NADPH Oxidase NOX4 Supports Renal Tumorigenesis by Promoting the Expression and Nuclear Accumulation...

2014;74:3501-3511. Published OnlineFirst April 22, 2014.Cancer Res Jennifer L. Gregg, Robert M. Turner II, Guimin Chang, et al.

αPromoting the Expression and Nuclear Accumulation of HIF2NADPH Oxidase NOX4 Supports Renal Tumorigenesis by

Updated version

10.1158/0008-5472.CAN-13-2979doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2014/04/22/0008-5472.CAN-13-2979.DC1.html

Access the most recent supplemental material at:

Cited Articles

http://cancerres.aacrjournals.org/content/74/13/3501.full.html#ref-list-1

This article cites by 44 articles, 19 of which you can access for free at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

[email protected]

To request permission to re-use all or part of this article, contact the AACR Publications Department at

on July 2, 2014. © 2014 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst April 22, 2014; DOI: 10.1158/0008-5472.CAN-13-2979



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-13-2979

http://cancerres.aacrjournals.org/content/suppl/2014/04/22/0008-5472.CAN-13-2979.DC1.html

http://cancerres.aacrjournals.org/content/74/13/3501.full.html#ref-list-1

http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

mailto:[email protected]

http://cancerres.aacrjournals.org/


Molecular and Cellular Pathobiology

NADPH Oxidase NOX4 Supports Renal Tumorigenesis byPromoting the Expression and Nuclear Accumulation ofHIF2a

Jennifer L. Gregg1, Robert M. Turner II1, Guimin Chang1, Disha Joshi1, Ye Zhan2, Li Chen1, andJodi K. Maranchie1

AbstractMost sporadically occurring renal tumors include a functional loss of the tumor suppressor von Hippel

Lindau (VHL). Development of VHL-deficient renal cell carcinoma (RCC) relies upon activation of the hypoxia-inducible factor-2a (HIF2a), a master transcriptional regulator of genes that drive diverse processes, includingangiogenesis, proliferation, and anaerobic metabolism. In determining the critical functions for HIF2aexpression in RCC cells, the NADPH oxidase NOX4 has been identified, but the pathogenic contributions ofNOX4 to RCC have not been evaluated directly. Here, we report that NOX4 silencing in VHL-deficient RCC cellsabrogates cell branching, invasion, colony formation, and growth in a murine xenograft model RCC. Thesealterations were phenocopied by treatment of the superoxide scavenger, TEMPOL, or by overexpression ofmanganese superoxide dismutase or catalase. Notably, NOX4 silencing or superoxide scavenging was sufficientto block nuclear accumulation of HIF2a in RCC cells. Our results offer direct evidence that NOX4 is critical forrenal tumorigenesis and they show how NOX4 suppression and VHL re-expression in VHL-deficient RCC cellsare genetically synonymous, supporting development of therapeutic regimens aimed at NOX4 blockade. CancerRes; 74(13); 3501–11. �2014 AACR.

IntroductionRenal cell carcinoma (RCC) is a common adult malignancy

with an estimated 65,150 new cases and 13,680 deaths in theUnited States in 2013 (1). Localized disease can be treated bysurgical resection alone, but advanced RCC is notoriouslyresistant to cytotoxic therapy or radiation. Immunotherapy,the mainstay of treatment for several decades, is curative infewer than 15% (2). Advances in the molecular genetics ofkidney cancer have led to FDA-approval of targeted agentswithgood clinical response rates (3). However, complete, durableresponses are rare, and novel therapeutic approaches are stilldesperately needed.More than 80% of clear cell RCCs have lost or mutated both

alleles of the von Hippel Lindau (VHL) tumor suppressor. VHL

is the binding subunit of an E3 ubiquitin ligase complex thattargets the a subunits of hypoxia-inducible transcriptionfactors 1 and 2 (HIFa) for ubiquitin-mediated, proteasomaldegradation. In the absence of VHL, HIFa accumulate in thecell, leading to increased transcription of more than 100 HIF-regulated genes involved in angiogenesis, anaerobic metabo-lism, proliferation, and other cell survival pathways. We andothers have shown that HIF2a is the relevant oncogenic targetof VHL degradation. Forced accumulation of HIF2a is suffi-cient to support xenograft growth of RCC cells despite rein-troduction of wild-type VHL, (4, 5) and specific HIF2a inhibi-tion suppresses tumor growth (6). In contrast, forced expres-sion of HIF1a suppresses xenograft growth, (4, 7), and specificHIF1a shRNA enhances xenograft growth (8). HIF1a andHIF2a have unique, nonoverlapping regulatory profiles sug-gesting a more proapoptotic rather than proproliferative rolefor the former (7, 9). In short, specific activation of HIF2aseems to be critical for renal tumorigenesis.

We previously reported that HIF2a expression and trans-activation are dependent upon expression of the NADPHoxidase 4 (Nox4; ref. 10). In the adult human, Nox4 is mosthighly expressed in the distal renal tubule where it generatesintracellular superoxide and is implicated in oxygen sensing forregulation of erythropoietin, a HIF-dependent gene (11). Incontrast to other Nox isoforms, Nox4 requires only p22phox forcoactivation (12). In renal cancer cells, Nox4 is a major sourceof intracellular reactive oxygen species (ROS; ref. 13). Wehypothesized that this heightened oxidative state might pro-mote HIF2a transactivation under normal oxygen conditions.

Authors' Affiliations: 1University of Pittsburgh, Department of Urology andUniversity of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania; 2Depart-ment of Surgery, University of Massachusetts, Worcester, Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

J.L. Gregg and R.M. Turner II contributed equally to this work.

Current address for Y. Zhan: Department of Biochemistry and MolecularPharmacology, University of Massachusetts, Massachusetts

Corresponding Author: Jodi K. Maranchie, University of Pittsburgh, 5200Centre Avenue, Suite 209, Pittsburgh, PA 15232. Phone: 412-623-3442;Fax: 412-605-3030; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-13-2979

�2014 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 3501




Consistent with this hypothesis, Nox4 silencing inhibits trans-activation of VEGF, Glut-1, and erythropoietin by greater than80% in 786-0 RCC cells. Furthermore, Nox4 siRNA suppressesHIF2a and VHL at the mRNA and protein levels (10). Nox4-dependent expression of HIF2a protein has been confirmed byothers (14, 15).

Thus, HIF2a is an established oncogene for clear cell kidneycancer, and Nox4 is critical for its expression and transactiva-tion in RCC. However, the contribution of Nox4 to renaltumorigenesis is not known. We report that in vitro branchingmorphogenesis and invasion are abrogated by Nox4 silencingand enhanced by Nox4 overexpression via generation of ROSand that in vivo RCC xenograft growth is suppressed by Nox4silencing. Further, we report that Nox4 regulates the intracel-lular distribution of HIF2a with abrogation of nuclear accu-mulation under both hypoxic and normal oxygen conditions.

Materials and MethodsCell lines and cultures

Established human conventional RCC lines, 786-0, RCC4,and Caki-1, were maintained in DMEM supplemented with10% FBS, penicillin, and streptomycin. 786-0 (WT) and 786-0(pRC), created by stable transfection of with wild-type VHL orempty pRC vector, respectively, were a gift from W. Kaelin,Dana Farber Cancer Institute, HarvardMedical School, Boston,MA (16). They were selected with G418 (500 mg/mL) everysixth passage. RCC4 was generously provided by M.C. Simon(Abramson Family Cancer Institute, University of Pennsylva-nia, Philadelphia, PA), and Caki1 cells were obtained fromATCC. Cell lines were routinely authenticated by DNA finger-printing at the start and twice annually for the duration ofthese studies by the core University of Pittsburgh CancerInstitute Cell Culture and Cytogenetics Facility.

Stable Nox4 knockdown was achieved for each cell line byexpressing two Nox4 shRNAs or a nontargeting shRNA inpSilencer 4.1-CMV puro (Ambion) as previously described(10). Stable transfectants were maintained in puromycin (1mg/mL). RT-PCR for Nox1–5, p22phox, p47phox, and p67phoxwas performed as described (17). Adenoviral vectors Ad-EGFP,Ad-MnSOD, and Ad-catalase were a generous gift of Dr. YongLee, Department of Surgery, University of Pittsburgh, Pitts-burgh, PA (18).

Adenoviral transduction was performed as previouslydescribed (19). Briefly, cells were infected at 100 or 200 mul-tiplicity of infection for 1.5 hours in DMEM. Assays wereperformed 48 hours after transduction. To overexpress Nox4,parental 786-0 cells were transfected with a pcDNA vectorexpressing the complete human Nox4 cDNA, and antibioticselection of stable clones was performed. Cells were pretreatedfor 4 hours with indicated concentrations of DL-Dithiothreitol(DTT; Promega) or 4-hydroxy-TEMPOL (Sigma-Aldrich) beforefixation or live cell assay. Drug was maintained in the mediathroughout live cell assays.

Quantitative RT-PCRTotal RNAwas extracted from 786-O, RCC4, and LNCap cells

with TRIzol reagent and RNeasyMini Kit (Qiagen). First strandcDNA was synthesized using the iScript cDNA synthesis Kit

(BIO-RAD). Gene-specific TaqMan Gene Expression Assaysprimer sets and Master Mix were used for quantitative PCRof NOX4 (Hs00418356), NOX1 (Hs00246589), and GAPDH(Hs99999905). Samples were then subjected to real-time PCRanalysis using the ABI StepOnePlus real-Time PCR System(Applied Biosystems). Relative mRNA expression of each tran-script was normalized against GAPDH.

Western blotProtein was extracted as previously described (4). Equal

amounts of protein were subjected to separation in a 4.5% to15% Tris-HCl gel, and the resolved proteins were transferred topolyvinylidene difluoride membrane. The blots were probedwith anti-Nox4 rabbit monoclonal Ab (1:2,000; Abcam) orb-actin Ab (1:1,000; Santa Cruz Biotechnology), followed byhorseradish peroxidase–conjugated secondary Ab. Bands werevisualized using SuperSignalWest FemtoMaximumSensitivitySubstrate (Thermo Scientific).

Measurement of NAD(P)H oxidase activitySuperoxide production from membrane fraction was deter-

mined by lucigenin chemiluminescence as described (20).Briefly, 20-mg protein was added to 200 mL of 50 mmol/Lphosphate buffer with 1 mmol/L ethylene glycol tetra aceticacid, 150 mmol/L sucrose, and 100 mmol/L NADPH. Lucigeninwas added, and chemiluminescence read every 30 seconds for20 minutes (SpectraMax Plus 384) and expressed as relativelight units (RLU)/mg protein. Alternatively, ROSs were mea-sured using 2070-dichlorofluorescin diacetate (Sigma-Aldrich)as described (21). Fluorescence at 530 nm was measured usinga Wallac Victor 1420 multilabel counter (Wallac Oy).

Branching and invasion assaysBranching morphogenesis assays were performed as pre-

viously described (22). Briefly, 60-mL cells at 3.5 � 105 cells/mL media were mixed 1:1 with Matrigel in six flat-bottomed96-well plates. After 30 minutes at 37�C, 120-mL DMEM with10% FBS and 40 ng recombinant hepatocyte growth factor(HGF; Sigma-Aldrich) was added, and cells were incubatedan additional 72 hours before determining the percentage ofbranched cells per well. Invasion assays were done in 8-mMatrigel 24-well invasion chambers (BD). In the top cham-bers, 1.5 � 104 cells were plated in 200-mL media in fivereplicate wells. The bottom wells contained 750 mL mediawith 20 ng/mL HGF. After 24 hours at 37�C, Matrigel andcells were removed from the top of the filter with a cottonswab, and the filter bottom was fixed and stained with Diff-Quick (IMEB Inc.). Stained cells were photographed at �100magnification and counted. Significance was determined bythe Student t test.

Immunofluorescence and confocal microscopyA total of 5� 104 cells were plated on cover-glass in 24-well

plates for 12 hours. They were then incubated 4 hours withindicatedmedia at 21%O2 or 1%O2. Cells were then fixed in 4%paraformaldehyde for 15 minutes, washed three times withPBS, then permeabilized with 0.1% Triton X-100 in PBS solu-tion for 15 minutes. Cover slides were blocked with 2% BSA for

Gregg et al.

Cancer Res; 74(13) July 1, 2014 Cancer Research3502




45 minutes and stained by primary antibodies for 60 minutes.After five washes with 0.5% BSA, secondary antibody wasapplied for 60 minutes, slides were counterstained with DAPIfor 30 seconds and mounted with gelvatol. Images were takenwith a FluoroView 1000 II confocal microscope.

Soft agar colony formationCells were grown in 0.3% bacto-agar (BD) on a cushion of

0.6% agar, in triplicate. Fresh 0.3% agar was applied weekly.After 30 days, colonies were photographed and counted at�40magnification.

Mouse xenograft assayOne million viable cells as determined by trypan blue

exclusion were suspended in 100-mL Hank's buffered salineand injected subcutaneously per flank of four 6-week femaleSCID beige mice (Charles River Laboratories). Tumors weremeasured twice weekly with digital calipers (VWR) in the twolargest dimensions by a technician blinded to the genotype.Mice were euthanized at 14 weeks and tumors harvested.

ImmunohistochemistryParaffin slides were deparaffinized and hydrated to deionized

water before heat-induced epitope retrieval by Diva antigenretrieval buffer (Biocare Medical). Endogenous peroxidase wasquenched with 3% hydrogen peroxide for 10 minutes followedbyTBSbuffer for 5minutes. In anDakoAutostainer Plus Stainer,slides were blocked 10minutes with CAS block (Invitrogen) andincubated 60 minutes with anti-HIF2a mouse monoclonal Ab1:500 (Novus Biologicals) or anti-Nox4 rabbit polyclonal Ab1:100 (Abcam). Slides were then rinsed twice with TBS bufferand incubated with Dako Envision Dual Link HRP (Dako NorthAmerica) for 30 minutes followed by Dakoþ Chromagen for 10minutes. After several rinses with deionized water, slides werecounterstained with Harris Hematoxylin for 10 seconds, rinsedwith tap water, dehydrated, cleared, and coverslipped. Allincubations were performed at room temperature.

Statistical analysisData were expressed as the mean � SE for at least three

independent experiments from separate harvests. Statisticalanalysis was performed using the Student t test. P values of<0.05 versus control group were considered significant.

ResultsNox4 shRNA selectively suppressed Nox4 mRNA andprotein expression and abrogated NADPH-dependentsuperoxide generationWe first assessed endogenous expression of Nox 1–5 and

coactivators in three human clear cell RCC cell lines, RCC4,786-0, and Caki1. RCC4 and 786-0 lack functional pVHL dueto a mutation in the VHL gene, whereas Caki1 cells expresswild-type VHL. By semiquantitative RT-PCR, all three linesabundantly expressed Nox4 and the cofactor p22phox (Fig.1). Nox2 was detectable in RCC4 and Caki1 cells but Nox3was seen only in Caki1 cells. In contrast to a prior report (14),we did not detect Nox1 in any of our cell lines (Supplemen-tary Fig. S1).

Silencing of Nox4mRNA and protein by specific shRNA (KD)relative to scramble control shRNA (NS) was confirmed byquantitative RT-PCR and Western blot, respectively. AlthoughNox4 expression was decreased by greater than 70%, Nox2 wasnot affected by silencing (Fig. 2A–C). A corresponding loss ofNADPH-dependent superoxide generation from the cell mem-brane fractionwasmeasured by lucigenin assay followingNox4silencing (Fig. 2D), consistent with its role as the major sourceof superoxide in these cells. Similar superoxide suppressionwas demonstrated by lucigenin in RCC4 and Caki-1 followingNox4 knockdown. Membrane fraction from both 786-0 andRCC4 demonstrated the highest superoxide generation (Sup-plementary Fig. S3). In summary, Nox4 was the dominantNADPH oxidase in three RCC cell lines. Nox4 shRNA silencingeffectively and selectively silenced Nox4 expression, leading toabrogation of NADPH-dependent superoxide generation.

Nox4 silencing inhibited branching morphogenesis andinvasion by VHL-deficient cells

We have reported that Nox4 is critical for expression andtransactivation of HIF2a in VHL-deficient RCC cells, suggest-ing that Nox4 silencing might phenocopy VHL reintroductionin these cells. To explore the impact of Nox4 expression onrenal tumorigenesis, we first assayed two established pheno-types of VHL-deficient renal cells: branching morphogenesisand invasion across a basement membrane. RCC cells dem-onstrate exuberant branching following exposure to HGF andwill migrate through a Matrigel-coated filter membranetoward an hepatocyte growth factor/scatter factor gradient.These behaviors are believed to reflect invasive andmetastaticpotential and are completely abrogated by reintroduction of awild-type copy of the VHL tumor suppressor (22). To test ourhypothesis thatNox4 expression is required to support branch-ing and invasion, we assayed VHL-deficient 786-0 and RCC4

Figure 1. Semiquantitative RT-PCR for NADPH oxidase family membersand cofactors was performed in three human clear cell renal cancercell lines, 786-0, RCC4, and Caki-1. The anticipated band sizes forNox1–5 are 247, 413, 458, 286, and 630 bp, respectively, and cofactorsp22phox, p47phox, and p67phox are 320, 771, and 760 bp, respectively.b-Actin served as an internal control.

NADPH Oxidase 4 Promotes Renal Tumorigenesiss

www.aacrjournals.org Cancer Res; 74(13) July 1, 2014 3503




cells expressing KD or NS. Nox4 knockdown decreased thefraction of branching cells by 83% (P < 0.001) and 93% (P < 0.01)relative to NS cells in 786-0 and RCC4 cells, respectively (Fig.3A–C), consistent with a critical requirement for Nox4 expres-sion. Similarly, Nox4 silencing decreased the number of inva-sive cells by 70% (P < 0.001) and 82% (P < 0.05) relative to NS in786-0 and RCC4 cells, respectively (Fig. 3D–F). Again, Nox4silencing recapitulated the wild-type VHL phenotype.

To determine if overexpression of exogenous Nox4 couldconversely enhance branching and invasion, we transfectedparental 786-0 cells with a pcDNA vector expressing thecomplete human Nox4 cDNA. Following selection, cells dem-onstrated altered morphology with smaller, rounded cells, butsimilar growth kinetics (Supplementary Fig. S2A and S2B).Although these changes may reflect effects of oxidative sig-naling on multiple pathways, they may also be attributed toincreased oxidative stress. Quantitative RT-PCR confirmed amarked increase in Nox4 mRNA expression (data not shown),which was confirmed at the protein level by Western blot (Fig.3G). Invasive cells increased nearly 2-fold (P < 0.05) followingexpression of pcDNA-Nox4 relative to empty vector (Fig. 3H–J).We did not observe increased branching in the Nox4-over-expressed cells, likely due to the overall morphologic changesnoted above. Taken together, these results indicate a regula-tory role for Nox4 on the invasive phenotype of RCC.

Superoxide scavenging with TEMPOL treatment orexpression of adenoviral-SOD recapitulated the effect ofNox4 silencing on branching and invasion

To determine if the impact of Nox4 on RCC behavior ismediated by generation of superoxide, we used two parallelstrategies. TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl) is a heterocyclic chemical oxidant that reacts withintracellular superoxide anion to form hydrogen peroxide.Treatment with TEMPOL has been shown to increase detect-able hydrogen peroxide (23) and thus, although it decreasesintracellular superoxide, it does not necessarily decrease theoverall oxidative status of the cell. TEMPOLexposure (0.125–10mmol/L) did not impact 786-0 or RCC4 cell viability by CellTitre Blue assay. When we treated parental 786-0 and RCC4cells, we observed suppression of branching comparable withNox4 silencing (P < 0.01; Fig. 4A). Similarly, treatmentwithTEMPOLdecreased invasion in a dose-dependentmanner(P < 0.01; Fig. 4B).

Although the major biologic functions of TEMPOL havebeen attributed to its ability to scavenge superoxide anion, it isnot a specific SODmimetic. It also has catalase-like activity andcan inhibit hydroxide generation via the Fenton reaction,making it a general purpose redox cycling agent. To reducethese off-target effects, we also examined branching andinvasion after transducing RCC4 and 786-0 cells with

Figure 2. A, semiquantitative RT-PCR for Nox isoforms 2–4 in 786-0,RCC4, andCaki-1 cells transfectedwith Nox4-specific siRNA (KD) ornontargeting siRNA (NS). b-Actinserved as an internal control. B,representative quantitative RT-PCR for Nox4 mRNA relative tob-actin in RCC4 cells expressingNox4 shRNA2 (KD) or scramble(NS). Columns, mean; bars, �SE;�, P < 0.01 relative to NS. C,Western blot analysis for Nox4protein in three RCC cell lines.786-0 indicates parental 786-0cells (ATCC) in contrast to the786-0 pRC (16) cells used forNS/KD. The upper band runsat the predicted 67 kD for full-length Nox4. D, lucigeninchemiluminescent assay forNADP(H)-dependent superoxidegeneration from isolated cellmembranes of 786-0 NS and KDcells. Luminescence followingaddition of lucinigen is measuredevery 20 seconds for 20 minutesand expressed as RLU.

Gregg et al.





adenoviral vectors expressing the ROS scavengers, manganesesuperoxide dismutase (Ad-SOD), or catalase (Ad-catalase).Nox4-generated superoxide cannot cross cell membranes, butis rapidly dismutated by SOD to hydrogen peroxide, whichfreely diffuses through the cell (24). Hydrogen peroxide is inturn reduced by catalase to water and oxygen. Ad-SOD alonemarkedly suppressed branching by 77% (P < 0.01) and 96% (P <0.01) relative to Ad-GFP in 786-0 and RCC4 cells, respectively.Comparable suppression was seen with cotransduction of Ad-catalase, or with Ad-catalase alone (Fig. 4C). Invasion assays(Fig. 4D) again revealed suppression of invasion by Ad-SODrelative to Ad-GFP in both 786-0 and RCC4 cells (P < 0.01 for

both). Cotransduction with Ad-catalase further decreasedinvasion in both cell lines (P < 0.01).

By 20,70-dichlorofluorescein (DCF) assay, Ad-SOD expressionalone had minimal impact on detectable ROS relative to Ad-GFP–transduced controls (Fig. 4E). This likely reflects the factthat DCF predominantly measures hydrogen peroxide, which isincreased by the antioxidant activity of manganese superoxidedismutase (MnSOD). Thus, DCF will underestimate the impactof specific superoxide suppression. In contrast, catalase is speci-fic for scavenging hydrogen peroxide and it is notable thattransduction with Ad-catalase alone or in combination leads toROS levels comparable with those observed with Nox4-silenced

Figure 3. A–C, branching assays.VHL-deficient cells (786-0 orRCC4) expressing control vector(NS; A) or Nox4 shRNA (KD; B),cultured 72 hours in Matrigel withHGF (0.33 mg/mL). Bar graph (C)depicts the mean percentage ofbranched cells. D–F, invasionassays. VHL-deficient cellsexpressing control vector (NS;D) orNox4-specific shRNA (KD; E) thathave crossed aMatrigel-coated, 8-m pore filter toward an HGFgradient (magnification, �100).Small circles, pores. Bar graph (F)shows mean crossing cells. G–J,exogenous Nox4 overexpression:Western blot for Nox4 expressionin lysates of parental 786-0stably transfected with pcDNA-Nox4 or empty vector. b-Actinserved as an internal loadingcontrol. Invasion assays wereperformed as described aboveusing pcDNA empty vector (H) orpcDNA-Nox4–transfected (I) cells.J, bar graph shows percentage ofbranching cells. Columns, mean;bars,� SE; �, P < 0.05; ��, P < 0.01.






KD cells, again demonstrating that Nox4 is a major source ofROS in these cells (Fig. 4E). Suppression of the superoxide burstfollowing exposure to TEMPOL was confirmed using a luci-genin chemiluminescent assay for NADP(H)-dependent super-oxide generation from isolated cell membranes (Fig. 4F).

To summarize, scavenging of intracellular superoxide byeither TEMPOL or expression of exogenous superoxide dis-mutase mimics Nox4 silencing with respect to suppression ofbranching and invasion of RCC cells. Further suppression bycatalase suggests that hydrogen peroxide may be an equally

Figure 4. Effects of ROS scavengers and DTT on RCC cell branching and invasion. A and B, branching assays were performed as in Fig. 3. Cells were treatedwith the indicated concentrations of TEMPOL or DTT (A) or transduced with adenoviral vectors expressing GFP (control), SOD, or catalase before Matrigelculture (B). C and D, invasion assays demonstrate the number of cells crossing a transwell membrane in the presence of TEMPOL or DTT (C), or aftertransduction of Ad-GFP, Ad-SOD, or Ad-catalase (D). Dichlorofluorescein assay (E) measures ROS produced by parental 786-0, 786-0 scramble (NS)or 786-0 expressing Nox4 shRNA (KD) cells transduced with adenoviral vectors expressing GFP (control), SOD, catalase, or both SOD and catalase. F,lucigenin chemiluminescent assay for superoxide on isolated cell membrane fraction of parental 786-0 cells with or without addition of TEMPOL. Columns,mean; bars, � SE; �, P < 0.02 relative to untreated or GFP controls.

Gregg et al.





important mediator of Nox4 signaling for branching andinvasion.

DTT induction of superoxide promoted cell branchingDTT is a strong antioxidant. However, under physiologic

conditions, the thiol-mediated antioxidant reaction results inintracellular generation of superoxide (25).We treated our cellswithDTT to determine if further induction of superoxide couldenhance branching and invasion relative to parental 786-0 andRCC cells. DTT treatment proved to be quite toxic, resulting inhigh cell death (Supplementary Fig. S4). The number of inva-sive cells was decreased in a dose-dependent fashion, likely dueto overall cell attrition. Despite this, we observed a nearly 2-foldincrease in the percentage of branching cells in both 786-0 andRCC4 cells (P ¼ 0.03 and P ¼ 0.017, respectively; Fig. 4A),consistent with our hypothesis that superoxide mediatesbranching morphogenesis.

Nox4 silencing blocked nuclear accumulation of HIF2aWehave reported that expression ofHIF2a at themRNAand

protein level is suppressed by Nox4 silencing (10). Others haveconfirmed that HIF2a protein expression is dependent upon

Nox4 expression (14, 15). However, we have observed suppres-sion inHIF2a transactivation even at highHIF2a protein levelswith Nox4 silencing. To test our hypothesis that Nox4 furthercontributes to activation of HIF2a, we used confocal micros-copy to examine HIF2a cellular localization in our Nox4-silenced 786-0 and RCC4 cells. Activation is amultistep processthat requires binding to the aryl hydrocarbon receptor nucleartranslocator, nuclear translocation, binding to CBP/p300 andother cofactors, and binding to promoter DNA. Under hypoxicconditions, this requires factor-inhibiting HIF (FIH)-mediatedhydroxylation of asparagine residues. As HIF2a has beenshown to be less dependent upon FIH than HIF1a for activa-tion (26), we hypothesize that Nox4 provides an alternateactivating signal for HIF2a.

Figure 5A shows representative confocal images of HIF2alocalization. We observed a Nox4-dependent distribution ofendogenous HIF2a protein. 786-0 and RCC4 NS cells showeddiffuse staining throughout the cytoplasm and nucleus undernormal oxygen conditions. As expected, culture at 1% oxygenresulted in nuclear concentration of HIF2a. However, Nox4 KDcells showed a striking nuclear exclusion of HIF2a withgranular perinuclear enhancement. Notably, this nuclear

Figure 5. Cellular distribution ofHIF2a. A, representative confocalmicroscopy images (magnification,�400) showing HIF2aimmunofluorescence, DAPInuclear staining, or mergedimages.Culturesweregrown in 1%oxygen for 8 hours before fixationand staining. Control (NS) or Nox4shRNA (KD) cells were imagedfollowing exposure to TEMPOL(0.25 mmol/L), DTT (1 mmol/L), ortransduction with Ad-SOD. B andC, Western blots for HIF2a proteinexpression in isolated nuclearfractions of 786-0 NS cells grownunder normal oxygen conditionswith serial dilutions of TEMPOL (B)or 786-0 KD cell grown undernormoxic conditions with serialdilutions of DTT (C). TATA bindingprotein (TBP) served as an internalcontrol for total loaded protein.






exclusion was seen under both normal oxygen (SupplementaryFig. S5A–S5D) and hypoxic conditions, suggesting that forHIF2a, there may be a superoxide requirement even forhypoxic activation. Consistent with superoxide as a signalingintermediary, pretreatment with TEMPOL or transductionwith Ad-SOD in NS cells mimicked the HIF2a distributionobserved with Nox4 silencing. Conversely, superoxide induc-tion with DTT rescued nuclear accumulation despite Nox4silencing. Western blot analysis of isolated nuclear fractionsconfirmed a dose-dependent decrease in nuclear HIF2aexpression following TEMPOL treatment, whereas nuclearHIF2a expression increased with DTT treatment (Fig. 5B andC). These findings support a role for Nox4-generated super-oxide in nuclear accumulation of HIF2a.

Nox4 silencing inhibits colony formation and xenografttumor growth

We next sought to determine if Nox4 silencing would besufficient to prevent tumor growth. Neoplastic cells exhibitanchorage-independent growth and can proliferate in theabsence of exogenous growth factors. This classic behavior,measured by the ability to form colonies in soft agar, wasdecreased by 94% (P ¼ 0.008) in 786-0 KD cells relative to NS(Fig. 6A). To determine if Nox4 expression is similarlyrequired to support in vivo tumor growth, we establishedxenografts of our 786-0 KD and control cells in SCID beigemice. 786-0 cells form tumors in immunocompromised micethat are abrogated by reintroduction of wild-type VHL (16).Attempts to establish subcutaneous xenografts with RCC4were unsuccessful. Tumor growth curves are presentedin Fig. 6B. Mice injected with VHL-deficient 786-0 cellsexpressing control shRNA (NS) developed palpable tumorsby week 5, whereas Nox4 knockdown (KD) tumors were notdetected before week 9. Further, KD tumors were signifi-cantly smaller at 14 weeks than NS (mean cross sectionalarea, 35 mm2 vs. 105 mm2; P < 0.01). Metastases to the brain,lungs, or abdominal organs were not observed in any mice.Interestingly, despite clear inhibition of tumor growth, KDtumors demonstrated similar growth kinetics to NS onceestablished. We postulate that the delayed tumors representselection and enrichment of a subpopulation of cells thathave escaped silencing—an effect that would lead to under-estimation of tumor inhibition. Consistent with this, immu-nohistochemical evaluation of the 14-week tumor explantsrevealed that protein expression of Nox4 and HIF2a was ashigh in the KD tumors as control (Supplementary Fig. S6).Regardless, this intermediate phenotype is consistent withour hypothesis that Nox4 silencing suppresses RCC tumorgrowth, and suggests that therapies designed to target Nox4or intracellular superoxide may have efficacy in RCC.

DiscussionLoss of the VHL tumor suppressor, resulting in abundant

HIFa protein and increased expression of HIF transcriptiontargets, occurs commonly in sporadic clear cell RCC. Wepreviously reported that expression and transactivation ofHIF2a in VHL-deficient RCC cells is critically dependent

upon expression of Nox4 (10). In the present study, we showthat Nox4 silencing inhibits morphogenesis, invasive poten-tial, colony formation, and xenograft tumor growth of VHL-deficient human RCC cells. Superoxide scavenging by treat-ment with TEMPOL or overexpression of superoxide dis-mutase or catalase mimicked the effects of Nox4 silencing,indicating a role for generation of superoxide and hydrogenperoxide in the tumorigenic RCC phenotype. Furthermore,Nox4 silencing abrogated nuclear accumulation of HIF2aunder both normal and hypoxic oxygen conditions demon-strating that Nox4 is an alternative activating signal forHIF2a translocation. To our knowledge, these data providethe first evidence that renal Nox4 expression is critical tosupport the renal tumorigenic phenotype, suggesting that itmay function as a renal oncogene.

HIF1a and HIF2a are both subject to VHL-mediatedoxygen-dependent degradation and recognize the same DNAresponse element. However, they have differential effects ongene expression such that a shift in balance toward HIF2apredominance promotes cell survival, whereas HIF1a pre-dominance favors apoptosis (27). In kidney cancer, HIFaexpression is biased toward HIF2a, and inhibition of HIF2asuppresses VHL �/� tumor growth (6, 28). We speculatethat heightened HIF2a transcriptional activity is due in part

Figure 6. Nox4 expression is required for RCC cell colony formation andxenograft tumor growth. A, soft agar colony formation in 786-0 cellswith Nox4 shRNA silencing (KD) or nonspecific control shRNA (NS).Colonies larger than 20 mmwere counted. B, mean cross-sectional areaof tumor xenografts from four SCID beige mice per cohort injectedsubcutaneouslywith 786-0NSor KDcellswith orwithout reexpression ofVHL. Tumor take rates were 100% for the 786-0 NS cohort and 75% forthe KD cohort. Columns, mean; bars, � SE; �, P < 0.008.

Gregg et al.





to constitutive, isoform-specific induction by highlyexpressed renal Nox4. In renal cells, HIF2a activity is heldin check only by VHL-mediated protein degradation, leadingto catastrophic proliferation and tumor formation followingthe loss of VHL. Consistent with this hypothesis, Nox4silencing in our VHL-deficient RCC cells phenocopied reex-pression of wild-type VHL.Our results are consistent with other reports indicating

that Nox4 modulates cellular phenotypic changes, includingmigration, invasion, and the epithelia-to-mesenchymal tran-sition. A recent report by Boudreau and colleagues foundthat inhibition of Nox4, either by shRNA knockdown orexpression of a dominant-negative form, abrogated woundhealing and cell migration in breast cancer epithelia (29).Similar to the results from this study, Yamaura and collea-gues demonstrated that inhibition of Nox4 by siRNAs inmelanoma cells decreased anchorage-independent growthand xenograft tumor growth in vivo (30). In keratinocytes,cell migration was inhibited by both diphenyliodonium, anonspecific flavoprotein inhibitor of Nox enzymes, and Nox4silencing (31). Nox4 has been implicated in the regulationof cell migration in nontumorigenic cell types as wellincluding myofibroblasts, endothelia, and vascular smoothmuscle cells (32–34).Although several sources of intracellular ROS exist, we and

others have shown that Nox4 is a major producer of ROS inrenal tumors (13). Importantly, there is growing evidence tosuggest that ROS, in conjunction with Nox expression, caninduce the tumorigenic phenotype via heightened oxidativestress. For example, ROS, as well as Nox4, is required forinvadopodia formation, an important step in invasion andmetastasis that acts as a catalyst for extracellular matrixdegradation (35, 36). Furthermore, ROSs have been shown toregulate the production of matrix metalloproteinases, criticalproteolytic enzymes of tumorigenic invasion, in several tumortypes including tumors of the pancreas and breast (37, 38) andglioblastomas (39).ROS-induced stabilization and induction of HIFa has been

described in a number of systems. Exogenous hydrogenperoxide stabilizes HIF1 a protein in normoxia (40). Expres-sion of exogenous MnSOD in MCF-7 cells suppresses hypoxicaccumulation of HIF1 a protein (41). MnSOD siRNA in thesecells increases both detectable superoxide and HIF1 aaccumulation, whereas TEMPOL decreased accumulation,implicating superoxide as the molecular effector (42). Quer-certin, a potent flavonoid antioxidant, induces normoxicaccumulation of HIF1 a that is reversed by iron chelation,leading investigators to conclude that it inhibited HIFaubiquitination by inactivating the HIF prolyl hydroxylase(PHD) and preventing binding of pVHL (43). Redox-medi-ated inactivation of PHD was also described in pulmonaryartery smooth muscle cells where overexpression or induc-tion of Nox4 stabilized HIF2 a in the setting of decreasedHIFa hydroxylation and pVHL binding (15).The mechanism of Nox4 regulation of HIF2a in VHL-

deficient RCC cells, however, remains unclear. Block andcolleagues describe increased expression p22(phox), lead-ing to inactivation of tuberin and downstream ribosomal

activation consistent with a translational pathway toincreased HIF2 a accumulation (44). Our finding of alterednuclear expression of HIF2a with Nox4 silencing or treat-ment with superoxide scavengers suggests an additionalrole for redox signaling on nuclear translocation or DNAbinding. We speculate that redox signaling may alter theHIF2a transcriptional complex and subsequent transacti-vation of target genes. Our study is limited by dependenceupon established RCC cell lines and by potential off-targeteffects of shRNA. However, suppression of the cancerphenotype was reproducible in two VHL-deficient lineswith independent shRNA sequences supporting our con-clusion that experimental effects were due to specific Nox4knockdown. Although we demonstrate that Nox4 silencingand TEMPOL treatment lead to specific superoxide sup-pression, our results cannot exclude the possibility thatNox4 signaling is also mediated by direct generation ofhydrogen peroxide.

In summary, we show that Nox4 plays a crucial permissiverole in the tumorigenic phenotype of VHL-deficient, humanRCC cells and that specific Nox4 suppression inhibits intra-cellular superoxide generation, prevents nuclear accumulationof HIF2a, and phenocopies reexpression of wild-type VHL.Furthermore, Nox4 silencing is mimicked by superoxide sca-vengers, TEMPOL or MnSOD, or by catalase, implicatingsuperoxide anion and hydrogen peroxide as mediators of Nox4regulation of HIF2a. Taken together, these support an onco-genic role for Nox4 in conventional RCC and suggest thatagents designed to target Nox4 might have clinical efficacyagainst kidney cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G. Chang, J.K. MaranchieDevelopment of methodology: G. Chang, D. Joshi, L. Chen, J.K. MaranchieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R.M. Turner II, G. Chang, D. Joshi, L. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J.L. Gregg, R.M. Turner II, G. Chang, D. Joshi, L. Chen,J.K. MaranchieWriting, review, and/or revision of the manuscript: J.L. Gregg, R.M. TurnerII, J.K. MaranchieAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J.L. Gregg, D. Joshi, L. ChenStudy supervision: J.K. Maranchie

AcknowledgmentsThe authors thank Dr. Simon Watkins for his assistance with confocal

microscopy imaging and Dr. Julie Eisemann for her invaluable assistance withmouse experiments and interpretation of the data.

Grant SupportThis work was supported by NIH grant DK064887 and American Cancer

Society grant 116627-RSG-09-023-01-CNE (J.K. Maranchie).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received October 18, 2013; revised April 15, 2014; accepted April 15, 2014;published OnlineFirst April 22, 2014.






References1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer

J Clin 2013;63:11–30.2. McDermott DF. Immunotherapy of metastatic renal cell carcinoma.

Cancer 2009;115:2298–305.3. Singer EA, Gupta GN, Srinivasan R. Update on targeted therapies for

clear cell renal cell carcinoma. Current opinion in oncology 2011;23:283–9.

4. Maranchie JK, Vasselli JR, Riss J, Bonifacino JS, Linehan WM, Klaus-ner RD. The contribution of VHL substrate binding and HIF1-alpha tothe phenotype of VHL loss in renal cell carcinoma. Cancer Cell2002;1:247–55.

5. KondoK,Klco J,Nakamura E, LechpammerM,KaelinWGJr. Inhibitionof HIF is necessary for tumor suppression by the von Hippel-Lindauprotein. Cancer Cell 2002;1:237–46.

6. Kondo K, Kim WY, Lechpammer M, Kaelin WG Jr. Inhibition ofHIF2alpha is sufficient to suppress pVHL-defective tumor growth.PLoS Biol 2003;1:E83.

7. Raval RR, Lau KW, Tran MG, Sowter HM, Mandriota SJ, Li JL, et al.Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2in von Hippel-Lindau-associated renal cell carcinoma. Mol Cell Biol2005;25:5675–86.

8. ShenC, BeroukhimR, Schumacher SE, Zhou J, ChangM, Signoretti S,et al. Genetic and functional studies implicate HIF1alpha as a 14qkidney cancer suppressor gene. Cancer discovery 2011;1:222–35.

9. HuCJ,Wang LY, Chodosh LA, Keith B, SimonMC. Differential roles ofhypoxia-inducible factor 1alpha (HIF-1alpha) and HIF-2alpha in hyp-oxic gene regulation. Mol Cell Biol 2003;23:9361–74.

10. Maranchie JK, Zhan Y. Nox4 is critical for hypoxia-inducible factor 2-alpha transcriptional activity in von Hippel-Lindau-deficient renal cellcarcinoma. Cancer Res 2005;65:9190–3.

11. GeisztM,KoppJB, Varnai P, Leto TL. Identification of renox, anNAD(P)H oxidase in kidney. Proc Natl Acad Sci U S A 2000;97:8010–4.

12. Martyn KD, Frederick LM, von Loehneysen K, Dinauer MC, Knaus UG.Functional analysis of Nox4 reveals unique characteristics comparedto other NADPH oxidases. Cell Signal 2006;18:69–82.

13. Fitzgerald JP, Nayak B, Shanmugasundaram K, Friedrichs W, Sudar-shan S, Eid AA, et al. Nox4 mediates renal cell carcinoma cell invasionthrough hypoxia-induced interleukin 6- and 8- production. PloS one2012;7:e30712.

14. Block K, Gorin Y, Hoover P, Williams P, Chelmicki T, Clark RA, et al.NAD(P)H oxidases regulate HIF-2alpha protein expression. J BiolChem 2007;282:8019–26.

15. Diebold I, Flugel D, Becht S, Belaiba RS, Bonello S, Hess J, et al. Thehypoxia-inducible factor-2alpha is stabilized by oxidative stressinvolving NOX4. Antioxid Redox Signal 2010;13:425–36.

16. IliopoulosO, Kibel A, Gray S, KaelinWGJr. Tumour suppression by thehuman von Hippel-Lindau gene product. Nat Med 1995;1:822–6.

17. Vaquero EC, Edderkaoui M, Pandol SJ, Gukovsky I, GukovskayaAS. Reactive oxygen species produced by NAD(P)H oxidase inhibitapoptosis in pancreatic cancer cells. J Biol Chem 2004;279:34643–54.

18. Song JJ, Lee YJ. Catalase, but not MnSOD, inhibits glucose depri-vation-activated ASK1-MEK-MAPK signal transduction pathway andprevents relocalization of Daxx: hydrogen peroxide as a major secondmessenger of metabolic oxidative stress. J Cell Biochem 2003;90:304–14.

19. Steiner MS, Zhang X, Wang Y, Lu Y. Growth inhibition of prostatecancer by an adenovirus expressing a novel tumor suppressor gene,pHyde. Cancer Res 2000;60:4419–25.

20. Mohazzab KM, Wolin MS. Sites of superoxide anion productiondetected by lucigenin in calf pulmonary artery smooth muscle. Am JPhysiol 1994;267:L815–22.

21. Goyal P, Weissmann N, Grimminger F, Hegel C, Bader L, Rose F, et al.Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species. Free RadicBiol Med 2004;36:1279–88.

22. KoochekpourS, JeffersM,WangPH,GongC, TaylorGA,Roessler LM,et al. The von Hippel-Lindau tumor suppressor gene inhibits hepato-cyte growth factor/scatter factor-induced invasion and branching

morphogenesis in renal carcinoma cells. Mol Cell Biol 1999;19:5902–12.

23. Chen Y, Pearlman A, Luo Z,Wilcox CS. Hydrogen peroxidemediates atransient vasorelaxation with tempol during oxidative stress. Am JPhysiol Heart Circ Physiol 2007;293:H2085–92.

24. Brown DI, Griendling KK. Nox proteins in signal transduction. FreeRadic Biol Med 2009;47:1239–53.

25. Clement MV, Sivarajah S, Pervaiz S. Production of intracellular super-oxide mediates dithiothreitol-dependent inhibition of apoptotic celldeath. Antioxid Redox Signal 2005;7:456–64.

26. Bracken CP, Fedele AO, Linke S, Balrak W, Lisy K, Whitelaw ML, et al.Cell-specific regulation of hypoxia-inducible factor (HIF)-1alpha andHIF-2alpha stabilization and transactivation in a graded oxygen envi-ronment. J Biol Chem 2006;281:22575–85.

27. Loboda A, Jozkowicz A, Dulak J. HIF-1 versus HIF-2–is one moreimportant than the other? Vascul Pharmacol 2012;56:245–51.

28. Turner KJ,Moore JW, JonesA, TaylorCF,Cuthbert-HeavensD,HanC,et al. Expression of hypoxia-inducible factors in human renal cancer:relationship to angiogenesis and to the von Hippel-Lindau genemutation. Cancer Res 2002;62:2957–61.

29. Boudreau HE, Casterline BW, Rada B, Korzeniowska A, Leto TL. Nox4involvement in TGF-beta and SMAD3-driven induction of the epithe-lial-to-mesenchymal transition and migration of breast epithelial cells.Free Radic Biol Med 2012;53:1489–99.

30. Yamaura M, Mitsushita J, Furuta S, Kiniwa Y, Ashida A, Goto Y, et al.NADPH oxidase 4 contributes to transformation phenotype of mela-noma cells by regulating G2-M cell cycle progression. Cancer Res2009;69:2647–54.

31. Nam HJ, Park YY, Yoon G, Cho H, Lee JH. Co-treatment with hepa-tocyte growth factor and TGF-beta1 enhances migration of HaCaTcells through NADPH oxidase-dependent ROS generation. Exp MolMed 2010;42:270–9.

32. Haurani MJ, Cifuentes ME, Shepard AD, Pagano PJ. Nox4 oxidaseoverexpression specifically decreases endogenous Nox4 mRNA andinhibits angiotensin II-induced adventitial myofibroblast migration.Hypertension 2008;52:143–9.

33. Pendyala S, Gorshkova IA, Usatyuk PV, He D, Pennathur A, LambethJD, et al. Role of Nox4 andNox2 in hyperoxia-induced reactive oxygenspecies generation and migration of human lung endothelial cells.Antioxid Redox Signal 2009;11:747–64.

34. Meng D, Lv DD, Fang J. Insulin-like growth factor-I induces reactiveoxygen species production and cell migration through Nox4 andRac1 in vascular smooth muscle cells. Cardiovasc Res 2008;80:299–308.

35. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK.Distinct subcellular localizations of Nox1 and Nox4 in vascu-lar smooth muscle cells. Arterioscler Thromb Vasc Biol 2004;24:677–83.

36. Diaz B, Shani G, Pass I, Anderson D, Quintavalle M, CourtneidgeSA. Tks5-dependent, nox-mediated generation of reactive oxy-gen species is necessary for invadopodia formation. Sci Signal2009;2:ra53.

37. Binker MG, Binker-Cosen AA, Richards D, Oliver B, Cosen-Binker LI.EGF promotes invasion by PANC-1 cells through Rac1/ROS-depen-dent secretion and activation of MMP-2. Biochem Biophys Res Com-mun 2009;379:445–50.

38. Pelicano H, LuW, Zhou Y, ZhangW, Chen Z, Hu Y, et al. Mitochondrialdysfunction and reactive oxygen species imbalance promote breastcancer cell motility through a CXCL14-mediated mechanism. CancerRes 2009;69:2375–83.

39. ChiuWT, Shen SC, Chow JM, Lin CW, Shia LT, Chen YC. Contributionof reactive oxygen species to migration/invasion of human glioblas-toma cells U87 via ERK-dependent COX-2/PGE(2) activation. Neuro-biol Dis 2010;37:118–29.

40. Chandel NS, McClintock DS, Feliciano CE, Wood TM, Melendez JA,Rodriguez AM, et al. Reactive oxygen species generated at mito-chondrial complex III stabilize hypoxia-inducible factor-1alpha dur-ing hypoxia: a mechanism of O2 sensing. J Biol Chem 2000;275:25130–8.


Gregg et al.




41. WangM,Kirk JS, VenkataramanS,DomannFE, ZhangHJ,Schafer FQ,et al.Manganese superoxide dismutase suppresses hypoxic inductionof hypoxia-inducible factor-1alpha and vascular endothelial growthfactor. Oncogene 2005;24:8154–66.

42. Kaewpila S, Venkataraman S, Buettner GR, Oberley LW. Manganesesuperoxide dismutase modulates hypoxia-inducible factor-1 alphainduction via superoxide. Cancer Res 2008;68:2781–8.

43. Park SS, Bae I, Lee YJ. Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation ofiron. J Cell Biochem 2008;103:1989–98.

44. Block K, Gorin Y, New DD, Eid A, Chelmicki T, Reed A, et al.The NADPH oxidase subunit p22phox inhibits the function ofthe tumor suppressor protein tuberin. Am J Pathol 2010;176:2447–55.






NADPH Oxidase NOX4 Supports Renal Tumorigenesis by Promoting the Expression and Nuclear Accumulation...

Documents

Transcript of NADPH Oxidase NOX4 Supports Renal Tumorigenesis by Promoting the Expression and Nuclear Accumulation...