sa-
wamiphak, et al. have shown that ephrinB2 can up-regulate vascular
endothelial growth factor receptor type 2 (VEGFR2) signaling through
PDZ interactions. Thus, ephrinB2 reverse signaling positively controls
sprouting and branching in pathological angiogenesis [58]. In a recent
study, Alam et al. demonstrated that ephrinB2 could induce ABCG2
expression in mutant p53 colorectal cancer cells, which promotes tu-
morigenesis and is responsible for the acquired chemo-resistance in the
cells [59]. The growth promoting effects of ephrinB2 are also found in
leukemia cell lines, indicating a role in the tumorigenesis [60].
3.2.3. Unconventional EphB4 receptor activities
Besides the kinase activity of EphB4, many studies have shown
ephrinB2 independent cancer promoting activity. Down regulation of
EphB4 by small interfering RNAs or antisense oligonucleotides has been
shown to decrease cancer cell malignancy in culture and mouse cancer
models [33,61]. In these studies, the EphB4 tyrosine was poorly phos-
phorylated indicating this receptor is independent of the ligand medi-
ated kinase activation [8,44]. Noren et al. demonstrated that down
regulation of EphB4 inhibits integrin-mediated cell substrate adhesion,
spreading and migration, and reduces beta1-integrin protein levels in
breast cancer cells, while low expression of the EphB4 ligand, ephrin-
B2, does not show these above effects [62].
Knockdown of EphB4 in prostate cancer causes a significant re-
duction in cell motility in vitro and tumor growth in vivo [9]. One of the
explanation from Mertens-Walker et al. is that lower expression of
EphB4 can lead to down regulation of integrin beta 8, which is
5
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
overexpressed and may play an unknown role in cancer progression
[63]. Rutkowski et al. showed the evidence that overexpression of
EphB4 leads to a transformed phenotype in MCF-10A cells and an in-
creased metastatic phenotype in 22Rv1 cancer cells. It is restrained by
ephrin-B2 stimulation due to reduce EphB4 levels [39]. Heroult, et al.
reported expression of EphB4 by tumor cells confers a preferential site-
specific metastasis phenotype to ephrinB2-expressing vascular beds
[64].
3.2.4. Regulation of angiogenesis by EphB4
Blood vessels are essential for tumor growth and represent an im-
portant venue for metastatic dissemination. Eph receptors and ephrins
promote angiogenesis by mediating communications among vascular
cells and with tumor cells. Moreover, the EphB4 extracellular domain
can induce angiogenic responses by stimulating ephrinB2 reverse sig-
naling in cultured endothelial cells [57]. During development, EphB4
receptor expression was detected in the endothelial cells of arteries and
veins. Suggestively, EphB4 regulates arterial-venous vessel segregation
and remodeling in vasculature [65,66]. Furthermore, the reverse sig-
naling medicated by ephrinB2 in endothelial tumor cells, pericytes, and
smooth muscle cells is important for blood vessel assembly and en-
largement, and for decreasing blood vessel permeability, as well as for
promoting the interaction between endothelial cells and pericytes or
vascular smooth muscle cells [57,67,68]. Additionally, ephrinB may
have other roles in the tumor-located endothelium, including enhan-
cing recruitment of bone marrow derived endothelial progenitor cells
that participate in tumor vascularization, which involves the EphB4
dependent up regulation [69]. Pfaff et al. showed that EphB-ephrinB
interactions during a cascade of events can lead to monocyte adhesion
and transmigration through the vascular endothelium [70].
4. EphB4 as a therapeutic target in cancer
Eph receptors and ephrins are promising new therapeutic targets in
cancer. Various strategies have been employed to evaluate the inter-
ference of tumor-promoting effects or the enhancement of tumor sup-
pressive effects. The inhibition of the Eph-ephrin system may be par-
ticularly useful for anti-angiogenic therapies. The EphB4/ephrinB2
bidirectional signaling has an established role in the formation of the
vascular system, as evidenced by embryonic lethality in knockout
mouse studies, which arise due to malformed vascular architecture
[71].
4.1. Regulation of expression
The EphB4-ephrinB2 interaction plays a distinct role in diverse
biological processes such as neuronal development, bone homeostasis,
angiogenesis, migration and tumor invasiveness [57,72]. Several stu-
dies point to significant levels of EphB4 expression in different malig-
nant tumors, whereas the inhibition of EphB4 expression inihibits
tumor progression [9,33,54]. The critical importance of EphB4 in tumor
progression is demonstrated by studies employing over expression and
knockdown strategies. The forced over-expression of EphB4 in non-tu-
morigenic MCF10A breast cells and in 22Rv1 prostate cancer cells led to
transformation in the MCF10A cell line and to an increase in the me-
tastatic phenotype of 22Rv1 cells [39]. Consequently, the knockdown
of EphB4 in several cancer cell lines consistently resulted in a 70–80%
reduction in cancer cell viability, an 8–16-fold increase in apoptosis,
and in an up to 80% reduction in cell migration and invasion [8,73].
The downregulation of EphB4 expression using siRNAs or antisense
oligonucleotides has been shown to inhibit malignant cell behavior in
different types of culture and tumor growth in vivo [8,9,61,74]. Kras-
noperov et al. reported that monoclonal antibodies inhibit tumor an-
giogenesis and growth by binding to fibronectin like domain 1. This
subsequently leads to the degration of EphB4 and inhibits the organi-
zation of human endothelial cell into tube-like structures in vitro [75].
Y. Chen et al.
However, the down regulation of EphB4 is not effective in preventing
ephrinB2 reverse signaling. Tumor cells expressing dominant negative
EphB4 were incapable of forward signaling, but could stimulate
ephrinB2, attract endothelial cells, and stimulate cell invasion, survival
and proliferation. This characteristic correlated with tumors with larger
blood vessels and higher blood content [57]. Therefore, anti-ephrinB2
antibodies have been proposed to reduce tumor growth by inhibiting
the formation of tumor blood and lymphatic vessels independent of
EphB4 [76].
4.2. Disrupting Eph-ephrin interactions in anti-angiogenic therapy
EphB4/ephrinB2 signaling is important for angiogenesis. Thus, po-
tential medical applications targeting the angiogenesis-related tumor
progress, such as tumor growth and metastasis, could be devised by
hindering this protein-protein interaction. EphB4-ephrinB2 binding
occurs at contact sites between cells. EphrinB2 induces EphB4 clus-
tering and increase kinase activity, thus generating downstream signals
that affect cellular behavior. Monoclonal antibodies and peptides that
block the EphB4-ephrinB2 interaction have been described, and some
have been utilized with high efficiency. Therefore, the disruption of
EphB4-ephrinB2 binding represents a potential avenue for anti-
angiogenic cancer therapies. Therefore, ephrinB2-Fc has also been used
as an agonist that slows breast cancer xenografts by activating anti-
oncogenic signaling [34,57]. Various peptides and competitive small
compound inhibitors have been designed to mimic the binding of
the15-amino acid long G-H loop of ephrins, which is the main region
mediating high affinity binding of the ephrins to the C-terminal pocket
of Eph receptors [21,77–79]. These EphB4 antagonistic compounds
may prove useful as a starting point for the generation more potent
small molecule derivatives that inhibit the binding of ephrinB2 to this
receptor.
The monomeric EphB4 ectodomain can inhibit tumorigenesis pre-
sumably by blocking the EphB4-ephrinB2 forward and reverse sig-
naling, resulting in the inhibition of angiogenesis. In fact, several
groups have demonstrated that the soluble monomeric extracellular
domain of EphB4 is able to block the interaction between ephrinB2and
its receptor, leading to the inhibition of angiogenesis and tumor growth
[80,81]. Soluble fusion proteins, containing the extracellular domain of
EphB4 that antagonize EphB4-ephrinB2 interaction could inhibit tumor
growth and angiogenesis in tumor xenograft models, thus confirming
the importance of EphB4 in oncology. Furthermore, the EphB4-human
serum albumin fusion protein has shown therapeutic promise by in-
hibiting EphB4-ephrinB2 signaling in tumors [82]. The extracellular
cysteine- rich region and fibronectin-like domains of EphB4 can be used
as epitope targets by antibodies, which may potentially affect the in-
teraction between EphB4 and ephrinB2 [75,83]. Thus, EphB4 agonists
that also antagonize ephrin binding may prove particularly beneficial,
by enhancing EphB4-dependent tumor suppression in cancer cells and
by inhibiting ephrinB2-dependent angiogenesis [34,57].
4.3. Kinase inhibitors for EphB4
The ephrin- binding pocket in the extracellular N-terminal domain
of Eph receptors and the ATP- binding pocket in the intracellular kinase
domain represent potential binding sites for peptides and small mole-
cules [84]. Efforts to identify small molecules that target the Eph kinase
domain have begun to yield some high-affinity inhibitors. Moreover,
some inhibitors designed to target other kinases are effective against
Eph receptors, due to the conserved structure of the kinase domain
[85,86]. Therefore, multi-targeted tyrosine kinase inhibitors could also
inhibit the kinase activity of EphB4; some of these, such as EXEL-7647
and dasatinib, are already being tested in clinical trials. Moreover, some
new inhibitors have been sourced from kinase inhibitor libraries pos-
sessing specificity for EphB4, for which the efficacy has already been
determined. The selective NVP-BHG712 EphB4 inhibitor has been
6
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
reported to inhibit VEGF driven angiogenesis in vivo [28,87].
Potent inhibitors of the kinase domain of EphB4 receptors have been
reported and the binding modes have been disclosed for these mole-
cules [88]. Type I inhibitors are ATP competitors that recognize the
activated state of the kinase [89–91]. Type II inhibitors interact with an
inactive conformation of the kinase and generally offer better se-
lectivity profiles. Like type I, type I1/2 inhibitors bind to the kinase in
the active DFG-in (DFG “in” is based on the position of the conserved
aspartate-phenylalanine-glycine triad at the beginning of the activation
loop) conformation, but extend their interactions to the hydrophobic
back pocket, establishing the characteristic hydrogen bonds present in
type II inhibitors [92]. Based on structural studies of EphB4 in complex
with its inhibitors, it is feasible to optimize the leading compound as
well as design better inhibitors. However, there are a few potential
problems regarding the use of these Eph kinase inhibitors, including
their anti-angiogenic activity that might also block possible Eph tumor
suppressive activities, since these compounds target the ATP- binding
site and therefore their selectivity tends to be low. Additionally, EphB4
kinase inhibitors will not be effective in preventing ephrinB2 reverse
signaling.
Thus, as Xiao et al. reported, EphB4 can promote or suppress ele-
ments of the signaling pathway, such as Ras/MEK/ERK, due to its
functional coupling to various downstream effectors in different cells
[37]. It represents a potential challenge in targeting EphB4 for cancer
therapy, due to its conflicting effects on cancer cells and endothelial cell
compartments.
5. Tumor inhibitors targeting EphB4
EphB4 inhibitors have been studied over the past several years, with
many different approaches used to test for inhibition of activity or
competitive binding affinity as a novel therapeutic modality to treat
malignancy. Several potent Eph kinase inhibitors were reported either
based on high molecular weight compounds like peptides [23,93–95],
which block the extracellular domain of the appropriate receptor, or on
small organic molecules, which bind to the intramolecular ATP- binding
pocket and inhibit EphB4 kinase activity [95].
5.1. Laboratorial or preclinical inhibitors of EphB4
5.1.1. Synthetic EphB4 kinase inhibitors or high-affinity ligands of the
EphB4 receptor
Small molecule inhibitors are a rapidly emerging area for dis-
covering potential therapeutic candidates [96]. The targeted synthesis
of specific EphB4 inhibitors has shown great promise. However, no
specific small-molecule EphB4 inhibitors exist, except for antibodies
and peptides, and most EphB4 inhibitors show multi-target effects.
A series of reports on EphB4 tyrosine kinase inhibitors emerged in
2008, 2010, and 2011 [97–100]. Among these, Bardelle, et al. reported
a series of synthetic inhibitors based on 2,4-bis-anilinopyrimidines and
3,5-bis substituted anilinopyrimidines, in two papers in 2008 [97,98].
Investigators such as Bardelle have used structural information from
two distinct species bound in the active site of EphB4 to generate a
novel series of 2,4-bis-anilinopyrimidine inhibitors. Structure–activity
relationship (SAR) studies around the C-2 hinge-binding aniline in-
dicated a strong preference for meta-substitution, particularly with re-
gard to electron-withdrawing groups. One example from this work,
compound 28, has shown promise in its selectivity and pharmacokinetic
profile for kinase inhibition and served as the basis for development.
Further optimization, based on non-benzodioxole, led to compound 20,
a potent inhibitor of EphB4 and Src kinases, with good pharmacoki-
netics in various preclinical species and high fraction unbound in
plasma [99]. The best IC50 obtained for EphB4 was 1 nM. Mamat et al.
presented the development of indazolylpyrimidine derivatives for
11
C-/18F-radiolabeling as high-affinity EphB4 receptor ligands [95].
Recently, a series of commercially available xanthine derivatives were
Y. Chen et al. Seminars in Cancer Biology xxx (xxxx) xxx–xxx

identified as micromolar inhibitors of EphB4 by high-throughput frag-
ment-based docking into the ATP-binding site of the kinase domain.
Karine et al. exploited the binding mode obtained by automatic docking
for the optimization of these EphB4 inhibitors by chemical synthesis.
Compound 66 employed threonine as the gatekeeper residue (Abl, Lck,
and Src) of all the inhibitors shows very high affinity for a few other
tyrosine kinases. On the other hand, it is selective against kinases with a
larger gatekeeper [86].
NVP-BHG712, a potent and selective inhibitor of the EphB4 receptor
tyrosine kinase, has an ED50 of 25 nM for the inhibition of EphB4 au-
tophosphorylation in cell-based assays, and is thereby estimated to be
roughly 200-fold more potent against EphB4 than against VEGFR2.
However, it cannot discriminate among different Eph receptor kinases
[87]. Moreover, NVP-BHG712 also displays a synergistic effect with
others chemotherapy drugs for solid tumors. Kathawala, et al. in-
vestigated the combination of NVP-BHG712 and paclitaxel, and showed
that the administration of NVP-BHG712 significantly increased the le-
vels of paclitaxel in tumors but not in plasma compared to paclitaxel
alone. The combination of NVP-BHG712 and paclitaxel could serve as a
novel and useful therapeutic strategy to attenuate paclitaxel resistance
mediated by the expression of the ABCC10 transporter [101].
The various synthesized tumor inhibitors targeting EphB4 are listed
in Table 1.
5.1.2. Natural compounds derived from plants
Natural products, with their extensive structural diversity, are a
major source for the currently available anticancer drugs. Several plants
derived natural products have been described as potential inhibitors.
However, we did not find any recent publications investigating EphB4
inhibitors from plants. Therefore, many potential screening studies
could be performed on plant products targeting EphB4. For instance,
the Astragalus polysaccharide (APS), obtained from Astragalus mem-
branaceus, displays a range of activities in many systems. Yang et al.
investigated the effects of APS on erythroid differentiation and its
mechanism of action in K562 cells using microarrays. Interesting, the
mechanism underlying the action of APS involved the down-regulation
of EphB4 mRNA [115].
5.1.3. Peptides
Many peptides that selectively bind to EphB4 have been identified
by phage display [78]. The inhibition of EphB4 signaling by using so-
luble extracellular domains of EphB4 has been shown to inhibit tumor

Table 1
Summary of laboratorial or preclinical studies of synthetic tumor inhibitors targeting EphB4.

Compounds Mechanism of action Pharmacodynamic effects on cancer Ref.

Inhibitors of kinase activity

Pyrazolo[3,4-d]pyrimidine (NVP-
BHG712)
Golvatinib
Dasatinib
Nilotinib
(18)F-labeled benzodioxolylpyrimidines
Imidazopyridines
Imidazo[1,2-a]pyrazine diaryl ureas
Quinazoline (EXEL-7647)
Pyrido[2–3]pyrimidine (PD173955)
Anilinopyrimidines derivatives
Xanthine derivatives
Quinoxalines derivatives
Trifluoromethylphenyl derivatives
Thieno[3,2-c]pyridine derivatives
Phenylthiopene derivatives
High selectivity for targeting the EphB4 kinase, inhibits
EphB4 and other kinases including c-Raf, c-Src and c-Abl
Inhibits kinase activities of c-Met, Tie2, and EphB4
A multi-targeted kinase inhibitor including EphB4
A selective Bcr-Abl kinase inhibitor; also inhibits EphB4
Inhibits both the EphB4 receptor tyrosine kinase and the
Src kinase, also inhibits the EphB4
Inhibits multiple receptor tyrosine kinases involved in
angiogenic and tumorigenic signaling
Multikinase inhibitor targets EphB4, EphA2, VEGF
receptor 2 and the Tie2 receptor
A potent inhibitor of EGFR, ErbB2, KDR, and EphB4
A potent inhibitor of several ephrin receptor tyrosine
kinases
EphB4 kinase inhibitors
Inhibits EphB4 by docking into the ATP-binding site of
the kinase domain
Inhibitor of the EphB4 tyrosine kinase
Inhibitors of kinase activity
ATP competitive kinase inhibitors against angiogenic
targets, VEGFR2, Tie-2, and EphB4
Inhibitor of the EphB4 tyrosine kinase
Inhibits RTK autophosphorylation in stable transfected A375 [86,87]
melanoma cells and A375 xenografts
Strongly inhibits tumor growth and tumor angiogenesis in [102,103]
xenograft models, and potently represses the growth of both c-
Met amplified tumor cells and that of endothelial cells
stimulated with either HGF or VEGF
Used in numerous cancers treatments, including advanced [104,105]
prostate cancer
Treatment of imatinib-resistant chronic myelogenous [106,107]
leukemia
Inhibits the growth of human melanoma cells [108]
More potent for the inhibition of endothelial tube formation [109]
in an in vitro angiogenesis co-culture model; inhibits the
VEGF-driven autophosphorylation of lung KDR in mice and
rats
—— [84,110]
Inhibits cellular proliferation and EGFR pathway activation in [91]
the erlotinib-resistant H1975 cell line in vitro and in vivo,
substantially inhibits the growth of H1975 and MDA-MB-
231xenograft tumors and reduces tumor vessel density
Antiproliferative effects on cell viability in KU812 leukemia [111]
cells
Inhibition of cancer cell viability [97–100]
Inhibits growth of the central nervous system (SNB-75), [1,86]
leukemia (K-562) and breast (HS578T) cancer cell lines;
inhibits tube formation of HUVECs
High antiproliferative activities against leukemia (K-562), [1]
lung (HOP-92), colon (HT-29), renal (A498) and breast cancer
cells (MDA-MB-231, HS 578T) in the low nM range; inhibits
the VEGF-A induced sproutin in HUVECs g; inhibits tumor
growth in MDA-MB-231 nude mice xenografts
—— [85]
—— [89]
—— [84,112]

Inhibitors of the Eph receptor-ephrin interaction

Indazolylpyrimidine derivatives for High-affinity EphB4 receptor ligands Inhibit cancer cell viability [95]
11
C-/18F-radiolabeling
Disalicylic acid-furanylderivative Binds to the ephrin-binding pocket of EphA4; targets Inhibits capillary-like tube formation on Matrigel as well as [113]
several other Eph receptors cell viability
Lithocholic acid A competitive and reversible Eph-receptor ligand, Lithocholic acid dose-dependently inhibits PC3 adhesion to [84,114]
inhibits ephrin binding to both EphA and EphB ephrinA1-Fc and antagonizes the EphA2 dependent PC3 cell
receptors; also inhibits the ephrin-induced EphA2 and rounding
EphB4 tyrosine phosphorylation

7
Y. Chen et al.
growth and angiogenesis with xenograft studies in vivo [80,81]. The
soluble monomeric derivative of the extracellular domain of EphB4
(sEphB4) was designed as an antagonist of EphB4- ephrinB2 signaling.
sEphB4 blocks the activation of EphB4 by ephrinB2, suppresses en-
dothelial cell migration, adhesion, and tube formation in vitro, and in-
hibits the angiogenic effects of various growth factors (VEGF and bFGF)
in vivo. sEphB4 also inhibits tumor growth in murine tumor xenograft
models [80]. sEphB4 is thus a therapeutic candidate for vascular pro-
liferative diseases and cancer.
TNYL (TNYLFSPNGPIA) was the most potent among several syn-
thetic peptides examined, with an IC50 value of 50- 150 μM for the
inhibition of EphB4–ephrin-B2 interaction in ELISA assays. However, a
modified version that contains the RAW motif found in other EphB4-
binding peptides (TNYL-RAW) at the C terminus has dramatically im-
proved potency [84]. Jill et al. reported the crystal structure of the
EphB4 receptor in complex with a highly specific antagonistic peptide
(TNYL-RAW) at a resolution of 1.65 Å. The structure provides a mole-
cular explanation for the remarkably high binding affinity of the pep-
tide. TNYL-RAW is localized to a hydrophobic cleft in EphB4, corre-
sponding to the cleft in EphB2 occupied by the ephrin-B2 G-H loop,
consistent with its antagonistic properties. The TNYL-RAW peptide is a
particularly effective antagonist because it interferes with ephrinB2
binding not only in the high- affinity binding cleft of EphB4, but also in
the regions adjacent to the dimerization interface. These studies reveal
structural features that will aid drug discovery initiatives, for devel-
oping EphB4 antagonists for therapeutic applications [21]. Liu et al.
produced an EphB4 inhibitor, sEphB4-HSA, which was prepared by
fusing monomeric soluble EphB4 to human serum albumin [52].
sEphB4-HSA was highly active as a single agent in inhibiting tumor
growth, that was accompanied by apoptosis in tumor cells and the in-
hibition of PI3K and Src signaling. A combination of sEphB4-HSA and
the anti-VEGF antibody (Bevacizumab) was superior to each agent
alone, and led to complete tumor regression [52].
5.2. Clinical studies on EphB4 inhibitors
None of the currently approved angiogenesis inhibitors have been
reported to inhibit EphB4. JI-101 is an oral multi-kinase inhibitor that
targets VEGFR2, platelet derived growth factor receptor β (PDGFR-β),
and ephrin type-B receptor 4 (EphB4) [116]. By targeting multiple
angiogenesis signaling pathways in tumor vessel beds, JI-101 has the
potential to inhibit multiple stages of tumor angiogenesis and thus
enhance its anti-tumor efficacy. JI-101 was previously studied in a
phase I trial in patients with advanced solid tumors [116]. Everolimus
is an FDA-approved drug for the treatment of advanced renal cell car-
cinoma and advanced neuroendocrine tumors. The combination of
these two agents may provide synergistic benefits to patients with
certain solid tumors.
XL647 is an oral small-molecule inhibitor of multiple receptor tyr-
osine kinases, including endothelial growth factor receptor (EGFR),
VEGFR2, HER2 and EphB4. XL647 inhibits cellular proliferation and
the activation of the EGFR-pathway in the erlotinib-resistant H1975 cell
line that harbors a double mutation (L858R and T790 M) in EGFR. In
vivo efficacy studies showed that XL647 substantially inhibited the
growth of H1975 xenograft tumors, and reduced both tumor EGFR
signaling and tumor vessel density. Further, XL647 demonstrated an
inhibitory effect on tumor cell growth and tumor vasculature in both
EGFR mutant xenografts and MDA-MB-231 xenografts that highly ex-
press VEGF [91]. Moreover, Pietanza et al. reported an open-label,
multi-institutional Phase II study to investigate the efficacy and safety
of XL647 for the treatment-naive non-small-cell lung cancer patients
that show an enrichment in EGFR mutations. Patients with EGFR mu-
tations had a 57% objective response rate when administered with
XL647 as a first-line treatment [117].
8
Seminars in Cancer Biology xxx (xxxx) xxx–xxx
6. Conclusions and perspective
EphB4 and its ligand ephrinB2 are increasingly being recognized as
therapeutic targets in cancer. However, we are still far from being able
to reliably decipher their mechanism of action and to precisely predict
the outcomes of their modulation. The uncertainty is due to the com-
plexity of the interacting cells that receive interdependent signals from
the same complex, EphB4 and ephrinB2, in association with other re-
ceptor tyrosine kinases that may promote or repress tumorigenesis. The
functional coupling of EphB4 with downstream effectors can be dra-
matically differentially activated in a cell or tissue context-dependent
manner. Meanwhile, responses after initiation could be due to kinase-
dependent or kinase- independent signaling pathways that are cell or
tissue specific- manner. Thus, inhibiting kinase activity could be ben-
eficial in some settings, whereas in others, promoting kinase activity
might be useful. The lack of specificity is always a challenge for ther-
apeutic targeting, which requires the targeting of pathological EphB4
functions, without inadvertently affecting unrelated tissues or organs.
An important step forward will involve understanding the EphB4-
ephrinB2 activities beyond bidirectional signaling and precisely eluci-
dating the crosstalk with oncogenic pathways. Systematic studies will
also be critical in providing a comprehensive overview of the effects of
the EphB4 bidirectional and unconventional signaling mechanisms in
cancer. Apart from their relatively well- understood role in angiogen-
esis, it will be interesting to elucidate how the EphB4-ephrinB2 system
influences metastasis, the epithelial-to-mesenchymal transition, tissue
invasion and other hallmarks of cancer.
For each type of target, there are some challenges: (1) the targets of
protein- protein interactions at the surface of the EphB4- ephrinB2
complex are typically hard to perceive and difficult to inhibit using
small molecules; (2) small molecule compounds targeting the ATP-
binding site could block the activity of other kinases, and therefore,
their selectivity tends to be low; (3) some Eph kinase inhibitors with
anti-angiogenic activity might also block potential EphB4 tumor sup-
pressive activities. Thus, despite the challenges presented by the com-
plex biology of the Eph receptor/ephrin system, exciting possibilities
exist for therapies exploiting these molecules. Multiple factors should
be taken into consideration such as tumor type, stage, and tumor mi-
croenvironment. The optimizing strategies will require more details
understanding of the signaling mechanisms via EphB4 in different
tumor cells. Thus, a more in depth understanding of the structure,
biogenesis, and mechanisms of EphB4 will provide invaluable in-
formation for functional studies and an increase in the efficiency of
rational drug design.
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgements
This work was supported by the National Natural Science
Foundation of China (Grant no. 81773772, 81370088, 81227802 and
81230079), the Fundamental Research Funds for the Central
Universities of Zhuizong.
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