Role of EphB4/ephrinB2 signaling
in breast cancer metastasis
Master thesis
Anneloes Dummer
a.dummer@students.uu.nl
3257339
Supervised by Stephin Vervoort
Examined by Paul Coffer and Madelon Maurice
2
Abstract
Eph receptors and ephrin ligands are the largest family of tyrosine receptor kinases and
the receptor and membrane bound ligand can signal to both the receptor and ligand
expressing cell. The Eph/ephrin family is involved in many developmental processes and
deregulation can result in tumor formation. Especially in breast cancer development,
involvement of Eph/ephrin signaling has been demonstrated with in particular EphB4 and
ephrinB2 as one of the major players. Moreover, EphB4 and ephrinB2 are also involved in
cancer progression towards metastases. However, data for the exact role of EphB4 and
ephrinB2 is extremely conflicting. EphB4/ephrinB2 signaling is demonstrated to be
involved in tumor suppression via induced adhesion and apoptosis although tumor
promoting roles via migration, proliferation, angiogenesis and stem cells has also been
demonstrated. In this thesis, current data is discussed and a model is proposed where
EphB4 and ephrinB2 can have distinct functions by signaling independent of binding to
each other. Moreover, the role of EphB4/ephrinB2 in EMT and cancer stem cell formation
and the effect of stromal cells facilitating adhesion of metastatic cells via EphB4/ephrinB2
signaling are discussed. With this model, where independent signaling of both EphB4 or
ephrinB2 can promote tumorigenesis and dependent signaling results in tumor
suppression, a therapy based on targeting EphB4/ephrinB2 signaling is challenging since a
severe side effect might be tumor induction after drug treatment. However, targeting the
stromal cells might be effective in blocking adhesion of circulating cells and will provide a
therapy in a later stage of cancer.
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4
Content
Abstract ....................................................................................................................................... 3
Abbreviations............................................................................................................................... 6
Introduction ................................................................................................................................. 7
The Eph/ephrin family ........................................................................................... 7
Complex formation and clustering of different Eph receptors ................................. 13
Breast cancer ...................................................................................................... 17
Breast cancer development .................................................................................. 18
The expression of Eph and ephrin in breast cancer ................................................ 19
EphB4/ephrinB2 in breast cancer metastasis ............................................................................... 23
The expression and regulation of EphB4 and ephrinB2 in normal mammary tissue and
breast carcinoma ................................................................................................ 23
Tumor suppressive roles for EphB4/ephrinB2 ........................................................ 25
Tumor promoting roles of EphB4 and ephrinB2 ..................................................... 28
Dependent and independent signaling of both EphB4 and ephrinB2 ....................... 32
Discussion .................................................................................................................................. 37
Distinct cellular responses of EphB4/ephrinB2 signaling dependent on complex
formation and clustering ..................................................................................... 37
A possible role for Eph/ephrin signaling in breast cancer progression by affecting
migration, EMT and tumor-stroma interactions. ................................................... 38
The effects of stromal cells on EphB4/ephrinB2 signaling ...................................... 42
Conclusion .......................................................................................................... 43
Summary for laymen .................................................................................................................. 44
References ................................................................................................................................. 46
5
Abbreviations
ECM
Extracellular matrix
EGF
Epidermal growth factor
EGFR
Epidermal growth factor receptor
EMT
Epithelial-to-mesenchymal transition
ER
Estrogen receptor
LBD
Ligand binding domain
MET
Mesenchymal-to-epithelial transition
MMPs
Matrix Metalloproteinases
PR
Progesterone receptor
SAM
Sterile α-motif
siRNA
Short interfering RNA
TRAIL
TNF-related apoptosis-inducing ligand
VEGFR
Vascular endothelial growth factor receptor
6
Introduction
Signal transduction and receptor tyrosine kinase signaling
The process of signal transduction is vital throughout cellular biology by converting extracellular
signals/cues into the correct intracellular response. The initial response to extracellular stimuli is
most commonly conveyed by the large and diverse group of membrane bound receptor tyrosine
kinases. Proper signaling through these receptors is crucial during development and cellular
homeostasis, as impaired signaling is implicated in numerous developmental defects and human
diseases. During tumorigenesis, receptor tyrosine kinases are frequently mutated and deregulated
resulting in an uncoupling between the extracellular cues and the intracellular response which
mediates cellular transformation and cancer progression.
The receptor tyrosine kinase family consists of 58 proteins in humans which are further subdivided
into approximately 20 groups. Interestingly, the Eph receptor tyrosine kinases and their ligands
(ephrin) constitute the largest subfamily of this receptor class (Robinson et al., 2000). The receptors
were discovered in 1987 in an Erythropoietin producing hepatocellular (Eph) carcinoma cell line
(Hirai et al., 1987) and their ligands as Eph receptor family interacting proteins (ephrin). In contrast
to the common theme of membrane bound receptors and soluble ligands, the ligands of the Eph
receptors are membrane bound. This provides juxtacrine signaling between neighboring cells, thus
restricting the interaction sites. Despite the large number of Eph receptors and ligands encoded in
the human genome, our knowledge about this important signaling pathway remains limited and is
largely overshadowed by the hormone, cytokine and growth factor receptor tyrosine kinases. In this
thesis, the role of Eph/ephrin signaling will be highlighted emphasizing the role during breast cancer
development.
The Eph/ephrin family
In humans, the family of Eph receptors consists of 14 members, which are divided into two
subclasses termed EphA and EphB based on sequence homology and binding preferences for
respectively ephrinA or ephrinB ligands. There are nine known members of EphA receptors (EphA1A8 and A10), six ephrinA ligands (ephrinA1-A6), five EphB receptors (EphB1-B4 and B6) and three
ephrinB ligands (ephrinB1-B3) (Gale et al., 1996; Eph Nomenclature Committee, 1997).The large
number of Eph receptors and ephrin ligands indicates that there is functional variation within the
family which relates to differences in tissue specific expression, protein structure, ligand binding and
intracellular signaling.
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The domain structure of Eph receptors and their ligands, ephrins
As mentioned previously Eph receptors are subdivided into two classes, which is solely based on their
sequence. Nevertheless, the structural composition of EphA and EphB receptors is very similar and
consists of three main parts: an intracellular, an extracellular and a transmembrane domain (Figure
1). The intracellular domain contains four functional units: the juxtamembrane domain, a classical
tyrosine kinase domain, a sterile α-motif (SAM) and a PDZ-domain binding motif. Ligand binding
results in dimerization and the formation of a tetraheterodimer consisting of two receptors and two
ligands (Himanen et al., 2001). When this dimerization occurs, cross phosphorylation takes place and
the receptor is phosphorylated at the conserved tyrosine residues in the juxtamembrane domain.
This phosphorylation leads to a conformation change and provides a binding place for adaptor
proteins such as PI3K and Abl through binding of their SH2 domain. In addition to autophosphorylation, the kinase domain of the receptor can phosphorylate effector proteins such as Src,
PI3K and Shp2 resulting in activation of downstream pathways (Pasquale, 2008).
Figure 1. Conserved structure of the
Eph receptor and the ephrin ligands.
The Eph receptors consists of an
extracellular domain containing a eprinbinding domain, a cysteine-rich region
with two additional low affinity ephrin
binding spots and two fibronectin typeIII repeats. The intracellular domain of
the
Eph
receptor
consists
of
a
juxtamembrane domain with conserved
tyrosines for cross-phosphorylation, a
kinase domain, a SAM domain and a
PDZ-binding domain.
EphrinB ligands contain an extracellular
part with the Eph-binding domain and a
cytoplasmic
tail
which
can
be
phosphorylated and have a PDZ-binding
domain. The ephrinA ligands have also a
Eph-binding domain in the extracellular
part but are anchored in the membrane
by a GPI tail and have no intracellular
domain.
8
In addition to the juxtamembrane domain, the PDZ-binding domain is important for downstream
signaling, as a variety of adaptor proteins are bound by this domain such as GRIP and Syntenin
(Torres et al., 1998). The SAM domain, which is not common for tyrosine kinase receptors, has been
proposed to play a role in receptor oligomerisation by binding to other SAM domains of other Eph
receptors or proteins (Stapleton et al., 1999; Nievergall et al., 2012). The extracellular domain is also
composed of four units: two fibronectin type III repeats, a cysteine-rich region and the globular
ligand-binding domain. The globular ligand-binding domain contains a high affinity binding site for
ephrin. In addition to provide low affinity, additional ephrin binding sides, the cysteine-rich region
has been proposed to play a role in oligomerisation (Kullander and Klein, 2002; Janes et al., 2012).
The
transmembrane
domain
can
stabilize
conformations
and
thereby
facilitate
the
heterodimerisation of the signaling complex. Additionally, it has been proposed that the
transmembrane domain can play a role in altering the conformation of the intracellular domains
resulting in an active conformation enabling signal transduction (Finger et al., 2009).
In contrast to the Eph receptors, the ephrin ligands differ greatly in their structural configuration. In
addition to the classification based on sequence homology and binding preferences, ephrins also
differ in their mode of membrane association. The ephrinA ligands are attached to the membrane by
a glycophosphatidylinositol (GPI) tail, whereas ephrinB ligands contain a classical transmembrane
domain and a intracellular domain which is the site for both tyrosine and serine phosphorylation and
contributes to intracellular signal transduction (see below) (Figure 1) (Kullander and Klein, 2002).
The preference for each subclass and the corresponding ligands is governed by their conformation
and thereby affinity (Chrencik et al., 2006). However, although all receptors and ligands have a
preferred binding partner in their own subclass, the Eph/ephrin family is highly promiscuous and
crosstalk between the subclasses has been described (Figure 2) (Pasquale, 2008; Bowden et al.,
2009). For example, EphB2 prefers binding ephrinB ligands but can also bind to ephrinA5 (Himanen
et al., 2004). The exception on the promiscuity is the EphB4 receptor, which essentially binds to only
ephrinB2 (Chrencik et al., 2006).
Signaling in both the Eph and ephrin expressing cell
Unlike the majority of receptor tyrosine kinases, Eph receptors are activated upon binding of
membrane bound ligands resulting in both forward and reverse signaling mediated by the receptor
and ligand, respectively. Forward signaling is initiated upon ligand binding of the Eph receptor and
results in a response in the Eph expressing cell. Pathways that can be turned on by Eph forward
signaling are PI3K, Abl, Ras and Rho-GTPases resulting in a wide variety of cellular responses such as
migration, adhesion and proliferation (Figure 3C).
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Figure 2. Overview of promiscuity between the Eph
receptors and their ligands. In general, ligands of the same
subclass prefer binding to the receptors of that subclass
(green and purple arrows). Exceptions are binding between
EphA4 receptor with all ephrinB ligands and the EphB2
receptor with the ephrinA5 ligand (blue arrows). The only
family member that does not show large promiscuity is
EphB4, which essentially prefers ephrinB2 binding (red
arrow) Adapted from Salvucci and Tosato, 2012.
In addition to the forward signal, the Eph/ephrin interaction can also stimulate a response in the cell
carrying the ephrin ligand resulting in a process termed reverse signaling which can be mediated by
both ephrinA and ephrinB ligands. Despite the fact that class A ephrins do not have an intracellular
domain, they are capable of attracting transmembrane adaptor proteins for intracellular signaling,
which interact most likely with proteins such as Fyn, a Src family member kinase (Davy et al., 1999)
and p120 to increase adhesion by integrin regulation. For example, activation of ephrinA3 results in
increased adhesion to Laminin mediated by phosphorylation of p120 (Figure 3A) (Huai and Drescher,
2001). Despite the fact that there is a strong correlation between ephrinA reverse signaling and
increased adhesion, the specific adaptor proteins remain unclear. EphrinB induced reverse signaling
is mediated by its intracellular domain, which can be phosphorylated upon binding to the EphB
receptor. As a result of this phosphorylation, a conformational change allows adaptor proteins such
as Grb4 to bind and transduce the reverse signaling (Figure 3B) (Cowan and Henkemeyer, 2001;
Essmann et al., 2008; Pasquale, 2010). This receptor-induced phosphorylation of ephrinB can be
mediated by members of the Src kinase family which in their turn probably are activated by
clustering of ephrinB (Palmer et al., 2002). The adaptor protein Grb4 induces actin cytoskeletal
movements through Abl, Cbl, β-catenin and Rac pathways resulting in both cell morphology changes
and disassembly of focal adhesions. On the other hand, both FAK and Paxilin can bind to the
phosphorylated tyrosines in the juxtamembrane domain of ephrinBs and are involved in the integrity
of focal ashesions (Cowan and Henkemeyer, 2001). Through Grb4 and FAK/Paxilin mediated
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signaling, ephrinB reverse signaling can regulate the focal adhesions and thereby cell-cell adhesion
and polarity in epithelial cells.
In addition to phosphorylation dependent signaling, ephrinB reverse signaling can switch to PDZdomain dependent signaling (Figure 3B). Upon recruitment of a PDZ domain-containing phosphatase,
PTP-BL, the intracellular domain of ephrinB is dephosphorylated, abrogating the phosphorylation
dependent signaling. Moreover, extended association of the phosphatase to ephrinB can facilitate
PDZ domain signaling, not only through the ephrinB PDZ domain, but by the PDZ domains of the
phosphatase as well (Palmer et al., 2002; Qiu et al., 2010). Since ephrinB ligands are capable of
reverse signaling, it is likely that the specific signal transduction is context dependent and thus cell
type specific depending on the Eph receptor on the adjacent cell.
Figure 3. Signaling of Eph receptors and ephrin ligands. EphrinA ligands can signal back via Fyn and P120 although they
lack an intracellular domain and the adaptor proteins are still unknown (A). EphrinB ligands can also signal back after
phosphorylation using Grb4 and Src. EphrinB ligands also can switch to PDZ-domain dependent signaling after
dephosphorylation by PTP-BL (B). Eph receptors can signal through Rho, Ras, Abl and PI3K with a variety of cellular
responses (C). See text for details.
Regulation of Eph and ephrin expression in cells and tissues
In addition to ligand activation regulated of Eph/Ephrin signaling, activation of this pathway can be
controlled through the regulation of Eph/ephrin expression on both the mRNA and protein level.
Since Eph/ephrin signaling is used for cell-cell communication, differential expression of Eph and
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ephrins may result in distinct cellular responses. Neighboring cells express either Eph or ephrin to
contribute to this juxtracrine signaling mechanism (Kullander and Klein, 2002; Miao and Wang, 2012).
To create correct cell-specific Eph signaling, Eph and ephrin expression must be regulated, although
little evidence is present on ephrin regulation, the expression of Eph receptors is under strict control.
Activation of growth factor pathways such as epidermal growth factor (EGF), Her2 and Wnt can result
in upregulation of Eph receptors (Somiari et al., 2004; Clevers and Batlle, 2006; Masood et al., 2006).
Whether this regulation occurs on the transcriptional or post-transcriptional level is not completely
clear. It seems that EphB receptors in the intestine are regulated at transcriptional level by Wnt and
by β-catenin/Tcf supporting the hypothesis that the transcriptional regulation appears to be
dominant (Clevers and Batlle, 2006). This hypothesis is also supported by results that demonstrate
EGFR mediated transcriptional upregulation of EphB receptors in head and neck carcinomas (Masood
et al., 2006) and no clear data is present about post-transcriptional regulation.
Another way to regulate Eph/ephrin signaling is by termination of active receptor-ligand complexes
through internalization of the receptor or protease mediated cleavage of the signaling complex.
Internalization of the receptor, occurring after activation of the complex, is mediated by Racdependent membrane ruffling which induces endocytosis of both the receptor and the ligand
(Marston et al., 2003; Zimmer et al., 2003). The complete complex is endocytosed in one of the cells,
including the full length receptor, full length ligand and a part of the plasma membrane from the
adjacent cell (Figure 4A). This internalization is actin dependent, since actin inhibition abrogates
internalization whereas inhibition of clathrin and caveolae-dependent internalization mechanisms do
not affect the Eph receptor internalization (Marston et al., 2003). After internalization, the complex
has been proposed to be degraded via the lysosomal pathway although this mechanism of
degradation has been shown only for EphB1 (Fasen et al., 2008). Especially the uptake and
internalization of the adjacent cell’s plasma membrane appears to be an exclusive process for
Eph/ephrin internalization and not yet described for other receptors (Marston et al., 2003; Zimmer et
al., 2003). This mechanism might be more widely conserved amongst tyrosine kinase receptors or is
exclusive for Eph/ephrin signaling. It might also contribute to persistent signaling after internalization
since the intracellular domain of the receptor or ligand is still cytoplasmic where the other
intracellular domain is sequestered from the original expressing cell resulting in signaling in only one
of the two cells as long as the endosome is not further transported towards a lysosome.
The other approach to terminate the activity of the signaling complex is cleavage of the Eph/ephrin
complex by proteases (Figure 4B). Downstream signaling of Eph/ephrin signaling can activate ADAM
proteases which are known to cleave Eph/ephrin complexes, thereby regulating the signal
transduction through an extracellular negative feedback loop (Solanas et al., 2011; Miao and Wang,
12
2012). Interestingly, the EphA/ephrinA signaling is typically terminated by proteases whereas the
EphB/ephrinB is generally regulated by internalization. Noteworthy is the observation that the ADAM
proteases are activated by the Eph/ephrinB class but preferentially cleave the Eph/eprhinA class
(Noren and Pasquale, 2004) indicating a mechanism of crosstalk between the two subclasses of
Eph/Ephrin pathways and pointing towards the possibility of mutual repression.
Complex formation and clustering of different Eph receptors
The activation of tyrosine kinase receptors is most often mediated through homo- or heterodimerization of the receptor upon ligand binding. However, the Eph/ephrin signaling provides new
insights in the possibilities, where Eph receptors form complexes with distinct Eph receptors,
resulting in distinct signaling events. For example, recent research demonstrated a role for EphB6, a
kinase dead family member of the EphB receptors which is able to dimerize with Eph receptors from
both the A and B class (Fox and Kandpal, 2011). Binding of an Eph receptor to EphB6 alters the final
cellular response since the binding partner cannot be phosphorylated by the kinase dead receptor
and sequester this Eph receptor to transduce the signal transduction (Figure 5). The binding of EphB6
may contribute to the contrasting roles of Eph/ephrin signaling in both tumor promotion and tumor
suppression (discussed below) (Fox and Kandpal, 2009). Moreover, this is the first evidence that the
Figure 4. Termination of the Eph/ephrin complexes. A. EphB/ephrinB complexes are generally internalized in one of the
cells, including the complete receptor, ligand and a part of the plasma membrane of the adjacent cell. Note that the
complete complex can also be internalized by the ephrin expressing cell. B. EphA/ephrinA complexes are generally cleaved
by ADAM proteases in the extracellular space to terminate signaling.
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Figure 5. Sequestering of Eph receptor signaling by EphB6. EphB6 is a kinase dead family member (C) of the Eph receptor
family and can dimerize with both A and B class receptors (B) after ligand binding (A). By binding to a receptor, cross
phosphorylation cannot take place, and the downstream signaling is inhibited.
different Eph receptor subclasses can dimerize and this concept might also be used among other
tyrosine kinase receptors.
Furthermore, clustering of multiple receptors can occur to regulate the final cellular response (Figure
6). In addition to the standard ligand binding domain, Eph receptors contain two low affinity binding
sites (Pasquale, 2008). When the ephrin ligand binds the receptor and the receptors dimerize and
become activated, these low affinity binding sites are able to bind each other and allow stable
oligomerisation resulting in a large complex with other Eph receptors (Himanen et al., 2010).
Moreover, additional Eph receptors are laterally attracted to the initial Eph/ephrin complex (Janes et
al., 2011) and the ligand binding domain and the cysteine rich domain appear to have a role in this
clustering and lateral attraction (Janes et al., 2012). Strikingly, clustering seems to be ligandindependent although ligand binding sites are involved (Wimmer-Kleikamp et al., 2004). In addition
to the extracellular domain, the transmembrane domains have also been described to contribute to
the cluster formation (Finger et al., 2009). A role for the intracellular domain has not yet been
described, although it is possible that downstream signaling events affect the clustering process by
mediating or inhibiting oligomerisation and thus contribute to a positive or negative feedback loop.
The formation of a large cluster of Eph receptors allows specified downstream signaling, probably
since multiple EphB and EphA receptors can both be involved in the cluster due to oligomerisation
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(Janes et al., 2012). One single ligand-receptor heterotetramer can have specific downstream effects
such as adhesion. However, if another Eph/ephrin combination is also activated, these downstream
effects can affect the same pathways in an opposite manner or activate a distinct pathway such as
inhibiting adhesion or facilitate migration. The balance of the combined signaling of all Eph/ephrin
combinations will determine the activated downstream pathways. Since each combination of
Eph/ephrin can have specific cellular responses, with the clearest differences between the A and B
subclass, there is a large amount of distinct signaling events can result from a limited number of
receptors and ligands. Taken together, a model can be envisaged where not one particular Ephephrin binding determines the effect, but the balance of different Eph and ephrins present on the
cell surfaces will define the final signal transduction (Figure 7). This might have implications when
investigating Eph/eprhin signaling in the context of cancer since not simply up- or downregulation of
one receptor or ligand will determine the final cellular respons.
Figure 6. Oligomerisation of Eph receptors. After ligand binding (A), receptors can dimerize (B) and thereby crossphosphorylate with their kinase domain the adjacent juxtamembrane domain. Mulitple dimers can also oligomerize and
form a large complex (C), including both A and B class receptors and ligands. This complex can also laterally attract other,
not ligand bound, receptors by using the transmembrane domain and the the low affinity binding spots in the SAM domain
(D). The combined cellular responses of all involved receptors define the final cellular response.
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Figure 7. Schematic representation of the balance
defining the final cellular response. When the
balance is even, or only one receptor/ligand couple
is signaling, the cellular response is defined by only
that couple (A). When multiple receptors are
active, here represented as multiple EphA/ephrinA
couples, the balance change to another cellular
response although EphB/ephrinB signaling is
present (B). This can also be the other way around,
that EphB/ephrinB signaling prevails (C). However,
this is not only possible with only the distinction
between A and B class, but probably also within
one class.
The role of Eph/ephrin in cellular homeostasis and development
Eph/ephrin signaling is involved in many processes including neural development (Klein, 2009;
Nomura et al., 2010), patterning of tissues during development such as hindbrain and neural crest
cells (Pasquale, 2008), tissue adhesion and integrity (Lin et al., 2012) and angiogenesis (Salvucci and
Tosato, 2012). During these processes the major roles of Eph/ephrin signaling are related to cell
morphology and regulation of adhesion and migration through the control of attraction and
repulsion. In addition, Eph/ephrin signaling has been described to control cell fate and stem cell
maintenance in a variety of tissues such as the intestine and neural system. During development of
the mammary gland, Eph/Ephrin signaling plays an important role by controlling correct tissue
branching and angiogenesis. For example, EphA2 signaling has been demonstrated to be necessary
for correct branching and proliferation as knock-out mice show deregulated mammary gland
development, due to loss of Rho-A inhibition after HGF stimulation which is used to induce branching
(Vaught et al., 2009). In addition, EphB4 has been shown to be essential for correct vascularization in
the mammary gland since transgenic EphB4 mice show enhanced and aberrant vascularization.
Moreover, EphB4 knockout mice are embryonic lethal and die after 10 days in utero due to the
absence of angiogenesis (Andres and Ziemiecki, 2003). Since multiple processes of normal
development are deregulated or altered in cancer, it is not surprising that Eph/ephrin signaling also
contributes to cancer development and progression in a variety of cancers including breast cancer.
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Breast cancer
Cancer is number one cause of death worldwide, responsible for 13% (7,6 million) of all deaths
(Globocan.iarc.fr). In most western countries, cancer is the second leading cause of death behind
vascular diseases (WHO). Cancer is not a simple disease but a multifactorial and complex process
which often takes years to arise. Every cancer is unique and has its own specific mutations,
development and progression and even within one tumor a large heterogeneity can be present.
Moreover, primary tumors are in general not lethal whereas metastases are. The cells that form the
origin of metastasis have successfully escaped physiologic defenses of the body and often escaped
initial treatment, resulting in an even larger heterogeneity and more aggressive nature of these cells.
Due to these variables and heterogeneity, cancer remains hard to treat and there is a pressing need
to elucidate the molecular mechanisms at the foundation of cancer development and progression to
improve future cancer therapy.
Breast cancer is the most common cancer in women affecting one in eight women and for young
women, under 55, breast cancer is number one in cause of death (KWF). Non-hereditary breast
cancer typically develops after the age of 50 and is an age-dependent cancer. However, several
hereditary mutations are known that contribute to an early onset of breast cancer. The best known
examples are mutations in BRCA1 (Ford et al., 1994), TP53 (Cattoretti et al., 1988) and Her2 (King et
al., 1985). Women carrying these mutations have a significantly higher chance to develop cancer..
Breast cancer can be classified to different subtypes by various characteristics as histology,
differentiation stage, distribution, mutation analysis and receptor expression. The major histological
subtypes are lobular carcinoma and ductal carcinoma characterized by their origin of development,
the mammary gland and the milk duct, respectively (Polyak, 2007). The differentiation stage is
characterized by the comparison to the normal breast tissue and cell types present whereas the
staging for distribution is determined by the presence of cancer cells at different locations around
the tumor origin (UK Cancer Research, 2013). On a molecular level, next generation sequencing can
reveal specific mutations leading to understanding the affected molecular mechanisms which can be
used for a personalized treatment. This, however, is still in research and is not yet being applied on a
large scale in the clinic. Another way to classify breast cancer on a molecular level is by the
assessment of the expression of a number of cellular receptors and this receptor status and is widely
used in the clinic to determine appropriate treatment. The expression level of three receptors is used
to subdivide the tumors, the estrogen receptor (ER), the progesterone receptor (PR) and growth
factor receptors (such as EGFR and HER2). According to the receptor status, cancer cells have a basal
like appearance when all receptors show a negative expression pattern (also called triple-negative)
and luminal when they express the ER, PR and or/EGFR. If the receptor status resembles the normal
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expression, the group is called ‘normal breast-like. (Over)expression of HER2 defines a subgroup on
its own and only these patients react to antibody treatment with trastuzumab (Hudis, 2007).
Due to these differences, not all subgroups show the same overall survival. For example, based on
the distribution stage, the overall survival positively correlates with diagnosis at early stages. This is
in agreement that early stages of breast cancer can be treated well whereas the more advanced
stages are more challenging to treat. Also the receptor status indicates overall survival chances since
upregulation of hormone receptors and growth receptors will make the tumor more sensitive for
growth factors resulting in faster progression. However, HER2 overexpression is an exception for a
poor prognosis based on growth factor expression levels. Although these tumors show enhanced
progression, treatment with the monoclonal antibody trastuzumab is extremely specific to HER2
overexpressing cancer cells and very effective when the tumor is detected in an early stage (Hudis,
2007). However, these personalized specific treatments also have disadvantages as toxic side effects
on other organs and tumor cell resistance can occur which may result in the recurrence of the
cancer.
Figure 8. Different stages of breast cancer. Normal ductal epithelial cells can show hyperplasia, which is benign. When this
hyperplasia continues to grow and disturbs the normal duct, it is the first stage of breast cancer. Since this tumor is stil
incapsulated by a membrane, it is called carcinoma in situ. The second stage is characterized by cells invadin the
surrounding tissue. The last stage is metastases (not shown in figure) when these invasive cells enter the circulation and
start a new tumor at a distant site. Adapted from (Mukhopadhyay et al., 2011)
Breast cancer development
Breast cancer development can be separated into three distinct stages based on morphology (Figure
8). The initial stage is characterized by tumor growth within a basal membrane and is referred to as
an in situ breast cancer. In this initial stage tumor cells acquire the capacity to proliferate
uncontrollably and avoid the induction of apoptosis, resulting in excess growth of the transformed
cells. It has been proposed that this primary tumor harbors cancer stem cells providing a continuous
source for many differentiated cancer cells. Additionally, the tumor induces angiogenesis to obtain
18
oxygen supply and nutrients. Angiogenesis is the process of formation of new blood vessels by
migrating endothelial cells, sprouting from existing blood vessels. In the second stage, the basal
membrane is degraded and the tumor cells are able to invade the surrounding tissue. This process
has been associated with a process called epithelial-to-mesenchymal transition (EMT) where the
differentiated epithelial cells activate genes to de-differentiate back to mesenchymal cells leading to
a more stem cell like phenotype. Moreover, the second stage is characterized by the increased ability
of the cancer cells to migrate and invade into the surrounding tissues through increased motility and
aberrant adhesion. Cell adhesion is necessary in normal tissue to maintain integrity and preserve
functionality and deregulation of cell adhesion and tissue integrity facilitates cancer progression. As
long as the tumor remains in the originating tissue, this is called the invasive stage. Cell motility is
necessary in normal tissues for tissue renewal and regeneration to transport ‘new’ cells to their final
destination. However, deregulated cell motility can facilitate cancer progression in a metastatic way.
The final stage is the metastatic phase, in which the tumor cells have acquired metastatic potential
and have spread through the body through lymph or blood vessels resulting in metastases at distant
sites (Polyak, 2007; Hanahan and Weinberg, 2011). In this final stage tumor cells have to evade the
immune system to survive in the circulation. In addition, tumor cells need to activate a distinct
adhesion mechanism to attach to the metastatic site to induce MET (mesenchymal-to-epithelial
transmission). Moreover, the cancer stem cells are probably involved in metastases by starting
proliferation of a new tumor cell population at a distinct site.
Recent studies have also highlighted a tumor promoting role for the tumor micro-environment, a
new emerging concept in cancer research. In this hypothesis, not only the tumor cells contribute to
the tumor mass but healthy cells as well. These cells support the tumor by providing survival signals
and cytokines, facilitating adhesion and supporting blood supply (Hanahan and Weinberg, 2011).
The expression of Eph and ephrin in breast cancer
Eph receptors and ephrin ligands play a role during various stages of breast cancer development and
have been shown to regulate adhesion and angiogenesis. Large screenings have been performed to
investigate the Eph/ephrin expression profiles in various stages of breast cancer development.
Assessment of the expression of Eph receptors and ephrin ligands in breast cancer patient material
demonstrated a correlation with clinical outcome in a prospective cohort study. Upregulation of
EphA2, EphA4, EphA7, EphB4 and EphB6 mRNA and protein have been demonstrated to be
significantly correlated to both decreased disease-free survival, metastatic-free survival and overall
survival in breast cancer patients (Brantley-Sieders et al., 2011). Moreover, examination of breast
cancer samples with immunohistochemistry and RT-PCR revealed that EphB2 receptor expression is
19
negatively correlated with the disease-free survival period, whereas increased EphB4 expression was
positively correlated to the tumor stage (Wu et al., 2004).
To support these in vivo observations, breast cancer cell lines MCF-10A, MCF-7 and MDA-MB-231
were used to represent the different stages of the normal breast, non-invasive breast tumor and
invasive breast tumor, respectively. Examination of these cell lines revealed that distinct Ephs and
ephrins are either up- or downregulated, depending on the stage of disease (Table 1). For some Ephs
and ephrins it is clear that up- or downregulation is correlated with progression of breast cancer for
example EphA10. However, many Eph receptors and ephrins do not show a clear expression profile
for example EphA2 which is downregulated in non-invasive breast cancer compared to normal breast
cells, but also compared to invasive breast cancer (Fox and Kandpal, 2004). These huge variations in
expression profiles upon all members indicate multiple possibilities for Eph/ephrin function and
implicate a complex regulation of the downstream effects. Another possibility is that there is a
balance between distinct Ephs and ephrins which determines the final cellular response as
mentioned before. In this case, small up- or downregulation can alter the cell response and activity
which can explain some apparent paradoxes.
Although the screening in patients and cell lines provides a good overview of the Eph/ephrin
expression profile in breast cancer, many other studies show more detailed roles of specific
Eph/ephrin family members. Unfortunately, not all members are investigated with the same depth
and their exact role is still unknown. Nonetheless, in breast cancer, EphA2 with ephrinA1/A2, and
EphB4 with eprhinB2, are the most studied receptor-ligand couples and the underlying signaling
mechanisms are slowly starting to be unraveled.
20
Table 1. Eph/ephrin expression profile in patient material and differences between breast cancer stages in breast cancer
cell lines. Upregulation (in green) and downregulation (in red) is shown compared to different stages of breast cancer.
Comparisons with no results are notated with a line (-). Data is based on RT-PCR results from the different cell lines MCF10A, MCF-7 and MDA-MB-231 representing normal, non-invasive and invasive breast cells respectively.
Patient tissue
Breast cancer cell lines (Fox and Kandpal,
2004)
Upregulation correlated with decreased Down
survival,
immunohistochemistry,
Down
Up
Up
Down
invasive cells
EphA2
Non-invasive vs.
-
normal cells
No data
Invasive vs.
EphA1
invasive cells
methods (references)
Normal vs non-
Effect of the Eph family member, used
cohort
study (Ogawa et al., 2000; Zelinski et al.,
2001; Brantley-Sieders et al., 2011)
EphA3
No data
Up
Down
Up
EphA4
Upregulation correlated with decreased Up
Down
Up
Up
Down
-
Up
Up
Up
survival, cohort study (Brantley-Sieders et
al., 2011)
EphA5
Downregulation during progression, DNA Down
methylation assay (Fu et al., 2010)
EphA6
No data
Up
EphA7
Upregulation correlated with decreased Up
survival, cohort study (Brantley-Sieders et
al., 2011)
EphA8
No data
Up
Down
Up
EphA10
No data
Up
Up
Up
EphB1
Downregulation during progression, GWAS -
-
-
Up
Down
study and microarray (Bonifaci et al., 2010)
EphB2
Upregulation
during
progression, Down
immunohistochemistry and cohort study
(Wu et al., 2004)
EphB3
No data
Down
Down
Up
EphB4
Upregulation correlated with decreased Down
Down
Up
21
survival,
immunohistochemistry,
cohort
study (Wu et al., 2004; Brantley-Sieders et
al., 2011)
Downregulation
leads
to
decreased
survival, immunohistochemistry (Berclaz et
al., 2002)
EphB6
Upregulation correlated with decreased Down
Down
Up
Down
Up
survival, cohort study (Brantley-Sieders et
al., 2011)
ephrinA1
Upregulation
during
immunohistochemistry
progression, Up
(Ogawa
et
al.,
2000)
ephrinA2
No data
Up
Up
Up
ephrinA3
No data
Up
-
Up
ephrinA4
No data
Up
-
Up
ephrinA5
No data
-
Up
Down
ephrinB1
No data
Down
Down
Up
ephrinB2
No data
Down
-
Down
ephrinB3
No data
Up
Up
Up
22
EphB4/ephrinB2 in breast cancer metastasis
As already suggested by the results of the screening to Eph/ephrin expression in breast cancer cell
lines, some of the Ephs and ephrins act as tumor suppressor genes, whereas others can act as
oncogenes. The main roles of Eph/ephrin in breast cancer are related to the processes of
proliferation, angiogenesis, cell motility and cell adhesion, although many Eph/ephrin pairs have not
yet been studied in breast cancer. Angiogenesis, cell motility and cell adhesion are important during
breast cancer development, in both the development of the primary tumor as well as during
metastasis. In addition to angiogenesis, EphB4/ephrinB2 regulates cell migration and adhesion,
whereas EphA2/ephrinA1 signaling activates survival pathways as PKB/mTOR (Kaenel, 2012). Since
EphB4/ephrinB2 regulates motility and adhesion, the emphasis here will lay on these signaling
partners and a discussion of their role during breast cancer progression. The focus will be in
particular on processes involved in regulating metastasis since EphB4/ephrin signaling is likely
involved in many processes facilitating metastasis.
Figure
9.
Morphology
mammary
of
gland
the
during
development (A) and pregnancy (B).
The ductal and luminal epithelial cells
are
covered
by
myoepithelial
a
layer
cells.
of
During
development (A), the cap cells grow
forward with concomitant apoptosis
of the body cells, creating a duct or
alveoli.
During
pregnancy,
this
process is restarted and branching is
expanded for more alveoli and ducts
capable of milk production and
drainage. Adapted from Gajewska
and Zielniok, 2013
The expression and regulation of EphB4 and ephrinB2 in normal mammary
tissue and breast carcinoma
Before understanding the role of EphB4/ephrinB2 during breast cancer development in detail, the
normal expression of EphB4 and ephrinB2 will be discussed. In normal mammary tissue, EphB4 is
expressed in myoepithelial cells of ducts and alveoli whereas ephrinB2 is expressed in the adjacent
cells, the luminal epithelium (Figure 9) (Nikolova et al., 1998; Vaught et al., 2009). EphB4 and
ephrinB2 are upregulated in the proliferative, estrogen dependent stage of the estrus cycle.
23
Moreover, both are regulated by estrogen since in the ovariectomized mice which cannot produce
estrogen, EphB4 and ephrinB2 are completely absent. Strikingly, the RNA level is not affected by
estrogen indicating that EphB4 is not regulated on the transcriptional level but rather seems to
depend on a post-transcriptional regulation (Nikolova et al., 1998).
Regulation of EphB4 on
transcriptional level is most predominantly regulated by EGFR through the Jak-Stat pathway and
PI3K, since inhibition of either EGFR, Jak or PI3K with chemical compounds in SKBR3 breast cancer
cells leads to a complete absence of the EphB4 receptor. Inhibition of Src, another downstream
target of EGFR, also leads to a reduction of EphB4 expression of around 75% indicating that EGFR is
indeed the dominant regulator. In addition to EGFR, EphB4 expression can be regulated by HER2 on
the transcriptional level since transfection of Her2 in mouse fibroblasts was sufficient to induce
expression of Ephb4 in these cells (Kumar et al., 2006). An association between EphB4 and another
major player in cancer development, Wnt, has been observed in colon cancer, where active Wnt
pathway has been demonstrated to enhance tumor growth and sustained proliferation by
upregulation of EphB4 (Clevers and Batlle, 2006). Although in breast cancer Wnt signaling also
contributes to tumor growth (Mohinta et al., 2007) an association with Eph/ephrin signaling has not
yet been discovered. In addition to aberrant regulation of EGFR and Her2 in breast cancer cells
(Kumar et al., 2006), overexpression of EphB4 in mammary tumor cells can be driven by gene
amplification (Somiari et al., 2004). In contrast to the knowledge of EphB4 expression, very little is
known about the transcriptional regulation of ephrinB2. Hif1 has been suggested to play a role in
ephrinB2 upregulation under hypoxic conditions during the development of the central nervous
system of the zebrafish embryo (Stevenson et al., 2012). However, this transcriptional regulation
occurs in neuronal development, and is not yet shown in epithelial cells. Although, HIF-1α does play a
role in breast cancer progression, where the hypoxic tumor environment and estrogen most likely
activate HIF1α (Kimbro and Simons, 2006), the link between HIF1α with either upregulation of EphB4
or ephrinB2 in breast cancer is not shown yet.
In breast tumors, the balance between EphB4 and ephrinB2 is deregulated. For example, in murine
mammary carcinomas, EphB4 shows an aberrant expression profile with expression at epithelial cells
instead of myoepithelial cells whereas expression of ephrinB2 is lost (Nikolova et al., 1998). Other
studies show seemingly contradictory results with loss of EphB4 in human tissue samples during
tumor progression (Berclaz et al., 2002) or an indication that in the majority of the tumor EphB4
expression is lost but upregulated in the periphery of human tumors (Kaenel, 2012).
Taken together, EphB4 and ephrinB2 are most likely regulated on a transcriptional level although the
exact mechanisms in breast cells have to be elucidated. Nevertheless, since breast cancer tissues
show aberrant expression of either EphB4 or ephrinB2, a role in breast cancer is likely.
24
The use of EphB4 and ephrinB2 as diagnostic marker
Several members of the Eph/ephrin family has been proposed as diagnostic markers in breast cancer
as their expression appears to correlate with tumor stage and patient survival and disease
progression. For this reason, breast cancer patients can be screened for their Eph/ephrin expression
profile to give an indication of the progression of the tumor. EphB4 is used as one of these
biomarkers and can predict clinical outcome (Brantley-Sieders et al., 2011). For these clinical
predictions, it is assumed that the increased expression of EphB4 is positively correlated with tumor
progression and thereby tumor stage, based on previous studies (Wu et al., 2004; Kumar et al., 2006;
Brantley-Sieders et al., 2011; Chen et al., 2011). However, in contrast to the evidence that
EphB4/ephrinB2 signaling can act to promote tumor development (Wu et al., 2004), it has also been
demonstrated to act in a tumor suppressive manner, with the expression of EphB4 being lost in late
stage breast cancer (Berclaz et al., 2002). It has to be noted however that the examination of protein
expression with immunohistochemistry on human tissue samples does not assess the activity of the
complex and therefore might not be sufficient to determine the actual role of this pathway in breast
cancer development. Similar to the contrasting data from patient material, a large number of in vitro
and in vivo studies have also yielded contradictory results on the role of this receptor ligand family in
breast cancer. To further investigate possible explanations for these contradictions, different aspects
of EphB4 and ephrinB2 signaling are discussed below.
Tumor suppressive roles for EphB4/ephrinB2
Since one of the functions of EphB4/ephrinB2 is maintaining tissue integrity, deregulation can
provide the basis for tumor progression (Noren and Pasquale, 2007). Despite the fact that Ephb4 is
upregulated in most breast cancers and breast cancer cell lines (Fox and Kandpal, 2004; Wu et al.,
2004; Kumar et al., 2006), it should be noted that the increased expression of EphB4 does not
directly link to activation of EphB4 signaling. The receptor activity depends on phosphorylation and
research has shown that overexpressed EphB4, in the absence of ephrinB2, is less phosphorylated in
MDA-MB-435, MCF7 and MDA-MB-231 cells and thus expression of this receptor does not correlate
to the activation status of this pathway (Noren et al., 2006).
The EphB4 receptor activates a tumor suppressive pathway directly in the presence of its ligand
ephrinB2. In a mouse xenograft model, non-invase MCF-7, treatment with ephrinB2-Fc reduced
tumor growth in vivo. These tumor cells, which express both ephrinb2 and Ephb4, display an active
Abl-Crk pathway, resulting in more adhesion, differentiation and less proliferation, whereas invasive
MDA-MB-435c tumor cells in a xenograft mouse model do not show an active Abl-Crk pathway. In
addition, activating the Abl-Crk pathway by EphB4 activation with eprhin-Fc in invasive MDA-MB25
Figure 10. Signaling of the EphB4 receptor via Abl-Crk pathway.
When EphB4 is phosphorylated, Abl is required for concomitant
Crk phosphorylation on Tyr221. Due to this phosphorylation, Crk
is no longer able to act as scaffold for other proteins since the
binding site is blocked resulting in inhibition of tumor growth.
When the EphB4 receptor is not able to phosphorylate Crk or Crk
is mutated, tumor growth is stimulated.
435c cells inhibits the proliferation, migration, invasion and viability of these cancer cells. This has
also been demonstrated in vitro in MDA-MB-435c, MCF7 and MDA-MB-231 cells, were treatment
with soluble ephrinB2-Fc induces EphB4 phosphorylation with concomitant Crk phosphorylation,
resulting in activation of the Abl-Crk pathway and reduced tumor growth (Figure 10) (Kumar et al.,
2006; Noren et al., 2006). Due to phosphorylation on Tyr221, Crk is no longer able to function as
adaptor protein, since the binding domain is no longer recognized, resulting in reduced proliferation
and migration of the cell. Two Abl family members, Abl and Arg, contribute to the phosphorylation of
Crk by EphB4 probably by acting as a scaffold through binding to a SH-binding site at the EphB4
receptor in addition to their contribution as directly activating kinase for Crk. Moreover, Abl is
required for the EphB4 dependent phosphorylation of Crk since Abl mutants can promote
tumorgenesis and in these cells no phosphorylated Crk is detected. Supportive data was obtained in
experiments with a constitutively active Crk mutant and knockdown of Abl with Gleevec. In all these
cases, active Crk is able to convey pro-migratory and survival signals and thus promote tumorigenesis
in the absence of the downstream effects of EphB4/ephrinB2 inhibitory signals (Noren et al., 2006).
Taken together, it is likely that the tumor suppressive effects of EphB4 and ephrinB2 are effective
through the Abl-Crk pathway.
The role of EphB4/ephrinB2 during a dhesion
Reduction of EphB4 receptor expression has been correlated to increased invasive characteristics of
both mammary tumors and breast cancer cell lines (Berclaz et al., 2002). EphB4 sequestering with
ephrinB2-FC both in vivo and in vitro in MDA-MB-435c cells resulted in reduction of active Crk and
induced a dramatic reduction of matrix metallo-proteinase 2 (MMP-2) expression (Noren et al.,
26
2006). Metallo-proteinases (MMPs) are secreted proteases that cleave adhesion molecules and
thereby facilitate cell migration (Vargová et al., 2012). When MMP-2 is present it degrades the
extracellular matrix and facilitates spreading, migration and invasion. Upregulation of MMP-2 is
mediated by Rac induced activation of Crk. As a consequence of inactivation by phosphorylation of
Crk by EphB4 inhibits MMP-2 expression resulting in a less invasive phenotype of the MDA-MB-435
cells (Noren et al., 2006). Moreover, in breast cancer cells ephrinB2-dependent phosphorylation of
EphB4 induces integrin mediated adhesion by upregulation of β1-integrin. Knockdown of EphB4 in
non-invasive MCF-7 breast cancer cells results in the acquisition of an invasive phenotype similar to
those observed in more metastatic MDA-MB-435 breast cancer cell lines (Noren et al., 2009).
Supportive for the tumor suppressive function of EphB4 is the fact that addition of ephrinB2 to
cancer cells overexpressing EphB4 leads to decreased proliferation of cells in the tumor mass by
inhibition of Ras (Noren et al., 2006). This has also been demonstrated to occur in leukemia cells
(Suzuki et al., 2010) and might thus be a more general mechanism of Ephb4 mediated tumor
suppression. Moreover, MDA-MB-435c, MDA-MB-231 and MCF7 cells show less motility and invasion
after treatment with ephrin-Fc and thereby more adhesion (Noren et al., 2006).
Induction of apoptosis by EphB4/ephrinB2 signaling
In addition to the reduced migratory phenotype observed in breast cancer, another study has shown
that activation of Ephb4 can contribute to the induction of apoptosis. Prolonged stimulation with the
soluble ephrinB2-Fc in EphB4 overexpressing MCF10A-B4 cells resulted in reduced proliferation, but
was also demonstrated to activate apoptotic pathways in these breast cancer cells. Stimulation with
ephrinB2-fc resulted in increased caspase-3/7 and 8 activity compared to non-treated control breast
cancer cells suggesting that stimulation of the EphB4 receptor induces apoptosis (Rutkowski et al.,
2012). This mechanism is similar to the role described in normal development of the mammary gland
where overexpression of EphB4 during pregnancy of mice induces apoptosis, although it has to be
noted that the expression of its ligand has not been studied (Munarini et al., 2002). Moreover,
spheroids, used to study three-dimensional organization, grown out of MDA-MB-435 breast cancer
cells treated with the soluble ephrinB2-Fc are smaller than non-treated spheroids and staining of
dead cells with Trypan blue, and observation of DNA fragmentation, revealed a high level of
apoptotic cells. This apoptosis is induced via the Abl-Crk pathway since inhibiting this pathway with
chemical compounds results in less apoptotic cells (Noren et al., 2006).
Together, these data indicates that functional EphB4/ephrinB2 signaling can inhibit tumor formation
by maintaining tissue integrity and homeostasis in a ligand dependent manner. It is thus possible that
through aberrant EphB4 expression and activity or down regulation of its ligand ephrinB2, cancer
cells evade the tumor suppressive functions of EphB4/ephrinB2 signaling.
27
Tumor promoting roles of EphB4 and ephrinB2
In contrast to a potential role as a tumor suppressor, EphB4/ephrinB2 is also associated with tumor
development and progression. As mentioned before, EphB4 is observed to be upregulated in a
majority of human breast tumors and in breast cancer cell lines (Kumar et al., 2006; Brantley-Sieders
et al., 2011). Since EphB4/ephrinB2 receptors and ligands has been demonstrated to be upregulated
in tumors, their signaling may actively contribute to tumor progression (Fox and Kandpal, 2004; Wu
et al., 2004).
Tumor promotion by enhanced proliferation and survival of tumor cells
One mechanism of tumor promotion by EphB4 is through enhanced proliferation modulated by
activation of the PI3K/PKB pathway after phosphorylation of Src by EphB4, leading to proliferative
downstream signals in MCF-7 breast cancer cells and MM1 endothelial cells. Activation of these
pathways was assessed by detection of induction phosphorylation of specific phospho-tyrosines on
PKB or Src after stimulation of EphB4 with ephrinB2-Fc (Steinle et al., 2002; Kumar et al., 2006). In
addition, also in ovarian cancer cells, suppression of EphB4 with antisense EphB4 leads to a
inactivation of the PI3K/PKB pathway as measured by the level of phosphorylated PKB (Ma et al.,
2012) indicating a general link between EphB4 activity and the PI3K/PKB pathway. Furthermore,
another study has shown increased proliferation of invasive MDA-MB-435 breast cancer cells after
stimulation with ephrinB2. Interestingly, these cells were transfected with an truncated form of
EphB4 missing the cytoplasmic domain suggesting a signaling pathway independent of the kinase
activity of the EphB4 receptor (see below) (Noren et al., 2004).
Furthermore, supportive data has been obtained by a depletion of EphB4 in MCF-7 breast cancer
cells, through siRNA mediated knockdown, which was demonstrated to result in decreased
proliferation and the induction of apoptotic pathways. The apoptosis induced through EphB4
knockdown is characterized by higher levels of the pro-apoptotic proteins caspase-8 and 9 and TRAIL
(TNF-related apoptosis-inducing ligand). In addition to increased pro-apoptotic factors, the antiapoptotic factors Mcl-1 and Bcl-XL are down-regulated resulting in a sensitization to apoptotic signals
after EphB4 knockdown. Similarly, in TRAIL sensitive cells A549 cells which do not have endogenous
EphB4 expression, transfection of Ephb4 increased resistance against TRAIL induced apoptosis
supporting the notion that EphB4 acts as survival factor for cancer cells (Kumar et al., 2006)
Increased migration of tumor cells by EphB4/ephrinB2 signaling
In addition to proliferation, EphB4 and ephrinB2 are linked to migration and metastasis. One of the
used mouse models to study human breast cancer are the NeuT transgenic mouse which have an
28
activated Neu oncogene (homologue of Her2) in only mammary gland tissue (Muller et al., 1988).
Transgenic NeuT mice show typical non-malignant tumor growth after a latency time of 6 months,
while upon overexpression of EphB4 this latency time is significantly decreased. Moreover, the
double transgenic NeuT/EphB4 mice show lung metastases while in normal NeuT mice metastases
are never observed. These results strongly suggest an oncogenic role for EphB4 signaling in both
tumor development, progression and metastasis (Munarini et al., 2002). In addition to EphB4,
overexpression of a truncated form of ephrinB2, only mediating forward signaling, in NeuT transgenic
mice also significantly increased primary tumor formation and subsequent metastases formation
(Haldimann and Custer, 2009). A possible metastatic role for EphB4 involves regulation of Ecadherins. Overexpression of EphB4 in transgenic NeuT mice results in cytoplasmic localization and
downregulation of E-cadherin and impaired polarization in the mammary gland (Haldimann and
Custer, 2009; Kaenel, 2012). This effect is mediated by EphB induced direct activation of the protease
ADAM10 resulting in cleavage of E-cadherin and shedding of E-cadherin which impairs cellular
adhesion. These results suggest that EphB4 affects adhesion and polarization. Interestingly, ADAM10
cleaves EphA receptors indicating that EphB receptors can modulate EphA receptor function,
suggesting cross talk between the A and B subclasses. For example, EphA2 is necessary for adhesion
where EphB4 activation of ADAM10 can interfere with this adhesion through cleavage of both EphA2
and E-cadherin and thereby facilitate migration (Kaenel, 2012).
However, EphB4 and ephrinB2 are not sufficient for tumor initiation since mice overexpressing only
EphB4 or ephrinB2 in the absence of NeuT never develop tumors (Munarini et al., 2002; Kaenel et al.,
2011). Together, these data indicate that overexpressed EphB4 is sufficient for malignant
transformation but not tumor initiation.
In addition to cell-cell contact, interaction with the extracellular matrix is important to facilitate
migration. Activation of the EphB4 receptor through ephrinB2 in vascular endothelial cells results in
migration through the ECM by upregulation of MMP2 and -9 (Steinle et al., 2002). The link between
EphB4 and MMPs has also been observed in breast cancer, where siRNA mediated knockdown of
EphB4 in non-invasive MCF-7 breast carcinoma cells resulted in a 60% reduction of MMP-2 and
MMP-9 on both protein and mRNA level (Kumar et al., 2006), which could be dependent on
activation of the PI3K/PKB pathway and subsequent MMP2 and MMP9 upregulation (Steinle et al.,
2002). This is in contrast to the study showing tumor suppressive roles by downregulation of MMPs
after EphB4 stimulation (Noren et al., 2006).
Another possible contribution of EphB4/ephrinB2 signaling to migration is through increased cell
motility. One of the major characteristics of motile and migrating cells is the formation of filopodia
and lamellipodia by actin cytoskeleton remodeling. Knockdown of ephrinB2 in zebrafish embryos
29
showed less filopodia and lamellipodia in migrating intersegmental vessel tip cells and was also
shown in cultured endothelial cells (Wang et al., 2010). Moreover, in prostate cancer cells
knockdown of either EphB4 or Cdc42, required for actin cytoskeleton remodeling, inhibited the
migration and showed less Cdc42 activity concomitant with less lamellipodia and filopodia (Wang,
2012).
In melanoma cells, EphB4 activity is also linked to migration. RhoA controls cell morphology by
remodeling the actin cytoskeleton and EphB4 activity increases RhoA activity through the receptor
kinase domain of EphB4. (Yang et al., 2006). However, a direct link with RhoA and EphB4 in breast
cancer cells is not yet described although EphB4 induced migration via RhoA in breast cancer cells
could be possible if this mechanism is conserved between migrating epithelial cells. In addition to a
likely connection between EphB4 and RhoA, EphB2 is linked to activation of Rho in breast cancer cells
(Noren and Pasquale, 2004) indicating possible redundant roles in the same pathways for distinct Eph
receptors.
EphB4 and ephrinB2 induce angiogenesis to promote tumor progression
Another function of EphB4/ephrinB2 activation is the regulation of angiogenesis. In the context of
migration and metastasis, angiogenesis is important as all tumors are reliant on blood supply for
oxygen and nutrients. Moreover, angiogenesis can facilitate metastasis by enabling extravasation of
tumor cells directly into the circulation. EphB4 is expressed on venous epithelial cells and ephrinB2
on arterial endothelial cells, arterial angioblasts and the mesenchyme cells (Wang et al., 1998;
Salvucci and Tosato, 2012). By mutual attraction and repulsion, EphB4 and ephrinB2 guide de novo
vessels through the tumor mass where ephrinB2 is expressed on blood vessels (Noren et al., 2004).
Both EphB4 and ephrinB2 have been shown to be essential for vessel formation since knockout mice
are embryonic lethal due to a severely impaired vascular system. Angiogenic sprouting is induced by
a VEGF-A gradient, which attracts the tip cell to the correct surrounding endothelial cells. EphrinB2
has been indicated as a regulator of tip cell migration by promoting the formation of filopodia. EphB4
regulates adhesion of the migrating tip cell by interacting with the ephrinB2 ligands (Salvucci and
Tosato, 2012). Moreover, EphB4 is linked to activation of cytoskeleton modulators such as the Ras
and Rho GTPases (Noren and Pasquale, 2004).
EphB4/ephrinB2 are also involved in proliferation and migration through regulation of the PI3K/PKB
pathway during angiogenesis. In the migrating phase of new vessels, the EphB4/ephrinB2 activate
MMP2 and –9 downstream of PI3K and PKB, facilitating the growth of the new vessels by disrupting
adhesion to the ECM (Steinle et al., 2002). This process during angiogenesis is similar to the process
of migration of cells during tumor development.
30
Metastasis takes place through lymph nodes or direct extravasation of cancer cells into the
circulation which can be facilitated by vessels nearby. Therefore, angiogenesis is not only necessary
for primary tumor growth but also in the process of metastasis. However, when normal NeuT tumors
and metastatic NeuT/EphB4 induced tumors are compared, the vascularization density was not
different and the vessels were adequately formed. This suggests that the angiogenic promoting
characteristic of EphB4/ephrinB2 signaling is not the main contributing factor for metastasis
formation (Kaenel, 2012).
EphB4 and ephrinB2 positively regulate stem cell maintenance
The cancer stem cell hypothesis assumes that a tumor is a largely heterogeneous and differentiated
cell population which is founded by specific cancer stem cells. Moreover, it is assumed that the
tumor cells with stem cell characteristics are capable of forming a new tumor and are therefore the
origin of metastasis
(Kaenel, 2012). Both Eph/ephrin A and B class play a role in stem cell
maintenance although distinct members have been implicated to be either positively or negatively
regulating the stem cell niche (Genander and Frisén, 2010). In the mammary gland, EphA1,2,3,
EphB2,3,4 and ephrinB1,2 are enriched in the mammary stem cell fraction (Ji et al., 2011). Since both
EphB4 and ephrinB2 are upregulated in the mammary stem cell fraction, the possibility arises that
EphB4/ephrinB2 can contribute to tumor progression by affecting the stem cell niche.
It has been shown that metastatic tumors in NeuT/EphB4 and NeuT/ephrinB2 transgenic mice
contain enriched fractions of cells with markers for undifferentiated progenitors such as CD24low and
CD49fhigh. Although primary mammary tumors also show an enrichment of stem cells, this
enrichment is even greater in metastatic tumors. Moreover, transgenic mice only overexpressing
EphB4 or ephrinB2 do not develop tumors but the mammary glands show an enriched population of
normal stem cells (Kaenel et al., 2011). Defective ephrinB2 signaling interferes with the stem cell
niche, leads to disturbed compartmentalization and a shift to more differentiated cells. Thus, the
reverse signaling is indispensable for maintaining the stem cell niche probably by maintaining correct
cell-cell contacts (Kaenel et al., 2012).
EphB/ephrinB are not only linked to stem cell homeostasis in breast cancer. In adult neural stem cells
a role for EphB4/ephrinB2 has also been described (Qiu et al., 2008, 2010; Nomura et al., 2010).
Moreover, also in colon cancer, EphB4 expression is positively associated with maintenance of the
stem cell niche which might indicate similar roles for EphB4/ephrinB2 signaling (Genander and
Frisén, 2010; Solanas et al., 2011). In addition to a role for maintaining the stem cell niche, a role in
cell identity, lineage specification (Nomura et al., 2010), regeneration and stem cell plasticity (Qiu et
al., 2010) has also been suggested. Since EphB receptors and their ligands are expressed in other
31
adult stem cell niches (Holmberg et al., 2006; Kaenel et al., 2011), a role in regeneration and
plasticity might be a more widely conserved theme in EphB/ephrinB signaling.
It is also possible that by deregulating the EphB4/ephrinB2 signaling, metastatic potential can be
acquired before tumor initiation (Kaenel et al., 2011, 2012; Kaenel, 2012). In this case, tumor cells
acquire stem cell properties and the tumor potential is more malignant from the start.
Taken together, EphB4 and ephrinB2 have an oncogenic role although there is seemingly a paradox
as there may be a role as tumor suppressor as well.
Dependent and independent signaling of both EphB4 and ephrinB2
To understand the contrasting data described above, it is relevant to examine the intracellular
signaling pathways activated by both the receptor and ligand. One possible explanation is that ligand
or receptor independent signaling is involved. In this case, the receptor can signal without the
presence of the ligand or the ligand can signal in the absence of receptor binding (Figure 11).
Physiological, independent signaling might occur when cells lose contact with the adjacent cell or
when an adjacent cell loses its receptor or ligand expression which results in the lack of normal
receptor-ligand interaction.
EphB4 can signal in a ligand dependent and independent manner
As described before, the tumor suppressive role of EphB4 was only observed in the presence of the
ligand ephrinB2 (Figure 11A) (Noren et al., 2006; Rutkowski et al., 2012) but never in the absence of
the ligand. This observation leads to the hypothesis that there might be ligand dependent and
independent signaling of the EphB4 receptor. Evidence to support this hypothesis is the fact that
highly expressed EphB4 is not phosphorylated in the absence of ephrinB2 (Figure 11D) (Noren et al.,
2006). However, another study demonstrated contrasting results where EphB4 lacking the ligand
binding domain, and thus cannot bind the ephrinB2 ligand, is phosphorylated at the same levels as in
the presence of ephrinB2 (Figure 11F). In this case phosphorylation might be triggered by receptor
clustering or by activation through distinct receptors (Noren et al., 2009). Strikingly, in both
observations where EphB4 is phosphorylated either in the presence or absence of ephrinB2, tumor
growth is induced.
This indicates that EphB4 can signal without ephrinB2 binding and can also be phosphorylated
without ephrinB2 binding although this phosphorylation may trigger different functional phenotypes.
A dominant negative, truncated ephrinB2, which is only capable of inducing forward signaling,
induces increased tumor progression in a transgenic NeuT mice suggesting that forward signaling of
the EphB4 receptor is essential for tumor promotion (Figure 11B) (Haldimann and Custer, 2009). In
contrast, when MCF10A tumor cells with overexpression of EphB4 are stimulated with soluble
32
Figure 11. Different studies show different cellular responses in EphB4/ephrin signaling. Summary of all described
controversies and studies. EphB4 and ephrinB2 show reduced tumor growth when they are both present and EphB4 is
phosphorylated after ephrinB2 binding (A). A truncated form of ephrinB2 is still capable of binding EphB4 and induce
phosphorylation dependent forward signaling of EphB4 resulting in increased tumor growth and metastasis (B) A truncated
form of EphB4 is still capable of bindin ephrinB2 and induce reverse signaling of ephrinB2 resulting in increased tumor
growth, but less metastasis (C). Only EphB4 expression in the absence of ephrinB2 results in increased tumor growth and
metastasis (D). Inhibiting the kinase function of EphB4 in the presence of ephrinB2 results in increased tumor formation (E).
EphB4 in the absence of ephrinB2 can also be phosphorylated by an unknown mechanism and results in increased
migration (F). Only ephrinB2 expression in the absence of EphB4 results in less survival and migration of breast cancer cells
and inhibits tumor growth (G). A combination of a truncated form of both the EphB4 receptor and the ephrinB2 ligand
results in no internalization and a strong adhesion (H). For details, see text.
33
ephrinB2, also only capable of inducing forward signaling, EphB4 is phosphorylated and showed
reduced cell migration and anchorage-independent growth (Figure 11A) (Rutkowski et al., 2012).
Nevertheless, when the kinase activity of EphB4 is blocked with PP2, a known inhibitor of Src kinases,
in human dermal microvascular endothelial cells, the migration inhibition by ephrinB2 is lost (Sturz et
al., 2004), although abolishing the kinase domain of EphB4 did not interfere with fibronectin
adhesion in Ba/F3 cells (Sakamoto et al., 2004), indicating that the kinase domain is necessary for
migration but not for adhesion (Figure 11E). Together, these observations indicate that the balance
of both forward and reverse signaling is important for tumor growth inhibition.
Adhesion appears to be ligand-dependent since injected transfected A375 cells expressing EphB4
preferentially spread and adhere to ephrinB2 expressing tissues in mice, thereby facilitating
metastasis by creating a niche (Héroult et al., 2010). Moreover, EphB4 signaling in the presence of
ephrinB2 mediates integrin adhesion (Figure 11A) whereas EphB4 signaling in the absence of
ephrinB2 inhibits integrin mediated adhesion (Figure 11D) by regulating the expression of integrins.
This was tested by treating MCF-7 cells with either soluble ephrinB2 to mimic the presence of
ephrinB2 or soluble EphB4 to sequester endogenous ephrinB2 sequentially with blocking the ephrin
binding site of EphB4 to mimic the absence of ephrin (Noren et al., 2009). In addition, EphB4 lacking
the intracellular domain increases tumor growth in MDA-MB-435 cells (Figure 11C) (Noren et al.,
2004) but metastasize less when transformed A375 cells with truncated EphB4 are injected in mice
compared to cells expressing the full length EphB4 receptors (Héroult et al., 2010). This indicates an
essential role for the intracellular domain in adhesion. However, which domain of the intracellular
part is necessary has not been examined and it is likely that different domains have different
contributions to metastasis.
It has been shown also in vitro that the transmembrane domains of all human tyrosine kinase
receptors can contribute to dimerization after ligand binding (Finger et al., 2009) although it is
possible that these domains facilitate dimerization only after a threshold amount of receptor is
reached and can cooperate in ligand independent signaling through clustering of receptors without
ligands. If the transmembrane domains contribute to lateral attraction, it is possible that distinct Eph
receptors mediate the ligand independent induced migration by clustering via their transmembrane
domains. However, this is not likely since a cell line generated which only express EphB4 and lacking
all other Eph receptors is capable of migration indicating that clustering of different classes of
receptors is not required for migration (Sturz et al., 2004).
One mechanism for ligand-independent signaling is that the EphB4 receptor could act as a scaffold
protein for proteins in the absence of ephrinB2 (Pasquale, 2008). One study shows that in Ba/F3 cells
34
non-phosphorylated EphB4 associates with p38 and p120, which activate other tyrosine kinases. In
this case, the kinetics of the reaction are slower but do initiate a response (Sakamoto et al., 2004).
All together, these studies suggest that EphB4 in the absence of ephrinB2 can act as tumor promoter
by inducing migration whereas the combination of EphB4 and ephrinB2 forward signaling by the
kinase domain acts tumor suppressive when activating adhesion. However, how these distinct
cellular responses exactly are regulated and if they are regulated by the same signaling pathways still
remain unclear.
EphrinB2 can signal independently of the EphB4 receptor
In addition to ligand-dependent and independent EphB4 signaling, receptor independent signaling of
ephrinB2 is also possible. Blocking ephrinB2 with specific antibodies inhibits tumor growth in
xenograft mice, associated with reduced angiogenesis. One study investigated two antibodies; one
blocks EphB4-ephrinB2 binding, whereas the other only blocks ephrinB2 reverse signaling.
Surprisingly, neither antibodies block phosphorylation of ephrinB2 indicating an alternative
mechanism of intracellular phosphorylation of ephrinB2. However, both antibodies reduce migration
and tumor size and increases apoptosis in the xenograft mice which indicates that the observed
effect must be independent of ephrinB2 by phosphorylation. A conformation change induced upon
antibody binding might explain the altered cellular response when treated with the antibodies
(Abéngozar et al., 2012). Moreover, monomeric soluble EphB4 also blocks ephrinB2 reverse signaling
and induces tumor growth inhibition in MCF-7 xenograft mice (Figure 11H) (Martiny-Baron et al.,
2004; Kertesz et al., 2006). A shift to PDZ domain dependent signaling of the ephrinB2 intracellular
domain (see introduction) might also contribute to receptor independent signaling although this is
not investigated yet.
On the other hand, EphB4 knockdown inhibits breast cancer cell survival, migration, invasion and
tumor growth in xenograft mice (Pasquale, 2008). Knockdown of only EphB4 indicates that ephrinB2
is still expressed and might be able to convey signals. Supporting this notion, overexpression of
ephrinB2 leads to less proliferation and delayed development of the mammary gland through
reduced initiation of differentiation. On the other hand, upon lactation, overexpression of ephrinB2
mammary glands results in increased proliferation (Figure 11G). However, this highly proliferating
tissue shows an adequate integrity suggesting overexpressed ephrinB2 still maintains the tissue
architecture including correct ducts and alveoli. Moreover, ephrinB2 overexpression in NeuT
transgenic mice does not increase tumor induction latency although some metastasis are observed in
contrast to normal NeuT mice (Haldimann and Custer, 2009). These results indicate that ephrinB2-
35
independent signaling is not tumor promoting although it might function in adhesion and thereby
facilitating metastases by adherence of circulating cells.
Knock-out of ephrinB2 in the mammary gland using Cre-Lox in mice showed increased apoptosis and
a concomitant increase of cell proliferation, which might indicate a role for ephrinB2 in regeneration
(Rohrbach et al., 2009). However, this observation was made during lactation and thus healthy
tissue. Nevertheless, in cancer more pathways are disturbed and the hormone levels differ from the
lactation period so it might be possible that this also occurs during cancer.
Nevertheless, overexpression of ephrinB2 might also induce overstimulation of EphB4 and the effects
must be ascribed to enhanced forward signaling. Taken together, ephrinB2 most likely acts as
survival factor for the cells with ephrinB2 expression and is therefore essential for tissue integrity.
However, most of the research focuses only on one component of the bidirectional signaling
pathway, either on EphB4 forward signaling or on ephrinB2 reverse signaling. Moreover, most
studies do not include proper experiments or controls for the other site of the signaling pathway
which might bias their results. Especially the exact role of reverse signaling in the absence of EphB4
should be examined in more detail to help clarify the controversial data.
In addition to correct distinguish the independent signaling, it is also important to test the actual
activity and functionality of the proteins instead of the levels. Almost all studies investigate only the
protein or mRNA levels but never take in account that mutations can also lead to altered signaling.
36
Discussion
Distinct cellular responses of EphB4/ephrinB2 signaling dependent on
complex formation and clustering
As described, EphB4/ephrinB2 signaling is complex and many studies show conflicting data. A
possible explanation for the different cellular responses of EphB4/ephrin signaling is the presence of
ligand-dependent and independent signaling. It has to be noted that the majority of studies highlight
only one side of the bidirectional pathway, making it difficult to develop one model for
EphB4/ephrinB2 signaling in breast cancer metastasis.
When combining the data of ligand and receptor dependent/independent signaling, described in the
previous chapter, the following model can be composed to explain the contrasting results. EphB4
receptor forward signals causes inhibition of migration in presence of ephrinB2, in a kinase
dependent manner (Figure 12A). This is based on the findings that EphB4 shows only tumor
suppressive effects in the presence of ephrinB2 (Noren et al., 2006; Rutkowski et al., 2012). However,
when ephrinB2 is absent, EphB4 forward signal induces migration (Figure 12B) as shown in different
studies with truncated ephrinB2. Moreover, it was shown that ephrinB2 is not necessary for
phosphorylation of EphB4 but that EphB4 can also signal in a kinase independent manner. On the
other side, EphrinB2 reverse signaling induces survival/adhesion cues in the presence of EphB4
(Figure 12C) but also causes adhesion cues without EphB4 (Figure 12D). The independent role of
ephrinB2 is particular shown during the normal development of the mammary gland where
overexpression is needed for tissue integrity (Haldimann and Custer, 2009). However, in breast
cancer models, this is not clearly defined.
Figure 12. Model of EphB4/ephrinB2 signaling in breast cancer metastasis. When EphB4 and ephrinB2 are both present,
both forward and reverse signaling send tumorsupressive signals to the adjacent cells. When EphB4 signals in the absence
of ephrinB2, migration is induced whereas ephrinB2 provide adhesion signals in the absence of EphB4.
37
In addition to dependent and independent signaling, clustering can also contribute to the contrasting
results observed for EphB4/ephrinB2 signaling in cancer. As mentioned before, Eph receptors are
capable of oligomerisation together with other Eph family members (Janes et al., 2012). Particularly
the observation of highly phosphorylated EphB4 in the absence of ephrinB2 (Noren et al., 2009) can
be explained by clustering. First of all, clustering of receptors via their transmembrane domains can
trigger autophosphorylation and exceed a receptor amount threshold where the kinase domains are
activated. This can explain the high phosphorylation in the absence of ligand binding. Another
function for clustering is the cellular response since phosphorylated EphB4 in the absence of
ephrinB2 results in a distinct cellular response. This signaling might be regulated by the involvement
of other Eph family members and the clustering makes other signaling pathways more dominant
(Figure 7). Moreover, from many Eph receptors and ephrin ligands it has been shown that they are
differentially regulated during tumor progression (Fox and Kandpal, 2004; Noren and Pasquale, 2007)
which can indicate a larger role for clustering.
Interestingly, EphB6 is a kinase dead tyrosine receptor which is phosphorylated by EphB4, Src and
EphB1. Activated EphB6 inhibits invasion and tumor progression in MDA-MB-231 cells. Moreover,
phosphorylated EphB6 activates the Cbl-Abl pathway resulting in actin dependent adhesion on the
ECM. By blocking the EphB4 kinase activity, no EphB6 activation is observed and cells show less
attachment, resulting in a tumor invasive phenotype (Truitt et al., 2010). However, since EphB6 is
kinase dead, this activity depends partly on the kinase domain of EphB4 and might thus also be
eprhinB2-dependent. On the other hand, upregulated EphB6 might be able to sequester
overexpressed EphB4 activity and thereby strictly regulating the cellular response by preventing
excess downstream signaling of overexpressed EphB4 (Figure 5). This might define slight differences
in cellular context and make the distinction between tumor promoting or suppressing pathways.
Receptor clustering makes Eph/ephrin signaling complex, but may explain the contradictions in
studies and dual functions as adhesion and migration within one tissue. This may also underscore the
assumption that Eph/ephrin signaling is highly cell type specific, since small differences in the
Eph/ephrin expression balance might be the main regulators of the final cellular response.
A possible role for Eph/ephrin signaling in breast cancer progression by
affecting migration, EMT and tumor-stroma interactions.
In addition to a functional, molecular model of EphB4 and ephrinB2, a model can be generated to
represent the physiological processes in which receptor/ligand signaling is involved (Figure 13).
These processes include migration, cancer stem cell formation through EMT and the influence of
stroma and adhesion which will discussed below.
38
Figure 13. Physiological model of EphB4/ephrinB2 dependent and independent signaling. When EphB4 and ephrinB2
both are present, tumorsupressive signals are provided for the adjacent cells resulting in adhesion (A). Signaling of solely
EphB4 can lead to migration (B) and might be involved in EMT and cancer stem cell formation (C). Only reverse signaling of
ephrinB2 can also be involved in cancer stem cell formation (D). For successful metastasis, circulating cells, either stem
cells or migrating cells need adhesion at a distant site (E) which can be facilitated by stromal cells expressing ephrinB2 (F).
Migration
One major requisite for a tumor cell to metastasize is migration. As described previously, EphB4 and
ephrinB2 overexpression are both already linked to migration. Moreover, the contrasting data that
EphB4 downregulation is linked to migration has been described. With the molecular model of
independent signaling, both results could be explained although most studies did not include both
components of the bidirectional pathway leading to data which is hard to compare. EphB4
overexpression can lead to migration since the tumor suppressive cellular response induced by
ephrinB2 is not activated. EphrinB2 overexpression can contribute to migration by increased survival
signaling in the tumor cell resulting in less cell death during migration. The loss of EphB4 can
contribute to migration by the loss of adherens junctions by upregulation of the MMPs and thereby
adhesion to adjacent cells. Moreover, EphB4 or ephrinB2 overexpressing tumor cells might use
stromal cells for adhesion and migration (discussed below).
39
In addition to breast cancer, in thyroid papillary carcinoma cells, EphB4 activation inhibits
phosphorylation of FAK and Paxillin in a kinase independent way. Both FAK and Paxillin are involved
in regulation of focal adhesions and interference with these proteins results in higher turnover of
focal adhesion, facilitating migration. However, a direct link between EphB4 and FAK and Paxillin is
not yet described (Xuqing et al., 2012). During migration of a cell, integrins are needed in focal
adhesions for adhesion to the ECM. Since EphB4 in the absence of ephrinB2 inhibits integrin
adhesion, this might favor migration since the turnover of focal adhesion is stimulated.
In addition to EphB4, due to the high homology among all Eph receptors, it can be helpful to include
other Eph receptors to elucidate the exact signaling. Activation of EphB2 has an opposite effect on
PC-3 prostate cancer cells to EphB4. Whereas EphB4 and EphB3 mediate invasion in an ephrinB2
environment, EphB2 acts together with ephrinB1 and inhibits migration (Astin et al., 2010). This
opposite role for EphB2 compared to EphB4 has also been described in a colon cancer cell line where
it induces E-Cadherin localization to membranes (Cortina et al., 2007) and activates integrins in
NG108 cells (Becker et al., 2000). This suggests that a similar mechanism might be present in breast
cancer cells. This is of interest since EphB2 and EphB4 are highly homologous, investigation of the
exact differences might elucidate how these similar receptors can act in an opposite manner.
EphB2 interacts with Rac1 and Cdc42, suggesting activity in the same pathways as EphB4 since EphB4
also interacts with Cdc42 (Noren and Pasquale, 2004). However, if Rac activation is tumor promoting
or suppression is not clear.
Taken together, it is likely that EphB4 and ephrinB2 contribute to breast cancer metastasis by the
migration of tumor cells.
Epithelial-to-mesenchymal transition and formation of cancer stem cells
In addition to migration, one of the other major changes cells undergo before metastasis is epithelialto-mesenchymal transition (EMT). During this process, the polarity of the cell changes and adhesion
junctions are remodeled. As result, the cell can gain more motility and invade surrounding tissue or
blood vessels. EMT is also considered necessary for de-differentiation and thus obtaining stem cell
properties. On the other hand, the reverse process of EMT, mesenchymal-to-epithelial transition
(MET) is needed during development and might be involved to form a micrometastase, small
beginning metastases which not develop all to a detectable metastase, although it is not required. By
initiation of adhesion and polarization of the circulating and/or migrating cells, a niche can be formed
facilitating metastatic growth (Hugo et al., 2007). However, if the transition is from migrating tumor
cells to cancer stem cells, from cancer stem cells to migrating cells or if these processes are
completely independent is not known. It might be possible that migrating cells undergo positive
40
selection and become more stem cell like, or that cancer stem cells are only successful when they
acquire migratory features. However, it is also possible that a migrating cancer cell can metastasize
without transforming to a cancer stem cell.
In a spontaneous Myc driven breast cancer model in transgenic mice, the primary tumors in the
mammary gland showed enhanced EMT markers and an EMT promoting environment with TAMs
(tumor associated macrophages) and higher levels of TGF, Pdgf and Egf. However, in contrast to the
Myc transgenic mice, the NeuT transgenic mice did not show enhanced EMT events (Trimboli et al.,
2008) suggesting that all models have variations and EMT is not always involved in breast tumor
development. Noteworthy is the fact that NeuT transgenic mice do not develop metastasis during
their life which might be the reason for the observed absence of EMT. However, in the NeuT/EphB4
and NeuT/ephrinB2 transgenic mice which showed enhanced metastases formation, EMT was not
examined. Since the stem cell fraction is enriched it would be interesting to test whether in these
models the primary tumors and metastases show enhanced EMT events which might be linked to the
enrichment of stem cells.
Direct links between the EphB4/ephrinB2 signaling and EMT are rare but there might be a link since
EphB4 and ephrinB2 are both located on cell-cell junctions such as tight junctions and cooperate in
adhesion integrity. To support the role, blocking the interaction between EphB4 and ephrinB2 results
in disruption of the cell-cell junctions (Miao and Wang, 2009). Moreover, phosphorylation of FAK and
paxillin has been shown to be involved in EMT in human lung cancer cells (Shah et al., 2012) and it
has been demonstrated in thyroid papillary carcinoma cells EphB4 activation inhibits this
phosphorylation (Xuqing et al., 2012). However, ephrinB2 is not included in this study, so it is unclear
of the inhibitory effects on EMT are dependent or independent of the presence of ephrinB2 and thus
hard to draw conclusions from this data.
It also has been shown that an active Crk pathway is necessary for EMT in T47D breast cancer cells
(Lamorte et al., 2002), and active EphB4/ephrinB2 signaling inhibits the Crk pathway. These results all
support the model where correct bidirectional signaling of EphB4/ephrinB2 contributes to adhesion
and is tumor suppressive by inhibiting EMT (Figure 11A,B).
Several Eph receptors have been linked to N-Cadherin regulation in normal tissue development by
recruiting β-catenin to N-cadherin to stabilize its function and inhibiting internalization of N-cadherin
(Pasquale, 2010). However, a direct link between N-cadherin and EphB4 has not yet been shown and
thus not clear if EphB4 regulates EMT.
Taken together, the direct link between EphB4/ephrinB2 signaling, the Crk pathway and EMT/MET
has not been described. It would be interesting to examine this pathway in the context of
EphB4/eprhinB2 deregulation and elucidate how they contribute to EMT. Remaining questions are
41
whether EphB4 and eprhinb2 are the main players involved in inducing EMT and does this lead to
more cancer stem cells. However, it is also possible that EphB4 and ephrinB2 are contributing factors
and act downstream of other EMT inducing processes instead of inducing factors and start the
process of EMT.
The effects of stromal cells on EphB4/ephrinB2 signaling
In addition to tumor cell - tumor cell interactions, tumor cell - stromal cell interactions might
contribute to metastasis. Since ephrinB2 has been described to be involved in adhesion,
metastasizing tumor cells in the circulation overexpressing EphB4 might recognize ephrinB2
expressing stromal cells (Héroult et al., 2010). Through EphB4/ephrinB2, circulating cells are capable
of adhesion and initiating a micrometastase in a foreign tissue. It has been shown that breast cancer
shows a preference to spread to bones, which also express ephrinB2 (Pennisi et al., 2009) which
might facilitate adhesion of EphB4 expressing circulating cells. Moreover, EphB expressing cells
spread to ephrinB1 sites both in vivo and in vitro indicating a preferentially spread of EphB expressing
migrating tumor cells to ephrin expressing sites. Also, within a tumor, compartmentalization
between EphB receptors and ephrin ligands is visible (Cortina et al., 2007) suggesting an repulsion
and attraction mechanism between Eph receptor expressing cells and ephrin ligand expressing cells.
Moreover, after activation of ephrinB1 by the EphB2 receptor, PDZ-RGS3 is activated and binds to
the CXCR4 receptor preventing signaling after activation by the chemokine SDF-1. Thereby, EphB2
inhibits chemo attraction during neuronal development (Noren and Pasquale, 2004). Furthermore, in
endothelial cells (HUVECs), EphB2 and EphB4 coordinate migration together with SDF-1 and CXCR4.
Forward signaling of EphB4 enhances SDF-1 activation (Salvucci et al., 2006) which might link the
signaling pathway not only to adhesion at distant sites but also to migration cues provided by the
stroma since it is known that SDF-1 and CXCR4 play a role in facilitating metastasis. CXCR4 expressing
MDA-231 cells preferentially spread to regions with high SDF-1 secretion in mice (Dewan et al.,
2006). Although the regulation of CXCR4 and SDF-1 by EphB4/ephrinB2 in breast cancer might have
other effects, the underlying mechanism of using the stromal cells as adhesion might be similar.
Therefore, it is interesting to investigate the link between EphB4/ephrinB2 and SDF-1 and CXCR4
during breast cancer metastasis.
EphrinB2 is expressed more on stroma than on prostate cancer cells when they are migrating
through tissue (Wang, 2012). This might suggest that the stroma facilitates migration since stromal
ephrinB2 induces tumor expressed EphB4 receptor activation resulting in Cdc42 activation and cell
motility. In prostate cancer, cells can switch between repulsion and attraction on the stroma by
expressing different sets of Eph receptors and ephrin ligands (Astin et al., 2010). This might suggest
42
that not a single Eph/ephrin couple is regulating the final cellular response, but the complete
expression profile and clustering of the different family members defines the signaling pathways. The
same mechanism might also be present in migrating breast cancer cells.
Conclusion
Taken together, the role of EphB4/ephrinB2 in breast cancer metastasis is complex and has multiple
cellular responses. There is data to support both tumor suppression and a tumor promotion. In
addition to the bidirectional signaling possibilities, ligand dependent and independent signaling plays
a major role as well. Due to this specific signaling, multiple processes can be different regulated by
EphB4 and ephrinB2, dependent on the cellular context. These processes include proliferation,
apoptosis, migration, adhesion, stem cell identity and angiogenesis. All processes have distinct
effects on tumor progression and might be a target for new therapies. However, since the exact
regulation of EphB4/ephrinB2 signaling and cooperating factors is unknown but likely very complex,
one simple drug against either EphB4 or ephrinB2 is not likely to be effective.
Since bidirectional and ligand/receptor independent signaling an anti-tumor drug might be tumor
promoting as well and the exact molecular composition of a tumor is needed to provide correct and
effective treatment. The possible involvement of EphB4 and ephrinB2 in stem cell identity is also a
risk if a drug against this pathway is developed. Interfering with the stem cell niche can have multiple
side effects in addition to tumor growth such as interfering with normal tissue homeostasis.
However, targeting the stroma instead might be effective to fight metastasis. If the cancer cells
indeed need the stroma for facilitating the migration, this can be a target to stop metastasis in an
early phase. However, if the stroma facilitates metastasis by expressing specific factors and thereby
creating a niche for circulating cells this might be a target in a later phase of metastasis. Taken
together, targeting EphB4/ephrinB2 signaling has many advantages and disadvantages since it might
be very specific for metastatic cells but side effects could accelerate tumor development and
metastasis rather than decrease it.
Despite the substantial amount of research on Eph/Ephrin signaling, the exact mechanisms of
Eph/Ephrin signaling is in most cases still a mystery and requires further investigation. Unraveling this
complex signaling pathway might yield novel therapeutic targets in the future.
43
Summary for laymen
During the development of all organisms that have more than one cell, such as fish, mice and
humans, correct communication between cells is essential. Because of this communication, all
organs and body parts are formed at the right place on the right time.
One way of cell
communication is by using receptors which can transduce a signal provided via a ligand. The
Eph/ephrin family is one of these receptors and is necessary in normal development by regulating
correct adhesion of tissues, blood vessel formation and neural development. The special feature of
Eph/ephrin signaling compared to other receptors is the fact that the ligand is also bound to a cell
and that it can signal back (reverse signaling), which is in contrast to soluble ligands or bound ligands
with no reverse signaling.
Cancer is one of deadliest diseases among humans and is caused when cells transform, cells start to
overproliferate, acquire mutations for malignant growth and eventually spread to other organs. Most
of the alterations in cancer cells are in processes that are normally used for correct development of
the organism, such as the Eph/ephrin signaling. The Eph/ephrin signaling is especially investigated in
breast cancer development with in particular the combination of two specific members, the EphB4
receptor and its ligand, ephrinB2.
For this pair, different roles in breast cancer are described. They can be involved in a tumor
suppressive way where EphB4 and ephrinB2 lead to adhesion of cells and thereby prevent migration
of cells. Moreover, they are also linked to apoptosis, a natural cellular program to induce cell death
when a cell is not needed anymore or becomes too aberrant compared to his actual function. This is
also a natural way to prevent cancer. By inducing apoptosis, EphB4/ephrinB2 can prevent cancer
development.
On the other hand, many studies also show a tumor promoting role for EphB4 and ephrinB2 where
activation of one of the components show more proliferation and survival of the cancer cells, show
enhanced migration of the cells, induce more blood vessel formation to provide nutrients and oxygen
to all tumor cells and show enriched stem cells contributing to more cells.
The contrasting data is not explained by most studies. However, almost no study investigates the
role of both sides of the communication pathway properly. It is possible that the receptor and the
ligand can not only signal when they are bind to each other, but that they are also capable of
different signaling when they act alone. To explain the contrasting data, a model is proposed where
both the receptor and the ligand have different roles when they are connected or signaling alone.
Moreover, this can also be translated into physiological processes involved in breast cancer
metastasis formation. These include the transformation of normal cells into migratory cells, the
44
change of normal cells into cancer stem cells and the possible effects of normal, healthy cells at
another place in the body to enable spreading to a specific site.
A treatment for cancer is not discovered yet, and the EphB4/ephrinB2 couple might be a new target.
However, it is probably difficult to use this as a target because it is not clear if a possible drug actually
induce the correct process: tumor suppression. It might have severe side effects when the drug
induces tumor growth, migration and metastasis formation. Nevertheless, if the normal, healthy cells
contribute specifically to metastasis formation, this might be an excellent option for a new
therapeutic target especially in later stages of cancer. In all cases, more research is needed to figure
out how EphB4 and ephrinB2 exactly communicate and what the effects on the cell are. Only then,
there is a real possibility that a perfect drug can be developed to fight cancer by targeting EphB4 and
ephrinB2.
45
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