The neuropilins and their role in tumorigenesis and tumor progression

Cancer Letters 231 (2006) 1–11 www.elsevier.com/locate/canlet

Mini review

The neuropilins and their role in tumorigenesis and tumor progression Noga Guttmann-Raviv, Ofra Kessler, Niva Shraga-Heled, Tali Lange, Yael Herzog, Gera Neufeld* Cancer and Vascular Biology Research Center, Rappaport Research Institute in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, 1 Efron St., P.O. Box 9679, Haifa 31096, Israel Received 17 December 2004; accepted 22 December 2004

Abstract The neuropilins were originally described as receptors for the six axon guidance factors belonging to the class-3 semaphorins. They were subsequently found to function in addition as receptors for specific splice forms of angiogenic factors belonging to the VEGF family. The neuropilins are expressed in many types of cancer cells, in endothelial cells and in additional many types of normal diploid cell types. Recent findings indicate that the neuropilins and their associated plexin and tyrosine-kinase VEGF receptors play a regulatory role in developmental angiogenesis as well as in tumor angiogenesis. The neuropilin ligands belonging to the semaphorin family as well as the various VEGF’s function as modulators of angiogenesis and tumor angiogenesis. Furthermore, since many types of cancer cells express neuropilins and neuropilin associated receptors, it is not surprising that various neuropilin ligands can modulate the behavior of cancer cells directly leading to the potentiation or inhibition of tumor progression. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tumor progression; Tumorigenesis; Semaphorin; Review; Neuropilin-1; Neuropilin-2; Plexin; Angiogenesis; Metastasis; Invasion; Semaphorin

1. Introduction The members of the neuropilin family were originally identified as receptors for the repulsive axon guidance factors belonging to the class-3 semaphorin1

* Tel.: C972 4 829 5430; fax: C972 4 852 3672. E-mail address: [email protected] (G. Neufeld). 1 The names of all semaphorins are abbreviated according to the following rules: s always appears as the first letter denoting a semaphorin. The number after the s designates the semaphorin to a specific class, and the final letter designates its place within the class. Thus, s3f means semaphorin-3F while s4d means semaphorin-4D.

sub-family [1,2]. However, several of these class-3 semaphorins such as s3b and s3f have been originally identified as tumor suppressors, indicating that neuropilins play a role in tumorigenesis. Recently, the neuropilins where also fund to function as splice form specific receptors for angiogenic factors belonging to the VEGF family, indicating that neuropilins could function as transducers of pro and anti-angiogenic signaling. It follows that neuropilin mediated signal transduction may regulate tumor progression by several concurrent mechanisms. We review current knowledge regarding the neuropilin structure, the interactions of the neuropilins with their various ligands

0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.12.047

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and associated cell surface receptors, and our current understanding of the role of the neuropilins in tumor progression.

2. The neuropilins, their ligands, and their associated membrane receptors 2.1. The neuropilins and their interactions The six class-3 semaphorins are secreted proteins that differ from other types of semaphorins by their inability to bind directly to plexins, although recently it was shown that contrary to this dogma, s3e binds directly to plexin-D1 but not to neuropilins [3]. The class-3 semaphorins bind to the neuropilin-1 (np1) or to neuropilin-2 (np2) receptors or to both of these receptors [1,2,4,5]. Even though the overall homology between the np1 and the np2 genes is only 44%, the neuropilins display an identical domain structure and their molecular mass is similar [4]. Important structural features of the neuropilins are

two complement binding (CUB) domains (a1 and a2 domains), two coagulation factor V/VIII homology domains (b1 and b2 domains), and a meprin (MAM) domain thought to be important for neuropilin dimerization and for the interaction of neuropilins with other membrane receptors [2] (Fig. 1). The intracellular domain of the neuropilins is short, and it was therefore assumed that it does not suffice to transduce biological signals. This view is supported by experiments that have shown that although np1 is required for s3a induced collapse of axonal growth cones, deletion of the cytoplasmic domain of np1 does not inhibit s3a activity, suggesting the existence of independent signal transducing moieties [6]. These were later found to be the products of genes encoding type-A plexins [7,8] (Fig. 1). Type-A plexins were found to form complexes with neuropilins, and to serve as the signal transducing components in these semaphorin holo-receptors [7–10]. Plexins belonging to the other three plexin subfamilies may also be able to form functional complexes with neuropilins, as demonstrated in the case of plexin-B1 and np1 [8,11],

CUB: Complement binding domain

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Sema domain

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MET-related sequence

Glycine-proline (G-P) rich motif

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Fig. 1. Shown is a scheme of np1, np2 and the A type plexin plexin-A1. The general domain structure of these receptors is depicted.

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and recently also in the case of plexin-D1 [12,13]. The assumption which predicts that neuropilins cannot transduce biological signals on their own due to their short intracellular domains was recently challenged in experiments in which the extracellular domain of the epidermal growth factor (EGF) receptor was fused to the intracellular and transmembrane domains of np1. These experiments indicate that the chimeric receptor can promote cell migration in response to EGF, suggesting that the intracellular domain of np1 does transduce biological signals independently [14]. The last three amino-acids of np1 (SEA-COOH), bind specifically to the PSD-95/Dlg/ZO-1 Domain of the NIP protein [15] and it is possible that this interaction is important for the induction of cell migration by the chimeric receptor. These experiments therefore indicate that the short intracellular domain of the neuropilins is not devoid of function. The neuropilins form complexes with additional cell surface molecules. Np1 was reported to form complexes with L1-CAM, an adhesion molecule which seems to be required under certain circumstances for s3a signal transduction. Interestingly, in the presence of L1-CAM, s3a attracts axon growth cones while in the absence of L1-CAM s3a repulses growth cones of responsive nerve cells [16,17]. This conversion of a repulsive signal to an attractive signal may be associated with changes in the intracellular levels of cGMP [18]. Both neuropilins were also found to form complexes with the VEGF tyrosinekinase receptor VEGFR-1 [19,20]. This interaction may be required for s3a induced repulsion of migrating neuronal progenitor cells which is dependent upon the simultaneous presence of VEGFR-1 and np1 [21]. It was also reported that np1 forms complexes with VEGFR-2 [22–24]. The formation of such complexes probably accounts, at least partially, for the np1 dependent potentiation of VEGF induced cell migration [25]. As with most receptors, alternative splicing also produces several secreted neuropilin forms that lack transmembrane and intra-cellular domains [26–28]. Soluble extracellular domains of membrane receptors are usually thought to be inhibitory, since they can function as ligand traps. However, in the case of the class-3 semaphorins it was shown that the binding of s3a to a soluble extracellular domain of np1 can lead to signal transduction, provided that a type-A plexin is

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present on the target cells [6]. Similarly, in the case of VEGF there is evidence suggesting that dimerized soluble extracellular domains of np1 potentiate VEGF signaling via the VEGFR-2 receptor while monomers act solely as VEGF traps to inhibit VEGF function [28,29]. 2.2. The plexins The plexin family contains nine vertebrate members segregated into four classes. The plexins are transmembrane proteins containing a cytoplasmic SP domain that includes putative tyrosine phosphorylation sites but no known enzymatic activity. Their extracellular domains are distinguished by the presence of a sema domain, by the presence of a Met related sequence (MRS) domain and by glycine– proline (G–P) rich motifs which the plexins share with the tyrosine-kinase receptors belonging to the Met receptor family (Fig. 1) [30]. Some vertebrate semaphorins belonging to the 4–7 classes have been shown to bind directly to plexins and to activate plexin mediated signal transduction. For example, semaphorin-4D (s4d) binds to plexin-B1 [31], s3e to plexin-D1 [3] and semaphorin-6D (s6d) binds to plexin-A1 [32]. In addition, semaphorin-7A as well as several viral semaphorins bind to plexin-C1 which is itself a virally encoded plexin like receptor [8]. Interestingly, the interaction between s6d and plexinA1 also results in retrograde signal transduction by s6d [33]. Type-A plexins associate with neuropilins to form functional receptors for class-3 semaphorins [7–10]. It was found that additional plexins such as plexin-D1 and plexin-B1 can also form complexes with neuropilins, and that in the case of plexin-D1 these complexes can convey semaphorin signals [12,13,34]. Plexin-B1 on the other hand, forms complexes with the hepatocyte growth factor (HGF) receptor MET. Interestingly, the MET tyrosine kinase can be activated following the binding of s4d to plexin-B1, resulting in the phosphorylation of both receptors and MET signal transduction in the absence of HGF [31]. Likewise, s6d potentiates the effects of VEGF as a result of complex formation between plexin-A1 and VEGF receptor-2 [32]. Plexins were found to associate with additional types of cell surface receptors. Plexin-A1 associates with the Off-Track

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(OTK) receptor [35] and the plexin-A1 ligand s6d can also induce OTK mediated signaling [32].

2–4 and 7) and thrombospondin repeats (class 5) (Fig. 2). The class-3 semaphorins are secreted proteins distinguished by the presence of a basic domain at their C-terminal. The active forms of the class-3 semaphorins are probably homo-dimeric as shown in the case of s3a [40,41]. Semaphorins have been mainly characterized as axon guidance factors [42]. However, it was recently realized that semaphorins play a role in many developmental processes outside of the nervous system, in particular as regulators of cell migration [43], immune responses [44] and organogenesis [45]. It is therefore not surprising that some of the semaphorins have been found to play important roles in tumor development and progression as well as in the control of angiogenesis.

2.3. Class-3 semaphorins The first neuropilin ligands to be characterized were the five proteins encoded by the class-3 semaphorin gene subfamily. These semaphorins are the only semaphorins that interact with neuropilins to activate signal transduction. In the literature the semaphorins are often referred to by an array of confusing designations. This situation was clarified several years ago by the adoption of a unified nomenclature for the semaphorins [36]. All the semaphorins are characterized by the presence of a w500 amino-acids long conserved sema domain which is usually located at their N-termini. The sema domain is essential for semaphorin signaling and determines receptor binding specificity [37]. The sema domains of two different semaphorins were recently characterized at atomic resolution revealing beta propeller topology [38,39]. In addition, semaphorins contain additional structural motifs such as immunoglobulin like domains (classes

2.4. The VEGF splice forms and their interaction with neuropilins The angiogenic factor VEGF (also known as VEGF-A) is considered to be a major angiogenic factor that plays an essential role in embryonic vasculogenesis and angiogenesis as well as in tumor

invertebrate Viral Semaphorins Semaphorins

Vertebrate Semaphorins

Sema domain Immunoglobulin loop Basic domain

Thrombospondin repeats

GPI anchor

Semaphorin class: 1

2

8

3

4

5

6

7

Fig. 2. The general structure of the semaphorin subfamilies along with their structural motifs is shown. Class 1 and class 2 semaphorins are invertebrate and class-8 semaphorins are viral. The only soluble, secreted type of vertebrate semaphorins are the class-3 semaphorins which interact with neuropilins. However, active soluble forms of the other semaphorin types can be generated as a result of proteolytic processing.

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The VEGF (VEGF-A) splice variants exons 1-5 exons 1-5

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7

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VEGF 189

exons 1-5

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VEGF 206

exons 1-5

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VEGF forms starting from alternative translation start sites

Fig. 3. The exon structure of the various splice forms of VEGF is shown. Exons are not drawn to scale. The main forms are labeled with stars. Of these forms one is inhibitory [88]. Alternative forms of VEGF that are translated from alternative CUG codons are probably nuclear [89]. Exons 6 and 7 encode independent heparin binding domains. Exon-7 is important for successful binding of VEGF165 to np1 [90].

angiogenesis [46]. Multiple forms of VEGF are produced as a result of alternative splicing (Fig. 3). three of these forms, VEGF121, VEGF165, and VEGF189 are commonly encountered. Most splice forms differ with respect to the expression of exons 6 and 7 of the VEGF gene. Exons 6 and 7 encode independent heparin binding domains that are incorporated into the longer VEGF forms. The shortest VEGF splice form, VEGF121, lacks exons 6 and 7 altogether and does not bind to heparin. VEGF165 is the form most commonly encountered and used. It includes the peptide encoded by exon 7 but not the peptide encoded by exon-6 which is expressed in VEGF145. VEGF189 includes both exons and interacts strongly with heparin and heparan-sulfates [46]. Despite the absence of these two exons, VEGF121 is still active as an inducer of angiogenesis in vivo and as a mitogen for endothelial cells [47–49]. However, VEGF121 alone cannot compensate for the lack of the other VEGF splice forms during embryonic development [50].

All the VEGF splice forms are able to bind to the VEGFR-1 and to the VEGFR-2 tyrosine-kinase VEGF receptors [46]. However, it was observed that human umbilical vein derived endothelial cells express VEGF receptors that bind VEGF165 but do not bind VEGF121 [51]. Similar receptors were subsequently found to be expressed in large amounts on several breast and prostate cancer derived cell lines lacking VEGFR-1 or VEGFR-2 [52]. Such cells were used as a source for the purification of these receptors, which were subsequently found to be the products of the np1 gene. It was found that the VEGF165 induced migration of cells expressing recombinant VEGFR-2 receptors was enhanced in the presence of np1. In-contrast, VEGF121 induced cell migration was unaffected by the presence of np1 [25]. Thus, np1 seems to function as an enhancer of VEGFR-2 activity in the presence of VEGF165. This effect is probably the result of complex formation between VEGFR-2 and np1 [22,23].

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The related np2 receptor [1,53] was also found to behave as a splice form specific VEGF receptor that binds VEGF165 but not VEGF121. This behavior resembles that of np1, but unlike np1, it was found that np2 is also able to bind the VEGF145 splice form of VEGF [54]. It was recently observed that mice lacking np2 do not respond efficiently to VEGF by induction of retinal neo-vascularization, indicating that np2 is probably important for the transduction of VEGF angiogenic signals in the retina [55]. Both neuropilins were also found to function as receptors for several additional members of the VEGF family. The heparin binding form of placenta growth factor (PlGF) binds to both np1 and np2 [54,56], VEGF-B binds to np1 [57] and VEGF-C was found to interact with np2 [58]. Interestingly, PlGF and VEGF-B are not able to bind to VEGFR-2, the tyrosine-kinase receptor that seems to be responsible for the transduction of VEGF induced proliferative and angiogenic responses [59,60]. Both VEGF-B and PlGF-2 bind to VEGFR-1, a tyrosine-kinase VEGF receptor that forms complexes with np1 and with np2 [19,20]. It is therefore likely that VEGFR-1 plays a role in neuropilin mediated VEGF and semaphorin signaling. An initial observation that agrees with this notion was provided when it was observed that the migration of DEV neuroectodermal progenitor cells is repulsed by sema-3A, and that the presence of both np1 and VEGFR-1 is required for the repulsion [21]. 2.5. Neuropilin-1 mediated effects on tumor progression and tumor angiogenesis The discovery of splice form specific vascular endothelial growth factor receptors on endothelial cells [51] and their subsequent identification as products of the np1 and np2 genes [25,54], indicated that neuropilins play a role in the mediation of VEGF induced angiogenesis. It was indeed observed that gene targeted mice lacking functional np1 display severe cardiovascular abnormalities in addition to neuronal abnormalities [61]. Since tumor expansion and tumor spread depend upon tumor angiogenesis [62], it follows that neuropilin mediated signaling may regulate tumor angiogenesis and consequently tumor expansion and progression.

2.6. The effects of soluble forms of neuropilin-1 on tumor progression The secreted soluble forms of np1 [26] may function as inhibitors of VEGF function by trapping VEGF and inhibiting its interaction with membrane receptors. This notion was supported by a study, which showed that a natural truncated, soluble form of np1 containing only the A and B domains but not the c domain, which is required for receptor dimerization inhibits VEGF activity. Tumors of rat prostate carcinoma cells expressing this recombinant soluble np1 were characterized by extensive hemorrhage, damaged vessels, and by the presence of apoptotic tumor cells. It therefore seems that this monomeric soluble np1 form functions as a VEGF165 antagonist and as an inhibitor of tumor angiogenesis and tumor progression [27]. In contrast, dimeric forms of soluble np1 are expected to enhance the activity of VEGF as recently demonstrated [29] and are expected to enhance VEGF induced tumor angiogenesis. 2.7. Effects of np1 over-expression in tumor cells on tumor progression Membrane bound np1 as well as soluble dimerized Np1 enhances VEGF induced signaling mediated by the VEGFR-2 VEGF receptor [29]. It follows that np1 expressed in tumor cells may be able to modulate VEGF induced signaling in adjacent endothelial cells. In agreement with this hypothesis it was found that inducible over-expression of np1 in AT2.1 prostate cancer derived cells led to faster tumor development even though it did not affect the proliferation rate of the cancer cells in vitro, indicating that the effects on tumor growth are not the result of a direct effect on the tumor cells. The enhancement in tumor growth rate was accompanied by an increased density of blood vessels in the tumors, presumably due to such trans effects on VEGF signaling [63]. Thus, even changes in neuropilin expression in tumor cells may be translated into enhanced angiogenesis. 2.8. Effects of class-3 semaphorins that bind to np1 on tumor progression The class-3 semaphorins function as repulsive factors during the development of the central nervous

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system [64]. Since neuropilins are expressed in endothelial cells of blood and lymph vessels, it is possible that class-3 semaphorins also regulate angiogenesis. S3a is a specific np1 agonist, and does not bind to np2. VEGF165 and s3a were found to compete for an overlapping binding site located in the extra-cellular domain of np1 [24]. S3a inhibits VEGF165 induced migration of endothelial cells as well as VEGF165 induced in vitro angiogenesis, presumably by inhibiting VEGF165 binding to np1 [65]. Subsequent experiments have shown that s3a can also inhibit developmental angiogenesis in chick embryos, a finding which suggests that s3a should also be able to inhibit tumor angiogenesis [66,67]. However, an effect of s3a on tumor angiogenesis has not yet been demonstrated. S3a may also affect directly the behavior of np1 expressing tumor cells in addition to angiogenesis. Np1 is expressed by tumor cells derived from prostate cancer [68], colon cancer [69], melanoma [70], pancreatic carcinoma [71] and breast cancer derived cells [52,72] to name a few examples. MDA-MB-231 breast cancer cells express large amounts of np1 and plexin-A1 and their migration and spreading are inhibited by s3a. VEGF165 competed with s3a for binding to np1 on these cells and abrogated the inhibitory effects of s3a, indicating that semaphorins such as s3a have the potential to function as antimetastatic agents, and that by this mechanism VEGF165 and semaphorins may affect directly the behavior of cancer cells [72].

3. Neuropilin-2 mediated effects on tumor progression and tumor angiogenesis Np2 was also found to behave as a splice form specific VEGF receptor and its VEGF binding characteristics closely resemble those of np1. However, np2 can also bind VEGF145, a VEGF splice form that is not recognized by np1 [54]. The binding of s3a to np1 is inhibited by VEGF165 but the binding of s3f to np2 is not inhibited by VEGF165 suggesting that the two np2 ligands bind to separate binding sites on np2 [19]. During early embryonic development np2 is expressed in veins while np1 is expressed in arteries [73]. Later, np2 is also expressed in lymph vessels suggesting that np2 ligands may affect

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lymphangiogenesis [74]. Indeed, np2 also functions as a receptor for the lymphangiogenesis inducer VEGF-C [58]. Np2 gene targeted mice are viable. They display neuronal abnormalities but the cardiovascular system is normal except for some abnormalities in peripheral lymph vessels [74]. However, mice in which both neuropilin genes were inactivated display cardiovascular defects that are much more acute than those generated by the inactivation of a single neuropilin. Such mice fail to develop any blood vessels and die in utero at day 8.5, thereby confirming a role for both neuropilins in vasculogenesis and developmental angiogenesis [75]. In agreement with this conclusion, it was recently reported that VEGF induced retinal angiogenesis in mice lacking a functional np2 gene is strongly inhibited. These experiments indicate that np2 has an important regulatory role as a regulator of VEGF function and as a regulator of angiogenesis [55] and that np2 ligands may affect tumor angiogenesis and tumor lymphangiogenesis. 3.1. The effects of np2 binding class-3 semaphorins on tumor progression Np2 receptors as well as multiple types of plexins are expressed in many types of tumor cells, and VEGF, VEGF-C or np2 binding class-3 semaphorins may therefore affect tumor progression by binding to np2 receptors of tumor cells. The gene encoding the np2 agonist s3f has been originally characterized a tumor suppressor of small cell lung carcinoma tumors [76,77]. Np2 is also able to bind to np1, although with a 10-fold lower affinity. However, no np1 mediated biological responses to s3f have been described so far [1,4,53]. Expression of recombinant s3f in small cell lung carcinoma cells inhibited colony formation in soft agar indicating that s3f affects directly the behavior of these cells [78]. This conclusion is supported by experiments showing that expression of recombinant s3f in MCF-7 breast cancer cells inhibits their adhesion and spreading [79]. The localization of s3f in s3f expressing tumor cells may be important, as it was shown that in lung tumors a cytoplasmic localization of s3f is correlated with high VEGF expression levels and increased tumorigenicity, suggesting that failure to secrete s3f may promote tumor progression instead of inhibiting it [80].

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Even though s3f does not complete with VEGF165 for binding to np2 [19], s3f was able to inhibit both VEGF and bFGF induced endothelial cell proliferation and angiogenesis, as well as the development of tumors from HEK293 cells engineered to over-express s3f even though the proliferation of these cells in cell culture was not inhibited by s3f. These experiments suggest that s3f can interfere with the pro angiogenic signaling of bFGF and VEGF165. Since bFGF does not interact with neuropilins and bFGF receptors do not bind s3f, these results suggest that s3f inhibits angiogenesis using a mechanism that does not require competition with angiogenic factors for binding to shared receptors. Rather, the experiments suggest that s3f binds to np2 receptors and initiates a signaling cascade that interferes with bFGF and VEGF angiogenesis promoting signal transduction [81]. Np2 is also expressed in lymphatic endothelial cells indicating that neuropilins may also play a role in the regulation of lymphangiogenesis. Recent evidence indicates that activation of np2 with s3f inhibits the migration of lymphatic endothelial cells. Furthermore, s3f was able to inhibit invasion and metastasis of melanoma derived cancer cells expressing functional np2 receptors, and to inhibit blood vessel growth into these tumors [82]. S3b, another class-3 semaphorin was also found to inhibit VEGF induced angiogenesis and tumor development by inhibiting VEGF activity [83]. The data regarding other class-3 semaphorins is scant, but indicates that some of these other class-3 semaphorins may promote tumor progression rather than inhibit tumor progression. Thus, S3c expression was found to be up-regulated in cis-diamminedichloroplatinum (II) (CDDP)-resistant ovarian cancer TYKnuR cells, in metastatic human lung adenocarcinoma cells, and in malignant melanoma cells indicating that upregulation of s3c expression may be linked to tumor progression [84–86]. Likewise, high level expression of s3e had been observed in several metastatic cell lines originating from mouse mammary adenocarcinoma tumors, indicating that high s3e expression levels are linked to tumor progression [87].

4. Conclusions The neuropilins have been shown to be expressed by many types of tumor cells as well as in endothelial

cells. It is therefore not surprising that the neuropilins have been implicated as important receptors that control angiogenesis as well as the behavior of neuropilin expressing cancer cells. The characterization of the possible role of the various class-3 semaphorins and the characterization of the mechanisms by which neuropilins modulate VEGF signaling will likely represent active topics of research in the near future.

Acknowledgements This work was supported by grants from the Israel Science Foundation (ISF), German–Israeli Binational Foundation (GIF), Israel Cancer Foundation and by the by the Rappaport Family Institute for Research in the Medical Sciences of the Faculty of Medicine at the Technion, Israel Institute of Technology (to G. N.).

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