title: C10ORF10/DEPP, a transcriptional target of FOXO3, regulates ROS-sensitivity in human neuroblastoma.
abstract: BACKGROUND
  FOXO 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.
  
  
  METHODS
  The 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Stefan
authors.initials: S
authors.lastname: Salcher
authors.forename: Judith
authors.initials: J
authors.lastname: Hagenbuchner
authors.forename: Kathrin
authors.initials: K
authors.lastname: Geiger
authors.forename: Maximilian A
authors.initials: MA
authors.lastname: Seiter
authors.forename: Johannes
authors.initials: J
authors.lastname: Rainer
authors.forename: Reinhard
authors.initials: R
authors.lastname: Kofler
authors.forename: Martin
authors.initials: M
authors.lastname: Hermann
authors.forename: Ursula
authors.initials: U
authors.lastname: Kiechl-Kohlendorfer
authors.affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Ausserlechner
authors.forename: Petra
authors.initials: P
authors.lastname: Obexer
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D001932:Brain Neoplasms / Q000235:genetics* / Q000473:pathology; D002374:Catalase / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D047908:Intracellular Signaling Peptides and Proteins; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D020675:Peroxisomes / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014158:Transcription, Genetic; D051176:beta Catenin / Q000378:metabolism
chemical_list: C506096:DEPP1 protein, human; C499517:FOXO3 protein, human; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051176:beta Catenin; D005047:Etoposide; D006861:Hydrogen Peroxide; D002374:Catalase