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Tumor and Stem Cell Biology

Aspirin Suppresses the Acquisition ofChemoresistance in Breast Cancer by Disruptingan NFkB–IL6 Signaling Axis Responsible for theGeneration of Cancer Stem CellsShilpi Saha1, Shravanti Mukherjee1, Poulami Khan1, Kirti Kajal1,Minakshi Mazumdar1, Argha Manna1, Sanhita Mukherjee2, Sunanda De3,Debarshi Jana3, Diptendra K. Sarkar3, and Tanya Das1

Abstract

Acquired chemoresistance has curtailed cancer survival sincethe dawn of chemotherapy. Accumulating evidence suggests amajor role for cancer stem cells (CSC) in chemoresistance,although their involvement in acquired resistance is stillunknown. The use of aspirin has been associated with reducedcancer risk and recurrence, suggesting that the anti-inflammatorydrug may exert effects on CSCs. In this study, we investigated thecontributionofCSCs to acquired chemoresistance of breast cancerand the avenues for reversing such effects with aspirin. Weobserved that the residual risk of recurrence was higher in breastcancer patients who had acquired chemoresistance. Treatment ofpreexisting CSCs with a genotoxic drug combination (5-fluoro-

uracil, doxorubicin, and cyclophosphamide) generated anNFkB–IL6–dependent inflammatory environment that imparted stem-ness to nonstem cancer cells, induced multidrug resistance, andenhanced themigration potential of CSCs. Treatmentwith aspirinprior to chemotherapy suppressed the acquisition of chemore-sistance by perturbing the nuclear translocation of NFkB inpreexisting CSCs. Therefore, disruptions to the NFkB–IL6 feed-back loop prevented CSC induction and sensitized preexistingCSCs to chemotherapy. Collectively, our findings suggest thatcombining aspirin and conventional chemotherapy may offer anew treatment strategy to improve recurrence-free survival ofbreast cancer patients. Cancer Res; 76(7); 2000–12. �2016 AACR.

IntroductionAlthough the advent of highly targeted therapeutics based on

small-molecule inhibitors andmAbs has revolutionized the treat-ment of some cancers, chemotherapy remains the mainstaytreatment for most tumors, especially for locally advanced breastcancer (LABC; ref. 1). Although a majority of LABC patientstreated with neoadjuvant chemotherapy (NACT) experience areduction in tumor size (1), a substantial number either exhibitssignificant intrinsic resistance or acquire characteristics of multi-drug resistance during chemotherapy, i.e., acquired resistance (2,3), thereby limiting the efficacy of chemotherapeutic regimens.

Several hypotheses have been proposed to explain this treat-ment failure and recurrence (4). In particular, it has been sug-gested that a small subpopulation of cells within tumors, called

cancer stem cells (CSCs), by virtue of their relative resistance tochemotherapy may contribute to tumor recurrence (5, 6). Con-sistent with these reports, several groups reported enrichment forCSCs when solid cancers were subjected to classical anticancertreatment (7–11). In accumulation, these studies suggest thatsignificant improvement in patient outcome will require drugsthat selectively target CSCs in combination with conventionalchemotherapeutic regimens that target rapidly proliferating cells.

Epidemiologic and clinical studies indicate that inflammationis correlated with an increased risk of recurrence in breast cancerpatients (12), and NFkB plays a causative role in inflammation(13). Recent literature highlights the importance of NFkB-con-trolled family of proinflammatory cytokines, significantly IL6, inCSC maintenance (14) and in regulating plasticity of neoplasticcells (15), thereby suggesting the possible involvement of NFkB–IL6 pathway in aggressive disease recurrence following NACT.

Interestingly,multiplemeta-analyses showed that patientswithcardiovascular diseases treated with FDA-approved anti-inflam-matory drug, aspirin, have reduced cancer risk (16, 17). Recentepidemiologic studies have found that women who took aspirinafter a diagnosis of breast cancer had a better prognosis thanwomen who did not take aspirin, indicating that aspirin wasassociated with a decreased risk of distant recurrence (18),although the underlying mechanism is still unclear. Consideringall this information, we envisaged the role of aspirin in improvingthe response of breast tumors to chemotherapeutic regimen tofinally improve recurrence-free survival of patients. We thereforeinvestigated whether aspirin could suppress acquisition of

1Division of Molecular Medicine, Bose Institute, Kolkata,West Bengal,India. 2Department of Physiology, Bankura Sammilani Medical Col-lege, Kenduadihi, Bankura, West Bengal, India. 3Department of Sur-gery, Seth Sukhlal Karnani Memorial Hospital, Kolkata, West Bengal,India.

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

Corresponding Author: Tanya Das, Bose Institute, P-1/12 CIT Scheme VII M,Kankurgachi, Kolkata, West Bengal 700054, India. Phone: 9133-2569-3257; Fax:9133-2355-3886; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1360

�2016 American Association for Cancer Research.

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resistance during standard chemotherapy by targeting CSC pool,which would be expected to diminish disease progression andconsequently improve overall and disease-free survival (DFS) ofLABC patients.

Materials and MethodsPreclinical study designRetrospective cohort observational study. A total of 203 patients,enrolled for treatment in either Bankura Sammilani MedicalCollege (BSMC; Bankura, West Bengal, India) or ComprehensiveBreast Service Clinic in IPGMER and SSKM Hospital, (Kolkata,West Bengal, India) between January 2010 and December 2013,with histologically or cytologically confirmed breast cancer recur-rence were retrospectively reviewed for their medical recordsand radiologic images to determine the DFS period and theirresponse to chemotherapy cycles. Patients havinghistologically orcytologically documented LABC characteristics, that is, primarytumors>5 cm,with skin or chest wall involvement orwithmattedaxillary adenopathy at the time of the first presentation of diseaseand grade III according to American Joint Committee on Cancer(AJCC) criteria (19) and were prescribed neoadjuvant 5-fluoro-uracil 500 mg/m2, doxorubicin 50 mg/m2, and cyclophospha-mide 500 mg/m2 per cycle (FAC) chemotherapeutic regimen,were eligible for this study, whereas previous solid tumors, age <18 years, distant recurrences, and neoadjuvant chemotherapydropouts were considered exclusion criteria of the study.

Prospective cohort experimental study. Fifty patients with histolog-ically or cytologically documented LABC characteristics, whoattended above-mentioned hospitals from January 2009 toDecember 2010, were assessed for their pathologic (20) andclinical (21) responses toward neoadjuvant FAC treatment. Onthe basis of their responses during the course of chemotherapy,patients were categorized into chemotherapy-sensitive (CS),acquired chemoresistant (CR), and inherent resistance group.Response of primary cells to treatments was also determined onthe basis of their estrogen receptor (ER), progesterone receptor(PR), andHER2 status. Tumor characteristics, that is, clinical (age,tumor, and node), histopathologic (grade, ER, PR, and HER2status, ductal, lobular, and invasive), andmolecular [luminal andbasal, by assessing their ER/PR status (22, 23)] characteristics ofeach patient were collected from the pathology departmentsof above-mentioned hospitals and tabulated for reference inSupplementary Table S1A. Patients with inherent chemotherapyresistance, previous solid tumors, <18 years, and breast cancerrecurrences or contralateral tumors were excluded from the study.All LABC patients were treated with FAC regime with a gap of 21days between two cycles, followed by surgery. After the comple-tion of treatment, patients were followed up monthly for firstthree months and then once in four months for a total period ofthree years to determine their "clinical outcome." DFS period wascalculated from the date of surgery to the date of recurrence ofdisease.

Ex vivo study. Patients having histologically or cytologically docu-mented LABC characteristics, and of grade III according to AJCCcriteria (19), luminal histotype, i.e., ERþ/PRþ and either negative(luminal A subtype) or positive (luminal B subtype) HER2 status(Supplementary Fig. S1A), and those not been treated withchemotherapy or radiotherapy were eligible for this study.

Selected cases consisted of 30 primary breast cancer patients. Basalhistotype breast cancers were excluded from the study as basalsubtypes displayed "inherent" resistance to chemotherapy (Sup-plementary Table S1A; ref. 24). Tumor characteristics (clinical andhistopathologic) were available and collected retrospectively fromthepathology departmentsof above-mentionedhospitals. Further-more, the response of primary cells to in vitro treatments was alsodetermined on the basis of their ER/PR/HER2 status. All thisinformation has been tabulated in Supplementary Table S1B.

All these studies were planned and approved by the HumanResearch Ethics Committees of BSMC, Bankura, India (approvalnumber: CNMC/ETHI/162/P) and IPGMER and SSKM Hospital(approval number: Inst/IEC/308), Kolkata, and associatedresearch and analyses were done in Bose Institute (Kolkata, India)in accordance with the Institutional Human Ethics Committee(approval number: BIHEC/2010-11/11). Written informed con-sent was obtained from all patients.

Primary human tissue cultureFor prospective cohort experimental study, ultrasound-guided

core biopsy samples were obtained at baseline before any che-motherapy, and thereafter, upon completion of treatment, sur-gically resected breast cancer tissue, tumor marginal tissue, andnormal breast tissue samples were obtained. For ex vivo preclinicalstudy, untreatedprimary humanbreast cancer tissue sampleswereprocured. All samples were mechanically disaggregated withcollagenase (25). Tumor cells were sorted twice after stainingwith APC-anti-CD44 and PE-anti-CD24. Nontumor cells weredepleted with FITC-tagged cocktail of lineage marker antibodies,and the purity was verified flow cytometrically (SupplementaryFig. S1B). Dilutions of antibodies are listed in SupplementaryTable S2.

Cell lines and cell cultureHuman breast cancer cell lines, MCF-7, T47D, and ZR-75-1

were obtained fromNationalCentre forCell Science (Pune, India)between 2012 and 2015, andMDA-MB-361 and BT-474, receivedin 2015, were kind gifts from Dr. A. Mal, Roswell Park CancerInstitute (Buffalo, NY). All cell lines were authenticated by shorttandem repeat analysis and each was passaged for fewer than 6months after resuscitation. Cells were routinely maintained inDMEM as described previously (26).

Antibodies and treatmentsCells were treated with chemotherapeutic regimen FAC (MP

Biomedicals) in 10:1:10 ratios and with 1 to 5 mmol/L aspirin(MP Biomedicals) for specific experiments. Because aspirin issparingly soluble in distilled water (27), the 1 mol/L stocksolutionwas prepared inDMSO. For all in vitro assayswith aspirin,DMSO was used as control. In selected experiments, cells wereincubated with IL6-neutralizing antibody (1.5 mg/mL), recombi-nant IL6 (rIL6, 1.2 ng/mL), Brefeldin A (1 mg/mL), PMA (500 ng/mL), ionomycin (500ng/mL; BDBiosciences), SN50 (18mmol/L)or MG132 (10 mmol/L; Calbiochem), and mitomycin c (10 mg/mL, MP Biomedicals).

In vitro tumorigenicity assay and mammosphere cultureDifferent breast cancer cells (2.5� 104 cells/well) were cultured

and propagated in suspension culture as described previously(28). Mammosphere-forming efficiency was determined by

Aspirin Sensitizes Breast Cancer Stem Cells

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counting the number of mammospheres (average diameter, 100mm) under a light microscope at 10�magnification and reportedas the number of mammospheres divided by the number of cellsseeded and expressed as percentage.

Flow cytometryHuman breast CSCmarkers, chemoresistance, EMT, and dedif-

ferentiation phenomena were analyzed flow cytometrically(FACSVerse, Becton Dickinson; ref. 29). Percent apoptosis ofCSCs/nonstem cancer cells (NSCC) were determined (26). Forassaying intracellular IL6, IL6-PE was used (30). Dilutions ofantibodies are listed in Supplementary Table S2. Positive stainingwas considered based on the negativity of an isotype control. Aminimum of 10,000 events were recorded for all samples. Mostphenotypic data were validated using the same antibodies withalternate labels.

Plasmid transfectionsCSCs were transfected separately with 300 pmol of IL6 shRNA

(Addgene). The pcDNA3.1 plasmid encoding IkBa-32A/36AcDNA (IkBa super-repressor, IkBa-SR; kind gift from Dr. J.Didonato, The Cleveland Clinic (Cleveland, OH) was transfectedin sorted CSCs for overexpression studies, and pd2EGFP-N1vector (Clontech) was used for labeling sorted NSCCs (29).

Confocal microscopyConfocalmicroscopy for the visualization ofNFkBwas done as

previously described (31).

Transwell migration assayCells (2.5 � 104) were seeded into 24-well cell culture inserts

with 8 mmpores (BD Falcon), andmigration of cells was recordedafter 12 hours (29).

ChIP assayChromatin immunoprecipitation (ChIP) assays were per-

formed using a ChIP Assay Kit (Millipore) according to themanufacturer's instructions. PCR assay for identification ofNFkB-binding region on ABCG2 and ABCC1 promoters wasperformed using specific primer sets. Primers are listed in Sup-plementary Table S2. The PCR products were analyzed by 2%agarose gel electrophoresis (32, 33).

Statistical analysisValues are shown as SEMs except otherwise indicated. Com-

parison of multiple experimental groups was performed by two-way ANOVA test. Kaplan–Meier method was used to plot DFScurves, and log-rank test was used to estimate the statisticaldifference between groups. Data were analyzed, and when appro-priate, significance of the differences between mean values wasdetermined by a Student t test. Results were considered significantat P � 0.05.

Other standard reagents and methods are provided in Supple-mentary Experimental Procedures.

ResultsResidual risk of recurrence was higher in LABC patients withacquired chemoresistance

Analysis of retrospective cohort observational study revealedthat the number of chemoresistant patients (n ¼ 153) with

recurrence of disease was higher than chemosensitive patients(n ¼ 50; Fig. 1A). Further scrutiny of the analysis revealed a closeassociation between locoregional recurrences (LCR) of diseasewith acquired resistance to therapy (Supplementary Fig. S2A).

These indications were confirmed by prospective cohort exper-imental study. According to clinical and pathologic responseguidelines (20, 21), 11 cases were categorized in acquired CR-group (CR-primary tumor), whereas 29 cases were categorized inCS-group (CS-primary tumor; Supplementary Table S1A). Inher-ently, CR patients were excluded from this study. Acquisition ofresistance by cells of CR-primary tissue was validated by theirhigher expression levels of chemoresistant markers, ABCG2 andABCC1, followingNACTwhen comparedwith cells ofCS-primarytissue (Supplementary Fig. S2B). The analysis of tumormolecularcharacteristics showed that all CS- andCR-primary tumorswere ofluminal histotype, that is, ERþ/PRþ, whereas inherently CRpatients were of basal histotype, i.e., ER�/PR� and were excludedfrom this study. Luminal primary tumorswere of two subtypes: (i)ERþ/PRþ/HER2� luminal A histotype and (ii) ERþ/PRþ/HER2þ

luminal B histotype. For this study, primary breast cancer tissues/cells of luminal histotype were included as a broad category,although the presence or absence of ER/PR and HER2 was pre-dictive of the response of tumor to chemotherapy (Supplemen-tary Table S1A). Follow-up studies further validated the relationbetween poor therapeutic response and LCR as confirmed byKaplan–Meier survival analysis (Fig. 1B). These findings con-firmed that the residual risk of breast cancer recurrence was higherin patients who acquire resistance with treatment as comparedwith CS patients.

Chemotherapy increases CSC pool in LABC patients withacquired chemoresistance

Next,we investigated theeffectof chemotherapyonCSCcontentofmammary epithelial tumors.Higher percentCSC content ofCR-tumors was detected as compared with CS-tumors (Fig. 1C).Moreover, increase in percent CSCs with chemotherapy wasobservedwhen paired specimens fromCRpatients, obtained priorto chemotherapy and at surgery following chemotherapy, werecompared (Fig. 1D and Supplementary Fig. S2C). Increase in theexpression levels of other CSC-relatedmarkers, Oct-4, Nanog, Sox-2, and Aldh1 in chemotherapy-treated CR-primary tumor sampleswhen compared with matched untreated samples, supportedabove-mentioned findings (Fig. 1E and Supplementary Fig. S2D).

In vitro validation of chemotherapy-mediated increase in CSCcontent

For in vitro validation of above findings, breast cancer cell lines,MCF-7, T47D, ZR-75-1, MDA-MB-361, and BT-474, mimickingthe characteristics of the primary cell lines, i.e., luminal histotype,were included in the study. Among these, MCF-7, T47D, and ZR-75-1 were ERþ/PRþ/HER2�, whereas MDA-MB-361 and BT-474were ERþ/PRþ/HER2þ. Percent CSCs, expression levels of CSC-related markers, and mammosphere-forming efficiency (Fig. 1F,Supplementary Fig. S2E) increased when these cells were treatedwith chemotherapeutic regimen FAC as administrated to thepatients included in the study (Supplementary Fig. S3A). Further-more, FAC treatment selectively killedNSCCs as opposed to CSCs(Fig 1G, top). These findings supported our postulation thatposttreatment increase in CSC repertoire can be "apparent" dueto relative decrease in NSCCs. Revalidating analysis, showingincrease in CSC number upon FAC treatment, ruled out the

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possibility that such increase in CSC repertoire was only due toselection of "preexisting" CSCs (Fig. 1G, bottom).

Therapy-generated CSC pool displays enhancedchemoresistance and migration potential

Next, CSCs purified from FAC-treated CR-primary tumors(Fig. 2A, right and Supplementary Fig. S3B) or MCF-7 cells(Supplementary Fig. S3D) demonstrated increase in chemore-

sistance markers when compared with CSCs from theiruntreated counterparts. However, CSCs from FAC-treatedCS-primary tumors failed to demonstrate such increase inchemoresistant markers (Fig. 2A, left). In addition, relativeincrease in migration property and associated markers con-firmed enhanced migration potential of FAC-treated CSCs ofprimary CR-tumors (Fig. 2B and Supplementary Fig. S3C) andMCF-7 cells (Supplementary Fig. S3E). Therefore, besides

Figure 1.Chemotherapy augments CSCs. A, during 2010–2013, patients with recurrence of disease were categorized as CS CR and represented graphically. B, graphicalrepresentation showing percentage of CS- and acquired CR-patients with LCR (left) and Kaplan–Meir survival analysis plot displaying DFS of these patients(right). C, percent CSCs in CS- and CR-primary tumors is represented as dot plots (left, each dot represents individual patient) and bar plots (right).D, representative flow cytometric density plots showing percent CSCs in pre and posttreatment primary tumors of CR-patients (left) and was represented as dotplot and bar plot (right). Baseline core biopsies from two patients containing inadequate tissue were excluded, leaving a total of 9 informative subjects. E, foldchange in the relative mean fluorescence intensities (in arbitrary units) of stemness markers in pre and posttreatment primary tumors of CR-patients is representedgraphically. F, flow cytometric determination of percent CSCs in pre and posttreatment breast cancer cells (top left) and mammosphere-forming efficiency ofthese cells (top right). Fold change in relative mean fluorescence intensities of stemnessmarkers (bottom) in pre and posttreatment cells. G, percent 7AAD-positiveNSCCs and CSCs of pre and posttreatment primary tumors of CR-patients (top left) and MCF-7 cells (top right). Changes in the number of NSCCs and CSCsin MCF-7 cells upon FAC treatment was determined by employing Trypan blue dye exclusion and flow cytometric assays (bottom). Data are presented asmean � SEM or representative of three independent experiments. � , P < 0.05; �� , P < 0.01; and �� , P < 0.001.






increasing CSC proportion, chemotherapy also enhanced theirchemoresistance and metastatic potential, thereby promotingthe chances of recurrence in CR-patients when compared withCS-patients.

Therapy-generated aggressive CSC pool enhances the risk ofLCR

Higher expression of CSC markers was observed in cells iso-lated from tumor marginal tissue (Fig. 2C, left) and normal

Figure 2.Therapy-generated aggressive CSC pool enhances risk of LCR in CR-breast cancer patients. Relative mean fluorescence intensities of ABCG2 and ABCC1 (A) inCSCs isolated from pre and posttreatment primary tumors of CS- (left) and CR-patients (right) and E-cadherin and vimentin (B) in CSCs isolated from preand posttreatment primary tumors of CR-patients (left). Graphical representation of migration of these cells in transwell migration assay (right). C, representativeflow cytometric density plots showing percent CSCs in tumor surgical margins (left) and normal breast tissue (right) of CS- and CR-patients are representedas dot plots and bar plots (bottom). Middle, illustration depicting tumor surgical margins and normal breast tissue. D, ratio of percent CSCs in normal breasttissue and primary tumor of CS- and CR-patients is represented graphically as dot plot (left) and as bar plot (right). E, relative mean fluorescence intensities (MFI) ofABCG2 and ABCC1 in normal breast tissue of CS- and CR-patients (left). Graphical representation of migration of CSCs purified from normal breast tissue ofCS- and CR-patients (right). Data are presented as mean � SEM or representative of three independent experiments. � , P < 0.05; �� , P < 0.01; and�� , P < 0.001. AU, arbitrary units.

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mammary tissue (Fig. 2C, right) of FAC-treated CR-patients ascompared with CS-patients. Such increase in CSCs might be dueto relative increase in CSC number in CR-primary tissue. How-ever, the fold change (normal to tumor) in CSC numbers, beingsignificantly higher in CR-set than in CS-set (Fig. 2D), nullifiedthis apprehension. CSCs isolated from normal tissue of CR-primary patients demonstrated enhanced expression of chemore-sistance markers and migration potential (Fig. 2E and Supple-mentary Fig. S3F) when compared with that of CS-patients. Thesefindings are in accordance with the recent reports that followingchemotherapy, the proportion of disseminated CSCs with mes-enchymal phenotype increases in the blood of patients (34, 35).

These findings indicate the possibility that aggressive CSCshiding in distant anatomic sites even after resection of primarytumor might play a role in disease recurrence at the local site inCR-patients (Fig. 1B).

Therapy induces expression of CSC phenotypic markers inNSCCs

FAC-induced increase inCSCsmight bedue to(i) proliferationofpreexisting CSC pool or/and (ii) conversion of NSCCs into a stem-like state. However, proliferation blocker mitomycin C failing tosignificantly inhibit FAC-induced increase in CSCs and the expres-sion of CSC-specific markers (Fig. 3A and Supplementary Fig. S4A)highlighted the second possibility. Interestingly, irrespective of thepresence or absence of mitomycin C, FAC treatment failed tosignificantly increase the number of purified CSCs (Fig. 3B). Thesefindings confirmed that induction of stemness in NSCCs was amajor cause underlying FAC-induced CSC expansion, although theinput of CSC proliferation was not completely negated.

Therapy-mediated induction of CSC phenotype in NSCCs isdriven by preexisting CSCs

In paradox with our previous findings, no significant inductionof CSCmarkers (Fig. 3C), chemoresistance, andmigration relatedmarkers (Fig. 3D and Supplementary Fig. S4B) was observed inFAC-treated NSCCs from pretreatment CR-primary tumors andMCF-7 cells (Supplementary Fig. S4C–S4E). Treatment-mediatedinduction of stem-like signature in NSCCs might thus be influ-enced by preexisting CSCs. To validate our hypothesis, (i) purifiedNSCCs were GFP-tagged and thereafter cocultured with untaggedCSCs (1:1) and (ii) CSCs and NSCCs (1:1) purified from MCF-7cells were cocultured in contact-independent manner. Appear-ance of GFP-CSCs in the mixed population postchemotherapy(Fig. 3E) confirmed the contribution of preexisting CSCs inNSCC-to-CSC conversion. Also, several fold increase in FAC-mediated CSC induction was observed in contact-independentNSCCs as compared with that in FAC-treated NSCCs (Fig. 3F).Increase in the expression levels of other CSC-specific markers(Fig. 3F and Supplementary Fig. S5A) only in NSCCs coculturedwith CSCs further validated preexisting CSC-mediated inductionof CSC phenotype in NSCCs.

Therapy triggers NFkB-dependent IL6 inflammatory feedbackloop in preexisting CSCs

Aforementioned results raised the possibility of the involve-ment of CSC-derived soluble factors in NSCC to CSC conversion.Recent reports have demonstrated NFkB-dependent IL6 activa-tion (13, 14) and the role of IL6 in inducible transition of NSCCsto CSCs (15). Our results showed FAC-induced significantincrease in nuclear NFkB in CR-primary tumors (Fig. 4A) and in

different breast cancer cells (Supplementary Fig. S5B). However,FAC-treated CS-primary tumors failed to demonstrate suchincrease (Fig. 4A). Interestingly, CSCs of MCF-7 cells displayedincreased levels of nuclear NFkB (Fig. 4Band Supplementary Fig.S5C) and its downstream inflammatory and antiapoptotic reg-ulators, Cox-2, Bcl-2, and XIAP (Fig. 4C), when compared withtheir corresponding NSCCs, and FAC treatment further increasedthe expressions. Posttreatment CR-primary tumors, but not CS-primary tumors, also demonstrated increase in IL6 mRNA whencompared with pretreatment ones (Fig. 4D). Time-dependentstimulation of intracellular IL6 was also observed in MCF-7 cellsupon FAC treatment (Fig. 4E, left) and also in different breastcancer cells upon 3 hours of FAC treatment (Fig. 4E, right). IL6secretion was higher from CSCs than NSCCs of MCF-7 cells andincreased further with FAC treatment (Fig. 4F). That FAC-medi-ated increase in CSC-shed IL6 was NFkB-dependent was con-firmed when blocking NFkB nuclear activity in CSCs significantlydecreased IL6 secretion (Fig. 4G, left). Interestingly, although IL6inhibitions failed to retain NFkB in nucleus in preexisting CSCseven upon FAC treatment, exposing CSCs to rIL6(1.2 ng/mL)enhanced the same (Fig. 4G, middle and right), confirming thatFAC treatment activated an NFkB–IL6–positive inflammatoryfeedback loop in preexisting CSCs.

Preexisting CSCs promote NSCC-to-CSC conversion in aparacrine manner

Next, NSCCs of MCF-7 cells were (i) cocultured with condi-tioned media (CM) of untreated/FAC-treated purified CSCs, or(ii) treated with rIL6 (Fig. 5A, left). After 72 hours, CM of FAC-treated CSCs or rIL6 was found to induce CD44þ/CD24�/low

phenotype in NSCCs of MCF-7 cells, while reversing such con-ditions resulted in significant inhibition, although not complete,in inducing CSC signature inNSCCs (Fig. 5A,middle). Increase inIL6 in the CM of CSCs also increased ABCG2 level in newlygenerated CSCs (Fig. 5A, right).

Therefore, FAC-triggered robust positive inflammatory feed-back loop in preexisting CSCs significantly contributed in thetransition of NSCCs-to-CSCs as well as in their attainment ofaggressive phenotype, although the involvement of other solublefactors could not be ruled out.

Aspirin inhibits chemotherapy-mediated de novo generation ofaggressive CSC pool

Next, preexisting CSCs, purified fromMCF-7 cells, were treatedwith plasma achievable dose range (1-5 mmol/L; ref. 36) ofaspirin. Results of Fig. 5B and C and Supplementary Fig. S6Adepicted that 2.5 mmol/L dose of aspirin efficiently downregu-lated nuclear NFkB as well as Bcl-2, and Cox2, in CSCs bydecreasing IkBa phosphorylation level and disrupting NFkB–IL6inflammatory signaling (Fig. 5D). Incubation of NSCCs with CMof CSCs treated with aspirin prior to FAC treatment decreased theinduction of CSC phenotype (Fig. 5E, left) and inhibited theupregulation of chemoresistance markers in newly generatedCSCs (Fig. 5F). In contrast, CM of aspirin-treated (i) NFkB-over-expressed or (ii) rIL6-exposed CSCs failed to resist in inducingCSC phenotype in NSCCs (Fig. 5E, left). Aspirin pretreatment wasalso effective when NSCCs were cocultured with CSCs, as evidentby decrease in GFP-CSCs in the mixed population postche-motherapy in comparison with FAC-treated sets (Fig. 5E, right).

In accumulation, pretreatment of aspirin disrupted NFkB–IL6inflammatory feedback loop in preexisting CSCs, thereby






hindering the expansion of chemotherapy-mediated de novo gen-eration of aggressive CSC pool.

Aspirin pretreatment sensitizes preexisting CSCsRecent report (37) demonstrating that aspirin at its 2.5mmol/L

(physiologically achievable) dose, fails to induce apoptosis inCSCs, prompted us to explore the possibility of its sensitizingCSCs towards chemotherapy. For this ex vivo study, 30 primary

human cell lines of luminal histotype, comprising of luminal Aand luminal B subtype cells (Supplementary Fig. S1A), derivedfrom untreated patients were included (Supplementary TableS1B). After assessing the in vitro expression levels of chemoresis-tant markers (Supplementary Fig. S6B), and response to in vitrocombinatorial therapy, 11 were categorized as CR and 19 as CS.ERþ/PRþ/HER2þ status was often predictive of poor response toin vitro chemotherapy, whereas ERþ/PRþ/HER2� status showed

Figure 3.Pre-existing CSCs drive therapy-mediated induction of CSC phenotype in NSCCs. A, flow cytometric analysis of percent CSCs (left), relative meanfluorescence intensities (MFI) of Aldh1 (middle), and Oct-4 (right) in MCF-7 cells under FAC/mitomycin c/mitomycin c–exposed FAC-treated conditions. B, changesin the number of CSCs purified from MCF-7 cells under the specified experimental conditions were determined by employing Trypan blue dye exclusion andflow cytometric assays. C, schematic representation of the experimental protocol (left). Flow cytometric analysis of percent CSCs in primary NSCCs upon FACtreatment (right). D, graphical representation of relativemean fluorescence intensities of ABCG2 andABCC1 (left) and E-cadherin and vimentin (middle) in untreatedand FAC-treated primary NSCCs. Migration of NSCCs purified from untreated and FAC-treated primary NSCCs (right). E, schematic representation of theexperimental protocol (top). Percent GFPþ CSCs in NSCCs of MCF-7 cells under the specified experimental conditions (bottom). F, illustration showingtranswell contact-independent coculture system (top). Percent CSCs (bottom left), relative mean fluorescence intensities of Aldh1 (bottom middle), andOct-4 (bottom right) in NSCCs of MCF-7 cells under the specified experimental conditions. Data are presented as mean � SEM or representative of threeindependent experiments. �� , P < 0.01 and �� , P < 0.001. AU, arbitrary units.

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Figure 4.Chemotherapy triggers NFkB-dependent IL6 inflammatory feedback loop in preexisting CSCs. A, immunohistology (brown color for antibody staining;counterstaining with hematoxylin) for NFkB in pre and posttreatment CS (top) and CR-primary tumor samples (bottom). Scale bar, 100 mm. B, nuclear andcytosolic NFkB in untreated and FAC-treated CSCs and NSCCs of MCF-7 cells (Western blot analysis data, left; quantitative data, right). C, Western blot dataof Bcl-2, Cox2, and XIAP in CSCs and NSCCs of pre and posttreatment primary tumors of CR-patients (left) and untreated and FAC-treated MCF-7 cells(right). Right panels of both represent quantitative data of respective Western blots. D, representative RT-PCR data of IL6 mRNA expression in pre andposttreatment primary tumors of CS- and CR-patient (left) and box-and-whisker plot of mean IL6/GAPDH ratio of all pre and posttreatment CS- and CR-tumors(right). E, relative mean fluorescence intensities (MFI) of IL6 levels in FAC-treated MCF-7 cells (left) and fold change of same in different cells upon 3 hoursof FAC treatment. F, ELISA of secretory levels of IL6 in spent media of untreated and FAC-treated CSCs and NSCC populations of MCF-7 cells. G, relative levelsof IL6 secreted in spent media of untreated and FAC-treated CSCs of MCF-7 cells under the indicated experimental conditions (left). Western blot analysisdata (middle) and quantitative data (right) of the same nuclear NFkB in CSCs of MCF-7 cells under the indicated experimental conditions; a-actin/histoneH1/GAPDH were internal loading controls. Data are presented as mean � SEM or representative of three independent experiments. � ,P < 0.05; �� , P < 0.01;and �� , P < 0.001. AU, arbitrary units.






Figure 5.Aspirin perturbs preexisting CSC-induced NSCC-to-CSC conversion by inhibiting NFkB-dependent IL6. A, diagrammatic representation of the experimentalprotocol (left). Percent CSCs in NSCCs of MCF-7 cells (middle) and relative mean fluorescence intensities (MFI) of ABCG2 in newly generated CSCs from NSCCs(right) when incubated with conditioned media of the indicated experimental sets. B, Western blot analysis of aspirin-treated CSCs of MCF-7 cells (top) andquantitative data of the same (bottom). C, time-dependent profiles of NFkB in nuclear and cytosolic fraction of CSCs isolated fromMCF-7 (top left) andWestern blotanalysis data of nuclear NFkB, Bcl-2, and Cox2 in primary CSCs (top right) upon treatment with 2.5 mmol/L aspirin. Bottom panels represent quantitativedata. D, relative levels of IL6 in spent media of untreated and DMSO/FAC/aspirin/combinatorial therapy–treated CSCs of MCF-7 cells by ELISA. E, percent CSCs inNSCCs of MCF-7 cells incubated with conditioned media of the indicated experimental sets (left) and percent GFPþ CSCs in NSCCs of MCF-7 cells under thespecified experimental conditions (right). F, relativemean fluorescence intensities of ABCG2 in newly generated CSCs fromNSCCswhen incubatedwith conditionedmedia of the indicated experimental sets; a-actin/histone H1 were internal loading controls. Data are presented as mean � SEM or representative of threeindependent experiments. �� , P < 0.01 and �� , P < 0.001. AU, arbitrary units.

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comparatively better response to treatment (Supplementary TableS1B). Thereafter, CSCs purified from CS- and CR-primary humancell lines and from different breast cancer cell lines, with almostsimilar characteristics as that of primary cell lines, were exposed toaspirin treatment prior to FAC. Supporting our hypothesis, resultsof aspirin-preexposed CSCs showed significant increase inAnnexin V positivity upon FAC treatment while aspirin alonefailed to do so (Fig. 6A and 6B, top and Supplementary Fig. S6C),indicating that aspirin sensitized CSCs towards chemotherapyregimen. Such combinatorial therapy also decreased CSC contenteven below the level present in their untreated counterparts (Fig.6A and 6B, bottomandSupplementary Fig. S6D). Further analysiswith MCF-7 cells revealed that such sensitization of preexistingCSCs resulted from significant decrease in the expression levels ofchemoresistance markers (Fig. 6C). Interestingly, aspirin-induced

decrease in chemoresistance of preexisting CSCs was inhibited byrIL6 or upon NFkB overexpression (Fig. 6D). ChIP assay furthershowed that although FAC treatment increasedNFkB recruitmenton the promoter regions of ABCG2 and ABCC1 in CSCs (Fig. 6E),such bindings were decreased in aspirin-pretreated sets. Theseresults signified that aspirin pretreatment decreased the expres-sion of drug efflux pumps, thereby sensitizing preexisting CSCs togenotoxic therapy.

All these results unveiled the hitherto unexplored role of aspirinin hindering the acquisition of aggressive phenotype by luminalhistotype breast cancer cells during the course of therapy by (i)perturbing preexistingCSC-assisted de novo conversionofNSCCs toaggressive CSCs and (ii) sensitizing inherently chemoresistantpreexisting CSCs. These findings not only identified preexistingCSCs as the probable candidates escalating therapy-induced

Figure 6.Aspirin chemosensitizes preexisting CSCs towards genotoxic therapy. Graphical representation of percent apoptosis (top) and percent CSCs (bottom) of CS- andCR-patients (A) and different cells (B) under the indicated experimental conditions. C, relative mean fluorescence intensities (MFI) of ABCG2 and ABCC1 inpreexisting untreated and DMSO/FAC/aspirin/combinatorial therapy–exposed CSCs of MCF-7 cells (left). The mRNA expression profiles of ABCG2 and ABCC1 inCSCs of MCF-7 cells under the indicated experimental conditions (middle) and quantitative data of the same (right). D, relative mean fluorescence intensitiesof ABCG2 in preexisting CSCs under the indicated experimental sets. E, pictorial representation of specific NFkB binding sites on ABCG2 and ABCC1 (left).ChIP assay of untreated and FAC/aspirin/combinatorial therapy–treated CSCs of MCF-7 cells (middle) and quantitative data of the same (right). Histone H1was used as an internal loading control. Data are presented as mean � SEM or representative of three independent experiments. � , P < 0.05; �� , P < 0.01; and�� , P < 0.001. AU, arbitrary units. IP, immunoprecipitation.






recurrence in breast cancer patients but also raised the possibility ofcombining aspirin with genotoxic therapies to efficiently improverelapse-free survival in NACT-treated breast cancer patients.

DiscussionResistance of breast tumors toward therapy is one of the major

causes of the incurability of the disease. In addition to beinginherently resistant to therapies during the course of chemother-apeutic treatments, tumors also acquire resistance towards suchtherapies. The consequences are 2-fold; first, the tumors acquireresistance with progressive treatment and second, provide impe-tus to the growth of recurrent tumors (1–4). CSC subpopulationof the tumor has beenheld responsible for both therapy resistanceand development of tumor recurrences (5–7, 26, 38). Therefore,the resistance of CSCs to chemotherapy/radiotherapy might be aplausible mechanism to explain breast cancer recurrence.

In keeping with these reports, we observed that acquiredchemotherapy–resistant patients are at a greater risk of developingtumor recurrence than chemotherapy-sensitive patients. An expla-nation for this observation is partly provided by existing reportsthat NACT leads to enrichment of CSCs (7–11, 39). However, thisexplanation alone is not enough to reason the recurrence oftumors as the CSC subpopulation, being a part of the primarytumor itself, must get removed during surgical resection of thetumor mass. In this preclinical study, we identified that in patientsamples and cell lines of luminal histotype, NACT treatmentresults in augmentation of aggressive CSC pool, which besidesimpeding therapeutic outcome, also helps them to hide in distantsites to escape surgery, thus providing anunintended advantage ofrecurrence.

Next, we sought to explore the mechanisms responsible for theenrichment of CSCs following NACT. Several studies have dem-onstrated that therapeutic intervention may contribute to CSCgenesis. In addition to earlier reports that suggest that the CSCenrichment after NACT results from selection as the NSCCs nat-urally succumb to these anticancer therapies, we found conversionof nonstem breast cancer population to CSCs, which also concur-rently acquire resistance and aggressive properties. These findingsare further supported by earlier reports describing the spontaneousconversion of nonstem tumor cells to CSCs (15, 40, 41).

An in-depth analysis to divulge themechanisms responsible forNACT-induced conversion of NSCCs-to-CSCs revealed that thiseffect was brought about through paracrine secretion of IL6,NFkBdownstream effector (13–15). Iliopoulos and colleagues (15)have shown that the spent media from CSCs promote non–CSC-to-CSC conversion in IL6-dependent manner. Interestingly,in many types of cancer, elevated level of serum IL6 was found(42), and the same was strongly correlated with poor overallsurvival and accelerated disease progression (43).

Interestingly, NFkB–IL6 signaling has been implicated in theself-renewal, chemoresistance, and tumorigenesis (14, 44–47).The involvement of NFkB–IL6 signaling in our ex vivo and in vitromodels prompted us to evaluate the role of aspirin in abrogatingthis pathway to chemosensitize resistant breast tumors. In ourstudy, physiologic doses of aspirin (36) efficiently perturbednuclear translocation of NFkB, resulting in the abrogation ofNFkB–IL6 feedback loop culminating in the inhibition of para-crine induction of NSCC-to-CSC conversion and also perturbingthe gain of chemoresistance and migration potential. Moreover,aspirin potentially chemosensitized preexisting CSC pool by

perturbing NFkB-mediated expression of chemoresistance pro-moting factors. Our study, therefore, signifies the possibility of acombination strategy that induces apoptosis in preexisting CSCsand NSCCs after FAC administration. This approach was furtherjustified by a recent study by Maity and colleagues (37), whereaspirin treatment prevented tumor growth in nudemice xenograftmodel by reducing the self-renewal capacity and growth of breastCSCs but failed to induce apoptosis by itself even in inherentlyresistant CSCs at physiologically achievable dose (37). Takentogether, our study thus provides a scientific rationale for com-bining aspirin with standard chemotherapy for successful target-ing of both CSC and NSCC pool to restrain the acquisition ofaggressive phenotype by breast cancer cells during the course ofchemotherapy. These data corroborated previous clinical studieswith breast cancer patients, in which a link between aspirin useand lower incidences of cancer initiation, recurrence, and metas-tasis, the three attributes associated with CSCs, has been found(16, 17, 48, 49).

In conclusion, tumor relapse is a common and devastatingphenomenon in clinical oncology following initial success withcytotoxic therapies. We provide evidence that aspirin-mediatedinhibition of NFkB–IL6 inflammatory signaling in preexistingCSCmeets the criteria for the development of potential treatmentstrategies by sensitizing preexisting CSCs in one hand and inhi-biting NSCC-to-CSC conversion on the other. Moreover, control-ling chemoresistance of CSCs and the generation of new CSCsduring chemotherapy treatment may ultimately improve curabil-ity and allow deescalation of currently given chemotherapeuticdose, thereby reducing acute and long-term adverse effects. There-fore, cotreatment of aspirin and chemotherapeutic regimen canbea potentially safe and attractive treatment strategy to tackle cancerin a multipronged approach, targeting both CSC and NSCCpopulations.

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

Authors' ContributionsConception and design: S. Saha, T. DasDevelopment of methodology: S. Saha, S. Mukherjee, T. DasAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): S. Saha, P. Khan, K. Kajal, M. Mazumdar, A. MannaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Saha, S.Mukherjee, S.Mukherjee,D.K. Sarkar, T.DasWriting, review, and/or revision of the manuscript: S. Saha, S. Mukherjee,T. DasAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): S. Mukherjee, S. De, D. Jana, D.K. SarkarStudy supervision: T. Das

AcknowledgmentsThe authors thank R. Dutta for technical help related to flow cytometry.

Grant SupportGrants were received from Council of Scientific and Industrial Research

(S. Saha, S. Mukherjee, and P. Khan), Department of Science and Tech-nology (K. Kajal, A. Manna, and T. Das), and Department of Biotechnology(M. Mazumdar), India.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 27, 2015; revised October 24, 2015; accepted December 28,2015; published OnlineFirst February 3, 2016.

Saha et al.





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2016;76:2000-2012. Published OnlineFirst February 3, 2016.Cancer Res Shilpi Saha, Shravanti Mukherjee, Poulami Khan, et al. the Generation of Cancer Stem Cells

IL6 Signaling Axis Responsible for−BκCancer by Disrupting an NFAspirin Suppresses the Acquisition of Chemoresistance in Breast

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