Preferential Estrogen Receptor b Ligands Reduce Bcl-2 ...B 0 50 100 150 200 Percentage of cell...

Cancer Biology and Signal Transduction

Preferential Estrogen Receptor b Ligands Reduce Bcl-2Expression in Hormone-Resistant Breast Cancer Cells toIncrease Autophagy

Samantha C. Ruddy1, Rosanna Lau1, Miguel A. Cabrita1, Chelsea McGregor1, Bruce C. McKay3,Leigh C. Murphy5, James S. Wright4, Tony Durst2, and M.A. Christine Pratt1

AbstractAcquired resistance to selective estrogen receptor (ER)modulators (SERM) and downregulators (SERD) is a

significant clinical problem in the treatment of estrogen (E2) receptor-positive (ERþ) breast cancers. There aretwo ER subtypes, ERa and ERb, which promote and inhibit breast cancer cell proliferation, respectively.

Although ERþ breast cancers typically express a high ratio of ERa to ERb, the acquisition of SERM resistance in

vitro and in vivo is associated with increased relative expression of the ERb. On some gene enhancers, ERb has

been shown to function in opposition to the ERa in the presence of E2. Here, we demonstrate that two different

ERb agonists,WAY-20070 and a novel "A-CD" estrogen called L17, produce amarked reduction inG2–Mphase

correlated with effects on cyclin D1 and cyclin E expression in a SERM/SERD-resistant breast cancer cell line.

ERb agonists recruited both the ERa and ERb to the Bcl-2 E2-response element strongly reducing Bcl-2mRNA

and protein in an ERb-dependent manner. L17 recruited RIP140 to the Bcl-2 promoter in cells overexpressing

ERb. Exposure to the ERb ligands also resulted in increased processing of LC3-I to LC3-II, indicative of

enhancedautophagic flux. The coaddition of ERb agonist and the autophagy inhibitor chloroquine resulted in a

significant accumulation of sub-G1 DNA which was completely prevented by the addition of the caspase

inhibitorZ-VAD-FMK.Wepropose that combined therapieswith anERb agonist andan inhibitor of autophagy

may provide the basis for a novel approach to the treatment of SERM/SERD-resistant breast cancers. Mol

Cancer Ther; 13(7); 1882–93. �2014 AACR.

IntroductionThe estrogen receptors (ER) are ligand-dependent

nuclear receptors that contain a DNA-binding domain,ligand-binding domain, an N-terminal transcriptionalactivating function AF-1, and a C-terminal AF-2 (1).There are two subtypes called ERa and ERb which caneither form homodimers or heterodimers to transacti-vate responsive genes in the presence of E2. Althoughthe ERa has a strong ligand independent AF-1 region,the ERb AF-1 function is weaker (2) and mediates adominant negative effect on ERa as a result of an N-

terminal repressor function (3). Chromatin immunopre-cipitation (ChIP) on chip analysis suggests that there isconsiderable overlap in DNA response element bindingbetween the two receptors (4). ERb activity is not onlycell type dependent, but also enhancer sequence andligand dependent (5). Several groups have demonstrat-ed that ectopic expression of the ERb in ERaþ breastcancer cells results in growth inhibition (6, 7) and pre-vents xenograft formation in nude mice in response toE2 (7). ERb regulates gene transcription in an E2-inde-pendent and dependent manner with downstreameffects impacting on cell-cycle progression (8, 9). Over-expression of ERb can activate p21 and p27 expressioncausing a G2 accumulation (3). Cyclin D1 is positivelyregulated by the ERa and negatively regulated by theERb in the presence of E2 (7).

In the normal human and rodent mammary gland, theERb is expressed at higher levels than theERa. This ratio istypically reversed in ER-positive (ERþ) breast cancer (10)although many breast cancers continue to express lowlevels of ERb. Reduced ERb expression in ERþ breastcancer cells is in part due to promoter methylation (11).

The standard of endocrine treatment for ERþ breastcancer is the selective ER modulator (SERM), tamoxifen(TAM). Unfortunately, the vast majority of responsivetumors eventually develop SERM resistance. TAM-

Authors' Affiliations: Departments of 1Cellular and Molecular Medicineand 2Chemistry; 3Cancer Therapeutics Program, Ottawa HospitalResearch Institute and the Departments of Medicine, and Cellular andMolecular Medicine, University of Ottawa; 4Department of Chemistry,Carleton University, Ottawa, Ontario; and 5Manitoba Institute of Cell Biol-ogy, University of Manitoba, Winnipeg, Manitoba, Canada

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

Corresponding Author: M.A. Christine Pratt, Department of Cellular andMolecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ONK1H 8M5. Phone: 613-562-5800 ext. 8366; Fax: 613-562-5636; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-1066

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 13(7) July 20141882

on March 7, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 30, 2014; DOI: 10.1158/1535-7163.MCT-13-1066

http://mct.aacrjournals.org/

resistant (Tam-R) cell lines remain sensitive to growthinhibition by selective ER downregulators (SERDs)including fulvestrant (ICI 182,780; refs. 12–15). About halfof patients respond to second-line endocrine therapyincluding aromatase inhibitors and fulvestrant (16); how-ever, fulvestrant-resistant cells emerge (17) representingthe limits of current endocrine therapy.SERM resistance can be either intrinsic to a subpopu-

lation of breast cancer cells within a tumor or can beacquired (18, 19). Typically, SERM resistance is not asso-ciated with loss of the ER (estimated at less than 25%;ref. 20). SERM resistance is complex and involves changesin intracellular signaling through growth factors or acti-vated oncogenes that is implicated in the ligand-indepen-dent activation of the ER (18, 21).Tamoxifen is a pure antagonist at the ERb (22) and some

reports have shown a correlation between lack of ERbexpression and de novo SERM resistance (23), while othersfound that SERM-resistant tumors have increased ERbmRNA expression (24). Derivatives of MCF-7 ERþ breastcancer cells have been selected for SERM/SERD resis-tance and demonstrate a net decrease in the expression ofthe ERa but not ERb (25). One such cell line, called LCC9,was derived from MCF-7 cells after long-term culture inthe presence of fulvestrant and displays cross-resistanceto tamoxifen (17).In previous work, we have designed and synthesized

new ligands that preferentially activate the ERb (26). Thestructure of L17 is based on theABCD-ring structure of E2but lacks the B ring. In the current study, we have inves-tigated the impact of L17 and a secondERb agonist,WAY-200070 (27) on SERD/SERM-resistant LCC9 cells. Ourresults show that ERb agonists inhibit LCC9 cell growthand induce an autophagic response associated withreduced Bcl-2 expression.

Materials and MethodsCell lines and cell cultureMCF-7/LCC1 andMCF-7/LCC9 and MCF-7CL paren-

tal cells (28) were obtained from Dr. Robert Clarke, Geor-getown University (Washington, DC) at low passage andwere routinely cultured in Dulbecco’s Modified EagleMedium (with or without phenol red) containing 5%unstripped or dextran charcoal-stripped FBS (CSS). Thegenetic relationship of the three cell lineswith the originalMCF-7 cell line was confirmed by DNA fingerprintingusing genetic markers at nine different loci (CSF1PO,TPOX, TH01, vWA, D16S539, D7S820, D13S317, D5S818,and the Y chromosome-specific amelogenin) in the Clarkelab. Cells were passaged a maximum of eight times fromthe point of receipt. Markers were not retested althoughSERM/SERD resistance of LCC9 cells was reconfirmed(Supplementary Data). MCF-7 cells (denoted MCF-7PL)were originally obtained from theAmerican TypeCultureCollection. They have not been retested for lineage mar-kers but continue to express the luminal markers ERa, E-cadherin, pS2, and CK18. The MCF-7 (rTA tet-ON ERb1)subclone was obtained from Dr. Leigh Murphy, Univer-

sity of Manitoba (Winnipeg, Manitoba, Canada), and wasderived from a clone of MCF-7 cells stably expressingreverse tetracycline transactivator (clone 89 rTA) trans-fected with doxycycline-inducible His-Xpress-ERb1expression (tagged-ERb1; ref. 29; and refs therein). Theseclones were not tested for genetic markers before or afterreceipt but were used exclusively for ChIP analysis afteracute treatmentwith ligands.All cellswere shown tobe freeof mycoplasma contamination by PCR.

Chemicals17b-estradiol (E2), 4-hydroxytamoxifen, WAY-200070,

and chloroquine were purchased from Sigma. Ligand 17(L17) was synthesized as previously described (26). Z-VAD-FMK was purchased from BD Biosciences.

AntibodiesAntibodieswere: anti-ERa (HC-20), anti-cyclinD1 (A-12)

SRC-3/AIB1 (sc-9119),RIP140 (sc-8997; SantaCruzBiotech-nology); anti-ERb for immunoblot analysis (Thermo-Scien-tific PAI-311; immunoblot analysis; Fig. 1)/anti-ERb(Novus Biologicals NBP-04936; all other immunoblot anal-yses); anti-ERb (GTX70182; GeneTex) for ChIP; anti-actin(A-2066; Sigma); anti-LC3 (Novus Biologicals), anti-Bcl-2(BD Biosciences), anti-cyclin E (ab7959), Abcam, Chrom-Pure rabbit immunoglobulinG (IgG),wholemolecule (011-000-003), peroxidase-conjugated goat anti-rabbit IgG(HþL) goat anti-mouse IgG (HþL), goat anti-chicken IgY(HþL; Jackson ImmunoResearch Inc.).

ERb knockdown and inductionLCC9 cells were transfected with 25 nmol/L siGen-

ome SMARTpool human ESR2 siRNA (DharmaconCat# M-003402–04) by reverse transfection using Dhar-mafect (Dharmacon) according to the manufacturer’sdirections. MCF-7(rTA tet-ON ERb1) was treated with 1mg/mL doxycycline (BioBasic) to induce overexpres-sion of ERb and reverse transfected with 25 nmol/LESR1 siRNA ON-TARGET plus (Dharmacon Cat# (L-003401-00-0005). Control transfections were done withsiCONTROL nontargeting (NT) siRNA #1 (Cat# D-001210-01-05). For some proliferation assays, LCC9cells were infected with retrovirus, pSUPER expressingpreviously verified ERb short hairpin RNA (shRNA;pERbshRNA; Addgene plasmid #35561) or nontarget-ing GFP-shRNA (shNT) vector (Addgene plasmid#30519).

Cellular proliferation/viability assayA total of 7.0 � 104 cells were plated in 60 mm dishes

then treated with ethanol (vehicle) or varying concentra-tions of E2, L17, and WAY. Treatment was performed intriplicate and experiments repeated three times. Viablecells were enumerated using Trypan blue exclusion or aVi-Cell XR cell viability analyzer (BeckmanCoulter). Insome experiments, chloroquine and Z-VAD-FMK wereadded at a final concentration of 33 and 100 mmol/L,respectively.

Targeting ERb in SERM-Resistant Breast Cancer

www.aacrjournals.org Mol Cancer Ther; 13(7) July 2014 1883




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Figure 1. Relative ER expression and effects of ERb agonists on LCC9 and MCF-7 cells. A, structures of E2, L17, and WAY200070. B, Western blot analysisshowing the expression levels of ERa and ERb in MCF-7 cells from our lab (MCF-7PL), two MCF-7 subclones that are hormone-independent (LCC1) andSERM/SERD-resistant (LCC9), and the MCF-7(LCC9) parental cell line obtained from the Clarke lab (MCF-7CL; lane 4). MCF-7 (C) and LCC9 (D) cellswere grown in phenol red-free medium with CSS and were treated with increasing concentrations of E2 or L17. Cells were enumerated using Trypan blueexclusionafter 3 and5days.Data represent themean from three separate experiments eachperformed in triplicate; error bars indicateSE. E,MCF-7 andLCC9cells were cultured in the presence of vehicle, 10 nmol/L E2, 10 nmol/L L17, or 10 nmol/L WAY for 3 days and viable cells were enumerated. Graph depictspercentage of vehicle-treated cell numbers� SE. F, LCC9 cells infected with retrovirus expressing shERb or shNTwere treated with 10 nmol/L L17 or vehiclefor 4 days and enumerated. Results shown are percentage of corresponding vehicle-treated cells. Bars are standard error of triplicate samples. Theexperiment was performed twice with similar results. G, anti-ERb immunoblot analysis of LCC9 cell lysates 72 hours after infection with or pSUPER-shERb orshNT. A cross-reactive band (�) is indicated. Actin was immunoblotted as a protein lysate loading control. Molecular weight marker (kDa) is shown on the left.

Ruddy et al.

Mol Cancer Ther; 13(7) July 2014 Molecular Cancer Therapeutics1884




Immunoblot analysisProteins were separated using SDS-PAGE and trans-

ferred to polyvinylidene difluoride membranes (Milli-pore). Immunoreactive bands were detected using sub-strate (Millipore) and quantified by densitometry usingImageJ v1.43.

Chromatin immunoprecipitationChIP assays were performed using a ChIP Assay Kit

(Upstate Biotechnology) according to the manufacturer’sinstructions with some modifications. Cells were treatedwith vehicle, 10 nmol/L E2, L17, or WAY for 30 minutes(ER ChIP) or 15 minutes (RIP140/SRC-3 ChIP). Chroma-tin was cross-linked with 1% formaldehyde then sonicat-ed and protein–DNA complexes were immunoprecipi-tated with anti-ERa, anti-ERb, anti-RIP140, anti-SRC-3,normal rabbit IgG, or no antibody (input) at 4�C over-night. Cross-linkswere reversed at 65�C for 4 hours. DNAwas purified by phenol/chloroform extraction, precipi-tated, and subjected to PCR analysis. PCR primersequences were the same as those used in quantitativereal-time PCR (qRT-PCR). Of note, 5 mL of DNA (noantibody) was used for the input PCR reaction. Productswere run on a 1% agarose gel and visualized by ethidiumbromide staining. Bandswere quantified by densitometryusing ImageJ v1.43 and IgG subtracted from test samplesthen results graphed as a percentage of input DNA.Additional Materials and Methods are included in

Supplementary Files.

ResultsERb agonists inhibit proliferation of LCC9 cellsTogether with structural differences (Fig. 1A), L17 and

WAY represent two different classes of ERb agonistsbased on selectivity and binding affinity. WAY-200070(WAY) is an aryl diphenolic azole ERb agonist. The ERbRBA of WAY is 133 (RBA ratio ERb:ERa ¼ 68; ref. 27),whereas the ERbRBAof L17 is 1.73 (RBA ratio ERb:ERa¼9.3; ref. 26). Because the ERb has been shown to mediaterepressive effects on proliferation, we determinedwheth-er ERb agonists would differentially affect cells with highERb:ERa expression. Figure 1B shows the relative level ofexpression of ERa and ERb protein in MCF-7 cells fromour laboratory (MCF-7PL) in comparisonwith LCC1 cells,LCC9 cells, andMCF-7 cells that are parental to LCC9 andLCC1 (MCF-7CL). The expression of ERa is similar in bothMCF-7 lines although the level of ERb is reduced inMCF-7CL cells relative to MCF-7PL cells demonstrating signif-icant variation across isolates of MCF-7 cells. ERa wasreduced in LCC1 cells, whereas the ERb was increasedrelative to parental MCF-7CL cells. LCC9 cells expresssubstantially lower levels of ERa but maintain expressionof ERb at levels similar to LCC1 cells and higher than thatin the parentalMCF-7 cells. Thus the ratio of ERa to ERb ismuch reduced in LCC9 cells compared with either MCF-7PL orMCF-7CL cells. The SERM/SERD sensitivity of theMCF-7 cells and resistance of the LCC9 cells used in thisstudywere verified in proliferation assays after treatment

with either tamoxifen or fulvestrant (SupplementaryFig. S1).

We next determined the effects of E2 and our novelligand, L17, on proliferation of MCF-7 cells and LCC9cells.We chose to useMCF-7PL cells because they expresslevels of the ERb comparable with LCC9 cells and there-fore serve as a more appropriate comparison than MCF-7CL cells. The results in Fig. 1C demonstrate that E2increased MCF-7 viable cell numbers in a concentra-tion-dependent manner while L17 had no significanteffect. In contrast, E2 had little overall effect on LCC9proliferation consistent with a previous report (30).Remarkably, L17 induced a dose-dependent decrease inviable cell numbers within 3 days which approached 40%within 5 days (Fig. 1D). WAY and L17 had a similargrowth inhibitory effect on LCC9 cells at 10 nmol/L (Fig.1E). To verify that growth inhibition induced by L17 wasmediated through the ERb, we treated LCC9 cells infectedwith a nontargeting retrovirus (shNT) or pSUPER-ERbshRNA (shERb; ref. 31) with L17 or vehicle. After 5 days,decreased proliferation was observed in L17-treatedshNT-infected cells, while shERb expression completelyprevented growth inhibition (Fig. 1F). Verification of ERb

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knockdown is shown in Fig. 1G. These results are consis-tent with L17 signaling in an ERb-dependent manner toinhibit LCC9 cell proliferation.

ERbagonists inhibitG1andSphaseexit inLCC9cellsWenext assessed the impact of L17 andWAYon the cell

cycle. After 3 days of culture in 10 nmol/L E2MCF-7 cellsdemonstrated small reduction in G1 and increase in Sphase cells (Fig. 2A and Supplementary Fig. S2A). Asexpected, tamoxifen produced a strong G1 arrest. Consis-tent with the results in Fig. 1, L17 and WAY had nosignificant effect on the cell-cycle distribution in MCF-7cells.

E2 (10nmol/L) induceda small (10%)decrease in theG1

and corresponding increase in the S phase of the cell cyclein LCC9 cells (Fig. 2B and Supplementary Fig. S2B),whereas tamoxifen had no effect. Remarkably, treatmentof these cellswith either L17 orWAY treatment resulted ina significant decrease in the G2–M phase with a corre-sponding increase in G1 and S phases of the cell-cycleaccumulation suggesting that ERb activation can inhibitboth progression into and exit from S phase.

Effects of ERb agonists on cell-cycle proteinsCyclinD1 is inducedbyE2via theERa and is negatively

regulated by the ERb (7). E2 led to an increased expression

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ActinFigure 3. Effects of ERb ligands oncell-cycle protein expression.Examples of immunoblot analysesfor cyclins and in lysates of MCF-7(A) and LCC9 (B) cells treated withthe indicated ligands over a 48-hour time course. Actin was usedas a protein loading control.Graphs showing meandensitometric values from threeexperiments for cyclin D1 (C), 48kDa cyclin E (D), and 50 kDa cyclinE (E). Bars, SE; P values werecalculated using a t test.

Ruddy et al.





of cyclin D1 and cyclin E that persisted up to 48 hours inMCF-7 cells, whereas L17 and WAY failed to elicit asimilar response (Fig. 3A). Although a lower molecularweight 48 kDa species of cyclin E was present in responseto E2 in LCC9 cells (Fig. 3B, D, and E), no induction ofcyclinD1wasdetected (Fig. 3B andC). Treatment of LCC9cells with L17 or WAY had comparably little effect on theexpression of cyclin D1 although increased cyclin E pro-tein was observed after 48 hours of treatment with eitherL17 orWAY (Fig. 3B and D). Overall, the pattern of cyclinD1andEexpression is consistentwith theobserved effectson proliferation and cell-cycle distribution.

L17 andWAY inhibit expression of Bcl-2 and activatean autophagic response in LCC9 cellsBcl-2 is a critical regulator of both apoptosis and autop-

hagy. LCC9 cells have been shown to express higher basallevels of Bcl-2 relative to parental MCF-7 cells, and RNAinterference (RNAi) for Bcl-2 induces an autophagicresponse in these cells (32). We previously reported thatBcl-2 is regulated in response to E2 (33) and consistentwith this, E2 strongly induced Bcl-2 expression in MCF-7cells (Fig. 4A and E). Both L17 and WAY transientlyinduced Bcl-2 within 24 to 48 hours and levels returnedto baseline by 72 hours. Bcl-2 can inhibit autophagythrough binding and inhibition of Beclin 1 (34). Phospha-tidylethanolamine conjugation and subsequent cleavageconvert LC3-I to II and can be used to assess autophagicflux (35). Immunoblot analysis of LC3 in MCF-7 cellsshowed that the overall basal level of LC3-I was highwith little or no LC3-II. Although LC3-I levels remainedconstant following E2 treatment of MCF-7 cells, we noteda small increase in LC3-II at 48 hours post L17 treatment,whereas WAY had little effect on LC3-II (Fig. 4A and F).Consistent with previous reports (32), Bcl-2 is highly

expressed in LCC9 cells and E2 treatment produced asmall decrease in Bcl-2 protein observed after 24 and 48hours (Fig. 4B and E). Remarkably, treatment with eitherL17 or WAY resulted in marked downregulation of theBcl-2 protein. UnlikeMCF-7 cells, baseline levels of LC3-IIwere detectable in LCC9 cells, however, LC3-II wasstrongly increased after treatment with E2, L17, or WAY(Fig. 4B and F). Knockdown of ERb using siRNA showedthat the L17-mediated decrease in Bcl-2 expression inLCC9 cells was ERb dependent (Fig. 4C and D). Thus,activation of the ERb, especially by ERb-preferential ago-nists, has opposite effects on Bcl-2 expression in LCC9 andMCF-7 cells that may be a consequence of the high ERb:ERa ratio in LCC9 cells.

ERb agonists recruit both the ERb and ERa to theBcl-2 estrogen response elementThe ability of ERb ligands to reduce Bcl-2 expression in

LCC9 cells might be the result of the association of ERbagonist-bound homodimers or ERb/ERa heterodimersto the Bcl-2 ERE. To address this question, we performeda ChIP analysis with antibodies against ERa and ERbfollowing a 1-hour exposure to each ligand. PCR using

primers adjacent to Bcl-2 ERE in exon 2 (36). We usedthe pS2 gene as a second ERE-containing E2-responsivegene since the ERb had previously been shown to interactwith its promoter region (5). Neither ER was present onthe Bcl-2 or pS2 promoter in vehicle-treated cells. Expo-sure to 10 nmol/L E2 for 1 hour resulted in recruitment ofboth the ERa and ERb on the Bcl-2 gene in LCC9 cells(Fig. 5A and B and Supplementary Fig. S3A). Interesting-ly, ERa and ERb were both present on the Bcl-2 ERE

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Figure 4. ERb agonists reduce Bcl-2 expression and increaseautophagy in LCC9 cells. Immunoblot analysis of Bcl-2 and LC3 inprotein lysates from MCF-7 (A) and LCC9 (B) cells. Cells were treatedwith 10 nmol/L E2, L17, WAY, or vehicle for the indicated times. C, Bcl-2 immunoblot analysis of lysate from LCC9 cells transfected with NTsiRNA or ERb siRNA and treated with vehicle or L17 for 48 hours. Actinwas detected as a loading control. D, ERb immunoblot analysis ofsiRNA-transfected LCC9 cell lysates. A nonspecific band (�) isindicated. Actin immunoreactivity was used as a loading control. Meandensities derived from three separate immunoblot analyses for Bcl-2(E) and LC3-II (F) are shown. Dashed lines indicate baseline expressionlevel set at value of 1 after 72 hours (vehicle). Bars, SE; P values werecalculated using a t test.






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following treatment with L17 or WAY in LCC9 cells withrelatively equivalent band amplification for all agonists.In contrast, the ERa was robustly recruited to the Bcl-2ERE inMCF-7 cells in the presence of E2, whereas L17 andWAY each recruited the ERa at significantly reducedlevels relative to E2 (Fig. 5C and D and SupplementaryFig. S3B). The ERb was present on the Bcl-2 promoterfollowing E2, L17, and WAY treatment of MCF-7 cellsalthough L17-induced recruitment was approximatelytwo-thirds that of the other ligands (Fig. 5B). Overall, bothL17 and WAY induced ERa and ERb recruitment to theBcl-2 and pS2 genes associated with transcriptionalrepression especially in LCC9 cells. In contrast, the stronginduction of Bcl-2 transcripts by E2 in MCF-7 cells (seebelow)was correlatedwith amuchgreater fold increase inERa recruitment to the Bcl-2 ERE by E2 relative to thatinduced by L17 and WAY.qRT-PCR analysis of transcripts induced after 1-hour

exposure to ligands showed that E2 significantly inducedBcl-2 mRNA by 5-fold in MCF-7 cells, whereas L17 andWAYreducedBcl-2 transcripts in both cell lines.Althoughthe latter reduction within the 1-hour period was nothighly significant, this may reflect the half-life of existingtranscripts. E2 also reduced Bcl-2 mRNA in LCC9 cellsalthough less than L17 and WAY (Fig. 5E).For comparison with another E2-responsive gene, we

analyzed the pS2 gene by ChIP and transcripts by qRT-PCR. All ligands recruited both ERa and ERb to the pS2promoter in LCC9 cells (Fig. 5A and B) which corre-sponded to weak induction (E2) or significant inhibitionof expression (L17 and WAY) following a 1-hour treat-ment (Fig. 5F). Although all three ligands also recruitedboth the ERa and ERb inMCF-7 cells (Fig. 5C andD), onlyE2 strongly increased pS2 mRNA (Fig. 5F).Wealso analyzedmRNA levels 24 hours after treatment

to assess the longer term effects of ligand activation ontranscript levels. Both Bcl-2 and pS2 mRNA remainedelevated after 24 hours in MCF-7 cells treated with E2,while the reduction in levels became less significant in L17and WAY-treated MCF-7 and LCC9 cells and in E2-trea-ted LCC9 cells (Fig. 5G). pS2 transcripts maintained asimilar pattern of expression after 24 hours although asmall rebound in pS2mRNAwas observed in L17-treatedcells, possibly due to receptor desensitizationwhich couldalleviate repression (Fig. 5H).

Overall, these results suggest that L17 andWAYbehaveas inverse agonists wherein association of L17/WAY-bound ERb mediates a reduction in basal transcriptionfrom the promoters regulated by these EREs. Transactiva-tion of genes by the E2-bound ERa is facilitated by inter-actionwith thep160 coactivators,while bindingof antago-nists results in the recruitment of corepressors (37). ChIPassays using antibodies against the RIP140 corepressorand SRC-3 coactivator were performed on Dox-treatedMCF-7(rTA tet-ON ERb) cells to increase the relativeexpression of ERb. Cells were transfected with a nontar-geting sequence (NT) or ERa siRNA as described inMaterials and Methods and treated for 10 minutes withvehicle, E2, or L17. The results in Fig. 5I show that RIP140was recruited to the Bcl-2 ERE by L17 in ERb overexpres-sing cells coexpressing endogenous ERa or after ERaknockdown. In contrast, E2 reduced baseline (vehicle)levels of RIP140 in siNT-transfected cells but stronglyrecruited RIP140 when the ERa was reduced. AlthoughE2 recruited SRC-3 onto the Bcl-2 ERE in the presence ofendogenous levels of ERa, this occupancywas reduced inthe siERa-transfected cells. By comparison, L17 did notrecruit SRC-3 in either siNT- or siERa-transfected cells(Fig. 5J). Expression of ERb mRNA and knockdown ofERawere confirmed as shown in Supplementary Fig. S4.Thus, the Bcl-2 ERE is negatively regulated by ligand-bound ERb in conjunction with RIP140 recruitment.

Finally, we expressed the ERa or ERb in HEK293 cells tocompare the effects of WAY and L17 with E2 on ERproteins. Maximal activation of transcription by the ERaresults in proteolytic degradation of the receptor (38).Consistent with this, the ERa protein was rapidly down-regulated following E2-mediated transactivation (Fig. 5K).L17 has a low level of ERa-binding activity and resulted ina weak reduction in the ERa protein. The ERb protein wasonly slightly reducedby bothE2andWAY, andL17hadnoeffect. This is consistent with the overall lack of transcrip-tional activation by the ERa in the presence of WAY andL17 as well as when all ligands bound to the ERb.

Chloroquine converts L17/WAY-induced autophagyto apoptosis

Chloroquine (CQ) prevents the acidification of lyso-somes to repress autophagy (39). Because WAY and L17reduced Bcl-2 and increased levels of LC3-II in LCC9 cells,

Figure 5. Ligand-dependent recruitment of the ERa and ERb and coregulators to the EREs of theBcl-2 and pS2 genes. ChIP assays for ERs bound to theBcl-2and pS2 EREs after a 1-hour treatment with vehicle, E2, L17, or WAY. Bcl-2 exon 4 primers were used as a negative control. Densitometric analysis of Bcl-2and pS2 EREPCRproducts from an ERaChIP expressed as a percentage of the input inMCF-7 cells (A) and LCC9 cells (B). The same analysis using the anti-ERb antibody in MCF-7 (C) and LCC9 (D) cells. The y-axis represents ImageJ quantification of the amount of specific PCR product expressed as thepercentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. The signal in the IgG lane wassubtracted from each sample. The fold-induction over control of Bcl-2 and pS2 mRNA in MCF-7 and LCC9 cells following a 1-hour (E and F) and 24-hourtreatment (G and H) with each ligand was measured by qRT-PCR. Dashed lines indicate baseline expression level set at value of 1 after 72 hours(vehicle). Data represent themean from twoseparate experiments eachperformed in triplicate; error bars indicateSE;P valueswere calculatedby t test. Darkerbars areMCF-7 and light bars areLCC9.Dox-treatedMCF-7 (rTA tet-ONERb1)were transfectedwith siNTor siERaand treatedwith vehicle (Veh/V), 10nmol/LE2 or L17 for 15 minutes. ChIP was performed as described in Materials and Methods using anti-RIP140 (I) or anti-SRC3 (J), followed by PCR for the Bcl-2ERE. PCRproducts quantified by ImageJ are expressed as a percentage of input. The graph is representative of three separate experiments. K, HEK293 cellswere transfected with ERa or ERb and treated for 3 hours with the indicated ligands. ERa and ERb immunoblot analysis of lysates is shown. Actinand a nonspecific band (�) interacting with anti-ERb serve as loading controls. U, untransfected; C, no treatment.






we tested the possibility that chloroquine might convertthis autophagic response to cell death. Figure 6A showsthat a 5-day treatment with 33 mmol/L chloroquine to E2-free cultures caused an approximate 20% decrease inLCC9 cells and 35% in MCF-7 cells compared with vehi-cle-treated cells (Fig. 6B). To confirm that the effects ofchloroquine in the presence of L17 were ERb dependent,LCC9 cells infected with shERb or control shNT weretreated with L17, WAY, or vehicle in the presence or

absence of chloroquine. Figure 6C shows that knockdownof ERb prevented the effects of chloroquine both in thepresence and absence of the ERb ligands. This resultsuggests that the ERbmay act as both a ligand-dependentand -independent mediator of autophagy.

Strikingly, the combination of chloroquine and L17 orWAY induced a dramatic 80% decrease in cell numberscompared with control, which correlated to induction ofsub-G1 DNA (Fig. 6D and Supplementary Fig. S5A).

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Figure 6. Inhibition of autophagyinduces apoptotic death whencombined with ERb agonists.LCC9 and MCF-7 cells weretreated with vehicle, 10 nmol/LL17, or WAY, with or withoutchloroquine for a total of 5 days.LCC9 (A) andMCF-7 (B) cells wereenumerated using Trypan blueexclusion. C, LCC9 cells wereinfected with pRS control virus orpSUPERshERb for 48 hours thentreated for 5 days with vehicle,10 nmol/L L17, 10 nmol/L WAYwith or without chloroquine(33 mmol/L). LCC9 (D) andMCF-7 (E) cells treated as in A andB were collected and sub-G1 DNAcontent was determined by flowcytometry. F, LCC9 cells weretreated as in D except that in somesamples 100 mmol/L Z-VAD-FMKwas included in the medium. Datarepresent the mean of triplicatedeterminations. Bars, SE.

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Chloroquine/ERb agonist treatment also reduced MCF-7viable cells in association with a sub-G1 peak (Fig. 6E andSupplementary Fig. S5B) albeit not as dramatically as inLCC9 cells. Thus, SERD-resistant LCC9 cells expressing ahigh ratio of ERb:ERa are preferentially sensitive to ERbagonist combined with autophagy inhibition.To distinguish whether the observed cell death was a

function of conversion of autophagy to apoptosis, werepeated the experiment in LCC9 cells the presence orabsence of the caspase inhibitor, Z-VAD-FMK (Fig. 6F andSupplementary Fig. S5C). Consistent with induction ofapoptosis, Z-VAD-FMK almost completely blocked celldeath in LCC9 cells induced by combination L17 andchloroquine.

DiscussionPrevious studies have demonstrated that overexpres-

sion of the ERb inMCF-7 breast cancer cells can inhibit cellgrowth in the presence of E2 (29) and prevent xenografttumor formation (7). In transfected HeLa cells, the ERbalone is unable to activate the transcription of the cyclinD1gene in thepresence of E2 andprevents E2 activation ofcyclin D1 in the presence of coexpressed ERa (40).Although ERb activation has antiproliferative effects, themajority of human ERþ breast tumors do not express highlevels ofERb relative toERaatdiagnosis. Remarkably, celllines with acquired resistance to SERMs express anincreased ratio of ERb to ERa (25).In this study, we found that L17 and WAY had mod-

ulatory effects on cell-cycle proteins and either reduced 50kDa cyclin E (MCF-7 cells) orweakly increased expressionof 48 kDa cyclin E (LCC9 cells). Both ligands reducedcyclin D1 expression. Multiple phosphorylated residueson cyclin E are required for recognition by the SCFFbw7

ubiquitin ligasewhich leads todegradationof cyclinE andprogression throughSphase (41). Further experiments arerequired to determinewhether the 48 kDa cyclin E proteinrepresents hypophosphorylated cyclin E, reflecting thelack of cell-cycle progression in E2 andERb ligand-treatedLCC9 cells.Results of ChIP-on-chip experiments have shown con-

siderable overlap between the enhancers that bind theERa and theERb (42). This study is thefirst todemonstratethatERb agonists downregulateBcl-2.Our results showedthat both the ERa andERb are recruited to theBcl-2ERE inboth MCF-7 and LCC9 cells by both L17 and WAY. WAYtreatment strongly recruited the ERb on the Bcl-2 ERE inMCF-7 cells, consistent with its high affinity for thisreceptor. In contrast, L17 which has a lower affinity forERb than WAY (26, 27), and retains low affinity for theERa, recruited the ERamore strongly thanWAY. Regard-less, both ligands decreasedBcl-2mRNA in both cell lines.Ligand-ER conformation on a DNA sequence elementdetermines transcriptional activation through coregulatorrecruitment. Our results suggest that, under conditionswhere the ratio of ERb:ERa is high, ERb agonists recruitcorepressors such as RIP140 to the Bcl-2 ERE resulting inreduced transcription of this gene. This result is consistent

with recent report that RIP140 is the preferential coregu-lator of the ERb (43). Because the liganded ER subtypedetermines chromatin binding (42), it seems that the Bcl-2and pS2 genes are negatively regulated by ligands inter-acting with ERb.

In LCC9 cells, E2 bound to ERa and/or ERb alsoreduced Bcl-2 transcription and protein expression. Infact, E2 can mediate growth inhibition and induce celldeath in long-term hormone-deprived and antiestro-gen-resistant breast cancer cells (44, 45). Notably, theBcl-2 expression level was found to be a critical deter-minant of the ability of E2 to induce apoptosis in thesecells (46). Indeed, it is possible that an increased ratio ofERb:ERa in SERM-resistant cells may play a significantrole in mediating the fundamentally different responseinduced by E2 in SERM-resistant cells (47) and also whyE2 fails to induce Bcl-2 in long-term E2-deprived breastcancer cells (48).

AlthoughMCF-7 cells express a lower constitutive levelof Bcl-2 relative to LCC9 TAM-R cells, they do not dem-onstrate the same autophagic flux seen in LCC9 cellswhich express a higher constitutive level of Bcl-2 protein.In LCC9 cells, autophagic flux is at least partially depen-dent on the presence of the ERb as demonstrated by thereduction in chloroquine sensitivity following KD of ERbin LCC9 cells. It is possible that ERb regulates other criticalcomponents of the autophagic response in LCC9 cells.Interestingly, LCC9 cells have also previously beenreported to contain a higher level of NF-kB activity rel-ative to parental cells (49) and NF-kB activation canpromote autophagy (50).

Clinical trials with agents which inhibit autophagy arebeing combined with chemotherapy and radiation torepress this prosurvival function (51). RNAi-mediatedknockdown of Bcl-2 results in induction of autophagy inMCF-7 cells (52). On the basis of the dominant expressionof the ERa in most ERþ primary breast cancers, ERbagonists may not be useful as first-line therapy. However,given that SERM/SERD-resistant cells often express ahigher ERb:ERa ratio (24, 53), and tamoxifen fails toreduce Bcl-2 in LCC9 cells (49), our novel finding thatERb agonists reduce Bcl-2 expression and promote autop-hagy in these cells suggests that combinationERb agonistsand autophagy inhibitors may represent a novel, relative-ly low toxicity therapeutic option in patients withacquired endocrine resistance.

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

Authors' ContributionsConception and design: M.A.C. PrattDevelopment of methodology: S.C. Ruddy, L.C. MurphyAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Lau, M.A. Cabrita, B.C. McKay, L.C. MurphyAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): S.C. Ruddy, R. Lau, M.A. Cabrita,B.C. McKay, M.A.C. PrattWriting, review, and or revision of the manuscript: S.C. Ruddy, R. Lau,M.A. Cabrita, B.C. McKay, L.C. Murphy, M.A.C. Pratt






Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S.C. Ruddy, C. McGregor,L.C. MurphyStudy supervision: M.A.C. PrattOther (computational analysis and design of model ligands): J.S. WrightOther (preparation of estrogen analogues): T. Durst

AcknowledgmentsThe authors thank Robert Clarke, Georgetown University, for the

generous gift of MCF-7/LCC9 cells and the parental MCF-7 control line.

Grant SupportThis work was supported by a grant from the Canadian Breast Cancer

Foundation (Ontario Region; to M.A.C. Pratt).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received December 11, 2013; revised April 10, 2014; accepted April 21,2014; published OnlineFirst April 30, 2014.

References1. Edwards DP. The role of coactivators and corepressors in the biology

and mechanism of action of steroid hormone receptors. J Mamm GlBiol Neopl 2000;5:307–24.

2. Delaunay F, Pettersson K, Tujague M, Gustafsson JA. Functionaldifferences between the amino-terminal domains of estrogen receptoralpha and beta. Mol Pharmacol 2000;58:584–90.

3. Hall JM, McDonnell DP. The estrogen receptor beta-isoform (ERbeta)of the human estrogen receptor modulates ERalpha transcriptionalactivity and is a key regulator of the cellular response to estrogens andantiestrogens. Endocrinol 1999;140:5566–78.

4. Liu Y, Gao H, Marstrand TT, Strom A, Valen E, Sandelin A, et al. Thegenome landscape of ERalpha andERbeta-bindingDNA regions. ProcNatl Acad Sci U S A 2008;105:2604–9.

5. Chang EC, Charn TH, Park S-H, Helferich WG, Komm B, Katzenel-lenbogen JA, et al. Estrogen receptorsa and b as determinants of geneexpression:Influence of ligand, dose and chromatin binding. MolEndocrinol 2008;22:1032–43.

6. Strom A, Hartman J, Foster JS, Kietz S,Wimalasena J, Gustafsson JA.Estrogen receptor beta inhibits 17-beta estradiol-stimulated prolifer-ation of the breast cancer cell line T47D. Proc Natl Acad Sci U S A2004;101:1566–71.

7. Paruthiyil S, Parmar H, Kerekatte V, Cunha GR, Firestone GL, LeitmanDC. Estrogen receptor b inhibits human breast cancer cell proliferationand tumor formation by causing a G2 cell cycle arrest. Cancer Res2004;64:423–8.

8. Vivar OI, Zhao X, Saunier EF, Griffin C, Mayba OS, Tagliaferri M, et al.Estrogen receptor b binds to and regulates three distinct classes oftarget genes. J Biol Chem 2010;285:22059–66.

9. Chang EC, Frasor J, Komm B, Katzenellenbogen BS. Impact ofestrogen receptor b on gene networks regulated by estrogen receptora in breast cancer cells. Endocrinol 2006;147:4831–42.

10. Park BW, Kim KS, Heo MK, Ko SS, Hong Sw, Wang WI , et al.Expression of estrogen receptor-beta in normal mammary and tumortissues:is it protective in breast carcinogenesis? Breast Cancer ResTreat 2003;80:79–85.

11. Zhao C, Lam EW, Sunters A, Enmark E, DeBella MT, Coombes RC,et al. Expression of estrogen receptor beta isoforms in normal breastepithelial cells and breast cancer:regulation bymethylation. Oncogene2003;22:7600–06.

12. Coopman P, Garcia M, Brunner N, Deroco D, Clarke R, Rochefort H.Anti-proliferative and anti-estrogenic effects of ICI 164,384 and ICI182,780 in 4-OH-tamoxifen-resistant human breast-cancer cells. Intl JCancer 1994;56:295–300.

13. Brunner N, Frandsen TL, Holst-Hansen C, Bei M, ThompsonEW, Wakeling AE, et al. MCF7/LCC2:a 4-hydroxytamoxifenresistant human breast cancer variant that retains sensitivityto the steroidal antiestrogen ICI 182,780. Cancer Res 1993;53:3229–32.

14. Hu XF, Veroni M, De Luise M, Wakeling A, Sutherland R, Watts CKW ,et al. Circumvention of tamoxifen resistance by the pure anti-estrogenICI182,780. Intl J Cancer 1993;55:873–6.

15. DeFriend DJ, Howell A, Nicholson RI, Anderson E, Dowsett M, ManselRE, et al. Investigation of a new pure antiestrogen (ICI 182,780) inwomen with primary receptor-positive breast Cancer. Cancer Res1994;54:408–14.

16. Osborne CK, Coronado-Heinsohn EB, Hilsenbeck SG, McCue BL,Wakeling AE,McClelland RA, et al. Comparison of the effects of a pure

steroidal antiestrogen with those of tamoxifen in a model of humanbreast cancer. J Natl Cancer Inst 1995;87:746–50.

17. Brunner N, Boysen B, Jirus S, Skaar TC, Holst-Hansen C, Lippman J ,et al. MCF7/LCC9: An antiestrogen-resistant MCF-7 variant in whichacquired resistance to the steroidal antiestrogen ICI 182,780 confersand early cross-resistance to the nonsteroidal antiestrogen Tamoxi-fen. Cancer Res 1997;57:3486–93.

18. Nicholson RI, Hutcheson IR, Hiscox SE, Knowlden JM, Giles M,Barrow D , et al. Growth factor signalling and resistance to selectiveoestrogen receptor modulators and pure anti-oestrogens: the use ofanti-growth factor therapies to treat or delay endocrine resistance inbreast cancer. Endocr Relat Cancer 2005;12 Suppl 1:S29–36.

19. Clarke R, Liu MC, Bouker KB, Gu Z, Lee RY, Zhu Y , et al. Antiestrogenresistance in breast cancer and role of the estrogen receptor signaling.Oncogene 2003;22:7316–39.

20. Bachleitner-Hofmann T, Pichler-Gebhard B, Rudas M, Gnant M, Tau-cher S, Kandioler D , et al. Pattern of hormone receptor status ofsecondary contralateral breast cancers in patients receiving adjuvanttamoxifen. Clin Cancer Res 2002;8:3427–32.

21. Santen RJ, Song RX, McPherson R, Kumar R, Adam L, JengMH , et al.The role of mitogen activated protein (MAP) kinase in breast cancer.J Steroid Biochem Mol Biol 2002;80:239–56.

22. Barkhem T, Carlsson B, Nilsson Y, Enmark E, Gustafsson JA, NilssonS. Differential response of estrogen receptora and estrogen receptor bto partial estrogen agonists/antagonists. Mol Pharmacol 1998;54:105–12.

23. Esslimanni-SahlaM, Simony-Lafontaine J, Kramar A, Lavaill R,MolleviC,WarnerM, et al. Estrogen receptorb (ERbeta) level but not its ERb cxvariant helps to predict tamoxifen resistance in breast cancer. ClinCancer Res 2004;10:5769–76.

24. Speirs V, Malone C, Walton DS, Kerin MJ, Atkin SL. Increased expres-sion of estrogen receptor beta in tamoxifen resistant breast cancerpatients. Cancer Res 1999;59:5421–4.

25. De Cremoux P, Tran-Perennou C, Brockdorff BL, Boudou E, BrunnerN, Magdelenat H , et al. Validation of real-time RT-PCR for analysis ofhuman breast cancer cell lines resistant or sensitive to treatment withantiestrogens. Endocr Rel Cancer 2003;10:409–18.

26. Wright JS, ShadniaH,Durst T, AsimM, El-SalfitiM,Choueiri C , et al. A-CD Estrogens:I. Substituent effects, hormone potency, and receptorsubtype selectivity in a new family of flexible estrogenic compounds.J Med Chem 2011;54:433–48.

27. Malamas MS, Manas ES, McDevitt RE, Gunawan I, Xu ZB, Collini MD ,et al. Design and synthesis of aryl diphenolic azoles as potent andselective estrogen-beta ligands. J Med Chem 2004;47:5021–40.

28. Brunner N, Boulay V, Fojo A, Freter CE, Lippman ME, Clarke R.Acquisition of hormone-independent growth in MCF-7 cells is accom-panied by increased expresssion of estrogen-regulated genes butwithout detectable DNA amplifications. Cancer Res 1993;53:283–90.

29. Murphy LC, Peng B, Lewis A, Davie JR, Leygue E, Kemp A , et al.Inducible upregulation of oestrogen receptor-beta1 affects oestrogenand tamoxifen responsiveness in MCF7 human breast cancer cells.J Mol Endocrinol 2005 34:553–66.

30. Klinge CM, Riggs KA, Wickramasinghe NS, Emberts CG, McCondaDB, Barry PN, et al. Estrogen receptor alpha 46 is reduced in tamoxifenresistant breast cancer cells and re-expression inhibits cell prolifera-tion and estrogen receptor alpha 66-regulated target gene transcrip-tion. Mol Cell Endocrinol 2010;323:268–76.


Ruddy et al.




31. Zhang G, Liu X, Farkas AM, Parwani AV, Lathrop KL, Lenzner D, et al.Estrogen receptor beta functions through nongenomicmechanisms inlung cancer cells. Mol Endocrinol 2009;23:146–56.

32. Crawford AC, Riggins RB, Shajahan AN, Zwart A, Clarke R. Co-inhibition of BCL-W and BCL2 restores antiestrogen sensitivitythrough BECN1 and Promotes an autophagy- associated necrosis.PloS ONE 2010;5:e8604.

33. Teixiera C, Reed JC, Pratt MAC. Estrogen promotes chemotherapeuticdrug resistance by a mechanism involving Bcl-2 proto-oncogeneexpression in human breast cancer cells. Cancer Res 1995;55:3902–7.

34. Pattingre S, TassaA, QuX,Garuti R, Liang XH,MizushimaN , et al. Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy. Cell2005;122:927–39.

35. Mizushima N, Yoshimori T. How to interpret LC3 immunblotting.Autophagy 2007;3:542–5.

36. Perillo B, Sasso A, Abbondanza C, Palumbo G. 17Beta-estradiolinhibits apoptosis in MCF-7 cells, inducing bcl-2 via two estrogen-responsive elements present in the coding sequence. Mol Cell Biol2000;20:2890–901.

37. Xu L, Glass CK, Rosenfeld MG. Coactivator and corepressor com-plexes in nuclear receptor function. Curr Opin Genet Dev 1999;9:140–7.

38. Shao W, Keeton EK, McDonnell DP, Brown M. Coactivator AIB1 linksestrogen receptor transcriptional activity and stability. Proc Natl AcadSci U S A 2004;101:11599–604.

39. Yang ZJ, Chee CE, Huang S, Sinicrope FA. The role of autophagy incancer:therapeutic implications. Mol Cancer Ther 2011;10:1533–41.

40. Liu MM, Albaneese C, Anderson CM, Hilty K, Webb P, Uht RM , et al.Opposing action of estrogen receptors a and b on cyclin D1 geneexpression. J Biol Chem 2002;277:24353–60.

41. Ye X, Nalepa G, Welcker M, Kessler BM, Spooner E, Qin J, et al.Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase. J Biol Chem 2004;279:50110–9.

42. Charn TH, Liu ET, Chang EC, Lee YK, Katzenellenbogen JA, Katze-nellenbogen BS. Genome-wide dynamics of chromatin binding ofestrogen receptors alpha and beta: mutual restriction and competitivesite selection. Mol Endocrinol 2010;24:47–59.

43. Madak-Erdogan Z, Charn T-H, Jiang Y, Liu ET, Katzenellenbogen JA,Katzenellenbogen BS. Integrative genomics of gene and metabolicregulation by estrogen receptors a and b, and their coregulators. MolSys Biol 2013;9:676.

44. Lewis JS, Meeke K, Osipo C, Ross EA, Kidawi N, Li T , et al. Intrinsicmechanism of estradiol-induced apoptosis in breast cancer cellsresistant to estrogen deprivation. J Natl Cancer Inst 2005;97:1746–59.

45. Jordan VC, Lewis JS, Osipo C, Cheng D. The apoptotic action ofestrogen following exhaustive antihormonal therapy: a new clinicaltreatment strategy. Breast 2005;14:624–30.

46. Song RX, Zhang Z, Mor G, Santen RJ. Down-regulation of Bcl-2enhances estrogen apoptotic action in long-term estradiol-depletedER(þ) breast cancer cells. Apoptosis 2005;10:667–78.

47. SwabyRF, JordanVC. Low-dose estrogen therapy to reverse acquiredantihormonal resistance in the treatment of breast cancer. Clin BreastCancer 2008;8:124–33.

48. Lewis-Wambi JS, Jordan VC. Estrogen regulation of apoptosis: howcan one hormone stimulate and inhibit? Breast Cancer Res 2009;11:206.

49. Nehra R, Riggins RB, Shajahan AN, Zwart A, Crawford AC, Clarke R.BCL2 and CASP8 regulation by NF-kappaB differentially affect mito-chondrial function and cell fate in antiestrogen-sensitive and -resistantbreast cancer cells. FASEB J 2010;24:2040–55.

50. BaldwinAS.Regulationof cell death andautophagyby IKKandNF-kB:critical mechanisms in immune function and cancer. Immunol Rev2012;246:327–45.

51. Chen N, Karantza V. Autophagy as a therapeutic target in cancer.Cancer Biol Ther 2011;11:157–68.

52. Akar U, Chaves-Reyez A, BarriaM, Tari M, Sanguino A, Kondo Y , et al.Silencing of Bcl-2 expression by small interfering RNA induces autop-hagic cell death in MCF-7 breast cancer cells. Autophagy 2008;45:669–79.

53. ShawLE, Sadler AJ, Pugazhendhi D,DarbrePD.Changes in oestrogenreceptor-a and -b during progression to acquired resistance to tamox-ifen and fulvestrant (Faslodex, ICI 182,780) in MCF7 human breastcancer cells. J Steroid Biochem Mol Biol 2006;99 19–32.






2014;13:1882-1893. Published OnlineFirst April 30, 2014.Mol Cancer Ther Samantha C. Ruddy, Rosanna Lau, Miguel A. Cabrita, et al. in Hormone-Resistant Breast Cancer Cells to Increase Autophagy

Ligands Reduce Bcl-2 ExpressionβPreferential Estrogen Receptor

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