Study of a new splice variant of Neuropilin-1: antagonistic functions in the regulation of cancer progression?

Neuropilin-1 (NRP1) is a transmembrane glycoprotein and a co-receptor for several growth factors, for example some variants of the Vascular Endothelial Growth Factor A (VEGF-A). It largely contributes to the regulation of angiogenesis but also to cancer formation. NRP1 can be considered as a proteoglycan as glycosaminoglycans side chains can be added on serine 612.
Currently, six splice variants of NRP1 have been described. An additional form was recently identified in our laboratory. Depending upon the cell types, it represents 20-30% of the total amount of NRP1. As compared to the full size NRP1 (NRP1-FS), 7 amino acids are deleted. As the missing sequence is located 2 amino acids downstream of the Ser612 required for glycosaminoglycans addition, this process could be somehow affected and the function of the protein could be modified. The glycosylation of NRP1-FS and -Δ7 was analyzed in different cells overexpressing each isoform. Western blotting analyses suggested that NRP1-Δ7 was less glycosylated than NRP1-FS.
Prostate cancer cells (PC3) were engineered to express NRP1-FS or –Δ7 only in the presence of doxycycline. The migration of these cells was analyzed by scratch assay, with or without doxycycline in the medium. As compared to controls and to NRP1-FS-expressing cells, production of NRP1-Δ7 was linked to a reduction of cell migration. A DNA dosage showed that NRP1-FS enhanced cell proliferation, while NRP1-Δ7 reduced it. Tumor growth was assessed in vitro by a culture in soft agar. As compared to control conditions, expression of NRP1-FS by doxycycline increased colonies formation. By contrast, NRP1-Δ7 inhibited colonies number, suggesting an inhibition of tumorigenesis by this variant. As PC3 cells express basal level of endogenous NRP1, this suggests some competitive inhibition of NRP1 functions by NRP1-Δ7.
Finally, the function of each variant was investigated in vivo in a model of injection in the flanks of nude mice of PC3 cells conditionally expressing NRP1-FS or -Δ7. As compared to the control, NRP1-FS increased tumor size and weight. By sharp contrast, the expression of NRP1-Δ7 was associated with a reduction of tumorigenicity. Cells with forced expression of NRP1-Δ7 also developed fewer blood vessels as compared to the control. These results suggest that NRP1-Δ7 have an antagonistic action on cancer formation and angiogenesis.