C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.

Stefan Salcher,Reinhard Kofler,Judith Hagenbuchner,Martin Hermann,Maximilian A Seiter,Kathrin Geiger,Ursula Kiechl-Kohlendorfer,Michael J Ausserlechner,Petra Obexer,Johannes Rainer

doi:10.1186/1476-4598-13-224

Stefan Salcher, Reinhard Kofler + Show 8 more

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https://doi.org/10.1186/1476-4598-13-224

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Abstract

BackgroundFOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. In the present study we investigated the regulation of C10orf10/DEPP by the transcription factor FOXO3. As a physiological function of C10orf10/DEPP has not been described so far we analyzed its effects on cellular ROS detoxification and death sensitization in human neuroblastoma cells.MethodsThe effect of DEPP on cellular ROS was measured by catalase activity assay and live cell fluorescence microscopy using the ROS-sensitive dye reduced MitoTracker Red CM-H2XROS. The cellular localization of DEPP was determined by confocal microscopy of EYFP-tagged DEPP, fluorescent peroxisomal- and mitochondrial probes and co-immunoprecipitation of the PEX7 receptor.ResultsWe report for the first time that DEPP regulates ROS detoxification and localizes to peroxisomes and mitochondria in neuroblastoma cells. FOXO3-mediated apoptosis involves a biphasic ROS accumulation. Knockdown of DEPP prevented the primary and secondary ROS wave during FOXO3 activation and attenuated FOXO3- and H2O2-induced apoptosis. Conditional overexpression of DEPP elevates cellular ROS levels and sensitizes to H2O2 and etoposide-induced cell death. In neuronal cells, cellular ROS are mainly detoxified in peroxisomes by the enzyme CAT/catalase. As DEPP contains a peroxisomal-targeting-signal-type-2 (PTS2) sequence at its N-terminus that allows binding to the PEX7 receptor and import into peroxisomes, we analyzed the effect of DEPP on cellular detoxification by measuring enzyme activity of catalase. Catalase activity was reduced in DEPP-overexpressing cells and significantly increased in DEPP-knockdown cells. DEPP directly interacts with the PEX7 receptor and localizes to the peroxisomal compartment. In parallel, the expression of the transcription factor peroxisome proliferator-activated receptor gamma (PPARG), a critical regulator of catalase enzyme activity, was strongly upregulated in DEPP-knockdown cells.ConclusionThe combined data indicate that in neuroblastoma DEPP localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification, which sensitizes tumor cells to ROS-induced cell death.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-224) contains supplementary material, which is available to authorized users.

Highlights

FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma
FOXO3 regulates Decidual Protein induced by Progesterone (DEPP) expression on mRNA and protein level in human neuroblastoma cells DEPP was identified as a FOXO3-induced gene by Affymetrix gene-chip expression profiling analysis in cells which stably express a 4-hydroxy-tamoxifen-inducible (4OHT), PKB-phosphorylation-independent FOXO3(A3) ERtm transgene
After activation of FOXO3 by treatment with 100 nM 4OHT for 3 hours, DEPP expression was induced in the neuroblastoma cell lines SH-EP/FOXO3 and NB15/FOXO3 171 and 87 fold compared to untreated controls and in the leukemia cell line CEM/FOXO3 50 fold (Figure 1a)

Summary

Introduction

FOXO transcription factors control cellular levels of reactive oxygen species (ROS) which critically contribute to cell survival and cell death in neuroblastoma. DEPP is regulated via progesterone in endometrial stromal cells and via insulin levels in adipose tissue and liver and is induced in malignant glioma cells in response to hypoxic stress. It is highly expressed in various tissues including placenta, ovary, kidney, white adipose and liver. Phosphorylation of FOXO3 by PKB at distinct amino acids leads to its association with 14-3-3 proteins, resulting in export from the nucleus and as a consequence thereof loss of target gene regulation in neuroblastoma cells [6]. Phosphorylation of FOXO3 by stress-induced kinases such as mammalian Ste20-like kinase (MST1) or c-Jun N-terminal kinase (JNK) in turn stimulates nuclear entry, leading to the activation or repression of target genes that affect growth, cell cycle progression, apoptosis and longevity [7,8,9]

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