Upregulation of ABCG2 by Romidepsin via the Aryl Hydrocarbon Receptor...

Signaling and Regulation

Upregulation of ABCG2 by Romidepsin via the ArylHydrocarbon Receptor Pathway

Kenneth K.W. To1,2, Robert Robey2, Zhirong Zhan2, Lois Bangiolo2, and Susan E. Bates2

AbstractHistone deacetylase inhibitors (HDACI) are promising anticancer agents and their use in combination with

conventional anticancer drugs is currently under investigation. We previously reported cell line–specificupregulation of ABCG2, a multidrug resistance transporter shown to control oral bioavailability and CNSpenetration, by the HDACI romidepsin, although the precise mechanism in a particular cell line remains to bedetermined. The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can be activatedby numerous environmental contaminants and has been shown to be a client protein of heat shock protein 90(Hsp90). A xenobiotic response element was defined in the ABCG2 promoter and was shown to mediate AhRsignaling. Activated AhR was found to be associated with the ABCG2 promoter only in cell line models thatrespond to romidepsin with ABCG2 upregulation. Our data suggest that romidepsin acetylated Hsp70 andinhibited the chaperone function of Hsp90, thereby allowing the dissociation of AhR from Hsp90. Thedissociation of AhR fromHsp90 may be a prerequisite for the differential upregulation of ABCG2 by romidepsin.Increasing our understanding of the mechanism(s) governing differential upregulation of ABCG2 in response toromidepsin could provide an insight into strategies needed to tackle resistance to HDACIs in cancer therapeutics.Mol Cancer Res; 9(4); 516–27. �2011 AACR.

Introduction

ABCG2 is a ubiquitous ATP-binding cassette (ABC)multidrug resistance transporter that plays a significant rolein normal tissue protection, stem cell maintenance, andclinical pharmacology (1). Its overexpression confers resis-tance in cancer cell lines to a variety of cancer chemother-apeutic agents such as mitoxantrone, topotecan, andmethotrexate (2–7). However, little is known about themechanisms underlying its regulation.Histone deacetylase inhibitors (HDACI) such as romi-

depsin (also known as FK228 or depsipeptide) and vorino-stat (suberoylanilide hydroxamic acid or SAHA) were foundto be potent anticancer agents, capable of inducing cell cyclearrest and apoptosis in cancer cells (8). A number ofHDACIs are currently in clinical trials as both monotherapyand in combination therapy because these compounds havebeen shown to potentiate the cytotoxic effects of other

anticancer drugs in experimental models. We and othershave shown that multidrug resistance transporters (includ-ing ABCG2 and MDR1) can be upregulated on treatmentwith various HDACIs (9–13), potentially hindering drugresponse in combination therapy. Therefore, a better under-standing about the mechanism for activation of thesetransporters on HDACI treatment could help developstrategies for the prevention of drug resistance.The aryl hydrocarbon receptor (AhR) is a ligand-activated

transcription factor that belongs to the basic helix-loop-helix/Per-ARNT-Sim (bHLH-PAS) family (14). It regulatesthe transcription of genes encoding xenobiotic metabolizingenzymes and also mediates the toxic effects caused byenvironmental carcinogens such as dioxins and polycyclicaromatic hydrocarbons. In the unliganded state, the AhR isassociated with 2 molecules of the chaperone heat shockprotein 90 (Hsp90), an immunophilin-like protein XAP2,and the Hsp90-interacting acidic protein p23 in the cyto-plasm. Ligand binding initiates a cascade of events leading toAhR translocation to the nucleus, release of Hsp90, anddimerization of AhR with nuclear translocator (Arnt). Theligand-bound AhR–Arnt complex subsequently recognizesand binds to its cognate binding site with aDNA sequence of50-TA/TGCGTG-30 (15), termed the AhR response ele-ment (or xenobiotic response element; XRE), located in thepromoter region of responsive genes, thereby modulatinggene transcription (16). Its ligands includemany natural andsynthetic compounds, some of which, such as polyhaloge-nated aromatic hydrocarbons and polycyclic aromatic hydro-carbons, are important environmental contaminants.

Authors' Affiliations: 1School of Pharmacy, The Chinese University ofHong Kong, Hong Kong and 2Molecular Therapeutics Section, MedicalOncology Branch, Center for Cancer Research, National Cancer Institute,NIH, Bethesda, Maryland

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Kenneth K.W. To, School of Pharmacy, TheChinese University of Hong Kong, Hong Kong. Phone: 852-26096860;Fax: 852-26035295. E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-10-0270

�2011 American Association for Cancer Research.

MolecularCancer

Research

Mol Cancer Res; 9(4) April 2011516

on April 8, 2019. © 2011 American Association for Cancer Research. mcr.aacrjournals.org Downloaded from

Published OnlineFirst February 25, 2011; DOI: 10.1158/1541-7786.MCR-10-0270

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Hsp90 is an ATP-dependent molecular chaperone thatcontrols the intracellular trafficking and folding of diversecellular proteins, particularly those involved in signal trans-duction, cell cycle regulation, and survival (17, 18). It plays akey role in the conformational maturation of oncogenicsignaling proteins including HER-2/ErbB2, Akt, and Raf-1(19–21). AhR is a known Hsp90 client protein. The associa-tion with Hsp90 is required for the AhR to assume aconformation that is optimal for ligand binding (22). Rever-sible acetylation is an important posttranslational modifica-tion ofHsp90 that is known to impair the chaperone functionof Hsp90 (23–25). This has been shown to cause dissociationof a number of client proteins such asHer-2& c-Raf (25, 26),causing their polyubiquitination and proteasomal degrada-tion. Importantly, Hsp90 acetylation has been detected incells treated with various HDACIs including romidepsin andvorinostat (27, 28). However, in the case of AhR, the effect ofHDACI treatment on chaperone association of AhR withHsp90, as well as the levels of AhR, has not been determined.Moreover, within the nucleus, the precise mechanisms med-iating the dissociation of AhR fromHsp90 and the formationof a heterodimer with Arnt is still not fully understood.In the present study, we sought to evaluate the involve-

ment of the AhR pathway in the activation of ABCG2 byromidepsin. Our data show that romidepsin disrupted thechaperone binding of Hsp90 with AhR through Hsp70acetylation and subsequently promoted the binding ofAhR–Arnt with the XRE element on the ABCG2 promoter.In cell lines that did not respond to romidepsin treatmentwith ABCG2 upregulation, Hsp70 acetylation was notobserved and AhR remained bound to Hsp90, therebyblocking the AhR-mediated ABCG2 upregulation.

Materials and Methods

Tissue cultureHuman colon cancer cell line S1, a derivative of LS180,

has been described previously (5). MCF-7, SF295, andSW620 cells were chosen and obtained from the NationalCancer Institute Tumor Drug Screen for this study. The celllines were maintained in Iscove's modified Eagle's medium(S1 and MCF-7) or RPMI medium (H460, SF295, andSW620) supplemented with 10% FBS, 100 units/mLstreptomycin sulfate, and 100 units/mL penicillin G sulfate,and incubated at 37�C in 5% CO2.

Drug treatmentTo test the effect of HDAC inhibition on ABCG2

expression, cells were treated with 10 ng/mL of romidepsin(also known as depsipeptide, FR901228, or NSC630176;Developmental Therapeutics Program, National CancerInstitute) supplemented with 5 mg/mL of verapamil (Sigma)for 24 hours. Verapamil was added to prevent the efflux ofromidepsin mediated by P-glycoprotein upregulation. Stocksolutions of romidepsin and verapamil were dissolvedin DMSO and water, respectively. For the study of theAhR pathway in the regulation of ABCG2, benzo(a)pyrene[B(a)P; Sigma] was used as the AhR agonist whereas

resveratrol (Sigma), kaempferol (Sigma), and salicylamide(Sigma) were used as AhR antagonists.

RNA interferenceTo construct a small interference hairpin-loop (sh) silen-

cing vector against the human AhR, 2 complementaryoligonucleotides were synthesized, annealed, and ligandedinto the BglII and EcoRI site of the pU6 vector (a kindgift from Dr Chen Yang Chao, School of BiomedicalSciences, Chinese University of Hong Kong, Hong Kong).The sequences were 50-GGTTTCAGCAGTCTGATGT-CttcaagagaGACATCAGACTGCTGAAACCCTTTTT-30(forward) and 50-AAAAAGGGTTTCAGCAGTCT-GATGTCtctcttgaaGACATCAGACTGCTGAAACC-30(reverse), which contained the oligonucleotide encoding a20-mer hairpin sequence specific to the human AhRmRNA(designed by using the siRNA Selection Program at theWhitehead Institute for Biomedical Research (http://jura.wi.mit.edu/siRNAext/; ref. 29), a ttcaagaga loop sequenceseparating the 2 complements, and a TTTTT terminator atthe 30-end. The various cell lines were transfected with thebackbone vector (pU6) or the shRNA vector specific againstAhR (pU6-AhR) by using Lipofectamine 2000 (Invitrogen)before evaluation for ABCG2 regulation by romidepsin.Silencing efficiency was assessed at the protein level byimmunoblot analysis.

Reverse transcription and real-time PCRTotal RNA was isolated by using the Trizol regent

(Invitrogen). RNA (1 mg) was reverse transcribed by usingTetro reverse transcriptase (Bioline). Quantitative real-time PCR was performed to quantify the change ofABCG2, MDR1, p21, and CYP1A1 transcript expres-sions by using the KAPA SYBR FAST qPCR Kit (Kapa-Biosystems) in a LightCycler 480 Instrument I (RocheApplied Science). The human glyceraldehyde 3 phosphatedehydrogenase (GAPDH) RNA was amplified in parallelas the internal control. Sequences of the specific primersused are listed in Table 1. PCRs were performed at 95�Cfor 10 minutes, followed by 50 cycles of 95�C for 10seconds and 60�C for 10 seconds. Fluorescent signal was

Table 1. Sequence of the primers used forreal-time PCR analysis

Gene Primer sequence

ABCG2 Forward: 50-CAATGGGATCATGAAACCTG-30

Reverse: 50-GAGGCTGATGAATGGAGAA-30

MDR1 Forward: 50-CTGTCAGTGTATTTTCAATGTTTCG-30

Reverse: 50-AATTTTGTCACCAATTCCTTCATTA-30

p21 Forward: 50-GAGCGATGGAACTTCGACTTT-30

Reverse: 50-GGGCTTCCTCTTGGAGAAGAT-30

CYP1A1 Forward: 50-CCATGACCAGAAGCTATGGGT-30

Reverse: 50-GCTCTCAAGCACCTAAGAGCG-30GAPDH Forward: 50-ACCACAGTCCATGCCATCAC-30

Reverse: 50-TCCACCACCCTGTTGCTGTA-30

Romidepsin Upregulates ABCG2 via the AhR Pathway

www.aacrjournals.org Mol Cancer Res; 9(4) April 2011 517




acquired at the end of the elongation step of every PCRcycle (72�C for 10 seconds) to monitor the increasingamount of amplified DNA. DCt was calculated by sub-tracting the Ct of GAPDH from the Ct of ABCG2,MDR1, p21, or CYP1A1. DDCt was then calculated bysubtracting the DCt of the parental cells from the DCt ofthe corresponding resistant cells. Fold change of geneexpression was calculated by the equation 2�DDCt.

Luciferase reporter assaysA series of human ABCG2 promoter constructs with

progressive deletions at the 50-end has been previouslydescribed (30). Two new reporter constructs were preparedwith the putative XRE sequences deleted from the full-length ABCG2 promoter (Fig. 2; D�60/�52 and D�198/�189). The ABCG2 promoter/firefly luciferase fusiongenes (200 ng DNA) were transfected in S1 cells by usingFugene 6 (Roche). The pGL3-Basic (promoterless) plasmid,encoding firefly luciferase (Promega), was used to determinethe basal levels. In each experiment, the phRG-Basic plas-mid (50 ng), encoding Renilla luciferase (Promega), wascotransfected for normalization purposes. Luminescencewas measured 48 hours after transfection by the Dual-Luciferase Reporter Assay System (Promega). In experi-ments involving drug treatment, the drugs were added24 hours after transfection. Reporter activity was normal-ized by calculating the ratio of firefly/Renilla values. Resultswere expressed as mean � SD of duplicate measurementsfrom 3 independent transfections.

Western blot analysisAhR repressor (AhRR) protein expression was examined

in S1, MCF-7, HepG2, SW620, A549, SF295, and HeLacells treated with or without romidepsin or B(a)P. Cellswere lysed in buffer containing 0.2 mol/L Tris (pH 7.4),1% Triton X-100, 0.02% b-mercaptoethanol, and com-plete mini EDTA-free protease inhibitor cocktail tablets(Roche), followed by sonication for homogenization.Whole cell lysates prepared were separated by SDS-PAGEand subjected to immunoblot analysis with the respectiveantibodies for AhRR (ab85666; Abcam) and GAPDH(American Research Products). The antigen–antibody com-plex was analyzed with an Odyssey (Li-Cor) infrared ima-ging system after incubation with a 1:10,000 dilution ofgoat anti-mouse (IRDye800CW) or anti-rabbit(IRDye680) secondary antibody (Li-Cor).

CoimmunoprecipitationFor coimmunoprecipitation experiments, cells were

lysed in IP buffer (50 mmol/L Tris-HCl, pH 8.0; 150mmol/L NaCl; 1% Triton X-100; and protease inhibitorcocktail tablets), homogenized by sonication, and centri-fuged at 13,000 rpm for 2 minutes at 4�C. Lysates (500mg) were precleared for 1 hour at 4�C then incubated withrabbit polyclonal anti-Hsp90 (SPA-846; Stressgen), rabbitpolyclonal anti-acetyl lysine antibody (ab21623; Abcam)or mouse monoclonal anti-AhR (RPT9; Abcam) antibodyovernight at 4�C. Protein and antibody were then

incubated with ImmunoPure Plus Immunobilized ProteinA (Pierce) for 2 hours at 4�C. Samples were washed 5times with 500 mL of IP buffer. The Protein A pellet wasresuspended in 35 mL of 1 � sample loading buffer andheated at 100�C for 5 minutes before being resolved by12% SDS–polyacrylamide gels and immunoblotted withmouse monoclonal antibodies for Hsp90 (AC88; Abcam),Hsp70 (5A5; Abcam), AhR (RPT1; Abcam), or Bcl-2(100/D5; Abcam). Results were determined by chemilu-minescence detection with an Odyssey (Li-Cor) infraredimaging system as described earlier.

ChIP assaysChromatin-immunoprecipitation (ChIP) was per-

formed by a ChIP assay kit (Upstate Biotechnology)according to the manufacturer's instructions, with somemodifications. Briefly, proteins were cross-linked withDNA in the cultured cells by using 1% formaldehydefor 10 minutes at 37�C and quenched with 0.125 mol/Lglycine for 5 minutes at room temperature. The cells werethen rinsed in ice-cold PBS containing 5 mmol/L sodiumbutyrate (Sigma), scraped, and resuspended in a lysisbuffer (Active Motif) with the addition of completeprotease inhibitor cocktail (Roche). The DNA–proteincomplexes were sheared by using an enzymatic shearing kit(Active Motif) under conditions that gave a range of DNAfragments from 200 to 600 bp, as determined by agarosegel electrophoresis. ChIP was then carried out overnight at4�C with either mouse monoclonal anti-AhR (RPT9;Abcam) or anti-AhRR (Abcam) antibody. The amountof immunoprecipitated DNA was assessed by semiquan-titative PCR, using primers spanning the distal region(distal, nt �1,527 to 1,268) or proximal regions (P3, nt�293 to �139) of the ABCG2 promoter (Fig. 3; ref. 12),and compared with the amount of input DNA beforeimmunoprecipitation.Taq DNA polymerase (Bioline) was used to amplify 1

mL of either the immunoprecipitated DNA by using thespecific antibody, the immunoprecipitate by using anormal immunoglobulin G (IgG) control antibody, or a1:10 dilution of input chromatin. Preliminary experi-ments were carried out to determine optimal PCR con-ditions so that the yield of PCR products was dependenton the amount of input DNA (data not shown). Theconditions for the PCR reactions were as follows: 94�C for3 minutes, 30 cycles at 94�C for 1 minute, 55�C for1 minute, 72�C for 1 minute, and a final extension at72�C for 7 minutes. PCR products were electrophoresedon 2% agarose gel and stained with ethidium bromide.Band intensity was quantified as mentioned earlier in thetext. Fold enrichment in each immunoprecipitation wasdetermined by quantifying the intensities of the PCRproduct in immunoprecipitated DNA versus inputDNA (total chromatin). Only 10% of the total inputwas used in the PCR reactions and they were run as the"input" lanes in gel electrophoresis. Percentage enrich-ment was calculated accordingly. ChIP assays wererepeated thrice by different chromatin preparations.

To et al.

Mol Cancer Res; 9(4) April 2011 Molecular Cancer Research518




Flow cytometryABCG2-mediated efflux activity in romidepsin-pre-

treated cells was determined by flow cytometric assaysas described previously (31). Cells after a 24-hour treat-ment of romidepsin (10 ng/mL) or vorinostat (5 mmol/L)were incubated in 1 mmol/L pheophorbide A (PhA) withor without 10 mmol/L ABCG2-specific inhibitor fumi-tremorgin C (FTC) in complete medium (phenol red–freeRPMI 1640 with 10% FBS) at 37�C in 5% CO2 for 30minutes. Subsequently, the cells were washed with coldcomplete medium and then incubated for 1 hour at 37�Cin PhA-free medium continuing with FTC to generatethe FTC/efflux histogram, or without FTC to generate theefflux histogram. The FTC-inhibitable PhA efflux wasdetermined as the difference in mean fluorescence inten-sity (DMFI) between the FTC/efflux and efflux histo-grams, which indicates the ABCG2-mediated transportactivity. Cells were finally washed with cold Dulbecco'sPBS and placed on ice in the dark until analysis by flowcytometry.Samples were analyzed on an LSRFortessa Cell Analyzer

(BD Biosciences). PhA fluorescence was detected witha 488-nm argon laser and a 670-nm bandpass filter. At least10,000 events were collected for all flow cytometry studies.

Results

Induction of ABCG2 by romidepsin or B(a)P inselected cancer cell linesPreviously, we showed that romidepsin upregulates

ABCG2 in selected cancer cell lines and this coincides withthe induction of permissive histone modification marks atthe ABCG2 promoter in these cells (12). In our continuedeffort aiming at understanding the regulation of ABCG2,we pursued an earlier report suggesting that ABCG2 couldbe regulated by AhR agonists (32). Our result showed thatABCG2 can be upregulated by B(a)P (an AhR agonist; 24hours) in several cell lines including S1, SW620, and MCF-7 cells (Fig. 1). This upregulation can be reversed in AhR-silenced cells (Fig. 1) or by concomitant treatment withknown AhR antagonists [Supplementary Fig. 1; resveratrol(33), kaempferol (34), or salicylamide (35)]. Interestingly,knockdown of AhR (Fig. 1) or AhR antagonists (Supple-mentary Fig. 1) could also inhibit the upregulation ofABCG2 in S1 by romidepsin. Consistent with our previousfindings, SW620, MCF-7, and SF-295 did not respond toromidepsin treatment with ABCG2 upregulation (Fig. 1;ref. 12). Similar results regarding ABCG2 regulation werealso obtained for other HDACIs including vorinostat andpanobinostat (LBH-589; Table 2). Of note, the cell lineswere chosen to cover a range of basal ABCG2 expressionlevels (Fig. 2). Although ABCG2 levels in S1 and SW620were marginally detectable, MCF-7 and SF295 expressmuch higher basal levels of ABCG2. As controls for com-parison, the upregulation of MDR1 and p21, 2 genescommonly found to be elevated by HDACIs, was notaffected by AhR knockdown (Fig. 1) or AhR antagonists(Supplementary Fig. 1) in all cell lines tested. Moreover,

both MDR1 and p21 levels were not changed by B(a)P,suggesting that the AhR-related ABCG2 upregulationobserved is specific. It is also interesting to note that atypical AhR-responsive gene, CYP1A1, was not affected byromidepsin treatment in all cell lines tested (Fig. 1).

An XRE cis-element is required for the activation ofABCG2 promoter activity by B(a)PBecause AhR was potentially involved in romidepsin-

mediated upregulation of ABCG2 in S1 cells (Fig. 1), therole of AhR in ABCG2 regulation was further examined. Aseries of 50-deletion reporter gene constructs were generatedfrom the ABCG2 promoter region with the 30-end termi-nating at þ396 bp (ref. 30; Fig. 3). Using a transient

Figure 1. Real-time PCR analysis showing the absolute requirement forAhR in the upregulation of ABCG2 by romidepsin or B(a)P.A, effective knockdown of AhR by pU6-AhRwas confirmed by immunoblotanalysis. Enhanced green fluorescent protein (EGFP) represents theloading control for the cotransfections. B, the cells were treated with eitherromidepsin or B(a)P for 24 hours, with or without the prior knockdown ofAhR (48 hours after transfection). The cell lines tested can be divided into 3categories according to the upregulation of ABCG2 on the drug treatment:romidepsin and B(a)P–-responsive (S1); romidepsin–-nonresponsive but B(a)P–-responsive (SW620 & MCF-7); and romidepsin and B(a)P—nonresponsive (SF295). MDR1 and p21 were also measured because theywere found to be universally upregulated by HDACIs. Expression level of atypical AhR-responsive gene CYP1A1 was also evaluated. EGFP (fromcotransfection) was used as the internal control for normalization. The datashown represent the fold increase in the levels of the various mRNAsrelative to the no-treatment control after normalization with EGFP. Mean�SD from 3 independent experiments is shown.






transfection assay system in S1 cells, all of the ABCG2promoter-luciferase constructs showed good activitiesabove the pGL3-Basic background (Fig. 3). Reporter activ-ity of all constructs was found to be activated on B(a)Ptreatment (24 hours), except the 2 shortest 50-deletedpromoter constructs (�105/þ396 and�31/þ 396). Usingthe PROMO virtual library for the identification of putativetranscription factor binding sites, 2 putative XRE elementswere predicted on the ABCG2 proximal promoter ("�60to �52" and "�198 to �189") based on the TRANSFAC database (36). B(a)P-mediated upregulation of ABCG2

promoter activity could be abolished in the �198/�189-deleted reporter construct (Fig. 3), suggesting that thisXRE region is important for ABCG2 regulation. More-over, activation of the ABCG2 promoter activity was alsoabolished in AhR-silenced cells (Supplementary Fig. 2).The reporter assay was also repeated in S1 cells treatedwith romidepsin (data not shown). As we have previouslynoted, romidepsin aberrantly increases the luciferase activ-ity of the Renilla vector (12). Therefore, the specific effectof romidepsin on the XRE could not be confirmed by thisassay.

Romidepsin increases the association of AhR with XREat the ABCG2 promoter in the responsive cell lineAn important step in the AhR pathway is the formation of

the AhR–Arnt heterodimer and the subsequent binding ofthis dimer to the XRE of its target gene promoter to regulate

Figure 2. Real-time PCR analysis comparing the basal ABCG2 mRNAexpression in the various cell lines used in this study (S1, SW620, MCF-7,and SF295). Relative ABCG2 transcript expression was shown afternormalization with GAPDH (the level in S1cells was set as 1 forcomparison). The level in SW620 was found to be nearly undetectable.

Table 2. Summary of real-time PCR analysis incells treated with romidepsin (10 ng/mL), vor-inostat (5 mmol/L), or panobinostat (100 nmol/L)

Cells ABCG2 MDR1

S1No treatment 1 1Romidepsin-treated 8.2 1.9Vorinostat-treated 6.5 2.2Panobinostat-treated 7.6 2.3

SW620No treatment ND 1Romidepsin-treated ND 4.8Vorinostat-treated ND 5.4Panobinostat-treated ND 4.9

SF295No treatment 1 1Romidepsin-treated 0.23 1.8Vorinostat-treated 0.2 1.9Panobinostat-treated 0.23 1.9

NOTE: Real-time PCR analysis was performed by the Uni-versal Probe Library (UPL) assays (Roche). The specificprobes used for ABCG2 and MDR1 are #56 and #49,respectively. Relative ABCG2 or MDR1 expression wasshown after normalization with GAPDH.Abbreviation: ND, not detectable.

Figure 3. ABCG2 promoter luciferase reporter assay. Left, schematicrepresentations of the 50-deletion ABCG2-promoter constructs. The 50-end of each of the constructs relative to the transcription start site (arrows)is indicated. The pGL3-basic (promoterless) vector, encoding fireflyluciferase, was used to determine the basal levels. The last 2 constructs(D�60/�52 and D�198/�189) were prepared with the putative XREelements deleted from the full-length ABCG2 promoter. Right, ABCG2transcriptional activity, with or without B(a)P treatment in S1 cellstransiently transfected with the various ABCG2 promoter constructs. Themean reporter activity � SD [firefly/Renilla luciferase units (RLU)] from 3independent experiments is presented. The reporter assay was alsorepeated in S1 cells treated with romidepsin (data not shown). As we havepreviously noted, romidepsin aberrantly increases the luciferase activity ofthe Renilla vector (12). Therefore, the specific effect of romidepsin on theXRE could not be confirmed by this assay.

To et al.





transcriptional activity. To find out whether this process candifferentiate the regulation of ABCG2 by romidepsin, weexamined the ability of romidepsin to affect the associationof the AhR to the ABCG2 proximal promoter by ChIPanalysis. ChIP assays measure the interaction of proteinwith DNA in a given chromatin region in vivo (37).Differential histone H3 hyperacetylation in the proximalABCG2 promoter has been shown in romidepsin-treatedcells with ABCG2 upregulation, indicating the activation oftranscription (12).DNA purified after immunoprecipitation with anti-

AhR antibody was evaluated by PCR by using primerstargeting the proximal (nt�293 to�139) or distal regions(nt�1,527 to�1,268) of the ABCG2 promoter (Fig. 4A).The relative amount of promoter-associated AhR wasquantified by measuring the PCR band intensity in a

molecular imager followed by normalization with theinput. Serial dilutions of the input DNA were amplifiedby PCR to determine the linearity of the PCR reactions.The levels of PCR amplifications for all ChIP sampleswere within this linear range. No signal was obtained fromimmunoprecipitated samples if normal IgG was used(Fig. 4B). The putative XRE element (nt �198 to�189) of the ABCG2 promoter was located within theproximal promoter fragment evaluated by ChIP (Fig. 4A).Romidepsin treatment (4 hours) increased the associationof AhR with the proximal ABCG2 promoter in theromidepsin-responsive S1 cells that upregulate ABCG2(Fig. 4B), but not in SW620 and SF295 cells. On thecontrary, an increase in AhR binding to the proximalABCG2 promoter following B(a)P was observed in bothS1 and SW620 cells but not in SF295 cells (4 hours),consistent with the upregulation of ABCG2 in these celllines. In contrast, there was negligible association of AhRwith the distal ABCG2 promoter, confirming the specificbinding of AhR to the XRE after romidepsin or B(a)Ptreatment in the corresponding respective cell lines(Fig. 4B).

Romidepsin decreased the binding of AhR to Hsp90 inthe responsive S1 cells, probably by inducing Hsp70acetylationHsp90 is a chaperone protein that is important in

maintaining its client protein in a functional conformationfor its biological function. Although somewhat controver-sial, previous reports have shown that the class I HDACinhibitor romidepsin induced acetylation of Hsp90, whichinhibits the ATP binding and chaperone association ofHsp90 with its client proteins, including Her-2, Akt,and c-Raf, leading to their polyubiquitylation and degrada-tion by the 20S proteasome (23, 25, 38). AhR is a knownHsp90 client protein and it is normally stabilized by thechaperone activity of Hsp90. Because AhR was found to beinvolved in the upregulation of ABCG2 by romidepsin,additional experiments were performed to find whetherromidepsin alters the ability of Hsp90 to interact withAhR, thereby regulating AhR stability. S1 and SW620 cellswere treated with 10 ng/mL romidepsin (þ5 mg/mL ver-apamil) or 10 mmol/L B(a)P for 16 hours. Hsp90 wasimmunoprecipitated with Hsp90 antibody and its asso-ciated proteins were determined by immunoblot analysis.In S1 cells, romidepsin or B(a)P treatment decreased the

association of AhR with Hsp90, which is believed to haveled to the lower protein stability of AhR (Fig. 5A,�MG132). However, in SW620 cells, the association ofAhR with Hsp90 could only be decreased by B(a)P but notby romidepsin. In romidepsin-treated SW620 cells, AhRprotein stability remained unchanged (Fig. 5A, �MG132,Input). This result suggested that the dissociation of AhRfrom Hsp90 may be a prerequisite for the upregulation ofABCG2 by romidepsin. Moreover, consistent with thefinding that resveratrol (an AhR antagonist) could inhibitABCG2 upregulation by romidepsin (SupplementaryFig. 1), the association between AhR and Hsp90 was

Figure 4.ChIP assay showing that theABCG2 promoter was enrichedwithAhR in romidepsin- or B(a)P-treated responsive cell lines. A, a schematicrepresentation of the ABCG2 promoter studied by ChIP analysis. Two setsof primers were used for amplification of the proximal (P3) and distalABCG2 promoter. Nucleotide positions are numbered relative to the majortranscription start site (þ1) defined in the published DNA sequence(Genbank accession no. AF151530). Big arrowhead, transcription startsite. B, ChIP analyses targeting the proximal and distal region of theABCG2 promoter in romidepsin or B(a)P-treated cells (4 hours), usingantibody against AhR. Input, DNA isolated from the lysate beforeimmunoprecipitation (only 10% of the total chromatin was used for thePCR reactions and it was run as the "input" in the gel electrophoresis); IgG,ChIP using normal IgG for immunoprecipitation. A representative set ofresult from 3 independent immunoprecipitations is shown.






restored in S1 cells treated with romidepsin (16 hours) inthe presence of resveratrol (Fig. 5B). A similar dissociationof AhR from Hsp90 was also observed in S1 or MCF-7 cellson B(a)P treatment (Fig. 5A; Supplementary Fig. 3, respec-tively), but not in the nonresponsive SF295 cells (Supple-mentary Fig. 3).Inhibition of the Hsp90 chaperone function causes

degradation of its client proteins via the ubiquitin-protea-some pathway. The protein expression of such a clientprotein, Bcl-2, and its binding to Hsp90 in the presenceof romidepsin was therefore also evaluated as a control.

Romidepsin remarkably reduced the binding of Bcl-2 toHsp90 and decreased the Bcl-2 protein expression in S1cells (Fig. 5A, �MG132). To ascertain that the observedAhR–Hsp90 dissociation was not because of artifacts attrib-uted by the decreased AhR stability, the immunoprecipita-tion experiments following romidepsin or B(a)P wererepeated in cells treated concurrently with the proteasomeinhibitor MG-132 (10 mmol/L; Calbiochem). AlthoughAhR was not degraded, AhR–Hsp90 dissociation was stillobserved in romidepsin/B(a)P-treated S1 cells, B(a)P-trea-ted SW620 cells, or B(a)P-treated MCF-7 cells (Fig. 5A andB, þMG132 versus �MG132; Supplementary Fig. 3).It is known that Hsp90 chaperone activity is regulated by

reversible acetylation and is controlled by the deacetylaseHDAC6 (39). As a class I HDACI, romidepsin has beenreported to have only a weak effect on HDAC 6 (27, 28) butit has been shown to disrupt the chaperone function ofHsp90 and induce apoptosis in human non–small cell lungcancer cells (23).Hsp70 is an important cochaperone proteinwithHsp90. It was reported that one of the class I HDACs isthe deacetylase of Hsp70 and that romidepsin treatment canincrease acetylation of Hsp70 (40). Of note, Hsp70 isrequired for the assembly ofHsp90–client protein complexes(41). More recently, it has been reported that romidepsinmay disrupt the chaperone function of Hsp90 indirectlythrough acetylation of Hsp70 (42). Therefore, immunopre-cipitation experiments were also performed to assess theeffect of romidepsin and vorinostat on acetylation of Hsp90/Hsp70 and the association between Hsp90 and AhR.As expected for the pan-HDACI, vorinostat induced

hyperacetylation of both Hsp90 and Hsp70 in S1 cells(Fig. 6A). On the contrary, romidepsin cannot inhibitHDAC6 and therefore it did not cause Hsp90 hyperacetyla-tion but instead only Hsp70 hyperacetylation in S1 cells(Fig. 6A). Consistent with the effect of Hsp90/Hsp70hyperacetylation onHsp90 chaperone function and agreeingwith the result in Figure 5A, romidepsin or vorinostattreatment (4 hours) was found to deplete the binding ofAhR to Hsp90 in S1 cells (Fig. 6B). Interestingly, no Hsp70and Hsp90 acetylation was observed in the nonresponsiveSW620 cells after exposing to romidepsin or vorinostat(Fig. 6A), which is believed to account for the unchangedassociation between AhR and Hsp90 (Fig. 6B). As a control,B(a)P treatment was able to dissociate AhR from Hsp90 inboth S1 and SW620 cells (Fig. 6B), consistent with the well-established biological effect of AhR ligand binding (16).

Romidepsin or B(a)P did not increase AhRR expressionor its binding to the ABCG2 promoter in thenonresponsive cell linesThe AhR repressor (AhRR) has been shown to inhibit

AhR signaling through a proposed negative feedbackmechanism involving induction of its constitution expres-sion and competition with AhR for dimerization to Arntand subsequently binding to AhR regulatory elements(XRE; ref. 43). To determine whether ABCG2 upregula-tion was constrained by AhRR in the nonresponsive celllines, AhRR protein expression and their association with

Figure 5. Immunoprecipitation assay showing that romidepsin led to adissociation of AhR from Hsp90 in the responsive cell lines. A, total celllysateswere prepared fromS1 or SW620 cells as described in theMaterialsand Methods. For each immunoprecipitation, a total of 500 mg of proteinwas incubated with rabbit anti-Hsp90 or control IgG antibody prebound toProtein A for 2 hours at 4�C. Proteins were resolved by SDS-PAGE andtransferred to nitrocellulose membrane. Hsp90, AhR, or Bcl-2 in theimmunoprecipitate was visualized by the respective antibodies. Toascertain that the observed AhR–Hsp90 dissociation was notbecause of artifacts attributed by the decreased AhR stability, theimmunoprecipitation experiments were carried out side-by-side in cellstreated concurrently with or without MG-132. It was noted that similarresults were obtained in both cases, except that AhR protein stability wasnot affected in cells treated with MG-132. B, dissociation of AhR fromHsp90 in the presence of romidepsin or B(a)P was inhibited by resveratrol(an AhR antagonist) in S1 cells. The immunoprecipitation experiment wasperformed as described in (A). Hsp90 was immunoprecipitated from celllysates after treatment of romidepsin or B(a)P, or a combination withresveratrol (in the presence or absence of MG132). AhR in theimmunoprecipitate was detected by immunoblot analysis as in (A). Arepresentative blot from 3 independent experiments is shown. GAPDHrepresents the loading control for the immunoprecipitation experiments.

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the ABCG2 promoter were evaluated by immunoblot andChIP analysis, respectively. Romidepsin or B(a)P (16 hours)decreased AhRR expression in HepG2 cells but had nodetectable effect in other cell lines tested (Fig. 7A). InSW620 (the romidepsin nonresponsive and B(a)P respon-sive cell line), no association between AhRR and theABCG2 promoter can be detected under either treatmentwith romidepsin or B(a)P (Fig. 7B). These results rule outthe involvement of AhRR in the lack of ABCG2 upregula-tion in the nonresponsive cell lines.

Romidepsin increased ABCG2 efflux activity only inthe responsive S1 cellsConsistent with the PCR analysis (Fig. 1; Table 2),

increased PhA efflux (as measured by DMFI) was also notedin romidepsin or vorinostat-treated S1 cells (Fig. 8). On thecontrary, PhA efflux remained unchanged or was slightlydecreased in SW620 and SF295 cells, respectively, onromidepsin or vorinostat treatment (Fig. 8).

Discussion

This study extends our earlier report about the modulationof the ABCG2 gene by HDACIs. We found that romidepsinincreased ABCG2 expression in selected cancer cell lines andthis modulation was accompanied by histone modificationssuch as an increase in histone acetylation at the ABCG2 pro-moter (12). UnlikeMDR1, the induction of ABCG2 follow-ing romidepsin exposurewas found tobe cell line specific (12).Although differential display (44) or microarray analyses (45)identified only 2% to 10% of expressed genes as sensitive toHDACIs, we have found a gene responding toHDAC inhibi-tion in some cell lines but not in others. The upregulation ofABCG2byHDACIsmay lead todrug resistancewhenused incombination with substrate drugs. Therefore, understandingthe differential effects by romidepsin could not only providethe guidance for the avoidance of HDACI-induced drugresistance, but also potentially provide insight into the induc-tion of therapeutically important genes byHDAC inhibition.

Figure 6. Immunoprecipitation assay showing that romidepsin decreasedthe binding of AhR to Hsp90 in S1 cells by inducing Hsp70 acetylation. A,comparison of the effects of romidepsin or vorinostat on Hsp90/Hsp70acetylation in S1 and SW620 cells. In S1 cells, vorinostat increased bothHsp90 and Hsp70 acetylation but romidepsin only induced Hsp70acetylation. On the contrary, no Hsp90/Hsp70 acetylation was detected inSW620 cells treated with either vorinostat or romidepsin. Cell lysatesimmunoprecipitated with anti-acetyl Lysine antibody were subjected toimmunoblot analysis by anti-Hsp90 or anti-Hsp70 antibody. The levels ofHsp90 and Hsp70 in the whole cell lysate not subjected toimmunoprecipitation were used as the loading controls. B, cell lysatesfrom S1 and SW620 cells were prepared as in Figure 5A andimmunoprecipitated with anti-AhR antibody. Hsp90 or Hsp70 in theimmunoprecipitate was visualized by the respective antibodies. Arepresentative blot from 3 independent immunoprecipitations is shown.GAPDH represents the loading control for the immunoprecipitationexperiments.

Figure 7. Immunoblot and ChIP analyses showing that AhRR was notinvolved in the lack of ABCG2 upregulation in SW620 cells treated withromidepsin or B(a)P. A, immunoblot analysis showing the cellular level ofAhRR in various cell lines indicated after treatment of romidepsin or B(a)P.HepG2 and HeLa cells were known to express detectable protein level ofAhRR, therefore they were included for comparison. GAPDH was used asa loading control. No activation of AhRR was detected in all the cell linestested. B, ChIP analyses targeting the proximal and distal region of theABCG2 promoter in romidepsin- or B(a)P-treated SW620 cells, using anti-AhRR antibody. Input, DNA isolated from the lysate beforeimmunoprecipitation (only 10% of the total chromatin was used for thePCR reactions and it was run as the "input" in the gel electrophoresis); IgG,ChIP using normal IgG for immunoprecipitation. A representative gelimage from 2 independent immunoprecipitations is shown.






The involvement of the AhR pathway in the upregulationof ABCG2has been suggested in studies showing that knownAhR agonists, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and B(a)P, elevated the levels of ABCG2mRNA and protein expression, resulting in increased trans-port of ABCG2 substrates (33, 46). In fact, during thepreparation of this manuscript, it was reported that AhRis a transcriptional activator of ABCG2 (47). In our con-tinued investigation into the molecular mechanisms thatregulate ABCG2, we confirmed and extended these findings,observing that ABCG2 was differentially upregulated incancer cell lines after treatment with B(a)P, which couldbe inhibited by concomitant treatmentwithAhR antagonists(resveratrol, kaempferol, and salicylamide) or by AhR knock-down. Upregulation of ABCG2 following romidepsin couldalso be abolished by chemically antagonizing AhR or geneticsilencing (Fig. 1; Supplementary Fig. 1). To this end,romidepsin is unlikely to be a direct AhR ligand becausethe typical AhR-responsive gene CYP1A1was not affected inall cell lines tested (Fig. 1). As additional controls, theuniversal upregulation of MDR1 and p21 by romidepsinin our study was not affected by chemical antagonists or AhRknockdown (Fig. 1; Supplementary Fig. 1), suggesting that

the observed AhR-mediated ABCG2 upregulation should bespecific. An XRE consensus sequence was identified andsubsequently confirmed at theABCG2 promoter close to thetranscription start site by a reporter gene assay (Fig. 3;Supplementary Fig. 2), which is consistentwith that reportedrecently by Tan and colleagues (47). Thus, we sought toelucidate the mechanisms for this differential effect byromidepsin on ABCG2 regulation.The formation of the AhR–Arnt heterodimer and its

subsequent binding to the XRE sequence at its target genesis critical for the activation of the AhR pathway. To studythe cell line–specific upregulation of ABCG2 by romidep-sin, we examined the association of AhR with the XRE onthe ABCG2 promoter. Results from ChIP analyses showedthat treatment with romidepsin or B(a)P induced theassociation of the AhR to XRE harbored in the ABCG2promoter only in the responsive cells (Fig. 4), which isconsistent with the cell line–dependent activation of theAhR pathway by drug treatment.Hsp90 is an important molecular chaperone playing a

critical role in maintaining the stability and function of itsclient proteins (17, 18), and Hsp90 acetylation has beenshown to impair the chaperone function of Hsp90 (23) andtarget the client proteins for degradation (48, 49). Hsp70 isan important cochaperone protein of Hsp90 and is requiredfor the assembly of Hsp90–client protein complexes (41). Ithas been reported that romidepsin may disrupt the chaper-one function of Hsp90 indirectly through acetylation ofHsp70 (42). Because the chaperone function of Hsp90 alsodetermines AhR protein stability, AhR protein expressionwas also assessed. Concurrent with release of AhR andtranslocation to the nucleus, AhR degradation also occursfollowing B(a)P exposure (50). We observed this phenom-enon following B(a)P exposure in both S1 and SW620 cells.Similarly, our data showed that AhR degrades substantiallyafter 16-hour exposure to romidepsin in S1 cells (Fig. 5A,�MG132, Input). In contrast, the Hsp90 chaperone wasfound to protect AhR from degradation in the romidepsinnonresponsive SW620 cells (Fig. 5A, �MG132, Input).Importantly, the AhR–Hsp90 dissociation was stillobserved in romidepsin/B(a)P-responsive cell lines whenthe proteasome inhibitor MG-132 was used to stabilize theAhR protein (Fig. 5A, þMG132 versus �MG132). Theseresults suggest that the observed lower AhR/Arnt/XREbinding in the responsive cells is not due to a decrease inthe cellular level of the AhR protein. Agonist-induceddegradation of the AhR protein is known to occur and itdoes not compromise the induction of AhR-regulatedgenes; rather, it is probably a regulated proteolytic eventto keep the gene induction under control (51).Romidepsin and vorinostat were found to cause Hsp90/

Hsp70 acetylation only in the responsive cell line S1 but notin the nonresponsive SW620 cells (Fig. 6A). It has beenrecently shown that Hsp90 dissociation from the AhR isessential for the formation of the AhR–Arnt heterodimer(52), which binds to the XRE of target genes and initiatestranscription. In S1 cells, in which the dissociation ofHsp90 and AhR was observed on romidepsin or vorinostat

Figure 8. Drug efflux assay showing the increase in ABCG2-mediatedefflux by romidepsin or vorinostat treatment in the S1 cells. Cells wereincubated with the fluorescent substrate PhA with or without 10 mmol/LFTC (ABCG2-specific inhibitor) for 30 minutes at 37�C, washed, and thenallowed to efflux for 1 hour at 37�C in substrate-free medium continuingwith (dashed lines, FTC/Efflux histogram) or without (solid lines, Effluxhistogram) FTC. Representative histograms from 3 independentexperiments are shown. DMFI was determined as the difference in meanfluorescence intensity between the FTC/efflux and efflux histograms,which indicates the ABCG2-mediated transport activity.

To et al.





treatment, there was a remarkable increase in the associa-tion between AhR and Hsp70 (Fig. 6B). Because Hsp70may also promote disaggregation and protein degradationthrough dissociation of Hsp90 and its client proteins (53),this could serve to prevent the excessive upregulation ofABCG2. In our model system (Fig. 5A, �MG132), romi-depsin disrupts the association of AhR from Hsp90 andleads to degradation of AhR. However, induction of Hsp70was not observed after romidepsin treatment in either S1(responsive) or SW620 (unresponsive) cells (Fig. 6A, lysate).Further, the IC50s of romidepsin in S1 and SW620 werefound to be similar (approximately 0.5 ng/mL in both celllines; ref. 54), suggesting that the differential Hsp90/Hsp70acetylation on romidepsin treatment in S1 and SW620 cellsapparently did not impact HDACI sensitivity. AlthoughHsp90/Hsp70 acetylation may be the critical determinantfor the differential upregulation of ABCG2 by romidepsin,the role of other cochaperone proteins such as p23 and XIAPshould not be excluded and deserves further investigation. Abetter understanding of the markers or cell phenotypes thatmake some cells responsive to HDACI with ABCG2 upre-gulation is needed to design strategies to modulate ABCG2by the AhR pathway.The romidepsin-induced dissociation of AhR from

Hsp90 was inhibited by cotreatment with resveratrol(Fig. 5B). In fact, resveratrol has been proposed to antag-onize the induction of AhR genes either by competing forAhR binding with the AhR ligands (55) or by inhibiting therecruitment of the AhR–Arnt heterodimer to the XRE (33).Our data suggest that resveratrol may prevent romidespin-mediated upregulation of ABCG2 by maintaining theassociation between Hsp90 and AhR.The negative feedback mechanism involving the AhRR

was also examined to determine its role in the lack ofABCG2 upregulation in the nonresponsive cell lines. Tothis end, the constitutive expression of AhRR was notaltered by romidepsin treatment (Fig. 7A). In the romi-depsin nonresponsive SW620 cell line, no AhRR bindingto the ABCG2 promoter can be detected (Fig. 7B). There-fore, AhRR is not likely to participate in the cell line–specific regulation of ABCG2 by romidepsin treatment.The biological/functional consequence of the cell line–

specific upregulation of ABCG2 was also investigated.Consistent with the ABCG2 mRNA analysis, an increasein ABCG2-mediated efflux was observed in romidepsin- orvorinostat-treated S1 cells (Fig. 8). It is noteworthy thatABCG2 protein expression or FTC-inhibitable efflux wasnot observed in S1 cells under basal condition. Given thatABCG2 upregulation may lead to treatment failure whenHDACI (romidepsin) is used in combination with otherchemotherapeutic agents, strategies could be designed tomodulate the AhR pathway therapeutically for the circum-vention of drug resistance. Moreover, because constitutiveoverexpression of AhR is commonly observed in severalcancer types including lung and esophageal cancers (56,57), the involvement of this AhR-mediated upregulation ofABCG2 in the acquired multidrug resistance and poorprognosis of these cancers may deserve investigation.

Taken together, the AhR signaling pathway was shown tobe involved in the cell line–specific upregulation of ABCG2by romidepsin treatment. A working model is illustrated inFigure 9. The AhR–Hsp90 interaction seems to represent anovel pathway for regulating ABCG2 expression in cancercells. Whether other therapeutic genes will be similarlyregulated is a subject for further study.

Disclosure of Potential Conflicts of Interests

No potential conflicts of interest were disclosed.

Grant Support

This research was supported by the Intramural Research Program of the NIH,National Cancer Institute, Center for Cancer Research and the Seed ResearchFunding provided by the School of Pharmacy, Chinese University of Hong Kong(K.K.W. To).

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

Received June 18, 2010; revised February 7, 2011; accepted February 8, 2011;published OnlineFirst February 25, 2011.

Figure 9. A working model for the cell line–specific ABCG2 upregulationvia the AhR pathway after romidepsin treatment. In romidepsin responsivecell lines, romidepsin treatment caused Hsp70 acetylation and impairedHsp90 chaperone activity. Although AhR protein expression wasdecreased because of the inhibition of Hsp90 chaperone activity, AhR–Hsp90 dissociation is a prerequisite for AhR–XRE binding and thesubsequent activation of ABCG2. ABCG2 upregulation in these cell lines isalso characterized by the enhanced enrichment of acetylated histone H3(H3 K9,14 Ac) and RNA polymerase II (Pol II) at the ABCG2 promoter (12).Regulation by other cochaperones may also play a role in this cell line–dependent ABCG2 regulation. In contrast, in romidepsin nonresponsivecell lines, although AhR was stabilized by Hsp90 because of the lack ofHsp90/Hsp70 acetylation, the AhR activation was hindered by theconstant association between Hsp90 and AhR.






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2011;9:516-527. Published OnlineFirst February 25, 2011.Mol Cancer Res Kenneth K.W. To, Robert Robey, Zhirong Zhan, et al. Hydrocarbon Receptor PathwayUpregulation of ABCG2 by Romidepsin via the Aryl

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