induction of udp-glucuronosyltransferase 2b15 gene...

1521-009X/43/6/889–897$25.00 http://dx.doi.org/10.1124/dmd.114.062935DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:889–897, June 2015Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics

Induction of UDP-Glucuronosyltransferase 2B15 Gene Expression bythe Major Active Metabolites of Tamoxifen, 4-Hydroxytamoxifen and

Endoxifen, in Breast Cancer Cells

Apichaya Chanawong, Dong Gui Hu, Robyn Meech, Peter I. Mackenzie, and Ross A. McKinnon

Department of Clinical Pharmacology and Flinders Centre for Innovation in Cancer, Flinders University School of Medicine, FlindersMedical Centre, Bedford Park, South Australia, Australia

Received December 22, 2014; accepted March 19, 2015

ABSTRACT

We previously reported upregulation of UGT2B15 by 17b-estradiolin breast cancer MCF7 cells via binding of the estrogenreceptor a (ERa) to an estrogen response unit (ERU) in theproximal UGT2B15 promoter. In the present study, we showthat this ERa-mediated upregulation was significantly reducedby two ER antagonists (fulvestrant and raloxifene) but was notaffected by a third ER antagonist, 4-hydroxytamoxifen(4-OHTAM), a major active tamoxifen (TAM) metabolite. Fur-thermore, we found that, similar to 17b-estradiol, 4-OHTAMand endoxifen (another major active TAM metabolite) elevatedUGT2B15 mRNA levels, and that this stimulation was signifi-cantly abrogated by fulvestrant. Further experiments using4-OHTAM revealed a critical role for ERa in this regulation.Specifically; knockdown of ERa expression by anti-ERa smallinterfering RNA reduced the 4-OHTAM–mediated induction of

UGT2B15 expression; 4-OHTAM activated the wild-type but notthe ERU-mutated UGT2B15 promoter; and chromatin immuno-precipitation assays showed increased ERa occupancy at theUGT2B15 ERU in MCF7 cells upon exposure to 4-OHTAM.Together, these data indicate that both 17b-estradiol and theantiestrogen 4-OHTAM upregulate UGT2B15 in MCF7 cells viathe same ERa-signaling pathway. This is consistent with previousobservations that both 17b-estradiol and TAM upregulate a commonset of genes in MCF7 cells via the ER-signaling pathway. As4-OHTAM is a UGT2B15 substrate, the upregulation of UGT2B15 by4-OHTAM in target breast cancer cells is likely to enhance localmetabolism and inactivation of 4-OHTAM within the tumor. Thisrepresents a potential mechanism that may reduce TAM therapeu-tic efficacy or even contribute to the development of acquired TAMresistance.

Introduction

Breast cancer is the most common cancer in women and a leadingcause of cancer mortality worldwide (Jemal et al., 2011; Youlden et al.,2012). The majority of breast cancers are estrogen receptor (ER)–positive and depend on ER signaling for their proliferation (Miller et al.,2008). Blockage of ER activation by antiestrogens (e.g., tamoxifen) and/or suppression of estrogen synthesis by aromatase inhibitors (e.g.,exemestane) represent the principal endocrine therapy for ER-positivebreast cancer (Burstein et al., 2014). Five years of tamoxifen therapy hasbeen the gold-standard adjuvant treatment of ER-positive breast cancerfor many decades (Burstein et al., 2014). Tamoxifen (TAM) is orallyadministered (20 mg daily) and is converted in vivo by cytochromeP450s (primarily CYP2D6 and CYP3A4) into several metabolites,including 4-hydroxytamoxifen (4-OHTAM) and 4-hydroxy-N-desme-thyltamoxifen (endoxifen) (Jacolot et al., 1991; Dehal and Kupfer,1997). Both 4-OHTAM and endoxifen exist as geometrical isomers and

have a likely trans-to-cis ratio of 70:30 at physiologic pH (Malet et al.,2002; Zheng et al., 2007). As both trans isomers of 4-OHTAM andendoxifen have similar antiestrogenic activity, which is nearly100-fold greater than that of TAM, it is believed that TAM elicits itsantiestrogenic activity primarily through its active metabolites4-OHTAM and endoxifen (Jordan et al., 1977; Furr and Jordan, 1984;Katzenellenbogen et al., 1984; Johnson et al., 2004; Lim et al., 2005;Borges et al., 2006). Although evidence exists supporting cis–4-OHTAM as a weak estrogenic compound (Williams et al., 1994),cumulative studies have provided strong evidence that, similar to trans–4-OHTAM, cis–4-OHTAM acts as an estrogen antagonist in normal andcancerous breast cells (Katzenellenbogen et al., 1984, 1985; Malet et al.,2002). A recent study has shown that both trans- and cis–4-OHTAMdisplayed roughly equipotent antiestrogenic effects on 17b-estradiol–induced progesterone receptor gene expression in MCF7 cells (Zhenget al., 2007).UDP-glucuronosyltransferases (UGTs), an enzyme superfamily, are

responsible for the glucuronidation of numerous therapeutic drugs,including TAM, 4-OHTAM, and endoxifen. N-glucuronidation of TAMand 4-OHTAM at the aminoethoxy side chain has been recentlyreported to be exclusively carried out by UGT1A4, and the resultingN-glucuronides were found to have ER binding affinities similar to thoseof their parent counterparts, suggesting that these N-glucuronides might

This work was supported by funding from the National Health and MedicalResearch Council of Australia [ID 1020931]. P.I.M. is a National Health andMedical Research Council Senior Principal Research Fellow. R.M. is an AustralianResearch Council Future Fellow.

dx.doi.org/10.1124/dmd.114.062935.

ABBREVIATIONS: ChIP, chromatin immunoprecipitation; E2, 17b-estradiol; ER, estrogen receptor; ERE, estrogen response element; ERU,estrogen response unit; FBS, fetal bovine serum; FUL, fulvestrant; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LBD, ligand-bindingdomain; MT, mutated construct; 4-OHTAM, 4-hydroxytamoxifen; PCR, polymerase chain reaction; siRNA, small interfering RNA; SULT,sulfotransferase; TAM, tamoxifen; UGT, UDP-glucuronosyltransferase; WT, wild-type.

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contribute to antiestrogenic activity of TAM in vivo (Kaku et al., 2004;Ogura et al., 2006; Sun et al., 2007; Zheng et al., 2007). By contrast,O-glucuronidation of 4-OHTAM and endoxifen at the 4-hydroxy positionhas been shown to be carried out by multiple UGTs (Nishiyama et al.,2002; Ogura et al., 2006; Sun et al., 2007). This reaction greatlydecreases the ER binding affinity of the resultant O-glucuronides andthus represents an inactivation pathway (Ogura et al., 2006; Zheng et al.,2007). Studies using recombinant human UGT isoforms expressed ininsect cells have shown O-glucuronidation of both 4-OHTAM isomersby six UGTs (1A1, 1A3, 1A8, 1A9, 2B7, and 2B15), with UGT2B7and UGT2B15 having the highest activity toward trans–4-OHTAM andcis–4-OHTAM, respectively (Nishiyama et al., 2002; Ogura et al.,2006). A later study using homogenates of individual UGT-overexpressing cell lines showed similar results and further revealedO-glucuronidation activity of 4-OHTAM by three other UGTs (1A7,1A10, and 2B17) (Sun et al., 2007). Functional polymorphisms in theregulatory and coding regions of three UGT genes (1A4, 1A8, and 2B7)have been shown to be related to differing glucuronidation activitytoward TAM and/or its metabolites (Sun et al., 2006; Blevins-Primeauet al., 2009; Greer et al., 2014).The majority of ER-positive breast cancers respond to TAM but often

develop resistance following prolonged TAM administration. This

acquired resistance remains a challenge for treating ER-positive breastcancer; however, the underlying molecular mechanisms are notcompletely known but are thought to be multifactorial, including lossof ER expression and function, altered expression of ER coregulators,activation of ER-independent signaling pathways, and altered metab-olism of tamoxifen in the tumor (Chang, 2012; Viedma-Rodriguezet al., 2014). With regard to metabolic changes, it has been reported thattamoxifen upregulates sulfotransferase 1A1 (SULT1A1) in breastcancer cells (Seth et al., 2002). As 4-OHTAM is a SULT1A1 substrate(Nishiyama et al., 2002), this upregulation promotes metabolism andclearance of 4-OHTAM through sulfation. In the present study, wedemonstrate that both 4-OHTAM and endoxifen upregulate UGT2B15in breast cancer cells, and we define the underlying molecularmechanisms of this regulation. Induction of UGT2B15 could facilitatemetabolism and clearance of 4-OHTAM through glucuronidation andmay contribute to acquired tamoxifen resistance.

Materials and Methods

Materials. Chemical compounds of analytical grade were purchased fromSigma-Aldrich (St. Louis, MO), including 17b-estradiol (E2), Z-4-hydrox-ytamoxifen (4-OHTAM), raloxifene hydrochloride, fulvestrant ((7a,17b)-7-{9-[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl}estra-1,3,5(10)-triene-3,17-diol), and

Fig. 1. Effects of 17b-estradiol and anties-trogens on UGT2B15 gene expression inMCF7 cells. MCF7 cells were treated intriplicate for 24 hours with 1 mM 4-OHTAM,10 nM E2, 1 mM raloxifene (RAL), 1 mMfulvestrant (FUL), 10 nM E2 plus one of thethree antiestrogens (4-OHTAM, RAL, FUL)at 1 mM, or vehicle (0.1% ethanol for4-OHTAM, 0.1% dimethylsulfoxide for bothRAL and FUL). Total RNA was extractedand then subjected to reverse-transcriptasequantitative real-time PCR to measure themRNA levels of UGT2B15 (A) and GREB1(B) as described in Materials and Methods.Data shown are the fold induction (mean 6S.E.M.) in target gene mRNA levels in drug-treated cells over those in vehicle-treated cells(set as a value of 1) from two (ii) or three(i and iii) independent experiments performedin triplicate. Statistical analyses used one-wayanalysis of variance followed by Tukey’spost-hoc multiple comparison test. #P ,0.05; $P , 0.01; *P , 0.001.

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(E/Z)-endoxifen hydrochloride hydrate. Primers were synthesized by Sigma-Genosys (Castle Hill, NSW, Australia).

Cell Culture, Drug Treatment, RNA Extraction, and Reverse-Transcriptase Quantitative Real-Time Polymerase Chain Reaction. Thehuman breast cancer MCF7 cell line was routinely maintained in RPMI 1640medium (Life Technologies, Grand Island, NY) supplemented with 5% (v/v)fetal bovine serum (FBS) at 37�C and 5% CO2. For ligand treatment, cells wereplated into six-well plates and cultured for 3–4 days to reach approximately70% confluence. Cells were further cultured in phenol red–free RPMI mediasupplemented with 5% dextran charcoal–stripped FBS for 2–3 days and thentreated with 17b-estradiol and/or antiestrogens in triplicate at concentrationsand lengths of time as indicated in the figures. Total RNA was isolated usingthe RNeasy MiniKit (Qiagen, Valencia, CA) or TRIzol reagent (LifeTechnologies) and converted to cDNA using reverse-transcription reagents(Invitrogen, Mulgrave, VIC, Australia) as previously reported (Hu andMackenzie, 2009). Quantitative real-time polymerase chain reaction (PCR)was performed using a RotorGene 3000 (Corbett Research, NSW, Australia)and either QuantiTect SYBR Green PCR master mix (Qiagen) or GoTaqqPCRmaster Mix (Promega, Madison, WI) in a 20-ml reaction containing ;60 ng ofcDNA sample and previously designed primers for target genes, includingUGT2B15, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), pS2, andGREB1 (Hu and Mackenzie, 2009, 2010; Hu et al., 2010). The target genemRNA levels relative to those of 18S ribosomal RNA were quantified using thedelta delta cycle threshold method (Livak and Schmittgen, 2001).

Glucuronidation Assay. MCF7 cells at approximately 80% confluence werecultured in phenol red–free RPMI media containing 5% dextran charcoal–stripped FBS for 3 days and then treated in triplicate with vehicle (0.1% ethanol),10 nM E2, or 1 mM 4-OHTAM for 72 hours. Cells were then harvested forpreparation of whole-cell lysates using TE buffer (10 mM Tris-HCl, 1 mMEDTA, pH 7.6). Protein concentrations of the resultant lysates were determinedusing the Bradford protein assay (Bio-Rad, Hercules, CA). Testosteroneglucuronidation assays were conducted at 37oC for 4 hours in a 100-ml reactioncontaining 200 mg of protein of each lysate, 2 mM UDP-glucuronic acid,

15 pmol/ml [14C]testosterone, 100 mM potassium phosphate (pH 7.5), and 4 mM ofMgCl2. Reactions were terminated by addition of 300 ml of chloroform. Freetestosterone was then extracted by vortex and centrifugation at 12,000 rpm for5 minutes. The aqueous phase containing glucuronide was transferred to a freshtube and diluted with an equal volume of ethanol (50% v/v). Eighty microliters ofthe resultant solution was applied to a thin layer chromatography (TLC) plate(250 mm silica gel; TLC Uniplates; Analtech, Newark, DE). Chromatographywas conducted in solvent containing chloroform, methanol, water, and acetic acid(65:25:4:2, v/v/v/v) for 1 hour. The dried TLC plate was exposed to a phosphorscreen (Molecular Dynamics; GE Healthcare Life Sciences) for 4 days and thenscanned using a Typhoon 9000 detector (GE Healthcare, Chalfont St. Giles, UK).Quantitation of band intensity and background subtraction were conducted usingImageQuant version 5.2 (Molecular Dynamics; GE Healthcare Life Sciences).The testosterone glucuronidation activity in the E2- or 4-OHTAM–treated cellswas presented as fold changes relative to the activity in vehicle-treated cells (setas a value of 1).

Knockdown of Gene Expression Using Small Interfering RNA.ON-TARGETplus nontargeting pool small interfering RNA (siRNA; controlsiRNA, negative control-siRNA) and ON-TARGETplus SMARTpool againstERa (ERa-siRNA) were purchased from Dharmacon RNAi Technologies(Lafayette, CO). MCF7 cells were transfected in quadruplicate in six-wellplates with either negative control-siRNA or ERa-siRNA at 100 nM usingLipofectamine 2000 (Invitrogen) as previously reported (Hu and Mackenzie,2009). Forty-eight hours postransfection, one well was harvested to preparewhole-cell lysates for Western blotting as described later, and the remainingwells were treated for 24 hours with vehicle, 1 nM E2, or 1 mM 4-OHTAM.After treatment, wells were harvested for total RNA, followed by reverse-transcriptase quantitative real-time PCR to quantify the mRNA levels of threetarget genes (UGT2B15, pS2, and GAPDH) as described earlier.

Western Blotting. Whole-cell lysates were prepared from drug-treated andsiRNA-transfected MCF7 cells in radioimmunoprecipitation assay buffer[50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 2 mM EDTA, 0.5% sodiumdeoxycholate, and 0.1% SDS]. Protein concentrations were determined using

Fig. 2. 4-Hydroxytamoxifen elevates UGT2B15 mRNA levelsand enzymatic activity in breast cancer MCF7 cells. (A) MCF7cells were treated in triplicate with vehicle (0.1% ethanol), 1 nME2, or 1 mM 4-OHTAM for 24 hours. Total RNA was extractedand then subjected to reverse-transcriptase quantitative PCR ofthree target genes (UGT2B15, pS2, and GAPDH) as describedin Materials and Methods. Data shown are the fold induction(mean6 S.E.M.) in target gene mRNA levels in cells treated withE2 or 4-OHTAM over those in vehicle-treated cells (set as a valueof 1) from three independent experiments performed in triplicate.(B) Cells were treated with vehicle (0.1% ethanol), 10 nM E2, or1 mM 4-OHTAM for 24 hours. Cell lysates were prepared andthen subjected to testosterone glucuronidation assays as describedin Materials and Methods. Data shown are the fold induction(mean 6 S.E.M.) of testosterone glucuronidation activity in cellstreated with E2 or 4-OHTAM compared with vehicle-treated cellsin two independent experiments, carried out in triplicate.Statistical analyses used one-way analysis of variance followed byTukey’s post-hoc multiple comparison test. *P , 0.001.

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the Bradford protein assay (Bio-Rad). Fifty micrograms of protein in eachsample was separated on SDS-polyacrylamide gels (10%) and transferred tonitrocellulose membranes for immunodetection. Membranes were probed firstwith an anti-ERa antibody (HC-20; Santa Cruz Biotechnology, Dallas, TX),and then with a horseradish peroxidase–conjugated donkey anti-rabbitsecondary antibody (NeoMarkers; Fremont, CA). The same membranes werereprobed with an antiactin antibody (Sigma-Aldrich) followed by thepreviously described anti-rabbit secondary antibody. Immunosignals weredetected with the SuperSignalWest Pico Chemiluminescent kit (Thermo FisherScientific, Waltham, MA) and an ImageQuant LAS 4000 luminescent imageanalyzer (GE Healthcare, Chalfont St. Giles, UK). Quantitation of bandintensity and background subtraction were conducted using MultiGaugeversion 3.0 (Fujifilm Corporation, Tokyo, Japan).

Transient Transfection and Luciferase Reporter Assay. The three pGL3luciferase UGT2B15 promoter constructs containing the region betweennucleotides 2458 and 23 relative to the translation start site used in this study

were reported previously (Hu and Mackenzie, 2009). As shown in Fig. 4A, theUGT2B15-458 wild-type (WT) construct had a functional wild-type estrogenresponse unit (ERU) containing two AP-1 sites, two estrogen response element(ERE) half sites, and an imperfect ERE full site (Hu and Mackenzie, 2009). ThisERU was mutated at three sites in two other constructs as indicated [UGT2B15-458-mutated construct 15 (MT15) and UGT2B15-458-MT16]. MCF7 cells (3 �104cells/well) were seeded into 96-well plates 24 hours prior to transfection. Eachwell was transfected with the transfection mixture in 50 ml of phenol red–freeRPMI containing pRL-null vector (2 ng), a UGT2B15 promoter construct (100 ng),and with or without an ERa expression vector (2.5 ng) using Lipofectamine2000 (Invitrogen). The ERa expression vector was the same as we previouslyreported (Hu and Mackenzie, 2009). Six hours posttransfection, the transfectionmedium was removed, and cells were then treated in triplicate in stripped FBScontaining RPMI media with vehicle (0.1% ethanol), 10 nM E2, or 1 mM4-OHTAM for 48 hours. After treatment, cells were lysed and analyzed usingthe Dual-Luciferase Reporter Assay System (Promega) as previously reported

Fig. 3. 4-Hydroxytamoxifen induces UGT2B15 gene expression via the ERa-signaling pathway in MCF7 cells. (A) MCF7 cells were treated in triplicate for 24 hours withvehicle, 1 mM fulvestrant (FUL), 1 nM E2, 1 mM 4-OHTAM, 1 nM E2 plus 1 mM FUL, or 1 mM 4-OHTAM plus 1 mM FUL and then harvested for total RNA, followed byreverse-transcriptase quantitative real-time PCR to quantify the mRNA levels of three target genes (UGT2B15, pS2, and GAPDH). Data shown are the fold induction (mean6 S.E.M.)in target gene mRNA levels in drug-treated cells over those in vehicle (ethanol)-treated cells (set as a value of 1) from two independent experiments performed in triplicate. Statisticalanalyses used one-way analysis of variance followed by Tukey’s post-hoc multiple comparison test. (B and C) MCF7 cells were cultured in six-well plates, and four wells weretransfected with nontargeting siRNA (negative control-siRNA) or siRNA targeting ERa (ERa-siRNA). Fort-eight hours post-transfection, one well from each transfection washarvested for Western blotting using an antibody recognizing ERa or actin; the remaining wells were treated for 24 hours with vehicle, 1 nM E2, or 1 mM 4-OHTAM. After 24-hourtreatment, wells were harvested for total RNA, followed by reverse-transcriptase quantitative PCR to quantify the mRNA levels of three target genes (UGT2B15, pS2, and GAPDH) asdescribed in Materials and Methods. Shown in (B) are ERa- and actin-immunosignals from a representative experiment of three independent Western blotting assays. Data shown in(C) are the fold induction (mean6 S.E.M.) in target gene mRNA levels in cells transfected and treated with NC-siRNA/E2, ERa-siRNA/E2, NC-siRNA/4-OHTAM, or ERa-siRNA/4-OHTAM over those in cells transfected and treated with NC-siRNA/vehicle (set as a value of 1) from two experiments performed in triplicate. Statistical analyses used the Kruskal-Wallis test followed by Mann-Whitney U Test. #P , 0.05; $P , 0.01; *P , 0.001.


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(Hu and Mackenzie, 2009). The firefly luciferase activity was first normalized tothe Renilla activity and then presented as fold changes relative to those observed incells transfected with the promoterless pGL3 basic vector and treated with the samechemical (set as a value of 1).

Chromatin Immunoprecipitation Assay and Quantitative Real-TimePCR. Chromatin immunoprecipitation (ChIP)–quantitative PCR was per-formed essentially as reported previously (Hu and Mackenzie, 2009). In brief,MCF7 cells were precultured in T175 flasks in phenol red–free RPMI mediacontaining 5% stripped FBS for 2 days and then treated in triplicate withvehicle (0.1% ethanol), 10 nM E2, or 1 mM 4-OHTAM for 2 hours. Followingtreatment, cells were cross-linked using 1% formaldehyde at 37�C for30 minutes, followed by quenching using 125 mM glycine solution at 37�C for10 minutes. Cells were lysed, sonicated, and then subjected to immunoprecip-itation with 8 mg of the rabbit preimmune IgG control (sc-2027; Santa CruzBiotechnology) or equivalent amounts of the anti-ERa antibody (HC-20).Precipitated chromatin was captured by Protein A Sepharose CL-4B beads (GEHealthcare) and subsequently eluted from the beads as described previously(Hu and Mackenzie, 2009). Cross-linking was reversed by heating the eluates at65�C overnight. The resulting DNA/protein precipitates were digested withproteinase K, followed by phenol-chloroform extraction and ethanol pre-cipitation. The DNA pellets were resuspended in 50 ml of Tris-EDTA buffer.Using 3 ml of each of the resultant DNA samples as template and previouslyreported primers (Carroll et al., 2006; Hu and Mackenzie, 2009), real-timequantitative PCR was conducted to quantify the UGT2B15 ERU region and twocontrol loci (ERE negative 1 and 2) that have been shown to have no ERa

binding upon E2 exposure in MCF7 cells. Data from ERE negative locus 1were used to normalize the starting amounts of immunoprecipitated DNAadded to each PCR. The ERa binding activity in ligand-treated cells at theUGT2B15 ERU and ERE negative locus 2 was presented as fold changesrelative to those observed in the respective vehicle-treated cells using the2-ΔΔCt method (Livak and Schmittgen, 2001).

Statistical Analysis. Statistical analysis was performed using the IBM(Armonk, NY) SPSS program (version 22) as we recently reported (Hu et al.,2015). In brief, log-transformed data met the assumption of equal variance ata significance level of less than 0.01 (Levene’s test) unless otherwise stated.Statistical analyses were conducted by one-way or two-way analysis ofvariance followed by Tukey’s post-hoc test or the Kruskal-Wallis test(nonparametric alternative for one-way analysis of variance) followed byMann-Whiteney U test as detailed in the legends of the figures. A P value lessthan 0.05 was considered statistically significant.

Results

4-Hydroxytamoxifen Elevates UGT2B15 mRNA Levels andEnhances Testosterone Glucuronidation Activity in Breast CancerCells. We previously reported that E2 induces UGT2B15 geneexpression via binding of the ERa to the UGT2B15 ERU at theproximal promoter in MCF7 breast cancer cells (Hu and Mackenzie,2009). In the present study, as shown in Fig. 1A, we found that this

Fig. 4. 17b-Estradiol and 4-hydroxytamoxifenactivate the UGT2B15 promoter through the ERUat the proximal promoter. (A) The UGT2B15promoter between nucleotides 2458/23 relative tothe translation start site has a functional ERU thatcontains two AP-1 binding sites, two half sites ofERE, and an imperfect ERE full site. Shown arethree UGT2B15 pGL3-drived promoter constructscontaining a wild-type ERU (UGT2B15-458WT)and two mutated ERU (UGT2B15-458-MT15 andUGT2B15-458-MT16). (B and C) MCF7 cellswere transfected in triplicate with the pRL-nullvector, one of the four promoter constructs (pGL3–basic vector, UGT2B15-458WT, UGT2B15-458-MT15, UGT2B15-458-MT16), with or without theERa expression vector, and subsequently treatedwith vehicle (0.1% ethanol), 10 nM 17b-estradiol,or 1 mM 4-hydroxytamoxifen for 48 hours. Aftertreatment, cells were lysed and analyzed using theDual-Luciferase Reporter Assay System (Promega)as described in Materials and Methods. The fireflyluciferase activity was first normalized to theRenilla activity and then presented as fold changesrelative to those observed in cells transfected withthe promoterless pGL3 basic vector and treatedwith the respective chemical (set as a value of 1).Data shown are the means 6 S.E.M. of fourexperiments performed in triplicate. Statisticalanalyses used two-way analysis of variancefollowed by Tukey’s post-hoc multiple comparisontest. $P , 0.01; *P , 0.001.


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E2-induced UGT2B15 gene expression was significantly abrogated bytwo ER antagonists (raloxifene and fulvestrant, both at 1 mM) but wasnot affected by a third ER antagonist, 4-OHTAM (1 mM) in MCF7cells. In contrast, all three antagonists inhibited E2-mediated inductionof GREB1, a well known ER target gene in MCF7 cells (Fig. 1B)(Deschenes et al., 2007). We further found that, similar to E2 at 1 nM,4-OHTAM at 1 mM significantly elevated UGT2B15 mRNA levelsbut had no significant effects on the mRNA levels of two known ERtarget genes (GREB1 and pS2) and the house-keeping gene GAPDH inMCF7 breast cancer cells (Figs. 1B and 2A). Of note, fulvestrantreduced basal UGT2B15 mRNA levels, and both 4-OHTAM andraloxifene decreased basal GREB1 mRNA levels. Consistent with thisobservation, 4-OHTAM–treated MCF7 cells showed significantlyhigher testosterone (a UGT2B15 substrate) glucuronidation activitycompared with vehicle-treated cells (Fig. 2B). Taken together, theseresults demonstrate that, similar to E2, 4-OHTAM increases theexpression and enzymatic activity of UGT2B15 in breast cancer cells.4-Hydroxytamoxifen Stimulates UGT2B15 Gene Expression via

the ERa-Signaling Pathway in MCF7 Cells. Previous studies haveshown that up to 23% of E2-induced genes are also induced by4-OHTAM via the ERa-signaling pathway in MCF7 cells (Frasoret al., 2004; Lim et al., 2006). The previously described induction ofUGT2B15 by both E2 and 4-OHTAM in MCF7 cells prompted us toinvestigate a potential role of ERa in 4-OHTAM–mediated inductionof UGT2B15. As expected, E2 (1 nM) elevated the mRNA levels ofboth UGT2B15 and pS2, and this elevation was completely abolishedby fulvestrant (Fig. 3A). Similarly, fulvestrant also completelyabrogated the induction of UGT2B15 by 4-OHTAM, suggesting a roleof ER signaling in mediating this induction (Fig. 3A). In support of thisrole, knockdown of ERa expression using siRNA (ERa-siRNA)completely abolished the induction of UGT2B15 by both E2 and4-OHTAM (Fig. 3, B and C). As expected, knockdown of ERaexpression also abolished the induction of pS2 by E2 but did not alterthe control GAPDH mRNA levels (Fig. 3, B and C). Collectively, ourresults demonstrate that, similar to E2, 4-OHTAM induces UGT2B15gene expression via the ERa-signaling pathway in MCF7 cells.4-Hydroxytamoxifen Activates the UGT2B15 Promoter and

Enhances ERa Binding to the ERU of the UGT2B15 ProximalPromoter in MCF7 Cells. We previously found that the upregulationof UGT2B15 by E2 is mediated by ERa binding to the UGT2B15ERU, which contains two AP-1 sites, two ERE half sites, and animperfect ERE full site (Fig. 4A) (Hu and Mackenzie, 2009). Wetested whether 4-OHTAM upregulated UGT2B15 via the same ERUusing luciferase reporter assays and ChIP assays. Consistent with ourprevious report (Hu and Mackenzie, 2009), E2 (10 nM) activated thewild-type ERU-containing UGT2B15 proximal promoter (UGT2B15-458WT), and ERa overexpression further enhanced this activation;however, this activation was completely abolished by mutation of thetwo AP-1 sites plus either the 39 ERE half site (UGT2B15-458-MT15)or the imperfect ERE full site (UGT2B15-458-MT16) (Fig. 4B).Similarly, 4-OHTAM (1 mM) alone or together with ERa over-expression enhanced the activity of the wild-type UGT2B15 promoter(UGT2B15-458WT) but did not stimulate the activity of the two ERU-mutated constructs (UGT2B15-458-MT15 and UGT2B15-458-MT16)(Fig. 4C). Furthermore, ChIP assays showed that treatment of MCF7cells with either E2 (10 nM) or 4-OHTAM (1 mM) enhanced ERabinding at the UGT2B15 ERU but not at the control negative ERE 2locus (Fig. 5). Of note, significant basal ERa occupancy at theUGT2B15 ERU was also observed. This is consistent with previousfindings that many ER sites have basal ER binding before ligandstimulation, presumably due to the presence of residual levels of activeestrogenic molecules in the stripped FBS added to the media or

synthesized by MCF7 cells under hormone-depleted culture conditions(Carroll et al., 2006). Taken together, our results indicate that, similarto E2, 4-OHTAM upregulates UGT2B15 via ERa binding to thepreviously reported UGT2B15 ERU in MCF7 cells.Endoxifen Stimulates UGT2B15 Gene Expression via the

ER-Signaling Pathway in MCF7 Cells. 4-OHTAM and endoxifenare the two major active TAM metabolites, and endoxifen is consideredas a new therapeutic agent for breast cancer (Ahmad et al., 2010a,b).Recent studies using MCF7 cells showed that endoxifen and 4-OHTAMhave similar effects on global gene expression patterns, and the majorityof the affected genes are estrogen-regulated genes (Lim et al., 2006). Assuch, we treated MCF7 cells with endoxifen to see whether endoxifencould also induce UGT2B15 gene expression via the ER-signalingpathway. As shown in Fig. 6, A and B, endoxifen at 1 mM significantlyincreased UGT2B15 mRNA levels but had no impact on the mRNAlevels of pS2 and GAPDH. Endoxifen slightly but significantly reducedE2-elevated UGT2B15 mRNA levels; however, the endoxifen-inducedUGT2B15 expression was completely abolished by fulvestrant,confirming a role for ER. Of note, the 3.4-fold induction of UGT2B15by endoxifen (1 mM) was lower than the 8-fold or 6-fold induction seenwith 4-OHTAM (1 mM) alone or 4-OHTAM (1 mM) together withendoxifen (1 mM). Furthermore, we demonstrated that, similar to4-OHTAM, endoxifen (1 mM) significantly activated the activity of theUGT2B15 promoter carrying a wild-type ERU (UGT2B15-458WT),and this activation was abolished by mutating the ERU (UGT2B15-458-MT15 or UGT2B15-458-MT16) (Fig. 6C). Collectively, our resultsindicate that, similar to 4-OHTAM, endoxifen induces UGT2B15 geneexpression via the UGT2B15 ERU.

Discussion

We recently reported that 17b-estradiol upregulates UGT2B15 inMCF7 breast cancer cells via ERa binding to an ERU in the proximal

Fig. 5. 4-Hydroxytamoxifen enhances the binding of the ERa at the UGT2B15ERU in MCF7 cells. MCF7 cells were treated for 45 minutes or 2 hours with vehicle(0.1% ethanol), 10 nM 17b-estradiol, or 1 mM 4-hydroxytamoxifen, and thensubjected to ChIP assays using an anti-ERa antibody (HC-20) or preimmune IgGserum, followed by real-time PCR to quantify the amount of precipitated DNAcorresponding to the UGT2B15 ERU or two negative control loci (ERE negativeloci 1 and 2) as described inMaterials and Methods. Data were normalized using theamplification values for ERE negative locus 1, and then expressed as the foldenrichment in DNA samples precipitated with the anti-ERa antibody relative to thatof the control DNA samples precipitated with equivalent amounts of the preimmuneIgG serum (set as a value of 1). Data shown are the means 6 S.E.M. offour experiments performed in triplicate. Statistical analyses used two-wayanalysis of variance followed by Tukey’s post-hoc multiple comparison test.*P , 0.001.


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promoter of UGT2B15 (Hu and Mackenzie, 2009). In the present study,we showed that, similar to 17b-estradiol, 4-OHTAM and endoxifen, thetwo major antiestrogenic metabolites of tamoxifen, induce UGT2B15gene expression in breast cancer MCF7 cells via ERa binding to thesame UGT2B15 ERU. This finding is consistent with previousobservations that about 74% of the genes regulated by both 4-OHTAMand endoxifen were regulated by 17b-estradiol via the ERa-signalingpathway in MCF7 cells (Lim et al., 2005). 4-OHTAM and raloxifeneare selective estrogen receptor modulators; however, they both actprimarily as ER antagonists in breast cancer cells, suppressing estrogen-mediated gene transcription and breast cancer cell proliferation (Frasoret al., 2004). Consistent with this notion, we showed in the present studythat, similar to the pure ER antagonist fulvestrant, both raloxifene and4-OHTAM significantly suppressed the 17b-estradiol–induced expressionof two ER target genes (i.e., GREB1 and PS2) in MCF7 cells. However,these three agents displayed differing effects at the UGT2B15 ERU inMCF7 cells. Specifically, fulvestrant and raloxifene acted as ERantagonists to repress the 17b-estradiol–induced UGT2B15 expression.4-OHTAM was not able to reduce this induction, and furthermore, it

acted as an ER agonist to stimulate UGT2B15 expression. As discussedin previous studies, whether 4-OHTAM acts as an ER agonist or an ERantagonist can depend on the target promoter structure and dynamicchanges of the ligand-bound ERa–ligand-binding domain (LBD) dimerat the promoter. Regarding the conformational changes of the ERa-LBD dimer following ligand binding, Chakraborty and Biswas (2014)recently showed that the binding of 4-OHTAM to the LBD remodelsthe conformational dynamics of the ERa-LBD dimer such that both theagonist and antagonist conformations are accessible. Results from thepresent study suggest that an agonist conformation of the 4-OHTAM–

bound ERa-LBD dimer is preferentially established at the UGT2B15ERU, followed by the assembly of an active transcriptional complexthat promotes UGT2B15 transcription. This assumption remains to bevalidated in future studies.Our study adds UGT2B15 regulation to the likely mechanisms

underlying acquired TAM resistance. Although both trans- and cis–4-OHTAM isomers are substrates of multiple UGTs, UGTs other thanUGT2B15 are expressed at low levels in breast cells or are not inducedby 4-OHTAM (data not shown). In contrast, UGT2B15 is highly

Fig. 6. Endoxifen stimulates the expression and promoter activity of the UGT2B15 gene in MCF7 cells. (A) Cells were treated in triplicate for 24 hours with vehicle, 1 mMendoxifen (END), 1 mM 4-OHTAM, 10 nM E2, 1 mM END plus 1 mM 4-OHTAM, or 1 mM END plus 10 nM E2. (B) Cells were treated in triplicate for 24 hours withvehicle (0.1% ethanol), 1 mM fulvestrant (FUL), 1 mM END, or 1 mM END plus 1 mM FUL. Total RNA was harvested from treated cells and subjected to reverse-transcriptase quantitative PCR to quantify the mRNA levels of target genes including UGT2B15, pS2, and GAPDH as described in Materials and Methods. The data werefirst normalized to those of 18S ribosomal RNA, and then presented as fold changes relative to those of vehicle-treated cells. Data shown are the means 6 S.E.M. of twoexperiments performed in triplicate. Statistical analyses used one-way analysis of variance followed by Tukey’s post-hoc multiple comparison test. (C) Cells were transfectedwith four reporter constructs (pGL3–basic vector, UGT2B15-458WT, UGT2B15-458-MT15, and UGT2B15-458-MT16) together with the internal control pRL-null vector,and then treated with vehicle (0.1% ethanol) or 1 mM END followed by luciferase assays, as described in Materials and Methods. The data were first normalized to theactivity of the pRL-null vector and then presented as fold changes relative to the activity of the pGL3–basic vector. Data shown are the means6 S.E.M. of three experimentsperformed in triplicate. Statistical analyses used two-way analysis of variance followed by Tukey’s post-hoc multiple comparison test. *P , 0.001.


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expressed in breast cancer tissues and cell lines (Nakamura et al., 2008;Ohno and Nakajin, 2009) and has the highest activity toward cis–4-OHTAM (Nishiyama et al., 2002; Ogura et al., 2006). Hence, theupregulation of UGT2B15 by 4-OHTAM and endoxifen in breast cancercells, as reported in the present study, would promote 4-OHTAMinactivation through glucuronidation. Consistent with this idea, a com-parison of gene expression profiles using DNA microarrays betweena TAM-sensitive breast cancer xenograft (MaCa 3366) and its TAM-resistant subline (MaCa 3366/TAM) showed significantly higherUGT2B15 mRNA levels in the resistant tumor compared with thesensitive tumor (Naundorf et al., 2000; Becker et al., 2005). Collectively,these results suggest a role of 4-OHTAM in autoinduction of its ownmetabolism through upregulation of UGT2B15 in breast cancer cells.UGT2B15 D85Y is a prevalent polymorphism (rs1902023), and the Y85allele confers a 2-fold higher catalytic activity compared with the D85allele (Levesque et al., 1997). The Y85 allele has been linked to anincreased risk of developing TAM resistance in a subset of genotype-phenotype association studies (Nowell et al., 2005; Dezentje et al., 2013;Markiewicz et al., 2013), although some other similar studies did not findan association (Wegman et al., 2007; Ahern et al., 2011; Brooks et al.,2013). A larger study using more patients will be required to clarify thislink. In addition to UGT2B15, other enzymes (i.e., SULT1A1 andCYP3A4) and the efflux transporter P-glycoprotein (also termedmultidrug resistance 1) that are involved in either metabolism ordisposition of TAM and/or its metabolites are also induced by4-OHTAM in MCF7 cells (Nishiyama et al., 2002; Seth et al., 2002;Nagaoka et al., 2006; Teft et al., 2011). Our new findings, together withthe previous studies described earlier, provide a possible explanation forprevious observations from studies of MCF7-derived breast cancerxenografts in nude mice that intratumoral concentrations of TAM and4-OHTAM in TAM-resistant tumors are significantly lower compared withconcentrations in TAM-sensitive tumors (Osborne et al., 1991).Collectively, these studies reaffirm the early hypothesis that reducedintratumoral concentrations of TAM and its active metabolites likelycontribute to the development of acquired tamoxifen resistance(Katzenellenbogen, 1991; Osborne et al., 1991; Johnston et al., 1993).In conclusion, we showed that, similar to 17b-estradiol, 4-OHTAM

and endoxifen, the two major antiestrogenic metabolites of tamoxifen,upregulate UGT2B15 in breast cancer cells via ER binding to thepreviously reported UGT2B15 ERU. This upregulation, together withthe previously reported upregulation of SULT1A1 and CYP3A4 bytamoxifen and/or 4-OHTAM, demonstrates induction of metabolism oftamoxifen and its active metabolites in breast cancer cells upon exposureto tamoxifen. If a similar induction occurs in vivo, this would likely leadto reduced tamoxifen therapeutic efficacy and could contribute to thedevelopment of acquired resistance to tamoxifen treatment.

Authorship ContributionsParticipated in research design: Chanawong, Hu, Mackenzie, McKinnon.Conducted experiments: Chanawong.Performed data analysis: Chanawong, Hu, Mackenzie.Wrote or contributed to the writing of the manuscript: Chanawong, Hu,

Mackenzie, Meech, McKinnon.

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Address correspondence to: Ross A. McKinnon, Flinders Centre for Innovationin Cancer, School of Medicine, Flinders University, Bedford Park SA 5042,Australia. E-mail: [email protected]


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