Chloroquine Inhibits Autophagy to Potentiate Antiestrogen Responsiveness in Erþ Breast Cancer

Clinical Cancer Cancer Therapy: Preclinical Research

Chloroquine Inhibits Autophagy to Potentiate Antiestrogen Responsiveness in ERþ Breast Cancer

Katherine L. Cook1, Anni Warri€ 1, David R. Soto-Pantoja2, Pamela AG. Clarke1, M. Idalia Cruz1, Alan Zwart1, and Robert Clarke1

Abstract Purpose: Estrogen receptor-a (ERa)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are þ used to treat ER 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. þ Experimental Design: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ER 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 ICIþCQ was 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 ICIþCQ was associated with ICI effects on cell-mediated immunity within the tumor microenvironment. The mouse chemokine KC (CXCL1) and IFNg were differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage þ development/activity. Consistent with these observations, TAMþCQ treatment increased tumor CD68 cells infiltration, whereas ICI and ICIþCQ reduced peripheral tumor macrophage 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 combination of TAMþCQ suggests a positive outcome for ongoing neoadjuvant clinical trials using þ this combination for the treatment of ER ductal carcinoma in situ lesions. Clin Cancer Res; 20(12); 3222–32. 2014 AACR.

Introduction selective ER downregulator Faslodex (fulvestrant, ICI), or One out of every eight American women will develop aromatase inhibitors that block the conversion of andro- invasive breast cancer over the course of her lifetime. An gens to estrogens. Even with the success of these drugs, some de novo estimated 230,000 new cases of breast cancer are diagnosed tumors fail to respond ( resistance) or acquire annually in the United States (1). Of these cases, 70% will resistance over time (2–4). express estrogen receptor-a (ERa). ER-a–targeted therapies Autophagy is a process by which a double membrane include the selective ER modulator tamoxifen (TAM), the vesicle surrounds cellular contents, such as damaged orga- nelles and misfolded or protein aggregates, and recycles the material through lysosomal degradation (5). Studies in 1 Authors' Afﬁliations: Department of Oncology and Lombardi Compre- breast cancer cells show that the induction of autophagy by hensive Cancer Center, Georgetown University Medical Center, Washing- ton, D.C.; and 2Department of Pathology, National Cancer Institute, NIH, various therapeutics is usually prosurvival (6–8). Further- þ Bethesda, Maryland more, TAM and ICI both induce autophagy in ER breast Note: Supplementary data for this article are available at Clinical Cancer cancer cells (6, 9–13). Antiestrogen-resistant cell lines Research Online (http://clincancerres.aacrjournals.org/). exhibit increased basal autophagy when compared with their antiestrogen-sensitive parental cells (10). Inhibiting Update: There was a nomenclature error in the published article. The primary chemical form of the drug used in each of the in vitro and in vivo autophagy through autophagy-related gene 5 (ATG5) experiments presented in the study was chloroquine (CQ) diphosphate and silencing potentiated antiestrogen-mediated cell death, not hydroxychloroquine (HCQ) diphosphate as reported. This version of the article contains the correct nomenclature. indicating that antiestrogen-stimulated autophagy is prosurvival and a critical mechanism of therapy resistance (10). Corresponding Author: Robert Clarke, W405A Research Building, Geor- getown University Medical Center, 3970 Reservoir Rd NW, Washington, DC Analysis of publically available human datasets indicates 20057. Phone: 202-687-8991; Fax: 202-687-2085: E-mail: that autophagy-related genes, ATG5, ATG7, and p62 [email protected] (SQSTM1), are elevated in early recurring breast cancer doi: 10.1158/1078-0432.CCR-13-3227 when compared with breast cancer that never recurs. More- 2014 American Association for Cancer Research. over, elevated p62 is signiﬁcantly correlated with poor

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xenografts. Furthermore, we found that the tumor micro- Translational Relevance environment plays an important role in the treatment of Breast cancer affects more than 230,000 American breast tumors with antiestrogens and CQ. We show that women each year. An estimated 70% of these cases have monocytes that express ERa, and differentiated macro- tumors that express the estrogen receptor and are eligible phages that express the estrogen and TAM-responsive G- to be treated with antiestrogens such as tamoxifen (TAM) coupled protein receptor 30 (GPR30), may mediate some of or, for the treatment of advanced postmenopausal dis- the estrogen agonist and tumor-inhibitory activities of TAM. ease, Faslodex. However, 50% of these breast cancers will In contrast, the antineoplastic activity of ICI was reduced by either fail to respond (de novo resistance) or lose therapy its ability to inhibit monocyte differentiation and macro- responsiveness over time (acquired resistance). We phage-mediated clearance of tumor cells. An ongoing clin- found that inhibiting autophagy through low-dose oral ical trial Preventing Invasive Neoplasia with Chloroquine chloroquine (CQ) administration increases the respon- (PINC) is investigating the ability of chloroquine alone or þ siveness of resistant mammary tumors to antiestrogens. in combination with TAM (in ER cases) to eliminate ductal Moreover, we show that the combination of TAM and carcinoma in situ (DCIS) progenitor cells, highlighting the CQ is more effective than that of Faslodex and CQ due to importance of our findings. activities within the tumor microenvironment. These preclinical data highlight the relevance of a clinical trial Materials and Methods combining chloroquine and TAM for the treatment of þ Materials ER in situ breast lesions (Preventing Invasive Neoplasia The following materials were obtained as indicated: 4- with Chloroquine trial) and suggest a clinical benefit of hydroxytamoxifen and CQ diphosphate salt (Sigma- the addition of CQ to antiestrogen therapy for the þ Aldrich); ICI 182,780 (Tocris Bioscience); Improved Mini- treatment of ER breast cancer. Furthermore, this study mal Essential Medium (IMEM; Gibco Invitrogen BRL,); FBS predicts a more modest result for combining Faslodex and bovine calf charcoal-stripped serum (CCS; Equitech-Bio and chloroquine-based therapies for prevention and/or Inc); crystal violet (Fisher Scientific). Growth factor reduced treatment of breast cancer. Matrigel (BD Biosciences). Antibodies were obtained from the following sources: LC3A/B, VEGFR2, phospho-VEGFR2 (Cell Signaling Technology); p62 [Western, BD Biosciences; immunohistochemistry (IHC), MBL International]; CD68 survival in patients with breast cancer (Supplementary Fig. (AbD Serotec); CD31 (Abcam); ERa, b-actin, and polyclonal S1), suggesting a role for autophagy in breast cancer recur- and horseradish peroxidase (HRP)-conjugated secondary rence (14–18). antibodies (Santa Cruz Biotechnology). CQ is a lysotropic chloroquine derivative that accumu- lates within lysosomes, resulting in lysosome neutralization Cell culture and the inhibition of autophagic flux. Originally applied as MCF7-RR and LCC9 human breast carcinoma cells an antimalarial medication, the use of chloroquine (or were grown in phenol-red free IMEM media containing chloroquine derivatives) to inhibit autophagy is currently 5% CCS. MCF7 and ZR-75-1/ICI-R cells were grown in being explored as possible chemotherapeutic interventions phenol-red IMEM media containing 5% FBS. MCF7 cells for the treatment of cancer (19). Here, we have explored the were obtained from Dr. Marvin Rich at the Michigan possible beneficial effect of combining antiestrogen thera- Cancer Foundation. MCF7-RR cells were obtained from pies with chloroquine (CQ) for the treatment of antiestro- þ Indiana University of Pennsylvania (Indiana, PA; ref. 21). gen-resistant ER breast cancers. We used several cellular þ LCC9 and ZR-75-1/ICI-R cells were generated by our models of endocrine resistance: MCF7-RR (ER , estrogen- group at Georgetown University (Washington, D.C.; independent, TAM-resistant, ICI-sensitive cells; derived ref. 22). Cells were grown at 37C in a humidified, 5% from MCF-7 cells selected against low serum and TAM; þ CO2:95% air atmosphere. refs. 20, 21), LCC9 (ER , estrogen-independent, ICI-resistant, TAM cross-resistant cells; derived from the estrogen- Crystal violet assay independent antiestrogen-sensitive LCC1 cells by selection Human breast cancer cells (3 103 cells/mL) in IMEM þ against ICI; ref. 22), and ZR-75-1/ICI-R (ER , ICI-resistant containing 5% CCS were plated in 24-well tissue culture cells; derived from estrogen-sensitive antiestrogen-sensitive plates. On day 1 after plating, and every 3 days, thereafter ZR-75-1 cells by selection against ICI). We also used two cells were treated with varying doses (10–1,000 nmol/L) of different categories of antiestrogen therapy: TAM, a selective either TAM or ICI and 1 mmol/L CQ. On day 6, media were estrogen receptor modulator (SERM) and ER partial agonist, aspirated and cells were stained with crystal violet. Cells and ICI, a selective estrogen receptor downregulator (SERD) were permeabilized using citrate buffer and absorbance was and ER antagonist. read at 480 nm on a plate reader. The combination of CQ with TAM partially resensitized resistant MCF7-RR and LCC9 tumors, whereas a combina- Western blot hybridization tion of ICIþCQ was not as effective as CQ given as a single- MCF7-RR and LCC9 cells were solubilized by sonication agent therapy in treating ICI-resistant LCC9 orthotopic in radioimmunoprecipitation assay buffer lysis buffer.

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Proteins were size fractionated by PAGE and then trans- using ImageJ and shown as average vessel diameter (Sup- ferred to a nitrocellulose membrane. Nonspecific binding plementary Fig. S3B). was blocked by incubation for one hour at room temperature with Tris-buffered saline containing 5% powdered Cytokine analysis milk and 1% Triton X-100. Membranes were incubated Plasma from treated mice was collected at necropsy and overnight at 4C with primary antibodies, followed by immediately frozen. Quansys Biosciences Q-Plex Array kits incubation with polyclonal HRP-conjugated secondary were used to measure the following mouse cytokines and antibodies (1:2000) for 1 hour at room temperature. Immu- chemokines: interleukin (IL) 1b, IL2, IL3, IL4, IL5, IL6, noreactive products were visualized by chemiluminescence IL12p70, IL17, TNFa, IFNg, MCP1, RANTES, Eotaxin, KC, (SuperSignal Femto West, Pierce Biotechnology) and quan- MDC, TARC, TCA3. tified by densitometry using the ImageJ digital densitometry software (http://rsbweb.nih.gov/ij/). Macrophage differentiation and cytotoxicity assay U937 human monocyte cells were treated in the presence Orthotopic human breast cancer xenografts of IFNg (to differentiate monocytes into activated macro- Five-week-old, intact, athymic nude mice (Harlan Labo- phages) and either 100 nmol/L TAM, 1 mmol/L CQ, 100 ratories) were injected with 1 106 LCC9 or MCF7-RR cells nmol/L ICI, or the combination of antiestrogen and CQ for in Matrigel:IMEM (50:50%). Cells were injected orthoto- 72 hours. Macrophages were counted and plated with 70% pically through a 3-mm skin cut into 4 different mammary confluent MDA-MB-231 (triple-negative, antiestrogen non- fat pads per mouse (one abdominal and one thoracic responsive breast cancer cells) in a 1:5 ratio of macrophages mammary gland on each side). For the MCF7-RR xeno- to cancer cells for 72 hours. Macrophage killing was then grafts, mice also received a low-dose 17b-estradiol pellet assessed by RTCA-ACEA xCELLigence system through (0.36 mg, 60-day release; Innovative Research of America) electrical impedance (26). implanted subcutaneously under isoflurane anesthesia. Tumors grew to approximately 25 to 35 mm2 before therapy Statistical analysis initiation; 1 to 2 tumors per mouse successfully reached the Data are presented as the mean SEM. For most studies, required size for treatment initiation. Mice were treated with Student t test (pairwise) or ANOVA followed by Bonferoni CQ, TAM, ICI, or a combination of an antiestrogen and CQ. posthoc tests (group wise) was used (Prism software). Sta- CQ was administered in the drinking water; 0.29 mg/mL tistical differences for in vivo tumor area were evaluated by results in approximately 1 to 2 mg daily dose per mouse Dr. Fang in the Department of Biostatistics, Bioinformatics (23). TAM was administered in a standard rodent chow and Biomathematics at Georgetown University Medical 5053 diet containing 400 ppm TAM citrate (Harlan-Tek- Center (Washington, D.C.). Tumor volumes were obtained lad), which corresponds to a TAM dose of approximately from measurements of the longest perpendicular axes. We 32 mg/kg/d. ICI was administered by subcutaneous injec- use the weighted F test for comparison between two groups tion 0.5 mg/mouse/week in 70% EtOH/30% NaCl. Mice with two tumor volumes because the behavior of two were euthanized after 5 weeks of treatment, and tumors tumors growing in a single mouse are not independent removed at necropsy, fixed in neutral-buffered 10% for- events (27). All computation was performed in the R- malin, and processed using routine histologic methods. environment. There were 26 mice for LCC9 xenografts, and The efficacy of antiestrogen treatment was confirmed by 19 mice for MCF7RR xenografts. Criterion for statistical staging of the estrus cycle; mice on TAM or ICI therapy did significance was set at P 0.05. not cycle. Results Tissue staining and immunohistochemistry Antiestrogen-sensitive MCF7 cells and antiestrogen-resis- Mammary tumors were fixed in 10% phosphate-buffered tant MCF7-RR (TAM resistant), ZR-75-1 ICI-R (ICI resis- formalin for 24 hours before embedding in paraffin. tant), and LCC9 (ICI resistant/TAM cross-resistant) breast Embedded tissues were cut into 5 mm thick sections and cancer cells were plated in 24-well dishes and treated with stained with hematoxylin and eosin (H&E) to determine vehicle, 1 mmol/L CQ, and/or various concentrations of morphology. Immunostaining was performed with an anti- TAM or ICI (vehicle, 10, 100, 1,000 nmol/L) for 6 days. The body to CD31 (1:100), pVEGFR2 (1:100), p62 (1:1000), effect on cell density was determined by crystal violet assays LC3 (1:100), CD68 (1:100) using the streptavidin-biotin (28). The combination of antiestrogen and CQ potentiated method. Stained sections were visualized and photo- TAM and ICI-mediated cell death in endocrine-sensitive graphed. Because CD31 and pVEGFR2 staining was hetero- MCF7 cells (Fig. 1A). Furthermore, CQ and antiestrogen geneous, data were quantified using a common method of therapy inhibited LCC9 cells (Fig. 1B), MCF7-RR (Fig. 1C), angiogenic marker quantification defined as "hotspot" and ZR-75-1 ICI-R (Fig. 1D). Moreover, the addition of CQ quantification. In summary, three "hotspots" in the tumor resulted in increased LC3-II formation (lipidated form of were selected, the number of vessels/microvessels in each LC3 that is a marker of autophagosome formation) and "hotspot" was counted at 200 magnification, and a final accumulation of p62 (marker of autophagosomal flux) in score was expressed as the mean vessel density (24, 25). The both MCF7-RR (Fig. 1E) and LCC9 (Fig. 1F) as determined þ longest diameter of each CD31 blood vessel was measured by Western blot hybridization. Increased LC3-II and p62

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AB Control 1 µmol/L CQ Control 1 µmol/L CQ Control 1 µmol/L CQ Control 1 µmol/L CQ Relative cell density Relative cell density

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Figure 1. CQ restores antiestrogen responsiveness in vitro. A, MCF7 cells were treated with 1 mmol/L CQ, 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 various doses (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 were treated 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 treated with 1 mmol/L CQ, and/or various doses (vehicle, 10, 100, 1,000 nmol/L) ICI for 6 days. Relative cell density was determined by 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 and CQ for 72 hours. Cells were harvested and Western blot hybridization was used to confirm levels of autophagy-related proteins LC3A/B and p62. Equivalence of protein loading onto gels was confirmed by measuring b-actin expression. n ¼ 3; , P < 0.05. expression is indicative of inhibited autophagic flux result- andB),butafter3weeksoftreatment,CQalonewasjust ing in the cellular accumulation of autophagosomes. Treat- as effective at inhibiting LCC9 as the TAMþCQ combi- ment of MCF7-RR cells with TAM or LCC9 cells with TAM or nation. Unexpectedly, the combination of ICIþCQ was ICI resulted in increased LC3-II with a corresponding less effective than either TAMþCQ or CQ treatment alone. decrease in p62 levels confirming previous studies that TAM or ICI treatment alone had no significant difference in antiestrogens stimulate autophagic flux (10). tumor area when compared with control tumors. When LCC9 and MCF7-RR cells were orthotopically injected tumor growth was measured as wet weight at necropsy, into the mammary fat pads of female athymic mice. LCC9 tumors treated with CQ alone, TAMþCQ, or ICIþCQ Tumors were grown to 20 to 35 mm2 before treatment had significantly reduced tumor wet-weight when com- with CQ and/or TAM (MCF7-RR), or with CQ and/or pared with control tumors (Fig. 2C). However, CQ alone TAM or ICI (LCC9). In LCC9 xenografts, TAMþCQ was was significantly more effective than ICIþCQ, suggesting a most effective at reducing initial tumor growth (Fig. 2A potentially antagonist interaction between ICI and CQ. In

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AB C

DE F

Figure 2. Low-dose oral CQ resensitizes resistant breast tumors to antiestrogen therapy. A, LCC9 orthotopic tumors were grown to 25 to 35 mm2 before treated with TAM, ICI, CQ, CQþICI, or CQþTAM for 5 weeks. Tumors were measured weekly with calipers; % change in tumor area (A) and tumor area (B) was calculated. C, LCC9 tumor weight upon completion of study. MCF7-RR orthotopic tumors were untreated (control) or treated with TAM, CQ, or CQþTAM for 5 weeks. Tumors were measured weekly with calipers and % change in tumor area (D) or tumor area growth curves (E) were calculated. F, average wet weight of MCF7-RR upon sacriﬁce. , P < 0.05.

MCF7-RR orthotopic xenografts, control tumors continued positive LC3 staining forming puncta-like structures to grow, whereas TAMþCQ significantly reduced tumor size broadly consistent with autophagy induction. Tumors (Fig. 2D and E). CQ or TAM alone had no significant effect from animals treated with ICI or TAM have reduced on MCF7-RR tumor growth when compared with controls. p62 expression, consistent with increased autophagic flux The combination of TAMþCQ significantly reduced MCF7- (Fig. 3C). In contrast, tumors from mice treated with CQ RR tumor wet weight when compared with control-treated have elevated p62 expression, suggesting a block in the tumor weight measured at necropsy (Fig. 2F). later stages of autophagy (29, 30). To corroborate the Formalin-fixed LCC9 tumors were embedded in paraf- systemic effect of CQ on autophagy, uterine tissue from fin and cut into 5 mm sections. Tumor sections were control mice or mice treated with CQ were isolated and stained with specific antibodies against either LC3 (Fig. Western blot hybridization was performed on protein 3A) or p62 (Fig. 3B) using an avidin-biotin technique that lysates for LC3-II and p62 levels. As shown in Fig. 3D, reacts with peroxidase-conjugated streptavidin substrate CQ dosing increases LC3-II formation and results in the to determine the effects of treatments on autophagy accumulation of p62, suggesting that CQ systemically markers. LCC9 tumors from mice treated with ICI, TAM, inhibits autophagy. or CQ show elevated LC3A/B staining(Fig.3C).Measur- To confirm antiestrogen drug effectiveness, LCC9 paraf- ing autophagic activity using immunohistochemistry can fin-embedded tumor sections were stained for ERa or be challenging often because it is difficult to differentiate progesterone receptor (PR; Supplementary Fig. S2). ICI between LC3 and LC3-II by IHC in tissue sections. How- reduces ERa staining and PR staining in LCC9 tumors, ever, the high level of magnification (1,000) shows consistent with the known effects of this drug. TAM

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A C

Figure 3. Immunohistochemical evaluation of autophagy in treated tumors. LCC9 tumor sections were stained using LC3A/B (A) or p62 (B) antibodies. LC3A/B is visualized at 1,000, whereas p62 is visualized IHC score at 400. C, quantification of LC3 and p62 IHC. Stained positive cells were scored and averaged 3 frames per tumor. n ¼ 5–8 tumors; , P < 0.05. D, uterine tissue from control and CQ-treated mice were BD harvested and Western blot hybridization was used to confirm levels of autophagy-related proteins LC3A/B and p62. Equivalence of protein loading u onto gels was confirmed by measuring b-actin expression. n ¼ 4; , P < 0.05. A d

treatment resulted in a cytoplasmic diffuse distribution of was collected at the time of euthanasia to measure various ERa and reduced PR staining, consistent with observed drug cytokine concentrations. We measured the mouse cytokines activity. and chemokines IL1b, IL2, IL3, IL4, IL5, IL6, IL12p70, IL17, Treatment of LCC9 cells with CQ, ICI, or TAM in vitro TNFa, IFNg, MCP1, RANTES, Eotaxin, KC, MDC, TARC, increased pVEGFR2 expression determined by Western and TCA3 using Q-Plex Array kits from Quansys Biosciences blot hybridization (Supplementary Fig. S3A), suggesting (Supplementary Figs. S4–S6). Although variability in plas- that antiestrogen therapy stimulates proangiogenic sigma cytokines is high, several cytokine trends are evident naling in drug-resistant breast cancer cells. Thus, LCC9 between treatment groups. Of particular interest, the mouse tumor sections were stained with antibodies against chemokine KC (human analog: chemokine C-X-C motif either CD31 (an endothelial cell marker) or phosphory- ligand 1; CXCL1) is elevated in the plasma of TAM and lated pVEGFR2. TAM and ICI treatment increased CD31- TAMþCQ mice and decreased in ICI and ICIþCQ-treated positive vessel staining (Fig. 4A and B) and elevated levels mice. Also, IFNg is reduced in ICI and ICIþCQ-treated of pVEGFR2 (Fig. 4C and D), implying increased angio- animals. genesis. To confirm the effect of antiestrogen therapy on Because CXCL1 is expressed by macrophages and IFNg angiogenesis, mice were injected with 500 mL of Matrigel stimulates macrophages, we investigated whether there between the abdominal wall and the skin to form a were differential effects of antiestrogens and CQ on macro- Matrigel plug. Mice were either untreated (as control) or phages. LCC9 tumor sections were stained with an antibody treated with TAM, ICI, CQ, ICIþCQ, or TAMþCQ. After against the macrophage marker CD68 (Fig. 6A). As quan- 5 days, the plug was removed, fixed in formalin, and tified in Fig. 6B, ICI and ICIþCQ decreased peripheral embedded in paraffin. Sections of Matrigel plug were tumor macrophage content when compared with control stained with H&E, as shown in Fig. 5A. Mice treated with tumors. Moreover, TAMþCQ-treated mice significantly ICI showed increased number of infiltrating cells into the increased infiltrating CD68-positive cells when compared Matrigel plug (Fig. 5B), which was not observed in Matri- with control mice, indicating increased tumor macrophage gel plugs from animals treated with TAM. However, infiltration. Next, U937 human monocyte cells were differ- treatment with CQ in combination with either antiestro- entiated into macrophages in vitro in the presence of IFNg gen had no effect on CD31 staining or pVEGFR2 staining, and protein lysates from the precursor monocytes and suggesting that the stimulation of angiogenesis likely does differentiated macrophages were collected. As shown not explain the differential effects observed between in Fig. 6C, MCF7-RR, LCC9, U937 undifferentiated mono- ICIþCQ and TAMþCQ treatment. cytes, and U937 differentiated macrophages were studied to Cytokine production and reticuloendothelial cell infil- confirm the presence of ERa and/or GPR30. The precursor trates are common in some breast cancers (31). Thus, blood U937 monocyte cells do not express GPR30 but express

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Figure 4. Antiestrogen therapy vd stimulates angiogenesis. A, LCC9 tumor sections from tumor treated with control, CQ, ICI, ICIþCQ, TAM, or TAMþCQ were stained for CD31 a marker of endothelial cells to visual blood vessels and quantified as average CDvessels per section (B). C, LCC9 tumors were also stained for pVEGFR2, visualized at 400, fi f and quanti ed as average positive cells per field (D). n ¼ 6–10; , P < 0.05.

ERa, albeit at a lower level than the breast cancer cells. Autophagy is a key pathway in the development of endo- Conversely, differentiated U937 macrophage cells do not crine resistance in breast cancer, and targeting autophagy express detectable ERa but express higher levels of GPR30 can reverse antiestrogen resistance (6). Chloroquine, an than MCF7-RR, LCC9, or their parental U937 monocyte antimalarial drug, inhibits autophagy by preventing degra- cells. dation of autolysosomes. Moreover, chloroquine deriva- To determine whether antiestrogens or CQ affect macro- tives, such as hydroxychloroquine (HCQ), in combination phages’ tumor cell killing capacity, U937 cells were differ- with antineoplastic chemotherapeutic drugs or radiothera- entiated in the presence of IFNg and vehicle control, py treatments inhibit multiple cancer cell types (32, 33). We 1 mmol/L CQ, 100 nmol/L ICI, 1 mmol/L CQþ100 nmol/L now show that using chloroquine therapy in combination ICI, 100 nmol/L TAM, or 1 mmol/L CQþ100 nmol/L TAM with antiestrogens increased the sensitivity of resistant for 72 hours. Macrophages were then collected, resus- breast cancer cells to endocrine therapies (Fig. 1A–C). pended in fresh media to remove drug, and added to Resensitization to antiestrogens is characterized by an preplated MDA-MB-231 breast cancer cells. Use of these increase in LC3-II levels, the lipidated form of LC3 that is ER-negative cells prevented any remaining antiestrogen found in the autophagosomal membrane, implying an from inhibiting the target cells. Monocytes, differentiated increase in the rate of autophagy initiation. In contrast, into macrophages in the presence of ICI, ICIþCQ, and TAM, increased levels of p62 indicate autophagosome accumu- had a decreased breast cancer cell killing capacity at 24 and lation (Fig. 1E–F) leading to undegraded autophagosome 48 hours after plating when compared with their vehicle- accumulation in the cytoplasm (34), suggesting that the treated controls (Fig. 6D). However, in the presence of CQ later steps in autophagy are not completed. When measured or TAMþCQ, macrophages had no significant difference in with changes in LC3, decreased p62 generally indicates that their cell killing capacity when compared with the vehicle- the autolysosome and its cargo have been degraded and that treated control macrophages. Seventy-two hours after the autophagic flux is intact. LCC9 or MCF7-RR cells treated addition of macrophages, only macrophages in the pres- with either TAM or ICI show increased LC3-II and reduced ence of ICI and ICIþCQ had significantly reduced breast p62 expression, consistent with prior reports that endo- cancer cell killing capacity as measured by cell index. crine-targeting therapy induces autophagic flux (6, 10, 35). A synthetic quinine analog often used for chloroquine- Discussion resistant malarial cases, mefloquine (Lariam), was also Breast cancer remains the most prevalent cancer in wom- shown to inhibit autophagy and induce cell death in en, with the majority of these tumors expressing ERa. MCF7 antiestrogen-sensitive breast cancer cells when Resistance to endocrine therapies remains a critical limita- given as a single agent (36). Chloroquine prevented DCIS tion in the ability of these agents to cure some patients. xenografts’ outgrowth in athymic mice (37, 38) and

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Figure 5. ICI treatment affects the extent of Matrigel plug cell invasion. A, Matrigel plug was injected subcutaneously in mice treated with control, CQ, ICI, ICIþCQ, TAM, TAMþCQ. After 5 days, plugs were fixed with formalin and embedded in paraffin. Matrigel plug sections were stained with H&E to show infiltrating cells. B, infiltrating cells into the Matrigel plugs were quantified as cells per section. n ¼ 3–6 Matrigel plugs; , P < 0.05. B f

inhibited N-methyl-N-nitrosurea–induced mammary car- ness of the CQþICI combination. LCC9 tumors from mice cinogenesis, suggesting chloroquine-based therapy as a treated with ICI, ICIþCQ, TAM, or TAMþCQ, each dis- possible agent in the prevention of initial premalignant played increased CD31 (Fig. 4A–B) and phospho-VEGFR2 lesions from progressing to breast cancer (39). (Fig. 4C–D) immunoreactivity. Thus, antiestrogens can We show that oral low-dose CQ given to tumor-bearing stimulate tumor angiogenesis in endocrine-resistant tumors mice in combination with an antiestrogen resulted in the and CQ has no effect on this response. Moreover, LCC9 cells resensitization of resistant MCF7-RR (Fig. 2A and C) and the treated with antiestrogens in vitro displayed increased potentiation of antiestrogen-mediated cell death in LCC9 expression of phospho-VEGFR2 (Supplementary Fig. S3), breast tumors (Fig. 2B and D). Overall, CQ is likely to be an suggesting that the effect of TAM and ICI on angiogenesis is effective chemotherapeutic agent in combination with spe- mediated directly by the tumor cells. Because both TAM and þ ciﬁc antiestrogen therapy for the treatment of some ER ICI stimulated angiogenesis, increased blood vessel forma- breast cancers. Nonetheless, because established tumors tion is unlikely to explain the differential antitumor effects were not eliminated completely, that is, "cured," during observed between TAMþCQ and ICIþCQ treatments. Pre- the 5-week treatment, improvements in overall response vious reports showed that TAM but not ICI increased rates to CQ in adjuvant settings may be meaningful but VEGFR2 expression in antiestrogen-sensitive MCF7 cells, modest. The efﬁcacy of CQ in prevention remains to be further suggesting that the increase in angiogenesis evaluated with the results of the ongoing PINC clinical trial. observed in both TAM and ICI-treated tumors is due to a Importantly, because increased LC3A/B and p62 were direct effect mediated by the tumor epithelial cells (40). To observed in LCC9 tumors from mice treated with CQ (Fig. determine the effect of antiestrogen therapy on cell inva- 3), consistent with an inhibition of autophagy, changes in sion, standard Matrigel plug assays were performed (Fig. 5). expression of these markers in tumors may be worth eval- Although both TAM and ICI stimulated tumor angiogenesis uating as possible biomarkers for predicting treatment (Fig. 4), only ICI and ICIþCQ-treated mice exhibited responses in patients. increased cell invasion into their Matrigel plugs. These data Although a combination of both CQþTAM and CQþICI suggest that ICI and TAM have different effects on the tumor resensitized resistant breast cancers to therapy, the combi- microenvironment. nation of CQ and ICI was less effective at reducing tumor Interestingly, analysis of circulating cytokines and che- burden than the combination of TAMþCQ at the doses mokines in mice treated with CQ and antiestrogens indicate tested. We hypothesized that systemic ICI therapy negative- that ICI and TAM may have a differential effect on the ly affects the tumor microenvironment to reduce effective- macrophage population (Supplementary Figs. S4–S6).

www.aacrjournals.org Clin Cancer Res; 20(12) June 15, 2014 3229

s A

Figure 6. Systemic ICI treatment inhibits macrophage activation. A, LCC9 tumor sections from tumor treated with control, CQ, ICI, ICIþCQ, TAM, or TAMþCQ were stained for CD68 a marker of macrophages. B, peripheral macrophages were quantiﬁed as cells per section. n ¼ 4–8; , P < 0.05. C, ERa and GPR30 B D di expression was determined in LCC9, MCF7-RR, U937 (undifferentiated monocytes), and di

fi U937 (differentiated macrophages) p protein lysates by Western blot hybridization. D, U937 cells

m differentiated into macrophages in + the presence of vehicle control, CQ, ICI, ICIþCQ, TAM, or TAMþCQ were added to preplated s cc MDA-MB-231 ER breast cancer cells (1:5 ratio macrophages: cancer cells) and cell index was measured by electrical impedance - over 72 hours. n ¼ 3; , P < 0.05.

Macrophages can be classiﬁed as M1-like (cytotoxic) or M2- gesting that systemic ICI treatment may reduce the cell- like (immunosuppressive) and often serve opposing roles, killing capacity of macrophages. As shown in Fig. 6C, highlighting the plasticity of these cell types (41). Estrogen human monocytes express ERa albeit at a reduced level was previously shown to affect the monocyte-macrophage than the breast cancer cells, whereas ERa expression is system (42, 43), suggesting that antiestrogen therapy may lost in the macrophages. Moreover, macrophages have also perturb these cells. When LCC9 tumors were stained for elevated GPR30 when compared with their parental the macrophage marker CD68 (Fig. 6A), mice treated with monocyte cells and breast cancer cells. GPR30 is a GPR ICI and ICIþCQ showed decreased staining when com- that may act as an ER and promotes cellular growth and pared with other treatments (Fig. 6B). Thus, ICI seems to activation. TAM’s partial estrogen agonist activity was effect negatively macrophage development and/or chemo- showntoalsoactivateGPR30(44), which may explain, taxis. Moreover, circulating levels of IFNg in mice treated in part, the elevated macrophage number in the tumors with ICI and ICIþCQ were reduced when compared with from TAMþCQ-treated animals. TAM and TAMþCQ-treated mice. IFNg can increase devel- To conﬁrm the effect of antiestrogen treatment on mac- opment of the M1-like cytotoxic macrophages (41), sug- rophage development and cytotoxicity, U937 monocytes

3230 Clin Cancer Res; 20(12) June 15, 2014 Clinical Cancer Research

were differentiated into macrophages in the presence of IFN apies with Faslodex. Future clinical trial study designs and either ICI, TAM, CQ, CQþTAM, or CQþICI. Macro- may consider using TAM as the preferred antiestrogen phages that were differentiated in the presence of ICI, to optimize the effects of an antiestrogen in the tumor ICIþCQ, or TAM had decreased tumor cell cytotoxicity, microenvironment. In our studies, a combination of indicating that systemic antiestrogen treatment reduces CQ þ TAM was more effective than either drug alone. macrophage activity (Fig. 6D). Furthermore, only ICI and ICIþCQ-treated macrophages had a significantly higher cell Disclosure of Potential Conflicts of Interest index (reduced cytotoxicity) at 72 hours postmacrophage No potential conflicts of interest were disclosed. addition to breast cancer cells, whereas TAM and TAMþCQ produced no significant difference in cell index when com- Authors' Contributions pared with vehicle-treated controls. These data suggest that Conception and design: K.L. Cook, R. Clarke € ICI but not TAM had deleterious effects on macrophage Development of methodology: K.L. Cook, A. Warri Acquisition of data (provided animals, acquired and managed patients, distribution, activation, and/or cytotoxicity that may result provided facilities, etc.): K.L. Cook, D.R. Soto-Pantoja, P.A.G. Clarke, in the potentially antagonist interaction seen with the ICI þ M.I.Cruz,A.Zwart Analysis and interpretation of data (e.g., statistical analysis, biosta- CQ combination therapy. tistics, computational analysis): K.L. Cook, D.R. Soto-Pantoja, R. Clarke An ongoing clinical trial is examining the effect of Writing, review, and/or revision of the manuscript: K.L. Cook, A. W€arri, combining TAM and chloroquine for the treatment of R. Clarke þ Study supervision: R. Clarke ER DCIS patients: PINC (see clinicaltrials.gov/show/ NCT01023477). Preclinical data showed that chloroquine treatment inhibited DCIS cell tumorgenicity in Grant Support This work was supported, in part, by awards from the US Department of NOD/SCID mice, indicating the possibility of using chlo- Health and Human Services (R01-CA131465 and U54-CA149147; to R. roquine as a chemopreventive drug for breast cancer (38). Clarke) and a DOD Breast Cancer Research Program Postdoctoral Fellowship In this study, we show that CQ in combination with (BC112023; to K.L. Cook). The costs of publication of this article were defrayed in part by the antiestrogens potentiates endocrine therapy sensitivity in payment of page charges. This article must therefore be hereby marked þ acquired resistant ER breast tumors. More importantly, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this study highlights a potentially nonbeneficial effect of this fact. Faslodex (ICI) on the tumor microenvironment that Received November 28, 2013; revised April 1, 2014; accepted April 9, reduces the value of combining chloroquine-based ther- 2014; published online June 13, 2014.

References 1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2013. CA Cancer 11. Samaddar JS, Gaddy VT, Duplantier J, Thandavan SP, Shah M, Smith J Clin 2013;63:11–30. MJ, et al. A role for macroautophagy in protection against 4-hydro- 2. Riggins RB, Bouton AH, Liu MC, Clarke R. Antiestrogens, aromatase xytamoxifen-induced cell death and the development of antiestrogen inhibitors, and apoptosis in breast cancer. Vitam Horm 2005;71: resistance. Mol Cancer Ther 2008;7:2977–87. 201–37. 12. Schoenlein PV, Periyasamy-Thandavan S, Samaddar JS, Jackson 3. Clarke R, Skaar TC, Bouker KB, Davis N, Lee YR, Welch JN, et al. WH, Barrett JT. Autophagy facilitates the progression of ERalpha- Molecular and pharmacological aspects of antiestrogen resistance. positive breast cancer cells to antiestrogen resistance. Autophagy J Steroid Biochem Mol Biol 2001;76:71–84. 2009;5:400–3. 4. Clarke R, Leonessa F, Welch JN, Skaar TC. Cellular and molecular 13. Cook KL, Soto-Pantoja DR, Abu-Asab M, Clarke PA, Roberts DD, pharmacology of antiestrogen action and resistance. Pharmacol Rev Clarke R. Mitochondria directly donate their membrane to form autop- 2001;53:25–71. hagosomes during a novel mechanism of parkin-associated mito- 5. Clarke R, Cook KL, Hu R, Facey CO, Tavassoly I, Schwartz JL, et al. phagy. Cell Biosci 2014;4:16. Endoplasmic reticulum stress, the unfolded protein response, autop- 14. Loi S, Haibe-Kains B, Desmedt C, Lallemand F, Tutt AM, Gillet C, et al. hagy, and the integrated regulation of breast cancer cell fate. Cancer Definition of clinically distinct molecular subtypes in estrogen recep- Res 2012;72:1321–31. tor-positive breast carcinomas through genomic grade. J Clin Oncol 6. Cook KL, Shajahan AN, Clarke R. Autophagy and endocrine resistance 2007;25:1239–46. in breast cancer. Expert Rev Anticancer Ther 2011;11:1283–94. 15. Loi S, Haibe-Kains B, Desmedt C, Wirapati P, Lallemand F, Tutt AM, 7. Thomas S, Thurn KT, Bicaku E, Marchion DC, Munster PN. Addition of et al. Predicting prognosis using molecular profiling in estrogen recep- a histone deacetylase inhibitor redirects tamoxifen-treated breast tor-positive breast cancer treated with tamoxifen. BMC Genomics cancer cells into apoptosis, which is opposed by the induction of 2008;9:239. autophagy. Breast Cancer Res Treat 2011;130:437–47. 16. Loi S, Haibe-Kains B, Majjaj S, Lallemand F, Durbecq V, Larsimont 8. Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA. Autophagy D, et al. PIK3CA mutations associated with gene signature of low facilitates the development of breast cancer resistance to the anti- mTORC1 signaling and better outcomes in estrogen receptor- HER2 monoclonal antibody trastuzumab. PloS One 2009;4:e6251. positive breast cancer. Proc Natl Acad Sci U S A 2010;107: 9. Clarke R, Shajahan AN, Riggins RB, Cho Y, Crawford A, Xuan J, et al. 10208–13. Gene network signaling in hormone responsiveness modifies apopto- 17. Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, et al. sis and autophagy in breast cancer cells. J Steroid Biochem Mol Biol Gene-expression profiles to predict distant metastasis of lymph-node- 2009;114:8–20. negative primary breast cancer. Lancet 2005;365:671–9. 10. Cook KL, Shajahan AN, Warri A, Jin L, Hilakivi-Clarke LA, Clarke R. 18. Zhang Y, Sieuwerts AM, McGreevy M, Casey G, Cufer T, Paradiso Glucose-regulated protein 78 controls cross-talk between apoptosis A, et al. The 76-gene signature defines high-risk patients that and autophagy to determine antiestrogen responsiveness. Cancer Res benefit from adjuvant tamoxifen therapy. Breast Cancer Res Treat 2012;72:3337–49. 2009;116:303–9.

www.aacrjournals.org Clin Cancer Res; 20(12) June 15, 2014 3231

19. Solomon VR, Lee H. Chloroquine and its analogs: a new promise of an 31. Clarke R, Dickson RB, Lippman ME. Hormonal aspects of breast old drug for effective and safe cancer therapies. Eur J Pharmacol cancer. Growth factors, drugs and stromal interactions. Crit Rev Oncol 2009;625:220–33. Hematol 1992;12:1–23. 20. Butler WB, Berlinski PJ, Hillman RM, Kelsey WH, Toenniges MM. 32. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn Relation of in vitro properties to tumorigenicity for a series of sublines A. Chloroquine sensitizes breast cancer cells to chemotherapy inde- of the human breast cancer cell line MCF-7. Cancer Res 1986;46: pendent of autophagy. Autophagy 2012;8:200–12. 6339–48. 33. Mukhopadhyay A, Helgason GV, Karvela M, Holyoake TL. Hydroxy- 21. Butler WB, Fontana JA. Responses to retinoic acid of tamoxifen- chloroquine for chronic myeloid leukemia: complete cure on the sensitive and -resistant sublines of human breast cancer cell line horizon? Expert Rev hematol 2011;4:369–71. MCF-7. Cancer Res 1992;52:6164–7. 34. He C, Klionsky DJ. Regulation mechanisms and signaling pathways of 22. Brunner N, Boysen B, Jirus S, Skaar TC, Holst-Hansen C, Lippman J, autophagy. Annu Rev Genet 2009;43:67–93. et al. MCF7/LCC9: an antiestrogen-resistant MCF-7 variant in which 35. Cook KL, Clarke R. Heat shock 70 kDa protein 5/glucose-regulated acquired resistance to the steroidal antiestrogen ICI 182,780 confers protein 78 "AMP"ing up autophagy. Autophagy 2012;8:1827–9. an early cross-resistance to the nonsteroidal antiestrogen tamoxifen. 36. Sharma N, Thomas S, Golden EB, Hofman FM, Chen TC, Petasis Cancer Res 1997;57:3486–93. NA, et al. Inhibition of autophagy and induction of breast cancer cell 23. Lewis MD, Pfeil J, Mueller AK. Continuous oral chloroquine as a novel death by meﬂoquine, an antimalarial agent. Cancer Lett 2012; route for Plasmodium prophylaxis and cure in experimental murine 326:143–54. models. BMC Res Notes 2011;4:262. 37. Espina V, Liotta LA. What is the malignant nature of human ductal 24. Soto-Pantoja DR, Menon J, Gallagher PE, Tallant EA. Angiotensin- carcinoma in situ? Nat Rev Cancer 2011;11:68–75. (1–7) inhibits tumor angiogenesis in human lung cancer xenografts 38. Espina V, Mariani BD, Gallagher RI, Tran K, Banks S, Wiedemann J, with a reduction in vascular endothelial growth factor. Mol Cancer et al. Malignant precursor cells pre-exist in human breast DCIS and Ther 2009;8:1676–83. require autophagy for survival. PloS One 2010;5:e10240. 25. Weidner N. Current pathologic methods for measuring intratumoral 39. Loehberg CR, Thompson T, Kastan MB, Maclean KH, Edwards DG, microvessel density within breast carcinoma and other solid tumors. Kittrell FS, et al. Ataxia telangiectasia-mutated and p53 are potential Breast Cancer Res Treat 1995;36:169–80. mediators of chloroquine-induced resistance to mammary carcino- 26. Martin-Manso G, Calzada MJ, Chuman Y, Sipes JM, Xavier CP, Wolf V, genesis. Cancer Res 2007;67:12026–33. et al. sFRP-1 binds via its netrin-related motif to the N-module of 40. Patel RR, Sengupta S, Kim HR, Klein-Szanto AJ, Pyle JR, Zhu F, et al. thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA- Experimental treatment of oestrogen receptor (ER) positive breast MB-231 breast carcinoma cell adhesion and migration. Arch Biochem cancer with tamoxifen and brivanib alaninate, a VEGFR-2/FGFR-1 Biophys 2011;509:147–56. kinase inhibitor: a potential clinical application of angiogenesis inhi- 27. Tan M, Fang HB, Tian GL, Houghton PJ. Small-sample inference for bitors. Eur J Cancer 2010;46:1537–53. incomplete longitudinal data with truncation and censoring in tumor 41. Laoui D, Movahedi K, Van Overmeire E, Van den Bossche J, Schouppe xenograft models. Biometrics 2002;58:612–20. E, Mommer C, et al. Tumor-associated macrophages in breast cancer: 28. Frandsen TL, Boysen BE, Jirus S, Spang-Thomsen M, Dano K, distinct subsets, distinct functions. Int J Dev Biol 2011;55:861–7. Thompson EW, et al. Experimental models for the study of human 42. Lang TJ. Estrogen as an immunomodulator. Clin Immunol 2004;113: cancer cell invasion and metastasis. Fibrinolysis 1992;6:71–6. 224–30. 29. Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena 43. Harkonen PL, Vaananen HK. Monocyte-macrophage system as a A, Adeli K, et al. Guidelines for the use and interpretation of assays for target for estrogen and selective estrogen receptor modulators. Ann monitoring autophagy. Autophagy 2012;8:445–544. N Y Acad Sci 2006;1089:218–27. 30. Schwartz-Roberts JL, Shajahan AN, Cook KL, Warri A, Abu-Asab M, 44. Du GQ, Zhou L, Chen XY, Wan XP, He YY. The G protein-coupled Clarke R. GX15-070 (obatoclax) induces apoptosis and inhibits receptor GPR30 mediates the proliferative and invasive effects cathepsin D- and L-mediated autophagosomal lysis in antiestrogen- induced by hydroxytamoxifen in endometrial cancer cells. Biochem resistant breast cancer cells. Mol Cancer ther 2013;12:448–59. Biophys Res Commun 2012;420:343–9.

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Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2014 American Association for Cancer Research. Correction Clinical Cancer Correction: Hydroxychloroquine Inhibits Research Autophagy to Potentiate Antiestrogen Responsiveness in ERþ Breast Cancer

In this article (Clin Cancer Res 2014;20:3222–32), which was published in the June 15, 2014, issue of Clinical Cancer Research (1), the corresponding author informed us of a nomenclature error in the published article. The primary chemical form of the drug used in each of the in vitro and in vivo experiments presented in the study was chloroquine (CQ) diphosphate and not hydroxychloroquine (HCQ) diphosphate as reported in the text. CQ is chloroquine diphosphate (Sigma-Aldrich, catalog number: C6628), i.e., N4-(7-chloro-4-qui- nolinyl)-N1,N1-dimethyl-1,4-pentanediamine diphosphate salt); Sigma does not sell HCQ diphosphate. Hydroxychloroquine is a hydroxyl derivative of chloroquine. The conclusions put forth in this article remain unchanged.

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

Reference 1. Cook KL, W€arri A, Soto-Pantoja DR, Clarke PAG, Cruz MI, Zwart A, et al. Hydroxychloroquine þ inhibits autophagy to potentiate antiestrogen responsiveness in ER breast cancer. Clin Cancer Res 2014;20:3222–32.

Published online June 1, 2016. doi: 10.1158/1078-0432.CCR-16-0644 Ó2016 American Association for Cancer Research.

www.aacrjournals.org 2825 Chloroquine Inhibits Autophagy to Potentiate Antiestrogen Responsiveness in ER + Breast Cancer

Katherine L. Cook, Anni Wärri, David R. Soto-Pantoja, et al.

Clin Cancer Res 2014;20:3222-3232.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/20/12/3222

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