Nuclear FOXO3 predicts adverse clinical outcome and promotes tumor angiogenesis in neuroblastoma

Neuroblastoma is the most frequent, extracranial solid tumor in children with still poor prognosis in stage IV disease. In this study, we analyzed FOXO3-phosphorylation and cellular localization in tumor biopsies and determined the function of this homeostasis regulator in vitro and in vivo. FOXO3-phosphorylation at threonine-32 (T32) and nuclear localization in biopsies significantly correlated with stage IV disease. DNA-damaging drugs induced nuclear accumulation of FOXO3, which was associated with elevated T32-phosphorylation in stage IV-derived neuroblastoma cells, thereby reflecting the in situ results. In contrast, hypoxic conditions repressed PKB-activity and caused dephosphorylation of FOXO3 in both, stroma-like SH-EP and high-stage-derived STA-NB15 cells. The activation of an ectopically-expressed FOXO3 in these cells reduced viability at normoxia, but promoted growth at hypoxic conditions and elevated VEGF-C-expression. In chorioallantoic membrane (CAM) assays STA-NB15 tumors with ectopic FOXO3 showed increased micro-vessel formation and, when xenografted into nude mice, a gene-dosage-dependent effect of FOXO3 in high-stage STA-NB15 cells became evident: low-level activation increased tumor-vascularization, whereas hyper-activation repressed tumor growth.The combined data suggest that, depending on the mode and intensity of activation, cellular FOXO3 acts as a homeostasis regulator promoting tumor growth at hypoxic conditions and tumor angiogenesis in high-stage neuroblastoma.