A Novel Physiological Glycosaminoglycan-Deficient Splice Variant of Neuropilin-1 Is Anti-Tumorigenic In Vitro and In Vivo

Abstract

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.

Multifaceted Role of Neuropilins in the Immune System: Potential Targets for Immunotherapy

“…Recombinant sIIINRP1 and sIVNRP1 act as ligand trap, antagonized the effect of NRP1 and affected breast cancer cell migration ( 12 ). Another splice variant of NRP1 was recently identified (lacking a small sequence of seven amino acids, located two residues downstream of the O -glycosylation site and hence, less glycosylated) which when overexpressed in prostate cancer cells in nude mice, significantly reduced tumor burden and decreased tumor cell proliferation and migration ( 13 ). However, further studies are required to fully understand why different splice forms exist under different conditions, whether they arise as a host response under specific conditions, and the signaling pathways they govern.…”

HLX affects cell cycle and proliferation in AML cells via the JAK/STAT signaling pathway

“…It was demonstrated that the knockdown of HLX resulted in a decreased expression of STAT5 at the protein level and of PAK1 and NRPl at the mRNA level, while BTG1 gene expression was increased. NRP1 is a receptor of VEGF 165 and can promote vascular proliferation via the PI3K/Akt, JAK/STAT and Notch signaling pathways (23)(24)(25). STAT5 is an important regulatory protein of the JAK/STAT signaling pathway and is closely associated with hematological malignancies (26).…”

“…The JAK/STAT signaling pathway plays an important role in the development and progression of AML. Epidermal growth factor receptor (EGFR), a co-receptor of NRP1, is a receptor tyrosine kinase located upstream of this signaling pathway (24). Within this pathway, STAT5 is an important regulatory protein, and PAK1 and c-Myc are notable downstream target genes; furthermore, BTG1 is involved in the translocation of c-Myc (27).…”

Alternative Splicing in Angiogenesis

“…More recently, a further splice isoform of NRP1 , NRP-1Δ7 , was discovered [42]. This isoform is formed by the deletion of just seven amino acids in exon 11 caused by the utilization of an alternative donor site located 21 bases upstream of the conventional splice site (Figure 4A).…”