Tumor and Stem Cell Biology

Combined Treatment with Epigenetic,Differentiating, and Chemotherapeutic AgentsCooperatively Targets Tumor-Initiating Cells inTriple-Negative Breast CancerVanessa F. Merino1, Nguyen Nguyen1, Kideok Jin1, Helen Sadik1, Soonweng Cho1,Preethi Korangath1, Liangfeng Han1, Yolanda M.N. Foster1, Xian C. Zhou1, Zhe Zhang1,RoisinM.Connolly1,VeredStearns1, SyedZ.Ali2,ChristinaAdams2,QianChen3,DuojiaPan3,David L. Huso4, Peter Ordentlich5, Angela Brodie6, and Saraswati Sukumar1

Abstract

Efforts to induce the differentiation of cancer stem cellsthrough treatment with all-trans retinoic acid (ATRA) haveyielded limited success, partially due to the epigenetic silencingof the retinoic acid receptor (RAR)-b. The histone deacetylaseinhibitor entinostat is emerging as a promising antitumor agentwhen added to the standard-of-care treatment for breast cancer.However, the combination of epigenetic, cellular differentia-tion, and chemotherapeutic approaches against triple-negativebreast cancer (TNBC) has not been investigated. In this study,we found that combined treatment of TNBC xenografts withentinostat, ATRA, and doxorubicin (EAD) resulted in significanttumor regression and restoration of epigenetically silencedRAR-b expression. Entinostat and doxorubicin treatment inhib-

ited topoisomerase II-b (TopoII-b) and relieved TopoII-b-mediated transcriptional silencing of RAR-b. Notably, EADwas the most effective combination in inducing differentiationof breast tumor–initiating cells in vivo. Furthermore, geneexpression analysis revealed that the epithelium-specific ETStranscription factor-1 (ESE-1 or ELF3), known to regulateproliferation and differentiation, enhanced cell differentiationin response to EAD triple therapy. Finally, we demonstrate thatpatient-derived metastatic cells also responded to treatmentwith EAD. Collectively, our findings strongly suggest thatentinostat potentiates doxorubicin-mediated cytotoxicity andretinoid-driven differentiation to achieve significant tumorregression in TNBC. Cancer Res; 76(7); 2013–24. �2016 AACR.

IntroductionTriple-negative breast cancers (TNBC) lack expression of estro-

gen receptor (ER), progesterone receptor (PR), and HER2, andcomprise approximately 15% to 20% of breast cancers. Theycontinue to be a clinical problem because of their relatively poorprognosis, aggressive behavior, and lack of targeted therapies,

leaving chemotherapy as the mainstay of treatment (1). Retinoicacid and its products, such as all-trans retinoic acid (ATRA),induce differentiation of various types of stem cells, includingthose that are present in breast cancer (2, 3). However, in clinicaltrials, ATRAhas shown limited therapeutic success (4) thatmay beattributed, in part, to frequent epigenetic silencing of the retinoicacid receptor (RAR)-b (5).We and others have shown that histonedeacetylase (HDAC) inhibitors cause reexpression of RAR-b andsensitize the cells to treatment (6, 7).

Acetylation of histone proteins controls transcription of genesinvolved in cell growth, and the expression of histone deacetylases(HDAC) is frequently upregulated in several malignancies (8).Although HDAC inhibitors showed limited effect as single agentsin breast cancer, their use in combination with other anticanceragents is currently being evaluated (9). Studies in advanced solidtumors in which HDAC inhibitors were combined either withdoxorubicin (10)orwithpaclitaxel and carboplatin (11) suggestedenhanced antitumor activity. The HDAC inhibitor entinostat usedin combination with retinoic acid in patients with advanced solidtumors was associated with prolonged stable disease (12).

Here, we show that a combination of entinostat, ATRA, anddoxorubicin effectively killed tumor cells in culture and decreasedtumor size of xenografts of TNBC cell lines, and present pilot dataon its effectiveness in metastatic ascites from patients. Further, weprovide insights into the mechanisms underlying the enhancedeffects observed with the drug combinations.

1Department of Oncology, Johns Hopkins University School of Med-icine, Baltimore, Maryland. 2Department of Pathology, Sol GoldmanPancreatic Cancer Research Center, Johns Hopkins University Schoolof Medicine, Baltimore, Maryland. 3Department of Molecular BiologyandGenetics, JohnsHopkinsUniversitySchoolofMedicine,Baltimore,Maryland. 4Department of Molecular and Comparative Pathobiology,Johns Hopkins University School of Medicine, Baltimore, Maryland.5SyndaxPharmaceuticals, Department of TranslationalMedicine,Wal-tham, Massachusetts. 6Department of Pharmacology and Experimen-tal Therapeutics, University of Maryland School of Medicine, Balti-more, Maryland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

V.F. Merino and N. Nguyen contributed equally to this article.

Corresponding Authors: Saraswati Sukumar, Johns Hopkins University Schoolof Medicine, 1650 Orleans Street, CRB1/Room 143, Baltimore, MD 21231. Phone:410-614-2479; Fax: 410-614-4073; E-mail: saras@jhmi.edu, and Vanessa F.Merino, Phone: 410-614-4075; E-mail: vmerino1@jhmi.edu

doi: 10.1158/0008-5472.CAN-15-1619

�2016 American Association for Cancer Research.

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Materials and MethodsDetails are provided in Supplementary Methods.

Patient samples, cell lines, constructs, and reagentsFreshly resected breast tissue of women undergoing reduction

mammoplasty, primary tumors, and pleural effusion from wom-en undergoing treatment, and also collected through the RapidAutopsy Tissue Donation Program, were provided by the JohnsHopkins Surgical Pathology Department under approved proto-cols. CD24þ and CD44þ cells were isolated from normal breasttissue as described previously (13).

Cell lines were recently obtained from the ATCC; SUM-149 andSUM-159 cells were obtained from Dr. S. Ethier, Medical Univer-sity of South Carolina, Charleston, SC. The cell lines were notauthenticated by us; however, early passages (p2–5) of the ATCCauthenticated cell lines were used. Sources of other reagents:siRNA to RAR-b (Dharmacon), topoisomerase II-b (TopoII-b)and ELF3 (Qiagen), ATRA, doxorubicin, and paclitaxel (SigmaChemicals), and carboplatin (Johns Hopkins Oncology Pharma-cy). Entinostat was provided by Syndax Pharmaceuticals, LLC.

Chromatin immunoprecipitation analysisThe chromatin immunoprecipitation (ChIP) assay was per-

formed essentially as described (14). Antibodies used wereagainst acetylated H3 (Millipore), RAR-a (Santa Cruz Biotech-nology), and TopoII-b (Santa Cruz Biotechnology).

Flow cytometryCells were stained with CD24-FITC (clone ML5), CD44-PE

(clone 515, BDPharmingen), CD326 (EpCAM)-APC (cloneHEA-125, Miltenyi Biotec), and 7AAD (BD Pharmingen), or AnnexinV–Alexa Fluor 488 and propidium iodide (PI; Molecular Probes),for quantification of apoptosis, necrosis, and analysis of the cellcycle.

Tumor sphere assayThe tumor sphere assay was performed as previously described

(15). Pleural effusion samples from breast cancer patients wereplated in serum-free Mammary Epithelial Growth Medium con-taining 10% pleural effusion supernatant and supplements (13).

Xenograft and limiting dilution assayAll animal studies were performed following approval of the

Animal Care Committee of the Johns Hopkins School of Medi-cine. First-generation xenografts ofMDA-MB-231 cells were estab-lished in athymic nude mice by injecting 2 � 106 tumor cellssubcutaneously (s.c.). The mice were treated for 4 weeks withentinostat (2.5mg/kg) 5 days/week per os (oral); ATRA (5mg/kg)5 days/week intraperitoneal (i.p.), doxorubicin (2 mg/kg) once aweek intravenous (i.v.), or carboplatin (50 mg/kg) single dosei.p. 3 days after the first entinostat treatment. For limiting dilutionassays, the tumors were digested with collagenase/ hyaluronidase(13). Single cells were injected at dilutions of 5 � 106 to 5 � 103

along with Matrigel into humanized mammary fat pads (16, 17).

Transcriptome arrayMDA-MB-231 cells were treated for 48 hours with entinostat

(2.5 mmol/L), ATRA (1 mmol/L), and doxorubicin (0.2 mmol/L)singly or in combination. RNA was extracted, and an Illumina

HumanHT12v4 gene expression array was performed. The GEOaccession number is GSE63351.

Statistical analysisCell line results were expressed as mean � SEM. Student t tests

were performed on pairwise combinations of data to determinestatistical significance defined as P < 0.05. qRT-PCR data of tumorxenografts and tumor volume results were analyzed using themedian and two-tailed Mann–Whitney U test. For in vivo studies,multilevel mixed-effects models were used using the Tukey pro-cedure. Tumor incidence of the secondary transplants followinginjection of cells at limiting dilutions was used to determine thetumor-initiating frequency by L-Calc software.

ResultsA combination of the HDAC inhibitor retinoids andchemotherapy decreases tumor size

The contribution of chemotherapeutic agents to the growth-inhibitory effects of HDAC inhibitors and retinoids on TNBCcells has not been previously studied. The combination ofentinostat (E), all-trans retinoic acid (A), and doxorubicin(D; EAD) resulted in the strongest growth inhibition of threeTNBC cell lines (MDA-MB-231, SUM-149, and SUM-159) inculture (Fig. 1A). Doxorubicin provided the most additivebenefit to the triple combination, and EAD was more cytotoxicto TNBC cells in comparison to combinations that includedpaclitaxel (EAP) or carboplatin (Supplementary Fig. S1Aand B).

We next evaluatedwhether the epigenetic combination therapycauses regression of TNBC xenografts in immunodeficient mice.EADwasmost effective in decreasing tumor volume ofMDA-MB-231 and SUM-159 xenografts (Fig. 1B and Supplementary TableS1 and S2). Again, the presence of doxorubicin in the triplecombination was associated with a higher additive effect ondecreasing tumor volume compared with carboplatin (Supple-mentary Fig. S1C and Supplementary Tables S3 and S4).

EAD therapy induces cell death by apoptosis and necrosisWe sought to investigate the mechanisms through which the

epigenetic combination therapy induced cell death. Because wefound that doxorubicin doses of 200 nmol/L caused cells to arrestat G2 phase (not shown), we determined cell viability followingtreatment with lower doses of doxorubicin (6.25–50 nmol/L).The addition of entinostat and ATRA to doxorubicin tended toinduce cell death even at these lower doses of doxorubicin(Supplementary Fig. S1D). We next treated MDA-MB-231 for48 hours and analyzed effects on apoptosis and necrosis 5 daysafter removal of the drug. EþD treatment doubled the incidenceof apoptotic (Annexin Vþ) andnecrotic cells (Annexin Vþ andPIþ

cells) compared with doxorubicin (12.5 and 25 nmol/L) orentinostat alone (Fig. 1CandSupplementary Fig. S1E).Comparedwith doxorubicin, EAD treatment induced slightly more necrosisthan EþD(Fig. 1C and Supplementary Fig. S1E). By assessing cellsin sub-G1 phase of the cell cycle, we confirmed in an independentassay that in comparison with doxorubicin, EþD and EADincreased the number of apoptotic cells (Fig. 1D). Significantly,we observed that epigenetic therapy induced necrosis and apo-ptosis in vivo. The study pathologist's quantification of necrosispresent in the tumor xenografts of mice treated with the eightdifferent modalities showed that EAD-treated tumors tended

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Figure 1.Entinostat, ATRA, and doxorubicin (EAD) combination therapy induces cell death by apoptosis and necrosis, and decreases tumor volume. A, cell growth inhibitionin MDA-MB-231 (a), SUM-149 (b), and SUM-159 (c) cells treated for 48 hours with entinostat (2.5 mmol/L), ATRA (1 mmol/L), and doxorubicin (0.2 mmol/L) singly and incombinations. B, mice bearing xenografts of MDA-MB-231 (a) and SUM-159 (b) were treated with entinostat (2.5 mg/kg oral), ATRA (5 mg/kg i.p.), or doxorubicin(2 mg/kg i.v.) singly, or in combinations for 4 weeks. Mean tumor volume of 12–14 tumors per group is reported � SEM. C, flow cytometry determination of apoptotic(% Annexin V–positive and PI-negative, blue) and necrotic cells (% double positive, red) (doxorubicin 25 nmol/L). D, cells at sub-G1 following 48-hour treatmentof MDA-MB-231 with the different drugs (doxorubicin 25 and 50 nmol/L) and 5 days of drug withdrawal. E, quantification of necrosis in xenografts (n¼ 7–10/ group; score0–3). F, cleaved caspase-3 IHC and quantification in treated xenografts. Cell viability and flow cytometry results were obtained from triplicate, and after Student t test,results are expressed as mean � SEM. Veh, vehicle; Ent (E), entinostat; A, ATRA; D, Dox, doxorubicin. � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.

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to have the highest necrosis score (Fig. 1E and SupplementaryTable S5). Further, by IHC, tumor xenografts from EþD– andEAD-treated mice showed increased cleaved caspase-3 (Fig. 1F),an indicator of increased apoptosis.

RAR-b is reactivated by a combination of entinostat, ATRA, anddoxorubicin

Preclinical studies showed that theHDAC inhibitor trichostatinA, in combination with retinoic acid, reactivated silenced RAR-band decreased tumor volume more efficiently than the singletreatments in ER-positive cells (18). To investigate if chemother-apy potentiates the effect of HDACi and retinoic acid on RAR-bexpression, we performed quantitative real-time PCR of RAR-b inTNBC cells. We found that EAD treatment was most effective ininducing reexpression of RAR-b (Fig. 2A and SupplementaryFig. S2A) and its downstream genes (Supplementary Fig. S2B;refs. 19–21). Doxorubicin potentiated EþA–induced reexpres-sion of RAR-b (Fig. 2A and Supplementary Fig. S2A), paclitaxelhad a small effect (Supplementary Fig. S2C, a), while carboplatinshowedno significant effect (Supplementary Fig. S2C, b–d). In thetumors of treated mice, in comparison with single and doubletreatments, EAD led to higher levels of expression of RAR-b (Fig.2B) and its target genes (Fig. 2C). These results confirmed thatreactivation of the RApathway occurs in the tumors and correlateswith the effectiveness of EAD.

RAR-b mediates EAD cytotoxicity and is transcriptionallyrepressed by topoisomerase II-b

Additional evidence for a critical role for RAR-b in EAD-medi-ated cytotoxicity in TNBC cells was sought by studying theirresponse to treatment following depletion of RAR-b. KnockdownofRAR-b in SUM-149,MDA-MB-231, and SUM-159 cells resultedin a significant decrease in the EAD-mediated induction of RAtarget genes (Supplementary Fig. S2D, a, b) and in cell death (Fig.2D and Supplementary Fig. S2E, a, b).

We next investigated molecular mechanisms underlying theeffect of EAD on expression of RAR-b. Compared with otherchemo-agents, doxorubicin exerted a greater effect when com-bined with entinostat and ATRA on RAR-b reexpression andinhibition of cancer cell growth. Intrigued by this finding, wesearched for targets of doxorubicin action. One mechanism ofcell death caused by doxorubicin is by stabilizing the topo-isomerase II complex after it has cleaved DNA strands forreplication, thereby halting cell division. In hepatocellularcarcinoma cells, entinostat caused degradation of TopoII-a,but not of TopoII-b (22). Interestingly, we found that in TNBCcell lines MDA-MB-231 and SUM-159, entinostat significantlyreduced both TopoII-a and TopoII-b protein (Fig. 2E, a), andmRNA levels (Supplementary Fig. S2F, a, b), in a dose- andtime-dependent manner. These results identified TopoII-b asa common target for both doxorubicin and entinostat inTNBC cells. As predicted, the EþD combination resulted in aslightly stronger inhibition of TopoII-b in comparison withtreatment with single agents (Fig. 2E, b and SupplementaryFig. S2F, c).

Based on published work on leukemia (23), we hypothesizedthat ATRA recruits TopoII-b and initiates low levels of RAR-btranscription in breast cancer cells. The EþDeffect on degradationof TopoII-b, in addition to entinostat-mediated degradation ofHDAC/NCOR, possibly potentiates RAR-b expression. To obtain

further evidence for TopoII-b occupancy at the RAR-b promoterwhile avoiding multiple off-target effects of entinostat, we usedT47D, a breast cancer cell line model with active RAR-b sig-naling. ATRA treatment induced binding of TopoII-b to theRAR-b promoter in T47D cells (Supplementary Fig. S2G, a),similar to effects seen in acute promyelocytic leukemia (APL)cells where TopoII-b occupies the RAR-b promoter (23). Inthese cells, early in RA signaling TopoII-b acts as an activator ofRAR-b, while at later time points, TopoII-b mediates repressionof RAR-b (23). To determine if the inhibitory action of TopoII-bon RAR-b expression was evident in TNBC cells, we usedchromatin immunoprecipitation assays to verify RAR-b andTopoII-b occupancy at their respective binding sites (23).RAR-a directly regulates the transcription of RAR-b (5). ChIPanalysis of three different putative RAR-a binding regionswithin the RAR-b locus was performed in SUM-159 cells (Fig.2F). Compared with sites 1 and 2, RAR-a was recruited atsignificantly higher levels to site 3 in SUM-159 cells treatedwith EAD (Fig. 2F, a and Supplementary Fig. S2G, b). TopoII-b(Supplementary Fig. S2G, c) and NCOR (Fig. 2F, b) levels weresignificantly decreased at site 3 in the RAR-b promoter uponEAD treatment, while the occupancy of H3K9-Ac (a marker oftranscriptional activation; Fig. 2F, c) was increased. To confirmthe specific contribution of TopoII-b in RAR-b expression, wedepleted TopoII-b in SUM-159 cells using siRNA (Supplemen-tary Fig. S2H, a). Silencing of TopoII-b resulted in increasedexpression of RAR-b (Fig. 2G) and its target gene, CYP26A1(Supplementary Fig. S2H, b) following treatment. Collectively,these results suggested that EAD enhanced RAR-b transcriptionthrough: (i) the binding of ATRA on RAR-a/RXR heterodimers,(ii) a TopoII-b inhibitory effect of doxorubicin and entinostat,and (iii) HDAC/ NCOR inhibition by entinostat at the RAR-bpromoter (Fig. 2H).

EAD therapy induces differentiation of breast tumor–initiatingcells in vitro

We next investigated whether, besides induction of celldeath, induction of differentiation is also part of the EAD'sdrug effects and is associated with decrease in tumor volume.Retinoids effectively induce the differentiation of breasttumor–initiating cells (2, 3, 24). Because we observed higherRAR-b expression following EAD treatment, we hypothesizedthat epigenetic therapy in combination with chemotherapeuticagents may potentiate the differentiation effects of ATRA onbreast tumor–initiating cells. We saw a marked dose-responseeffect of doxorubicin (6.25–50 nmol/L) in potentiating enti-nostat-mediated (EþD) decrease in CD44þ/CD24�/Epcamþ

breast tumor–initiating cells (25) in three different TNBC celllines, MDA-MB-231 (Fig. 3A), SUM-149 (Fig. 3B), andHCC1937 (Supplementary Fig. S3A). Doxorubicin reducedtumor sphere formation in semisolid medium by 32% incomparison with entinostat or ATRA (18%), EþA (34%), EþD(85%), and EAD (90%) (Fig. 3C). Compared with EþD, EADfurther decreased (2-fold) the number of tumor-initiating cellsas shown in the tumor sphere formation assay (Fig. 3C),suggesting that EAD may institute a program of differentiationin the tumor cells.

To further investigate EAD's action,we evaluated the expressionof several breast differentiationmarkers by qRT-PCR andWesternblot analysis. In MDA-MB-231 cells, compared with entinostatalone, EAD increased mRNA expression of basal lineage markers

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such as cytokeratins CK5 and CK15 (Fig. 3D and SupplementaryFig. S3B, b), the luminal markers ER and PR (SupplementaryFig. S3B, a), and CK19 (Fig. 3D and Supplementary Fig. S3B, b).EAD also increased mRNA levels of epithelial cell–specific genessuch as claudins CLDN 1, 2, 4, and 7 (Supplementary Fig. S3B, c),

occludin (OCLN; Supplementary Fig. S3B, d), and E-cadherin(CDH1; Supplementary Fig. S3B, e), and decreased the levelof the mesenchymal marker vimentin (VIM; SupplementaryFig. S3B, d). MDA-MB-231 cells treated with entinostat aloneshowed a significant increase in the epithelial proteins, CLDN1

Figure 2.EAD potentiates expression of RAR-b and its depletion decreases drug sensitivity, and directs transcriptional regulation of RAR-b by TopoII-b. QuantitativeRT-PCR of RAR-b expression in MDA-MB-231 cells (A) and xenografts (B) treated with indicated drugs. C, RA target genes in MDA-MB-231 xenografts.D, cellular viability assay was performed in SUM-149 cells depleted of RAR-b and treated for 48 hours with vehicle and EAD combination (2.5 mmol/Lentinostat, 1 mmol/L ATRA, and 0.2 mmol/L doxorubicin). E, Western blot analysis of TopoII-a and -b (a) in MDA-MB-231 (top) or SUM-159 (bottom) for12, 24 and 48 hours, following entinostat (1.25 and 2.5 mmol/L) and TopoII-b (b) following the indicated treatments for 48 hours. b-Actin, loading control.F, diagrammatic representation of the putative RAR binding sites (RARE) in the RAR-b promoter (top). ChIP and q-PCR analysis was performed usingeither an anti–RAR-a antibody or control IgG to sites 1–3 (a), NCOR (b), and H3K9Ac (c). G, qRT-PCR analysis of RAR-b in SUM-159 cells followingTopoII-b knockdown and treatment. H, model of the proposed mechanism of RAR-b-induced expression by EAD. Student t test was performed, andthe mean (� SEM) is shown.

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Figure 3.EAD combination decreases stemness in vitro. Flow cytometry determination, in quadruplicate, of the CD44þ/CD24�/Epcamþ population in MDA-MB-231 (A) andSUM-149 (B) cells treated for 48 hours with entinostat (2.5 mmol/L), ATRA (1 mmol/L), and increasing doses of doxorubicin (6.25–50 nmol/L) either alone or incombinations. The percentage of CD44þ/CD24� cells is shown on the right. C, tumor sphere formation and quantification of MDA-MB-231, treated as above(doxorubicin, 25 nmol/L) in duplicate, for 48 hours in 2D culture, followed by 30 days of drug withdrawal in 3D. P values (�) of each group, in relation to EAD,are shown. D, quantitative RT-PCR of keratin gene expression in MDA-MB-231 treated for 48 hours with the indicated drugs (doxorubicin, 12.5 nmol/L). P values (�)of each group, in relation to entinostat, are shown. E, Western blot (left) and quantification (right) of CLDN1, occludin, and Zeb1 proteins in MDA-MB-231–treatedcells as above (doxorubicin, 12.5 and 200 nmol/L). GAPDH, loading control. Student t test was performed, and the mean (�SEM) is shown.

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andOCLN, aswell as a decrease in themesenchymal protein Zeb1(Fig. 3E and Supplementary Fig. S3C).

EAD therapy induces differentiation of breast tumor–initiatingcells in vivo

Our observations raised the possibility that EAD treatment alsotargets breast tumor–initiating cells in vivo. MDA-MB-231 xeno-grafts were excised from mice treated with the different single,double, and triple drug combinations after 4 weeks (Fig. 1B) anddigested to single cells (schema in Fig. 4A). Secondary tumortransplants were significantly smaller in the EAD-treated groupcompared with the other groups (Fig. 4B and Supplementary Fig.S4). EAD treatment significantly decreased tumor-initiating fre-quencies ofMDA-MB-231 tumor cells that were transplanted intosecondary hosts at limiting dilutions (Table 1). The frequency ofbreast tumor–initiating cells in EAD-treated tumor cells wasdetermined to be 1 in 236,570 compared with 1 in 10,818 invehicle-treated mice (Table 1, week 2.5). The EAD effect onreducing the frequency of breast tumor–initiating cells was alsoobserved in an independent replicate (Supplementary Table S6).Following EAD, EþA was the second most effective treatment for

targeting breast tumor–initiating cells in vivo (1 in 150,721; Table1). The tumor-initiating cell frequency in EAD-treated tumors wassignificantly lower than in EþA–treated tumors (P < 0.001). Thesedata support the efficacy of the EAD combination in targetingtumor initiating cells.

Interestingly, tumor cells isolated from first-generation xeno-grafts treatedwith doxorubicin as a single agent or in combinationwere arrested at the G2 phase of the cell cycle (Fig. 4C). On theother hand, tumor cells from the secondary transplants of ATRA-containing groups alone sustained arrest in the G2 phase (Fig.4D). These data strongly suggest that ATRA treatment had a long-term effect on in vivo induction of tumor cell arrest, providingevidence for ATRA's beneficial effects and contribution in thiscombination therapy.

ELF-3 mediates epigenetic therapy-induced differentiationTo gain insight into global changes brought about by the

combined therapeutic strategy, we performed gene expressionarray analysis ofMDA-MB-231 cells treatedwith entinostat, ATRA,and doxorubicin singly or in combination. To identify putativecandidates that might mediate the EAD-induced differentiation

Figure 4.EAD combination decreases stemnessin vivo. A, schematic outline of thetumorigenicity assays. B, median tumorvolume of MDA-MB-231 secondaryxenografts established with 5 � 105

cells (at week 2.5). Mann–WhitneyU test was performed, and P values (�)of each group, in relation to EAD, areshown. Flow cytometry determinationof cell-cycle distribution in TNBC cellsisolated from the first- (C) and thesecond (D)-generation (B) xenografts.The P values (�) of each group inrelation to doxorubicin are shown. NS,not statistically significant.

Table 1. EAD decreases tumor incidence at limiting dilution

Week 2.5 Vehicle Ent ATRA EþA Dox AþD EþD EAD

5 � 106 0/0 0/0 0/0 0/0 0/0 4/4 4/4 6/65 � 105 3/3 4/4 2/2 3/4 5/5 3/3 8/8 5/75 � 104 3/3 2/4 1/3 4/5 5/7 6/6 6/8 3/75 � 103 1/3 1/4 1/3 0/3 2/4 2/3 1/4 1/4SC frequency 1 in 10,818 1 in 53,192 1 in 66,908 1 in 150,721 1 in 29,438 1 in 4,548 1 in 32,989 1 in 236,570

Week 3.5 Vehicle Ent ATRA EþA Dox AþD EþD EAD5 � 106 0/0 0/0 0/0 0/0 0/0 4/4 4/4 6/65 � 105 3/3 4/4 2/2 4/4 5/5 3/3 8/8 6/75 � 104 3/3 4/4 3/3 4/4 7/7 6/6 6/8 5/75 � 103 3/3 2/4 3/3 1/3 4/4 3/3 3/4 3/4SC frequency 1 1 in 7,121 1 1 in 10,544 1 1 1 in 23,663 1 in 123,342

NOTE: Tumor incidence at week 2.5 (top) and week 3.5 (bottom) of secondary transplants of MDA-MB-231 at limiting dilutions. The table shows tumors/numbers ofmice/group. The bottom row indicates the estimated breast tumor–initiating/stem cell (SC) frequencies.

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effect, we examined genes that are differentially upregulated byEAD in comparison with EþD. Among others, we identifiedELF-3, IL1b, and DHRS3. It was reported that human embry-onic stem cells (hESC) upregulate ELF-3 and RAR-b uponretinoic acid treatment (26). Treatment of nasopharyngealsquamous cell carcinoma cells (27) and MCF7 breast cancercells (28) with retinoids also induced expression of ELF-3. qRT-PCR validation of ELF-3 and its inducer IL1b (29) in MDA-MB-231 cells treated with the different drugs showed that theirexpression was higher in EAD than in EþD–treated cells, andwas also induced by ATRA alone and EþA (Fig. 5A and Sup-plementary Fig. S5A). Also, ELF-3 mRNA was significantlyhigher in EAD treated MDA-MB-231 xenografts (Fig. 5B, a).Investigating human tissues, we found that in comparison withnormal breast epithelial organoids, primary TNBCs had signif-

icantly lower expression of ELF-3 mRNA. Further, comparedwith primary TNBC, distant metastasis showed even lowerlevels of i (Fig. 5B, b). Because metastatic tumors displayenhanced cancer stem cell properties (30), and we saw a drasticdecrease of ELF-3 in metastasis, we hypothesized that breasttumor–initiating cells will have lower expression of these genesand that our combination therapy might restore their expres-sion. We observed that differentiated CD24þ cells from breastorganoids had significantly higher levels of ELF-3 mRNA com-pared with CD44þ (Fig. 5B, c). Tumor-initiating cells isolatedbased on CD44þ/CD24�/Epcamþ cell-surface markers fromMDA-MB-231 cells (25), on the other hand, showed 20-foldlower levels of ELF-3 mRNA in comparison with CD24þ cells(Fig. 5C, a). Interestingly, EþA and EAD treatments, whichshowed more potent differentiation effects in vivo, were also the

Figure 5.ELF-3 is increased in differentiatedstem cell and mediates differentiationeffect. A, qRT-PCR of ELF-3 mRNAlevels in MDA-MB-231 treated withentinostat (2.5 mmol/L), ATRA(1 mmol/L), and doxorubicin (12.5 and200 nmol/L) combinations. B, ELF-3 intumor xenografts treated as indicated(a); normal breast ORG, primary andmetastatic samples fromTNBCpatients(b); and differentiated (CD24þ) andstem cells (CD44þ) isolated fromnormal breast organoids (c). C, ELF-3 inthe differentiated (CD44þ/CD24þ) andbreast tumor–initiating (CD44þ/CD24�) cells isolated from MDA-MB-231 cells, following DMSO treatment(a) and the fold change (in comparisonwith DMSO) following the differenttreatments (doxorubicin, 25 nmol/L; b).D and E, flow cytometry determinationof CD44þ/CD24�/Epcamþ breasttumor–initiating cells in MDA-MB-231cells transfected with scramble orELF-3 siRNA (D) or infected withlentivirus containing H163-vector orH163-ELF-3 (E) and treated with thedifferent drugs (doxorubicin,25 nmol/L). Mann–Whitney U test wasperformed, and the median of ELF-3expression in xenograft and primarysamples is shown. Student t test wasperformed, and the mean (�SEM) isshown.

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most effective in restoring ELF-3 mRNA in the breast tumor–initiating cell population (Fig. 5C, b).

Finally, to examine the contribution of ELF-3 in epigenetictreatment-mediated induction of differentiation, we studied theeffect of silencing andoverexpressing ELF-3 inMDA-MB-231 cells.We observed that depleting ELF-3 with siRNA decreased thedifferentiation effect, abrogating the decrease of CD44þ/CD24�/Epcamþ cells in entinostat-containing groups (Fig. 5Dand Supplementary Fig. S5A). On the other hand, ELF-3 over-expression enhanced the differentiation effect in entinostat-con-taining groups (Fig. 5E and Supplementary Fig. S5B), supportingthe role of ELF-3 as one of the mediators of the therapy-induceddifferentiation.

Loss of RAR-b and ELF-3 is associated with aggressive breastcancer

Wenext investigated if there is a correlation betweenRAR-b andELF-3 gene expression with survival in patients with breast cancer.Higher expression of RAR-b correlated with increased recurrence-free survival in patients with tumors of all subtypes (Fig. 6A).Upon examination of stem cell–specific gene expression data sets,we found that ELF-3 expression was significantly lower in thebreast cancer stem cell population (31, 32). Interestingly, tumorsin patients enriched for themesenchymal stem-like TNBC subtype(33) also expressed significantly lower levels of ELF-3 (Fig. 6B).

EAD targets metastatic cells in patient samplesFinally, to validate our findings in patient-derived samples,

we tested the effect of the novel combination therapy onpatient-derived breast metastatic cells. Pleural effusion sampleswere collected from patients with treatment-refractory breastcancer (Supplementary Table S7), grown as tumor spheres, andtreated with the various drugs alone and in combination. EADtreatment tends to be the most effective in decreasing thenumber of tumor spheres compared with vehicle control,DMSO (Fig. 6C and Supplementary Fig. S6A). Entinostat treat-ment showed the strongest long-term suppressive effect onsphere formation, and cells exposed to entinostat-containingcombinations did not form spheres even at first passage (P1;Supplementary Fig S6A). Moreover, ELF-3 mRNA expressionwas induced in metastatic cells treated with EþA and EAD(Fig. 6D and Supplementary Fig. S6B), providing additionalevidence from patient-derived tumor cells that ELF-3 could beinvolved in the killing effect of EAD.

Thus, as shown in the schema in Fig. 6E, our comprehensivemolecular and phenotypic analysis of the effect of entinostat,ATRA, and doxorubicin as single and combined agents revealedthat entinostat and doxorubicin targeted TopoII-b and togetherwith ATRA reactivated RAR-b gene transcription. The addition ofATRA to entinostat and doxorubicin (EAD) induced breast cancercell death and tumor-initiating cell differentiation, contributingto a significant reduction in tumor size.

DiscussionCombining HDAC inhibitors with either doxorubicin (34) or

retinoids (12) has been shown to result in increased cancer cellcytotoxicity. HDAC inhibitors also potentiate retinoid effects onRAR-b expression (18) and thereby could overcome retinoidresistance attributable to an epigenetically silenced receptor.Here,we report that a combination of the HDAC inhibitor entinostat, a

retinoid, ATRA, and doxorubicin (EAD) resulted in the greatestinhibition of xenografts of TNBC cells, and provide further insightinto the mechanism of their action alone and in combination ontumor regression.

The increase in cancer cell cytotoxicity by EAD was partiallymediated by the induction of RAR-b and expression of its targetgenes. The highest induction of RAR-b expression and inhibi-tion of cancer cell growth was observed mainly in the presenceof doxorubicin in the combination therapy, and less so withother chemotherapeutic agents. Because doxorubicin is aninhibitor of TopoII-b, we addressed the role of TopoII-b in theEAD effect. We found that in TNBC cells, both doxorubicin andentinostat caused degradation of TopoII-b. Previously, a directbinding of TopoII-b to RAR-b promoter was reported in APLcells (23). Here we show, for the first time, evidence for thebinding of TopoII-b to the RAR-b promoter and inhibition of itstranscription in TNBC cells. EAD treatment effectivelydecreased occupancy of TopoII-b and NCOR and increasedbinding of H3K9-Ac to the RAR-b promoter, thereby possiblyincreasing RAR-b expression.

Conclusions on the effect of retinoids on tumor initiatingcells are mainly based on observations in cell culture (2, 3).Recently, an HDAC inhibitor, abexinostat, was shown to inducetumor-initiating cell differentiation (35). In our study, ATRAand entinostat as single agents had comparable effects on thedecrease of TNBC-initiating cells in vivo, and were superior todoxorubicin. Most importantly, the combination of EþA wasmore effective in targeting breast tumor-initiating cells thansingle agents. Further, the addition of doxorubicin to EþA(EAD) achieved the optimal combination in targeting breasttumor-initiating cells both in vitro and in vivo. Thus, EAD's effecton breast tumor–initiating cell differentiation is likely to haveprofound and lasting effects on the decrease of tumor size andrecurrence.

In addition to the role of RA/RAR-b in cancer stem cell differ-entiation (2, 5), we identified ELF-3 as a novel mediator ofthe differentiation effect of epigenetic therapy. E74-like factor 3(ELF-3) belongs to the E26 transformation–specific family oftranscription factors and is known to regulate epithelial celldifferentiation (36). Interestingly, we found that the ELF-3mRNAlevel was lower in the progenitor cell populations from normalbreast and tumors and was restored mainly following EþA andEAD. The increase in ELF-3 levels in the breast tumor–initiatingcells together with the functional assays suggests that ELF-3 is, inpart, a mediator of EAD's effects on differentiation.

We observed that high levels of RAR-b expression in tumorscorrelated with a better outcome in patients with breast cancer,suggesting that they may benefit from the combination therapy.Also, low ELF-3 expression was observed in tumor-initiating cellsisolated from patients after neoadjuvant chemotherapy or hor-mone therapy (32), and with the claudin-low breast tumorsubtype, known to be enriched in tumor-initiating cell features(31). We showed that EAD and entinostat targeted the self-renewal ability of metastatic ascites cells, supporting the possi-bility of developing alternative treatments to target treatment-refractory disease.

In summary, using a number of culture and xenograft modelsystems and primary and metastatic samples from patients, wehave demonstrated the effectiveness of cooperative targeting ofbothdifferentiated and tumor-initiating cell populations inbreastcancer using a combination of DNA damaging agents, epigenetic

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Figure 6.RAR-b and ELF-3 are associated with better outcome of breast cancer patients, and EAD targets metastatic samples. A, overall survival Kaplan–Meier curvesof 633 patients from The Cancer Genome Atlas breast cancer cohort (all subtypes), separated by median RAR-b expression. B, ELF-3 log2 expression inLehmann and colleagues (33) patients (n¼ 225) of different TNBC subtypes, including twobasal-like (BL1 andBL2), an immunomodulatory (IM), amesenchymal (M),a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. A Welch modified t test was used. C, pleural ascites cells from six metastaticbreast cancer patients (TNBC; a) grown in 3D low adhesion cultures for 15 days, with combination treatments of entinostat (2.5 mmol/L), ATRA (1.0 mmol/L),and doxorubicin (25 nmol/L), and quantification (b). P0, cell passage as mammospheres. Not all treatments could be tested in 2 of 6 patient samples. For PE#6,a composite figure, all at the same magnification, is shown because tumor spheres were scattered. D, qRT-PCR of ELF-3 expression in the metastatic patientsamples following combination therapies. E, summary of findings on the effect of EAD combination therapy in TNBCs. Student t test was performed and themean (� SEM) is shown.

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and differentiation agents to achieve an optimal therapeuticoutcome.

Disclosure of Potential Conflicts of InterestV. Stearns reports receiving commercial research grant from Abbvie, Celgene,

Pfizer, Novartis, Puma, Merck, and Medimmune. P. Ordentlich has ownershipinterest (including patents) in Syndax Pharmaceuticals. Nopotential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: V.F. Merino, N. Nguyen, L. Han, R.M. Connolly,S. SukumarDevelopment of methodology: V.F. Merino, N. Nguyen, K. Jin, S.Z. Ali,Q. Chen, D. PanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V.F. Merino, N. Nguyen, H. Sadik, P. Korangath,C. Adams, Q. Chen, D.L. Huso, A. Brodie, S. SukumarAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): V.F. Merino, N. Nguyen, H. Sadik, S. Cho, X.C. Zhou,Z. Zhang, R.M. Connolly, V. Stearns, S.Z. Ali, Q. Chen, D.L. Huso, P. Ordentlich,A. Brodie, S. Sukumar

Writing, review, and/or revision of the manuscript: V.F. Merino, N. Nguyen,Z. Zhang, R.M.Connolly, V. Stearns, S.Z. Ali, D.L.Huso, P.Ordentlich, A. Brodie,S. SukumarAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): V.F. Merino, N. Nguyen, D. PanStudy supervision: V.F. Merino, S. SukumarOther (helped in preparing samples and reagents andperformedpreliminaryepigenetic experiments to inform the study): Y.M.N. Foster

AcknowledgmentsThe authors thank Dr. Unutmaz for providing H163 cells and Dr. Kumar for

the pcDNA-ELF-3 construct.

Grant SupportThis work was funded by the DOD BCRP Center of Excellence Grant

W81XWH-04-1-0595 to S. Sukumar, and DOD BCRP, W81XWH-09-1-0499to V.F. Merino, and the SKCCC Core grant P30 CA006973.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 16, 2015; revised December 21, 2015; accepted January 11,2016; published OnlineFirst January 19, 2016.

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