Chloroquine Inhibits Autophagy to Potentiate Antiestrogen ... · in Matrigel:IMEM (50:50%). Cells were injected orthoto-pically through a 3-mm skin cut into 4 different mammary fat

Cancer Therapy: Preclinical

Chloroquine Inhibits Autophagy to Potentiate AntiestrogenResponsiveness in ERþ Breast Cancer

Katherine L. Cook1, Anni W€arri1, David R. Soto-Pantoja2, Pamela AG. Clarke1, M. Idalia Cruz1,Alan Zwart1, and Robert Clarke1

AbstractPurpose: Estrogen receptor-a (ERa)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are

used to treat ERþ breast cancers. Up to 50% of tumors will acquire resistance to these interventions.

Autophagy has been implicated as a major driver of antiestrogen resistance. We have explored the ability of

chloroquine (CQ), which inhibits autophagy, to affect antiestrogen responsiveness.

Experimental Design: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ERþ breast

cancer cells were injected into mammary fat pads of female athymic mice and treated with TAM and/or ICI

in combination with oral low-dose CQ.

Results: We show that CQ can increase antiestrogen responsiveness in MCF7-RR and LCC9 cells and

tumors, likely through the inhibition of autophagy. However, the combination of ICIþCQwas less effective

than CQ alone in vivo, unlike the TAMþCQ combination. Antiestrogen treatment stimulated angiogenesis in

tumors but did not prevent CQ effectiveness. The lower efficacy of ICIþCQwas associatedwith ICI effects on

cell-mediated immunity within the tumormicroenvironment. Themouse chemokine KC (CXCL1) and IFNgwere differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage

development/activity. Consistent with these observations, TAMþCQ treatment increased tumor CD68þ cells

infiltration,whereas ICI and ICIþCQreducedperipheral tumormacrophage content.Moreover,macrophage

elimination of breast cancer target cells in vitro was reduced following exposure to ICI.

Conclusion: CQ restores antiestrogen sensitivity to resistant tumors. Moreover, the beneficial combi-

nation of TAMþCQ suggests a positive outcome for ongoing neoadjuvant clinical trials using

this combination for the treatment of ERþ ductal carcinoma in situ lesions. Clin Cancer Res; 20(12);

3222–32. �2014 AACR.

IntroductionOne out of every eight American women will develop

invasive breast cancer over the course of her lifetime. Anestimated 230,000 new cases of breast cancer are diagnosedannually in the United States (1). Of these cases, 70% willexpress estrogen receptor-a (ERa). ER-a–targeted therapiesinclude the selective ER modulator tamoxifen (TAM), the

selective ER downregulator Faslodex (fulvestrant, ICI), oraromatase inhibitors that block the conversion of andro-gens to estrogens. Evenwith the success of these drugs, sometumors fail to respond (de novo resistance) or acquireresistance over time (2–4).

Autophagy is a process by which a double membranevesicle surrounds cellular contents, such as damaged orga-nelles and misfolded or protein aggregates, and recyclesthe material through lysosomal degradation (5). Studies inbreast cancer cells show that the induction of autophagy byvarious therapeutics is usually prosurvival (6–8). Further-more, TAM and ICI both induce autophagy in ERþ breastcancer cells (6, 9–13). Antiestrogen-resistant cell linesexhibit increased basal autophagy when compared withtheir antiestrogen-sensitive parental cells (10). Inhibitingautophagy through autophagy-related gene 5 (ATG5)silencing potentiated antiestrogen-mediated cell death,indicating that antiestrogen-stimulated autophagy is pro-survival and a criticalmechanismof therapy resistance (10).Analysis of publically available human datasets indicatesthat autophagy-related genes, ATG5, ATG7, and p62(SQSTM1), are elevated in early recurring breast cancerwhen compared with breast cancer that never recurs. More-over, elevated p62 is significantly correlated with poor

Authors' Affiliations: 1Department of Oncology and Lombardi Compre-hensive Cancer Center, Georgetown University Medical Center, Washing-ton, D.C.; and 2Department of Pathology, National Cancer Institute, NIH,Bethesda, Maryland

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

Update: There was a nomenclature error in the published article. Theprimary chemical form of the drug used in each of the in vitro and in vivoexperiments presented in the studywas chloroquine (CQ) diphosphate andnot hydroxychloroquine (HCQ) diphosphate as reported. This version of thearticle contains the correct nomenclature.

Corresponding Author: Robert Clarke, W405A Research Building, Geor-getownUniversityMedicalCenter, 3970Reservoir RdNW,Washington,DC20057. Phone: 202-687-8991; Fax: 202-687-2085: E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-3227

�2014 American Association for Cancer Research.

ClinicalCancer

Research

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survival in patients with breast cancer (Supplementary Fig.S1), suggesting a role for autophagy in breast cancer recur-rence (14–18).CQ is a lysotropic chloroquine derivative that accumu-

lateswithin lysosomes, resulting in lysosomeneutralizationand the inhibition of autophagic flux. Originally applied asan antimalarial medication, the use of chloroquine (orchloroquine derivatives) to inhibit autophagy is currentlybeing explored as possible chemotherapeutic interventionsfor the treatment of cancer (19). Here, we have explored thepossible beneficial effect of combining antiestrogen thera-pies with chloroquine (CQ) for the treatment of antiestro-gen-resistant ERþ breast cancers. We used several cellularmodels of endocrine resistance: MCF7-RR (ERþ, estrogen-independent, TAM-resistant, ICI-sensitive cells; derivedfrom MCF-7 cells selected against low serum and TAM;refs. 20, 21), LCC9 (ERþ, estrogen-independent, ICI-resis-tant, TAM cross-resistant cells; derived from the estrogen-independent antiestrogen-sensitive LCC1 cells by selectionagainst ICI; ref. 22), and ZR-75-1/ICI-R (ERþ, ICI-resistantcells; derived from estrogen-sensitive antiestrogen-sensitiveZR-75-1 cells by selection against ICI). We also used twodifferent categories of antiestrogen therapy: TAM, a selectiveestrogen receptormodulator (SERM)andERpartial agonist,and ICI, a selective estrogen receptor downregulator (SERD)and ER antagonist.The combination of CQ with TAM partially resensitized

resistant MCF7-RR and LCC9 tumors, whereas a combina-tion of ICIþCQwas not as effective as CQ given as a single-agent therapy in treating ICI-resistant LCC9 orthotopic

xenografts. Furthermore, we found that the tumor micro-environment plays an important role in the treatment ofbreast tumors with antiestrogens and CQ. We show thatmonocytes that express ERa, and differentiated macro-phages that express the estrogen and TAM-responsive G-coupled protein receptor 30 (GPR30),maymediate someofthe estrogen agonist and tumor-inhibitory activities of TAM.In contrast, the antineoplastic activity of ICI was reduced byits ability to inhibit monocyte differentiation and macro-phage-mediated clearance of tumor cells. An ongoing clin-ical trial Preventing Invasive Neoplasia with Chloroquine(PINC) is investigating the ability of chloroquine alone orin combinationwith TAM(in ERþ cases) to eliminate ductalcarcinoma in situ (DCIS) progenitor cells, highlighting theimportance of our findings.

Materials and MethodsMaterials

The following materials were obtained as indicated: 4-hydroxytamoxifen and CQ diphosphate salt (Sigma-Aldrich); ICI 182,780 (Tocris Bioscience); Improved Mini-mal Essential Medium (IMEM; Gibco Invitrogen BRL,); FBSand bovine calf charcoal-stripped serum (CCS; Equitech-BioInc); crystal violet (Fisher Scientific). Growth factor reducedMatrigel (BD Biosciences). Antibodies were obtained fromthe following sources: LC3A/B, VEGFR2, phospho-VEGFR2(Cell Signaling Technology); p62 [Western, BD Biosciences;immunohistochemistry (IHC), MBL International]; CD68(AbDSerotec); CD31 (Abcam); ERa,b-actin, and polyclonaland horseradish peroxidase (HRP)-conjugated secondaryantibodies (Santa Cruz Biotechnology).

Cell cultureMCF7-RR and LCC9 human breast carcinoma cells

were grown in phenol-red free IMEM media containing5% CCS. MCF7 and ZR-75-1/ICI-R cells were grown inphenol-red IMEM media containing 5% FBS. MCF7 cellswere obtained from Dr. Marvin Rich at the MichiganCancer Foundation. MCF7-RR cells were obtained fromIndiana University of Pennsylvania (Indiana, PA; ref. 21).LCC9 and ZR-75-1/ICI-R cells were generated by ourgroup at Georgetown University (Washington, D.C.;ref. 22). Cells were grown at 37�C in a humidified, 5%CO2:95% air atmosphere.

Crystal violet assayHuman breast cancer cells (3 � 103 cells/mL) in IMEM

containing 5% CCS were plated in 24-well tissue cultureplates. On day 1 after plating, and every 3 days, thereaftercells were treated with varying doses (10–1,000 nmol/L) ofeither TAM or ICI and 1 mmol/L CQ. On day 6, media wereaspirated and cells were stained with crystal violet. Cellswere permeabilized using citrate buffer and absorbance wasread at 480 nm on a plate reader.

Western blot hybridizationMCF7-RR and LCC9 cells were solubilized by sonication

in radioimmunoprecipitation assay buffer lysis buffer.

Translational RelevanceBreast cancer affects more than 230,000 American

women each year. An estimated 70% of these cases havetumors that express the estrogen receptor and are eligibleto be treatedwith antiestrogens such as tamoxifen (TAM)or, for the treatment of advanced postmenopausal dis-ease, Faslodex.However, 50%of these breast cancerswilleither fail to respond (de novo resistance) or lose therapyresponsiveness over time (acquired resistance). Wefound that inhibiting autophagy through low-dose oralchloroquine (CQ) administration increases the respon-siveness of resistant mammary tumors to antiestrogens.Moreover, we show that the combination of TAM andCQ ismore effective than that of Faslodex andCQdue toactivities within the tumor microenvironment. Thesepreclinical data highlight the relevance of a clinical trialcombining chloroquine and TAM for the treatment ofERþ in situ breast lesions (Preventing Invasive Neoplasiawith Chloroquine trial) and suggest a clinical benefit ofthe addition of CQ to antiestrogen therapy for thetreatment of ERþ breast cancer. Furthermore, this studypredicts a more modest result for combining Faslodexand chloroquine-based therapies for prevention and/ortreatment of breast cancer.

Chloroquine Inhibits Breast Cancer

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Proteins were size fractionated by PAGE and then trans-ferred to a nitrocellulose membrane. Nonspecific bindingwas blocked by incubation for one hour at room temper-ature with Tris-buffered saline containing 5% powderedmilk and 1% Triton X-100. Membranes were incubatedovernight at 4�C with primary antibodies, followed byincubation with polyclonal HRP-conjugated secondaryantibodies (1:2000) for 1hour at room temperature. Immu-noreactive products were visualized by chemiluminescence(SuperSignal FemtoWest, Pierce Biotechnology) and quan-tified by densitometry using the ImageJ digital densitometrysoftware (http://rsbweb.nih.gov/ij/).

Orthotopic human breast cancer xenograftsFive-week-old, intact, athymic nude mice (Harlan Labo-

ratories) were injected with 1� 106 LCC9 orMCF7-RR cellsin Matrigel:IMEM (50:50%). Cells were injected orthoto-pically through a 3-mm skin cut into 4 different mammaryfat pads per mouse (one abdominal and one thoracicmammary gland on each side). For the MCF7-RR xeno-grafts, mice also received a low-dose 17b-estradiol pellet(0.36 mg, 60-day release; Innovative Research of America)implanted subcutaneously under isoflurane anesthesia.Tumors grew to approximately 25 to 35mm2before therapyinitiation; 1 to 2 tumors per mouse successfully reached therequired size for treatment initiation.Micewere treatedwithCQ, TAM, ICI, or a combination of an antiestrogen andCQ.CQ was administered in the drinking water; 0.29 mg/mLresults in approximately 1 to 2 mg daily dose per mouse(23). TAM was administered in a standard rodent chow5053 diet containing 400 ppm TAM citrate (Harlan-Tek-lad), which corresponds to a TAM dose of approximately32 mg/kg/d. ICI was administered by subcutaneous injec-tion 0.5 mg/mouse/week in 70% EtOH/30% NaCl. Micewere euthanized after 5 weeks of treatment, and tumorsremoved at necropsy, fixed in neutral-buffered 10% for-malin, and processed using routine histologic methods.The efficacy of antiestrogen treatment was confirmed bystaging of the estrus cycle; mice on TAM or ICI therapy didnot cycle.

Tissue staining and immunohistochemistryMammary tumors were fixed in 10% phosphate-buffered

formalin for 24 hours before embedding in paraffin.Embedded tissues were cut into 5 mm thick sections andstained with hematoxylin and eosin (H&E) to determinemorphology. Immunostaining was performedwith an anti-body to CD31 (1:100), pVEGFR2 (1:100), p62 (1:1000),LC3 (1:100), CD68 (1:100) using the streptavidin-biotinmethod. Stained sections were visualized and photo-graphed. Because CD31 and pVEGFR2 staining was hetero-geneous, data were quantified using a common method ofangiogenic marker quantification defined as "hotspot"quantification. In summary, three "hotspots" in the tumorwere selected, the number of vessels/microvessels in each"hotspot" was counted at �200 magnification, and a finalscore was expressed as themean vessel density (24, 25). Thelongest diameter of eachCD31þ blood vessel wasmeasured

using ImageJ and shown as average vessel diameter (Sup-plementary Fig. S3B).

Cytokine analysisPlasma from treated mice was collected at necropsy and

immediately frozen. Quansys Biosciences Q-Plex Array kitswere used to measure the following mouse cytokines andchemokines: interleukin (IL) 1b, IL2, IL3, IL4, IL5, IL6,IL12p70, IL17, TNFa, IFNg , MCP1, RANTES, Eotaxin, KC,MDC, TARC, TCA3.

Macrophage differentiation and cytotoxicity assayU937 humanmonocyte cells were treated in the presence

of IFNg (to differentiate monocytes into activated macro-phages) and either 100 nmol/L TAM, 1 mmol/L CQ, 100nmol/L ICI, or the combination of antiestrogen and CQ for72 hours. Macrophages were counted and plated with 70%confluent MDA-MB-231 (triple-negative, antiestrogen non-responsive breast cancer cells) in a 1:5 ratio ofmacrophagesto cancer cells for 72 hours. Macrophage killing was then

assessed by RTCA-ACEA xCELLigence� system throughelectrical impedance (26).

Statistical analysisData are presented as the mean� SEM. For most studies,

Student t test (pairwise) or ANOVA followed by Bonferoniposthoc tests (group wise) was used (Prism software). Sta-tistical differences for in vivo tumor area were evaluated byDr. Fang in the Department of Biostatistics, Bioinformaticsand Biomathematics at Georgetown University MedicalCenter (Washington, D.C.). Tumor volumes were obtainedfrom measurements of the longest perpendicular axes. Weuse the weighted F test for comparison between two groupswith two tumor volumes because the behavior of twotumors growing in a single mouse are not independentevents (27). All computation was performed in the R-environment. There were 26mice for LCC9 xenografts, and19 mice for MCF7RR xenografts. Criterion for statisticalsignificance was set at P � 0.05.

ResultsAntiestrogen-sensitive MCF7 cells and antiestrogen-resis-

tant MCF7-RR (TAM resistant), ZR-75-1 ICI-R (ICI resis-tant), and LCC9 (ICI resistant/TAM cross-resistant) breastcancer cells were plated in 24-well dishes and treated withvehicle, 1 mmol/L CQ, and/or various concentrations ofTAM or ICI (vehicle, 10, 100, 1,000 nmol/L) for 6 days. Theeffect on cell density was determined by crystal violet assays(28). The combination of antiestrogen and CQ potentiatedTAM and ICI-mediated cell death in endocrine-sensitiveMCF7 cells (Fig. 1A). Furthermore, CQ and antiestrogentherapy inhibited LCC9 cells (Fig. 1B), MCF7-RR (Fig. 1C),and ZR-75-1 ICI-R (Fig. 1D). Moreover, the addition of CQresulted in increased LC3-II formation (lipidated form ofLC3 that is a marker of autophagosome formation) andaccumulation of p62 (marker of autophagosomal flux) inboth MCF7-RR (Fig. 1E) and LCC9 (Fig. 1F) as determinedby Western blot hybridization. Increased LC3-II and p62

Cook et al.

Clin Cancer Res; 20(12) June 15, 2014 Clinical Cancer Research3224



expression is indicative of inhibited autophagic flux result-ing in the cellular accumulation of autophagosomes. Treat-ment ofMCF7-RR cellswith TAMor LCC9 cells with TAMorICI resulted in increased LC3-II with a correspondingdecrease in p62 levels confirming previous studies thatantiestrogens stimulate autophagic flux (10).LCC9 and MCF7-RR cells were orthotopically injected

into the mammary fat pads of female athymic mice.Tumors were grown to 20 to 35 mm2 before treatmentwith CQ and/or TAM (MCF7-RR), or with CQ and/orTAM or ICI (LCC9). In LCC9 xenografts, TAMþCQ wasmost effective at reducing initial tumor growth (Fig. 2A

and B), but after 3 weeks of treatment, CQ alone was justas effective at inhibiting LCC9 as the TAMþCQ combi-nation. Unexpectedly, the combination of ICIþCQ wasless effective than either TAMþCQ or CQ treatment alone.TAM or ICI treatment alone had no significant difference intumor area when compared with control tumors. Whentumor growth was measured as wet weight at necropsy,LCC9 tumors treatedwith CQ alone, TAMþCQ, or ICIþCQhad significantly reduced tumor wet-weight when com-pared with control tumors (Fig. 2C). However, CQ alonewas significantly more effective than ICIþCQ, suggesting apotentially antagonist interaction between ICI and CQ. In

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Figure 1. CQ restores antiestrogen responsiveness in vitro. A,MCF7 cells were treatedwith 1mmol/LCQ, and/or various doses (vehicle, 10, 100, 1,000 nmol/L)of either TAM or ICI for 6 days. Relative cell density was determined by crystal violet assay. B, LCC9 cells were treated with 1 mmol/L CQ, and/or variousdoses (vehicle, 10, 100, 1,000 nmol/L) of either TAM or ICI for 6 days and cell density determined by crystal violet assay. C, MCF7-RR cells weretreated with 1 mmol/L CQ, and/or various doses (vehicle, 10, 100, 1,000 nmol/L) TAM for 6 days. Relative cell density was determined by crystal violet assay.D, ZR-75-1 ICI-R cells were treatedwith 1 mmol/LCQ, and/or various doses (vehicle, 10, 100, 1,000 nmol/L) ICI for 6 days. Relative cell densitywas determinedby crystal violet assay. MCF7-RR (E) or LCC9 (F) cells were treated with 1 mmol/L CQ, 100 nmol/L TAM or ICI, or a combination of antiestrogen andCQ for 72 hours. Cells were harvested andWestern blot hybridizationwas used to confirm levels of autophagy-related proteins LC3A/B and p62. Equivalenceof protein loading onto gels was confirmed by measuring b-actin expression. n ¼ 3; �, P < 0.05.





MCF7-RR orthotopic xenografts, control tumors continuedto grow,whereas TAMþCQ significantly reduced tumor size(Fig. 2D and E). CQ or TAM alone had no significant effecton MCF7-RR tumor growth when compared with controls.The combination of TAMþCQ significantly reducedMCF7-RR tumor wet weight when compared with control-treatedtumor weight measured at necropsy (Fig. 2F).

Formalin-fixed LCC9 tumors were embedded in paraf-fin and cut into 5 mm sections. Tumor sections werestained with specific antibodies against either LC3 (Fig.3A) or p62 (Fig. 3B) using an avidin-biotin technique thatreacts with peroxidase-conjugated streptavidin substrateto determine the effects of treatments on autophagymarkers. LCC9 tumors from mice treated with ICI, TAM,or CQ show elevated LC3A/B staining (Fig. 3C). Measur-ing autophagic activity using immunohistochemistry canbe challenging often because it is difficult to differentiatebetween LC3 and LC3-II by IHC in tissue sections. How-ever, the high level of magnification (�1,000) shows

positive LC3 staining forming puncta-like structuresbroadly consistent with autophagy induction. Tumorsfrom animals treated with ICI or TAM have reducedp62 expression, consistent with increased autophagic flux(Fig. 3C). In contrast, tumors from mice treated with CQhave elevated p62 expression, suggesting a block in thelater stages of autophagy (29, 30). To corroborate thesystemic effect of CQ on autophagy, uterine tissue fromcontrol mice or mice treated with CQ were isolated andWestern blot hybridization was performed on proteinlysates for LC3-II and p62 levels. As shown in Fig. 3D,CQ dosing increases LC3-II formation and results in theaccumulation of p62, suggesting that CQ systemicallyinhibits autophagy.

To confirm antiestrogen drug effectiveness, LCC9 paraf-fin-embedded tumor sections were stained for ERa orprogesterone receptor (PR; Supplementary Fig. S2). ICIreduces ERa staining and PR staining in LCC9 tumors,consistent with the known effects of this drug. TAM

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Figure2. Low-doseoralCQ resensitizes resistant breast tumors to antiestrogen therapy.A, LCC9orthotopic tumorsweregrown to25 to35mm2before treatedwith TAM, ICI, CQ, CQþICI, or CQþTAM for 5 weeks. Tumors were measured weekly with calipers; % change in tumor area (A) and tumor area (B) wascalculated. C, LCC9 tumorweight upon completion of study.MCF7-RRorthotopic tumorswere untreated (control) or treatedwith TAM,CQ, or CQþTAM for 5weeks. Tumors were measured weekly with calipers and % change in tumor area (D) or tumor area growth curves (E) were calculated. F, average wetweight of MCF7-RR upon sacrifice. �, P < 0.05.

Cook et al.




treatment resulted in a cytoplasmic diffuse distribution ofERa and reduced PR staining, consistentwith observed drugactivity.Treatment of LCC9 cells with CQ, ICI, or TAM in vitro

increased pVEGFR2 expression determined by Westernblot hybridization (Supplementary Fig. S3A), suggestingthat antiestrogen therapy stimulates proangiogenic sig-naling in drug-resistant breast cancer cells. Thus, LCC9tumor sections were stained with antibodies againsteither CD31 (an endothelial cell marker) or phosphory-lated pVEGFR2. TAM and ICI treatment increased CD31-positive vessel staining (Fig. 4A and B) and elevated levelsof pVEGFR2 (Fig. 4C and D), implying increased angio-genesis. To confirm the effect of antiestrogen therapy onangiogenesis, mice were injected with 500 mL of Matrigelbetween the abdominal wall and the skin to form aMatrigel plug. Mice were either untreated (as control) ortreated with TAM, ICI, CQ, ICIþCQ, or TAMþCQ. After5 days, the plug was removed, fixed in formalin, andembedded in paraffin. Sections of Matrigel plug werestained with H&E, as shown in Fig. 5A. Mice treated withICI showed increased number of infiltrating cells into theMatrigel plug (Fig. 5B), which was not observed in Matri-gel plugs from animals treated with TAM. However,treatment with CQ in combination with either antiestro-gen had no effect on CD31 staining or pVEGFR2 staining,suggesting that the stimulation of angiogenesis likely doesnot explain the differential effects observed betweenICIþCQ and TAMþCQ treatment.Cytokine production and reticuloendothelial cell infil-

trates are common in some breast cancers (31). Thus, blood

was collected at the time of euthanasia to measure variouscytokine concentrations.Wemeasured themouse cytokinesand chemokines IL1b, IL2, IL3, IL4, IL5, IL6, IL12p70, IL17,TNFa, IFNg , MCP1, RANTES, Eotaxin, KC, MDC, TARC,and TCA3usingQ-Plex Array kits fromQuansys Biosciences(Supplementary Figs. S4–S6). Although variability in plas-ma cytokines is high, several cytokine trends are evidentbetween treatment groups. Of particular interest, themousechemokine KC (human analog: chemokine C-X-C motifligand 1; CXCL1) is elevated in the plasma of TAM andTAMþCQ mice and decreased in ICI and ICIþCQ-treatedmice. Also, IFNg is reduced in ICI and ICIþCQ-treatedanimals.

Because CXCL1 is expressed by macrophages and IFNgstimulates macrophages, we investigated whether therewere differential effects of antiestrogens and CQ on macro-phages. LCC9 tumor sectionswere stainedwith an antibodyagainst the macrophage marker CD68 (Fig. 6A). As quan-tified in Fig. 6B, ICI and ICIþCQ decreased peripheraltumor macrophage content when compared with controltumors. Moreover, TAMþCQ-treated mice significantlyincreased infiltrating CD68-positive cells when comparedwith control mice, indicating increased tumor macrophageinfiltration. Next, U937 human monocyte cells were differ-entiated into macrophages in vitro in the presence of IFNgand protein lysates from the precursor monocytes anddifferentiated macrophages were collected. As shownin Fig. 6C, MCF7-RR, LCC9, U937 undifferentiated mono-cytes, andU937 differentiatedmacrophages were studied toconfirm the presence of ERa and/or GPR30. The precursorU937 monocyte cells do not express GPR30 but express

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Figure 3. Immunohistochemicalevaluation of autophagy in treatedtumors. LCC9 tumor sections werestained using LC3A/B (A) or p62 (B)antibodies. LC3A/B is visualized at�1,000, whereas p62 is visualizedat �400. C, quantification of LC3and p62 IHC. Stained positive cellswere scored and averaged 3frames per tumor. n ¼ 5–8 tumors;�, P < 0.05. D, uterine tissue fromcontrol and CQ-treated mice wereharvested and Western blothybridization was used to confirmlevels of autophagy-relatedproteins LC3A/B and p62.Equivalence of protein loadingonto gels was confirmed bymeasuring b-actin expression.n ¼ 4; �, P < 0.05.





ERa, albeit at a lower level than the breast cancer cells.Conversely, differentiated U937 macrophage cells do notexpress detectable ERa but express higher levels of GPR30than MCF7-RR, LCC9, or their parental U937 monocytecells.

To determine whether antiestrogens or CQ affect macro-phages’ tumor cell killing capacity, U937 cells were differ-entiated in the presence of IFNg and vehicle control,1 mmol/L CQ, 100 nmol/L ICI, 1 mmol/L CQþ100 nmol/LICI, 100 nmol/L TAM, or 1 mmol/L CQþ100 nmol/L TAMfor 72 hours. Macrophages were then collected, resus-pended in fresh media to remove drug, and added topreplated MDA-MB-231 breast cancer cells. Use of theseER-negative cells prevented any remaining antiestrogenfrom inhibiting the target cells. Monocytes, differentiatedintomacrophages in thepresence of ICI, ICIþCQ, andTAM,had a decreased breast cancer cell killing capacity at 24 and48 hours after plating when compared with their vehicle-treated controls (Fig. 6D). However, in the presence of CQor TAMþCQ, macrophages had no significant difference intheir cell killing capacity when compared with the vehicle-treated control macrophages. Seventy-two hours after theaddition of macrophages, only macrophages in the pres-ence of ICI and ICIþCQ had significantly reduced breastcancer cell killing capacity as measured by cell index.

DiscussionBreast cancer remains the most prevalent cancer in wom-

en, with the majority of these tumors expressing ERa.Resistance to endocrine therapies remains a critical limita-tion in the ability of these agents to cure some patients.

Autophagy is a key pathway in the development of endo-crine resistance in breast cancer, and targeting autophagycan reverse antiestrogen resistance (6). Chloroquine, anantimalarial drug, inhibits autophagy by preventing degra-dation of autolysosomes. Moreover, chloroquine deriva-tives, such as hydroxychloroquine (HCQ), in combinationwith antineoplastic chemotherapeutic drugs or radiothera-py treatments inhibitmultiple cancer cell types (32, 33).Wenow show that using chloroquine therapy in combinationwith antiestrogens increased the sensitivity of resistantbreast cancer cells to endocrine therapies (Fig. 1A–C).

Resensitization to antiestrogens is characterized by anincrease in LC3-II levels, the lipidated form of LC3 that isfound in the autophagosomal membrane, implying anincrease in the rate of autophagy initiation. In contrast,increased levels of p62 indicate autophagosome accumu-lation (Fig. 1E–F) leading to undegraded autophagosomeaccumulation in the cytoplasm (34), suggesting that thelater steps in autophagy are not completed.Whenmeasuredwith changes in LC3, decreased p62 generally indicates thatthe autolysosome and its cargohave beendegraded and thatautophagic flux is intact. LCC9 or MCF7-RR cells treatedwith either TAM or ICI show increased LC3-II and reducedp62 expression, consistent with prior reports that endo-crine-targeting therapy induces autophagic flux (6, 10, 35).A synthetic quinine analog often used for chloroquine-resistant malarial cases, mefloquine (Lariam), was alsoshown to inhibit autophagy and induce cell death inMCF7 antiestrogen-sensitive breast cancer cells whengiven as a single agent (36). Chloroquine prevented DCISxenografts’ outgrowth in athymic mice (37, 38) and

vd

f

A B

C D

Figure 4. Antiestrogen therapystimulates angiogenesis. A, LCC9tumor sections from tumortreated with control, CQ, ICI,ICIþCQ, TAM, or TAMþCQ werestained for CD31 a marker ofendothelial cells to visual bloodvessels and quantified as averagevessels per section (B). C, LCC9tumors were also stained forpVEGFR2, visualized at �400,and quantified as averagepositive cells per field (D).n ¼ 6–10; �, P < 0.05.

Cook et al.




inhibited N-methyl-N-nitrosurea–induced mammary car-cinogenesis, suggesting chloroquine-based therapy as apossible agent in the prevention of initial premalignantlesions from progressing to breast cancer (39).We show that oral low-dose CQ given to tumor-bearing

mice in combination with an antiestrogen resulted in theresensitizationof resistantMCF7-RR (Fig. 2A andC) and thepotentiation of antiestrogen-mediated cell death in LCC9breast tumors (Fig. 2B andD). Overall, CQ is likely to be aneffective chemotherapeutic agent in combination with spe-cific antiestrogen therapy for the treatment of some ERþ

breast cancers. Nonetheless, because established tumorswere not eliminated completely, that is, "cured," duringthe 5-week treatment, improvements in overall responserates to CQ in adjuvant settings may be meaningful butmodest. The efficacy of CQ in prevention remains to beevaluated with the results of the ongoing PINC clinical trial.Importantly, because increased LC3A/B and p62 wereobserved in LCC9 tumors from mice treated with CQ (Fig.3), consistent with an inhibition of autophagy, changes inexpression of these markers in tumors may be worth eval-uating as possible biomarkers for predicting treatmentresponses in patients.Although a combination of both CQþTAM and CQþICI

resensitized resistant breast cancers to therapy, the combi-nation of CQ and ICI was less effective at reducing tumorburden than the combination of TAMþCQ at the dosestested. We hypothesized that systemic ICI therapy negative-ly affects the tumor microenvironment to reduce effective-

ness of the CQþICI combination. LCC9 tumors from micetreated with ICI, ICIþCQ, TAM, or TAMþCQ, each dis-played increased CD31 (Fig. 4A–B) and phospho-VEGFR2(Fig. 4C–D) immunoreactivity. Thus, antiestrogens canstimulate tumor angiogenesis in endocrine-resistant tumorsandCQhas no effect on this response.Moreover, LCC9 cellstreated with antiestrogens in vitro displayed increasedexpression of phospho-VEGFR2 (Supplementary Fig. S3),suggesting that the effect of TAM and ICI on angiogenesis ismediated directly by the tumor cells. Because both TAMandICI stimulated angiogenesis, increased blood vessel forma-tion is unlikely to explain the differential antitumor effectsobserved between TAMþCQ and ICIþCQ treatments. Pre-vious reports showed that TAM but not ICI increasedVEGFR2 expression in antiestrogen-sensitive MCF7 cells,further suggesting that the increase in angiogenesisobserved in both TAM and ICI-treated tumors is due to adirect effect mediated by the tumor epithelial cells (40). Todetermine the effect of antiestrogen therapy on cell inva-sion, standardMatrigel plug assays were performed (Fig. 5).Although both TAMand ICI stimulated tumor angiogenesis(Fig. 4), only ICI and ICIþCQ-treated mice exhibitedincreased cell invasion into their Matrigel plugs. These datasuggest that ICI and TAMhave different effects on the tumormicroenvironment.

Interestingly, analysis of circulating cytokines and che-mokines inmice treatedwithCQand antiestrogens indicatethat ICI and TAM may have a differential effect on themacrophage population (Supplementary Figs. S4–S6).

f

A

B

Figure 5. ICI treatment affects theextent of Matrigel plug cellinvasion. A, Matrigel plug wasinjected subcutaneously in micetreated with control, CQ, ICI,ICIþCQ, TAM, TAMþCQ. After 5days, plugs were fixed withformalin and embedded in paraffin.Matrigel plug sections werestained with H&E to showinfiltrating cells. B, infiltrating cellsinto the Matrigel plugs werequantified as cells per section.n ¼ 3–6 Matrigel plugs; �, P < 0.05.





Macrophages can be classified asM1-like (cytotoxic) orM2-like (immunosuppressive) and often serve opposing roles,highlighting the plasticity of these cell types (41). Estrogenwas previously shown to affect the monocyte-macrophagesystem (42, 43), suggesting that antiestrogen therapy mayalso perturb these cells.WhenLCC9 tumorswere stained forthe macrophage marker CD68 (Fig. 6A), mice treated withICI and ICIþCQ showed decreased staining when com-pared with other treatments (Fig. 6B). Thus, ICI seems toeffect negatively macrophage development and/or chemo-taxis. Moreover, circulating levels of IFNg in mice treatedwith ICI and ICIþCQ were reduced when compared withTAM and TAMþCQ-treated mice. IFNg can increase devel-opment of the M1-like cytotoxic macrophages (41), sug-

gesting that systemic ICI treatment may reduce the cell-killing capacity of macrophages. As shown in Fig. 6C,human monocytes express ERa albeit at a reduced levelthan the breast cancer cells, whereas ERa expression islost in the macrophages. Moreover, macrophages haveelevated GPR30 when compared with their parentalmonocyte cells and breast cancer cells. GPR30 is a GPRthat may act as an ER and promotes cellular growth andactivation. TAM’s partial estrogen agonist activity wasshown to also activate GPR30 (44), which may explain,in part, the elevated macrophage number in the tumorsfrom TAMþCQ-treated animals.

To confirm the effect of antiestrogen treatment on mac-rophage development and cytotoxicity, U937 monocytes

sfi

m

+

cc -

s

A

B

C

D di

dip

Figure 6. Systemic ICI treatmentinhibits macrophage activation. A,LCC9 tumor sections from tumortreated with control, CQ, ICI,ICIþCQ, TAM, or TAMþCQ werestained for CD68 a marker ofmacrophages. B, peripheralmacrophages were quantified ascells per section. n ¼ 4–8;�, P < 0.05. C, ERa and GPR30expression was determined inLCC9, MCF7-RR, U937(undifferentiated monocytes), andU937 (differentiated macrophages)protein lysates by Western blothybridization. D, U937 cellsdifferentiated into macrophages inthe presence of vehicle control,CQ, ICI, ICIþCQ, TAM, orTAMþCQwere added to preplatedMDA-MB-231 ER� breast cancercells (1:5 ratio macrophages:cancer cells) and cell index wasmeasured by electrical impedanceover 72 hours. n ¼ 3; �, P < 0.05.

Cook et al.




were differentiated intomacrophages in the presence of IFNand either ICI, TAM, CQ, CQþTAM, or CQþICI. Macro-phages that were differentiated in the presence of ICI,ICIþCQ, or TAM had decreased tumor cell cytotoxicity,indicating that systemic antiestrogen treatment reducesmacrophage activity (Fig. 6D). Furthermore, only ICI andICIþCQ-treatedmacrophages had a significantly higher cellindex (reduced cytotoxicity) at 72 hours postmacrophageaddition to breast cancer cells, whereas TAM and TAMþCQproduced no significant difference in cell index when com-pared with vehicle-treated controls. These data suggest thatICI but not TAM had deleterious effects on macrophagedistribution, activation, and/or cytotoxicity that may resultin the potentially antagonist interaction seen with the ICIþCQ combination therapy.An ongoing clinical trial is examining the effect of

combining TAM and chloroquine for the treatment ofERþ DCIS patients: PINC (see clinicaltrials.gov/show/NCT01023477). Preclinical data showed that chloro-quine treatment inhibited DCIS cell tumorgenicity inNOD/SCID mice, indicating the possibility of using chlo-roquine as a chemopreventive drug for breast cancer (38).In this study, we show that CQ in combination withantiestrogens potentiates endocrine therapy sensitivity inacquired resistant ERþ breast tumors. More importantly,this study highlights a potentially nonbeneficial effect ofFaslodex (ICI) on the tumor microenvironment thatreduces the value of combining chloroquine-based ther-

apies with Faslodex. Future clinical trial study designsmay consider using TAM as the preferred antiestrogento optimize the effects of an antiestrogen in the tumormicroenvironment. In our studies, a combination ofCQ þ TAM was more effective than either drug alone.

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

Authors' ContributionsConception and design: K.L. Cook, R. ClarkeDevelopment of methodology: K.L. Cook, A. W€arriAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): K.L. Cook, D.R. Soto-Pantoja, P.A.G. Clarke,M.I. Cruz, A. ZwartAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): K.L. Cook, D.R. Soto-Pantoja, R. ClarkeWriting, review, and/or revision of the manuscript: K.L. Cook, A. W€arri,R. ClarkeStudy supervision: R. Clarke

Grant SupportThis work was supported, in part, by awards from the US Department of

Health and Human Services (R01-CA131465 and U54-CA149147; to R.Clarke) andaDODBreastCancer ResearchProgramPostdoctoral Fellowship(BC112023; to K.L. Cook).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 28, 2013; revised April 1, 2014; accepted April 9,2014; published online June 13, 2014.

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Cook et al.



Correction

Correction: Hydroxychloroquine InhibitsAutophagy to Potentiate AntiestrogenResponsiveness in ERþ Breast CancerIn this article (Clin Cancer Res 2014;20:3222–32), which was published in theJune 15, 2014, issue of Clinical Cancer Research (1), the corresponding authorinformed us of a nomenclature error in the published article. The primarychemical form of the drug used in each of the in vitro and in vivo experimentspresented in the study was chloroquine (CQ) diphosphate and not hydroxy-chloroquine (HCQ) diphosphate as reported in the text. CQ is chloroquinediphosphate (Sigma-Aldrich, catalog number: C6628), i.e., N4-(7-chloro-4-qui-nolinyl)-N1,N1-dimethyl-1,4-pentanediamine diphosphate salt); Sigma does notsell HCQ diphosphate. Hydroxychloroquine is a hydroxyl derivative of chloro-quine. The conclusions put forth in this article remain unchanged.

The online version of this article has been changed to reflect the correct nomenclatureand no longer matches the print version. The authors regret this error.

Reference1. Cook KL, W€arri A, Soto-Pantoja DR, Clarke PAG, Cruz MI, Zwart A, et al. Hydroxychloroquine

inhibits autophagy to potentiate antiestrogen responsiveness in ERþ breast cancer. Clin Cancer Res2014;20:3222–32.

Published online June 1, 2016.doi: 10.1158/1078-0432.CCR-16-0644�2016 American Association for Cancer Research.

ClinicalCancerResearch

www.aacrjournals.org 2825

2014;20:3222-3232. Clin Cancer Res Katherine L. Cook, Anni Wärri, David R. Soto-Pantoja, et al.

Breast Cancer+Responsiveness in ERChloroquine Inhibits Autophagy to Potentiate Antiestrogen

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Chloroquine Inhibits Autophagy to Potentiate Antiestrogen ... · in Matrigel:IMEM (50:50%). Cells were injected orthoto-pically through a 3-mm skin cut into 4 different mammary fat