C10ORF10/DEPP-mediated ROS accumulation is a critical modulator of FOXO3-induced autophagy

S Salcher,P Obexer,M J Ausserlechner,U Kiechl-Kohlendorfer,M Hermann

doi:10.1186/s12943-017-0661-4

Abstract

BackgroundNeuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance.We demonstrated before that C10ORF10/DEPP, a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification. In the present study, we investigated the impact of FOXO3 and DEPP on the regulation of autophagy. Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles.MethodsThe effect of FOXO3 and DEPP on autophagy induction was analyzed using live cell fluorescence microscopy and immunoblot analyses of SH-EP cells transfected with a plasmid for EYFP-LC3 and with siRNAs specific for LC3, respectively. ROS steady-state levels were measured with reduced MitoTrackerRed CM-H2XROS. Cellular apoptosis was analyzed by flow cytometry and the caspase 3/7 assay.ResultsWe report for the first time that DEPP induces ROS accumulation and thereby mediates the formation of autophagosomes as inhibition of ROS formation by N-acetyl-cysteine completely blocks autophagy. We further demonstrate that H2O2-treatment triggers autophagy-induction by FOXO3-mediated DEPP expression. Importantly, knockdown of DEPP was sufficient to efficiently inhibit autophagy-induction under different stress conditions such as serum starvation and genotoxic stress, suggesting that DEPP expression is critical for the initiation of autophagy in neuroblastoma. FOXO3-triggered autophagy partially protects neuroblastoma cells from cell death. Consistent with this concept, we demonstrate that inhibition of autophagy by LC3-knockdown significantly increased etoposide- and doxorubicin-induced apoptosis. These results were also confirmed by the use of the autophagy-inhibitor chloroquine that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in neuronal tumor cells.ConclusionTargeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy.

Highlights

Neuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system
DEPP expression induces autophagy in human neuroblastoma cells To investigate whether DEPP expression induces autophagy in neuroblastoma cells, we measured the formation of LC3-containing autophagosomal vesicles, which is a critical marker for autophagic activity
To analyze whether DEPP affects LC3, the pLIB-EYFP-LC3-iresPuro plasmid was transiently transfected into SH-EP/tetCtr and SH-EP/tetDEPP cells and ectopic DEPP expression was induced by treatment of the cells with 200 ng/ml doxycycline

Summary

Introduction

Neuroblastoma is the most common solid tumor in childhood and develops from undifferentiated progenitor cells of the sympathetic nervous system. In neuronal tumor cells DNA-damaging chemotherapeutic agents activate the transcription factor FOXO3 which regulates the formation of reactive oxygen species (ROS) and cell death as well as a longevity program associated with therapy resistance. Phosphorylation of FOXO3 by PKB induces its association with 14-3-3 proteins and its export from the nucleus and repression of target gene transcription [2,3,4,5]. Oxidative- and genotoxic stress triggers FOXO3-phosphorylation on distinct threonine residues by mammalian sterile 20-like kinase 1 (MST1) and c-Jun N-terminal kinase (JNK) causing its translocation into the nucleus even in the presence of growth factor signaling [6, 7]. In high-stage neuroblastoma tumors nuclear FOXO3 contributes to death resistance under cellular stress conditions that occur during serum starvation or chemotherapy in the tumor tissue [8]

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