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

// Judith Hagenbuchner 1, * , Martina Rupp 1, 2, * , Christina Salvador 2 , Bernhard Meister 2 , Ursula Kiechl-Kohlendorfer 1 , Thomas Müller 2 , Kathrin Geiger 3 , Consolato Sergi 4 , Petra Obexer 1, 3 , Michael J. Ausserlechner 2, 3 1 Departments of Pediatrics II, Medical University Innsbruck, Innsbruck, Austria 2 Pediatrics I, Medical University Innsbruck, Innsbruck, Austria 3 Tyrolean Cancer Research Institute, Innsbruck, Austria 4 Walter C. Mackenzie Centre, University of Alberta, Edmonton, Canada * These authors have contributed equally to this work Correspondence to: Michael J. Ausserlechner, email: Michael.J.Ausserlechner@i-med.ac.at Petra Obexer, email: Petra.Obexer@i-med.ac.at Keywords: chorioallantoic membrane (CAM), hypoxia, chemotherapy Received: June 07, 2016     Accepted: October 03, 2016     Published: October 18, 2016 ABSTRACT 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.