C10ORF10/DEPP-mediated ROS accumulation is a critical modulator of FOXO3-induced autophagy
© The Author(s). 2017
Received: 25 November 2016
Accepted: 15 May 2017
Published: 25 May 2017
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.
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.
The 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.
We 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.
Targeting FOXO3/DEPP-triggered autophagy is a promising strategy to sensitize neuroblastoma cells to chemotherapy.
Neuroblastoma, a pediatric tumor of the sympathetic nervous system, covers a broad spectrum of clinical outcome. The majority of neuroblastoma patients with high stage tumors respond poorly to radio- or chemotherapy and show a high relapse rate with a 5-year survival rate of less than 40% . In neuroblastoma, the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB) signaling cascade is frequently deregulated. The transcription factor FOXO3 acts as a critical death-inducing factor downstream of this signaling pathway. Phosphorylation of FOXO3 by PKB induces its association with 14-3-3 proteins and consequently its export from the nucleus and repression of target gene transcription [2–5]. However, 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 .
We recently demonstrated that the Decidual Protein induced by Progesterone (C10ORF10/DEPP), a transcriptional target of FOXO3, localizes to peroxisomes and mitochondria and impairs cellular ROS detoxification . Watanabe et al. first described DEPP as a protein that is induced by progesterone in endometrial stromal cells during decidualization . Others found DEPP expression significantly induced by hypoxia in glioblastomas and in human endothelial cells [11, 12] by UV and ionizing radiation  or by energy deprivation [14–16], indicating that DEPP might participate in the cellular stress response. Oxidative stress is accompanied by increased ROS accumulation that can lead to mitochondrial dysfunction and cell injury . Under conditions of cellular stress, elevated cellular ROS levels contribute to the induction of autophagy [18, 19]. However, the detailed molecular mechanisms that induce autophagy via ROS are still not well understood (reviewed in ).
Autophagy serves to reduce oxidative damage as it triggers a self-degradative process for the removal of aggregated or misfolded proteins and damaged organelles like mitochondria and peroxisomes by sequestration within a double-membrane structure called the autophagosome and successive delivery to lysosomes for degradation [20, 21]. The autophagic process requires a number of autophagy-related genes (ATGs) and is divided into an initiation-, nucleation- and elongation step. To initiate autophagy Unc-51 like kinase 1 (ULK1) phosphorylates Atg13, and focal adhesion kinase family interacting protein of 200 kD (FIP200)  which leads to activation of class III phosphatidylinositol-3-kinase (PI3K) and nucleation through BECN1/Beclin-1. Finally Atg5, Atg12, and the microtubule-associated protein light chain 3 (MAP1ALC3/LC3) control the closure of the isolation membrane (reviewed in ). In cancer, the tumor-promoting and tumor-suppressing properties of autophagy are still under debate (reviewed in ), however, recent studies revealed that the induction of autophagy enhances the resistance of tumors to different anticancer therapies like chemotherapy or radiation therapy [25, 26].
Oehme et al. provided evidence that highly aggressive neuroblastoma cells use autophagy to overcome stress caused by cytotoxic agents, resulting in resistance to chemotherapy . In addition, autophagy regulates the ROS-mediated induction of the cell cycle inhibitor CDKN1A/p21 , which controls cellular proliferation, contributes to therapy resistance in cancer [29, 30] and controls phosphorylation of extracellular signal-regulated kinase MAPK1/ERK , which is a core regulator of p21 [32, 33].
The FOXO protein family members FOXO1 and FOXO3 modulate autophagy in different cell types [34–39]. FOXO3 transcriptionally controls autophagy by regulating ATG genes and autophagy regulatory genes like LC3 and the GABA[A] receptor-associated protein like 1 (Gabarapl1) [38, 40] and thereby contributes to cell survival under stress conditions [36, 41, 42].
As FOXO3 controls cellular ROS steady-state levels via DEPP expression , the present study was designed to investigate the impact of FOXO3 and DEPP on autophagy and associated effects on ROS-mediated cell death as well as therapy resistance in human neuroblastoma.
Cell lines, culture conditions, and reagents
The neuroblastoma cell line STA-NB15 (termed NB15) was isolated at the St. Anna Children’s Hospital (Vienna, Austria) . The neuroblastoma cell line SH-EP was kindly provided by N. Gross, Lausanne, Switzerland . The cell lines were cultured in RPMI 1640 (Lonza, Basel, Switzerland) containing 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Gibco BRL, Paisley, GB) at 5% CO2 and 37 °C in saturated humidity. Phoenix™ packaging cells for helper-free production of amphotropic retroviruses  and HEK293T packaging cells for production of lentiviruses were cultured in DMEM (Lonza, Basel, Switzerland). All cells were routinely tested for mycoplasma contamination using the VenorRGeM-mycoplasma detection kit (Minerva Biolabs, Germany). MnTBAP was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Texas, USA). All other reagents were purchased from Sigma-Aldrich (Vienna, Austria) unless indicated otherwise. For each experiment, mid-log-phase cultures were seeded in fresh medium.
Production of retroviruses and lentiviruses for infection of neuroblastoma cells
6 × 105 Phoenix™ packaging cells were transfected with 2 μg of retroviral vectors and 1 μg of a plasmid coding for VSV-G protein using Lipofectamine2000. The vectors pLIB-FOXO3(A3)-ER-iresNeo, pQ-tetCMV-SV40Neo, pQ-tetCMV-EGFP-SV40Neo, pQ-tetCMV-DEPP-SV40-Neo, pQ-tetCMV-EYFP-DEPP-SV40-Neo, pLIB-EYFP-LC3-iresPuro, the lentiviral vectors coding for human DEPP-specific shRNA, FOXO3-specific shRNA and the control vector pLKO.1 have been described previously [2, 3, 9, 46] For expression of YFP-tagged LC3, mus-LC3 was amplified from pEYFP-C1-mus-LC3 (kindly donated from Prof. Noboru Mizushima, Tokyo Medical and Dental University). To generate the vector pLIB-EYFP-LC3-iresPuro the PCR-product was inserted into the EcoR1 and BamH1 site of the previously described pLIB-EYFP-MCL1JAM-iresPuro  thereby replacing Mcl1LJAM by LC3. The tandem-fluorescent pQCXI-Neo-DsRed-LC3-GFP construct and the Lamp1-RFP plasmid were purchased from Addgene (Addgene, Cambridge, GB).
Genetically modified neuroblastoma cell lines
The cell lines SH-EP/tetCtr, SH-EP/tetEGFP, SH-EP/tetDEPP, SH-EP/tetEYFP-DEPP, SH-EP/FOXO3-shCtr, SH-EP/FOXO3-shDEPP-10, SH-EP/FOXO3-shDEPP-12, SH-EP/FOXO3-shDEPP-13, SH-EP/shCtr, SH-EP/shFOXO3-17, NB15/shCtr, NB15/shDEPP, and NB15/shFOXO3 have been described previously [3, 9, 48]. SH-EP/FOXO3 cells were infected with retroviral pLIB-EYFP-LC3-iresPuro supernatants to generate SH-EP/FOXO3-EYFP-LC3 cells.
Cells were lysed on ice in lysis-buffer (50 mM TRIS/HCl, 1 mM EDTA, 150 mM NaCl, 1% IPEGAL, 0.25% Deoxycholic acid sodium salt) with protease and phosphatase inhibitors. To analyze LC3 conversion the cells were incubated with 10 μg/ml pepstatin A and 10 μg/ml E-64d. Protein concentration was determined using Bradford-Reagent (BioRad Laboratories, Munich, Germany). Total protein samples (20–50 μg/lane) were separated by SDS-PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Little Chalfont, GB) by a semi-dry blotting device (Hoefer TE70, Amersham Biosciences). Membrane blocking was performed with PBS blocking buffer containing 0.1% Tween20 and 5% nonfat dry milk, incubated with primary antibodies specific for DEPP, LC3, GAPDH (Novus, Littleton, USA), Gabarapl1 (Abcam, Cambridge, UK), CDK6, p62, ERK1/2, phosphorylated pThr202/Tyr204-ERK1/2 (Cell Signaling, Danvers, USA), p21, p27, Cyclin D1 and CDK4 (BD Biosciences, Heidelberg, Germany). After incubation with anti-mouse or anti-rabbit horseradish-peroxidase-conjugated secondary antibodies the blots were analyzed by the enhanced chemiluminescence substrate ECL Select™ (Amersham Biosciences, Little Chalfont, GB) according to the manufacturer’s instructions and detected with the AutoChemiSystem (UVP, Cambridge, GB). Quantification of protein expression was done with the ImageJ 1.48 software.
Live cell ROS staining
For ROS measurements, cells were grown on ibidi μ-slide 8 well™ slides (Ibidi, Munich, Germany) and incubated with reduced MitoTrackerRed CM-H2XROS (200 nM, Invitrogen, Carlsbad, CA, USA) for 20 min according to the manufacturer’s instructions. Cells were analyzed by live confocal microscopy using an inverted microscope (Zeiss Observer.Z1; Zeiss, Oberkochen, Germany) in combination with a spinning disc confocal system (UltraVIEW VoX; Perkin Elmer, Waltham, MA, USA). All images were acquired using a 63x oil immersion objective.
Live cell analysis of autophagic cells
Cells were grown on ibidi μ-slide 8 well™ slides (Ibidi, Munich, Germany) and transfected with 1 μg pLIB-EYFP-LC3-iresPuro, pQCXI-Neo-DsRed-LC3-GFP, or Lamp1-RFP using jetPrime reagent according to the manufacturer’s instructions (PeqLab, Erlangen, Germany). Images were collected with an Axiovert200M microscope equipped with filters for EYFP (exitation: BP500/20, emission: BP535/30) and a 63x oil objective (Zeiss, Oberkochen, Germany) or by live confocal microscopy using an inverted microscope (Zeiss Observer.Z1; Zeiss, Oberkochen, Germany) in combination with a spinning disc confocal system (UltraVIEW VoX; Perkin Elmer, Waltham, MA, USA) and a 63x oil immersion objective.
Quantification of EYFP-LC3 dots per cell was done with the ImageJ 1.48 software.
Quantitative RT-PCR analyses
To quantify DEPP mRNA levels, we designed “real-time” RT-PCR assays, using GAPDH as reference gene. Total RNA was prepared from 5×106 cells using TRIzol™ Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the Revert H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Huntsville, USA). Quantitative RT-PCR was performed as described previously  using DEPP (forward ACTGTCCCTGCTCATCCATTCTC and reverse AGTCATCCAGGCTAGGAGAGGG), LC3 (forward AGCAGCATCCAACCAAAATC and reverse CTGTGTCCGTTCACCAACAG), Gabarapl1 (forward ATGAAGTTCCAGTACAAGGAGGA and reverse GCTTTTGGAGCCTTCTCTACAAT), and GAPDH-specific oligonucleotides (forward TGTTCGTCATGGGTGTGAACC and reverse GCAGTGATGGCATGGACTGTG). After normalization on GAPDH expression, regulation was calculated between treated and untreated cells.
Gene-silencing by siRNA
2×105 cells were seeded in 6-well plates and incubated overnight to reach a confluence of 50%. Cells were transfected with 100 nM siRNA oligonucleotides using jetPrime reagent according to the manufacturer’s instructions (PeqLab, Erlangen, Germany). Twenty-four hours after transfection, cells were plated for the different experiments. SignalSilence® LC3B siRNA I and SignalSilence® Control siRNA were purchased form Cell Signaling (Danvers, USA).
Determination of apoptosis by flow cytometry
Apoptosis was measured by staining the cells with propidium-iodide (PI) and forward/sideward scatter analysis using a CytomicsFC-500 Beckman Coulter. 2×105 cells were harvested and incubated in 500 μl hypotonic PI solution containing 0.1% Triton X-100 for 4 to 6 h at 4 °C. Stained nuclei in the sub-G1 marker window were considered to represent apoptotic cells.
Caspase 3/7 assay
Caspase 3/7 activity was determined using Caspase-Glo 3/7 assay kit according to the manufacturer’s instructions (Promega, Madison, USA). Briefly, cells were cultured at 80% confluence in 96-wells containing 50 μl media. Lyophilized Caspase-Glo 3/7 substrate was resuspended in Caspase-Glo 3/7 buffer and 50 μl of this reagent were added to each 96-well and incubated at room temperature for 1 hour. Luminescence of each sample was analyzed with a Hidex Sense microplate reader (Hidex, Turku, Finland). Caspase 3/7 activity was calculated between treated and untreated cells.
Analysis of cell viability and proliferation
Viability of cells was determined with the AlamarBlue assay (AbD Serotec, Kidlington, UK) in a Hidex Sense microplate reader (Hidex, Turku, Finnland) according to the manufacturer’s instructions. Proliferation of cells was analyzed with a BrdU cell proliferation ELISA kit (Abcam, Cambridge, UK) in a Benchmark Microplate Reader (BioRad Laboratories, Munich, Germany) according to the manufacturer’s instructions. The amount of incorporated BrdU was calculated between treated and untreated cells.
Statistical significance of differences between controls and treated cells were calculated using unpaired t-test. All statistical analyses were performed using the Graph Pad Prism 6.0 software.
DEPP expression induces autophagy in human neuroblastoma cells
We have shown before that DEPP expression affects cellular ROS detoxification capacities in neuroblastoma cells . Thus, we measured ROS steady state levels in SH-EP/tetEGFP and SH-EP/tetEYFP-DEPP cells treated with doxy. Expression of the EYFP-DEPP fusion protein, which localizes to mitochondria and peroxisomes in neuroblastoma cells , caused a significant increase of cellular ROS as shown by live-cell imaging analyses using a reduced, non-fluorescent version of the MitoTrackerRed CM-H2XROS that fluoresces upon oxidation (Fig. 1c). As ROS, especially hydrogen peroxide (H2O2), mediate the induction of autophagy in different cell types (reviewed in ), we analyzed whether the DEPP-triggered LC3 conversion is mediated by ROS in neuronal cells. Therefore, we treated SH-EP/tetEYFP-DEPP cells with doxy for 8 h to induce DEPP expression, while ROS formation was inhibited with the ROS scavenger N-acetyl cysteine (NAC). We detected a significant reduction of DEPP-induced LC3 lipidation due to ROS inhibition (Fig. 1d), which suggests that DEPP initiates the formation of autophagosomes by increasing cellular ROS steady-state levels in neuronal cells. In line, DEPP-triggered LC3-II expression was efficiently inhibited using the superoxide dismutase (SOD) mimetic MnTBAP (Additional file 3a). MnTBAP is a potent superoxide anion and peroxynitrite scavenger, but does not scavenge nitric oxide, supporting the notion that intracellular ROS, including superoxides and peroxynitrite, contribute to the induction of DEPP-triggered autophagy.
FOXO3 induces autophagy through induction of DEPP
As the transcription factor FOXO3 is involved in the modulation of autophagy [37, 38, 55] and DEPP is a transcriptional target of FOXO3 , we wondered whether FOXO3 induces autophagy in neuroblastoma cells and whether this process is mediated via DEPP. Therefore, we used SH-EP/FOXO3-shCtr cells that stably express a 4-hydroxy-tamoxifen-inducible (4OHT), PKB-phosphorylation-independent FOXO3(A3)ERtm transgene . DEPP expression was knocked down by lentiviral expression of DEPP-specific shRNAs in these cells .
To determine whether LC3 and Gabarapl1 are also transcriptionally regulated by FOXO3 in neuroblastoma cells we measured the mRNA expression of LC3 and Gabarapl1 in SH-EP/FOXO3-shCtr and SH-EP/FOXO3-shDEPP-13 cells by quantitative RT-PCR analyses. FOXO3 activation caused the upregulation of LC3 (2.2-fold) and Gabarapl1 (4.8-fold) in SH-EP/FOXO3-shCtr cells. However, the FOXO3-triggered induction of these autophagy-related genes was diminished as a consequence of DEPP knockdown in SH-EP/FOXO3-shDEPP-13 cells (Fig. 2c). Together, these data demonstrate that FOXO3 mediates autophagic flux via regulation of DEPP expression in neuronal cancer cells.
FOXO3 triggered ROS accumulation induces autophagy
We demonstrated before that FOXO3 induces a biphasic ROS accumulation in neuroblastoma cells , which is significantly reduced by DEPP knockdown due to an increased capacity to detoxify cellular ROS, especially H2O2 . Luo et al. found that H2O2-induced autophagy depends on the induction of intracellular production of ROS , raising the question whether FOXO3-mediated autophagy is a consequence of cellular ROS accumulation.
To further prove that FOXO3 triggers autophagy via induction of ROS in neuroblastoma cells we performed immunoblot analysis on LC3 conversion in SH-EP/FOXO3 and NB15/FOXO3 cells treated with 4OHT and with NAC to scavenge ROS. NAC-treatment prevented the FOXO3-mediated LC3 conversion in both cell lines (Fig. 3b). In line, also MnTBAP efficiently abolished FOXO3-mediated LC3-II expression (Additional file 3b), indicating that FOXO3-mediated ROS accumulation triggers autophagy in neuroblastoma cells.
Cellular stress induces DEPP expression and autophagy
To assess, whether cellular stress induces autophagy by activating FOXO3 and DEPP, we transiently transfected the pLIB-EYFP-LC3-iresPuro plasmid into SH-EP/shCtr, SH-EP/shDEPP as well as SH-EP/shFOXO3-17 cells and performed live-cell imaging analyses. Both, H2O2-treatment and serum starvation (media supplemented with 0.5% FCS) induced the formation of LC3-II punctate in SH-EP/shCtr cells. Importantly, knockdown of DEPP as well as of FOXO3 efficiently impaired the formation of autophagosomes under cellular stress conditions (Fig. 4b). Also immunoblot analyses revealed that H2O2-treatmentand starvation-triggered autophagy depends on the expression of DEPP and FOXO3 in neuronal cells. We found a marked increase in LC3 conversion during H2O2-treatment and serum starvation in SH-EP/shCtr and NB15/shCtr cells (Fig. 4c). Knockdown of DEPP prevented LC3 conversion under stress conditions in both neuroblastoma cell lines. Consistently, knockdown of FOXO3 impaired both stress-triggered LC3 lipidation and induction of DEPP expression in SH-EP/shFOXO3-17 as well as in NB15/shFOXO3 cells (Fig. 4c).
Taken together, these results indicate that oxidative and metabolic stress induce the expression of DEPP via FOXO3 and thereby cause the formation of autophagosomes. Importantly, inhibition of the FOXO3/DEPP axis is sufficient to efficiently abrogate the induction of autophagy during cellular stress conditions in neuronal cells.
DEPP reduces cellular proliferation through LC3-mediated ERK phosphorylation and upregulation of the cell cycle inhibitor p21
As the autophagy regulatory gene LC3-II is required for ERK phosphorylation in NIH/3 T3 cells  and DEPP-induced autophagy is triggered by ROS accumulation, we wondered whether DEPP mediates ERK phosphorylation and p21 induction via ROS and LC3-II conversion in neuroblastoma cells. By immunoblot analyses of SH-EP/tetDEPP cells transiently transfected with scrambled or LC3-specific siRNAs we found that LC3 knockdown and ROS inhibition by NAC attenuated DEPP-triggered ERK1/2 phosphorylation and p21 induction indicating that DEPP-driven ROS accumulation induces autophagy and thereby ERK1/2 phosphorylation and p21 upregulation (Fig. 5b).
As DEPP modulates the expression of the cell cycle inhibitor p21 we assessed the effect of DEPP on the number of viable cells. The AlamarBlue viability assay (Fig. 5c) indicated a significant reduction of living cells due to DEPP expression in SH-EP/tetDEPP cells. In line, we found that forced DEPP expression decreased the amount of incorporated BrdU into SH-EP/tetDEPP cells, indicating reduced proliferation (Additional file 4). In neuroblastoma, ectopic DEPP expression does not induce apoptosis per se , supporting the notion that DEPP inhibits cellular proliferation. Importantly, ERK1/2 inhibition by PD98059 was sufficient to prevent DEPP-triggered reduction of proliferation (Fig. 5c). Together, these results indicate that DEPP impairs proliferation of neuronal tumor cells by modulating LC3 and ERK/p21 signaling.
Autophagy inhibits FOXO3-induced apoptosis in neuroblastoma cells
Inhibition of autophagy sensitizes neuroblastoma cells to etoposide- and doxorubicin-induced cell death
Inhibition of autophagy by CQ caused a highly significant increase in etoposide- as well as doxorubicin-induced apoptosis and caspase-3/7 activity in SH-EP/shCtr as well as in NB15/shCtr cells (Fig. 7d and e). The increase in apoptosis and caspase-3/7 activity due to autophagy inhibition was significantly reduced by DEPP knockdown, both in SH-EP/shDEPP and in NB15/shDEPP cells (Fig. 7d upper panel and e) as well as by FOXO3 knockdown in SH-EP/shFOXO3-17 and NB15/shFOXO3 cells (Fig. 7d lower panel and e).
These data show that the FOXO3/DEPP-mediated ROS accumulation simultaneously mediates an autophagy and apoptosis program, which counteract each other. The FOXO3/DEPP-triggered autophagy pathway thereby partially protects against chemotherapy-induced cell death and attenuates apoptosis induction in neuroblastoma. The inhibition of autophagic flux by CQ abrogates protective autophagy and might cause additional cellular stress due to accumulation of autophagosomes which sensitizes neuroblastoma cells to chemotherapeutic agent-induced cell death.
In this study, we demonstrate for the first time that the FOXO3-regulated gene DEPP modulates the induction of autophagy in human neuroblastoma. We found that ectopic DEPP expression induces the conversion of LC3-I into its active, membrane-bound form LC3-II, reduces the protein level of p62 (Fig. 1b), and causes the formation of autophagolysosomes as visualized by the colocalization of LC3 with LAMP1 (Additional file 1b and c), demonstrating autophagic flux. Stepp et al. observed that DEPP expression induces autophagy in HEK293 cells . However, the detailed mechanism how DEPP contributes to the activation of autophagy is not described so far. DEPP is induced by different forms of cellular stress [11–16] and impairs the cellular ROS-detoxification capacity , which results in increased cellular ROS levels (Fig. 1c). As ROS accumulation contributes to mitochondrial dysfunction, cell injury  and to the induction of autophagy (reviewed in ), we hypothesized that DEPP might modulate autophagy by increasing ROS levels. Indeed, a marked reduction in DEPP-triggered LC3 conversion was observed due to ROS inhibition by NAC and MnTBAP (Fig. 1d and Additional file 3a).
In neuronal cells, FOXO3 directly regulates DEPP expression by three putative FOXO3 binding sites located on the DEPP promoter . Here we report that FOXO3 leads to autophagic flux via regulation of DEPP as knockdown of DEPP completely abrogated the FOXO3-triggered lipidation of LC3, induction of Gabarapl1, and degradation of p62 (Fig. 2). FOXO3 is a transcriptional activator for autophagy-related genes in different cell types (reviewed in ). FOXO3 directly regulates LC3 gene expression in muscle cells  and binds to the promoter sequence of Gabarapl1 thereby inducing starvation-mediated autophagy in cardiomyocytes . As DEPP triggers autophagy via intracellular ROS we hypothesized that also FOXO3-mediated LC3 lipidation was ROS- dependent. ROS - inhibition by NAC and MnTBAP prevented FOXO3-triggered autophagy (Fig. 3 and Additional file 3b). In agreement, DEPP knockdown completely inhibited FOXO3-triggered ROS accumulation due to increased cellular ROS detoxification capacities via an increase of the CAT/catalase enzyme activity . When DEPP is expressed, increased cellular ROS steady-state levels might act via a feedback loop and thereby amplify the FOXO3-regulated autophagy program. These data show that FOXO3-triggered autophagy depends on the expression of DEPP and associated ROS accumulation.
Autophagy is described as a cellular rescue pathway during stress conditions as it reduces levels of ROS to protect cell integrity via selective degradation of damaged mitochondria (mitophagy)  or peroxisomes (pexophagy) . Li et al. reported that mitochondrial ROS induce autophagy mediated by the AMP-activated protein kinase (AMPK) pathway under starvation conditions . Of note AMPK is a direct regulator of FOXO3 . We recently provided evidence that DEPP expression is regulated via FOXO3 on mRNA and protein level under growth factor withdrawal , indicating a possible role of FOXO3/DEPP expression in stress-induced autophagy. DEPP expression is elevated on mRNA- and protein level by oxidative stress triggered by H2O2-treatment (Fig. 4a and c). Oxidative stress induces nuclear translocation of FOXO transcription factors and functional activation of their target genes [7, 67]. DEPP induction by H2O2 was strongly reduced by stable FOXO3 knockdown suggesting that DEPP regulation by stress signaling almost exclusively relies on FOXO3 (Fig. 4a). Oxidative and metabolic stress induced the lipidation of LC3 (Fig. 4c) and the formation of autophagosomes (Fig. 4b). Importantly, DEPP- and FOXO3 knockdown experiments revealed that the induction of the FOXO3/DEPP axis by cellular stress is essential to induce autophagy in neuroblastoma (Fig. 4b and c). Also, Warr et al. demonstrated that FOXO3 controls a pro-autophagy gene expression program that specifically directs hematopoietic stem cells to a protective autophagic response upon metabolic stress . However, our studies provide significant new insights into the role of DEPP as a critical link between cellular stress and the induction of autophagy in neuronal tumor cells.
Luo et al. found that intracellular ROS mediate H2O2-induced autophagy, which is followed by posttranscriptional upregulation of the cell cycle inhibitor p21 and reduced cell proliferation in human fetal lung WI-38 cells . In neuroblastoma, DEPP elevated p21 protein expression (Fig. 5a), which is a result of DEPP-triggered ROS as inhibition of ROS by NAC impaired DEPP-mediated p21 upregulation (Fig. 5b). Of note, autophagy-related proteins including LC3-II stimulate ERK1/2 activity which is a critical upstream regulator of p21 . Martinez-Lopez et al. propose that LC3-II-positive membranes serve as scaffolds for efficient spatial coordination of the Raf-MEK-ERK cascade and thus enable ERK phosphorylation . Several studies have demonstrated that hyperactivation of ERK1/2 causes cell cycle arrest by inducing the expression of p21 (reviewed in ), indicating a possible link between DEPP, ERK1/2, and p21 expression. Indeed, conditional induction of DEPP induces ERK1/2 phosphorylation and p21 upregulation (Fig. 5a). Importantly, inhibition of LC3 lipidation by transient expression of LC3-specific siRNA impaired DEPP-triggered ERK1/2 activation as well as p21 induction (Fig. 5b). ERK1/2 inhibition by the MAPK inhibitor PD98059 also prevented p21 upregulation, whereas other cell cycle related proteins were not affected (Fig. 5a). Consistent with the increased p21 expression we found a marked reduction of BrdU incorporation (Additional file 4) and number of viable cells due to increased DEPP expression, which was abolished by ERK1/2 inhibition (Fig. 5c). Together, these results provide first evidence that DEPP-mediated ROS accumulation induces LC3 lipidation, ERK1/2 phosphorylation, and p21 expression, which results in reduced proliferation.
We have shown that FOXO3 causes ROS accumulation, which in turn triggers cell death in neuroblastoma . However, here we report for the first time that FOXO3 simultaneously modulates an autophagy program that partially protects neuroblastoma cells from undergoing apoptosis, suggesting a fragile balance between survival and death controlled by FOXO3. Inhibition of autophagosome formation by LC3 knockdown resulted in a significant increase in FOXO3-triggered activation of caspase 3/7 (Fig. 6c) as well as induction of apoptosis (Fig. 6b). This protective effect of autophagy during stress conditions is in line with several recent studies that describe autophagy as a modulator of resistance to chemotherapy in different types of cancer (reviewed in ). Consistent with this concept, the inhibition of autophagy by LC3 knockdown also significantly increased etoposide- and doxorubicin-mediated apoptosis in neuronal cells (Fig. 7c). Both chemotherapeutic agents induce autophagy and autophagic flux in neuroblastoma (Fig. 7b and Additional file 5) similar to what has been published for other cancer types [69, 70]. The idea that autophagic processes are essential for eliminating damaged proteins and organelles to preserve tumor cell life also during chemotherapy was further supported by using the autophagy-inhibitor CQ that significantly enhanced the chemotherapeutic effect of etoposide and doxorubicin in SH-EP and NB15 cells (Fig. 7d and e). Of note, the FDA-approved substance CQ has shown antitumor activity in different cancer types (reviewed in ) and might be a useful drug to overcome autophagy-mediated death resistance of neuroblastoma tumors in vivo.
Taken together, in this paper we delineate a complex cellular network based on the FOXO3 - DEPP - ROS axis that steers the life - death balance in response to chemotherapy and we reveal that autophagy serves as an essential degradation pathway that can be targeted for chemosensitization by the FDA-approved compound CQ.
We would like to thank David Sabatini for providing the pQCXI-DsRed-LC3-GFP-puro plasmid and Walther Mothes for providing the Lamp1-RFP plasmid.
This work was supported by grants from the “Kinderkrebshilfe Tirol und Vorarlberg”, the “Südtiroler Krebshilfe”, the “Kinderkrebshilfe Südtirol-Regenbogen”, the “SVP-Frauen-Initiative”, the “MFF Tirol” (project Nr 246 and Nr 208), and the “Provita Kinderleukämie Stiftung”. The Tyrolean Cancer Research Institute and this study are supported by the “Tirol-Kliniken GmbH” and the “Tyrolean Cancer Society”.
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SS performed all the experiments and drafted the manuscript. HM performed the live confocal imaging analysis. KKU participated in the coordination of the study. AMJ and OP conceived, designed and coordinated the study, and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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