Nrf2 Prevents Initiation but Accelerates Progression ... · Tumor and Stem Cell Biology Nrf2 Prevents Initiation but Accelerates Progression through the Kras Signaling Pathway during

Tumor and Stem Cell Biology

Nrf2 Prevents Initiation but Accelerates Progression throughthe Kras Signaling Pathway during Lung Carcinogenesis

Hironori Satoh1,2, Takashi Moriguchi1, Jun Takai1, Masahito Ebina2, and Masayuki Yamamoto1

AbstractNrf2 (Nfe2l2) governs cellular defenses against oxidative and electrophilic stresses and protects against

chemical carcinogenesis. However, many cancers have been found to accumulate NRF2 protein, raisingquestions of precisely how Nrf2 contributes to carcinogenesis. In this report, we explored such questions inan established urethane-induced multistep model of lung carcinogenesis. Consistent with earlier observa-tions, Nrf2-deficient (Nrf2�/�) mice exhibited a relative increase in tumor foci by 8 weeks after urethaneadministration. However, after 16 weeks, we observed a relative reduction in the number of tumors with moremalignant characteristics in Nrf2�/� mice. Furthermore, all Nrf2þ/þ tumors harbored activated mutations inKras, whereas Nrf2�/� tumors were rarely associated with similar Kras mutations. Overall, our resultsestablished that Nrf2 has two roles during carcinogenesis, one of which is preventive during tumor initiationand the second that promotes malignant progression. These findings establish Nrf2 inhibitors as rationaltools to prevent malignant progression in lung cancer, whereas Nrf2 activators are more suited for lungcancer prevention. Cancer Res; 73(13); 4158–68. �2013 AACR.

IntroductionNrf2 (Nuclear factor-erythroid derived 2-like 2, Nfe2l2) is a

leucine zipper transcription factor and plays an important rolein the maintenance of redox balance and cytoprotectionagainst chemical carcinogens (1, 2). When subjected to oxida-tive and electrophilic stress conditions, Nrf2 is released fromKeap1 (Kelch-like ECH-associated protein 1)-mediated rapiddegradation. Nrf2 is stabilized, accumulated, and translocatesto the nucleus, where Nrf2 dimerizes with a small Maf protein(sMaf). The Nrf2–sMaf heterodimer binds to a specific DNAsequence, referred to as the antioxidant/electrophile responseelement (ARE/EpRE), and induces the expression of a cohort ofcytoprotective enzyme genes, such as Nqo1, HO-1, Gclc, andGstp1/p2 (1, 2).

Previous reports on chemically induced carcinogenesis haveshown that Nrf2-deficient (Nrf2�/�) mice tend to form a largernumber of tumors than wild-type (Nrf2þ/þ) mice, indicatingthat the perturbation in the carcinogen detoxification systeminNrf2�/�mice leads to cancer susceptibility in various tissues(3–6). However, recent studies have revealed that the NRF2

protein is significantly accumulated in many types of humancancers through several independent mechanisms. Forinstance, somatic mutations in the interface of KEAP1 andNRF2 (7) or epigenetic modifications in the promoter region ofKEAP1 genes (8) gives rise to the accumulation of NRF2. Asthese changes are often related to accelerated cancer cellgrowth and poor clinical prognosis, the oncogenic side ofNRF2 function has been attracting considerable attention.Furthermore, we recently showed that the Nrf2�/� micetransplanted with Lewis lung carcinoma (3LL) cells provideamore tumor-permissive immunemicroenvironment than theNrf2þ/þ mice (9). These observations raise a fundamentalquestion of whether Nrf2 exerts cancer-preventive or -promo-tive activity in each stage of the carcinogenic process. Toaddress this issue, we used the urethane (ethyl carbamate)-induced lung carcinogenesis model, a well-known multistepmurine carcinogenesis model (10).

Urethane has been extensively used as an inducer of chem-ical lung carcinogenesis (11). Administration of urethaneevokes hyperplasia or adenoma formation in the lung andeventually leads to adenocarcinomas in a time-dependentmanner (10). Through cytochrome P450 2E1 (Cyp2e1)-medi-ated oxidation, urethane is converted to vinyl carbamateepoxide, which serves as an ultimate carcinogen by inducingDNA-, RNA-, or protein–adduct formation in airway epithelialcells (12). In the detoxification process, microsomal epoxidehydrolase (mEH) converts vinyl carbamate epoxide into 1,2-dihydroxyethyl carbamate, which subsequently undergoes theglutathione conjugation catalyzed by Gstp1/p2 (13) and isexcreted into urine (14).

In the present study, we found that Nrf2�/�-mutant micedeveloped a large number of urethane-induced lung micro-nodules in the early phase after urethane administration.

Authors' Affiliations: Departments of 1Medical Biochemistry and 2Respi-ratory Medicine, Tohoku University Graduate School of Medicine, Sendai,Japan

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

Corresponding Authors: Masayuki Yamamoto, Tohoku University Grad-uate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575,Japan. Phone: 81-22-717-8084; Fax: 81-22-717-8090; E-mail:[email protected]; and Takashi Moriguchi, E-Mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-4499

�2013 American Association for Cancer Research.

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However, in the later stages, Nrf2þ/þ mice developed a highernumber of Kras-mutated adenocarcinomas than did Nrf2�/�

mice. These results show that Nrf2 deficiency leads to anincreased susceptibility to chemical carcinogens and resultanthigh-level tumor initiations, whereas Nrf2 serves as an onco-genic factor that accelerates malignant progression of Kras-mutated adenocarcinomas in the later stages of lungcarcinogenesis.

Materials and MethodsExperimental animalsNrf2�/� mice with an ICR/CD-1 genetic background (out-

bred) were used in this study (2, 15). Age-matched (5–9 weeks)Nrf2þ/þmice were used as concurrent controls. Themice weremaintained in a facility free of specific pathogens (SPF). Nudemice (8- to 9-weeks old) were purchased from CLEA Japan. Allanimal experiments were conducted with the approval of theTohoku University Animal Care Committee.

Lung carcinogenesis experimentsMouse lung tumors were induced by the intraperitoneal

injection of urethane (1 g/kg body weight; refs. 16, 17). For theenumeration of lung surface tumors, the lungs were removedand the total number of lung surface nodules per mouse wascounted macroscopically.

Kras mutation analysisPCR-amplified DNA samples from the urethane-induced

lung tumors and intact stromal tissues were subjected tosequencing analysis to detect Kras mutations. The primersamplifying the nucleotide sequences in the second exon ofKrasgene, which contains codon 61, are listed in SupplementaryTable S1.

Microarray analysisSurface lung tumors were excised, and surrounding tissues

were carefully removed under a observation via a stereo-microscope. The lung tumors and nontumor regions ofNrf2þ/þ and Nrf2�/�mice that had been treated with urethane(8 mice/group) were pooled and subjected to a whole-mousegenome microarray analysis (4 � 44 k; Agilent Technologies).The expression data were analyzed with GeneSpring software(Silicon Genetics). Heatmaps were generated using Cluster 3.0(http://bonsai.hgc.jp/~mdehoon/software/cluster/) and JAVATreeview 159 (http://jtreeview.sourceforge.net/). The classifi-cation of the selected genes according to their biologic andtoxicologic functions was done using Ingenuity Pathway Anal-ysis (IPA) software (Ingenuity system). P value, represented asthe negative log ratio of the IPA results, is the probability basedon Fisher exact test. The GEO accession number for themicroarray data is GSE46048.

Flow cytometryAnalyses of the bone marrow cells were conducted using

FACS-Caliber (BD Pharmingen). Quantification of reactiveoxygen series (ROS) level with 2,7-dichlorodihydrofluoresceindiacetate (DCFDA), separation of myeloid-derived suppressorcells (MDSC), and T cells has been described previously (9).

Immunoblotting analysisNuclear extracts were prepared fromNIH3T3 cells that were

treated with the indicated concentrations of urethane for 6hours. The mouse lung nuclear extracts were prepared fromtheNrf2þ/þmice administered either vehicle (PBS) or urethane(1 g/kg body weight) for the indicated time periods. Immuno-blotting analysis was conducted using anti-Nrf2 and anti-laminB antibodies (Santa Cruz Biotechnology) as described previ-ously (9).

Statistical analysesThe data are expressed as the mean � SD. The statistical

differences were determined using Student t test or theMann–Whitney U test. The values for either the incidence of lungnodules or large tumors were analyzed using the Fisher exactprobability test. P values less than 0.05 were consideredsignificant.

See Supplementary Materials and Methods for furtherdetails.

ResultsUrethane induces accumulation of Nrf2 anddetoxification enzymes in the lung

Although urethane exerts carcinogenic activity throughelectrophilicity of its metabolites (18), it remains unclearwhether urethane induces Nrf2 accumulation. To address thisquestion, we examined Nrf2 accumulation in NIH/3T3 cellstreated with urethane (10 or 50 mmol/L for 6 hours). Upontreatment with urethane, Nrf2 accumulated in nuclear fractionof NIH/3T3 cells (Fig. 1A). In addition, we found that Nrf2accumulated in the lung tissues 3 hours after intraperitonealinjection of urethane (1 g/kg body weight) into Nrf2þ/þ mice(Fig. 1B). As the Nrf2 accumulation could be monitored by theimmunohistochemistry for Nrf2–b-galactosidase fusion pro-tein expressed from the Nrf2-targeted allele (9), we conductedanti-b-galactosidase antibody staining with paraffin-embed-ded lung sections. The Nrf2–b-galactosidase fusion proteinpredominantly accumulated in the bronchial epithelium of theurethane-treated mice (arrows in Fig. 1C). These results showthat urethane induced Nrf2 accumulation in the nucleus ofairway epithelial cells.

To clarify downstream events upon the urethane treatment,we next examinedmRNA expression of Nrf2 target genes in thelung. Expression levels of Nrf2 target genes, i.e.,Nqo1,HO-1, andGclc, were markedly induced in the Nrf2þ/þ mice in a time-dependent manner (Fig. 1D). In contrast, inducible expressionof the Nrf2 target genes was attenuated in the Nrf2�/� mice.These observations show that urethane-treatment increasedthe expression of Nrf2-target genes through the induction ofNrf2 protein accumulation in normal lung tissue.

Cyp2e1 expression is independent of Nrf2 activityIt has been shown that Cyp2e1-mediated oxidation plays an

essential role in urethane-induced carcinogenicity (Supple-mentary Fig. S1A) and, indeed, Cyp2e1-deficient mice areresistant to urethane-induced lung carcinogenesis (14). Toclarify whether the Nrf2-deficiency affects the urethane bioac-tivation to its carcinogenic metabolite, we examined the

Nrf2 in Kras-Mutant Lung Cancer

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Cyp2e1 level in Nrf2�/�mouse lungs. Basal Cyp2e1mRNA leveldid not differ substantially between Nrf2�/� and Nrf2þ/þmice,and induced Cyp2e1 mRNA expression after the urethaneadministration was comparable between the 2 genotypes(Supplementary Fig. S1B). Cyp2e1 immunoreactivity in theairway epithelial cells was also comparable between the 2genotypes, regardless of the urethane treatment (Supplemen-tary Fig. S1C). These results indicate that the basal and inducedexpression of Cyp2e1 is independent of the Nrf2 activity. Inaddition, we examined mRNA expression of the enzymesinvolved in the detoxification process of vinyl carbamateepoxide. We found that urethane-induced expression of mEHwas diminished in Nrf2�/� mice in comparison with those ofNrf2þ/þmice (Supplementary Fig. S1D).Gstp1/p2 expression inNrf2�/� mice at both basal and urethane-induced states waslower than that in Nrf2þ/þ mice. Together, these observationssupport our contention that both, maintained Cyp2e1-medi-ated oxidation and attenuated mEH-Gst detoxification inNrf2�/� mice, lead to the accumulation of a higher amountof vinyl carbamate epoxide than that in Nrf2þ/þmice upon theurethane treatment.

Urethane elicits acute inflammatory response inNrf2-deficient mouse lung

Nrf2�/� mice are susceptible to a number of oxidative orelectrophilic insults, including butylated hydroxytoluene andbleomycin, and the mice are more susceptible to pneumonia,

fibrosis, and inflammatory cell infiltration than Nrf2þ/þ mice(19). Given this, we next examined inflammatory status of thelungs after the urethane treatment. We observed a largenumber of inflammatory cell foci in the pulmonary perivas-cular region of the Nrf2�/� mice 24 hours after urethaneadministration, whereas such inflammatory cell infiltrationwas not observed in Nrf2þ/þmouse lungs (Supplementary Fig.S2A). These cell clusters mainly consisted of CD3þ T-lympho-cytes. Pan-cytokeratinþ epithelial cells were also involvedfrequently (Supplementary Fig. S2B). Notably, we observed agreater increase in Ki-67–positive cells in the cell clusters ofNrf2�/� mice, which presumably represent increased prolifer-ation of the epithelial cells (Supplementary Fig. S2B). There-fore, we surmised that Nrf2�/� mice were susceptible to theurethane-induced inflammatory lung injury; thereafter, theinflammation-induced proliferating pulmonary epithelial cellsmight give rise to high frequency of lung tumorigenesis.

Nrf2�/� mice are susceptible to urethane-induced lungcarcinogenesis in early stage

To examine susceptibility to lung cancer, we adopted ure-thane-induced carcinogenesis experiments with Nrf2�/� andNrf2þ/þ mice in the following 4 different observation periods:(i) very short-term observation (4 weeks); (ii) short-termobservation (8 weeks); (iii) middle-term observation (16weeks); and (iv) long-term observation (24 weeks; Supplemen-tary Fig. S3). At 4 weeks after the intraperitoneal urethane

Figure 1. Urethane administration activates Nrf2 and its downstream genes in vitro and in vivo. A and B, Nrf2 expression was detected by Western blotting inNIH3T3 cells and urethane-treated mouse lungs. Diethyl maleate (DEM) and PBS were used as positive and negative controls, respectively. Anti-lamin Bantibody staining was used for equal nuclear protein loading control. C, induction of Nrf2-LacZ protein by urethane treatment in lung epithelial cells inNrf2�/�

andNrf2þ/þmice. Arrows indicate positive cells. The black box indicates a highmagnification region. Black scale bar, 20 mm; red scale bar; 5 mm.D, inductionof Nrf2-target genes by urethane treatment in lung. mRNA expression of Nrf2-target genes was examined by qRT-PCR analysis using lung tissues fromNrf2�/� andNrf2þ/þmice after the urethane treatment. The expression level of eachmRNAwas normalized to the b-actin abundance. The data are presentedas the mean � SD. The significant differences determined by Student t test are indicated (�, P < 0.05 and ��, P < 0.01).

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injection, Nrf2�/� mice showed a much larger number ofmicroscopic nodules (average 11.7, n ¼ 3; P < 0.05) than theNrf2þ/þmice (average 1.67, n¼ 3; Fig. 2A and B), whereas bothgroups rarely showed gross surface tumors. At 8 weeks afterurethane administration, all the urethane-treated Nrf2�/�

mice developedmacroscopic (f > 0.5mm) lung surface tumors,whereas only half of the Nrf2þ/þ mice developed grosssurface tumors (Fig. 2C and D, Table 1, A). Furthermore,Nrf2�/� mice had a much higher number of lung surfacenodules than Nrf2þ/þ mice. These results indicate that Nrf2contributes to the prevention of urethane-induced carcino-genesis in the early tumorigenic stages (4 or 8 weeks).

Nrf2�/� mice are resistant to urethane carcinogenesis inthe middle-term observation period

Given the oncogenic function of NRF2 in human cancers(20), we hypothesized that Nrf2�/� tumor cells might have alower proliferative potency than Nrf2þ/þ tumor cells. To testthis hypothesis, we conducted a middle-term observation (16weeks; Supplementary Fig. S3). The total number of grosssurface tumors (f > 0.5 mm) per mouse was comparablebetween 2 genotypes (Fig. 2F and Table 1, B). However, whenwe determined the diameter of the largest tumors in theindividual lung, we found that, while all Nrf2þ/þ mice devel-oped large tumors (f> 1.5mm), only 1 out of the 8Nrf2�/�miceharbored such large tumors (Fig. 2E andG). Furthermore, whenwe measured all tumors in the individual lung, the averagediameter of Nrf2þ/þ tumors tended to be larger (average 1.2mm) than those of Nrf2�/� mice (average 0.9 mm; Fig. 2H).These results show that the Nrf2�/� mice showed a lowersusceptibility to urethane-induced carcinogenesis than theNrf2þ/þ mice at 16 weeks after the urethane administration.

Nrf2-deficient mice are resistant to urethanecarcinogenesis in the long-term observation period

An observation at an even late stage revealed more signif-icant difference in the cancer progression betweenNrf2�/� andNrf2þ/þ mice. At 24 weeks after the urethane administration(Supplementary Fig. S3D), Nrf2�/� mice exhibited a markedlyreduced number of tumors compared with the Nrf2þ/þ mice(Fig. 3A andBandTable 1, C). In addition, the size of the tumorswas larger inNrf2þ/þmice than inNrf2�/�mice. Indeed, 60%ofmice in theNrf2þ/þ group exhibited large palpable nodules (f >2 mm), including some that were very large (f > 5 mm), whilenoNrf2�/�mice formed such large tumors (Fig. 3B). Consistentwith this observation, the average tumor diameter of Nrf2þ/þ

tumors was larger than that of Nrf2�/� mice (1.2 vs. 0.8 mm,respectively; Fig. 3C). Importantly, the heterozygous (Nrf2þ/�)mice showed an increased number of lung surface tumorscompared with the Nrf2�/� mice (Fig. 3B), suggesting that theNrf2 abundance is a critical determinant of the lung cancergrowth.

Showing very good agreement with themacroscopic obser-vations, histologic examinations revealed that the Nrf2þ/þ

mice developed malignant adenocarcinomas at a higherfrequency than the Nrf2�/� mice (Fig. 3D and E). Tumors inthe Nrf2þ/þ mice exhibited extensive invasion into the sur-rounding tissues, but Nrf2�/� tumors were minimally inva-sive with a clear border. Tumors in the Nrf2þ/þ mice seemedto show a higher number of Ki-67–positive cells than those inthe Nrf2�/� mice (Fig. 3D and F). Nrf2þ/þ tumors exhibitedaccumulation of mucosubstances stained with periodic acid-Schiff (PAS; Fig. 3D), which correlates with progression to

Figure 2. Urethane-induced lung tumorigenesis in the short- and middle-term observation protocols. A–D, micro- and macroscopic examinationof precancerous nodules at 4 and 8 weeks after urethane administration.A, histologic examination of precancerous lesions in the lung of Nrf2þ/þ

and Nrf2�/� mice. Lung sections from mice at 4 weeks after urethaneadministration were stained with hematoxylin and eosin. Scale bar, 100mm. B, number of microscopic nodules in Nrf2þ/þ and Nrf2�/� mice fourweeks after urethane administration. C, representative gross observationof lung surface tumors inNrf2þ/þ andNrf2�/�mice. Lungs frommice at 8weeks after urethane administration were examined. Arrowheads showthe surface tumors. Scale bar, 10 mm. Bottom, representativehematoxylin and eosin-stained sections. Scale bar, 100mm.D, number oflung surface tumors in eachmouseof both genotypes. E–H,macroscopicexamination of surface tumors at 16weeks after urethane administration.E, representative gross pictures of lung surface tumors in Nrf2þ/þ andNrf2�/� mice. Arrows indicate the lung surface tumors. Scale bars,10 mm. F, numbers of surface tumors in lungs of Nrf2þ/þ and Nrf2�/�

mice. Each dot represents total number of macroscopic tumors (f >0.5mm) in individualmouse.G, number of large-size nodules (f > 1.0mm)in lungs of Nrf2þ/þ and Nrf2�/� mice. The color of dots indicates size ofthe largest tumor in each mouse lung. H, average of tumor diameters inlungs ofNrf2þ/þ andNrf2�/�mice. The significant differences by Studentt test are indicated (�, P < 0.05 and ��, P < 0.01).






adenocarcinoma (21). Taken together, these observationsshow that Nrf2 plays important roles for the malignanttransformation of lung adenomas in the late stage ofcarcinogenesis.

Urethane-induced lung tumors of Nrf2�/� mice failed toengraft in nude mice

One of the standard approaches in testing the tumorigenic-ity of cancer cells is to transplant the cells into immunode-ficient mice (22). When highly metastatic 3LL cells weretransplanted into Nrf2�/� mice, Nrf2 deficiency generated amore permissive microenvironment for cancer cell growththan in Nrf2þ/þ mice (9). We, therefore, hypothesized that theless proliferative Nrf2-deficient tumor cells might be able togrow in a tumor-permissive microenvironment in the Nrf2�/�

mice. To compare the cell-autonomous proliferative ability ofNrf2þ/þ and Nrf2�/� tumor cells by excluding the host envi-ronmental factors, we excised lung tumors of approximatelyequal sizes (f¼ 1mm) from theNrf2�/� andNrf2þ/þmice andtransplanted these tumors into nude mice. During a 5-monthobservation period, Nrf2�/� tumors failed to engraft and growin the nude mice, whereas Nrf2þ/þ tumors grew progressively

to an approximately 50-fold increase in volume (Fig. 3G andH).These results show that Nrf2�/� tumor cells suffer from a cell-autonomous growth defect.

Nrf2-deficiency decreased the malignancy-risk genesignature

To clarify whether Nrf2 is functionally activated in urethane-induced tumors, we examined the expression of glutathioneperoxide (Gpx2) and multidrug resistance–associated protein4 (Mrp4) mRNAs, as both Gpx2 and Mrp4 contribute to thepromotion of cancer (23, 24). The Gpx2 and Mrp4 mRNAexpressions were significantly increased in the tumors com-pared with the normal lung tissues in Nrf2þ/þ mice (Fig. 4A),supporting the notion that accumulated Nrf2 contributes tothe proliferation of urethane-induced adenocarcinomas. Tocomprehensively examine changes in the gene expressionprofile, we conducted microarray analyses using the compa-rable size of lung tumors (1.0 mm < f < 1.5 mm) and intactnontumor lung tissues derived either from Nrf2þ/þ (n ¼ 8) orNrf2�/�mice (n¼ 8) at 16 weeks after the urethane treatment.

Of the 114 genes increased in the lung tumor tissues, 57geneswere increased solely in theNrf2þ/þ tumors and 25 genes

Table 1. Summary of urethane-induced lung carcinogenesis experiments

A. Short-term observation (8 weeks) after single urethane administration in Nrf2þ/þ and Nrf2�/� mice

Incidence of lung surface tumorsAverage number of lung surface tumors

per mouse

Tumor size (mm) 0.5 < f

Nrf2þ/þ (n ¼ 10) 5/10 (50.0%) 0.8 � 0.5 (n ¼ 10)Nrf2�/� (n ¼ 9) 9/9b (100.0%) 3.1 � 1.1a (n ¼ 9)

B. Middle-term observation (16 weeks) after single urethane treatment in Nrf2þ/þ and Nrf2�/� mice

Incidence of lung surface tumorsAverage number of lung surface tumors

per mouse

Tumor size (mm) 0.5 < f 1.5 < f 0.5 < f 1.5 < f

Nrf2þ/þ (n ¼ 7) 7/7 (100.0%) 7/7 (100.0%) 18.1 � 13.3 4.7 � 3.4Nrf2�/� (n ¼ 8) 8/8 (100.0%) 1/8a (12.5%) 11.3 � 7.0 0.125 � 0.3a

C. Long-term observation (24 weeks) after four weekly contiguous urethane-treatment in Nrf2þ/þ and Nrf2�/� mice

Incidence of lung surface tumorsNumber of lung surface tumors

per mouse

Tumor size (mm) 0.5 < f < 2.0 2.0 < f 0.5 < f 2.0 < f

Nrf2þ/þ (n ¼ 10) 10/10 (100.0%) 6/10a (60.0%) 35.7 � 22.5a 4.9 � 6.59a

Nrf2þ/� (n ¼ 5) 5/5 (100.0%) 2/5b (40.0%) 37.0 � 10.9a 0.4 � 0.55b

Nrf2�/� (n ¼ 10) 10/10 (100.0%) 0/10 (0.0%) 16.4 � 12.6 0.0 � 0.0

aP < 0.01 compared with wild-type mice.bP < 0.05 compared with wild-type mice.

Satoh et al.





were increased solely in the Nrf2�/� tumors, whereas 32 geneswere increased in both tumor genotypes (Fig. 4B and C;Supplementary Tables S2A–S2C for details). Lung adenocarci-nomas tend to be associated with increased expression of avariety of lung development-related genes (21, 25). In theexpression array data, we noticed upregulation of a series oflung development-related genes, including Sox9 (21, 26), Id2(21), Nkx2-1 (27), Foxa2 (28), and N-myc (29) in the Nrf2þ/þ

tumors compared with the Nrf2�/� tumors (Fig. 4D). It hasbeen reported that Sox9 accelerates malignant transformationthrough an increase in Cdk4 expression (26). Indeed, theincreased expression of Sox9 in the Nrf2þ/þ tumor was accom-panied by an increase in Cdk4 expression. Similarly, cyclin D1

expression was more significantly increased in the Nrf2þ/þ

than in the Nrf2�/� tumors, and this observation is consistentwith the theory that many oncogenic signaling pathwaysconverge to elevate cyclin D1 expression at the transcriptionlevel (30).

Consistent with these results, we noticed that majority ofthe genes preferentially expressed in the Nrf2þ/þ rather thanin the Nrf2�/� tumor are classified into the Cellular Devel-opment, Cell Growth and Proliferation, and EmbryonicDevelopment categories by the Ingenuity Pathway Analysis(IPA) software analysis. Furthermore, this pathway analysisrevealed that the Nrf2þ/þ tumors were associated with moreprominent activation of a number of carcinogenic or stem

Figure 3. Urethane-induced lungcarcinogenesis in the long-termobservation protocol. A, grossobservation of lungs (left) andhematoxylin and eosin staining oflung sections (right) of Nrf2þ/þ andNrf2�/� mice. Black scale bar, 10mm; blue scale bar, 400 mm. B,number of lung surface tumors (f > 1mm) in the Nrf2þ/þ, Nrf2þ/�, andNrf2�/�-mice. The color of dotsindicates size of the largest tumor ineach mouse lung. C, average tumordiameter in each mouse group. D,representative pathologicphotomicrographs of the urethane-induced lung tumors in the Nrf2þ/þ

andNrf2�/�mice byhematoxylin andeosin (top), Ki-67 (middle), and PASstaining (bottom). Highermagnification images of eachgenotype are in the inlets. Blackscale bars, 50 mm; blue scale bars,10 mm. E, number of totaladenocarcinomas in a section fromeach mouse. F, number of Ki67-positive cells in similar size tumors(f 100 mm) of Nrf2þ/þ and Nrf2�/�

mice. G and H, Nrf2 is necessary forcolonization and growth of urethane-induced tumors in nude mice. G,reduced growth of Nrf2�/� tumorscompared with Nrf2þ/þ tumorssubcutaneously transplanted in nudemice. H, representative photographs(top) and photomicrographs (middleand bottom) from each genotype.Black scale bar, 10 mm; blue scalebar, 1 mm; white scale bar, 20 mm.The significant differences byStudent t test are indicated(�, P < 0.05 and ��, P < 0.01).






cell–related pathways, including Wnt/b-catenin signalingand Notch signaling compared with the Nrf2�/� tumors,suggesting substantial contribution of Nrf2 to cancer pro-gression (Supplementary Fig. S4A and S4B).

Low frequency of constitutively active Kras mutations inNrf2-deficient mice

Krasmutations are strongly associated with the progressionof adenocarcinomas (29, 31) and Kras activation induces Nrf2mRNA expression to exert its oncogenic activity as summa-rized in Fig. 5A (32). However, frequency of the Krasmutationsin theNrf2�/� cancer cells has not been evaluated. As urethaneis known to evoke constitutively active mutations in Kras,particularly at codon 61 (CAA!CGA; Gln!Arg) in the second

exon (33), we sequenced the second exon of theKras gene in theNrf2�/� lung tumors to clarify the somatic Kras mutationstatus (Fig. 5C). Importantly, all the Nrf2þ/þ tumors exhibitedcodon 61 substitution, whereas only 1 out of 13Nrf2�/� tumorsshowed a Krasmutation (Fig. 5D). We concomitantly observedan increase of mRNA abundance of Kras signaling pathwaygenes, including Erk1 and c-Myc, in the Nrf2þ/þ lung tumors,whereas the mRNA levels of these genes were not significantlyincreased in the Nrf2�/� tumors (Fig. 5B). These resultsrevealed that the Nrf2-deficient tumors were associated witha decreased frequency of Kras-activatingmutations anddimin-ished expression of the Kras pathway genes.

Secreted phosphoprotein 1 or osteopontin (Spp1) is asecreted glycoprotein highly expressed in several types of

Figure 4. Differential gene expression in the urethane-induced lung tumors (4 months after urethane treatment) of Nrf2�/� mice. A, expression ofGpx2 and Mrp4 in the tumors and nontumor regions of Nrf2�/� and Nrf2þ/þ mice. B, Venn diagram of the differentially expressed genes in the Nrf2þ/þ andNrf2�/� tumors. The numbers of genes highly expressed in tumors compared with nontumor tissues of both genotype mice are shown together with theoverlap in both genotypes of mice. C, clustering analysis of differentially expressed genes in the lung tumors of Nrf2þ/þ and Nrf2�/� mice. Geneexpression values (shown below B) using the color scheme (green-black-red) indicate low-moderate-high gene expression levels compared with thecorresponding nontumor tissues. Heat map comparisons of genes preferentially expressed inNrf2þ/þmouse tumors (left) and those preferentially expressedin Nrf2�/� tumors (right) are shown. D, qRT-PCR analysis of representative mRNAs for cancer-related and lung development-related genes. Data arepresented as mean � SD. The significant differences by Student t test are indicated (�, P < 0.05 and ��, P < 0.01).

Satoh et al.





cancers and precancerous lesions. The high-level expression ofSpp1 is frequently associated with a high-grade malignancy oflung adenocarcinomas and poor clinical prognosis of thepatients (34). Spp1-deficient mice are resistant to chemicallyinduced skin tumorigenesis, suggesting the oncogenic functionof Spp1 (35).We found that Spp1 expressionwas 2.5-fold higherin Nrf2�/� tumors than in Nrf2þ/þ tumors (Fig. 4D). A micro-array analysis revealed that a number of other lung cancer–related genes, such as Grin2A (36), Itgav (37), and Ptk6 (38),were preferentially induced in the Nrf2�/� tumors. These datasuggest that the Nrf2�/� cancer cells develop through Kras-independent oncogenic pathways.

Nrf2-deficient cancers show reduced potency foractivation of MDSCsMDSCs are potent immunosuppressor cells, which are

increased in many types of cancer hosts and create immunetolerance to cancers (39). The intracellular ROS level primarilydetermines the immunosuppressive activity of MDSCs, whichdecreases theCD8-mediated cancer immune response throughperoxynitrite modification of the T-cell receptor (TCR)–CD8complex (40). We previously found that transplantation of 3LLcells induced abundant ROS accumulation in the MDSCspopulation of the Nrf2�/� host mice, which led to the gener-ation of a tumor-permissive microenvironment (9). To delin-eate changes in the host immune microenvironment afterurethane treatment, we examined ROS levels in the MDSCs'fraction (MDSCs-ROS) and population of CD8þ-T-cells at 16weeks after the urethane administration, when the urethane-induced tumor numbers were comparable between Nrf2�/�

and Nrf2þ/þ mice. The tumor-bearing Nrf2þ/þ mice showedincreased MDSC-ROS levels compared with the concurrentlyvehicle-treated mice (Fig. 6A). However, the MDSC-ROS levels

in the tumor-bearingNrf2�/�micewere only slightly increasedrelative to the vehicle-treated mice.

Consistent with the increase of MDSC-ROS levels, the num-ber of splenic CD8þ-T-cells was markedly decreased in thetumor-bearing Nrf2þ/þ mice (Fig. 6C and E). In contrast, theCD8þ-T-cell population was not changed substantially intheNrf2�/�mice regardless of tumor progression. The numberof CD4þ-T-cells was almost equivalent, irrespective of tumorbearing in both mouse genotypes (Fig. 6C and D). We normal-ized the population of CD8þ-T-cells with that of CD4þ-T-cellsand further confirmed the preferential reduction of CD8þ-T-cell population in the tumor-bearing Nrf2þ/þ mice (Fig. 6F).These observations emphasize that MDSCs predominantlysuppress CD8þ-T-cell proliferation in the tumor-bearingNrf2þ/þ mice (9).

It has been reported that malignant cancers release a set ofsoluble factors, including granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulatingfactor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin (IL)-3, IL-6, and VEGF, which facilitate therecruitment and ROS accumulation in MDSCs (41). Weobserved that GM-CSF expression was more highly increasedin Nrf2þ/þ tumors than in Nrf2�/� tumors (Fig. 6B). Overall,these results argue that Nrf2�/� tumors have reduced potencyfor the induction of ROS accumulation in MDSCs and, there-fore, the CD8þ-T-cell immunity is barely diminished in thetumor-bearing Nrf2�/� mice.

DiscussionIn this study, we have shown that Nrf2�/� mice are

sensitive to chemical carcinogens and exhibit a high-levelof cancer initiation in the early stages of urethane-inducedlung carcinogenesis. In stark contrast, in the late stages of

Figure 5. Constitutively active Kras mutation frequently observed in the lung tumors of urethane-treated mice. A, schematic diagram of the Krassignaling pathway. Blue arrows indicate the genes highly induced in the Nrf2þ/þ tumor. B, expression of mRNAs coding for representative Kras downstreamfactors. mRNAswere quantified by qRT-PCR analysis using lung tumors fromNrf2�/� andNrf2þ/þmice 16weeks after urethane treatment. C, left, the A-to-Gsubstitution at the second nucleotide of codon 61 in the second exon of the Kras gene in the Nrf2þ/þ tumors. Right, the Nrf2�/� tumors showed intactnucleotide sequences of codon 61. D, frequency of Kras codon-61 mutation (CAA!CGA; Gln!Arg) detected in the tumors from Nrf2�/� or Nrf2þ/þ mice.Significant differences were determined using Fisher exact test (��, P < 0.01).






urethane carcinogenesis, the Nrf2�/� mice developed can-cers that were significantly less malignant than did thecontrol Nrf2þ/þ mice. These results indicate that the Nrf2activity accelerates the malignant transformation of benignadenoma to adenocarcinoma. The molecular basis of theNrf2 contribution to the urethane carcinogenesis is sum-marized in Supplementary Fig. S5.

The constitutively active Kras mutations have been identi-fied in many types of human cancers, including those of thelung, pancreas, and colon, as well as in experimental cancermodels of rodents (42). Urethane is known to cause Kras codon61 mutations in multiple mouse strains (33). An importantobservation is that Kras activates Nrf2 and leads to the cancerby malignant transformation (32). Therefore, in the Nrf2�/�

mice, even if lung cells suffer a large number of initial genetichits by the urethane metabolites, subsequent progression of

malignant transformation is markedly attenuated because ofthe lack of Nrf2 activity.

In contrast, several chemical carcinogens are known torarely induce activating Kras mutations. Such carcinogensinclude N-nitrosobutyl (4-hydroxybutyl) amine, azoxymeth-ane, and 7,12-dimethyl-benz[a]anthracene. Notably, these car-cinogens are found to provoke cancers more abundantly in theNrf2�/� mice than Nrf2þ/þ mice (3, 6, 43). We surmise thatthese Kras-independent carcinogens evoke a higher number ofcancers in the Nrf2�/� mice as a direct consequence of theincreased chemical susceptibility and activation of other onco-genic pathways.

Spp1, an integrin-binding and cell transformation-relatedprotein, highly associates with the malignancy of non–smallcell lung carcinoma (34). We found that Spp1 is much moreabundantly expressed in Nrf2�/� than in Nrf2þ/þ tumors,

Figure 6. Nrf2-deficient cancersshow reduced potency foractivation of MDSCs. Nrf2�/� miceharboring urethane-induced lungtumors exhibit a lower level of ROSaccumulation in MDSCs. A,quantification of ROS levels of theMac1þGr1þ MDSCs fraction in thebone marrow cells from Nrf2�/� orNrf2þ/þ mice harboring lungtumors (left). Data are presented asthe median � SD (��, P < 0.01 byStudent t test; n¼ 3 in each group).Representative histogram of ROSlevel analysis with DCFDA inMDSCs from bone marrow ineach group (right). B, GM-CSFexpression level was significantlyrepressed in the Nrf2�/� tumors incomparison with the Nrf2þ/þ

tumors. C–F, splenic CD8þ-T-cellpopulation was decreased in thetumor-bearing Nrf2þ/þ micecompared with the tumor-bearingNrf2�/� mice. C, representativeflow cytometric dot plots of spleencells using CD4 and CD8antibodies. The green framesindicate the CD8þ-T-cellpopulation. D, the CD4þ-T-cellpopulation was comparablebetween both genotype groups.E, splenic CD8þ-T-cell populationwas decreased in the Nrf2þ/þ micebearing tumors more significantlythan in the vehicle-treated Nrf2þ/þ

mice. F, population of CD8þ-T cellsare normalized with that of CD4þ-Tcells. Data are presented as themedian � SD (�, P < 0.05 usingStudent t test; n¼ 3 in each group).

Satoh et al.





suggesting that Spp1 contributes to lung tumorigenesis inNrf2�/� mice. Because forced expression of Nrf2 in a chon-drocyte cell line significantly decreased Spp1mRNA expression(44), Nrf2 may act as a negative regulator for Spp1 geneexpression. These results, thus, imply that Spp1may compriseone alternative oncogenic pathway that replaces the Kras-Nrf2 pathway.Nrf2 activators have been shown to exert preventive effects

against various types of carcinogens in animal models andhumans (45, 46). In addition, therapeutic efficacy of a potentNrf2 activator, bardoxolonemethyl (CDDO-Me), was shown forthe treatment of chronic kidney disease and diabetic nephrop-athy in a large clinical trial (47). Our results unequivocallyshowed that Nrf2 is critical for the prevention of the initiationstep of lung carcinogenesis. Therefore, preventive treatmentthat attains Nrf2 activation seems to reduce the cancer initi-ation andwould be of particular value for those who are at highrisk of lung cancers, such as those with a history of heavysmoking and inhalation of asbestos. On the other hand, treat-ment with Nrf2 inhibitors through the lung cancer-targeteddrug delivery system would reduce the cancer lesions associ-ated with oncogenic Kras mutation. Overall, this study hasrevealed that Nrf2 is a prime candidate of personalized cancertreatment in the near future, and development of Nrf2 inhi-bitors and activators, as well as accurate diagnostic proceduresquantifying Nrf2 expression levels in lung cancer cells, arecritically important for this purpose.

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

Authors' ContributionsConception and design: H. Satoh, T. Moriguchi, M. YamamotoDevelopment of methodology: H. Satoh, T. Moriguchi, J. TakaiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Satoh, T. Moriguchi, J. TakaiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Satoh, T. Moriguchi, J. TakaiWriting, review, and/or revision of the manuscript: H. Satoh, T. Moriguchi,M. YamamotoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Satoh, T. MoriguchiStudy supervision: T. Moriguchi, M. Ebina, M. Yamamoto

AcknowledgmentsThe authors thank Drs. Hozumi Motohashi, Michito Hamada, and Jon Maher

for their insightful advice and helpful discussions.

Grant SupportThis work was financially supported in part by Grants-in-Aid for Scientific

Research from the Ministry of Education, Culture, Sports, Science, and Tech-nology and the Japan Society for Promotion of Science (T. Moriguchi and M.Yamamoto), Scientific Research on Priority Areas (M. Yamamoto), and SpeciallyPromoted Research (M. Yamamoto). H. Satoh was supported by ResearchResident Fellowship from the Promotion of Cancer Research Japan for the ThirdTerm Comprehensive 10-Year Strategy for Cancer Control.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 9, 2012; revised March 14, 2013; accepted April 1, 2013;published OnlineFirst April 22, 2013.

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2013;73:4158-4168. Published OnlineFirst April 22, 2013.Cancer Res Hironori Satoh, Takashi Moriguchi, Jun Takai, et al. Kras Signaling Pathway during Lung CarcinogenesisNrf2 Prevents Initiation but Accelerates Progression through the

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