Neuropilin and Its Ligands in Normal Lung and Cancer

Roche J, Drabkin H, Brambilla E.

Publication Details

Summary

Neuropilins (NRPs) are receptors for class 3 Semaphorins and function as co-receptors for vascular endothelial growth factor isoforms, VEGF165 and VEGF145 and related molecules. NRPs are expressed in a variety of neural and non-neural tissues and are required for normal development. Interestingly, class 3 Semaphorins and VEGF compete for common NRP binding. As a consequence, Semaphorins and VEGF appear to be mutually antagonistic. In the lung, NRP levels increase during development and NRPs and Semaphorins are involved in lung branching, probably by altering cell morphology or by regulating cell motility and migration. During lung tumorigenesis, both NRP and VEGF expression increase on dysplastic lung epithelial cells; SEMA3F expression is reduced and SEMA3F protein is delocalized from the membrane to the cytoplasm. In lung cancers, SEMA3F staining correlates inversely with tumor stage with high SEMA3F associated with less aggressive tumors. Conversely, more aggressive tumors are associated with increased VEGF staining and a corresponding loss in membranous SEMA3F.

Introduction

Neuropilin (NRP) 1 and 2 are transmembrane glycoproteins involved in neuronal cell guidance, axon growth and fasciculation.1 In addition to its role in the nervous system, NRP1 is expressed in the developing heart, vasculature, skeleton and lung. NRP2 has a similar expression profile. Neuropilins are receptors for two types of very different ligands: semaphorins2,3 and vascular endothelial growth factor, VEGF.4

Semaphorins are a large family of secreted and membrane associated molecules containing a characteristic 500 amino acid Sema domain. They have been classified into eight groups based on their overall similarity and structural features.5 Collapsin, now known as Sema3A, was originally identified on the basis of its chemorepellent activity. Secreted semaphorins from class 3 are the only semaphorins that bind neuropilins and have been implicated in axon steering, fasciculation, branching and synapse formation.6 While Sema3A only binds NRP1, Sema3C binds NRP1 and NRP2 equally whereas Sema3F has greater affinity for NRP2 than NRP1.7 This binding is essential for semaphorin function2,3,8and NRP2 is the functional receptor for Sema3F in the nervous system.9–12 Other molecules are necessary to transduce semaphorin signals which include plexins13 and collapse response mediator protein CRMP.14

VEGF, a 40-45 kDa homodimeric protein, regulates normal embryonic vasculogenesis, physiological angiogenesis and tumor angiogenesis. Originally defined as an endothelial cell (EC) mitogen and chemotactic factor, there is now growing evidence that VEGF stimulates non-EC cells.15–18 Five different isoforms of VEGF monomers consisting of 121, 145, 165, 186 and 206 amino acids produced by alternative splicing have been identified with VEGF121 and VEGF165 being the most abundant.

NRP1 was identified as a receptor for VEGF165 but not for VEGF121.4 NRP2 binds both VEGF165 and VEGF145.19 Importantly, the presence of NRP1 together with the high affinity VEGF receptor 2 (KDR/flk-1) result in greater tyrosine kinase activity. Further support for the role NRPs in cardiovascular development comes from studies utilizing transgenic and knock-out mice. Mice that overexpress NRP1 develop excess capillaries and blood vessels, dilatation of blood vessels and heart defects in addition to neurological abnormalities.20 When deleted for NRP1, mutant mice die during the second half of gestation. In addition to neurological defects, NRP1 −/− mice exhibit severe defects in the cardiovascular system reflecting either a requirement for Semaphorin signaling and/or the presence of NRP1 as a receptor for critical VEGF isoforms.8 Disruption of Sema3A also causes severe abnormalities in neural and non-neural tissues including hypertrophy of the right ventricle and dilatation of the right atrium.21 The discovery that Neuropilins were capable of binding two distinct ligands suggested that class 3 Semaphorins and VEGF might compete. A competitive interaction was documented between Sema3A and VEGF in endothelial cells.22,23

In addition to their role in the nervous system and in angiogenesis, Neuropilins and Semaphorins have been implicated in other developmental processes and in tumorigenesis. For the remainder of this discussion, we will focus on these molecules in the development of normal lung and lung cancer.

Neuropilin and Semaphorin in Normal Mice Lung Development

Fetal lung development involves coordinated cell proliferation, migration, branching morphogenesis and differentiation and normal development depends on reciprocal induction between epithelial and mesenchymal cells. Several growth factors are known to affect lung epithelial cell proliferation. For example, epidermal growth factor and fibroblast growth factors positively influence proliferation, whereas bone morphogenetic protein BMP-4 and TGFβ have negative effects.24–28 Many factors which affect branching morphogenesis are also becoming elucidated. Guidance molecules such as Semaphorins are likely candidates to affect these processes as both neuropilins and semaphorins are expressed in the lung.2,3,11,21,29,30 Moreover, rCRMP-2 which is an intra-cellular protein required for Sema3A signaling14 is expressed in the lung31 as it is also the case for CRMP-1.32

Only a few lung effects have been reported in mice overexpressing NRP1 or in mice with a NRP1 knock-out. When the lung was examined in NRP1 homozygous null (nrp1−/−) animals, it was found to be smaller and the number of branches in the left lung significantly lower than in wild-type (nrp1+/+) or heterozygous (nrp1+/-) animals.33 In contrast to nrp1−/− mice, manynrp2−/− mice survive into adulthood despite the existence of numerous neurological deficits, some of which are complementary to those observed previously in NRP1 mutants.34,35 However, no effects involving the lungs were reported for either the nrp2−/− mice or for Sema3A knock-outs.21,36,37

The absence of severe developmental effects in the lung may be the result of redundancy. Other data indicate that semaphorins and neuropilins are likely to be critically involved in lung development.33,38 Expression studies indicate that Sema3A is expressed at high levels mainly in the distal mesenchyme around the airway epithelium and this expression decreases with time. In epithelial cells, Sema3A is strongly expressed in intermediate bronchioles at E15.5. Expression of Sema3C was restricted predominantly to the lobar bronchus. These expression patterns overlapped or were adjacent to regions expressing NRP1 and NRP2. In contrast, SEMA3F expression was weak and diffuse in the distal epithelium and in the surrounding mesenchyme in early stages, but became confined to the terminal epithelium by E15.5. NRP1 levels rise dramatically during development along with CRMP-2 immunoreactivity in developing and adult alveolar epithelium.

Ito et al.33 treated lung explant culture with different semaphorins. A striking feature of early lung development is the budding and branching which is retained even in culture. Treatment of explant cultures with Sema3A resulted in fewer terminal buds. Co-treatment with a soluble form of NRP139 lacking the transmembrane and intracellular regions attenuated the Sema3A effects whereas sNRP1 alone had no activity. The reduction in terminal buds was not attributable to growth as no effects were detected with BrdU incorporation. In contrast, Sema3C and Sema3F stimulated branching morphogenesis in lung explants from fetal mice and these effects were blocked with sNRP1 or sNRP2, respectively.38 Cell proliferation was stimulated as shown by BrdU labeling. These data indicate that multiple semaphorins exert counterbalancing effects on branching morphogenesis, constituting a novel regulatory system in lung development. Dual effects of Semaphorins were first reported in the nervous system10,12,40 which, in at least some cases, are the result of different cGMP concentrations.41

How do Semaphorins/Neuropilins affect lung branching? One hypothesis from Kagoshima et al.38 is that Semaphorins could promote or inhibit airway branching by the alteration of cell morphology or by the regulation of cell motility and migration. Alterations of cell morphology were seen in COS cells transfected with Sema3A42 and in mammary adenocarcinoma cells transfected with SEMA3F (Nasarre et al., submitted). In the later case, transfected cells rounded up and detached. Cell motility and migration might also be involved as Sema3A inhibits endothelial cell motility22 and has been shown to regulate neural crest migration.43 Likewise, in C. elegans, Semaphorin-2a prevents ectopic cell contacts during epidermal morphogenesis.44 We also demonstrated that SEMA3F is localized in motile regions such as in leading edges or ruffling membranes of lamellipodia in HeLa cells.45

Neuropilins and Its Ligands in Human Lung Tumor

Following the cloning of SEMA3F, by our group and others, from a recurrent homozygous deletion region in small-cell lung cancer (SCLC)46–48 and SEMA3B48, we were intrigued by the possibility that neural guidance molecules such as semaphorins could be involved in lung tumorigenesis. This chromosomal region is well known for loss of heterozygosity (LOH) as an early event in lung tumors and was postulated to contain a tumor suppressor gene.49,50 More direct evidence for such an activity came from the transfection of P1 clones containing SEMA3F into a mouse tumor cell line51 and it was also shown that SEMA3F by itself suppresses tumor formation in nude mice.52 It is also notable that another 3p homozygous deletion region, identified in the SCLC cell line U2020 encodes a repulsive neural guidance molecule DUTT1 (Deleted in U Twenty-Twenty).53 DUTT1 is the probable human homologue of the Drosophila gene Roundabout (Robo)54 which is the receptor for the midline ligand, Slit.

Several reports in the literature have implicated semaphorins in cancers as survival factors with increased metastatic ability. SEMA3E was identified in human cancer to confer non-MDR (Multi Drug Resistance) resistance55 and was also overexpressed in metastatic human lung adenocarcinomas.56 Similarly, Sema3E expression has been correlated with the metastatic ability of certains tumors.57 SEMA4D (CD100) downregulation occurs in non-Hodgkin's B-cell lymphomas and has been postulated to regulate adhesiveness and metastatic potential.58 Therefore some semaphorins show overexpression in tumors whereas others are downregulated. This may reflect the bifunctional effects of semaphorins previously observed in the nervous system.

In normal lung, we studied SEMA3F expression using a specific affinity purified antibody.59 SEMA3F expression was found in epithelial cells. In large bronchi, there was strong membrane staining in addition to mild diffuse cytoplasmic staining.45 In bronchioles, SEMA3F was restricted to basal epithelial cells. Endothelial cells of the alveolar capillary bed did not express SEMA3F, whereas about 20% of vessels more than 100 mm diameter were positive for expression. In lung tumors, SEMA3F localization was predominantly cytoplasmic and the overall levels were reduced (Fig. 1). In resected NSCLC cancers (Non Small Cell Lung Cancer), low levels of SEMA3F correlated with higher stage (more aggressive) disease. In all lung cancer subtypes, an exclusive cytoplasmic localization of SEMA3F was associated with VEGF overexpression which suggested that SEMA3F could compete with VEGF for binding to cell surface NRP receptors.45 These studies have now been expanded to include 112 lung cancers and 50 preneoplasic lesions (Lantuejoul et al., submitted). In preneoplasic lesions, SEMA3F was low indicating that loss of SEMA3F protein, like the previous LOH studies would predict, is an early event in lung tumorigenicity. Recently, expression of CRMP-1, a mediator in the Semaphorin pathway, was found to be inversely correlated with the invasive capability of lung cancer cell lines.32 In normal lung, we found NRP1 and NRP2 expressed in bronchial basal cells (Lantuejoul et al., submitted). In preinvasive bronchial lesions NRP1 and NRP2 expression was significantly increased from hyperplastic mucosa to moderate dysplasia with a plateau reached in severe dysplasia (Fig. 1). Increased neuropilin staining was also observed in conjunction with increased VEGF.

Figure 1. SEMA3F, VEGF, Neuropilins and CRMP-1 levels during lung tumor progression.

Figure 1

SEMA3F, VEGF, Neuropilins and CRMP-1 levels during lung tumor progression. SEMA3F is present in normal lung but its level decreases in preneoplasic lesions indicating that loss of SEMA3F is an early event. In addition, SEMA3F is found in the cytoplasm (more...)

Interestingly, we observed using a wound assay of HeLa cells that cells at the border of the wound had increased staining for NRP1 but NRP1 was translocated to the cytoplasm (Fig. 2). Since cells at the wound border are apparently stimulated to migrate, up-regulation of NRP1 and translocation to the cytoplasm would be expected to facilitate this process (Lantuejoul et al., submitted). NRP1 has been previously implicated in tumor progression through its effects on angiogenesis and NRP1 overexpression likely represents a biomarker for tumor aggressiveness. In prostate carcinoma AT2.1 cells, overexpression of NRP1 resulted in increased basal cell motility and VEGF165 binding.60 Furthermore, the tumors were enlarged in vivo and showed increased microvessel density, proliferation of endothelial cells, dilated blood vessels and, notably, less tumor cell apoptosis.60 The expression of NRP1 in Neuropilin-deficient breast carcinoma cells protects them from apoptosis.61 NRP1 expression has also been correlated with an advanced stage of prostatic cancer and malignant behavior in astrocytomas.62,63 Likewise, NRP1 was higher in rat estrogen-induced pituitary tumors and promoted angiogenesis.64 In addition, experimentally overexpressed soluble NRP1 (sNRP1), a naturally occurring antagonist, leads to tumors which are apoptotic, hemorrhagic and full of disrupted blood vessels.65

Figure 2. NRP1 staining at the border of a wound in a confluent HeLa cell culture.

Figure 2

NRP1 staining at the border of a wound in a confluent HeLa cell culture. Cells were immunostained with a polyclonal anti-NRP1 antibody provided by A Kolodkin (dilution 1/1000). Only some cells are positive in a confluent culture (A) but cells at the border (more...)

VEGF is expressed in normal lung by bronchial basal cells as well as hyperplastic type II pneumocytes in addition to endothelial cells. Expression includes a frequent reinforcement at the membrane. We found that VEGF increased significantly with the histological grades of preneoplasic lesions and culminated in corresponding invasive carcinoma in parallel to neuropilins (Fig. 1). Lung tumors stained positively for VEGF with more intense staining at the periphery of tumor lobules than inside. VEGF may stimulate an autocrine signaling pathway, independent of angiogenesis, to maintain cell survival as it was proposed for NRP1 expressing breast carcinoma cell lines.61 Surprisingly, we also found isolated clusters of tumors cells in lung tumors which stained strongly for SEMA3F, Neuropilins and VEGF (Fig. 3). While as yet unproven, this raises the possibility that Semaphorin expression may be dynamic as has been reported for β-catenin and E-cadherin in colon tumors66 and in breast cancers undergoing migration in vitro.67 However, overexpression of SEMA3B in lung carcinoma cell lines induces apoptosis68 and Semaphorins were also described as death inducers in sensory neurons69 and neural progenitors.23 Therefore, extra SEMA3F would rather lead to elimination of transformed cells but there is a balance between SEMA3F and VEGF and it is hard to predict on which side, proliferation or apoptosis, cells will go.

Figure 3. Squamous cell carcinoma with small clusters of cell isolated in the stroma, evading from the tumor bulk on serial sections.

Figure 3

Squamous cell carcinoma with small clusters of cell isolated in the stroma, evading from the tumor bulk on serial sections. Samples were incubated with rabbit polyclonal NRP1 and NRP2 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA USA) at 1/100 (more...)

A model for these various interactions is shown in Figure 4. Normal stationary cells in the lung express Neuropilins and a substantial amount of SEMA3F. During the process of tumor development, VEGF and Neuropilin expression increase while SEMA3F binding to the surface of epithelial cells declines. Further downregulation of SEMA3F occurs at the transcriptional level. It is possible that hypoxia may regulate components of the system other than VEGF but this has not been reported. Not only does the reduction in SEMA3F levels facilitate growth or survival activities of VEGF on primary tumors, which appears to occur even in the absence of VEGFR2 (Vascular endothelial growth factor Receptor 2/ KDR/ Flk-1), but increased VEGF levels compete for Semaphorin binding and overcome its inhibitory actions. With this scenario, we would anticipate that semaphorin replacement combined with anti-VEGF therapies should be additive or even synergistic in the treatment of established tumors or preneoplastic lesions.

Figure 4.. Model of how cells may shift from non proliferation to tumorigenecity or apoptosis.

Figure 4.

Model of how cells may shift from non proliferation to tumorigenecity or apoptosis. Increasing VEGF and NRP levels along with decreases in SEMA3F promote tumorigenesis, migration and survival. In contrast, a high level of SEMA3F with a low level of VEGF (more...)

Acknowledgments

This work was supported by CNRS, ARC and Ligue Nationale Contre le Cancer for JR, by the University of Colorado Lung Cancer SPORE CA5187-07 for HD and by INSERM, Ligure Nationale Contre le Cancer and PHRC 1999 for EB.

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