Background FOXO transcription factors control programmed cell death, stress resistance and longevity in normal and malignant cells. We investigated the expression, subcellular localization and phosphorylation of FOXO3 in tumour sections of post chemotherapy neuroblastoma (NB) patients and analysed the effects of FOXO3 in cultured NB cells.
Methods Paraffin-embedded sections from patients were analysed for FOXO3 expression, localization and phosphorylation. Effects of chemotherapeutics on FOXO3 subcellular shuttling were assessed by live cell fluorescence imaging in ECFP-FOXO3 transgenic cells. To study how FOXO3 modulates survival we generated cell lines expressing a conditional PKB-independent FOXO3 allele (FOXO3(A3)ERtm) that can be activated by 4OH-tamoxifen and studied the effects of FOXO3-activation in vitro by clonagenic survival and propidium iodide FACS-analyses and in vivo by xenograft transplantation into nude mice.
Results We found that FOXO3 was localised in the nucleus in tumour sections from high-risk NB patients. FOXO3 nuclear localization and phosphorylation significantly correlated with reduced patient survival. The chemotherapeutics etoposide and doxorubicin led to rapid nuclear accumulation and increased phosphorylation of FOXO3. After low activation of FOXO3 increased clonagenic survival was observed in NB8/FOXO3 cells in combination with chemotherapeutic drugs whereas NB15/FOXO3 cells underwent spontaneous apoptosis. When transplanting NB15/FOXO cells into nude mice, basal FOXO3 activity induced angiogenesis of NB tumours in vivo, whereas full activation eradicated the tumour.
Conclusions The combined data suggest that FOXO3 is activated in high risk NB tumours and depending on the level of its activation, contributes to chemotherapy resistance and tumour angiogenesis or acts as a tumour suppressor.
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