Drug Resistance Updates 29 (2016) 1–12

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Drug Resistance Updates

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

The semaphorins and their receptors as modulators of tumor

progression
Gera Neufeld ∗ , Yelena Mumblat, Tanya Smolkin, Shira Toledano, Inbal Nir-Zvi,
Keren Ziv, Ofra Kessler
The Cancer Research and Vascular Biology Center, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa 31096, Israel

a r t i c l e i n f o a b s t r a c t

Article history: The semaphorins were initially characterized as repulsive axon guidance factors. However, they are cur-
Received 22 May 2016 rently also recognized as important regulators of diverse biological processes which include regulation of
Received in revised form 31 July 2016 immune responses, angiogenesis, organogenesis, and a variety of additional physiological and develop-
Accepted 23 August 2016
mental functions. The semaphorin family consists of more than 20 genes divided into seven subfamilies,
all of which contain the sema domain signature. They usually transduce signals by activation of receptors
Keywords:
belonging to the plexin family, either directly, or indirectly following the binding of some semaphorins to
Semaphorins
receptors of the neuropilin family which subsequently associate with plexins. Additional receptors which
Angiogenesis
Cancer
form complexes with these primary semaphorin receptors are also frequently involved in semaphorin sig-
Lymphangiogenesis nalling, and can strongly influence the nature of the biological responses of cells to semaphorins. Recent
evidence suggests that semaphorins play important roles in the etiology of multiple forms of cancer.
Some semaphorins such as some semaphorins belonging to the class-3 semaphorin subfamily, have been
found to function as bona fide tumor suppressors and to inhibit tumor progression by various mech-
anisms. Because these class-3 semaphorins are secreted proteins, these semaphorins may potentially
be used as anti-tumorigenic drugs. Other semaphorins, such as semaphorin-4D, function as inducers
of tumor progression and represent targets for the development of novel anti-tumorigenic drugs. The
mechanisms by which semaphorins affect tumor progression are diverse, ranging from direct effects on
tumor cells to modulation of accessory processes such as modulation of immune responses and inhibition
or promotion of tumor angiogenesis and tumor lymphangiogenesis. This review focuses on the diverse
mechanisms by which semaphorins affect tumor progression.
© 2016 Elsevier Ltd. All rights reserved.

1. The semaphorins and their receptors
1.1. The semaphorins
Members of the semaphorin family are divided into 8 subclasses
of which subclasses 1 and 2 contain invertebrate semaphorins,
whereas subclasses 3-7 contain the 22 vertebrate semaphorins
and subclass 8 contains viral semaphorins. In early publications,
semaphorins were assigned confusing names. This situation was
rectified by the adoption of a unified semaphorin nomenclature
in which sema is followed by the subclass number and by alpha-
betic designation within the subclass (Goodman et al., 1999).
The semaphorins as well as their plexin receptors are character-
ized by a ∼500 amino acids-long sema domain located close to
∗ Corresponding author.
E-mail address: gera@tx.technion.ac.il (G. Neufeld).
their N-termini, and by a plexin-semaphorin-integrin (PSI) domain
located downstream to the sema domain. The sema domain has
a ␤ propeller topology (Love et al., 2003; Antipenko et al., 2003;
Liu et al., 2010), is essential for semaphorin activity, and deter-
mines to some extent, the receptor binding specificity (Feiner
et al., 1997). Different semaphorin subclasses can be distinguished
by class-specific structural motifs. Thus, vertebrate semaphorins
belonging to classes 4 and 7 contain immunoglobulin-like domains,
class-5 semaphorins contain thrombospondin repeats and class-
3 semaphorins contain a basic domain. Class-3 semaphorins are
the only vertebrate semaphorins produced as secreted proteins
while other vertebrate semaphorins are membrane anchored.
Some membrane-anchored semaphorins can be further processed
into soluble forms by proteolytic cleavage (Fig. 1). Some membrane
anchored semaphorins may also be able to function as signal trans-
ducing proteins (Toyofuku et al., 2012; Segarra et al., 2012). The
active forms of several class-3 and class-6 semaphorins are homod-
imers (Klostermann et al., 1998; Liu et al., 2010; Janssen et al.,

http://dx.doi.org/10.1016/j.drup.2016.08.001
1368-7646/© 2016 Elsevier Ltd. All rights reserved.
2 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12

Fig. 1. The vertebrate semaphorins: (A) The structural elements of vertebrate semaphorin subclasses are shown. All semaphorins feature the signature N-terminal sema
domain. A conserved stretch of amino-acid residues near the C-terminal of the sema domain bears homology to the N-terminal of ␤-integrins and is designated as the PSI
domain. Class-3 semaphorins are the only secreted semaphorins and are distinguished by a conserved basic domain at their C-termini. Class 4-7 semaphorins are membrane-
anchored. Class 5 semaphorins are distinguished by thrombospondin repeats. All the vertebrate semaphorins except for the class-5 and 6 semaphorins also contain an
immunoglobulin-like domain. The extracellular domains of class-4 semaphorins can be cleaved with metalloproteases generating active soluble extracellular domains.

2010; Nogi et al., 2010), suggesting that the active forms of all the
semaphorins are dimeric.
1.2. Plexin family receptors
The receptors of the plexin family are segregated into four
groups consisting of four Type-A plexins, three Type-B plexins,
and single C and D plexins (Negishi et al., 2005a; Gutmann-Raviv
et al., 2006). Most semaphorins bind to one or to several plexins
(Hota and Buck, 2012). For example, plexin-B1 is a receptor for
sema4D (Tamagnone et al., 1999), plexin-A2 and plexin-A4 func-
tion as sema6A and sema6B receptors (Suto et al., 2007; Suto et al.,
2005) and plexin-D1 is a receptor for sema3E and sema4A (Gu et al.,
2005; Toyofuku et al., 2007) (Fig. 2). The sema domain found in the
extracellular domain of the plexins serves as an auto-inhibitory
domain in the basal, non-activated state of the receptor and the
inhibition is removed following a conformational change induced
by the binding of a semaphorin (Takahashi and Strittmatter, 2001).
The intracellular domains of plexins are characterized by the pres-
ence of a conserved GTPase activating (GAP) domain (Oinuma et al.,
2004; He et al., 2009; Sakurai et al., 2010). Most of the develop-
mental effects of plexin-D1 and plexin-B1 are lost when the GAP
domain is mutated suggesting that it plays an essential role in
plexin mediated semaphorin signal transduction (Worzfeld et al.,
2014). Type-A plexins associate spontaneously to form homod-
imers (Janssen et al., 2010; Nogi et al., 2010) or heterodimers (Kigel
et al., 2011). Recent data indicates that activation of plexin signal-
ing by semaphorins that bind directly to plexins such as sema6A is
associated with a conformational change that shifts the conforma-
tion of the dimer from the inactive to the active conformation (Liu
et al., 2010; Nogi et al., 2010).
Activation of plexin signaling by semaphorins activates the GAP
domain leading to the inactivation of R-ras, resulting in the subse-
quent inactivation of beta1-integrin, and finally reduced adhesion
(Negishi et al., 2005b; Toyofuku et al., 2005; Sakurai et al., 2010;
Sakurai et al., 2011). Activation of type-A plexins was also found
to induce the activation of enzymes of the Mical family. These
enzymes perform reduction-oxidation (redox) enzymatic reactions
and oxidize actin subunits leading to the disassembly of actin
fibers and to the localized collapse of the actin cytoskeleton of
axonal growth cones, thereby contributing to growth cone guid-
ance (Terman et al., 2002; Hung et al., 2010; Hung et al., 2011; Hung
et al., 2013). Activation of plexin signaling by semaphorins also
results in the activation of various intracellular tyrosine-kinases
(Franco and Tamagnone, 2008) and to the inactivation of small
GTPases that control the polymerization of the actin cytoskeleton
such as Rho as a result of the activation of regulators of Rho activ-
ity including the p190 Rho-GTPase and Rho guanine nucleotide
exchange factors (Puschel, 2007; Worzfeld et al., 2014). However,
semaphorin induced signal transduction is far from being com-
pletely understood and its thorough description is beyond the
scope of the present review.
1.3. Neuropilins
Six of the seven class-3 semaphorins are unable to bind to plex-
ins directly but instead bind to one or to both receptors of the
neuropilin family (Gu et al., 2005; Neufeld and Kessler, 2008).
Because of their short intracellular domains, neuropilins are not
able to transduce semaphorin signals independently, and asso-
ciate with type-A plexins or with plexin-D1 after binding a class-3
semaphorin to transduce semaphorin signals (Tamagnone et al.,
1999; Takahashi et al., 1999; Gitler et al., 2004). Recent structural
studies indicate that functional class-3 semaphorin receptors con-
sist of a tetramer containing a neuropilin homodimer and a plexin
homodimer that are linked together by the binding of a class-3
semaphorin homodimer (Janssen et al., 2012). However, there is
evidence indicating that functional class-3 semaphorin receptors
can also contain plexin heterodimers since inhibition of the expres-
sion of either neuropilin-1 or plexin-A1 or plexin-A4 in endothelial
cells was found to be sufficient to completely abrogate sema3A sig-
nal transduction suggesting that the receptor complex in these cells
contains plexin-A4 as well as plexin-A1 in addition to neuropilin-
1 (Kigel et al., 2011). Similarly, both plexin-A2 and plexin-A4 are
required for sema3B induced signal transduction suggesting that
functional sema3B receptors also contain more than one type of
plexin (Sabag et al., 2014).
The neuropilins can perhaps be best described as “scaffold
receptors” since they seem to bind to and modulate the activities of
diverse types of receptors and ligands but do not appear to trans-
duce signals independently. In addition to class-3 semaphorins,
the neuropilins also function as receptors for several types of
growth factors including several members of the vascular endothe-
lial growth factor (VEGF) family (Gitay-Goren et al., 1996; Soker
et al., 1998; Makinen et al., 1999; Karpanen et al., 2006; Migdal
et al., 1998), hepatocyte growth factor (HGF) (Sulpice et al.,
2007) and TGF-␤ (Glinka and Prud’homme, 2008) to name but
a few. The VEGF-A binding domain of neuropilin-1 seems to be
 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12 3

Fig. 2. Semaphorin receptors: The different semaphorins are described using a three letter code in which the S stands for semaphorin, the number designates the subfamily
and the following letter the specific sub-family member. Thus, S3a stands for sema3A. The binding receptors for the different semaphorins are shown. Most of the class-3
semaphorins bind to receptors composed of neuropilins and plexins but the primary binding site is located on the neuropilins.

distinct from its semaphorin binding domain (Appleton et al.,
2007) and it was indeed observed that sema3A and sema3F can
inhibit VEGF induced activation of ERK1/2 without inhibition
of VEGF induced auto-phosphorylation of the VEGFR2 receptor
which associates with neuropilin-1 (Guttmann-Raviv et al., 2007),
lending support to these observations. However, there is also
evidence suggesting that some class-3 semaphorins may com-
pete with members of the VEGF family for binding to neuropilins
(Mumblat et al., 2015) and that post-translational modifications of
semaphorins such as cleavage by furin like pro-protein convertases
(Adams et al., 1997) may modulate their neuropilin binding ability
and their ability to compete with VEGFs for binding to neuropilins
(Guo et al., 2013).
In addition to binding several types of diverse ligands,
neuropilins are also able to form complexes with diverse
membrane-anchored receptors in addition to plexins. In this
respect, the best studied interaction is with the VEGFR-2 tyrosine-
kinase receptor in which neuropilin-1 functions as an amplifier
that enhances VEGF induced pro-angiogenic signaling mediated by
this receptor (Soker et al., 1998). Similarly, neuropilin-2 interacts
with the VEGF-C receptor VEGFR-3 and is required for efficient
transduction of VEGF-C induced pro-lymphangiogenic signals (Xu
et al., 2010; Karpanen et al., 2006; Favier et al., 2006). Similarly,
neuropilins form complexes and modulate signal transduction
mediated by several additional receptors including TGF-␤ recep-
tors (Glinka et al., 2011), platelet derived growth factor (PDGF)
receptors (Cao et al., 2010), neurotrophin receptors (Ben-Zvi et al.,
2007), the epidermal growth factor (EGF) receptor (Rizzolio et al.,
2012) and the hepatocyte growth factor receptor (Matsushita
et al., 2007).
Mice lacking functional neuropilin-1 display major defects
in the organization of their nervous system and in their blood
vessels resulting in embryonic lethality. Mice lacking functional
neuropilin-2 developed almost normally but have defects in
lymph vessels (Yuan et al., 2002). The intracellular domain of
neuropilin-1 contains a PDZ binding domain which binds synectin
(also known as GIPC or NIP) which is important for the formation
of complexes with VEGFR-2 (Cai and Reed, 1999; Prahst et al.,
2008). The neuropilins can also form complexes with adhesion
receptors such as L1-CAM which associates with neuropilin-1
or with Nr-CAM which associates with neuropilin-2, and these
interactions were found to modulate signal transduction induced
by class-3 semaphorins such as sema3A and sema3F (Castellani
et al., 2002; Falk et al., 2005). Interestingly, the interactions of
the neuropilins are not limited to interactions with proteins.
Thus, neuropilin-1 was recently found to bind to rare 3-O-sulfate
moieties of heparan sulfate containing proteoglycans thereby
inhibiting sema3A induced signal transduction in axonal growth
cones of sema3A responsive neurons (Thacker et al., 2016).
1.4. Additional semaphorin receptors
Some semaphorins bind to additional types of receptors besides
plexins and neuropilins. Thus, sema4A also signals using the Tim-2
4 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12

Fig. 3. The dual role of sema3E in tumor progression: Like all class-3 semaphorins, sema3E contains a conserved cleavage site for furin-like pro-protein convertases (FPPC)
which when cleaved generates the active N-terminal fragment p61-Sema3E (Upper Panel). In contrast to uncleaved full length sema3E, P61-Sema3E induces the association
of the sema3E plexin-D1 receptor with ErbB2 on tumor cells, thus activating “in-trans” ErbB2 mediated signal transduction that promotes tumor metastasis. Full length
sema3E does not promote this association and only inhibits angiogenesis after it binds to its plexin-D1 receptor. FPPC expression is strongly up-regulated in malignant tumor
cells causing most of the sema3E produced by the tumor cells to be cleaved to p61-Sema3E. As a result p61-Sema3E efficiently competes with the remaining full length
sema3E for binding to plexin-D1, resulting in the induction of ErbB2 mediated tumor metastasis.

receptor, a member of the family of T-cell immunoglobulin domain
and mucin domain (Tim) proteins that is expressed on activated
T cells (Kumanogoh et al., 2002a). Sema4D was also found to bind
to the lymphocyte receptor CD-72 (Kumanogoh et al., 2000) in
addition to plexin-B1 and plexin-B2. Sema5A and sema3A also
bind to chondroitin sulfate proteoglycans, an interaction that can
convert sema5A from an attractive to an guidance inhibitory cue
(Kantor et al., 2004; Dick et al., 2013). Sema4B also binds to a
CLCP1, a receptor that displays a structural similarity to neuropilins
(Nagai et al., 2007). Finally, sema7A binds to ␣-1/␤-1 integrin to
modulate inflammatory responses mediated by these integrins
(Suzuki et al., 2007) (Fig. 2).
2. Modulation of angiogenesis by semaphorins
2.1. Anti-angiogenic semaphorins
Vascular endothelial growth factor (VEGF-A) had been exten-
sively characterized as a major angiogenesis promoting factor
(Gospodarowicz et al., 1989; Leung et al., 1989; Keck et al., 1989).
VEGF-A is produced in several forms as a result of alternative
splicing. VEGF-A signals are transduced by two tyrosine kinase
receptors (VEGFR-1 and VEGFR-2) which bind all the VEGF-A
splice forms. This indicated that there may exist receptors that
are able to distinguish between different VEGF-A splice forms.
Such receptors were indeed first identified in binding/cross-linking
experiments in endothelial cells (Gitay-Goren et al., 1996). They
were subsequently identified as the products of the neuropilin-
1 (Soker et al., 1998) and neuropilin-2 (Gluzman-Poltorak et al.,
2000) genes. Since neuropilins have already been identified as
class-3 semaphorin receptors (Kolodkin et al., 1997; He and
Tessier-Lavigne, 1997; Chen et al., 1997), it followed that class-3
semaphorins could perhaps modulate VEGF-A induced angiogene-
sis. Since angiogenesis is usually required for the development of
solid tumors (Folkman, 1990), it followed that if the semaphorins
do modulate angiogenesis, then they will likely affect tumor
progression. This logic led initially to the identification of the
class-3 semaphorin sema3F as an inhibitor of angiogenesis and
angiogenesis-dependent tumor progression (Kessler et al., 2004;
Bielenberg et al., 2004). The identification of sema3F as an anti-
angiogenic semaphorin lead to the characterization of additional
class-3 semaphorins, acting as inhibitors of angiogenesis. Indeed,
the class-3 semaphorins sema3A (Acevedo et al., 2008), sema3B
(Varshavsky et al., 2008), sema3C (Yang et al., 2015; Mumblat
et al., 2015), sema3D (Sabag et al., 2012) and sema3E (Casazza
et al., 2010) were found to function as inhibitors of angiogene-
sis. It is not completely clear how the class-3 semaphorins inhibit
angiogenesis. They function on the one hand as repulsive guidance
factors and it was found, for example, that expression of sema3E in
somites of developing embryos repels blood vessels and keeps the
somites avascular (Gu et al., 2005). On the other hand, prolonged
stimulation of endothelial cells to sema3A and sema3F induces
 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12
apoptosis and it is possible that part of the anti-angiogenic effect
of semaphorins is due to induction of apoptosis (Guttmann-Raviv
et al., 2007; Reidy et al., 2009; Maione et al., 2009; Shirvan et al.,
1999).
The development of the retinal vasculature serves as a major
developmental angiogenesis model because the whole network of
blood vessels can be easily observed in retinal whole-mounts. New-
born babies as well as newborn mouse pups exposed to high partial
oxygen pressure develop blindness when shifted back to normoxia
(retinopathy of prematurity (ROP)), because of wild growth of new
blood vessels that is driven by the acute hypoxia felt by astrocytes
following the sudden drop in oxygen partial pressure which induces
the astrocytes to express high levels of VEGF (Alon et al., 1995;
Stone et al., 1995). Sema3A functions as an inhibitor of the wild
angiogenesis induced by a shift to normoxia as well as an inhibitor
of laser coagulation induced choroidal angiogenesis (Yu et al., 2013;
Bai et al., 2014). Tip cells are endothelial cells located at the tips
of the growing angiogenic sprouts. These cells send out filopodia
and lamellipodia to guide the growing sprout while the stalk cells
which are the endothelial cells that form the main body of the grow-
ing sprout do not extend such filopodia (Siekmann et al., 2008;
Eilken and Adams, 2010). Sema3A expressed by endothelial cells
was found to inhibit filopodia formation by vascular tip cells in
angiogenic sprouts. Consequently, knock-out of the sema3A gene
in endothelial cells causes a significant increase in the number and
the length of tip cell filopodia at the leading edge of angiogenic
sprouts of developing retinal vessels (Ochsenbein et al., 2016). Like-
wise, sema3E was also found to inhibit angiogenesis in the ROP
model as well as laser- induced choroidal angiogenesis (Suda et al.,
2014; Fukushima et al., 2011). Activation of VEGFR-2 in tip cells by
VEGF also induces the expression of the notch ligand Dll4 in the
tip cells. Dll4 then activates signaling by notch receptors in stalk
cells located adjacent to the tip cells in angiogenic sprouts. This in
turn down-regulates the expression of VEGFR-2 in the stalk cells,
resulting in the maintenance of their stalk cell identity (Hellstrom
et al., 2007; Lobov et al., 2007). Interestingly, VEGF also induces
the expression of the plexin-D1 receptor in tip cells of sprouting
angiogenic retinal blood vessels. Sema3E produced by retinal gan-
glion cells acts specifically on the plexin-D1 expressing tip cells to
inhibit the VEGF induced expression of Dll4 causing a cell fate shift
that favours tip cells identity. Thus, sema3E expression is part of
a feedback mechanism by which neuronal cells of the retina regu-
late the formation of the developing vascular network (Kim et al.,
2011). Sema4A, a membrane anchored semaphorin that also uti-
lizes plexin-D1 as its receptor also functions as an anti-angiogenic
factor (Toyofuku et al., 2007) although it is not known if it is also part
of a similar feedback mechanism. Finally, sema3F was also found
to function as a regulator of angiogenesis during the development
of the retinal vasculature in which sema3F produced by RPE cells
in the outer retina forms an anti-angiogenic barrier that inhibits
the growth of choroidal and retinal blood vessels into these layers
(Buehler et al., 2013). Thus, several class-3 semaphorins function
as natural anti-angiogenic agents during embryonic development.
Taken together, these observations suggest that anti-angiogenic
class-3 semaphorins may function as endogenous inhibitors of
tumor angiogenesis whose function is lost during tumor progres-
sion, enabling the angiogenic switch thus allowing developing
tumors to initiate angiogenesis-driven growth (Hanahan and
Folkman, 1996). This hypothesis predicts that class-3 semaphorins
may function as tumor suppressors. Indeed, both sema3F and
sema3B have been characterized as bona fide tumor suppressor
genes (Tomizawa et al., 2001; Sekido et al., 1996; Xiang et al., 1996;
Xiang et al., 2002). These observations further suggest that class-3
semaphorins could perhaps be utilized as drugs for the treatment of
solid tumor development and of diseases characterized by rampant
deregulated angiogenesis. Indeed, several class-3 semaphorins
5
such as sema3A (Casazza et al., 2011), sema3B (Varshavsky et al.,
2008) sema3D (Sabag et al., 2012) and sema3F (Kessler et al., 2004;
Bielenberg et al., 2004) inhibit tumor angiogenesis and tumor
development. Down-regulation of sema3A expression in tumor
cells promotes tumor angiogenesis and tumor progression in
many types of solid tumors (Vacca et al., 2006; Maione et al., 2009;
Barresi and Tuccari, 2010; Song et al., 2012; Jiang et al., 2015;
Tang et al., 2014; Jiang et al., 2015; Zhou et al., 2014) suggesting
that sema3A does indeed function as an endogenous negative
regulator of the angiogenic switch (Hanahan and Folkman, 1996).
Likewise, over-expression of sema3A in tumor cells or addition
of exogenous sema3A can inhibit tumor angiogenesis and tumor
progression (Kigel et al., 2008; Acevedo et al., 2008; Casazza et al.,
2011; Chakraborty et al., 2012; Sabag et al., 2012). Sema3F and
sema3B expression in tumor cells also inhibits tumor angiogenesis
and tumor development (Kessler et al., 2004; Ikegami et al.,
2004; Varshavsky et al., 2008). In the case of sema3F it was also
found that its expression is under the control of p53. Loss of p53
activity which is frequently encountered in tumor cells results in
down-regulation of sema3F expression thus alleviating sema3F
induced inhibition of angiogenesis and promoting tumor growth,
suggesting that sema3F also functions as an endogenous inhibitor
of angiogenesis (Futamura et al., 2007).
All class-3 semaphorins contain conserved cleavage sites for
proteases belonging to the furin-like pro-protein convertase family
(Adams et al., 1997). The expression of some furin-like pro-
protein convertases is up-regulated by hypoxia (McMahon et al.,
2005) and their expression is up-regulated in malignant cells
(Bassi et al., 2005). Cleavage by furin-like pro-protein conver-
tases can be a modality for the inactivation of class-3 semaphorins
thereby relieving anti-angiogenic inhibition as in the case of
sema3B (Varshavsky et al., 2008). However, it should be noted
that in addition to their direct effects on endothelial cells, anti-
angiogenic semaphorins such as sema3B can also activate biological
responses in additional types of cells and these may indirectly
modulate the anti-angiogenic activity of these semaphorins. For
example, sema3B can also induce the expression of interleukin-
8, which in turn, induces the recruitment of tumor-associated
macrophages and promotes the metastatic dissemination of tumor
cells (Rolny et al., 2008). Interleukin-8 is also a well character-
ized pro-angiogenic factor (Huang et al., 2002) and it is also likely
that induction of interleukin-8 expression induced by sema3B
may counteract the direct anti-angiogenic effects of sema3B. Sim-
ilarly, sema3A was reported to recruit, to developing tumors, a
neuropilin-1 expressing subpopulation of bone marrow-derived
monocytes that promotes the recruitment of pericytes and the sta-
bilization and normalization of tumor blood vessels thus protecting
them against angiogenesis inhibitors (Zacchigna et al., 2008; Carrer
et al., 2012).
In contrast with class-3 semaphorins such as sema3A or
sema3F which were characterized primarily as anti-tumorigenic
semaphorins, the class-3 semaphorin sema3E was paradoxically
found to promote tumor progression despite its anti-angiogenic
activity (Christensen et al., 1998). Like other class-3 semaphorins,
sema3E is cleaved by furin-like pro-protein convertases. One of the
cleavage products is a shorter peptide (p61-Sema3E) which also
displays anti-angiogenic properties. However, unlike full- length
sema3E, p61-Sema3E also promotes the association of the sema3E
receptor plexin-D1 with ErbB2 receptors on tumor cells, resulting
in the “in-trans” activation of ErbB2 and promotion of tumor metas-
tasis (Casazza et al., 2010). In contrast to p61-Sema3E, full-length
sema3E rendered resistant to cleavage by furin-like pro-protein
convertases by the introduction of point mutation into the con-
served furin cleavage sites, retained the anti-angiogenic activity
of full length sema3E, but was unable to activate ErbB2 and did
not promote tumor metastasis. Furthermore, this point mutant
6 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12
inhibited the binding of p61-Sema3E to plexin-D1, and thereby
prevented the activation of ErbB2 and the induction of tumor
metastasis by p61-Sema3E while simultaneously inhibiting tumor
angiogenesis and tumor development (Casazza et al., 2012) (Fig. 3).
Similarly, sema3C was also characterized as a promoter of tumor
progression (Miyato et al., 2012; Man et al., 2014) even though it
too functions as an anti-angiogenic factor that inhibits, for exam-
ple, oxygen-induced retinal angiogenesis (Yang et al., 2015). In this
case too, a point mutated sema3C resistant to cleavage by furin
like pro-protein convertases displayed anti-angiogenic properties
and inhibited tumor growth and metastasis. Preliminary evidence
suggests that the p65-Sema3C peptide produced from sema3C as
a result cleavage by furin-like pro-protein convertases functions
as a promoter of tumor cell survival (Mumblat et al., 2015). It is
thus possible that in the case of sema3C too, cleavage by furin-
like pro-protein convertases is responsible for the activation of
pro-tumorigenic functions.
2.2. Pro-angiogenic semaphorins
In contrast to the class-3 semaphorins, which function primar-
ily as inhibitors of angiogenesis, Sema4D (also known as CD100)
functions as an inducer of angiogenesis and as a promoter of tumor
progression that plays a central role in the development and tumor
progression of head and neck squamous cell carcinomas (Basile
et al., 2004; Basile et al., 2006). Sema4D is a membrane-bound
class-4 semaphorin that binds to plexin-B1, plexin-B2 and to the
CD-72 receptors (Tamagnone et al., 1999; Kumanogoh et al., 2000)
(Fig. 2). The extracellular domain of sema4D can be cleaved and
released from producing cells by membrane type-1 matrix met-
alloproteinase (MT1-MMP) and by the metalloprotease ADAM17
(TACE). These two metalloproteases are up-regulated in many types
of malignant cells (Strongin, 2010; Zhu et al., 2007; Arribas and
Esselens, 2009). Sema4D is stored in platelets and its extracellu-
lar domain can be released from platelets (Zhu et al., 2007). The
cleaved soluble extracellular domain of sema4D retains the biolog-
ical activity of the full length sema4D (Basile et al., 2007b) and was
used extensively to study the role of sema4D in immune reactions,
tumor progression and the control of angiogenesis.
The tyrosine-kinase receptor Met is the receptor for hepatocyte
growth factor/scatter factor (HGF/SF), a potent inducer of tumor
cell invasiveness and angiogenesis (Bussolino et al., 1992; Trusolino
et al., 2010). The soluble extracellular domain of sema4D was found
to bind to plexin-B1 and to induce the association of the sema4D
receptor plexin-B1 with Met. This association then promotes “in-
trans” auto-phosphorylation of the Met receptor and induction
of tumor cells invasiveness (Giordano et al., 2002). It was subse-
quently observed that sema4D can also transactivate the related
macrophage stimulating protein (MSP) receptor Ron (Yao et al.,
2013), and that all three type-B plexins are able to form complexes
with the Met and Ron receptors (Conrotto et al., 2004; Conrotto
et al., 2005). Sema5A, a semaphorin that binds to the plexin-B3
receptor, can also transactivate Met similarly to sema4D (Artigiani
et al., 2004). Since HGF/SF is also a potent inducer of angiogenesis,
these observations suggested that sema4D may also function as a
pro-angiogenic factor and thereby further promote tumor progres-
sion as was indeed demonstrated (Conrotto et al., 2005).
However, sema4D was also found to induce angiogenesis inde-
pendently of Met by utilizing plexin-B1 induced Rho dependent
mechanisms (Basile et al., 2004). This mechanism involves the acti-
vation of the PI3K/Akt pathway following the binding of sema4D
to plexin-B1. Activated plexin-B1 activates in turn an intracellu-
lar tyrosine kinase cascade that involves the sequential activation
of PYK2 and Src which results in the tyrosine phosphorylation
of Plexin-B1, recruitment of a multimeric signaling complex that
includes PYK2, Src, and PI3K to Plexin-B1 and activation of the
Akt signaling pathway (Basile et al., 2005; Basile et al., 2007a).
It was recently reported that this Met independent activity is
mediated, in addition, by Rho/Rho Kinase (ROK) dependent genera-
tion of PI(4,5)P(2) upon treatment of endothelial cells with Sema4D
(Binmadi et al., 2011; Binmadi et al., 2012). In addition, activation of
plexin-B1 by sema4D in endothelial cells can result in the activation
of NF-kappaB and subsequently in the expression of the angiogenic
factor IL-8 (Yang et al., 2011; Strieter et al., 1992). Because sema4D
activates angiogenesis using the plexin-B1 receptor and not the
VEGFR-2 receptor used by VEGF, it follows that sema4D can act
additively with VEGF and that inhibition of sema4D signaling can
represent an alternative anti-angiogenic treatment strategy (Zhou
et al., 2012). In addition, it was observed that sema4D also regulates
the permeability of blood vessels by enhancing the expression of
PDGF-B and angiopoietin like protein-4 in endothelial cells, result-
ing in the dissociation of pericytes (Zhou et al., 2013).
Studies of sema7A focused primarily on its immune modulating
activity. However, recent evidence indicates that sema7A also
functions as a pro-tumorigenic and pro-angiogenic factor. Thus, its
expression is up-regulated in transformed mammary tumor cells.
Macrophages exposed to sema7A expressed elevated levels of
CXCL2/MIP-2 and tumor cells in which the expression of sema7A
was inhibited formed less angiogenic tumors (Garcia-Areas et al.,
2014). Sema7A expression is associated with ductal carcinoma in
situ (DCIS) progression. Enhanced sema7A expression occurs in a
large percentage of breast cancers and is associated with decreased
overall and distant metastasis-free survival and similar results
were reported with regard to oral cancer (Black et al., 2016; Saito
et al., 2015).
3. Modulation of lymph vessels-mediated tumor
progression by semaphorins
Lymph vessels drain excessive fluids from tissues. Lymphangio-
genesis is the process by which new lymph vessels grow out of an
existing bed of lymph vessels. Tumor development is frequently
accompanied by lymphangiogenesis that is induced by tumor
derived lymphangiogenic factors such as VEGF-C and VEGF-D, and
in certain forms of cancer such as in head and neck carcinomas or
in breast cancer tumor cells invade these new lymph vessels and
utilize them to migrate to sentinel lymph nodes in which they form
secondary tumors and from these sites further migrate to distant
organs (Li and Li, 2014; Podgrabinska and Skobe, 2014; Karaman
and Detmar, 2014; Alitalo and Detmar, 2012).
The discovery of anti-angiogenic semaphorins suggests that
some semaphorins may also function as regulators of lymph ves-
sels function and lymphangiogenesis. Indeed, sema3A and its
neuropilin-1 and plexin-A1 are required for correct development
of lymph vessel valves. In utero treatment of mice with an antibody
that blocks Sema3A binding to neuropilin-1, but not treatment
with an antibody that blocks VEGF-A binding to neuropilin-1, com-
promised the function of lymph vessels, and resulted in abnormal
lymph vessel and lymphatic valve morphology (Jurisic et al., 2012;
Bouvree et al., 2012).
Sema3F repels cultured lymphatic endothelial cells suggest-
ing that it may function as an anti-lymphangiogenic agent in vivo
(Ikegami et al., 2004). Indeed, it was recently demonstrated that
sema3F inhibits tumor lymphangiogenesis and tumor metastasis of
head and neck squamous cell carcinomas, a tumor type that metas-
tasizes primarily via lymph vessels (Doci et al., 2015; Zhang et al.,
2014). Likewise, it was recently found that sema3C also functions
as a repellant of lymphatic endothelial cells. A mutated form of
sema3C resistant to cleavage by furin-like pro-protein convertases
strongly reduced the density of lymph vessels in tumors derived
from breast cancer cells and strongly inhibited the spontaneous
 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12
formation of lymph node metastases (Mumblat et al., 2015). In con-
trast, enhanced expression of sema4D is associated with enhanced
expression of VEGF-C and VEGF-D and with increased lymph node
metastasis of cervical cancer and colon cancer (Liu et al., 2014; Mu
et al., 2014). Likewise, expression of sema7A was also reported to
be associated with increased metastasis and increased lymphan-
giogenesis of breast cancer tumors (Black et al., 2016).
4. Direct effects of semaphorins on tumor cells and tumor
progression
In addition to their effects on angiogenesis and lymphangio-
genesis, semaphorins can also affect the behavior of tumor cells
directly. Thus, sema3A inhibited the proliferation of gastric can-
cer cells and their migration in “in vitro” assays, indicating that in
addition to its anti-angiogenic effects it can also affect the behavior
of tumor cells directly (Tang et al., 2014). Sema3A also inhibited
the anchorage-independent proliferation of triple negative breast
cancer cells as did additional class-3 semaphorins such as sema3D
and sema3F but not sema3E (Kigel et al., 2008). The sema3B gene
was identified along with sema3F as a tumor suppressor whose
function is lost in small cell lung carcinoma cells by a variety of
mechanisms that include promoter methylation and loss of het-
erozygosity (Tomizawa et al., 2001; Kuroki et al., 2003; Nair et al.,
2007; Campioni et al., 2008; Chen et al., 2014; Loginov et al.,
2015; Gao et al., 2015). Single nucleotide polymorphisms in the
sema3B gene were also found to be associated with poor prog-
nosis of prostate cancer (Beuten et al., 2009). Lung cancer and
breast cancer cells express neuropilins, and it found that sema3B
induces apoptosis of breast and lung cancer cells grown in cell
culture. This pro-apoptotic effect was inhibited by VEGF165 which
binds to neuropilins but not by VEGF121 which does not, suggest-
ing that VEGF165 functions as a survival factor for these cells and
that sema3B can inhibit this effect of VEGF165 (Castro-Rivera et al.,
2004). Similarly, sema3F causes non small cell lung carcinoma cells
to round up and lose extracellular contacts (Kusy et al., 2005) and
inhibited the anchorage-independent growth of breast cancer cells
(Kigel et al., 2008). In breast cancer cells, the expression of sema3F is
regulated by retinoid orphan nuclear receptor ␣ (ROR␣). Reduced
ROR␣ expression correlated with diminished sema3F expression
and poor prognosis while restoration of ROR␣ expression repro-
grammed breast cancer cells to form non-invasive structures in 3D
culture (Xiong et al., 2012). Likewise Sema3F inhibited the attach-
ment and spreading of MCF-7 breast cancer cells and inhibited the
expression of E-cadherin thus contributing to epithelial to mes-
enchymal transition (EMT) (Perl et al., 1998), a process of cardinal
importance for the conversion of tumor cells into metastatic cells
(Nasarre et al., 2003; Nasarre et al., 2005). Knockdown of SEMA3F
also promoted the self-renewal and tumorigenicity of colorectal
cancer cells and increased the expression of stemness-associated
genes while over-expression of SEMA3F reduced the expression
of stemness genes (Rao et al., 2014). In addition, Sema3F inhib-
ited integrin-␤1-mediated attachment of A375 melanoma cells by a
neuropilin-2-mediated mechanism and suppressed the metastatic
spread of cells from tumors derived from these cells (Bielenberg
et al., 2004). SEMA3F inhibits phosphatidyl inositol-3 kinase (PI3K)
and Akt activity in a variety of target cells including endothelial
cells and several tumor cell types. These responses were associated
with the disruption of mTOR/rictor assembly and mTOR-dependent
activation of the RhoA GTPase (Nakayama et al., 2015).
In contrast with the above mentioned class-3 semaphorins,
sema4D acts as a pro-tumorigenic factor. Sema4D can also pro-
mote tumor progression in “trans” by activation of the Met and Ron
tyrosine-kinase receptors and induction of epithelial to mesenchy-
mal transition (EMT) following the binding of sema4D to tumor cell
7
surface plexin-B1 receptors (Giordano et al., 2002; Yao et al., 2013).
Likewise, sema5A, a semaphorin that binds to the plexin-B3 recep-
tor, can also transactivate Met similarly to sema4D (Artigiani et al.,
2004). These observations lead to the development of inhibitory
humanized antibodies that are developed as anti-tumorigenic bio-
logicals that target sema4D (Patnaik et al., 2015). However, there
are also opposite observations. In melanocytes and in malignant
melanoma cells sema4D inhibits HGF-induced activation of Met
and the inhibition of plexin-B1 expression in these cells leads to
the activation, rather than to the inactivation of Met (McClelland
et al., 2011; Soong et al., 2012; Soong and Scott, 2012). In breast car-
cinoma cells, plexin-B1 and plexins-B2 also form complexes with
the ErbB2 tyrosine-kinase receptor, and sema4D as well as sema4C
are both able to induce ErbB2 phosphorylation “in-trans” following
their binding to plexins-B1 or plexins-B2 receptors (Swiercz et al.,
2004). In these cells, the binding of sema4D to plexin-B1 which
is associated with ErbB2, induces cell migration and metastasis;
whereas the binding of sema4D to plexin-B1 associated with Met
causes inhibition of cell migration, indicating that the exchange of
the two receptor tyrosine kinases is sufficient to convert the cellu-
lar response of Sema4D from pro- to anti-migratory and vice versa
(Swiercz et al., 2008). Similar observations were also reported in
prostate cancer cells (Damola et al., 2013). It was recently observed
that the activation of plexin-B1 by Sema4D in breast carcinoma
cells results in tyrosine phosphorylation of plexin-B1 by Met, thus
creating a docking site for the SH2 domain of growth factor recep-
tor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1
receptor complex and through its SH3 domain, interacts with p190
RhoGAP and mediates RhoA deactivation and leads to the subse-
quent inhibition of breast carcinoma cells motility (Sun et al., 2012).
Sema6D is a membrane-anchored semaphorin that binds to the
plexin-A1 receptor. It induces the association of plexin-A1 with
the VEGFR-2 VEGF tyrosine-kinase receptors and activates signal
transduction by this receptor (Toyofuku et al., 2004). Sema6D
functions as a survival and metastasis inducing gene in malignant
pleural mesothelioma in a plexin-A1 and VEGFR-2-dependent
manner (Catalano et al., 2009). In addition, sema6D is important
for tumor progression in a subtype of triple negative breast cancer
(Chen et al., 2015). Another family member, sema6B, was recently
described as an inducer of metastasis in gastric cancer (Ge et al.,
2013) but was also reported as a possible inhibitor of breast
cancer progression (D’Apice et al., 2013). Sema5A is a semaphorin
that binds to the plexin-A1, plexin-A3 and plexin-B3 receptors
(Matsuoka et al., 2011; Artigiani et al., 2004). It was recently found
to promote tumor metastasis in gastric cancer (Pan et al., 2012)
and in pancreatic cancer (Sadanandam et al., 2012) but inhibited
the motility of glioma cells (Li and Lee, 2010). Sema5B may play
a role in renal cell carcinoma since down-regulation of sema5B
expression in renal cell carcinoma cells significantly compromises
their viability (Hirota et al., 2006).
5. Modulation of tumor progression by semaphorins that
alter immune responses or by semaphorins expressed in
immune cells
Tumor associated macrophages (TAMs) secrete a variety of pro-
angiogenic factors including VEGF and PlGF, which render them key
promoters of tumor angiogenesis. Normally TAMs are character-
ized as M1 TAMs that express CD11c which function as inhibitors
of tumor progression. However, in the tumor microenvironment
they frequently change their gene expression profile and behave
as M2 macrophages that secrete pro-angiogenic factors such as
VEGF, PlGF and sema4D to promote tumor angiogenesis and tumor
progression (Sierra et al., 2008; Mantovani and Sica, 2010). M2
macrophages also suppress anti-tumor immunity by preventing
8 G. Neufeld et al. / Drug Resistance Updates 29 (2016) 1–12
activation of dendritic cells (DCs), cytotoxic T lymphocytes (CTLs),
and natural killer (NK) cells (Mantovani and Sica, 2010).
Treatment of macrophages and monocytes with thioglycolate
or LPS induces the activation of toll like receptors (TLRs) and
increases the expression levels of SEMA4A and its receptors Plexin-
B2 and plexin-D1 specifically in Ly6Chigh inflammatory monocytes
but not in Ly6Clow resident monocytes. In turn, Sema4A increases
the migratory capacity of macrophages and induces VEGF expres-
sion as a result of the activation of plexin-D1, thereby enhancing
angiogenesis. Since inflammation and tumor progression are inti-
mately linked (Ben-Neriah and Karin, 2011; Mantovani et al., 2008),
these observations suggest that sema4A produced by recruited
monocytes and macrophages can also indirectly promote tumor
progression. Unlike other class-4 semaphorins, sema4A also uti-
lizes neuropilin-1 as a binding/signaling receptor. Neuropilin-1
is required by Treg cells to limit anti-tumor immune responses.
Sema4A binds to neuropilin-1 expressed on T-regulatory cells
(Tregs) and as a result supports their survival (Delgoffe et al., 2013).
An important aspect of tumor progression is the ability of
cancer cells to escape detection and clearance by the immune sys-
tem. Interestingly, some tumor cells express sema3A despite its
anti-angiogenic properties. T cells express the neuropilin-1 and
plexin-A4 sema3A receptors, and Sema3A containing conditioned
medium from such tumor cells was observed to inhibit the prolif-
eration of these cells and their activation by anti-Cd3 antibodies. In
contrast, suppression of Sema-3A expression in tumor cells using
a specific siRNA augmented T-cell activation (Catalano et al., 2006;
Yamamoto et al., 2008). Thus, sema3A seems to interfere with the
recognition of tumor cells by the immune system.
Sema4D was also identified as a critical regulator of T cell func-
tion. In the tumor microenvironment Sema4D is mainly expressed
by TAMs. CD4+ T-cells derived from mice lacking a functional
sema4D gene display impaired proliferation and impaired cytokine
production in response to antigen stimulation (Kumanogoh et al.,
2002b), while transgenic mice overexpressing a soluble extra-
cellular domain of sema4D display enhanced T-cell responses
(Watanabe et al., 2001). Likewise, down-regulation of sema4D
expression in CD8+ T-cells is correlated with impaired T-cells func-
tion. Dendritic cells (DC) express the sema4D receptor CD-72 and
the binding of sema4D to DC induces their maturation which in
turn results in enhanced activation of T-cells (Ishida et al., 2003;
Li et al., 2006). Natural killer cells express sema4D as well as
CD-72, and stimulation by sema4D enhanced target cell killing
(Lammie et al., 1991). These activities of sema4D are apparently
in conflict with the pro-angiogenic and pro-tumorigenic functions
of sema4D and how these activities are balanced remains to be
elucidated. Interestingly, sema4D not only affects the activities of
immune cells but also their recruitment to the tumor microenvi-
ronment. Strong expression of SEMA4D at the invasive margins of
actively growing tumors influences the infiltration and distribu-
tion of leukocytes in the tumor microenvironment. Neutralization
of sema4D function by blocking antibodies disrupts this gradient
of expression and enhances recruitment of activated monocytes
and lymphocytes into the tumor. This shifts the balance of cells
and cytokines in a pro-inflammatory and anti-tumorigenic direc-
tion and was found to be associated with durable tumor rejection in
murine Colon26 and ERBB2+ mammary carcinoma models (Evans
et al., 2015). The migration of immune cells can also be controlled
by other semaphorins such as sema3F which was recently found to
repulse thymocytes and T-cells (Mendes-Da-Cruz et al., 2014).
Another semaphorin that has distinct roles in immune
responses is sema7A. Activated T-cells express sema7A, and
sema7A in turn induces the expression of pro-inflammatory fac-
tors in macrophages (Suzuki et al., 2007; Holmes et al., 2002).
Sema7A produced by T-cells inhibits the activation of T-cells
since T-cells lacking sema7A display enhanced antigen induced
proliferation. Sema7A also plays a role in the adaptive immune sys-
tem in vitro by promoting transmigration of poly morphonuclear
granulocytes across endothelial cells into hypoxic areas via acti-
vation of its Plexin-C1 receptor. Thus up-regulation of sema7A
appears to enhance the immune response at hypoxic sites such as
those found in tumors by enhancing the accumulation of polymor-
phonuclear granulocytes (Morote-Garcia et al., 2012).
6. Conclusions and future perspectives
Initially it was thought that the semaphorins would function
primarily as inhibitors of tumor progression and tumor angiogen-
esis. This turned out not to be the case and nowadays several
semaphorins have been found to promote tumor progression
and to enhance angiogenesis. Furthermore, several semaphorins
were reported to both induce and inhibit tumor progression.
These different activities seem context-dependent and evidence
exists suggesting that interactions between semaphorin receptors
and apparently unrelated receptors such as various tyrosine-
kinase receptors as well as post-translational modifications of the
semaphorins and their receptors can profoundly affect their biolog-
ical activities as exemplified in the case of sema3E (Casazza et al.,
2010; Chauvet et al., 2007; Luchino et al., 2013; Rizzolio et al., 2012).
These interactions and modifications can in turn profoundly affect
the course of diseases such as cancer, and a better understanding of
these interactions and post translational modifications is required
if one considers the development of anti-tumorigenic and anti-
angiogenic therapeutic agents that target or utilize semaphorin
signal transduction. Thus, research aimed at a better understand-
ing of the processing of semaphorins and their receptors and better
characterization of the cross-talk between semaphorins and their
receptors and other signal transduction pathways is likely to be a
focus of attention for some time to come. In addition to cancer, it
seems that semaphorins play major regulatory roles in the devel-
opment and maintenance of the vascular and neuronal networks of
organs such as the retina and the kidney as well as in the immune
system. It is likely that the study of the role of the semaphorins in
the development of vascular diseases such as complications of dia-
betes including diabetic retinopathy or diabetic nephropathy will
also become a focus of intensive research in the near future.
Conflict of interest
The authors report no conflict of interest.
Acknowledgements
This work was supported by grants from the Israel Science Foun-
dation (ISF) and by a grant from the Rappaport Family Institute for
Research in the Medical Sciences.
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