Neuroblastoma (NB) is a pediatric malignancy that originates from precursors of the sympathetic neurons and expresses neuronal proteins, such as neuropeptide Y (NPY). NPY is a neurotransmitter released from sympathetic nerves, acting via its Y1-Y5 receptors (Y1R-Y5R). NB cells constitutively express Y2R, while Y5R is induced in pro-apoptotic conditions. Our previous data indicated that Y2R is involved in NB cell proliferation and angiogenesis, while Y5R acts as a pro-survival factor. In NB patients, elevated serum NPY levels are associated with poor prognosis and metastasis. Interestingly, in NB tissues, tumor cells with angioinvasive phenotype have a strong expression of Y5R on the leading edge of the penetrating cell. Thus, the goal of our study was to determine the role of NPY and its receptors in NB dissemination. To this end, we assessed the effect of NPY system perturbations on NB cell motility (IncuCyte Live Cell Analysis system), cytoskeleton organization (phalloidin staining) and activity of a cytoskeleton regulator, RhoA (ICC, pull-down assay). The role of particular NPY receptors in these processes was validated by their overexpression in CHO-K1 cells. Treating NB cells with NPY stimulated NB cell migration in a dose-dependent manner, with a peak of activity at 10-8M and lower effects at higher and lower peptide concentrations. This effect was inhibited by Y5R antagonist, while in some cell lines further reduction was observed when combining Y5R and Y2R antagonists. The stimulatory effect of NPY on cell migration was associated with cytoskeleton remodeling, which included a higher number of filopodia positive for Y5R and co-localization of Y5R with an active form of RhoA at both leading and trailing edges of a migrating cell. In line with this role for Y5R in NB motility, expression of this receptor was up-regulated by hypoxia, a known pro-metastatic factor. Consistent with the results on native NB cells, overexpression of Y5R in CHO-K1 cells significantly increased their motility, while transfection with Y1R or Y2R had no such effect. Stimulation of CHO-K1/Y5R cells with NPY increased migration in a dose-dependent manner, while blocking Y5R inhibited cell motility. Moreover, NPY exerted a chemotactic effect for CHO-K1/Y5R cells. Like in NB cells, in CHO-K1 transfectants, Y5R co-localized with active RhoA in both leading and trailing edges of migrating cells. In line with this, CHO-K1/Y5R cells had elevated basal RhoA activity, while NPY stimulation further increased RhoA-GTP levels. Altogether, our data implicate Y5R as the main NPY receptor mediating its stimulatory effect on cell motility. In NB cells, Y2R may further enhance this effect. The Y5R-induced cell motility is associated with cytoskeleton remodeling driven by RhoA activation. The concentration-dependent activity of NPY and its chemotactic effect indicates that the peptide may spatially regulate NB cell migration within tumor tissue.

Citation Format: Nouran Abualsaud, Lindsay Caprio, Abrar Bakr, Lamia Alamri, Ewa Krawczyk, Susana Galli, Sung Hyeok Hong, Joanna Kitlinska. Neuropeptide Y stimulates neuroblastoma cell migration via Y5R/RhoA pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3663.