Class 3 semaphorins (SEMA3) were first identified as glycoproteins that negatively mediate neuronal guidance by binding to neuropilin and repelling neurons away from the source of SEMA3. However, studies have shown that SEMA3s are also secreted by other cell types, including tumor cells, where they play an inhibitory role in tumor growth and angiogenesis (specifically SEMA3B and SEMA3F). SEMA3s primarily inhibit the cell motility and migration of tumor and endothelial cells by inducing collapse of the actin cytoskeleton via neuropilins and plexins. Besides binding to SEMA3s, neuropilin also binds the protumorigenic and proangiogenic ligand vascular endothelial growth factor (VEGF). Although some studies attribute the antitumorigenic and antiangiogenic properties of SEMA3s to competition between SEMA3s and VEGF for binding to neuropilin receptors, several others have shown that SEMA3s display growth-inhibitory activity independent of competition with VEGF. A better understanding of these molecular interactions and the role and signaling of SEMA3s in tumor biology will help determine whether SEMA3s represent potential therapeutic agents. Herein, we briefly review (a) the role of SEMA3s in mediating tumor growth, (b) the SEMA3 receptors neuropilins and plexins, and (c) the potential competition between SEMA3s and VEGF family members for neuropilin binding. (Clin Cancer Res 2009;15(22):676370)

Translational Relevance

Semaphorins are glycoproteins that were originally described as mediators of neuronal guidance. However, recent studies show that other cell types, including tumor cells, also secrete semaphorins. In particular, class 3 semaphorins (SEMA3) serve as key regulators of cellular processes such as endothelial and tumor cell survival, proliferation, apoptosis, and migration. Additionally, they function as potent inhibitors of tumor growth and vessel density, although the mechanisms by which these effects are mediated remains to be elucidated. Given the results from numerous preclinical forced expression studies, it is clear that exogenous SEMA3s may offer a new class of drugs that can be used for the treatment of cancer. In this review article, we will highlight current knowledge regarding the SEMA3s and their role in tumor progression.

Semaphorins were first described in 1993 as negative mediators of axonal guidance in the central nervous system (1). However, the discovery of more widespread expression of class 3 semaphorins (SEMA3) by various tumor cell types has suggested a role for these ligands in tumor biology. SEMA3 mediates its actions by binding to neuropilin, cell surface receptors that complex with plexins to mediate downstream signaling. Neuropilins additionally serve as receptors for vascular endothelial growth factor (VEGF; refs. 2, 3), whereby they serve to promote VEGF-induced tumor angiogenesis and growth. Because SEMA3s are understood to inhibit angiogenesis and tumor growth through neuropilins, it has been suggested that perhaps they act by merely competing with VEGF and thus inhibiting its proangiogenic and protumorigenic effects. Despite numerous studies investigating the interaction between SEMA3s, neuropilins, and VEGF, it is not clear whether SEMA3s inhibit tumor growth by simply competing with VEGF for neuropilin ligand-binding sites, whether they act independently of VEGF or both (4,6). Therefore, from a therapeutic standpoint, it is not clear whether semaphorins represent (a) simply another approach to blocking VEGF activity or (b) an antitumorigenic/antiangiogenic therapy that works through alternative pathways. The rationale for developing semaphorin-based anticancer therapy will ultimately depend on a better understanding of the precise roles of SEMA3s, neuropilins, and VEGF in tumor biology. In this review, we present evidence for a role for SEMA3s in tumor biology with a specific focus on the possible effects of SEMA3s on the VEGF/neuropilin system.

Semaphorins, also known as collapsins, were first identified as a family of genes encoding guidance molecules for the embryologic development of the nervous system (1). They were found to induce collapse of growth cones of neurons and to repel axons from growing sensory, sympathetic, and motor neurons. We now know that semaphorins can affect other cell lineages such as endothelial cells, inflammatory cells, vascular smooth muscle cells, and various tumor cells (reviewed in ref. 7).

SEMA3s comprise one of five vertebrate families of semaphorins and are known to play an important role in tumor biology (8). The SEMA3 class consists of seven soluble proteins of 100 kDa (designated by the letters A-G), which are secreted by cells of multiple lineages, including epithelial cells, neurons, and specific tumor cells. SEMA3s act in a paracrine fashion by binding to neuropilins via a highly conserved amino-terminal 500amino acid region in the SEMA3 protein called the Sema domain (9). After binding to SEMA3s, neuropilins complex with another family of proteins, the plexins, to mediate downstream signaling. Plexins are large 200-kDa transmembrane receptors that primarily play a role in cellular organization and migration, although the exact mechanism remains unclear (reviewed in ref. 10). The seven SEMA3s have different binding affinities to neuropilins; hence, each SEMA3 has a distinct biological function (Fig. 1A). Whereas SEMA3A preferentially binds to NRP1, SEMA3F and SEMA3G have a higher binding affinity to NRP2. SEMA3B, SEMA3C, and SEMA3D bind to both isoforms (7); SEMA3E binds to neither neuropilin but instead binds directly to plexin D1 (11). Additionally, each SEMA3-neuropilin complex recruits specific plexins to mediate downstream signaling (Fig. 1B).

Fig. 1.

Binding profile of semaphorins. A, members of the SEMA3 family bind to neuropilin with varying affinities and then complex with plexin. Whereas SEMA3A binds to NRP1-plexins A1 to A4, SEMA3F and SEMA3G bind to NRP2-plexins A1 to A4. SEMA3B, SEMA3C, and SEMA3D bind to both NRP1-plexin and NRP2-plexin. B, SEMA3s signal through various plexins. All SEMA3s can signal through plexins A1 to A4 with the exception of SEMA3E, which binds directly to plexin D1 to mediate its effects. SEMA3E does not require neuropilins.

Fig. 1.

Binding profile of semaphorins. A, members of the SEMA3 family bind to neuropilin with varying affinities and then complex with plexin. Whereas SEMA3A binds to NRP1-plexins A1 to A4, SEMA3F and SEMA3G bind to NRP2-plexins A1 to A4. SEMA3B, SEMA3C, and SEMA3D bind to both NRP1-plexin and NRP2-plexin. B, SEMA3s signal through various plexins. All SEMA3s can signal through plexins A1 to A4 with the exception of SEMA3E, which binds directly to plexin D1 to mediate its effects. SEMA3E does not require neuropilins.

Close modal

In the vascular system, SEMA3s impair endothelial cell adhesion, migration, tube formation, sprouting, branching, survival, permeability, and angiogenesis (reviewed in ref. 12). In tumor biology, however, SEMA3s function as repellants, inhibitors of migration, and inducers of apoptosis for both endothelial cells and specific tumor cells [e.g., breast cancer (13, 14) and lung cancer (15)]. Therefore, SEMA3s play a dual inhibitory role of impeding tumor angiogenesis and tumor growth (specifically SEMA3B and SEMA3F). It should be noted, however, that the inhibitory effects of SEMA3s cannot be generalized to all members of the SEMA3 family. Although most members of the SEMA3 family have been shown to be inhibitory (SEMA3A, SEMA3B, SEMA3D, SEMA3F, and SEMA3G), some promote tumor angiogenesis, growth, and metastasis (SEMA3C and SEMA3E; refs. 16, 17).

Neuropilins

Neuropilins (NRP1 and NRP2) are transmembrane glycoproteins with short cytoplasmic domains (42-44 amino acids) and no known signaling motifs of their own; however, some researchers have refuted this and suggested that the last three amino acids (S-E-A) of either neuropilin may participate in signaling via G-interacting proteininteracting proteins (18). Although neuropilins were originally identified as key mediators of axonal guidance (via semaphorins), they are now known to also play a major role in vasculogenesis, angiogenesis, and tumor growth (reviewed in ref. 19). In addition to serving as coreceptors for SEMA3s, neuropilins are also coreceptors for VEGF family members, which are structurally unrelated to SEMA3s. VEGF family members mediate their downstream effects by binding to neuropilins and forming complexes with VEGF receptors (VEGFR) analogous to the SEMA3-neuropilin-plexin complex (2, 20). Neuropilins are expressed on various cell lineages, including neurons, endothelial cells, inflammatory cells, vascular smooth muscle cells, and tumor cells. This expression profile allows VEGF and SEMA3s to have wide-ranging effects on multiple cell lineages. Neuropilins are differentially expressed in the vascular system: whereas NRP1 is mainly expressed on arterial endothelial cells, NRP2 is primarily expressed on venous and lymphatic endothelial cells (21,23). This specificity forms the basis for how different combinations of SEMA3 and VEGF family members play tightly regulated roles in lymphangiogenesis and vasculogenesis through their interactions with specific neuropilins (21, 24).

Neuropilins are 130-kDa single-spanning transmembrane glycoproteins composed of five distinct domains: three extracellular domains (a1a2, b1b2, and c), one transmembrane domain, and one intracellular domain (19, 25). The a1a2 domain typically binds to SEMA3s, whereas the b1b2 domain binds to VEGF family members. Just as neuropilins have different binding affinities for SEMA3 family members, neuropilins have varying affinities for VEGFs (Fig. 2). Both NRP1 and NRP2 can bind to several different isoforms of VEGF-A, particularly to those that contain the neuropilin-binding domain in exon 7 (26, 27). In addition, NRP1 binds to VEGF-B and placental growth factor-2 (PlGF-2) and complexes with either VEGFR-1 or VEGFR-2 after ligand binding. NRP2 can bind to VEGF-C, VEGF-D, and PlGF-2 and can complex with VEGFR-1, VEGFR-2, or VEGFR-3 (19). The result of these biophysical differences is that various VEGFR/VEGF/neuropilin and SEMA3/neuropilin/plexin complexes are generated that each may exert unique influences on not only physiologic processes but also on tumor angiogenesis and subsequent tumor growth.

Fig. 2.

Binding profile of VEGF ligands. NRP1 and NRP2 function as coreceptors for VEGFRs and affect the binding affinity of VEGF ligands. Pink circles on VEGFRs indicate immunoglobulin loops.

Fig. 2.

Binding profile of VEGF ligands. NRP1 and NRP2 function as coreceptors for VEGFRs and affect the binding affinity of VEGF ligands. Pink circles on VEGFRs indicate immunoglobulin loops.

Close modal

Plexins

The primary cellular consequence of semaphorin signaling is cytoskeletal collapse mediated by plexins, which are part of the SEMA3-neuropilin ligand-receptor complex. Plexins are large transmembrane receptors with highly conserved cytoplasmic domains (structure reviewed in ref. 7). The plexin family consists of nine members: four type A plexins (A1, A2, A3, and A4), three type B plexins (B1, B2, and B3), and plexins C1 and D1. The SEMA3s mediate their actions either via plexins type A or D1, which then employ a distinct GTPase-activating intracellular domain to affect actin depolymerization (5, 28). Miao et al. first showed that Sema3A binds to endothelial NRP1 and inhibits endothelial cell migration and capillary sprouting by causing retraction of endothelial cell lamellipodia (5). Despite extensive research in this field, there is no general consensus on the exact intracellular signaling pathway employed by plexins to cause actin depolymerization and cellular collapse (28,30). In addition, Serini et al. recently showed that SEMA3s are able to inhibit integrin activation in endothelial cells, a phenomenon required for endothelial cells to adhere to fibronectin and vitronectin (components of the extracellular matrix) to migrate (31). These inhibitory effects of SEMA3s on integrins also seem to be dependent on plexins (32). Although no clear mechanism has been elicited for plexin-mediated inhibition of tumor growth and angiogenesis, it is universally accepted that SEMA3s inhibit cellular motility and migration by mediating its actions via plexins (33).

As discussed above, the functions of SEMA3s in guiding endothelial cells during vascular development are dependent on receptor complexes that vary according to tissue-specific expression as well as coreceptor combinations. Yet another level of complexity is added when variability among SEMA3 family members is taken into account. Among the SEMA3s, whereas SEMA3A and SEMA3F have been shown to have antiangiogenic properties (31, 3437), SEMA3C is considered to be a proangiogenic factor (16). Numerous studies have shown that both SEMA3A and SEMA3F are able to inhibit in vitro endothelial cell proliferation, survival, and cord formation, although the exact mechanism remains unclear. Some studies have suggested that SEMA3A and SEMA3F inhibit migration of endothelial cells by inhibiting activation of integrins and thereby disrupting tumor angiogenesis (31, 37). In contrast, other studies have shown that SEMA3A and SEMA3F inhibit VEGF-induced and basic fibroblast growth factorinduced angiogenesis (36, 37). SEMA3C, on the other hand, enhances endothelial cell proliferation, migration, and adhesion in renal glomeruli, which can be attributed to its ability to activate integrins and alter cytoskeletal dynamics (16).

The inhibitory effects of SEMA3A and SEMA3F on tumor angiogenesis have been explored by several investigators as potential therapeutic agents for cancer. A recent study by Kigel et al. has shown successful inhibition of tumor growth by coexpressing SEMA3A in tumor cells expressing NRP1 (38). Furthermore, the results of that study showed that SEMA3A, SEMA3D, SEMA3E, and SEMA3G can function as potent inhibitors of tumor growth and affect vessel density. Another two seminal studies by Bielenberg et al. (35) and Kessler et al. (37) recently showed that melanoma cells and tumorigenic baby hamster kidney-21 cells transfected with SEMA3F, respectively, produced tumors with reduced microvessel density. These studies suggested that exogenous SEMA3A and SEMA3F can inhibit tumor angiogenesis and can potentially be used therapeutically to inhibit tumor angiogenesis.

Table 1 provides a summary of the function of the different members of the SEMA3 family in tumor biology. Of the SEMA3 family members, SEMA3A, SEMA3B, and SEMA3F have the best-described roles in tumor growth and metastasis. One of the observations suggesting that SEMA3s may be a type of tumor suppressor gene came from studies showing that that loss of heterozygosity of the 3p21.3 region correlated with the development of lung cancer (39, 40). This region was later discovered to correspond to a deletion of SEMA3B and SEMA3F genes that are now known to behave as tumor suppressor genes (41,44). To this end, loss of SEMA3F has been shown to correlate with advanced tumor stage in lung cancer and the development of metastasis in melanoma (35, 45).

Table 1.

Functional significance of SEMA3s in cancer biology (preclinical studies only)

MoleculeTarget cellFunctionRefs.
SEMA3A Endothelial cells Impairs endothelial cell adhesion and migration by inhibiting integrin activation (31, 36, 66) 
T lymphocytes Inhibits T-cell activation, leading to T-cell dysfunction (67) 
Human umbilical vein endothelial cells Induces disappearance of endothelial cell focal contacts, which is followed by collapse of actin cytoskeleton; repels endothelial cells; inhibits proliferation and survival of endothelial cells; and promotes apoptosis of endothelial cells (36) 
Breast cancer Inhibits in vitro cell migration and inhibits tumor growth in vivo (9, 38) 
Prostate cancer Overexpression correlates with decreased invasion and adhesion in vitro (46) 
SEMA3B Lung and breast cancers Induces apoptosis and antiproliferative effects in tumor cells (15, 47) 
Ovarian cancer Reduces anchorage-independent growth and in vivo tumor growth (43) 
SEMA3C Glomerular endothelial cells Promotes glomerular endothelial cell proliferation, adhesion, and migration (16) 
Prostate cancer Overexpression correlates with increased invasion and adhesion in vitro (46) 
SEMA3D Breast cancer Overexpression inhibits tumor development and angiogenesis in vivo (38) 
SEMA3E Prostate cancer Overexpression correlates with decreased invasion and adhesion in vitro (68) 
Breast cancer Processed SEMA3E promotes in vitro growth and migration and induces metastasis to lungs (69) 
SEMA3F Human umbilical vein endothelial cells Additive effect with SEMA3A to impair endothelial cell adhesion and migration (36) 
Human umbilical vein endothelial cells Inhibits VEGF and basic fibroblast growth factorinduced proliferation of human umbilical vein endothelial cells (37) 
Endothelial cells Induces chemorepulsion and inhibits adhesion of endothelial cells in vitro and in vivo angiogenesis (31, 35, 37) 
Lung cancer Inhibits 53 integrin activation (48) 
Melanoma Inhibits integrin function, motility, and cellular adhesion to fibronectin (35) 
Breast cancer Inhibits cell attachment, spreading, proliferation, and cell contacts (13, 14) 
Lung cancer (nonsmall cell lung cancer) Overexpression inhibits tumor growth by inhibiting angiogenesis; tumor suppressor gene (41, 48, 70) 
Melanoma Induces a benign phenotype in vivo: encapsulated, nonmetastatic tumor with increased apoptosis and diminished vascularity (35) 
Fibrosarcoma and ovarian adenocarcinoma SEMA3F-expressing clones suppressed tumor formation in vivo and induced growth arrest with chemotherapy (44) 
SEMA3G Breast cancer Overexpression inhibits angiogenesis in vivo (38) 
MoleculeTarget cellFunctionRefs.
SEMA3A Endothelial cells Impairs endothelial cell adhesion and migration by inhibiting integrin activation (31, 36, 66) 
T lymphocytes Inhibits T-cell activation, leading to T-cell dysfunction (67) 
Human umbilical vein endothelial cells Induces disappearance of endothelial cell focal contacts, which is followed by collapse of actin cytoskeleton; repels endothelial cells; inhibits proliferation and survival of endothelial cells; and promotes apoptosis of endothelial cells (36) 
Breast cancer Inhibits in vitro cell migration and inhibits tumor growth in vivo (9, 38) 
Prostate cancer Overexpression correlates with decreased invasion and adhesion in vitro (46) 
SEMA3B Lung and breast cancers Induces apoptosis and antiproliferative effects in tumor cells (15, 47) 
Ovarian cancer Reduces anchorage-independent growth and in vivo tumor growth (43) 
SEMA3C Glomerular endothelial cells Promotes glomerular endothelial cell proliferation, adhesion, and migration (16) 
Prostate cancer Overexpression correlates with increased invasion and adhesion in vitro (46) 
SEMA3D Breast cancer Overexpression inhibits tumor development and angiogenesis in vivo (38) 
SEMA3E Prostate cancer Overexpression correlates with decreased invasion and adhesion in vitro (68) 
Breast cancer Processed SEMA3E promotes in vitro growth and migration and induces metastasis to lungs (69) 
SEMA3F Human umbilical vein endothelial cells Additive effect with SEMA3A to impair endothelial cell adhesion and migration (36) 
Human umbilical vein endothelial cells Inhibits VEGF and basic fibroblast growth factorinduced proliferation of human umbilical vein endothelial cells (37) 
Endothelial cells Induces chemorepulsion and inhibits adhesion of endothelial cells in vitro and in vivo angiogenesis (31, 35, 37) 
Lung cancer Inhibits 53 integrin activation (48) 
Melanoma Inhibits integrin function, motility, and cellular adhesion to fibronectin (35) 
Breast cancer Inhibits cell attachment, spreading, proliferation, and cell contacts (13, 14) 
Lung cancer (nonsmall cell lung cancer) Overexpression inhibits tumor growth by inhibiting angiogenesis; tumor suppressor gene (41, 48, 70) 
Melanoma Induces a benign phenotype in vivo: encapsulated, nonmetastatic tumor with increased apoptosis and diminished vascularity (35) 
Fibrosarcoma and ovarian adenocarcinoma SEMA3F-expressing clones suppressed tumor formation in vivo and induced growth arrest with chemotherapy (44) 
SEMA3G Breast cancer Overexpression inhibits angiogenesis in vivo (38) 

Over the past decade, the role of SEMA3s in the pathogenesis of multiple malignancies has also been investigated in preclinical studies. SEMA3A has been implicated in the inhibition of tumor cell migration and chemotaxis in breast cancer cells (9). In prostate cancer, SEMA3A-transfected cells differentially regulate adhesion of cells together and have been shown to exhibit decreased invasion and adhesion (46). Similarly, SEMA3B transfection has been shown to inhibit in vitro tumor cell growth and to induce apoptosis in lung and breast cancer cells (15, 47). In ovarian cancer, transfection and overexpression of SEMA3B has been correlated with decreased anchorage-independent growth in vitro and with tumor formation in vivo (43). In lung cancer, overexpression of SEMA3F has been correlated with a decrease in tumorigenicity (48). These findings have been confirmed in two other studies in which lung cancer cell lines overexpressing SEMA3F produced smaller tumors than did control cells (41, 49). In breast cancer cells, exposure to SEMA3F has been shown to inhibit cell attachment and lamellipodia extension, the two processes necessary to mediate cellular migration (13, 14). Additionally, transfection of SEMA3F in fibrosarcoma cell lines has been shown to result in complete loss of tumorigenicity (44). Likewise, melanoma cells overexpressing SEMA3F exhibited decreased in vitro migration and resulted in a benign tumor phenotype (highly encapsulated and poorly vascularized tumors) in vivo (35). Taken together, these findings suggest that SEMA3A, SEMA3B, and SEMA3F inhibit tumor growth.

The prognostic role of SEMA3s has been further elucidated in several clinical studies. For example, Osada et al. showed that a high VEGF:SEMA3 ratio is an adverse prognostic factor for patients with ovarian cancer (50). Additionally, whereas high levels of SEMA3A have been correlated with poor prognosis in patients with pancreatic cancer, low levels of SEMA3F have been correlated with an advanced stage of melanoma (35, 51). However, melanoma clones overexpressing SEMA3F have been shown to show decreased potential to spontaneously metastasize to both lymph nodes and lungs in vivo (35). Numerous pathologic studies have reported a correlation with prognosis in cancer patients with variable expression levels of SEMA3s (summarized in Table 4 in ref. 52), suggesting that SEMA3s may play a major role in tumor growth and that they can potentially be used as therapeutic targets or agents depending on the specific SEMA3 ligand and tumor type.

As discussed previously, SEMA3 ligands have been hypothesized to exert their antineoplastic behavior by competing with VEGF for their binding site on neuropilin receptors (5). This raises the possibility that exogenously administered SEMA3s may simply serve as a form of anti-VEGF therapy. VEGF is the prototypical member of a family of angiogenic factors that mediate a variety of biological effects on endothelial cells and tumor cells via receptor tyrosine kinases (VEGFR; ref. 26). The mammalian VEGF family consists of five distinct glycoproteins (VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PlGF-2; ref. 53), each of which has unique receptor-binding and action profiles (Fig. 2). VEGF-A, the best-characterized molecule in the VEGF family, binds to VEGFR-1, VEGFR-2, NRP1, and NRP2. These receptors, expressed on tumor cells and endothelial cells, mediate downstream signaling after binding to VEGF-A and thereby promote tumor growth (reviewed in ref. 54). VEGF-B is presumed to mediate effects similar to those of VEGF-A and is known to bind to VEGFR-1 and NRP1, but the precise role of VEGF-B in malignancy has yet to be elucidated. VEGF-C and VEGF-D primarily play roles in lymphangiogenesis and bind to VEGFR-2, VEGFR-3, and NRP2, although VEGF-C may also be angiogenic (53). PlGF is a VEGF homologue that stimulates angiogenesis, local inflammatory response, tumor growth, and stromal cell migration (55,57). Differential splicing of the human PlGF gene generates two secreted PlGF proteins, PlGF-1 and PlGF-2. PlGF-2, the heparin-binding isoform, mediates its actions via binding to VEGFR-1 directly or by binding to the VEGFR-1 coreceptors NRP1 and NRP2 (58). Clinical and experimental data have suggested that the VEGF signaling pathways in tumor cells are important for their survival, migration, and invasion (59,61) as well as for the development of metastases (61,63). These observations have led to the development and validation of anti-VEGF therapies for advanced malignancies either as a single agent or combined with chemotherapy. However, although anti-VEGF therapy has been shown to be of benefit for patients with certain tumor types, resistance, either inherent or acquired remains an obstacle for long-term survival (26). Therefore, alternative approaches to anti-VEGF therapy are warranted that might provide alternative therapeutic regimens for patients who do not respond well to anti-VEGF therapy.

Semaphorins offer a novel approach in which the addition of a protein can inhibit tumor growth and angiogenesis. The mechanism(s) by which SEMA3s exert antagonistic effects on endothelial cells and tumor cells remains to be elucidated, and it is still not understood whether they compete with VEGF for neuropilin binding (Fig. 3). Miao et al. first showed that when SEMA3A complexes with the NRP1 a1a2 domain, the carboxyl-terminal immunoglobulin domain of SEMA3A, blocks the VEGF b1b2-binding domain of NRP1 (5). In terms of the consequence of this steric hindrance to function, Miao et al. suggested that SEMA3A competitively inhibits the binding of VEGF to NRP1, thus inhibiting endothelial cell motility (5). Similarly, Geretti et al. showed that VEGF can compete with SEMA3F and that SEMA3F can compete with VEGF for the binding of NRP2, suggesting that VEGF and SEMA3F are two competitive NRP2 ligands (64). In contrast, other investigators have shown that SEMA3F inhibits the VEGF-induced survival and proliferation of human umbilical vein endothelial cells in vitro and VEGF-induced angiogenesis in vivo; however, this inhibition is not mediated via competition between SEMA3F and VEGF but instead is regulated at an intracellular level that requires active SEMA3F-mediated signaling (37).

Fig. 3.

Neuropilin structure. Neuropilins bear two binding sites, one for SEMA3 molecules (a1a2 domain) and another for VEGF (b1b2 domain). Whether these two molecules compete for binding to neuropilin is unclear, and contrasting results have been published.

Fig. 3.

Neuropilin structure. Neuropilins bear two binding sites, one for SEMA3 molecules (a1a2 domain) and another for VEGF (b1b2 domain). Whether these two molecules compete for binding to neuropilin is unclear, and contrasting results have been published.

Close modal

Despite other studies suggesting that the antitumor effects associated with SEMA3s are partially due to competition with VEGF (4, 65), structural studies have suggested that VEGF and SEMA3A have distinctly separate binding sites and therefore do not compete for NRP1 binding (25). According to this structural model, the NRP1 a1a2 domain is an exclusive binding region for SEMA3A, whereas the b1b2 domain is an exclusive binding region for VEGF-165. This structural model is further supported by evidence that SEMA3A-mediated collapse of axonal growth cones from dorsal root ganglia occurs even at increasing concentrations of VEGF-165 (25). Likewise, Pan et al. showed that saturating concentrations of SEMA3A do not seem to affect VEGF-induced endothelial cell migration (6). However, Miao et al. clearly showed that greater concentrations of SEMA3A are required to achieve the same level of growth cone collapse in the presence of VEGF, suggesting that VEGF competes with SEMA3A to induce the collapse of dorsal root ganglia (5). Given that experimental support exists for both of the competing hypotheses, further studies need to be done to better elucidate the true interaction between SEMA3s and various VEGF isoforms. The question of competitive inhibition versus independent antagonistic effects of SEMA3s and VEGF is important, because it offers investigators a novel approach to develop anticancer therapies.

Semaphorins, a secreted class of proteins originally described as guidance cues for developing neurons, have emerged as key regulators of vasculogenesis, angiogenesis, and tumorigenesis. Targeting endothelial cells and tumor cells with different members of the SEMA3 family may represent a promising new type of therapy for preventing tumor angiogenesis, growth, and metastasis. Additionally, if further investigation supports the hypothesis that SEMA3s compete with VEGF for binding to neuropilins, it might be possible to use SEMA3 overexpression to inhibit the proangiogenic and mitogenic effects of VEGF. Additionally, exogenous SEMA3 therapy may have additive or synergistic activity when added to anti-VEGF therapy (6). Further studies are warranted to elucidate the interactions between VEGF, neuropilins, and SEMA3s that inhibit tumor growth and progression.

L.M. Ellis, commercial research grant, Sanofi-Aventis, ImClone Systems; consultant, Schering-Plough, Roche, Amgen.

We thank Virginia M. Mohlere (Department of Scientific Publications) and Rita Hernandez (Department of Surgical Oncology) for editorial assistance and Kristin Johnson (Vascular Biology Program, Children's Hospital) for graphic design.

1
Luo
Y
,
Raible
D
,
Raper
JA
. 
Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones
.
Cell
1993
;
75
:
217
27
.
2
Gluzman-Poltorak
Z
,
Cohen
T
,
Herzog
Y
,
Neufeld
G
. 
Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165
.
J Biol Chem
2000
;
275
:
29922
.
3
Soker
S
,
Miao
HQ
,
Nomi
M
,
Takashima
S
,
Klagsbrun
M
. 
VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding
.
J Cell Biochem
2002
;
85
:
357
68
.
4
Gu
C
,
Limberg
BJ
,
Whitaker
GB
, et al
. 
Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165
.
J Biol Chem
2002
;
277
:
18069
76
.
5
Miao
HQ
,
Soker
S
,
Feiner
L
,
Alonso
JL
,
Raper
JA
,
Klagsbrun
M
. 
Neuropilin-1 mediates collapsin-1/semaphorin III inhibition of endothelial cell motility: functional competition of collapsin-1 and vascular endothelial growth factor-165
.
J Cell Biol
1999
;
146
:
233
42
.
6
Pan
Q
,
Chanthery
Y
,
Liang
WC
, et al
. 
Blocking neuropilin-1 function has an additive effect with anti-VEGF to inhibit tumor growth
.
Cancer Cell
2007
;
11
:
53
67
.
7
Neufeld
G
,
Kessler
O
. 
The semaphorins: versatile regulators of tumour progression and tumour angiogenesis
.
Nat Rev Cancer
2008
;
8
:
632
45
.
8
Raper
JA
. 
Semaphorins and their receptors in vertebrates and invertebrates
.
Curr Opin Neurobiol
2000
;
10
:
88
94
.
9
Bachelder
RE
,
Lipscomb
EA
,
Lin
X
, et al
. 
Competing autocrine pathways involving alternative neuropilin-1 ligands regulate chemotaxis of carcinoma cells
.
Cancer Res
2003
;
63
:
5230
3
.
10
Tamagnone
L
,
Comoglio
PM
. 
To move or not to move? Semaphorin signalling in cell migration
.
EMBO Rep
2004
;
5
:
356
61
.
11
Gu
C
,
Yoshida
Y
,
Livet
J
, et al
. 
Semaphorin 3E and plexin-D1 control vascular pattern independently of neuropilins
.
Science
2005
;
307
:
265
8
.
12
Serini G, Maione F, Giraudo E, Bussolino F. Semaphorins and tumor angiogenesis. Angiogenesis 2009;12:187-93.
13
Nasarre
P
,
Constantin
B
,
Rouhaud
L
, et al
. 
Semaphorin SEMA3F and VEGF have opposing effects on cell attachment and spreading
.
Neoplasia
2003
;
5
:
83
92
.
14
Nasarre
P
,
Kusy
S
,
Constantin
B
, et al
. 
Semaphorin SEMA3F has a repulsing activity on breast cancer cells and inhibits E-cadherin-mediated cell adhesion
.
Neoplasia
2005
;
7
:
180
9
.
15
Tomizawa
Y
,
Sekido
Y
,
Kondo
M
, et al
. 
Inhibition of lung cancer cell growth and induction of apoptosis after reexpression of 3p21.3 candidate tumor suppressor gene SEMA3B
.
Proc Natl Acad Sci U S A
2001
;
98
:
13954
9
.
16
Banu
N
,
Teichman
J
,
Dunlap-Brown
M
,
Villegas
G
,
Tufro
A
. 
Semaphorin 3C regulates endothelial cell function by increasing integrin activity
.
FASEB J
2006
;
20
:
2150
2
.
17
Martin-Satue
M
,
Blanco
J
. 
Identification of semaphorin E gene expression in metastatic human lung adenocarcinoma cells by mRNA differential display
.
J Surg Oncol
1999
;
72
:
18
23
.
18
Cai
H
,
Reed
RR
. 
Cloning and characterization of neuropilin-1-interacting protein: a PSD-95/Dlg/ZO-1 domain-containing protein that interacts with the cytoplasmic domain of neuropilin-1
.
J Neurosci
1999
;
19
:
6519
27
.
19
Ellis
LM
. 
The role of neuropilins in cancer
.
Mol Cancer Ther
2006
;
5
:
1099
107
.
20
Soker
S
,
Takashima
S
,
Miao
HQ
,
Neufeld
G
,
Klagsbrun
M
. 
Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor
.
Cell
1998
;
92
:
735
45
.
21
Herzog
Y
,
Kalcheim
C
,
Kahane
N
,
Reshef
R
,
Neufeld
G
. 
Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins
.
Mech Dev
2001
;
109
:
115
9
.
22
You
LR
,
Lin
FJ
,
Lee
CT
,
DeMayo
FJ
,
Tsai
MJ
,
Tsai
SY
. 
Suppression of Notch signalling by the COUP-TFII transcription factor regulates vein identity
.
Nature
2005
;
435
:
98
104
.
23
Yuan
L
,
Moyon
D
,
Pardanaud
L
, et al
. 
Abnormal lymphatic vessel development in neuropilin 2 mutant mice
.
Development
2002
;
129
:
4797
806
.
24
Karpanen
T
,
Heckman
CA
,
Keskitalo
S
, et al
. 
Functional interaction of VEGF-C and VEGF-D with neuropilin receptors
.
FASEB J
2006
;
20
:
1462
72
.
25
Appleton
BA
,
Wu
P
,
Maloney
J
, et al
. 
Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding
.
EMBO J
2007
;
26
:
4902
12
.
26
Ellis
LM
,
Hicklin
DJ
. 
VEGF-targeted therapy: mechanisms of anti-tumour activity
.
Nat Rev Cancer
2008
;
8
:
579
91
.
27
Pan
Q
,
Chathery
Y
,
Wu
Y
, et al
. 
Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting
.
J Biol Chem
2007
;
282
:
24049
56
.
28
Aizawa
H
,
Wakatsuki
S
,
Ishii
A
, et al
. 
Phosphorylation of cofilin by LIM-kinase is necessary for semaphorin 3A-induced growth cone collapse
.
Nat Neurosci
2001
;
4
:
367
73
.
29
Atwal
JK
,
Singh
KK
,
Tessier-Lavigne
M
,
Miller
FD
,
Kaplan
DR
. 
Semaphorin 3F antagonizes neurotrophin-induced phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase signaling: a mechanism for growth cone collapse
.
J Neurosci
2003
;
23
:
7602
9
.
30
Jin
Z
,
Strittmatter
SM
. 
Rac1 mediates collapsin-1-induced growth cone collapse
.
J Neurosci
1997
;
17
:
6256
63
.
31
Serini
G
,
Valdembri
D
,
Zanivan
S
, et al
. 
Class 3 semaphorins control vascular morphogenesis by inhibiting integrin function
.
Nature
2003
;
424
:
391
7
.
32
Barberis
D
,
Artigiani
S
,
Casazza
A
, et al
. 
Plexin signaling hampers integrin-based adhesion, leading to -kinase independent cell rounding, and inhibiting lamellipodia extension and cell motility
.
FASEB J
2004
;
18
:
592
4
.
33
Shimizu
A
,
Mammoto
A
,
Italiano
JE
 Jr.
, et al
. 
ABL2/ARG tyrosine kinase mediates SEMA3F-induced A inactivation and cytoskeleton collapse in human glioma cells
.
J Biol Chem
2008
;
283
:
27230
8
.
34
Acevedo
LM
,
Barillas
S
,
Weis
SM
,
Gothert
JR
,
Cheresh
DA
. 
Semaphorin 3A suppresses VEGF-mediated angiogenesis yet acts as a vascular permeability factor
.
Blood
2008
;
111
:
2674
80
.
35
Bielenberg
DR
,
Hida
Y
,
Shimizu
A
, et al
. 
Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype
.
J Clin Invest
2004
;
114
:
1260
71
.
36
Guttmann-Raviv
N
,
Shraga-Heled
N
,
Varshavsky
A
,
Guimaraes-Sternberg
C
,
Kessler
O
,
Neufeld
G
. 
Semaphorin-3A and semaphorin-3F work together to repel endothelial cells and to inhibit their survival by induction of apoptosis
.
J Biol Chem
2007
;
282
:
26294
305
.
37
Kessler
O
,
Shraga-Heled
N
,
Lange
T
, et al
. 
Semaphorin-3F is an inhibitor of tumor angiogenesis
.
Cancer Res
2004
;
64
:
1008
15
.
38
Kigel
B
,
Varshavsky
A
,
Kessler
O
,
Neufeld
G
. 
Successful inhibition of tumor development by specific class-3 semaphorins is associated with expression of appropriate semaphorin receptors by tumor cells
.
PLoS ONE
2008
;
3
:
e3287
.
39
Roche
J
,
Boldog
F
,
Robinson
M
, et al
. 
Distinct 3p21.3 deletions in lung cancer and identification of a new human semaphorin
.
Oncogene
1996
;
12
:
1289
97
.
40
Sekido
Y
,
Bader
S
,
Latif
F
, et al
. 
Human semaphorins A(V) and IV reside in the 3p21.3 small cell lung cancer deletion region and demonstrate distinct expression patterns
.
Proc Natl Acad Sci U S A
1996
;
93
:
4120
5
.
41
Futamura
M
,
Kamino
H
,
Miyamoto
Y
, et al
. 
Possible role of semaphorin 3F, a candidate tumor suppressor gene at 3p21.3, in p53-regulated tumor angiogenesis suppression
.
Cancer Res
2007
;
67
:
1451
60
.
42
Kusy
S
,
Potiron
V
,
Zeng
C
, et al
. 
Promoter characterization of semaphorin SEMA3F, a tumor suppressor gene
.
Biochim Biophys Acta
2005
;
1730
:
66
76
.
43
Tse
C
,
Xiang
RH
,
Bracht
T
,
Naylor
SL
. 
Human semaphorin 3B (SEMA3B) located at chromosome 3p21.3 suppresses tumor formation in an adenocarcinoma cell line
.
Cancer Res
2002
;
62
:
542
6
.
44
Xiang
R
,
Davalos
AR
,
Hensel
CH
,
Zhou
XJ
,
Tse
C
,
Naylor
SL
. 
Semaphorin 3F gene from human 3p21.3 suppresses tumor formation in nude mice
.
Cancer Res
2002
;
62
:
2637
43
.
45
Lantuejoul
S
,
Constantin
B
,
Drabkin
H
,
Brambilla
C
,
Roche
J
,
Brambilla
E
. 
Expression of VEGF, semaphorin SEMA3F, and their common receptors neuropilins NP1 and NP2 in preinvasive bronchial lesions, lung tumours, and cell lines
.
J Pathol
2003
;
200
:
336
47
.
46
Herman
JG
,
Meadows
GG
. 
Increased class 3 semaphorin expression modulates the invasive and adhesive properties of prostate cancer cells
.
Int J Oncol
2007
;
30
:
1231
8
.
47
Castro-Rivera
E
,
Ran
S
,
Thorpe
P
,
Minna
JD
. 
Semaphorin 3B (SEMA3B) induces apoptosis in lung and breast cancer, whereas VEGF165 antagonizes this effect
.
Proc Natl Acad Sci U S A
2004
;
101
:
11432
7
.
48
Kusy
S
,
Nasarre
P
,
Chan
D
, et al
. 
Selective suppression of in vivo tumorigenicity by semaphorin SEMA3F in lung cancer cells
.
Neoplasia
2005
;
7
:
457
65
.
49
Potiron
V
,
Nasarre
P
,
Roche
J
,
Healy
C
,
Boumsell
L
. 
Semaphorin signaling in the immune system
.
Adv Exp Med Biol
2007
;
600
:
132
44
.
50
Osada
R
,
Horiuchi
A
,
Kikuchi
N
, et al
. 
Expression of semaphorins, vascular endothelial growth factor, and their common receptor neuropilins and allelic loss of semaphorin locus in epithelial ovarian neoplasms: increased ratio of vascular endothelial growth factor to semaphorin is a poor prognostic factor in ovarian carcinomas
.
Hum Pathol
2006
;
37
:
1414
25
.
51
Muller
MW
,
Giese
NA
,
Swiercz
JM
, et al
. 
Association of axon guidance factor semaphorin 3A with poor outcome in pancreatic cancer
.
Int J Cancer
2007
;
121
:
2421
33
.
52
Potiron
VA
,
Roche
J
,
Drabkin
HA
. 
Semaphorins and their receptors in lung cancer
.
Cancer Lett
2009
;
273
:
1
14
.
53
Hicklin
DJ
,
Ellis
LM
. 
Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis
.
J Clin Oncol
2005
;
23
:
1011
27
.
54
Dallas
NA
,
Fan
F
,
Gray
MJ
, et al
. 
Functional significance of vascular endothelial growth factor receptors on gastrointestinal cancer cells
.
Cancer Metastasis Rev
2007
;
26
:
433
41
.
55
Luttun
A
,
Brusselmans
K
,
Fukao
H
, et al
. 
Loss of placental growth factor protects mice against vascular permeability in pathological conditions
.
Biochem Biophys Res Commun
2002
;
295
:
428
34
.
56
Luttun
A
,
Tjwa
M
,
Carmeliet
P
. 
Placental growth factor (PlGF) and its receptor Flt-1 (VEGFR-1): novel therapeutic targets for angiogenic disorders
.
Ann N Y Acad Sci
2002
;
979
:
80
93
.
57
Luttun
A
,
Tjwa
M
,
Moons
L
, et al
. 
Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1
.
Nat Med
2002
;
8
:
831
40
.
58
Fischer
C
,
Jonckx
B
,
Mazzone
M
, et al
. 
Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels
.
Cell
2007
;
131
:
463
75
.
59
Fan
F
,
Wey
JS
,
McCarty
MF
, et al
. 
Expression and function of vascular endothelial growth factor receptor-1 on human colorectal cancer cells
.
Oncogene
2005
;
24
:
2647
53
.
60
Wey
JS
,
Fan
F
,
Gray
MJ
, et al
. 
Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines
.
Cancer
2005
;
104
:
427
38
.
61
Yang
AD
,
Camp
ER
,
Fan
F
, et al
. 
Vascular endothelial growth factor receptor-1 activation mediates epithelial to mesenchymal transition in human pancreatic carcinoma cells
.
Cancer Res
2006
;
66
:
46
51
.
62
Bates
RC
,
Goldsmith
JD
,
Bachelder
RE
, et al
. 
Flt-1-dependent survival characterizes the epithelial-mesenchymal transition of colonic organoids
.
Curr Biol
2003
;
13
:
1721
7
.
63
Maeda
K
,
Chung
YS
,
Ogawa
Y
, et al
. 
Prognostic value of vascular endothelial growth factor expression in gastric carcinoma
.
Cancer
1996
;
77
:
858
63
.
64
Geretti
E
,
Shimizu
A
,
Kurschat
P
,
Klagsbrun
M
. 
Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F
.
J Biol Chem
2007
;
282
:
25698
707
.
65
Narazaki
M
,
Tosato
G
. 
Ligand-induced internalization selects use of common receptor neuropilin-1 by VEGF165 and semaphorin3A
.
Blood
2006
;
107
:
3892
901
.
66
Serini
G
,
Napione
L
,
Arese
M
,
Bussolino
F
. 
Besides adhesion: new perspectives of integrin functions in angiogenesis
. Cardiovasc Res 2008;78:213-22.
67
Catalano
A
,
Caprari
P
,
Moretti
S
,
Faronato
M
,
Tamagnone
L
,
Procopio
A
. 
Semaphorin-3A is expressed by tumor cells and alters T-cell signal transduction and function
.
Blood
2006
;
107
:
3321
9
.
68
Herman
JG
,
Meadows
GG
. 
Transferrin reverses the anti-invasive activity of human prostate cancer cells that overexpress sema3E
.
Int J Oncol
2007
;
31
:
1267
72
.
69
Christensen
C
,
Ambartsumian
N
,
Gilestro
G
, et al
. 
Proteolytic processing converts the repelling signal Sema3E into an inducer of invasive growth and lung metastasis
.
Cancer Res
2005
;
65
:
6167
77
.
70
Potiron
VA
,
Sharma
G
,
Nasarre
P
, et al
. 
Semaphorin SEMA3F affects multiple signaling pathways in lung cancer cells
.
Cancer Res
2007
;
67
:
8708
15
.