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NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis

Open ArchivePublished:January 26, 2017DOI:https://doi.org/10.1016/j.jid.2016.12.027
      The nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes are involved in several physiological functions. However, their roles in keratinocyte responses to UV radiation have not been clearly elucidated. This study shows that, among other NOX family members, UVB irradiation results in a biphasic activation of NOX1 that plays a critical role in defining keratinocyte fate through the modulation of the DNA damage response network. Indeed, suppression of both bursts of UVB-induced NOX1 activation by using a specific peptide inhibitor of NOX1 (InhNOX1) is associated with increased nucleotide excision repair efficiency and reduction of apoptosis, which is finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation is blocked, both nucleotide excision repair efficiency and apoptosis are decreased. Our results show that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced skin cancer in nucleotide excision repair-proficient and -deficient patients.

      Abbreviations:

      CPD (cyclobutane pyrimidine dimer), DDR (DNA damage response), InhNOX1 (peptide inhibitor of NOX1), NADPH (nicotinamide adenine dinucleotide phosphate), NER (nucleotide excision repair), NOX (nicotinamide adenine dinucleotide phosphate oxidase), ROS (reactive oxygen species), SCC (squamous cell carcinoma), sh (short hairpin), XPC (xeroderma pigmentosum type C)

      Introduction

      Solar UVB radiation is the primary environmental risk factor responsible for the induction of nonmelanoma skin cancers, including basal cell carcinomas and squamous cell carcinomas (SCCs), the most common types of human malignancies worldwide. An estimated 5.4 million cases of nonmelanoma skin cancers were affecting 3.3 million patients among the US population in 2012 (
      • Rogers H.W.
      • Weinstock M.A.
      • Feldman S.R.
      • Coldiron B.M.
      Incidence estimate of nonmelanoma skin cancer (keratinocyte carcinomas) in the US population, 2012.
      ). Unlike other cancers, where the incidence has stabilized or decreased, the rate of nonmelanoma skin cancers continues to rise (
      • Donaldson M.R.
      • Coldiron B.M.
      No end in sight: the skin cancer epidemic continues.
      ). Thus, the identification of new therapeutic targets in nonmelanoma skin cancers has significant clinical implications. To this end, relationships between the various signaling networks that regulate cellular responses to UV irradiation need a more in-depth understanding. Indeed, the natural skin photoprotection barrier comprises several factors, including constitutive and UV-induced pigmentation, stratum corneum thickness, UV-induced immune responses, UV-induced apoptosis, antioxidant defense, and DNA repair systems (
      • Matsumura Y.
      • Ananthaswamy H.N.
      Short-term and long-term cellular and molecular events following UV irradiation of skin: implications for molecular medicine.
      ,
      • Sander C.S.
      • Chang H.
      • Hamm F.
      • Elsner P.
      • Thiele J.J.
      Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis.
      ). Among these factors, reactive oxygen species (ROS) could play a key role in the regulation of cellular responses to UVB by affecting various components of the skin photoprotection barrier via their dual effects (i.e., acting as cellular toxicants or signaling molecules) (
      • Afanas'ev I.B.
      Signaling by reactive oxygen and nitrogen species in skin diseases.
      ,
      • Bickers D.R.
      • Athar M.
      Oxidative stress in the pathogenesis of skin disease.
      ,
      • Kammeyer A.
      • Luiten R.M.
      Oxidation events and skin aging.
      ,
      • Van Laethem A.
      • Garmyn M.
      • Agostinis P.
      Starting and propagating apoptotic signals in UVB irradiated keratinocytes.
      ).
      We and others have already shown that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) could be the source of UVB-induced ROS generation (
      • Rahman M.
      • Kundu J.K.
      • Shin J.W.
      • Na H.K.
      • Surh Y.J.
      Docosahexaenoic acid inhibits UVB-induced activation of NF-kappaB and expression of COX-2 and NOX-4 in HR-1 hairless mouse skin by blocking MSK1 signaling.
      ,
      • Rezvani H.R.
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      • Cario-Andre M.
      • Pain C.
      • Ged C.
      • Taieb A.
      • et al.
      Protective effects of catalase overexpression on UVB-induced apoptosis in normal human keratinocytes.
      ,
      • Rezvani H.R.
      • Dedieu S.
      • North S.
      • Belloc F.
      • Rossignol R.
      • Letellier T.
      • et al.
      Hypoxia-inducible factor-1alpha, a key factor in the keratinocyte response to UVB exposure.
      ,
      • Ryu H.C.
      • Kim C.
      • Kim J.Y.
      • Chung J.H.
      • Kim J.H.
      UVB radiation induces apoptosis in keratinocytes by activating a pathway linked to “BLT2-reactive oxygen species”.
      ). However, it has not yet been fully defined which members of the NOX family are responsible for UVB-induced ROS generation in keratinocytes and what precise role NOX might play in UVB-induced carcinogenesis. The NOX family includes seven members (NOX1 through NOX5, Duox1, and Duox2), which are involved in several physiological functions including host defense, posttranslational processing of proteins, cellular signaling, regulation of gene expression, and cell differentiation. However, they differ in their tissue distribution and their activation mechanisms. For instance, NOX1–3 are inactivated in resting cells. To be active, they need to be assembled with the membrane-bound p22phox and their respective cytoplasmic subunits (i.e., NOX activator, NOX organizer, and perhaps small guanosine triphosphate-ase Rac). In particular, p22phox, NOXA1, NOXO1, and the small guanosine triphosphate-ase Rac are necessary for the activation of NOX1 and NOX3. The activated NOX2 complex consists of p22phox, p47 phox, p67 phox, p40 phox, and the small guanosine triphosphate-ase Rac. Unlike NOX1, NOX2, and NOX3, NOX4 is constitutively active, and its activity has been shown to be positively regulated by p22phox. Rac may also be implicated in the function of NOX4 (reviewed in
      • Altenhofer S.
      • Radermacher K.A.
      • Kleikers P.W.
      • Wingler K.
      • Schmidt H.H.
      Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement.
      ,
      • Bedard K.
      • Krause K.H.
      The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology.
      ,
      • Sumimoto H.
      Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species.
      ).
      UVB-induced genomic insults elicit the DNA damage response (DDR) network, which orchestrates cell cycle progression and DNA repair machineries to trigger the appropriate responses, including repair of DNA damage, cell cycle delay, senescence, and/or apoptosis. The ultimate fate of cells with damaged DNA is, indeed, dependent on the type and extent of damage and DNA repair capacity (
      • Branzei D.
      • Foiani M.
      Regulation of DNA repair throughout the cell cycle.
      ,
      • Lagerwerf S.
      • Vrouwe M.G.
      • Overmeer R.M.
      • Fousteri M.I.
      • Mullenders L.H.
      DNA damage response and transcription.
      ,
      • Surova O.
      • Zhivotovsky B.
      Various modes of cell death induced by DNA damage.
      ). If not repaired or if misrepaired, UVB-induced DNA damage can ultimately contribute to the development of skin cancers. Among DNA repair systems, nucleotide excision repair (NER) is the major pathway for repairing numerous types of damage induced by UVB irradiation, including cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts, and some ROS-induced damage (
      • Hosseini M.
      • Ezzedine K.
      • Taieb A.
      • Rezvani H.R.
      Oxidative and energy metabolism as potential clues for clinical heterogeneity in nucleotide excision repair disorders.
      ,
      • Marteijn J.A.
      • Lans H.
      • Vermeulen W.
      • Hoeijmakers J.H.
      Understanding nucleotide excision repair and its roles in cancer and ageing.
      ). Defective NER is associated with several human diseases, such as xeroderma pigmentosum, which has a high incidence of skin cancers, Cockayne syndrome, and trichothiodystrophy (
      • Hosseini M.
      • Ezzedine K.
      • Taieb A.
      • Rezvani H.R.
      Oxidative and energy metabolism as potential clues for clinical heterogeneity in nucleotide excision repair disorders.
      ,
      • Marteijn J.A.
      • Lans H.
      • Vermeulen W.
      • Hoeijmakers J.H.
      Understanding nucleotide excision repair and its roles in cancer and ageing.
      ).
      There is an increasing body of evidence on the critical role of NOX-derived ROS in the DDR network. Indeed, NOX1 and NOX4 have been shown to play a key role in oncogene-induced tumoral transformation and senescence (
      • Kodama R.
      • Kato M.
      • Furuta S.
      • Ueno S.
      • Zhang Y.
      • Matsuno K.
      • et al.
      ROS-generating oxidases Nox1 and Nox4 contribute to oncogenic Ras-induced premature senescence.
      ,
      • Park M.T.
      • Kim M.J.
      • Suh Y.
      • Kim R.K.
      • Kim H.
      • Lim E.J.
      • et al.
      Novel signaling axis for ROS generation during K-Ras-induced cellular transformation.
      ). Similarly, we have previously shown that NOX1 activation in xeroderma pigmentosum type C (XPC)-deficient cells triggers neoplastic transformation of keratinocytes and premature skin aging (
      • Hosseini M.
      • Mahfouf W.
      • Serrano-Sanchez M.
      • Raad H.
      • Harfouche G.
      • Bonneu M.
      • et al.
      Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.
      ,
      • Rezvani H.R.
      • Kim A.L.
      • Rossignol R.
      • Ali N.
      • Daly M.
      • Mahfouf W.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas.
      ,
      • Rezvani H.R.
      • Rossignol R.
      • Ali N.
      • Benard G.
      • Tang X.
      • Yang H.S.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.
      ). Moreover, up-regulation of NOXs in several cancers (
      • Liu-Smith F.
      • Dellinger R.
      • Meyskens Jr., F.L.
      Updates of reactive oxygen species in melanoma etiology and progression.
      ,
      • Roy K.
      • Wu Y.
      • Meitzler J.L.
      • Juhasz A.
      • Liu H.
      • Jiang G.
      • et al.
      NADPH oxidases and cancer.
      ), as well as the capability of NOXs to modulate the proliferative capacity and replicative senescence in various cells (
      • Block K.
      • Gorin Y.
      Aiding and abetting roles of NOX oxidases in cellular transformation.
      ,
      • Ogrunc M.
      Reactive oxygen species: The good, the bad, and the enigma.
      ), support this notion. In this study, we report that NOX1 is up-regulated after acute and chronic UVB irradiation and that inhibition of its activation modulates the DDR network.

      Results

      UVB irradiation induces a biphasic activation of NOX1

      We first measured the activity of NOX at different time points after UVB irradiation. Results showed that UVB irradiation induced an immediate strong activation of NOX, which rapidly declined to basal levels 1 hour after irradiation. A second, more moderate increase in NOX activation was observed from 9–13 hours after irradiation (Figure 1a). Western blotting showed that NOX1, NOX2, and NOX4 (Figure 1b) but not the others (data not shown) were expressed in keratinocytes. However, modification in their protein expression was not correlated with the kinetics of NOX activation (Figure 1b). These results are not surprising, because activation of most NOX proteins depends on the translocation of their cytosolic subunits to the cell membranes. To determine whether UVB irradiation affects the assembly of NOX complexes, co-immunoprecipitation experiments were performed immediately (∼0.5 hours) and 10 hours after irradiation using antibodies against p22phox, NOX1, and NOX2. Co-precipitation of both NOXO1 and NOXA1 with p22phox and NOX1 was increased at both tested postirradiation time points compared with nonirradiated cells (Figure 1c and d). In contrast, the co-precipitation levels of p67phox with p22phox and NOX2 in irradiated cells were similar to those found in nonirradiated cells (Figure 1c and e). Similarly, the co-precipitation levels of NOX4 with p22phox were not modified upon UVB irradiation (Figure 1c). These results suggest that the NOX1 assembly is responsible for the observed UVB-induced NOX overactivation. In support of this notion, NOX overactivation was blocked only in short hairpin (sh) NOX1-transduced cells (Figure 1a).
      Figure 1
      Figure 1Biphasic effect of UVB irradiation on NOX activity. (a) NOX activity was measured at indicated time points. The results are expressed as mean ± standard deviation of three independent experiments. P < 0.05 and ∗∗P < 0.01 versus cells before irradiation. (b) Total protein extracts were assessed for the presence of different subunits of NOX1, NOX2, and NOX4 complexes by Western blot. GAPDH was used as a loading control. (c–e) The cell lysates were immunoprecipitated with (c) anti-p22phox, (d) anti-NOX1, and (e) anti-NOX2 and analyzed by Western blot for co-immunoprecipitated levels of different components of NOX complexes. Part of the cell lysate was used as an input to show the endogenous level of each protein, and immunoprecipitation with IgG was used as negative control. Ctrl, control; GADPH, glyceraldehyde-3-phosphate dehydrogenase; h, hour; HC, heavy chain of immunoprecipitating antibody; LC, light chain of immunoprecipitating antibody; nIr, nonirradiated; NOX, nicotinamide adenine dinucleotide phosphate oxidase; sh, short hairpin.

      Competitive inhibition of NOXO1 and NOXA1 interaction blocks NOX1 activity specifically

      To identify the respective roles of each peak in keratinocyte responses to UVB irradiation, we sought to develop a specific NOX1 inhibitor by targeting NOXO1 and NOXA1 interaction. It has been shown that the proline-rich region of human NOXO1 interacts with the SH3 domain of NOXA1 (
      • Sumimoto H.
      Structure, regulation and evolution of Nox-family NADPH oxidases that produce reactive oxygen species.
      ). Therefore, we tested various peptide inhibitors targeting the proline-rich region of human NOXO1 or the SH3 domain of human NOXA1 (peptides A1 to A11 and O1 to O11; see Supplementary Table S1 online). Initially, we assessed the cytotoxic effects of each peptide 24, 48, and 72 hours after treatment using the Trypan blue exclusion assay (see Supplementary Figure S1 online) and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (i.e., MTT) assay (see Supplementary Figure S2 online). The peptides, which had no significant cytotoxicity when used up to 50 μmol/L, were then evaluated for their efficiency and specificity in inhibition of NOX1 activity. To this end, NOX activity and ROS levels were first assessed in HT29 cells in which NOX1 was shown to be the only active member of the NOX family (
      • Gianni D.
      • Bohl B.
      • Courtneidge S.A.
      • Bokoch G.M.
      The involvement of the tyrosine kinase c-Src in the regulation of reactive oxygen species generation mediated by NADPH oxidase-1.
      ). Treatment with O-1, O-2, O-9, and O-13 resulted in a significant reduction in NOX activity and ROS levels (see Supplementary Figure S3a online). The efficiency and specificity of selected peptides were then tested on keratinocytes in which the expression of endogenous NOX1, NOX2, and NOX4 proteins were inhibited by using shRNA technology (see Supplementary Figure S3b). These four peptides blocked NOX1 activity efficiently in human keratinocytes. Among them, O-1 peptide, hereafter called InhNOX1, was found to be more efficient than the others (see Supplementary Figures S3c and d). By comparing NOX activity and ROS levels among shNOX1-transduced cells, InhNOX1-treated cells, and shNOX1-transduced cells treated with InhNOX1, we found that InhNOX1 inhibited NOX1-dependent ROS generation with very high (near 100%) efficiency and specificity (see Supplementary Figures S3c and d).

      Inhibition of NOX1 activation increases removal of UVB-induced DNA damage and decreases apoptosis

      Because accumulating evidence suggests that NOX-derived ROS contribute to the DDR network (
      • Block K.
      • Gorin Y.
      Aiding and abetting roles of NOX oxidases in cellular transformation.
      ,
      • Ogrunc M.
      Reactive oxygen species: The good, the bad, and the enigma.
      ), we wondered what impact NOX could have in the repair of UVB-induced DNA damage and apoptosis. Keratinocytes were treated before exposure to UVB with InhNOX1 or GKT137831, a pharmacological NOX inhibitor developed by GenKyoTex (Geneva, Switzerland) that has been shown to inhibit NOX1 and NOX4 with high affinity and NOX2 with lower potency and that is almost without any affinity for xanthine oxidase and other ROS-producing and redox-sensitive enzymes (
      • Aoyama T.
      • Paik Y.H.
      • Watanabe S.
      • Laleu B.
      • Gaggini F.
      • Fioraso-Cartier L.
      • et al.
      Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent.
      ,
      • Gaggini F.
      • Laleu B.
      • Orchard M.
      • Fioraso-Cartier L.
      • Cagnon L.
      • Houngninou-Molango S.
      • et al.
      Design, synthesis and biological activity of original pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives as novel dual Nox4/Nox1 inhibitors.
      ). Inhibition of NOX1 resulted in a significant decrease in baseline NOX activity in nonirradiated conditions (Figure 2a). Pretreatment of keratinocytes with InhNOX1 and GKT137831 prevented both initial and late UVB-induced increases in NOX activity (Figure 2a). Co-immunoprecipitation experiments confirmed that although InhNOX1 treatment efficiently prevented UVB-induced assembly of the NOX1 complex, GKT137831 did not affect it (Figure 2b), suggesting that the inhibitory effect of GKT137831 was independent of the NOX1 complex assembly.
      Figure 2
      Figure 2Treatment of keratinocytes before exposure to UVB with InhNOX1 or GKT137831 leads to increased removal rate of UVB-induced DNA damage and decreased apoptosis. (a) NOX activity and (b) co-immunoprecipitated levels of different components of NOX1 complexes were assessed. The percentages of (c) 6-4PPs and (d) CPD remaining at various time points after irradiation was evaluated by comparison with the initial levels. (e) The quantity of CPD was assessed by immunodot blot. SYBR green was used as a loading control. (f) The relative activities of caspase-3, -8, and -9 were measured. The activity of each caspase in the scrambled-treated cells was arbitrarily set to 100. (g) Total protein extracts were assessed for cell-cycle regulators and activation of caspases. β-actin was used as a loading control. The results are expressed as the mean ± standard deviation of three independent experiments. P < 0.05 and ∗∗P < 0.01 for treated cells at the indicated time points versus corresponding scrambled-treated cells. 6-4PP, 6-4 photoproducts; CPD, cyclobutane pyrimidine dimer; h, hour; HC, heavy chain of immunoprecipitating antibody; InhNOX, peptide inhibitor of NOX1; IP, immunoprecipitate; Ir, irradiated; LC, light chain of immunoprecipitating antibody; NAPDH, nicotinamide adenine dinucleotide phosphate; nIr, nonirradiated; NOX, nicotinamide adenine dinucleotide phosphate oxidase; p-, phosphorylated.
      To examine whether NOX inhibition might affect NER efficiency, we then evaluated the repair kinetics of 6-4 photoproducts and CPDs, the most frequent types of photolesions removed primarily by NER with different kinetics (
      • Moser J.
      • Volker M.
      • Kool H.
      • Alekseev S.
      • Vrieling H.
      • Yasui A.
      • et al.
      The UV-damaged DNA binding protein mediates efficient targeting of the nucleotide excision repair complex to UV-induced photo lesions.
      ,
      • Rezvani H.R.
      • Mahfouf W.
      • Ali N.
      • Chemin C.
      • Ged C.
      • Kim A.L.
      • et al.
      Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.
      ). Pretreatment of keratinocytes with InhNOX1 or GKT137831 resulted in an increased rate of 6-4 photoproduct and CPD repair (Figure 2c and d). In agreement with this, immunodot blotting showed that the CPD level was much lower in InhNOX1- and GKT137831-pretreated cells than in control keratinocytes (Figure 2e). Because apoptosis is a key determinant affecting the ultimate fate of cells with damaged DNA, we then examined the effect of NOX inhibition on UVB-induced apoptosis 20 hours after irradiation. Inhibition of NOX reduced the UVB-induced activation of caspase-3, -8 and -9 (Figure 2f and g).
      We next examined the effect of NOX inhibition on cell cycle progression. The positive cell cycle regulators, namely CDK4, CDK6, CDC25C, cyclin B, cyclin D1, and cyclin E, were up-regulated at 20 hours after irradiation compared with nonirradiated cells and that their increase was more pronounced in InhNOX1- and GKT137831-pretreated cells than in control keratinocytes (Figure 2g). On the contrary, the negative cell cycle regulators, namely p16 and p21WAF1, were increased more markedly in scrambled-treated cells than in keratinocytes treated with NOX inhibitors. UVB irradiation-mediated up-regulation of phosphorylated ATR and CHK1 levels was lower in cells treated with NOX inhibitors compared with scrambled-treated keratinocytes (Figure 2g). These results suggest that inhibition of NOX leads to increased DNA repair efficiency and thus to decreased UVB-induced apoptosis.
      Given that UVB irradiation induced NOX activity at two different times, we then sought to understand what role the late increased activity of NOX might play in DNA repair and apoptosis. To this end, we suppressed the second peak of UVB-induced biphasic NOX activation, without abrogation of the first burst, by adding InhNOX1 and GKT137831 one hour after exposure to UVB irradiation (Figure 3a). The repair kinetics of 6-4 photoproducts and CPDs showed that the removal of both photolesions was slowed down in InhNOX1- and GKT137831-treated cells compared with the control counterparts. (Figure 3b–d). The activations of caspase-3 and -9 (but not caspase-8) were also reduced by about 46% (Figure 3e and f). Western blot analysis of the positive and negative cell cycle regulators (Figure 3f) suggested that the decrease in DNA repair efficiency and the subsequent accumulation of DNA damage in cells treated with NOX inhibitors 1 hour after irradiation possibly delayed the cell cycle progression.
      Figure 3
      Figure 3Treatment of keratinocytes with InhNOX1 or GKT137831 1 hour after exposure to UVB results in decreased removal rate of UVB-induced DNA damage and apoptosis. (a) NOX activity and the levels of (b) 6-4PPs and (c) CPDs were assessed at various time points after irradiation by comparison with the initial levels. (d) The quantity of CPD was evaluated by immunodot blot. SYBR green was used as a loading control. (e) The relative activities of caspase-3, -8, and -9 were measured at 20 hours after irradiation. The activity of each caspase in the scrambled-treated cells was arbitrarily set to 100. (f) Total protein extracts were assessed for cell cycle regulators and activation of caspases by Western blot analysis. β-actin was used as a loading control. Results are expressed as the mean ± standard deviation of three independent experiments. P < 0.05 and ∗∗P < 0.01 for treated cells versus scrambled-treated cells at corresponding time point. 6-4PP, 6-4 photoproducts; CPD, cyclobutane pyrimidine dimer; h, hour; InhNOX, peptide inhibitor of NOX1; Ir, irradiated; NAPDH, nicotinamide adenine dinucleotide phosphate; nIr, nonirradiated; p-, phosphorylated.
      Altogether, these results indicate that inhibition of NOX1 before exposure to UVB irradiation results in up-regulation of the DNA repair machinery and down-regulation of apoptosis, suggesting that NOX1 inhibitors might prevent UVB-induced carcinogenesis.

      InhNOX1 and GKT137831 inhibit NOX1 activation efficiently in mouse skin

      To determine the effects of InhNOX1 and GKT137831 on the kinetics of UVB-induced NOX activation in vivo, mice were first treated with different doses of InhNOX1 and GKT137831 and then irradiated with UVB. Measurement of NOX activity in mouse skin showed that UVB irradiation induced a biphasic NOX activation similar to that found in human keratinocytes (see Supplementary Figure S4a online). Of note, the second NOX up-regulation in mouse skin after irradiation occurred some hours later than that in irradiated human keratinocytes (compare Supplementary Figure S4A with Figures 1a). Topical application of InhNOX1 at 1.36 and 5.44 μmol/kg reduced the baseline level of NOX activity by about 41%. These concentrations efficiently blocked both peaks of UVB-induced NOX activation in mouse skin (see Supplementary Figure S4a). It appeared that InhNOX1 blocks NOX1 activity efficiently up to 48 hours after its application (see Supplementary Figure S4a). Although oral administration of GKT137831 at 20 and 60 mg/kg blocked only the first peak of UVB-induced NOX activation in mouse skin, its administration per day inhibited both bursts of NOX activation (see Supplementary Figure S4a). Similar to human keratinocytes, immunodot blotting and immunohistochemical staining showed that epidermal CPD levels were much lower in InhNOX1- and GKT137831-treated mice than in the control counterparts (Figure S4b–d). Evaluation of caspase-3, -8, and -9 activation also showed that NOX1 inhibition attenuated UVB-induced apoptosis (Figure S4e and f).

      Inhibition of UVB-induced NOX activation reduces photocarcinogenesis in XPC- proficient and -deficient mice

      To study the effect of NOX1 inhibition on UVB-induced SCC, we used hairless SKH-1 mice, a well-defined murine model for the study of photocarcinogenesis that closely mimics photocarcinogenesis in humans (
      • DiGiovanni J.
      Multistage carcinogenesis in mouse skin.
      ). After chronic UVB-irradiation, these mice develop skin tumors, including papilloma and malignant SCC. GKT137831 was administered daily, and InhNOX1 was administered topically three times per week and 10 minutes before each UVB exposure. We first monitored whether chronic treatment with InhNOX1 and GKT137831 could affect the skin architecture. Histological analysis of the skin showed a normal epidermis with no obvious abnormality in epidermal differentiation or proliferation (Figure 4a). To determine the photoprotective effects of NOX inhibition, the number and size of tumors were measured weekly. Because there were no meaningful differences in outcome between mice receiving InhNOX1 or GKT137831, hereinafter only the results of InhNOX1 are shown. Treatment with both InhNOX1 and GKT137831 resulted in a significant decrease in the number and size of tumors (Figure 4b–d). Analysis of tumor volumes indicated that about 60% of the tumors in InhNOX1-treated mice measured less than 5 mm3, compared with only 8% in the scrambled-treated mice (Figure 4e).
      Figure 4
      Figure 4NOX1 inhibition increases removal rate of UVB-induced DNA damage and reduces photocarcinogenesis in XPC-proficient and -deficient mice. (a) One-month-old Xpc+/+ mice were treated with GKT137831 or InhNOX1 for 4 months. Histopathology of epidermis was evaluated. Scale bars = 50 μm. (b–e) Xpc+/+ and Xpc–/– mice were subjected to chronic UVB irradiation. (b) Variable numbers of tumors of variable size that are mostly ulcerated are visible on the back of mice. The (c) number and (d) volume of tumors per mouse were assessed. (e) The tumor volume distribution was evaluated at week 24. (f) NOX activity was evaluated in nonirradiated (nIr) mouse skin, non-tumor bearing and tumors. (g) The CPD level was evaluated, quantified densitometrically, and normalized to SYBR green intensity. (h) Relative activity of caspase-3 was measured. Results are expressed as the mean ± standard deviation. n = 6 mice per group in a, n = 20 in be, n = 8 in f and h, and n = 15 in g. CPD, cyclobutane pyrimidine dimer; h, hours; H&E, hematoxylin and eosin; InhNOX, peptide inhibitor of NOX1; Ir, irradiated; K, keratin; min, minutes; NAPDH, nicotinamide adenine dinucleotide phosphate; NOX, nicotinamide adenine dinucleotide phosphate oxidase; XPC, xeroderma pigmentosum type C.
      Measurement of NADPH oxidase activity showed the significant effectiveness of InhNOX1 and GKT137831 in preventing UVB-induced NOX activation. The activity of NADPH oxidase was reduced 65% in non–tumor-bearing skin and 62% in tumors isolated from mice treated with InhNOX1 compared with scrambled-treated mice (Figure 4f).
      Given that NOX1 inhibition affected apoptosis and DNA repair efficiency in acute UVB irradiation, we wondered whether these processes were affected in chronic irradiation. The level of CPD and the activity of caspase-3 were much lower in InhNOX1- and GKT137831-treated mice than in their control counterparts (Figure 4g and h).
      To determine whether NOX1 inhibition had a more pronounced photoprotective effect in a DNA repair-deficient context, XPC-deficient mice treated with InhNOX1 and GKT137831 were exposed to chronic UVB irradiation. The hairless SKH-1 Xpc-knockout model was chosen because we have previously shown that up-regulation of NOX1 in XPC-deficient cells triggers neoplastic transformation of keratinocytes and premature skin aging (
      • Hosseini M.
      • Mahfouf W.
      • Serrano-Sanchez M.
      • Raad H.
      • Harfouche G.
      • Bonneu M.
      • et al.
      Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.
      ,
      • Rezvani H.R.
      • Kim A.L.
      • Rossignol R.
      • Ali N.
      • Daly M.
      • Mahfouf W.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas.
      ,
      • Rezvani H.R.
      • Rossignol R.
      • Ali N.
      • Benard G.
      • Tang X.
      • Yang H.S.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.
      ). As expected, Xpc–/– mice were more prone to UVB-induced cancer than their wild-type counterparts (Figure 4c and d). Treatment with InhNOX1 or GKT137831 resulted in a significant decrease in the number and size of tumors in XPC-deficient mice (Figures 4b–d). However, NOX1 inhibition reduced UVB-induced skin SCCs with the same efficacy (Figures 4b–d).
      Altogether, these results suggest that NOX1 plays a key role in the determination of the ultimate fate of UVB-irradiated cells by affecting DNA repair and apoptotic machineries as a part of the DDR signaling network.

      NOX1 inhibition reduces the growth of UVB-induced SCC in XPC-proficient and -deficient mice

      We then sought whether NOX1 could play a role in SCC progression for the following three reasons: First, NOX-derived ROS are thought to play a central role in different steps (initiation, promotion, and progression) of carcinogenesis by modulating invasion, proliferation, adhesion, and angiogenesis (reviewed in
      • Block K.
      • Gorin Y.
      Aiding and abetting roles of NOX oxidases in cellular transformation.
      ). Second, NOX activity was 2.56-fold higher in tumors than in normal skin (Figure 4f). Third, the antioxidant capacity and the activity of catalase, CuZnSOD, and MnSOD, three important antioxidant enzymes, were much lower in SCC than in nonirradiated skin (Figure 5a–d), suggesting a potential harmful effect of up-regulation of NOX and a potential protective effect of NOX inhibition.
      Figure 5
      Figure 5NOX1 inhibition slows down the growth of UVB-induced skin tumors in XPC-proficient and -deficient mice. (a–d) Xpc+/+ and Xpc–/– mice were subjected to chronic UVB irradiation. The mice received scrambled peptide or InhNOX1 topically 10 minutes before each irradiation. Antioxidant (a) Trolox capacity, (b) catalase, (c) CuZnSOD, and (d) MnSOD activities in mouse skin were evaluated. ∗∗∗P < 0.005 versus nonirradiated XPC-proficient mice conditions; °P < 0.05 tumors versus non-tumor bearing skin, P < 0.05 versus nonirradiated XPC-deficient mice. (e–g) Effects of InhNOX1 treatment on the growth of UVB-induced skin tumors. (e) Drawing showing experimental protocol design. The effects of InhNOX1 treatment on the (f) number and (g) volume of tumors per mouse were assessed. P < 0.5 versus scrambled-treated counterpart controls at corresponding time point. Magenta and blue-violet P < 0.05 for InhNOX1-treated mice at indicated time versus starting point. (a–d) n = 20 and (e–g) 15 mice per group. InhNOX, O-1 peptide; h, hour; nIr, nonirradiated; XPC, xeroderma pigmentosum type C.
      To verify this hypothesis, XPC-proficient and -deficient mice were irradiated for 22 and 13 weeks, respectively. Mice were then divided into three groups treated topically every 2 days for 6 weeks with either the scrambled peptide or two different concentrations of InhNOX1 (Figure 5e). The number of tumors increased only in XPC-deficient mice in the first week after discontinuation of UVB irradiation, whereas InhNOX1 treatment had no effect (Figure 5f). Although SCCs in scramble-treated mice increased continuously in size, InhNOX1 treatment slowed down tumor growth in both Xpc+/+ and Xpc–/– mice (Figure 5g). InhNOX1 treatment had a very pronounced effect on tumor growth in both Xpc+/+ and Xpc–/– mice within the first week of its application. Furthermore, a slight increase in tumor size was observed at the 6th week of treatment, which could be in relation to InhNOX1 resistance.

      Discussion

      Oncogene activation is a critical step in cellular transformation (
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      ). However, ectopic expression of several oncogenes has been shown to trigger cellular senescence (
      • Campisi J.
      • d'Adda di Fagagna F.
      Cellular senescence: when bad things happen to good cells.
      ,
      • Halazonetis T.D.
      • Gorgoulis V.G.
      • Bartek J.
      An oncogene-induced DNA damage model for cancer development.
      ). To explain this, it was recently shown that oncogene activation in normal cells leads to an initial transient hyperproliferative phase, which is followed by the permanent establishment of cellular senescence (
      • Di Micco R.
      • Fumagalli M.
      • Cicalese A.
      • Piccinin S.
      • Gasparini P.
      • Luise C.
      • et al.
      Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.
      ) through a ROS/DDR-dependent process. Indeed, it has been proposed that the increased ROS levels in cells expressing an oncogene act as mitogenic signaling molecules and stimulate cell proliferation (
      • Irani K.
      • Xia Y.
      • Zweier J.L.
      • Sollott S.J.
      • Der C.J.
      • Fearon E.R.
      • et al.
      Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts.
      ,
      • Ogrunc M.
      Reactive oxygen species: The good, the bad, and the enigma.
      ,
      • Park M.T.
      • Kim M.J.
      • Suh Y.
      • Kim R.K.
      • Kim H.
      • Lim E.J.
      • et al.
      Novel signaling axis for ROS generation during K-Ras-induced cellular transformation.
      ). The promitotic effect of NOX-mediated ROS could be related to the transient inactivation of protein tyrosine phosphatase through the oxidation of redox-sensitive cysteine residues (
      • Caputo F.
      • Vegliante R.
      • Ghibelli L.
      Redox modulation of the DNA damage response.
      ,
      • Sancho P.
      • Fabregat I.
      NADPH oxidase NOX1 controls autocrine growth of liver tumor cells through up-regulation of the epidermal growth factor receptor pathway.
      ). Shifting the balance between protein tyrosine phosphatase and kinase in favor of the latter finally results in the activation of growth factors such as EGFR (
      • Cheng Y.
      • Wang Y.
      • Wang H.
      • Chen Z.
      • Lou J.
      • Xu H.
      • et al.
      Cytogenetic profile of de novo acute myeloid leukemia: a study based on 1432 patients in a single institution of China.
      ,
      • Sancho P.
      • Fabregat I.
      NADPH oxidase NOX1 controls autocrine growth of liver tumor cells through up-regulation of the epidermal growth factor receptor pathway.
      ). However, excess ROS induces DNA lesions and consequently triggers DDR network activation (
      • Bartkova J.
      • Rezaei N.
      • Liontos M.
      • Karakaidos P.
      • Kletsas D.
      • Issaeva N.
      • et al.
      Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.
      ,
      • Di Micco R.
      • Fumagalli M.
      • Cicalese A.
      • Piccinin S.
      • Gasparini P.
      • Luise C.
      • et al.
      Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.
      ) that finally drives senescence. DDR inactivation allows the oncogene-induced senescence to be bypassed and thus induces the proliferation of oncogene-expressing cells and their transformation (
      • Bartkova J.
      • Rezaei N.
      • Liontos M.
      • Karakaidos P.
      • Kletsas D.
      • Issaeva N.
      • et al.
      Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.
      ,
      • Di Micco R.
      • Fumagalli M.
      • Cicalese A.
      • Piccinin S.
      • Gasparini P.
      • Luise C.
      • et al.
      Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication.
      ). In support of this notion, inhibition of NOX-induced ROS production was reported to be an effective strategy to reduce hyperproliferation and to prevent DDR activation and oncogene-induced senescence in oncogenic Ras-expressing cells (
      • Kodama R.
      • Kato M.
      • Furuta S.
      • Ueno S.
      • Zhang Y.
      • Matsuno K.
      • et al.
      ROS-generating oxidases Nox1 and Nox4 contribute to oncogenic Ras-induced premature senescence.
      ,
      • Ogrunc M.
      Reactive oxygen species: The good, the bad, and the enigma.
      ). On the contrary, NOX1 overexpression was shown to enhance the tumorigenic conversion of NIH3T3 cells (
      • Suh Y.A.
      • Arnold R.S.
      • Lassegue B.
      • Shi J.
      • Xu X.
      • Sorescu D.
      • et al.
      Cell transformation by the superoxide-generating oxidase Mox1.
      ) and to increase neoplastic progression of immortalized human keratinocytes (
      • Chamulitrat W.
      Role of gp91phox homolog nox1 in induction of premalignant spindle phenotypes of HPV 16 E6/E7-immortalized human keratinocytes.
      ). Because the activation of the NOX family has been shown in many cancer cells (
      • Roy K.
      • Wu Y.
      • Meitzler J.L.
      • Juhasz A.
      • Liu H.
      • Jiang G.
      • et al.
      NADPH oxidases and cancer.
      ), blocking of NOX activation could be a relevant therapeutic target. NOX4 inhibition has been shown to be a potent inhibitor of cancer cell growth for pancreatic tumor cells harboring mutant Ras (
      • Ogrunc M.
      Reactive oxygen species: The good, the bad, and the enigma.
      ). Silencing of Nox4 expression in melanoma cells by small interfering RNAs decreased ROS production and thereby inhibited anchorage-independent cell growth and tumorigenicity in nude mice (
      • Liu-Smith F.
      • Dellinger R.
      • Meyskens Jr., F.L.
      Updates of reactive oxygen species in melanoma etiology and progression.
      ,
      • Yamaura M.
      • Mitsushita J.
      • Furuta S.
      • Kiniwa Y.
      • Ashida A.
      • Goto Y.
      • et al.
      NADPH oxidase 4 contributes to transformation phenotype of melanoma cells by regulating G2-M cell cycle progression.
      ). Up- and down-regulation of NOX1 have been shown to result in increased and decreased melanoma cell invasion, respectively (
      • Liu-Smith F.
      • Dellinger R.
      • Meyskens Jr., F.L.
      Updates of reactive oxygen species in melanoma etiology and progression.
      ). Consistent with this, our results show that NOX is overactivated in UVB-induced tumors and that NOX1 inhibition reduces the growth of these tumors.
      Our studies indicate that UVB-induced NOX1 activation plays an important role in defining the ultimate fate of keratinocytes. Indeed, suppression of both bursts of the UVB-induced NOX1 activation was associated with increased NER repair efficiency and reduced apoptosis, which finally translated into decreased photocarcinogenesis. On the contrary, when only the second peak of UVB-induced NOX1 activation was blocked, both NER repair efficiency and apoptosis declined. To explain these results, we propose that the first and second peaks of NOX1 activation might reduce NER efficiency and apoptotic cell death, respectively, through as yet unknown mechanisms. Therefore, inhibition of the first peak leads to increased removal of UVB-induced DNA damage and subsequently to decreased apoptosis (Figures 2c–g, and see Supplementary Figure S4b–f). When the second burst of NOX1 activation is inhibited (in the absence of abrogation of the first peak), reduction in UVB-induced apoptosis results in accumulation of cells harboring DNA damage, which reflects the decreased NER efficiency (i.e., increased number of CPD-positive cells and CPD quantity) observed in our experiments (Figure 3b–f).
      Although the mechanism of immediate UVB-induced activation of NOX1 remains to be elucidated, the second burst of NOX activation might depend on the remaining UVB-induced DNA damage. In fact, DNA damage from various sources has been shown to increase intracellular levels of ROS (
      • Kang M.A.
      • So E.Y.
      • Simons A.L.
      • Spitz D.R.
      • Ouchi T.
      DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway.
      ,
      • Nair R.R.
      • Bagheri M.
      • Saini D.K.
      Temporally distinct roles of ATM and ROS in genotoxic-stress-dependent induction and maintenance of cellular senescence.
      ), which in turn regulates cell death or senescence through different mechanisms. Indeed, several components of the DDR network such as p53, p21, ATM (
      • Caputo F.
      • Vegliante R.
      • Ghibelli L.
      Redox modulation of the DNA damage response.
      ), and H2AX (
      • Kang M.A.
      • So E.Y.
      • Simons A.L.
      • Spitz D.R.
      • Ouchi T.
      DNA damage induces reactive oxygen species generation through the H2AX-Nox1/Rac1 pathway.
      ) have been shown to be sensitive to oxidative stress (
      • Caputo F.
      • Vegliante R.
      • Ghibelli L.
      Redox modulation of the DNA damage response.
      ). In line with this, we have already shown that accumulation of unrepaired damaged DNA bases in XPC-knockdown cells triggers NOX1 activation through the DNA-dependent protein kinase (DNA-PK)/protein kinase B (PKB) axis (
      • Rezvani H.R.
      • Kim A.L.
      • Rossignol R.
      • Ali N.
      • Daly M.
      • Mahfouf W.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations that drive the formation of squamous cell carcinomas.
      ,
      • Rezvani H.R.
      • Rossignol R.
      • Ali N.
      • Benard G.
      • Tang X.
      • Yang H.S.
      • et al.
      XPC silencing in normal human keratinocytes triggers metabolic alterations through NOX-1 activation-mediated reactive oxygen species.
      ).
      In conclusion, we have shown that UVB irradiation results in the biphasic activation of NOX1, which plays a critical role in defining the fate of keratinocytes. Our results suggest that inhibition of NOX1 activation could be a promising target for the prevention and treatment of UVB-induced cancers in both NER-proficient and -deficient patients. Because UVA is generally stronger than UVB in inducing ROS production, a pivotal role of NOX1-mediated ROS generation in the response of skin to solar radiation may reasonably anticipated.

      Materials and Methods

      Transgenic mice and irradiation procedure

      Female SKH-1 hairless mice were purchased at 4–6 weeks of age from Charles River (L’arbresle, France). Hairless Xpc mutant mice (a generous gift of N. de Wind, Leiden University Medical Center, Leiden, The Netherlands) were generated (
      • Cheo D.L.
      • Ruven H.J.
      • Meira L.B.
      • Hammer R.E.
      • Burns D.K.
      • Tappe N.J.
      • et al.
      Characterization of defective nucleotide excision repair in XPC mutant mice.
      ) and bred into a SKH-1 hairless background as described previously (
      • van Oosten M.
      • Rebel H.
      • Friedberg E.C.
      • van Steeg H.
      • van der Horst G.T.
      • van Kranen H.J.
      • et al.
      Differential role of transcription-coupled repair in UVB-induced G2 arrest and apoptosis in mouse epidermis.
      ). Mice were bred and maintained in a pathogen-free mouse facility at Bordeaux University. All mouse experiments were carried out with the approval of the Bordeaux University Animal Care and Use Committee.

      UV light source and tumor study

      A UV Irradiation Unit (Daavlin, Bryan, OH) equipped with an electronic controller to regulate the dosage was used. The UV dose was quantified with an X-96 Dosimeter (Daavlin). Mice were irradiated with 150 mJ/cm2 (corresponding to one minimal erythemal dose) three times per week. Tumor numbers and volume were recorded every week. Tumor volume was estimated by measuring the shortest (width, W) and longest (length, L) axes, based on the volume of a cylinder with hemispherical ends, according to the following equation: Calculated volume (mm3) = [π(W/2)2(LW)] + [43π(W/2)3].

      NOX inhibitor GKT137831

      The NOX inhibitor GKT137831 is a member of the pyrazolopyridine dione family and is a specific inhibitor of NADPH isoforms NOX1 and NOX4 (NOX1 inhibitory constant [Ki] = 110 nmol/l, Nox4 Ki = 140 nmol/l) and has been shown to inhibit NOX1- and NOX4-derived ROS in vitro and in vivo (
      • Aoyama T.
      • Paik Y.H.
      • Watanabe S.
      • Laleu B.
      • Gaggini F.
      • Fioraso-Cartier L.
      • et al.
      Nicotinamide adenine dinucleotide phosphate oxidase in experimental liver fibrosis: GKT137831 as a novel potential therapeutic agent.
      ,
      • Gaggini F.
      • Laleu B.
      • Orchard M.
      • Fioraso-Cartier L.
      • Cagnon L.
      • Houngninou-Molango S.
      • et al.
      Design, synthesis and biological activity of original pyrazolo-pyrido-diazepine, -pyrazine and -oxazine dione derivatives as novel dual Nox4/Nox1 inhibitors.
      ). For the purpose of oral administration, GKT137831 was solubilized in 1.2% methyl cellulose (Sigma-Aldrich, Saint Quentin Fallavier, France) and 0.1% polysorbate 80 (Sigma) in deionized water. This solution without GKT137831 was used as placebo treatment.
      Detailed information on viability tests, co-immunoprecipitation, Western blotting procedure, lentiviral vector constructs and keratinocyte transduction, determination of skin antioxidant capacity, measurement of NADPH oxidase activity, and determination of 6-4 photoproducts and CPD repair kinetics are described in the Supplementary Materials online.

      Statistics

      Student t test was applied for statistical evaluation, and a P-value less than 0.05 was considered significant. Results are presented as mean ± standard deviation.

      Conflict of Interest

      None of the authors have any financial interest related to this work. A patent is pending on InhNOX1 with HR, GH, and AT as inventors and Bordeaux University as owner of the patent.

      Acknowledgments

      The authors acknowledge the patients’ support group “Les Enfants de La Lune.” HRR gratefully acknowledges support from the ARC “Association pour la Recherche sur le Cancer,” the Institut National du Cancer “INCA_6654,” the Fondation Maladies Rares and SATT aquitaine “la Société d'Accélération du Transfert de Technologies de la région Aquitaine.” They wish to thank Pierre Cau for helpful discussions and are grateful to P. Costet (University of Bordeaux) for his valuable expertise. The authors thank M.T. Quinn and F. Morel for providing antibodies against p22phox and pg91phox. HR and GH were financed by grants from “La ligue contre le cancer.”

      Supplementary Material

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      Linked Article

      • Redox-Redux and NADPH Oxidase (NOX): Even More Complicated than We Thought it Might Be
        Journal of Investigative DermatologyVol. 137Issue 6
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          The NOX (nicotinamide adenine dinucleotide phosphate oxidase) family includes seven unique members that are involved in a multitude of physiological functions, including extensive interaction with UVR and the skin. NOX1 is uniquely present and activated by UVB radiation with biphasic expression of the enzyme immediately and then after a several-hour delay. Specific inhibition of both early and late NOX1 activation leads to evidence of decreased photocarcinogenesis in in vitro keratinocytes and in well-characterized mouse models in which antitumor efficacy has been shown; inhibiting only late NOX activation does not exhibit such effects.
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      • Expression of Concern
        Journal of Investigative DermatologyVol. 142Issue 5
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          NADPH Oxidase-1 Plays a Key Role in Keratinocyte Responses to UV Radiation and UVB-Induced Skin Carcinogenesis
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