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Published OnlineFirst August 3, 2010; DOI: 10.1158/1535-7163.MCT-10-0108
Published OnlineFirst on August 3, 2010 as 10.1158/1535-7163.MCT-10-0108
Molecular
Cancer
apeutics

linical Development

ression of ABCG2 (BCRP) Is Regulated by Nrf2 incer Cells That Confers Side Population and

Ther

moresistance Phenotype

ingh1, Hailong Wu1, Ping Zhang1, Christine Happel1, Jinfang Ma1, and Shyam Biswal1,2

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-binding cassette, subfamily G, member 2 (ABCG2) is expressed in both normal and cancer cells anda crucial role in side population (SP) formation and efflux of xenobiotics and drugs. Nrf2, a redox-g transcription factor, on constitutive activation in non–small-cell lung cancer cells upregulates a wideum of genes involved in redox balance, glutathione metabolism, and drug detoxification, which con-to chemoresistance and tumorigenicity. This study examined the mechanism underlying Nrf2- depen-xpression of ABCG2 and its role in the multidrug resistance phenotype. In silico analysis of the 5′-ter flanking region of ABCG2 identified an antioxidant response element (ARE) at −431 to −420 bp.iled promoter analysis using luciferase reporter assays showed that ARE at −431 to −420 bp is criticalNrf2-mediated expression in lung cancer cells. Electrophoretic mobility shift assays and chromatin

noprecipitation assays revealed that Nrf2 interacts with the ABCG2 ARE element at −431 to −420 bpo and in vivo. Disruption of Nrf2 expression in lung and prostate cancer cells, by short hairpin RNA,ated the expression of ABCG2 transcript and protein, and dramatically reduced the SP fraction in Nrf2-ed cancer cells. Moreover, depleted levels of ABCG2 in these Nrf2 knockdown cells sensitized them tontrone and topotecan, two chemotherapy drugs detoxified mainly by ABCG2. As expected, overex-on of Nrf2 cDNA in lung epithelial cells led to an increase in ABCG2 expression and a 2-fold higher SP
pressi
fraction. Thus, Nrf2-mediated regulation of ABCG2 expression maintains the SP fraction and confers che-moresistance. Mol Cancer Ther; 9(8); 2365–76. ©2010 AACR.

Reccancewhichcells.criticaand thare ofcuraticanceroblas

duction

g cancer is the leading cause of cancer-relateds in both men and women in the United Stateshe prognosis for lung cancer remains poor, witherall 5-year survival of 14%. The death toll causedg cancer alone counts more than that of breast, co-al, and prostate cancers combined. Non–small cellcarcinoma (NSCLC) constitutes ∼85% of all lungrs (1). Chemotherapy is the standard treatment for

LC patients, but chemotherapy resistancetacle and leads to mortality.

cancecell potanceof whcancein theulatiothat HcancecassetABCG(BCRPbreastthe ch(3, 6).matio

ns: 1Department of Environmental Health Sciences,l of Public Health and 2Department of Oncology,omprehensive Cancer Center, Johns Hopkins

re, Maryland

ary material for this article is available at Molecularcs Online (http://mct.aacrjournals.org/).

nd P. Zhang contributed equally to this work.

uthor: Shyam Biswal, Johns Hopkins University,of Public Health, E7624, 615 North Wolfe Street, Bal-. Phone: 410-955-4728; Fax: 410-955-0116. E-mail:

7163.MCT-10-0108

ssociation for Cancer Research.

ls.org

on April 4, 2020. © 2010mct.aacrjournals.org ed from

ent discoveries have provided clear evidence thatrs may develop from rare self-renewing stem cells,are biologically distinct from differentiated cancer

The eradication of these cancer stem cells is likely al component of any successful anticancer strategy,is may explain why conventional cancer therapiesten effective in reducing tumor burden but are rarelyve. Cancer stem cells have been identified in severalrous tissues, such as acute myelogenic leukemia, neu-toma, lung, colon, and breast cancers (2–4). Theser stem cells represent only a small percentage of totalpulations. They show distinct features such as resis-to irradiation and chemotherapy, and reconstitutionole populations after irradiation (3, 5). Interestingly,r stem cells efficiently efflux Hoechst dye, resultingdye-negative phenotype, also known as the side pop-n (SP) phenotype (3). Further investigations revealedoechst dye efflux and the SP formation capacity ofr stem cells are largely attributable to ATP-bindingte, subfamily G, member 2 (ABCG2) molecule (6–8).2, also known as breast cancer resistance protein), was originally cloned from multidrug-resistantcancer cells (9). Its upregulation has been linked toemoresistance phenotype in various cancer cells
It was shown that ABCG2 is responsible for SP for-n in lung cancer cells (10, 11).
2365

American Association for Cancer Research.

http://mct.aacrjournals.org/

Nrffactordativtransctectivtion feffluxmembber 2(KEAsteadytion b(15, 16zygosin canmutasquamtion igenesand ttanceThe

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2, a cap ‘n’ collar basic leucine zipper transcription, protects against environmental toxicants, oxi-e injury, inflammation, and apoptosis throughriptional induction of a broad spectrum of cytopro-e genes involved in electrophile/drug detoxifica-unction, including several ATP-dependent drugpumps (e.g., ATP-binding cassette, subfamily C,er 1, and ATP-binding cassette, subfamily C, mem-; refs. 12–14). Kelch-like ECH-associated proteinP1) is a cytoplasmic anchor of Nrf2 and maintains-state levels of Nrf2 and Nrf2-dependent transcrip-y signaling Nrf2 for proteosomal degradation). Somatic mutations in KEAP1 and loss of hetero-ity at KEAP1 locus result in loss of KEAP1 functioncer cells and gain of Nrf2 function (17). Activatingtions in Nrf2 have been recently reported inous cell lung carcinomas (18). Gain of Nrf2 func-n lung cancer cells upregulates the expression ofinvolved in protection against oxidative stress

hereby promotes tumorigenicity and chemoresis-(17, 19–22).ABCG2 gene is highly expressed in the plasmarane of several drug-resistant cell lines, where iten shown to transport antitumor drugs, includingantrone, topotecan, doxorubicin, and daunorubicin23). ABCG2 has also been identified as a protectiveagainst endogenous and exogenous toxic agents.az and tert-butylhydroquinone, which are knownivate Nrf2-dependent gene expression, upregulated2 expression in primary human hepatocytes andn hepatocellular carcinoma cell lines, respectively5). Because Nrf2 is a stress-inducible transcription, which regulates the expression of several cytopro-e genes and drug detoxification enzymes through aon antioxidant response element (ARE) located inromoter, we decided to investigate whether Nrf2tes the expression of ABCG2 as well. A better un-anding of the role of Nrf2 in the regulation of2 expression in cancer cells will help elucidate itsn promoting the multidrug resistance phenotypecer cells. Here, we show that Nrf2 controls ABCG2ssion at the transcriptional level and is requiredaintaining SP in A549 and H460 lung cancer cellsll as in prostate cancer cells. Reduced Nrf2 expres-esults in enhanced sensitivity to mitoxantrone andcan in both A549 and H460 lung cancer cells.

rials and Methods

ulture and reagents9, H460, H23, and Du145 cells were purchasedthe American Type Culture Collection and wereained in DMEM with 10% fetal bovine serum andillin/streptomycin. The cell lines were routinelyfor Mycoplasma contamination. However, the

rs did not attempt to authenticate the cell lines
. Generation of A549 and H460 cells constitutivelyssing short hairpin RNA (shRNA) against Nrf2 or
wild-tmoter

ancer Ther; 9(8) August 2010

on April 4, 2020. © 2010mct.aacrjournals.org Downloaded from

l luciferase shRNA cells with stable expression ofshRNA or control nontargeting luciferase shRNAmaintained in DMEM containing 0.2 μg/mL ofycin (Roche). The human airway epithelial cell linederived from normal lung, was grown in a serum-edium, bronchial epithelial growth medium

M, serum-free), from Cambrex, made of BEBMmedium and SingleQuot additives supplemented50 μg/mL G-418. Details on the generation ofLuchsRNA, A549-Nrf2shRNA, H460-LucshRNA,-Nrf2shRNA, Du145-LucshRNA, and Du145RNAused in this article have been published (21, 22).equence of Keap1 shRNA used to downregulate1 expression was 5′-CCGGGCCTTAATTCAGCT-TGTTCTCGAGAACACTCAGCTGAAT-GCTTTTTTG-3′ (Sigma-Aldrich). Lentiviral Nrf2ssion vector (PLOHS_100067113) was purchasedOpen Biosystems. Mitoxantrone was obtainedSigma-Aldrich, and topotecan was purchased fromaboratories.

ification of ARE(s) in the promoter of ABCG2identify the presence and location of AREs in the2 promoter, a 600-bp upstream region sequencethe transcription start site and exons 1 and 2 wasloaded from the National Center for Biotechnologyation database (Human Genome resources). Thisnce was screened for ARE binding sites with thef Genamics Expression 1.1 software using the pri-core sequence of ARE (RTGABNNNGCR; ref. 26)probe.

ids and mutagenesis5′ flanking region of human ABCG2 promoter re-−496 bp to +198 bp) was PCR amplified from hu-genomic DNA using high-fidelity Taq polymeraseied Biosystems). The primers used for amplificationas follows: forward, 5′-CACTTTCTCAGAATCC-CAC-3′; reverse, 5′-GAACCTTTTGAGTGGGCA--3′. The isolated PCR product was ligated to.1 vector (Invitrogen), and a KpnI-XhoI fragmentthis construct was cloned into the pGL3 basic vectorega). A deletion construct (−310 bp to +198 bp) wasated from the full-length promoter construct. Tothe ARE enhancer sequence in pTAL vector, thebinding site with minimal flanking region wasified using the following primers: forward, 5′-AAAGGTACCATCCCATTCACCAGAAACCA-3′;e primer, 5′-AAAAAACTCGAG CGAACGGAAT-CCAGAGT-3′ . Mutant ARE sequences wereated by using a site-directed mutagenesis kitStratagene. Primers containing the mutant AREnces (GCAGCGCTTGgGcCTGGGCAACCTGT-C) were used for PCR amplification of the mutant2 ARE binding site in the promoter, and PCR pro-were digested with DpnI for 1 hour to cleave the
ype promoter template. The sequence of each pro-construct was verified by sequencing.
Molecular Cancer Therapeutics



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transfection and luciferase activityls were transfected at 75% to 85% confluency usingctamine 2000 (Invitrogen). Briefly, cells were seed-24-well plates at a density of 2 × 105 cells/mL andn overnight. After ∼12 hours, the medium wased, and transfection complex containing 800 ngsmid DNA, 40 ng of pRL-TK plasmid (Promega)atio of 20:1, and transfection reagent were addedch well in the presence of fetal bovine serum.were incubated for another 36 hours, and werelysed and assayed. Renilla and Firefly luciferaseties were measured using the dual luciferase as-it (Promega) with a luminometer (EG&G). Fireflyrase activity was normalized to Renilla luciferasety for calculation of relative reporter activity forconstruct. Results were plotted from three inde-nt experiments, with each assay conducted inate.

cytometrylysis for SP formation was carried out followingotocol of Goodell's laboratory with minor modifica-(27). Briefly, cells (1 × 106/mL) were incubatedC for 60 minutes with 5 μg/mL Hoechst 33342a-Aldrich), washed, and resuspended in ice-coldwith 2% FCS and 2 μg/mL propidium iodidea-Aldrich). Fumitremorgin C (FTC, 10 μmol/L), at and specific inhibitor of ABCG2 activity, was usedositive control for the assay. Side population waszed with fluorescence-activated cell sorting (FACS)ge (Becton Dickinson).

ern blotstern blot was carried out using the protocol pub-by Singh et al. (17). Primary antibody incubationarried out at 4°C for 16 hours with a mouse anti-2 antibody (Sigma-Aldrich) diluted at 1:300 in 3%e serum albumin, followed by incubation withradish peroxidase–conjugated horse anti-mouse sec-y antibody, and developed using an enhancediluminescence detection system (GE Healthcare).α-tubulin and anti–glyceraldehyde-3-phosphate de-genase antibodies were used as the loading controlunoblot assays (Santa Cruz Biotechnology).

roliferation (MTT) assaysmotherapy drug treatments were done followingcols published by Singh et al. (17). The in vitro drugivity experiments were carried out by using a celleration assay kit (Roche) according to the manufac-instructions.

time reverse transcriptase-PCRl-time reverse transcriptase-PCR (RT-PCR) reac-were carried out using a protocol published byet al. (17). Briefly, 500 ng of total RNAwere reverse
ribed using a high-capacity cDNA synthesis kitApplied Biosystems. An aliquot of diluted cDNA
NaCl]solub

acrjournals.org


sed to measure human ABCG2 (Hs01053790_m1)rf2 (Hs00232352_m1), NQO1 (Hs00168547_m1),CLm (Hs00157694_m1) gene expression using

an primer and probe mixes from Applied Biosys-The assays were done using the ABI 7000 TaqMan(Applied Biosystems). β-Actin (Hs99999903_m1)

sed for normalization.

rophoretic mobility shift assaysble-stranded DNA oligonucleotides correspondingconsensus ARE sequence (5′-GCGCTTGTGA-GCAACCTGTGC-3′) in the ABCG2 gene promotersynthesized commercially from Integrated DNAologies. The duplex was end-labeled using T4e (Promega) in the presence of [γ-32P]dCTP (MPemicals). The probe was purified once with NAP-mn (GE Healthcare) according to the manufacturer'sction. The binding reactions were carried out withof nuclear protein isolated from A549-LucshRNAor A549-Nrf2shRNA cells and 32P-labeled probe05 cpm/reaction) in a 20 μL reaction mixture con-g 5 mmol/L HEPES (pH 7.9), 100 mmol/L NaCl,mmol/L EDTA, and 0.25 mmol/L DTT. Polyyinosinic-deoxycytidylic) acid 50 μg/mL (Roche)ncluded in each reaction as nonspecific carrier. The mixture was placed on ice for 30 minutes.n-DNA complexes were separated by electrophore-a nondenaturing 5% PAGE gel at 200 V with a run-buffer consisting of 25 mmol/L Tris, 25 mmol/Lacid, and 1 mmol/L EDTA. Gels were dried, anddioactive bands were visualized by autoradiogra-or competition reactions, either 25- or 50-fold excessunlabeled oligonucleotides harboring the wild-typesequence of the ABCG2 gene or corresponding mu-RE (5′-GCGCTTGGGCCTGGGCAACCTGTGC-3′)dded during the preincubation period. Unlabeleducleotides containing AP1 or NF-κB binding se-e were used as nonspecific competitors.

matin immunoprecipitation assayomatin immunoprecipitation (ChIP) assays werected using the ChIP assay kit from Upstate Cellling according to the manufacturer's instructions.(∼107) were harvested and chromatin was cross-by adding formaldehyde to the cell culture medi-a final concentration of 1% and incubating the

re for 10 minutes at 37°C. Cells were washed twicece-cold PBS containing a protease inhibitor cocktaile) and were suspended in 0.2 mL of lysis buffermol/L Tris-HCl (pH 8.1), with 1% SDS andol/L EDTA]. Samples were sonicated on ice to ange length of 500 to 1,000 bp (four pulses of 10 sec-each) and were centrifuged at 10,000 × g. Solubi-chromatin was diluted 10-fold with ChIP dilution[16.7 mmol/L Tris-HCl (pH 8.1), with 0.01% SDS,

Triton X-100, 1.2 mmol/L EDTA, and 167 mmol/L
and was used for ChIP assays. An aliquot of thele fraction was saved for total chromatin input.
Mol Cancer Ther; 9(8) August 2010 2367



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Singh et al.

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atin was precleared with salmon sperm DNA/in A–agarose for 30 minutes and then incubatedeither antibody to (Nrf2 sc-722X; Santa Cruz Bio-logy), and rabbit IgG for 18 hours at 4°C with ro-. Immunoprecipitation, washing, and elution wered out according to the manufacturer's instructions.-linked immunoprecipitates and total chromatinwere reverse cross-linked, and samples wered with proteinase-K (Sigma) and extracted withl-chloroform-isoamyl alcohol. DNAwas precipitat-d resuspended in 100 μL of TE (10 mmol/L Tris-Cl.1 mmol/L EDTA). Then, 1 μL of DNAwas used for(35 cycles) with primers specific for the ABCG2oter. For PCR amplification, forward primer 5′-CATTCACCAGAAACCA-3′ and reverse primerTCTGGTTCATTCCGTTCG-3′ were used.

tical analysisa are presented as the mean ± SD. To assesstical significance of differences, Student's t teste-way ANOVA was conducted. P values <0.05considered statistically significant as indicatedterisks. IC50 values in drug sensitization studiesobtained by using GraphPad Software.

lts

uation of Nrf2 expression in lung cancer cellsto a decline in ABCG2 mRNA andin levelstudy the Nrf2-dependent regulation of ABCG2 ex-on, we selected A549 and H460 lung cancer cells asel system because they harbor the KEAP1 gene mu-that leads to a high basal level of Nrf2 pathway

ty and are drug resistant (17, 22). We have estab-and characterized A549 and H460 cells constitu-expressing shRNA targeting Nrf2 (17, 22). Shortn targeting luciferase shRNA-expressing cells weres nontargeting control. A549 and H460 cells stablyssing Nrf2 shRNA (A549-Nrf2shRNA, H460-hRNA) showed significant reduction in Nrf2 mRNAssion and a parallel decrease in ABCG2 transcriptcompared with cells expressing luciferase shRNALucshRNA and H460 LucshRNA; Fig. 1A). Immu-t analysis revealed diminished levels of ABCG2n in Nrf2 knockdown A549 and H460 cells com-with control cells (Fig. 1B). The expression of

and ABCG2 did not change significantly betweenntrol cells transfected with luciferase shRNA andntransfected parent cancer cells (Supplementary1).

directly regulates transcriptional expressionCG2nvestigate whether ABCG2 is a direct transcription-get of Nrf2, we used promoter reporter assays to
ABCG2 promoter regulation in Nrf2-proficientcontrol cells or parental A549 cells) and Nrf2-
Nrf2(Fig.



ted (A549-Nrf2shRNA) lung cancer cells. Two2 promoter deletion constructs were made accord-the in silico analysis of ABCG2 promoter that

fied a putative ARE located at −431 to −420 bp up-of the ABCG2 transcription start site (TSS). The

ngth reporter construct contained the putative(−496 bp to +198 bp), whereas truncated reporteruct did not (−310 to +198 bp; Fig. 2A, schematic).wo reporter constructs were transfected into A549l and A549-Nrf2shRNA cells, and luciferase report-ivity was measured. As shown in Fig. 2A, promoterty of the ABCG2 full-length construct (−496 to +198as significantly reduced in Nrf2-depleted A549-hRNA cells compared with Nrf2-proficient A549ol cells. The shorter promoter deletion constructg the putative ARE (−310 to +198 bp) exhibitedr activity in both Nrf2-depleted and control cells.ther confirm that Nrf2-dependent luciferase report-ivity was suppressed in A549Nrf2shRNA cells com-with control cells, a Nrf2-dependent NQO1 basal

oter reporter construct (28) was included in the pro-r reporter studies. The NQO1 reporter plasmided significantly reduced luciferase activity in A549

pressing control luciferase shRNA and Nrf2 shRNA.

1. Nrf2-dependent ABCG2 expression in lung cancer cells.ive expression of ABCG2 in A549 and H460 cells constitutivelying Nrf2shRNA analyzed by real-time RT-PCR. β-Actin was usedalization. Data are presented as fold change calculated using

xpression levels in control luciferase shRNA cells as baseline.unoblot analysis of ABCG2, Nrf2, and glyceraldehyde-3-ate dehydrogenase (GAPDH) expression in A549 and H460

shRNA cells compared with control A549 cells2A). These data strongly suggest that the ARE




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g site in the ABCG2 promoter region is active andons as a transcriptional enhancer. To further con-hat the ARE sequence in ABCG2 promoter func-as an enhancer, we cloned the ABCG2 promotersequence with sufficient flanking sequence into a-Luc reporter vector containing a minimal promoterke a ABCG2 ARE construct, and cloned ABCG2
oter fragment with mutant ARE into a pTAL-Luc site in
e control (lanes 8–11). Nuclear extracts from A549 control cells were used in botcomplex; FP, free probe. D, ChIP assays were done in A549 cells stably expres

acrjournals.org


). Reporter assays results showed that mutation ofRE core sequence significantly inhibited the Nrf2-dent luciferase reporter activity in A549 cells2B). These observations suggest that the AREnt located at −431 to −420 bp is involved in Nrf2-dent regulation of ABCG2.further verify that Nrf2 binds to the ARE binding
the ABCG2 promoter in vitro and in vivo, we per-
uct to make a mutant ARE construct (Fig. 2B, sche- formed electrophoretic mobility shift assays. As shown in

2. Nrf2 regulates ABCG2 transcription through an ARE element in the ABCG2 promoter. A, a putative ARE at −431 to −420 bp in the proximaler region of the human ABCG2 gene was identified by in silico analysis and promoter reporter assay. Left, schematic representation of the twocts with and without putative ARE. The 5′ end of each of the constructs relative to the transcription start site (arrows) is indicated. These constructsansfected into A549 control and A549-Nrf2shRNA cells, and luciferase activity was measured. Luciferase activities were normalized relative toilla luciferase activity. NQO1 basal promoter construct was included as a positive control for measuring Nrf2-dependent reporter expression.G2 promoter fragment containing the ARE core sequence was cloned upstream of heterologous minimal TATA-like promoter driving the luciferaseion (pTAL vector). Mutations in the ARE core sequence (shown on top) were introduced by site-directed mutagenesis. These constructs wereted into A549 cells, and luciferase activity was measured. *, P < 0.05, significant when compared with wild-type ARE (analyzed by Student's t test).tro DNA binding activity of Nrf2 to ABCG2 ARE using electrophoretic mobility shift assays. Nuclear proteins from Nrf2-depleted cells (lane 2) andA549 cells (lane 3) were incubated with 32P-labeled oligonucleotides harboring the ARE consensus sequence of ABCG2 gene. The resultingxes were resolved by nondenaturing PAGE and analyzed. For competition assays, a 25- and 50-fold excess of unlabeled oligonucleotides harboring-type ARE (lanes 4 and 5) of ABCG2 gene or a 25- to 50-fold excess of unlabeled oligonucleotides with the mutated ARE sequence was addedand 7) during the preincubation period. Nonspecific unlabeled oligonucleotides that contain AP1 and NF-κB binding sequences were used as a

h specific and nonspecific competitions (lanes 4–11). Arrow, AREsing Nrf2shRNA and control cells expressing luciferase shRNA.




Fig. 2Cmatiodupletrol Athe Nwhennucleacompdepenand 5tidesthat tpleteltidesless elanesAP1 oNext,Nrf2promtitutivin ABA549Nrf2sreducmoterfailedregioenrichindicain thereportreveathroufrom

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Singh et al.

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, there was a marked Nrf2-dependent complex for-n when 32P-labeled ABCG2 ARE oligonucleotidexes were incubated with nuclear extract from con-549-LucshRNA cells (double arrows). In contrast,rf2-dependent complex formation was reducedthe 32P-labeled ARE probes were incubated withr extracts from Nrf2-deficient A549 cells (Fig. 2C,are lane 3 with lane 2). The specificity of Nrf2-dent complex formation was tested using 25×0× excess of unlabeled ABCG2-ARE oligonucleo-as cold competitor. The competition results showedhe Nrf2-dependent complex formation was com-y abolished by cold wild-type ARE oligonucleo-(Fig. 2C, lanes 4 and 5), but was competed to axtent by mutant ARE oligonucleotides (Fig. 2C,6 and 7) and was poorly competed by nonspecificr NF-κB oligonucleotides (Fig. 2C, lanes 8–11).we conducted ChIP assays to examine whetherconstitutively binds to the endogenous ABCG2oter in cancer cells. As shown in Fig. 2D, cons-e recruitment of Nrf2 to the ARE element locatedCG2 promoter was observed in Nrf2-proficientLucshRNA control cells. As expected, in A549-hRNA cells, Nrf2 was present at low levels anded amplification was detected using ABCG2 pro-primers. Immunoprecipitations with control IgGto enrich the ARE-containing ABCG2 promoter

n (Fig. 2D), suggesting that the sites were noted in a nonspecific fashion. Overall, these resultste that Nrf2 specifically binds to the ARE elementABCG2 promoter. In conclusion, these promoterer studies and in vitro and in vivo binding assaysled that Nrf2 regulates ABCG2 promoter activitygh the ARE element located at −431 to −420 bpthe transcription start site.

asing Nrf2-dependent ABCG2 expression byA results in loss of the SP phenotypeG2 activity is required for maintaining the SP phe-e in cancer cells. To investigate whether reduced2 expression affects SP formation in Nrf2-deficientand H460 cells versus control cells, Hoechst dye ex-n assays were conducted. The results show thatare ∼58% cells that belong to SP fraction in controlLucshRNA cells; on the contrary, the percentage ofops to <2% in Nrf2-knockdown A549 cells. Similar-content in Nrf2-knockdown H460 cells are ∼0.18%ared with ∼1.3% in control H460-LucshRNA cells). To confirm the specificity of the Hoechst dye ef-ssay, we used fumitremorgin C (FTC), a specific in-r of ABCG2. The cells were incubated with Hoechstthe presence of FTC followed by propidium iodideg and flow cytometric analysis. The SP phenotypeompletely blocked by FTC treatment in both A549460 cells (Fig. 3; Supplementary Fig. S2). To deter-if antibiotic selection during generation of stable
es altered the percentage of cells in the SP, we com-the SP in A549-parent cells, A549-LucshRNA, and
SP inDu14



Nrf2shRNA cells (Supplementary Fig. S3A). We de-∼1.75- to 2-fold higher numbers of cells in the SP ofLucshRNA relative to A549-parent cells. Similar re-were obtained with H460-parent, H460-LucshRNA,460-Nrf2shRNA cells (Supplementary Fig. S3B).ver, Nrf2-depleted cells A549 and H460 had dra-ally smaller fraction of cells in SP.

iting Nrf2-dependent ABCG2 expression byA causes chemosensitization to itst anticancer drugsCG2 is crucial for detoxification of several chemo-eutic drugs; thus, its increased expression confersoresistance. It has been reported that efflux ofantrone and topotecan depends on ABCG2 (9, 23).termine whether decreased expression of ABCG2sensitize A549 and H460 cells to mitoxantrone-

opotecan-induced cytotoxicity, we incubated bothdepleted and control A549 and H460 cells with mi-trone and topotecan for 2 to 4 days, and measuredercentage of viable cells using the MTT assay. Asted, the Nrf2-deficient A549 and H460 cells (A549-hRNA and H460-Nrf2shRNA), with low endoge-levels of ABCG2, displayed enhanced sensitivitytoxantrone and topotecan in vitro (Fig. 4). Theof mitoxantrone in A549 LucshRNA and A549-hRNA cells was 31.38 and 23.09 nmol/L, respec-. The IC50 of mitoxantrone in H460-LucshRNA460-Nrf2shRNA cells was 22 and 6 nmol/L, re-vely. Similar to mitoxantrone, Nrf2-depleted A549460 cells showed lower IC50 values for topotecan-LucshRNA, 35.68 nmol/L; A549-Nrf2shRNA,nmol/L; H460-LucshRNA, 78.8 nmol/L; andNrf2shRNA, 30.1 nmol/L).

dependent ABCG2 expression in prostater cellshave established and characterized Du145 cellsitutively expressing shRNA targeting Nrf2ript (Du145-Nrf2shRNA) and the control DU145xpressing shRNA against luciferase gene (Du145-RNA; ref. 21). Du145 cells stably expressinghRNA (Du145-Nrf2shRNA) showed >85% reduc-n Nrf2 mRNA expression and a parallel decreaseCG2 transcript levels compared with cells expres-uciferase shRNA (Du145-LucshRNA; Fig. 5A). Im-blot analysis revealed diminished levels of Nrf2BCG2 protein in Nrf2 knockdown Du145 cells com-with parent Du145 cells and Du145-LucshRNA

Fig. 5B and C). The expression of Nrf2 and ABCG2145-LucshRNA cells was similar to that of the par-u145 cells. To determine whether Nrf2-dependent2 expression in prostate cancer cells affects SP for-n, we used the Hoechst dye exclusion assay. Thenoblotting data presented above show that Du154xpress low levels of ABCG2 protein. Analysis of
Du145-parent cells, Du145-LucshRNA, and
5-Nrf2shRNA cells revealed very low SP fraction




FigureNrf2-deH460 cdye andby flowcells wthe prepercentReactiorepeated three times.


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3. Alteration of the SP phenotype inpleted lung cancer cells. A549 andells were incubated with Hoechst 33342propidium iodide, and were analyzedcytometry. To show assay specificity,ere incubated with Hoechst dye insence of FTC. The SP is shown as aage of the whole viable cell population.ns were done in triplicates and were


April 4, 2020. © 2010 American Association for Cancer Research.


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145 cells. We detected no cells in the SP fraction of-Nrf2shRNA cells (Supplementary Fig. S4). Thus,ar to Nrf2-depleted lung cancer cells, Du145-hRNA cells showed reduced ABCG2 mRNA, pro-xpression, and SP compared with Nrf2 proficientollectively, these results suggest that transcriptionaltion of ABCG2 in lung and prostate cancer cells isted by Nrf2.

activation induces ABCG2 expression in lungelial cellst, we examined whether activation of Nrf2 induces2 expression in nontumorigenic airway epitheliali.e., NuLi cells). We used a shRNA targeting Keap1ibit Keap1 expression and activate Nrf2-dependentexpression. Luciferase shRNA was used as nontar-control shRNA. As shown in Fig. 6A, reduction intranscript levels by Keap1 shRNA resulted in a

el increase in NQO1, GCLm, and ABCG2 transcript. We observed an inverse correlation betweenexpression and Nrf2-dependent ABCG2 expres-

n lung epithelial cells.how that commonly used oxidative stress–inducinglike cigarette smoke condensate and tert-butyl hy-

inone, known to upregulate Nrf2-dependent signal-lso upregulate ABCG2 expression, we treated NuLiith these agents and measured gene expression. Theere exposed to cigarette smoke condensate (100 μg/nd tert-butyl hydroquinone (20 μmol/L) for 24, and induction of ABCG2 in response to theseents was quantified by real-time RT-PCR. Pretreat-with Nrf2-activating agents upregulated the expres-of ABCG2 and other classic Nrf2 target genes,
sting that Nrf2 mediates inducible expression of2 (Fig. 6B).
SP-poity of



ernatively, we overexpressed Nrf2 cDNA in a lungcarcinoma cell line, H23, harboring wild-typeand Nrf2 activity. H23 cells were transduced with

iral particles containing Nrf2 cDNA or the controlvector. Four to 6 days posttransduction, cells were

sted and expression of Nrf2 and its target genes, in-g ABCG2, was measured by real-time RT-PCR andnoblotting. As expected, Nrf2 overexpression re-in upregulation of ABCG2 expression at the

A and protein levels (Fig. 6C). Analysis of SP inells revealed that cells overexpressing Nrf2 (H23-cDNA) had a 2-fold higher SP fraction compared23 empty vector control cells (Fig. 6D). In summa-

e have convincingly shown that Nrf2 regulates2 gene expression and SP fraction in cancer cells.

ssion

he present study, we show that Nrf2 regulates theriptional expression of humanABCG2 gene throughE element located at the −431 to −420 bp regionpromoter, and Nrf2 is essential for maintaining2 expression and function in lung cancer and pros-ancer cells. Nrf2 depletion resulted in dramatic re-n of SP formation and reversal of chemoresistance–small-cell lung cancer cell lines. Similarly, upregu-of Nrf2 activity induced the expression of ABCG2creased the SP fraction in lung epithelial cells.e of the defining characteristics of cancer stem cellsr ability to transport Hoechst dye, leading to the SPtype, which is attributable to the expression of2 gene (3, 6). A study by Scharenberg et al. (11)ed that A549 cells contain an increased fraction of

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4. Reversal ofresistance in Nrf2-depletedncer cells. A549 and H460ere treated withtrone and topotecan forays, and cell viability wasd by MTT assays. Resultsare median values obtainedree independentents with eight replicatesh dose. Results wered by one-way ANOVA

chst dye efflux capac-trongly with ABCG2

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ty. Transcriptome analyses of embryonic, hemato-c, and neural stem cells revealed a common signa-
f gene expression, which includes transcripts thaton as a cytoprotective factor against environmental
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idativtionalenzymwell athe glase faeffluxtein fativityNrf2-NSCLOur pin thesubstof KEcell limutatwithtion fa(18, 3ported36, 37gulatieral Apumpsistanwerecells,pendeer repthat NABCGcorresformaABCGtate cRec

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enobiotic stress (29). Studies showed that stemexpress high levels of drug detoxification en-s such as P-glycoprotein, BCRP (ABCG2), glutathi--transferases, and glutathione peroxidase (6, 30,nterestingly, several of these cytoprotective anddetoxification genes are classic Nrf2-dependent.2, a bZIP transcription factor, protects cells from ox-e stress and xenobiotics by induction of a transcrip-program that includes major antioxidants such ases in the glutathione and thioredoxin pathways, ass xenobiotic detoxification enzymes that includeutathione S-transferase and UDP glycosyltransfer-milies, aldehyde dehydrogenase, and various drugpumps—members of the multidrug resistance pro-mily (32–34). KEAP1 negatively regulates Nrf2 ac-by targeting it for proteasomal degradation (15, 16).KEAP1 interactions are frequently dysfunctional inC, leading to constitutive activation of Nrf2 (17).revious studies have shown that point mutationsKEAP1 gene lead to nonconservative amino acid

itutions and nonsense mutations, resulting in lossAP1 function in a high percentage of the NSCLCnes and primary tumors (17). Recently, two Nrf2ion “hotspots” were identified in ∼10% of patientssquamous lung carcinoma, enabling the transcrip-ctor for the evasion of KEAP1-mediated repression5). Furthermore, mutations in KEAP1 have been re-in breast, gall bladder, and prostate cancers (21,

). High Nrf2 activity in cancer cells results in upre-on of phase II drug detoxification enzymes and sev-TP-dependent multidrug-resistant drug effluxs (e.g., ABCC1 and ABCC2) and promotes drug re-ce (12, 38–42). ABCG2 transcript and protein levelsdramatically reduced in Nrf2-depleted lung cancersuggesting that ABCG2 gene expression is Nrf2 de-nt in lung cancer cells. Attenuated ABCG2 promot-orter activity in the absence of Nrf2 activity impliesrf2 regulates ABCG2 transcription by modulating2 promoter activity. Loss of Nrf2 expression and aponding decrease in ABCG2 expression reduced SPtion in lung cancer cells. Similar Nrf2-dependent2 expression was observed in Du145 cells, a pros-ancer cell line.ently, it has been reported that the expression of2 along with some other efflux pumps was de-d in hepatocytes of Nrf2 null mice compared withf wild-type mice, which indicates that Nrf2 isved in physiologic regulation of ABCG2 (43).y-butylhydroquinone, an agent known to activateby inducing oxidative stress, upregulated the ex-ion of ABCG2 in hepatocellular carcinoma cellsIn another study, Jigorel et al. (24) reported thataz, a small-molecule activator of Nrf2, upregulatedpression of ABCG2 in primary human hepatocytes.es focused on regulation of the expression of
2 revealed that estrogen receptor–, hypoxia-, and
5. Attenuated expression and activity of ABCG2 in Nrf2-deficiente cancer cells. A, relative expression of ABCG2 and Nrf2 inparent cells and Du145-LucshRNA and Du145-Nrf2shRNA cellsd by real-time RT-PCR. Data are presented as fold changeted using gene expression levels in control luciferase shRNA cells, immunoblot analysis of Nrf2 and ABCG2, expression in Du145, densitometric quantitation of Nrf2 and ABCG2 relative

isome proliferator-activated receptor γ–inducible




FigureABCG2expressGCLM,treatmecondencompaof Nrf2with the

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6. Nrf2 activation induces ABCG2 expression in nontumorigenic lung epithelial cells and tumorigenic lung epithelial cells. A, upregulation ofexpression in cells expressing Keap1 shRNA. Total RNA from NuLi cells expressing Keap1 shRNA and luciferase shRNA was isolated, andion of Keap1 and ABCG2 was determined by real-time RT-PCR. Downregulation of Keap1 expression led to an increase in Nrf2-dependent NQO1,and ABCG2 expression. *, P < 0.05, significant when compared with Luc-shRNA group. B, induction of ABCG2 expression in response tont with multiple oxidative stress–inducing agents. NuLi cells were treated with tert-butylhydroquinone (TBHQ; 20 nmol/L) and cigarette smokesate (CSC; 100 μg/mL) for 24 hours, and expression of NQO1, GCLM, and ABCG2 was measured by real-time RT-PCR. *, P < 0.05, significant whenred with vehicle-treated sample. C, ecotopic expression of Nrf2 in lung cancer cells upregulates Nrf2-dependent ABCG2 expression. Overexpression
cDNA in H23 lung cancer cells resulted in activation of Nrf2 target gene expression including ABCG2. *, P < 0.05, significant when comparedempty vector group. D, enforced expression of Nrf2 cDNA in H23 cells increases the proportion of SP cells.
ancer Ther; 9(8) August 2010 Molecular Cancer Therapeutics

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ments are located at the 5′ promoter region of2 gene in normal SP-positive cells (44–46).reasing the expression of the redox master regula-rf2 potentially affects cellular protection against xe-tics and drugs (17). The attenuation in ABCG2ssion in Nrf2-deficient lung cancer cells leads tosensitization (22). Cancer cells with reduced Nrf2

ty and a parallel decline in ABCG2 expression dis-d enhanced sensitivity to mitoxantrone and topote-owever, besides reduced ABCG2 levels, alternate

anisms such as decrease in glutathione level or/educed drug detoxification may also be involvedtoxantrone and topotecan sensitization of Nrf2-ated lung cancer cells. We recently reported thatinhibition in lung cancer cells results in enhancedellular accumulation of carboplatin and etoposide,us, enhanced carboplatin- and etoposide-induced

eath (21, 22). Interestingly, no significant differencef2-dependent sensitivity to another drug, metho-e, was observed (data not shown). These findingsthat reversal of drug resistance by mitigating

dependent drug detoxification genes is specificelies on different Nrf2 downstream executors inntext of different chemotherapeutic agents. Onengly obvious advantage of targeting the Nrf2 mol-to increase sensitivity to chemotherapeutic drugsdifferent scenarios, is that electrophile drug detox-on pathways involved in detoxification of a wideum of chemotherapy drugs are tackled at the samethus reducing the possibility of alternative or re-ant detoxification pathway activation in targeting
ne or few detoxification and/or drug pump path- Rece
ancer stem-like cells in the C6 glioma cell line. Proc Natl Acad SciA 2004;101:781–6.

yle LA, Yang W, Abruzzo LV, et al. A multidrug resistance trans-

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onclusion, the loss of KEAP1 function and aberranttion of Nrf2-dependent pathways are frequentlyed in lung cancer and other solid tumors (17, 21,). Upregulated Nrf2 activity in cancer cells/tissuestially stimulates ABCG2 expression and promotesultidrug resistance phenotype. Because ABCG2with other cytoprotective genes are upregulatedcer stem cells and protect against stress, it remainsdetermined whether Nrf2 and its downstreamling is amplified in cancer stem cells and whetherting Nrf2 activity in cancer stem cells can be anve strategy for overcoming multidrug resistance.

osure of Potential Conflicts of Interest

swal: a pending Patent Cooperation Treaty application.

owledgments

hank Dr. Joseph B. Margolick and Hao Zhang at the Departmentcular Microbiology and Immunology, Bloomberg School of Public, Johns Hopkins University, for assistance with side populations.

Support

grants P50 CA058184, RO1 CA140492, and P30ES03819; develop-grant from Prostate Cancer Specialized Programs of Research Ex-P50 CA58236 (S. Biswal) and Flight Attendant Medical Research

e (S. Biswal and A. Singh).costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 02/02/2010; revised 06/18/2010; accepted 06/24/2010;
molecules alone. published OnlineFirst 08/03/2010.
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Published OnlineFirst August 3, 2010.Mol Cancer Ther Anju Singh, Hailong Wu, Ping Zhang, et al. PhenotypeCells That Confers Side Population and Chemoresistance Expression of ABCG2 (BCRP) Is Regulated by Nrf2 in Cancer

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