title: The P2X7 receptor is a key modulator of the PI3K/GSK3β/VEGF signaling network: evidence in experimental neuroblastoma.
abstract: Neuroblastoma (NB) is an aggressive pediatric tumor, responsible for 15% of cancer-related deaths in childhood, lacking an effective treatment in its advanced stages. The P2X7 receptor for extracellular ATP was associated to NB cell proliferation and recently emerged as a promoter of tumor engraftment, growth and vascularization. In an effort to identify new therapeutic options for neuroblastoma, we studied the role of P2X7 receptor in NB biology. We first analyzed the effect of P2X7 activation or down-modulation of the main biochemical ways involved in NB progression: the PI3K/Akt/GSK3β/MYCN and the HIF1α/VEGF pathways. In ACN human NB cells, P2X7 stimulation enhanced PI3K/Akt, while decreasing GSK3β activity. In the same model, P2X7 silencing or antagonist administration reduced the activity of PI3K/Akt and increased that of GSK3β, leading to a decrease in cellular glycogen stores. Similarly, P2X7 downmodulation caused a reduction in HIF1α levels and vascular endothelial growth factor (VEGF) secretion. Systemic administration of two different P2X7 antagonists (AZ10606120 or A740003) in nude/nude mice reduced ACN-derived tumor growth. An even stronger effect of P2X7 blockade was obtained in a syngeneic immune-competent neuroblastoma model: Neuro2A cells injected in AlbinoJ mice. Together with tumor regression, treatment with P2X7 antagonists caused downmodulation of the Akt/HIF1α axis, leading to reduced VEGF content and decreased vessel formation. Interestingly, in both experimental models, P2X7 antagonists strongly reduced the expression of the probably best-accepted oncogene in NB: MYCN. Finally, we associated P2X7 overexpression with poor prognosis in advanced-stage NB patients. Taken together, our data suggest that P2X7 receptor is an upstream regulator of the main signaling pathways involved in NB growth, metabolic activity and angiogenesis, and a promising therapeutic target for neuroblastoma treatment.
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Amoroso
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Capece
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Rotondo
authors.affiliation: Laboratory of Molecular Biology, Giannina Gaslini Institute, Genoa, Italy.
authors.forename: D
authors.initials: D
authors.lastname: Cangelosi
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: M
authors.identifier: http://orcid.org/0000-0002-1595-6887
authors.initials: M
authors.lastname: Ferracin
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Franceschini
authors.affiliation: Laboratory of Oncology, Giannina Gaslini Institute, Genoa, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Raffaghello
authors.affiliation: Laboratory of Oncology, Giannina Gaslini Institute, Genoa, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Pistoia
authors.affiliation: Laboratory of Molecular Biology, Giannina Gaslini Institute, Genoa, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Varesio
authors.affiliation: Department of Morphology, Surgery and Experimental Medicine, Section of Experimental Pathology, Oncology and Biology, University of Ferrara, Ferrara, Italy.
authors.forename: E
authors.identifier: http://orcid.org/0000-0001-8129-9929
authors.initials: E
authors.lastname: Adinolfi
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009447:Neuroblastoma / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D058486:Receptors, Purinergic P2X7 / Q000502:physiology*; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D058486:Receptors, Purinergic P2X7; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3