title: Anti-migratory effect of vinflunine in endothelial and glioblastoma cells is associated with changes in EB1 C-terminal detyrosinated/tyrosinated status.
abstract: We previously showed that vinflunine, a microtubule-targeting drug of the Vinca-alkaloid family exerted its anti-angiogenic/anti-migratory activities through an increase in microtubule dynamics and an inhibition of microtubule targeting to adhesion sites. Such effect was associated with a reduction of EB1 comet length at microtubule (+) ends. In this work we first showed that the pro-angiogenic vascular endothelial growth factor VEGF suppressed microtubule dynamics in living Human Umbilical Vein Endothelial Cells (HUVECs), increased EB1 comet length by 40%, and induced EB1 to bind all along the microtubules, without modifying its expression level. Such microtubule (+) end stabilization occurred close to the plasma membrane in the vicinity of focal adhesion as shown by TIRF microscopy experiments. Vinflunine completely abolished the effect of VEGF on EB1 comets. Interestingly, we found a correlation between the reduction of EB1 comet length by vinflunine and the inhibition of cell migration. By using 2D gel electrophoresis we demonstrated for the first time that EB1 underwent several post-translational modifications in endothelial and tumor cells. Particularly, the C-terminal EEY sequence was poorly detectable in control and VEGF-treated HUVECs suggesting the existence of a non-tyrosinated form of EB1. By using specific antibodies that specifically recognized and discriminated the native tyrosinated form of EB1 and a putative C-terminal detyrosinated form, we showed that a detyrosinated form of EB1 exists in HUVECs and tumor cells. Interestingly, vinflunine decreased the level of the detyrosinated form and increased the native tyrosinated form of EB1. Using 3-L-Nitrotyrosine incorporation experiments, we concluded that the EB1 C-terminal modifications result from a detyrosination/retyrosination cycle as described for tubulin. Altogether, our results show that vinflunine inhibits endothelial cell migration through an alteration of EB1 comet length and EB1 detyrosination/retyrosination cycle.
authors.affiliation: Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale UMR_S 911, Marseille, France.
authors.forename: Amandine
authors.initials: A
authors.lastname: Rovini
authors.forename: Géraldine
authors.initials: G
authors.lastname: Gauthier
authors.forename: Raphaël
authors.initials: R
authors.lastname: Bergès
authors.forename: Anna
authors.initials: A
authors.lastname: Kruczynski
authors.forename: Diane
authors.initials: D
authors.lastname: Braguer
authors.forename: Stéphane
authors.initials: S
authors.lastname: Honoré
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D005909:Glioblastoma / Q000473:pathology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry* / Q000378:metabolism*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology; D014747:Vinblastine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; C111217:vinflunine; D014747:Vinblastine