VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC.These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Neuropilin-1 (NRP1) is a transmembrane protein acting as a co-receptor for several growth factors and interacting with other proteins such as integrins and plexins/semaphorins. It is involved in axonal development, angiogenesis and cancer progression. Its primary mRNA is subjected to alternative splicing mechanisms generating different isoforms, some of which lack the transmembrane domain and display antagonist properties to NRP1 full size (FS). NRP1 is further post-translationally modified by the addition of glycosaminoglycans (GAG) side chains through an O-glycosylation site at serine612. Here, we characterized a novel splice variant which has never been investigated, NRP1-Δ7, differing from the NRP1-FS by a deletion of 7 amino acids occurring two residues downstream of the O-glycosylation site. This short sequence contains two aspartic residues critical for efficient glycosylation. As expected, the high molecular weight products appearing as a smear in SDS-PAGE and reflecting the presence of GAG in NRP1-FS were undetectable in the NRP1-Δ7 protein. NRP1-Δ7 mRNA was found expressed at an appreciable level, between 10 and 30% of the total NRP1, by various cells lines and tissues from human and murine origin. To investigate the biological properties of this isoform, we generated prostatic (PC3) and breast (MDA-MB-231) cancer cells able to express recombinant NRP1-FS or NRP1-Δ7 in a doxycycline-inducible manner. Cells with increased expression of NRP1-Δ7 were characterized in vitro by a significant reduction of proliferation, migration and anchorage-independent growth, while NRP1-FS had the expected opposite “pro-tumoral” effects. Upon VEGF-A165 treatment, a lower internalization rate was observed for NRP1-Δ7 than for NRP1-FS. Finally, we showed that NRP1-Δ7 inhibited growth of prostatic tumors and their vascularization in vivo. This report identifies NRP1-Δ7 as a splice variant displaying anti-tumorigenic properties in vitro and in vivo, emphasizing the need to consider this isoform in future studies.

Neuropilin-1 (NRP1) is a transmembrane glycoprotein functioning as a co-receptor for several soluble factors, including some variants of the Vascular Endothelial Growth Factor A (VEGF-A). Therefore it contributes to regulating angiogenesis but its expression is also associated with cancer. Neuropilin can be also considered as a proteoglycan as it can be modified by the addition glycosaminoglycans on serine 612. Six splice variants of NRP1 have been described in the literature. An additional form was recently identified in our laboratory. Depending upon the cell types, it represents 5-20% of the total amount of NRP1. As compared to the full size NRP1 (NRP1-FS), it lacks the 7 amino acids encoded by the last 21 bases of exon 11. As the missing sequence is located 2 amino acids downstream of the Ser612 required for glycosaminoglycan side chain formation, it could possibly affect the glycosylation process and modify the function of the entire molecule. The glycosylation of NRP1-FS and NRP1-Δ7 was analyzed in HEK293 cells engineered to overexpress each isoforms. As expected, Western blotting analyses suggested that NRP1-Δ7 was less glycosylated than NRP1-FS. Experiments with deglycosylating enzymes are in progress to confirm the differential glycosylation of these two variants. Prostate Cancer cells (PC3) expressing a tetracycline-sensitive repressor were further transfected with an appropriate expression vector (PC3/TR/NRP1-FS) or (PC3/TR/NRP1-Δ7) allowing the conditional expression recombinant NRP1-FS or NRP1-Δ7 only in the presence of doxycycline. Anchorage-independent growth was analyzed by seeding these cells on soft agar. As compared to control conditions, forced expression of NRP1-FS by doxycycline stimulated cell growth and colony formation. By contrast, the expression of NRP1-Δ7 induced a sharp reduction of cell proliferation and colony number, suggesting an inhibition of tumorigenesis by this variant. The same cells were further evaluated in a migration scratch assay, with or without doxycycline in the medium. As compared to controls and to NRP1-FS expressing cells, a decrease migration rate was observed with cells producing NRP1-Δ7. As PC3 cells express basal level of endogenous NRP1, this suggests a mechanism of competitive inhibition of NRP1 functions by NRP1- Δ7. Finally, the involvement of the two Neuropilin-1 variants in tumorigenesis was assessed in vivo in a model of injection in the flanks of nude mice of PC3 cells conditionally expressing NRP1-FS or -Δ7. Increased tumor size and weight were observed using cells expressing NRP1-FS as compared to the control condition. By sharp contrast and in agreement with the in vitro observations, the expression of NRP1-Δ7 induced a reduction of tumor growth. These results suggest that NRP1–Δ7, though lacking 7 amino acids as compared to the full size variant, can have specific and perhaps antagonistic functions. Citation Format: Céline Hendricks, Lauriane Janssen, Romain Delcombel, Johanne Dubail, Christophe Deroanne, Alain Colige. Study of a new splice variant of Neuropilin-1: Antagonistic functions in the regulation of tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2015-5135