title: 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.
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Céline
authors: C
authors: Hendricks
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Johanne
authors: J
authors: Dubail
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Laura
authors: L
authors: Brohée
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Yves
authors: Y
authors: Delforge
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Alain
authors: A
authors: Colige
authors: Laboratory of Connective Tissues Biology, Tour de Pathologie, GIGA-Cancer, University of Liège, Sart-Tilman, Belgium.
authors: Christophe
authors: C
authors: Deroanne
mesh_terms: D017398:Alternative Splicing; D000818:Animals; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D006025:Glycosaminoglycans / Q000172:deficiency*; D006031:Glycosylation; D064593:Heterografts; D006801:Humans; D051379:Mice; D023421:Models, Animal; D009389:Neovascularization, Pathologic / Q000235:genetics; D039942:Neuropilin-1 / Q000235:genetics*; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
chemical_list: D006025:Glycosaminoglycans; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; D039942:Neuropilin-1