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

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

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.

Conclusion: 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.

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Related in: MedlinePlus

Knockdown of DEPP reduces FOXO3-mediated apoptosis. a) SH-EP/FOXO3 (SH-EP/FOXO3-shDEPP clone-10, -12 and -13; left panel) and NB15/FOXO3 (NB15/FOXO3-shDEPP bulk; right panel) cells were infected with vectors coding for DEPP-specific shRNA. Knockdown efficiency was verified by immunoblot (upper panel) and quantitative RT-PCR (lower panel). Cells were treated with 50 nM 4OHT to induce DEPP expression. b) SH-EP/FOXO-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells were treated with 50 nM 4OHT for the indicated time points. PI-FACS analyses were performed to detect apoptotic cells. Shown is the mean ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025; ***P < 0.01 compared to corresponding controls. c) ROS accumulation was detected using MitoTrackerRed CM-H2XROS by live-cell imaging of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as of NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells treated with 50 nM 4OHT for 4 and 16 hours and for 12 and 48 hours respectively to induce FOXO3 dependent biphasic ROS accumulation. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software (Axiovision 4.6). Each panel represents the mean ± s.e.m. of 50 to 80 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. d) Immunoblot analysis of p66/SHC1 and phosphorylated pSer36-p66/SHC1 expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points. GAPDH served as loading control.
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Fig3: Knockdown of DEPP reduces FOXO3-mediated apoptosis. a) SH-EP/FOXO3 (SH-EP/FOXO3-shDEPP clone-10, -12 and -13; left panel) and NB15/FOXO3 (NB15/FOXO3-shDEPP bulk; right panel) cells were infected with vectors coding for DEPP-specific shRNA. Knockdown efficiency was verified by immunoblot (upper panel) and quantitative RT-PCR (lower panel). Cells were treated with 50 nM 4OHT to induce DEPP expression. b) SH-EP/FOXO-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells were treated with 50 nM 4OHT for the indicated time points. PI-FACS analyses were performed to detect apoptotic cells. Shown is the mean ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025; ***P < 0.01 compared to corresponding controls. c) ROS accumulation was detected using MitoTrackerRed CM-H2XROS by live-cell imaging of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as of NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells treated with 50 nM 4OHT for 4 and 16 hours and for 12 and 48 hours respectively to induce FOXO3 dependent biphasic ROS accumulation. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software (Axiovision 4.6). Each panel represents the mean ± s.e.m. of 50 to 80 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. d) Immunoblot analysis of p66/SHC1 and phosphorylated pSer36-p66/SHC1 expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points. GAPDH served as loading control.

Mentions: FOXO3 activation has been shown to induce apoptosis in neuroblastoma cells[5]. To study a possible effect of DEPP on FOXO3-mediated apoptosis, the DEPP expression was knocked down by lentiviral expression of DEPP-specific shRNA as shown in Figure 3a. Three individual clones of SH-EP/FOXO3-shDEPP (Figure 3a, left panel) and bulk-selected NB15/FOXO3-shDEPP (Figure 3a, right panel) were analyzed by immunoblot and quantitative RT-PCR analysis. Propidium iodide-(PI) FACS-analysis showed significantly reduced FOXO3-mediated apoptosis in shRNA-expressing neuroblastoma cell lines (Figure 3b).Figure 3


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

Salcher S, Hagenbuchner J, Geiger K, Seiter MA, Rainer J, Kofler R, Hermann M, Kiechl-Kohlendorfer U, Ausserlechner MJ, Obexer P - Mol. Cancer (2014)

Knockdown of DEPP reduces FOXO3-mediated apoptosis. a) SH-EP/FOXO3 (SH-EP/FOXO3-shDEPP clone-10, -12 and -13; left panel) and NB15/FOXO3 (NB15/FOXO3-shDEPP bulk; right panel) cells were infected with vectors coding for DEPP-specific shRNA. Knockdown efficiency was verified by immunoblot (upper panel) and quantitative RT-PCR (lower panel). Cells were treated with 50 nM 4OHT to induce DEPP expression. b) SH-EP/FOXO-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells were treated with 50 nM 4OHT for the indicated time points. PI-FACS analyses were performed to detect apoptotic cells. Shown is the mean ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025; ***P < 0.01 compared to corresponding controls. c) ROS accumulation was detected using MitoTrackerRed CM-H2XROS by live-cell imaging of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as of NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells treated with 50 nM 4OHT for 4 and 16 hours and for 12 and 48 hours respectively to induce FOXO3 dependent biphasic ROS accumulation. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software (Axiovision 4.6). Each panel represents the mean ± s.e.m. of 50 to 80 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. d) Immunoblot analysis of p66/SHC1 and phosphorylated pSer36-p66/SHC1 expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points. GAPDH served as loading control.
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Fig3: Knockdown of DEPP reduces FOXO3-mediated apoptosis. a) SH-EP/FOXO3 (SH-EP/FOXO3-shDEPP clone-10, -12 and -13; left panel) and NB15/FOXO3 (NB15/FOXO3-shDEPP bulk; right panel) cells were infected with vectors coding for DEPP-specific shRNA. Knockdown efficiency was verified by immunoblot (upper panel) and quantitative RT-PCR (lower panel). Cells were treated with 50 nM 4OHT to induce DEPP expression. b) SH-EP/FOXO-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells were treated with 50 nM 4OHT for the indicated time points. PI-FACS analyses were performed to detect apoptotic cells. Shown is the mean ± s.e.m. of three independent experiments; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025; ***P < 0.01 compared to corresponding controls. c) ROS accumulation was detected using MitoTrackerRed CM-H2XROS by live-cell imaging of SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 as well as of NB15/FOXO3-shCtr and NB15/FOXO3-shDEPP (bulk) cells treated with 50 nM 4OHT for 4 and 16 hours and for 12 and 48 hours respectively to induce FOXO3 dependent biphasic ROS accumulation. Relative ROS staining was quantified using the Axiovert200M fluorescence microscope software (Axiovision 4.6). Each panel represents the mean ± s.e.m. of 50 to 80 cells; statistical analysis was done with the Student’s unpaired t-test, *P < 0.05; **P < 0.025 compared to corresponding controls. d) Immunoblot analysis of p66/SHC1 and phosphorylated pSer36-p66/SHC1 expression in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-clone-10, -12 and -13 cells treated with 50 nM 4OHT for the indicated time points. GAPDH served as loading control.
Mentions: FOXO3 activation has been shown to induce apoptosis in neuroblastoma cells[5]. To study a possible effect of DEPP on FOXO3-mediated apoptosis, the DEPP expression was knocked down by lentiviral expression of DEPP-specific shRNA as shown in Figure 3a. Three individual clones of SH-EP/FOXO3-shDEPP (Figure 3a, left panel) and bulk-selected NB15/FOXO3-shDEPP (Figure 3a, right panel) were analyzed by immunoblot and quantitative RT-PCR analysis. Propidium iodide-(PI) FACS-analysis showed significantly reduced FOXO3-mediated apoptosis in shRNA-expressing neuroblastoma cell lines (Figure 3b).Figure 3

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics I, Medical University Innsbruck, Anichstraße 35, 6020 Innsbruck, Austria. michael.j.ausserlechner@i-med.ac.at.

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus