roles of keap1 nrf2 system in upper aerodigestive tract ... · hypothesize that a mechanical...

Research Article

Roles of Keap1–Nrf2 System in Upper Aerodigestive TractCarcinogenesis

Akira Ohkoshi1,2, Takafumi Suzuki1, Masao Ono3, Toshimitsu Kobayashi2, and Masayuki Yamamoto1

AbstractCancers in the upper aerodigestive tract, including cancers of the tongue and the esophagus, are the third

leading cause of cancer-relateddeaths in theworld, andoxidative stress iswell recognized as oneof themajor

risk factors for carcinogenesis. The Keap1–Nrf2 system plays a critical role in cellular defense against

oxidative stress, but little is known about its associationwith upper aerodigestive tract carcinogenesis. In this

study, we examined whether loss of Nrf2-function exacerbates carcinogenesis by using an experimental

carcinogenesis model that is induced by 4-nitroquinoline-1-oxide (4NQO). We found that Nrf2-knockout

(Nrf2-KO)mice weremore susceptible to 4NQO-induced tongue and esophageal carcinogenesis thanwild-

type mice, which suggests that Nrf2 is important for cancer prevention. We also examined how the

suppression of Keap1 function or the induction of Nrf2 activity affected 4NQO carcinogenesis. Keap1-

knockdown (Keap1-KD) mice were resistant to 4NQO-induced tongue and esophageal carcinogenesis.

Importantly, no growth advantagewas observed in tongue tumors in theKeap1-KDmice. These results show

that the Keap1–Nrf2 system regulates an important defense mechanism against upper aerodigestive tract

carcinogenesis. In addition to several important functions of Nrf2 that lead to cancer chemoprevention, we

hypothesize that a mechanical defense of thickened keratin layers may also be a chemopreventive factor

because thickened, stratified, squamous epitheliumwas found on the tongue of Keap1-KDmice.Cancer Prev

Res; 6(2); 149–59. �2012 AACR.

IntroductionCancers in the upper aerodigestive tract, including can-

cers in the oral cavity, oropharynx, hypopharynx, larynx,and esophagus, are the third leading cause of cancer-relateddeaths in the world (1). Tongue and esophageal cancers arethe most frequent among them. Because both the tongueand the esophagus are covered by stratified squamousepithelium (2), squamous cell carcinoma is the most com-mon cancer type in these regions. Important risk factors fortongue and esophageal cancers include tobacco smokingand alcohol use, and the cancer risk attributable to bothfactors is estimated to be more than 60% (3). It has beenreported that carcinogenesis induced by tobacco and alco-hol is mediated, at least in part, by oxidative stress (4).However, while the importance of oxidative stress in oraland esophageal carcinogenesis is well recognized, little isknown about the mechanisms mediating how stress pro-

vokes carcinogenesis or how cancer prevention is attainedthrough targeting oxidative stresses.

The transcription factor Nrf2 plays a pivotal role incellular defense against toxic electrophiles and oxidativestresses (5). Nrf2 regulates both basal and inducibleexpression of antioxidative and detoxifying enzymes (6),including NAD(P)H quinone oxidoreductase 1 (NQO1),glutathione S-transferases (GST), glutamate–cysteine ligasecatalytic subunit (GCLC), and hemoxygenase-1 (HO-1).Due to a lack of activation of these genes, Nrf2-knockout(Nrf2-KO) mice are susceptible to various chemicals,including carcinogens for stomach (7), bladder (8), skin(9), liver (10), colon (11), and breast (12).

We have been studying the molecular basis of Nrf2activation in response to environmental insults (13). Wefound that Keap1 regulates Nrf2 stability and subsequentactivity (14). Keap1 is a subunit of the ubiquitin E3 ligasecomplex (15). Under nonstressful conditions, Keap1 con-stitutively and efficiently ubiquitinates Nrf2 and promotesits rapid degradation via the proteasome pathway. Understressful conditions, Keap1 is inactivated, and Nrf2 is sta-bilized andactivated. Reactive cysteine residues ofKeap1 aremodified by electrophiles or reactive oxygen species (ROS),which leads to Nrf2 stabilization and accumulation in thenucleus and subsequently activates Nrf2 (15, 16). Whilesome antioxidant chemicals have cancer preventive effectsin an adequate range of doses, there are concerns that anoverdose of these chemicals could have some degree oftoxicity due to electrophilic insults (17, 18). Another hurdle

Authors' Affiliations: Departments of 1Medical Biochemistry, 2Otorhino-laryngology, Head and Neck Surgery, and 3Histopathology, Tohoku Uni-versity Graduate School of Medicine, Sendai, Japan

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

Corresponding Author: Masayuki Yamamoto, Tohoku UniversityGraduate School of Medicine, 2-1 Seiryo-cho, Aoba-ku, Sendai 980-8575, Japan. Phone: 81-22-717-8084; Fax: 81-22-717-8090; E-mail:[email protected]

doi: 10.1158/1940-6207.CAPR-12-0401-T

�2012 American Association for Cancer Research.

CancerPreventionResearch

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Research. on January 20, 2021. © 2013 American Association for Cancercancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst December 18, 2012; DOI: 10.1158/1940-6207.CAPR-12-0401-T

http://cancerpreventionresearch.aacrjournals.org/

for the Nrf2-inducing chemicals is that the pharmaceuticalNrf2 activation is only transient (19). Therefore, 1 of theimportant strategies for cancer prevention is promotingconstitutive Nrf2 activation by Keap1 suppression usingnonelectrophilic molecules.

Recent studies have found that there are somatic muta-tions of KEAP1 in various human cancer cells that causeconstitutive NRF2 activation and promote cancer malig-nancy (20, 21). Another intriguing observation is thatKeap1-knockout mice die at weaning due to malnutritioncaused by severe hyperkeratosis of the upper digestive tract(22). Considering these effects of Keap1 loss-of-function, itremains to be seen whether suppression of Keap1 functionis beneficial for cancer prevention. Recently, we generatedKeap1-knockdown (Keap1-KD) mice that are viable withconstitutive Nrf2 activation (23). Thesemice are resistant tosome types of stresses (24, 25) and provide an excellentmodel system to studywhether Keap1-KDmice are resistantto carcinogens.

To examine the cancer preventive effect of Keap1-KD inthe tongue and the esophagus, we used the water-solublecarcinogen 4-nitroquinoline-1-oxide (4NQO) for experi-mental carcinogenesis (26).We selected this systembecausethe animalmodel of oral carcinogenesis with 4NQO allowsfor the reproducible isolation of all stages of tongue andesophageal carcinogenesis, and the tumors have histologicand molecular changes that are similar to human cancer(27). 4NQO generates oxidative stress, and its metabolitecovalently binds to DNA. In this study, we found that Nrf2-KO mice are susceptible to tongue and esophageal carcino-genesis induced by 4NQO, showing the importance of Nrf2for the preventionof tongue and esophageal cancer.We alsoshowed that Keap1-KD mice are resistant to tongue andesophageal carcinogenesis. No growth advantage of 4NQO-induced cancer cells in Keap1-KDmicewas observed, show-ing that the suppression of Keap1 activity has potential forcancer prevention without significant adverse impacts.

Materials and MethodsChemicals

4NQO was purchased from Sigma-Aldrich for experi-mental carcinogenesis and from Wako for experiments incultured cells. Diethyl maleate (DEM) was purchased fromWako.

Cell cultureThe human oral squamous cell carcinoma cell line HSC3

was kindly provided in 2011 by Dr. Saiki (Tohoku Univer-sity). Cells were regularly tested for mycoplasma contam-ination using the e-Myco Mycoplasma PCR Detection Kit(iNtRON Biotechnology). No cell authentication was doneby the authors. Cells were maintained in Dulbecco’s mod-ified Eagle’s medium (DMEM) with 10% FBS and supple-mented with antibiotics.

MiceSix- to 7-week-old femalewild-type (WT), Keap1-KD, and

Nrf2-KO mice on the C57BL/6J genetic background were

used for this study. Themiceweremaintained under specificpathogen-free conditions in the animal facility at TohokuUniversity. All of the animal experiments were executedwith the approval of the Tohoku University Animal CareCommittee.

Immunoblot analysisCultured cells were collected and lysed in SDS sample

buffer and stored at �80�C (16). The samples were sub-jected to immunoblot analysis using anti-Nrf2 (clone 103;ref. 28), anti-phosphorylated histone H2AX (Millipore),anti-histone H2AX (Calbiochem), and anti-tubulin (Sig-ma) antibodies.

Real-time PCR analysisTotal RNA was isolated from either cultured cells or

tongues and esophagi from mice using the Isogen RNAextraction kit (Nippon Gene). cDNA was synthesized from1 mg total RNA using Superscript III (Invitrogen). Quanti-tative real-time PCR (qRT-PCR) analysis was conductedusing the ABI7300 system (Applied Biosystems). The pri-mers and probes for detecting human NQO1, GCLC,GSTP1, GPX2, and mouse Nqo1, Gclc, Gstp1, Gpx2, G6pd,Pgd, Tkt, Taldo1,Me1, and 18S ribosomal RNA are describedin Supplementary Table S1.

Carcinogen treatment4NQOwas dissolved in dimethylsulfoxide (DMSO) at 10

mg/mL and diluted into the drinking water to 100 mg/mL.WT, Keap1-KD, and Nrf2-KO mice were subdivided into 3groups. Five mice per genotype received tap water for 24weeks as the control groups. For the experimental groupstreated with 4NQO, at least 20 mice per genotype weretreated first with 100mg/mL4NQO in the drinkingwater foreither 8 (short term) or 16 weeks (long term) and subse-quently with tap water for either 16 (short term) or 8 weeks(long term). All of the mice were allowed to drink 4NQO-containing water or tap water ad libitum in all periods of theexperiments. All of themicewereweighed and checkedoncea week. 4NQO-containing water and tap water were chan-ged fresh everyweek. At the endof the experiments, themicewere sacrificed after anesthetization, and tongues andesophagi were dissected. Whole esophagi were openedlongitudinally, and tongue and esophageal tumors largerthan 1 mm diameter were counted.

For the analysis of blood counts, WT and Nrf2-KO micewere treated with 100 mg/mL 4NQO in the drinking waterfor 8 or 12 weeks, and peripheral blood taken from theretro-orbital plexus was analyzed by a hemocytometer(Nihon Kohden).

Histologic analysisTissue from the mice was fixed with Mildform 10N

(Wako), embedded into paraffin, and cut into 5-mm thicksections. After staining with hematoxylin and eosin (H&E),a histopathological diagnosis of the tongues and the esoph-agi was carried out by a pathologist without knowledge ofthe genotypes and the carcinogen experiment. The sections

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Cancer Prev Res; 6(2) February 2013 Cancer Prevention Research150




were graded as normal, dysplasia, or squamous cell carci-noma using established criteria (29). For immunohisto-chemical analysis, an anti-Ki-67 antibody (DAKO) wasused. Ki-67-positive cells in tumors or progenitor cells inthebasal layer of the tongueswere countedusing the Image Jsoftware for the analysis of cell growth.

Statistical analysisSignificant differences were determined using the Stu-

dent’s t test or the Mann–Whitney test. The values arepresented as the means � SEM.

Results4NQO does not activate NRF2 either in vitro or in vivoTo explore the roles the Keap1–Nrf2 system plays in

upper aerodigestive tract carcinogenesis, we used a 4NQOexperimental carcinogenesis model. While Nrf2 is activated

in response to various environmental stresses (30), therelationship between 4NQO and Nrf2 activation has notbeen elucidated. Therefore, to delineate whether 4NQOdirectly activates NRF2, we examined the response of HSC3human oral cancer cells to 4NQO by measuring NRF2protein expression. While DEM, an NRF2 inducer, nicelyincreased NRF2 protein expression, 4NQO did not. Whenwe tested 1 and 3 mmol/L 4NQO, 3 mmol/L 4NQO treat-ment was sufficient to promote the phosphorylation ofhistone H2AX, a DNA damage marker (Fig. 1A). Consistentwith the observation that 4NQO did not change NRF2expression, 4NQO also did not increase the mRNA expres-sion of either NQO1 or GCLC, while DEM stronglyincreased the expression of both genes (Fig. 1B).

To examine the in vivo response to 4NQO, we treated6-week-old female mice with either tap water or 4NQO(100 mg/mL)-containing water for 24 and 48 hours and

Figure 1. 4NQO does not activateNrf2 in vitro or in vivo. A, the levels ofNRF2, TUBULIN, phosphorylatedhistone H2AX (gH2AX), and H2AX inHSC3 cells was determined byimmunoblot analysis. HSC3 cellswere treated either with or without 1or 3 mmol/L 4NQO and 100 mmol/LDEM for 3 hours. B, the relativeexpression levels of NQO1 andGCLC mRNA in HSC3 cells wasexamined by quantitative real-timePCR (qRT-PCR). HSC3 cells weretreated with or without 1 to 3 mmol/L4NQO and 100 mmol/L DEM for 12hours. The expression values arerelative to HSC3 cells withouttreatment. The error bars indicatestandard error (n ¼ 4). Asterisksindicate statistically significantdifferences compared with thecontrol (�, P < 0.05; ��, P < 0.01).C, the expression levels of Nqo1,Gclc, Gstp1, and Gpx2 mRNA in thetongues of 6-week-old female WTmice were examined by qRT-PCR.The mice were treated with100 mg/mL 4NQO in the drinkingwater or tap water ad libitum for 24 or48 hours. The expression values arerelative to the mice treated with tapwater. The error bars indicatestandard error (n ¼ 3). D, the relativeexpression levels of Nqo1, Gclc,Gstp1, and Gpx2 mRNA in thetongues ofWT, Keap1-KD, and Nrf2-KO mice were examined by qRT-PCR. The expression values arerelative to those of WT mice. Theerror bars indicate standard error(n¼ 3). Asterisks indicate statisticallysignificant differences comparedwith control mice (�, P < 0.05;��, P < 0.01; ��, P < 0.001).

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examined the expression of Nrf2 target genes in the tongue.The expression of Nqo1, Gclc, Gstp1, and Gpx2 was notincreased in the tongues at either 24 or 48 hours after theadministration of 4NQO (Fig. 1C). These results show that4NQO does not activate NRF2 in either human oral cells inculture or mouse tongue in vivo.

Nrf2 target genes are highly expressed in the tongueand esophagus of Keap1-KD mice

It has previously been reported that Nrf2 target genes arehighly expressed in the liver and lungs of Keap1-KD mice(23). Consistent with this observation, high levels ofNqo1,Gclc, and Gstp1 were also observed in the tongues andesophagi of Keap1-KD mice (Fig. 1D left and right, respec-tively). These results suggest that Nrf2 can be stabilized andactivated in the tongues and esophagi of Keap1-KD mice.

We also observed that the Nqo1mRNA expression in theNrf2-KO mouse tongues was lower than that in the WTmouse tongues, and the expression ofNqo1,Gclc,Gstp1, andGpx2 mRNA in the esophagi of Nrf2-KO mice was lowerthan that observed in the WT mouse esophagi (Fig. 1D).These results show that Nrf2 is expressed at a basal level, butthe suppression of Keap1 causes a strong activation of Nrf2in the tongue and esophagus.

Nrf2-KO promotes and Keap1-KDprevents tongue andesophageal carcinogenesis induced by 4NQO

To examine the contribution ofNrf2 andKeap1 to tongueand esophageal carcinogenesis, we conducted 2 series of4NQO carcinogenesis experiments using Nrf2-KO andKeap1-KD mice. In 1 series of experiments, we fed thesemutant lines of mice with 4NQO-containing water for 8weeks. These mice were subsequently fed with tap water for16 weeks and then sacrificed for analysis. We refer to thisexperiment as the short-term 4NQO experiment (Fig. 2A).Macroscopically, we found that Nrf2-KO mice had manytongue and esophageal tumors, while Keap1-KD miceshowed almost no tongue and esophageal tumors (Fig.2B). WT mice showed no tongue tumors and a very smallnumber of esophageal tumors.

Statistical analyses revealed that the number of tonguetumors in the Nrf2-KOmice (1.43� 0.22) was significantlyhigher than that in the WT mice (0.29 � 0.12), while therewere no significant differences in the numbers of tonguetumors between Keap1-KD andWTmice (Fig. 2C, left). Thenumber of esophageal tumors in Nrf2-KO mice (5.48 �0.45) was significantly higher than that in the WT mice(1.76 � 0.24), while the number of esophageal tumors inKeap1-KDmice (0.20� 0.09)was significantly smaller thanthat in the WT mice (Fig. 2C, right).

Histologic analyses revealed that therewas both dysplasiaand squamous cell carcinoma in the tongues of Nrf2-KO,Keap1-KD, and WT mice, which suggests that the tonguetumors from the 4NQO model experiments progressedtoward tumorigenesis. Representative histologic sections ofthe tongues, including dysplasia in WT mice (left) andsquamous cell carcinoma in Nrf2-KO mice (right), areshown in Fig. 2D. The analyses revealed that 13 of 21

Nrf2-KO mice had dysplasia (62%) and 5 Nrf2-KO micehad squamous cell carcinoma (24%) in the tongue. Thesenumbers were markedly higher than the numbers observedinWTmice (dysplasia 7/21, 33%; squamous cell carcinoma1/21, 5%), indicating that tongue tumors from Nrf2-KOmice show more malignant progression than those fromWT mice (Table 1A). There was no such difference in thetongues from Keap1-KD and WT mice (Table 1A).

Similarly, there was both dysplasia and squamous cellcarcinoma in the esophagi from these mutant mice. Rep-resentative histologic sections of the esophagi are shownin Supplementary Fig. S1A (dysplasia in WT mice andsquamous cell carcinoma in Nrf2-KO mice). Statisticalanalyses revealed significantly more advanced esophagealsquamous cell carcinomas in Nrf2-KO mice (18/21 weresquamous cell carcinoma) compared with the WT mice (1/21was squamous cell carcinoma, Table 1B). In contrast, thehistologic stage of the esophageal carcinomas in Keap1-KDmice was significantly less advanced than those in the WTmice (Table 1B).

Despite the low tumor incidence in the esophagus,Keap1-KD mice drank significantly larger amounts of the4NQO-containing water per body weight than theWTmicedid (Supplementary Fig. S2). In contrast, the Nrf2-KOmiceconsumed amuch smaller amount of the 4NQO-water thanthe WT mice did (Supplementary Fig. S2). This result isparticularly interesting if we consider the fact that the highincidence of squamous cell carcinoma was observed in thetongues and esophagi of Nrf2-KO mice. Thus, these short-term 4NQO experiments show that Nrf2 loss-of-functionpromotes 4NQO-induced tongue and esophageal carcino-genesis, whereas Nrf2 activation by Keap1-KD preventstumorigenesis in the esophagus.

Keap1-KD prevents carcinogenesis in the tongue andthe esophagus in a long-term experiment with 4NQO

Because we were unable to fully evaluate carcinogenesisin the tongues of Keap1-KD and WT mice due to a lowincidence of tumors in the short-term experiment with4NQO, we decided to use more stringent conditions in along-term experiment with 4NQO. In the long-term exper-iment, the mice were treated with 4NQO for 16 weeksinstead of 8 weeks (Fig. 3A). In the long-term experiment,all of the mice lost body weight (Supplementary Fig. S3A–D). While the majority of the WT and the Keap1-KD micesurvived until the end of the experiment, most of the Nrf2-KO mice lost more than 20% of their body weight, and theexperiment was stopped by euthanizing the animals (Sup-plementary Fig. S4A). To identify the reason why the Nrf2-KO mice had such a severe decrease in body weight, weexamined Nrf2-KO and WT mice after 8- or 12-week-treat-ments with 4NQO and found that only a few tumors wereobserved in the Nrf2-KO and WT mice (data not shown).We believe that the stringent conditions of the long-term4NQO experiment likely produced severe acute toxicity inthe Nrf2-KO mice (31). However, we did not find a differ-ence in hematologic indices (Supplementary Fig. S2) orhistologic damage of the liver (Supplementary Fig. S4B) in

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the Nrf2-KO mice in this experimental condition. We sur-mise that, as we stopped the 4NQO experiments at 20%body-weight-loss point for animal protection reasons,severe toxicitymight not be apparent at this timing. Becausethe focus of this study is on cancer prevention, we did notconduct further examination about the acute toxicity of4NQO. For these reasons, we excluded the Nrf2-KO micefrom this long-term experiment.We continued the evaluation of tumor incidence in Keap1-

KDmice andWTmice. This long-term treatment with 4NQOcaused tongue and esophageal tumors in almost all the WTmice but only in half of the Keap1-KD mice (Fig. 3B and C).Statistical analyses revealed that thenumber of tongue tumorsin Keap1-KD mice (0.83 � 0.62) was significantly smallerthan that in the WT mice (1.68 � 0.95; Fig. 3C, left panel).The numbers of esophageal tumors in Keap1-KD mice(0.94� 1.51)were also significantly smaller than that observ-

ed in the WT mice (3.68 � 1.39; Fig. 3C, right). Histologicanalysis revealed that squamous cell carcinomawas observedin the tongues of only 1 of 18 Keap1-KD mice, while morethanhalf ofWTmice had squamous cell carcinoma (Table 1Cand Fig. 3D). It should also be noted that squamous cellcarcinoma was not observed in the esophagi of Keap1-KDmice, while 14 of 22 WT mice had squamous cell carcinomain the esophagus (Table 1D and Supplemental Fig. S1B).These results show that Keap1-KD improves incidence andprevents the progression of 4NQO-induced carcinogenesis.

Keap1-KDdoes not promote cell growth indysplasia ofthe tongue

There still remain concerns thatNrf2 activitymay confer agrowth advantage to cancer cells (20, 32). To evaluate thegrowth ability of cancer cells, we examined the density ofKi-67-positive cells (Fig. 4A and B) by using Ki-67 as a

Figure 2. Loss of Nrf2 promotestongue and esophagealcarcinogenesis in a short-termexperiment with 4NQO. A, theexperimental design for the short-term experiment with 4NQO. Micewere treated with 100 mg/mL 4NQOin the drinking water ad libitum for 8weeks, followed by regular water for16 weeks and then analyzed. B,representative macroscopic imagesof tongues and esophagi from theshort-term experiment with 4NQO.At the end of the experiment, thetongues and esophagi weredissected from the mice sacrificedafter anesthetization. Wholeesophagi were openedlongitudinally. Yellow arrow headsindicate tumors. C, the number oftongue tumors thatwere larger than1mm was counted in WT, Keap1-KDand Nrf2-KO mice (left). The numberof esophageal tumors that werelarger than 1mmwas counted (right).Asterisks indicate statisticallysignificant differences comparedwith the WT mice (��, P < 0.001).D, representative hematoxylin–eosinstained sections of tumors from theshort-term experiment with 4NQO.Dysplasia in the tongues from WTmice and squamous cell carcinomain the tongues fromNrf2-KOmice areshown.

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proliferationmarker (33). We found that the Ki-67-positivecell densitywas significantly higher in tongue squamous cellcarcinoma than in tongue dysplasia in both Nrf2-KO andWT mice (Fig. 4C). Although we could not determine theproliferative ability of squamous cell carcinoma in Keap1-KD mice due to its rare incidence, the Ki-67-positive celldensity was not higher in the tongue dysplasia in Keap1-KDmice than that observed in WT mice (Fig. 4C). In contrast,there was no difference in the Ki-67-positive cell density ineither tongue dysplasia or squamous cell carcinomabetween Nrf2-KO and WT mice (Fig. 4C). These resultssupport the hypothesis that Nrf2 activation or deficiencydoes not affect the growth of cells in 4NQO-inducedtumors.

Keap1-KD promotes epithelial cell growth andincreases epithelial and keratin layer thickness

In Keap1-KD mice, the cancer preventive effects seem tobe due to the high expression of antioxidant anddetoxifyingenzymes (8, 10, 11).Wehypothesized that thickening of theepithelia in the tongue and esophagus might protect epi-thelial cells from exposure to carcinogens. Keap1-KD micehave been shown to have hyperkeratosis in these tissues(23), and we surmised that this might be an additionalmechanism to protect epithelial cells from carcinogens.

To address this hypothesis, we measured the thickness ofthe tongue epithelia and its keratin layers (34). We foundthat both the tongue epithelia and its keratin layer in Keap1-KD mice were significantly thicker than those in the WTmice (bothP<0.05; Fig. 5A andB).On the other hand, boththe tongue epithelia and its keratin layer in Nrf2-KO micewas thinner than those in the WT mice (both P < 0.05; Fig.5A and B). It has been reported that the epithelial thicknessof stratified squamous epithelium is regulated by the growthof progenitor cells in the basal layer (2). Therefore, we

examined Ki-67-positive cells in the tongue. The density ofKi-67-positive cells in the basal layer was significantlyhigher in Keap1-KDmice than in theWTmice (P < 0.05; Fig.5C andD).On the contrary, while the Ki-67 density inNrf2-KOmicewas lower than that observed in theWTmice, itwasnot statistically significant (P, 0.09; Fig. 5C and D).

We recently found that Nrf2 regulates antioxidant anddetoxifying enzymes and growth-related genes that providegrowth advantages to cells by increased anabolism andreinforcement of metabolic reprogramming (35). There-fore, we examined the expression of glucose-6-phosphatedehydrogenase (G6pd), phosphogluconate dehydrogenase(Pgd), transketolase (Tkt), transaldolase 1 (Taldo1), andmalic enzyme 1 (Me1) in the tongue. We found markedlyhigher expression ofG6pd, Pgd, and Taldo1 in the tongues ofKeap1-KDmice comparedwith theWTmice (Fig. 5E). Theseresults support the idea that suppressing Keap1 increasesNrf2,which accelerates progenitor cell growth and increasesepithelium thickness and may, in part, contribute tomechanical protection against carcinogens.

DiscussionIn this study, we found that Nrf2-KO mice are more

susceptible to 4NQO-induced carcinogenesis in the tongueand esophagus than WT mice. Because 4NQO does notactivateNrf2 effectively, this result suggests thatNrf2 plays aprotective role against carcinogenesis, even in uninducedconditions. Keap1 is a repressor ofNrf2 (36), andKeap1-KDor derepression of Nrf2 is found to elevate the expression ofdetoxifying and antioxidant enzymes in the tongue andesophagus ofmice. Because Nrf2 acts as an oncogenic factorin cancer cells (37), there are controversial interpretationswith regard to the cancer preventive effects observed inKeap1-KD mice. In this study, we have shown that Keap1suppression prevents 4NQO-induced carcinogenesis in the

Table 1 Histology of the tongues and esophagi in 4NQO-treated mice.

Genotype Number of mice Dysplasia Squamous cell carcinoma P value

A. Histology of the tongues in short-term experiment with 4NQOWild-type 21 7 (33%) 1 (5%) �Keap1-KD 20 3 (15%) 2 (10%) 0.48Nrf2-KO 21 13 (62%) 5 (24%) 0.0013

B. Histology of the esophagi in short-term experiment with 4NQOWild-type 21 18 (86%) 1 (5%) �Keap1-KD 20 6 (30%) 0 (0%) <0.001Nrf2-KO 21 3 (14%) 18 (86%) <0.001

C. Histology of the tongues in long-term experiment with 4NQOWild-type 22 9 (41%) 12 (55%) �Keap1-KD 18 9 (50%) 1 (6%) <0.001

D. Histology of the esophagi in long-term experiment with 4NQOWild-type 22 8 (36%) 14 (64%) �Keap1-KD 18 8 (44%) 0 (0%) <0.001

NOTE: P value evaluated in comparison with WT mice.

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tongue and esophagus without showing any adverse effects.The cancer preventive effects observed in the Keap1-KDmice seem to be due to the high expression of Nrf2 andthe subsequent expression of detoxifying enzymes specificfor 4NQO. Alternatively, thickened stratified squamousepithelium might contribute to the mechanical defenseagainst 4NQO (Supplementary Fig. S5).The carcinogenicity of 4NQO could be elicited by 2

pathways; 1 is the ROS-mediated oxidative DNA damage(38) mechanism, while the other is the direct DNA-adduct formation by 4-hydroxyaminoquinoline-1-oxide(4HAQO), a carcinogenic metabolite of 4NQO (27). Oneof the important characteristics of 4NQO is the inabilityto activate Nrf2, while Nrf2 is often activated by variousenvironmental insults, including carcinogens (30). Theincapability of Nrf2 activation suggests that 4NQO is not

able to generate ROS sufficient to activate Nrf2, and thecarcinogenicity of 4NQO is mainly mediated by the directDNA-adduct formation by 4HAQO. Observations in theNrf2-KO mice suggest that the basal expression of Nrf2 iscritical for cancer prevention in tongue and esophagus.Indeed, the basal expression of Nqo1, Gclc, Gstp1, andGpx2 in the esophagi of Nrf2-KO mice is significantlylower than that in WT mice.

The detoxification of 4NQO is mostly mediated by glu-tathione conjugation (27, 39).Of theNrf2 target genes,Gclcincreases glutathione synthesis and Gstp1 catalyzes forma-tion of 4NQO-glutathione (39). In contrast, Nqo1 is thecandidate enzyme that converts 4NQO to 4HAQO (40).Therefore, there was an alternative hypothesis suggestingthat Nrf2 acts to stimulate 4NQO carcinogenesis. However,our current results clearly show that Nrf2 prevents

Figure 3. Keap1 knockdownprevents tongue and esophagealcarcinogenesis in a long-termexperiment with 4NQO. A, theexperimental design of the long-termexperiment with 4NQO. Mice weretreated with 100 mg/mL 4NQO in thedrinking water ad libitum for 16weeks and, then, were given regularwater for 8weeks andanalyzed at theend of the time course. B,representative macroscopic imagesof tongues and esophagi from thelong-term 4NQO experiment.Whole esophagi were openedlongitudinally. Yellow arrow headsindicate tumors. C, the number oftongue tumors thatwere larger than1mm was counted in WT and Keap1-KD mice (left). The number ofesophageal tumors that were largerthan 1 mm was counted inWT and Keap1-KD mice (right).Asterisks indicate statisticallysignificant differences compared tothe WT mice (��, P < 0.001). D,representative hematoxylin–eosin-stained sections of tumors from thelong-term 4NQO experiment.squamous cell carcinoma in tonguesfrom WT mice and dysplasia intongues from Keap1-KD mice areshown.

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carcinogenesis, which suggests that Nrf2 targets gene pro-ducts other than Nqo1 contribute to the cancer prevention.We surmise that Gclc and Gstp1 are 2 important targets inthis regard. Alternatively, Nqo1may not convert 4NQO to acarcinogen. In human epidemiology, decreased expressionof NQO1, GCLC, and GSTP1 by polymorphisms is inde-pendently related to an increased incidence of oral oraerodigestive tract cancer (41–43), which suggests thatNqo1, Gclc, and Gstp1 are critical among the Nrf2 targetgenes for preventing carcinogenesis.

Because loss of Keap1 function in cancer cells bysomatic mutations or other mechanisms activatesNrf2 constitutively and promotes malignancy, cancersderived from the Keap1-KD mice appear to be malignant(21, 44). However, our study showed that suppressingKeap1 activity prevented 4NQO-induced carcinogenesis.Constitutive Nrf2 activation does not affect the growthand development of tongue and esophageal tumors in

this model. While it has been reported that pharmaco-logic and transient Nrf2 activation prevents chemicalcarcinogenesis (7, 8), our study shows, for the first time,that constitutive Nrf2 activation by Keap1 suppressionis a cancer prevention mechanism. In contrast, Nrf2promotes Ras-mediated (45) or urethane-induced lungcarcinogenesis (32). These observations show that theinfluence of Nrf2 activation depends on the context.Accumulated lines of evidence support the notion thatsuppressing Keap1 activity or inducing Nrf2 activity iseffective for preventing the initial stage of carcinogenesis.

One of the most plausible explanations for the mecha-nism underlying how Nrf2 prevents upper aerodigestivetract carcinogenesis is that Nrf2 induces the expression ofantioxidant and detoxifying enzymes and renders cellsresistant to carcinogens (8, 10, 11). In addition, themechanical defense of thickened stratified squamous epi-thelium may also be an important mechanism to prevent

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Figure 4. Keap1 knockdown doesnot promote cell growth indysplasia of the tongue. A,representative pictures of tonguedysplasia stained with a Ki-67antibody. B, representativepictures of tongue squamous cellcarcinoma stained with a Ki-67antibody in WT and Nrf2-KO mice.C, the density of Ki-67-positivecells in tongue dysplasia from WT,Keap1-KD, and Nrf2-KO mice, andtongue squamous cell carcinomafrom WT and Nrf2-KO mice isshown. The error bars indicatestandard error (dysplasia: WT,n¼ 10; Keap1-KD, n¼ 6; Nrf2-KO,n ¼ 10; squamous cell carcinoma:WT, n ¼ 10; Nrf2-KO, n ¼ 5). N.D.means not determined due to itssmall number.

Ohkoshi et al.





tissue exposure to carcinogens. Nrf2 regulates growth-relat-ed genes and increases the growth of progenitor cells in thebasal layer, leading to thickened epithelia. In humans, theupper aerodigestive tract is covered by stratified squamousepithelium, and the growth of progenitor cells in the basallayer causes thickening of the epithelia. It is conceivable thatthe epithelial thickness regulates resistance to chemical andmechanical insults (2, 34). This function may be physio-logically critical as the defense mechanism. Another possi-ble mechanism is that Nrf2 may prevent the creation of thecancer microenvironment that supports metastasis in thelung (46).In conclusion, we have shown that Nrf2-KO mice are

susceptible to 4NQO carcinogenesis, while Keap1-KDmice are resistant to 4NQO-induced tongue and esoph-ageal carcinogenesis. Because 4NQO does not activate or

induce Nrf2, the importance of the basal Nrf2 activity oncancer prevention can be identified. We have also shownthe increase in progenitor cells and the thickened epithe-lium in the tongues of Keap1-KD mice, which shows theunique protective mechanism of stratified squamousepithelium.

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

Authors' ContributionsConception and design: A. Ohkoshi, T. Suzuki, T. Kobayashi, M.YamamotoDevelopment ofmethodology:C.Mascaux, J.I. Eckelberger,W.A. Franklin,F.R. HirschAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Ohkoshi, M. Ono

Figure 5. Keap1 knockdownpromotes epithelial cell growth andincreases epithelial and keratin layerthickness. A, representative picturesof the tongue epithelium withoutcancerous or precancerous lesionsstained with hematoxylin–eosin.Yellow and blue lines indicate theepithelial layer and the keratin layer,respectively. B, the graph indicatesthe thickness of the epithelium andkeratin layer. The values are relativeto those of WT mice. The error barsindicate standard error (WT, n ¼ 8;Keap1-KD, n ¼ 8; Nrf2-KO, n ¼ 7).Asterisks indicate statisticallysignificant differences comparedwith WT mice (�, P < 0.05). C,representative pictures of the tongueepithelium stained with Ki-67. D, thegraph indicates the density of Ki-67-positive cells in the basal layer of thetongue from WT, Keap1-KD, andNrf2-KO mice. The error barsindicate standard error (WT, n ¼ 8;Keap1-KD, n ¼ 8; Nrf2-KO, n ¼ 7).E, the relative expression of G6pd,Pgd, Tkt, Taldo1, and Me1 mRNA inthe tongues of WT, Keap1-KD, andNrf2-KO mice was examined byqRT-PCR. The expression values arerelative to those of WT mice. Theerror bars indicate standard error(n¼ 3). Asterisks indicate statisticallysignificant differences comparedwith the WT mice (�, P < 0.05).

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Analysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Ohkoshi, M. OnoWriting, review, and/or revision of the manuscript: A. Ohkoshi, T.Suzuki, T. Kobayashi, M. YamamotoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M. YamamotoStudy supervision: T. Suzuki, M. Yamamoto

AcknowledgmentsThe authors thank Ms. Eriko Naganuma for technical assistance.

Grant SupportThis work was financially supported in part by Grants-in-Aids for Creative

Scientific Research and Scientific Research from JSPS, JST CREST, the TohokuUniversity Global COE Program for Conquest of Signal Transduction Dis-eases with "Network Medicine", the NAITO Foundation, and the TakedaScience Foundation.

Received September 24, 2012; revised December 3, 2012; acceptedDecember 8, 2012; published OnlineFirst December 18, 2012.

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2013;6:149-159. Published OnlineFirst December 18, 2012.Cancer Prev Res Akira Ohkoshi, Takafumi Suzuki, Masao Ono, et al. Carcinogenesis

Nrf2 System in Upper Aerodigestive Tract−Roles of Keap1

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