her2-associated radioresistance of breast cancer stem ... · 6634 clin cancer res; 18(24) december...

Cancer Therapy: Preclinical

HER2-Associated Radioresistance of Breast Cancer StemCells Isolated from HER2-Negative Breast Cancer Cells

Nadire Duru1, Ming Fan1, Demet Candas1, Cheikh Menaa1, Hsin-Chen Liu1, Danupon Nantajit1, Yunfei Wen6,Kai Xiao2, Angela Eldridge1,3, Brett A. Chromy3,5, Shiyong Li7, Douglas R. Spitz8, Kit S. Lam2,4, Max S. Wicha9,and Jian Jian Li1,4

AbstractPurpose:Tounderstand the role ofHER2-associated signalingnetwork inbreast cancer stemcells (BCSC)

using radioresistant breast cancer cells and clinical recurrent breast cancers to evaluate HER2-targeted

therapy as a tumor eliminating strategy for recurrent HER2�/low breast cancers.

Experimental Design: HER2-expressing BCSCs (HER2þ/CD44þ/CD24�/low) were isolated from

radiation-treated breast cancer MCF7 cells and in vivo irradiated MCF7 xenograft tumors. Tumor aggres-

siveness and radioresistance were analyzed by gap filling, Matrigel invasion, tumor-sphere formation, and

clonogenic survival assays. The HER2/CD44 feature was analyzed in 40 primary and recurrent breast cancer

specimens. Protein expression profiling in HER2þ/CD44þ/CD24�/low versus HER2�/CD44þ/CD24�/low

BCSCs was conducted with two-dimensional difference gel electrophoresis (2-D DIGE) and high-perfor-

mance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) analysis and HER2-mediated

signaling network was generated by MetaCore program.

Results: Compared with HER2-negative BCSCs, HER2þ/CD44þ/CD24�/low cells showed elevated

aldehyde dehydrogenase (ALDH) activity and aggressiveness tested by Matrigel invasion, tumor sphere

formation, and in vivo tumorigenesis. The enhanced aggressive phenotype and radioresistance of the

HER2þ/CD44þ/CD24�/low cells were markedly reduced by inhibition of HER2 via siRNA or Herceptin

treatments. Clinical breast cancer specimens revealed that cells coexpressing HER2 and CD44 were more

frequently detected in recurrent (84.6%) than primary tumors (57.1%). In addition, 2-D DIGE and HPLC/

MS-MS of HER2þ/CD44þ/CD24�/low versus HER2�/CD44þ/CD24�/low BCSCs reported a unique HER2-

associated protein profile including effectors involved in tumor metastasis, apoptosis, mitochondrial

function, and DNA repair. A specific feature of HER2–STAT3 network was identified.

Conclusion: This study provides the evidence that HER2-mediated prosurvival signaling network is

responsible for the aggressive phenotype of BCSCs that could be targeted to control the therapy-resistant

HER2�/low breast cancer. Clin Cancer Res; 18(24); 6634–47. �2012 AACR.

IntroductionDespite advances in early diagnosis and treatment, breast

cancer–related death remains significantly high due toresistance of metastatic and recurrent tumors to currentanticancer regiments; with as many as 40% relapsing withmetastatic disease (1, 2). Accumulating evidence of tumorheterogeneity and the presence of cancer stem cells (CSC)detected in many tumors with the stem cell–like character-istics offer new paradigms to understand and generateeffective targets to treat recurrent and metastatic tumors(3, 4). Breast cancer cells that are able to propagate asmammospheres andpossessCSCproperties aremore radio-resistant (5) and the population of breast cancer stem cells(BCSC) is increased after chemotherapy (6). Breast cancercells surviving radiation show enhanced clonogenic surviv-al indicating the enrichment of radioresistant cells (7, 8).

HER2 belongs to the HER family of transmembraneglycoproteins, which consists of 4 homologous receptors(9). About 25%of patients with breast cancer are diagnosed

Authors'Affiliations: Departments of 1RadiationOncology, 2Biochemistryand Molecular Medicine, and 3Pathology and Laboratory Medicine,University of California Davis School of Medicine; 4NCI-Designated Com-prehensive Cancer Center, University of California Davis, Sacramento;5Physical and Life Sciences Directorate, Lawrence Livermore NationalLaboratory, Livermore, California; 6Department of Gynecologic Oncology,MD Anderson Cancer Center, University of Texas, Houston, Texas;7Department of Pathology and Laboratory Medicine, Emory UniversitySchool of Medicine, Atlanta, Georgia; 8Free Radical and Radiation BiologyProgram, Department of Radiation Oncology, Holden ComprehensiveCancer Center, The University of Iowa, Iowa City, Iowa; and 9Universityof Michigan Comprehensive Cancer Center, Ann Arbor, Michigan

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

Current address for N. Duru: California National Primate Research Center,University of California Davis, Davis, CA 95616.

Corresponding Author: Jian Jian Li, Department of Radiation Oncology,University of California Davis School of Medicine, 1136 Oak Park Building,2700 Stockton Boulevard, Sacramento, CA 95817. Phone: 916-703-5174;Fax: 916-734-4107; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-1436

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(24) December 15, 20126634

on December 9, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst October 22, 2012; DOI: 10.1158/1078-0432.CCR-12-1436

http://clincancerres.aacrjournals.org/

as HER2-amplified status (HER2þ) associated with a highrisk of relapse (10, 11) and targeting HER2 expressioninhibits tumor aggressiveness (12, 13). Clinical data showthat anti-HER2, trastuzumab, treatment reduces tumorrecurrence when estrogen receptor–positive (ERþ) tumorsbecome resistant to hormonal therapy (14, 15), suggestingthat HER2 expression is activated as an escape prosurvivalpathway. Consistent with this result, inhibition of HER2increases tumor cell killing (16, 17). Importantly, over-expression of HER2 is able to increase the CSC populationexpressing aldehyde dehydrogenase (ALDH) withenhanced invasiveness and tumorigenesis (18). The highestHER2 expression level is detected in tumor-initiating cells ofHER2þ breast cancer cell lines (19). We have reported thatthe overexpression of HER2 in HER2�/low MCF7 cellsenhances their radioresistance (7) and a NF-kB–bindingsite in theHER2 promoter region is identified to be respon-sible for HER2 transactivation in radiation-treatedHER2�/low breast cancer cells (20). However, the exactmechanisms involved in HER2-mediated repopulation ofBCSCs under radiation treatment, especially in HER2�/low

breast cancer remain to be elucidated.Here, we identified that HER2-overexpressing BCSCs

are responsible for the radioresistance of HER2�/low

breast cancer. BCSCs with the feature of HER2þ/CD44þ/CD24�/low, compared with the counterpart HER2�/CD44þ/CD24�/low cells, showed an increased aggres-siveness, tumorigenesis, and radioresistance that can bereducedby siRNA- orHerceptin-mediatedHER2 inhibition.Clinical study revealed that HER2 protein expression wasenhanced more frequently in the recurrent tumors thanprimary cancers with an increased rate of coexpression ofHER2 and CD44. Proteomics and connective map studiesrevealed a unique cluster of HER2-associated effectorsincluding elements in tumor metastasis, redox imbalance,mTOR signaling, and DNA repair. A connective networkbetween HER2 and STAT3 is created. Altogether, our results

suggest that HER2-initiated proliferative network is respon-sible for the resistant phenotype of BCSCs that are enrichedin the therapy-resistant breast cancer.

Materials and MethodsCell culture

Humanbreast cancerMCF7 cells (American TypeCultureCollection), radioresistant MCF7/C6 cells (20), and MCF7cells transfected with HER2, MCF7/HER2 (7) were main-tained as described earlier (20) in Eagle’s minimum essen-tial medium (EMEM), supplemented with 10% FBS(HyClone), 5% sodium pyruvate, 5% nonessential aminoacid (NEAA), penicillin (100 U/mL), and streptomycin(100 mg/mL) in a 37�C incubator (5% CO2).

Irradiation of xenograft tumor and cellsA standard cell inoculation was used to generate mouse

breast cancer xenograft tumors using MCF7 cells asdescribed (20) following the protocol approved by theInstitutional Animal Care andUse Committee of Universityof California Davis (IACUC No. 15315, Davis, CA). Eight-weeks old female athymic nude mice (Jackson Laboratory)were pretreated for 5 days with estrogen pellets (InnovativeResearch of America) followed by inoculation with 5� 106

MCF7 cells. Three weeks after cell inoculation, the averagetumor size was 2,000 to 2,300mm3. Themice were dividedinto 2 groups; one received sham radiation and the otherexposed to local tumor radiation with fractionated doses(5 � 2 Gy) delivered by GR-12 irradiator of 60Co g-rays,under anesthetic agents using ketamine. Twenty hours afterthe last radiation treatment, mice were sacrificed and tumorcells suspensionwasprepared for fluorescence-activated cellsorting (FACS). Radiation was delivered to cultured cellsusing a Cabinet X-rays System Faxitron Series (dose rate:0.997 Gy/min; 130 kVp; Hewlett Packard). Cells shelteredfrom radiation were included as the sham-IR control.

Western blottingTotal cell lysates (20 mg) were separated by SDS-PAGE

and blotted onto polyvinylidene difluoridemembrane. Themembrane was incubated with specific primary antibodyovernight at 4�C, followed by the horseradish peroxidase(HRP)-conjugated secondary antibody, and visualized bythe ECL Western blotting detection system (Amersham).STAT3 antibodywas purchased fromSantaCruz (SC-8019).HER2 (MS-730-P) and b-actin antibodies were purchasedfrom Lab Vision Corporation and Sigma, respectively.

Clonogenic survival assayStandard radiation clonogenic survival assays were con-

ducted as previously described (7) following either 0Gy (sham) or 5 Gy radiation in combination with orwithout HER2 inhibition by siRNA or Herceptin treatment(10 mg/mL for 5 days with refreshed Herceptin every48hours). For siRNA treatment, cells were seeded to achieve30% to 50% confluence on the day of transfection andsiRNA (20 nmol/L) was transfected using LipofectamineRNAiMAX reagent (Invitrogen) in antibiotic-free medium

Translational RelevanceDespite a trend toward overall improvement in early

detection and therapy outcomes, breast cancer mortalityremains unacceptably high due to the ineffectiveness ofthe control over the recurrent and metastatic lesions,especially for HER2-negative breast cancer. Herein, weidentified a population of breast cancer stem cells(BCSC) expressing HER2 (HER2þ/CD44þ/CD24�/low)isolated from radioresistantHER2-negative breast cancercells, and the HER2þ/CD44þ/CD24�/low was more fre-quently detected in the recurrent tumors as comparedwith the primary breast cancer. A HER2-linked proteinnetwork, such as HER2–STAT3, was detected in theHER2-postive BCSCs by proteomics analysis. Thus,the HER2-associated signaling network may be an effec-tive target to treat resistant breast tumorswithHER2�/low

status.

HER2-Mediated Aggressiveness in Breast Cancer Stem Cells

www.aacrjournals.org Clin Cancer Res; 18(24) December 15, 2012 6635




for 24 hours. Scrambled RNA Duplex (Ambion) and mul-tiple HER2 siRNA were tested and served as negative con-trols. All transfectants were maintained in antibiotic-freecomplete medium and replaced with fresh medium beforeradiation. The colonies were fixed and stained with Coo-massie blue and colonies containing more than 50 cellswere counted as surviving clones and normalized to theplating efficiency of cells without radiation.

Invasion assayAliquots (0.4 mL) of Matrigel (BD Biosciences) were

diluted in serum-free minimum essential medium (MEM;Life Technologies) to the final concentration of 3 mg/mL.Matrigel was then loaded into the upper chamber of 24-welltranswell (Costar) and incubated at 37�C for 1 hour beforecells (105/mL) were added. The lower chamber was filledwith 600 mL of MEMmedia containing 5 mg/mL fibronectinas an adhesive substrate (Santa Cruz Biotechnology). Thetranswell with differently treated cells was incubated forvaried times and stained with Diff-Quick Stain (FisherScientific), and the invasive cells were counted using lightmicroscopy.

Gap-filling rate assayGap-filling capacitywas conducted as described (21)with

3 � 105 cells grown in each of 6-well plates to 100%confluence followed by culture in medium without serumfor 48 hours for cell starvation. The gap was created byscraping the cells diagonally with a sterile pipette’s tip andthe filling capacity was monitored at different times, asspecified in results.

Tumor sphere formationTumor sphere assay was conducted as described (22).

Cells were sieved with 40 mm-cell strainers (Fisher) andsingle-cell suspensions were seeded into ultra low-attach-ment 60-mmPetri dishes at a density of 1,000 cells/mL. Thecells were grown in serum-free mammary epithelial basalmedium (MEBM, Lonza), supplemented with B27 (LifeTechnology), 20 ng/mL EGF (Biovision), 20 ng/mL basic-fibroblast growth factor (FGF), and 4 mg/mL heparin(VWR). Cells were cultured for 10 days and tumor sphereswere counted under light microscopy.

Orthotopic mammary fat tumor formationTumor-initiating test was conducted following the

described methods (23, 24), and the protocol was reviewedand approved by the IACUC at the University of CaliforniaDavis (IACUC No. 15315). Eight-weeks old female non-obese diabetic/severe combined immunodeficient (NOD/SCID)mice (Jackson Laboratory) were pretreated for 5 dayswith estrogen pellets (Innovative Research of America), andfreshly sorted BCSCs were resuspended and diluted inserum-free PBS/Matrigel mixture (1:1 v/v) and inoculatedinto the mammary pads side by side of the animal using27G needle (500 cells/injection). Tumorigenesis wasassessed twice a week with palpation. Tumor sizes weremeasured using a caliper and experiments were terminated

when tumors reached approximately 1.2 cm in the largestdiameter.

Mitochondrial membrane potential (Dcm) andapoptosis

Irradiated cells were incubated with 2 mg/mL of JC-1 for30minutes. The fluorescence intensity of the red precipitate(JC-1 red), and green monomer (JC-1 green) were deter-mined by a plate reader (Spectra Max M2e, MolecularDevices Co.) at 485 nm/595 nm or 485 nm/525 nm(excitation/emission). The ratio of JC-1 red (595)/JC-1green (525) was calculated as DYm. For apoptotic analysis,cells were harvested 24 hours postirradiation and wereincubatedwithAnnexin-Vfluorescein isothiocyanate (FITC;Biosource) and propidium iodide (PI; Sigma) for 15 min-utes before analysis with FACS-Canto (Becton Dickinson).

BCSC sortingFollowing described procedures (3), cell suspension was

rinsed with PBS containing 2% FBS, suspended in PBScontaining 0.5% FBS and PI (0.5 mg/mL), and sorted usingthe Cytopeia inFlux Cell Sorter (BD Biosciences). The anti-bodies applied for sorting were anti-HER2/neu conjugatedto allophycocyanin (APC; BD Biosciences), anti-humanCD44 conjugated to FITC and human CD24 conjugatedto phycoerythrin (PE; Invitrogen). Cell viability wasassessed by 7-AA staining during cell sorting, and by Trypanblue exclusion after sorting.

ALDH activityCellswere suspended (106 cells/mL) inALDEFLUORassay

buffer containing activated ALDH substrate BAAA (boron-dipyrromethene–aminoacetaldehyde) with or without 50mmol/LDEAB(diethylaminobenzaldehyde, a specificALDHinhibitor) and incubated at 37�C for 40 minutes and ana-lyzedbyFACS.Thegateswerenormalized tocells treatedwithALDEFLUOR-DEAB.

siRNA-mediated target gene inhibitionThe siRNA targeting HER2 was synthesized using the

Silencer siRNA Construction Kit (Ambion) with 2 targetingsequences: 50 AACCTGGAACTCACCTACCTG30 and50 AAG-CCTCACAGAGATCTTGAA 30. Cell transfection was con-ducted at 30% to 50% cell confluence using LipofectamineRNAiMAX reagent (Invitrogen). Scrambled RNA Duplex(Ambion) was used for specificity as a negative control.

FISH and IHC of breast cancer specimensThe pathology database was searched for invasive breast

cancer, and cases with clinical follow-up were retrievedfollowing Institutional Review Board’s approval (EmoryUniversity, Atlanta, GA, IRB#: 901-2004). Total 40 clinicalsamples were grouped according to pathologic classifica-tion. Fluorescence in situ hybridization (FISH) was con-ducted using the PathVysion kit (Vysis, Inc.). A centromere17:HER2 probe signal ratio ofmore than 2.2 obtained from30 interphase nuclei was considered positive forHER2 geneamplification. Immunohistochemical staining (IHC) was

Duru et al.

Clin Cancer Res; 18(24) December 15, 2012 Clinical Cancer Research6636




conducted using UltraVision LP Detection System HRPPolymer & DAB Plus Chromogen staining kit (ThermoScientific) after heat-induced epitope retrieval. The slideswere incubated with anti-HER2 (Sigma, Cat. E2777), anti-CD44 (R&D Systems, Cat. BBA10), and anti-CD24 (SantaCruz, Cat. Cs-11406) primary antibodies for overnight at4�C, followed by 3 washes, and then incubation with thesecondary antibody for 1 hour at room temperature. Theslides were then incubated with 40,6-diamidino-2-pheny-lindole (DAPI; 300 nmol/L in PBS) for 5 minutes at roomtemperature, followed by 2 washes and then sealed andanalyzed with a Nikon microscope (Eclipse, E1000M).Nikon microscope (Eclipse, E1000M) and graded asHER2þ (IHC 2þ and 3þ) or amplified (FISH, HER2: cen-tromere 17 signal ratio >2.2); HER2� (IHC 0) or nonam-plified (FISH, HER2: centromere 17 signal ratio <1.8); andHER2 equivocal (IHC 1þ and FISH, HER2: centromere 17signal ratio ¼ 1.8–2.2).

Two-dimensional difference gel electrophoresisTotal cellular protein (50 mg) was minimally labeled with

Cy3 and Cy5 following the reported protocol (25, 26).Labeled samples were mixed together and diluted to 450 mLin rehydration buffer, applied to a 24 cm pH 3 to 10 NL IPGstrips (GE Life Sciences) and isoelectric focusing conducteduntil 62,500 Vh was reached. The second dimension sepa-rationwas then conducted using 12.5% Tris–Glycine precastgel for 16 hours at 2 W per gel. CyDye protein spots wereimaged on a Typhoon 9410 and differential protein expres-sion analyzedusingDeCyder 6.5 software.Differential In-gelAnalysis (DIA) module was used to conduct spot detection,quantitation and Cy3/Cy5 volume ratio calculations, foldchanges greater than 1.5 were considered significant.

High-performance liquid chromatography tandemmass spectrometryFor large scale analysis, 1 mg proteins, for each 3 biologic

replicate samples, were precipitated and processed withstrong cation exchange (SCX) column as described (27) fol-lowed by a 120-minute reverse-phase gradient to separatepeptides based on their hydrophobicity. The LTQ OrbitrapXL (Thermo Scientific) coupled to reverse phase high-perfor-mance liquid chromatography (RP-HPLC) systemwith a lowflow ADVANCED Michrom MS source, was set to scan theprecursors with a top 4 data-dependent MS/MS method inthe profile mode on the precursors and centroid mode ontheMS/MSspectra. Tandemmass spectrawere analyzedusingMascot database search engine (MatrixScience) with masstolerances set to 1 Da for parent ions and 0.3 Da for MS/MSions and allowance of up to 1 missed cleavages. All searcheswere done against the International Protein Index humandatabase and proteins were considered significant when theymet the MASCOT significance threshold of P > 0.05.

Gene ontology and protein-differential expression assayThe MS and MS/MS data were submitted to Sorcerer

Enterprise v.3.5 release (Sage-N Research Inc.) for proteinidentification against the International Protein Index

human protein database. The relative abundance of eachidentified protein in different samples were analyzed byQTools and Prolucid search algorithm andDTASelect usingIP2 (Integrated Proteomics Pipeline) server and AmazonCloud Computing. Comparative peptide abundance wasevaluated by IP2-Census, a Label-free analysis for automat-ed differential peptide/protein spectral counting analysis.Using MetaCore Version 6.9, a cluster of proteins andeffecters were extracted from the data and identified tointeract with both STAT3 and HER2.

Statistical analysisThe significance of the data was analyzed using an analy-

sis of variance single-factor test and the 2-tailed Student ttest. A difference was considered significant when P < 0.05.

ResultsEnhanced aggressiveness of radioresistant MCF7 cellsdue to HER2 induction

To identify the key factors responsible for tumor resis-tance, we first assessed the question of whether HER2expression is enhanced in the radiation-treatedHER2�/low breast cancer cells MDA-MB231 cells and aradioresistant cell line (MCF7/C6) that survived a treatmentcourse using fractionated doses of ionizing radiation(8, 20). Consistent with previous observations (7, 20),substantial amount of HER2 proteins was detected in theirradiated MDA-MB231 and the radioresistant cell lineMCF7/C6 (Fig. 1A; refs. 20, 28). To further elucidate themechanism of radiation-induced tumor radioresistance, westudied HER2 expression in MCF7/C6 cell line with MCF7/HER2 and MCF7 wt cells as positive and negative controls,respectively. The induced HER2 protein expression in theMCF7/C6 cells could even reach to the level of HER2expression in MCF7 cells stably overexpressing HER2 gene(MCF7/HER2). In addition, cell invasiveness of MCF7/C6cells in Matrigel analysis (Fig. 1B and Supplementary Fig.S1C) was also significantly enhanced as compared with wtMCF7 cells. Interestingly, the gap-filling capacity remainedin a similar rate in all cells except a tendency of increasedgap-filling rate of MCF7/C6 and MCF7/HER2 cells at 96hours (Fig. 1B and Supplementary Fig. S1A). Using tumorsphere formation assay (22), we found that, in addition toincreased sizes of tumor spheres, the number of tumorspheres was increased 3.48- and 3.79-folds in MCF7/C6and MCF7/HER2 cells, respectively, as compared withMCF7 cells (Fig. 1B and Supplementary Fig. S1D). Theclonogenicity was increased 2.85- and 2.62-folds in theMCF7/C6 and MCF7/HER2, respectively (Fig. 1B). Theseresults raise the possibility that the expression ofHER2 thatwas silenced in many HER2�/low breast cancers could beinduced in therapy-resistant fraction of cancer cells leadingto the overall aggressiveness of recurrent tumors.

HER2þ/CD44þ cells enriched in irradiated MCF7 cellswith increased radioresistance

Significant reduction in cell sensitivity to IR-inducedapoptosis (Fig. 1C) and increased clonogenic survival






D

A

B

0

10

20

30

40

50

60

Ce

ll m

ark

er

(%)

0 Gy

5x2 Gy

** **

**

Ce

ll m

ark

er

(%)

0

5

10

15

20

25

30

35

0 Gy

CD44+ /C

D24–/lo

w

HER2+ /C

D44+

HER2+ /C

D24–/lo

w

CD44+ /C

D24–/lo

w

HER2+ /C

D44+

HER2+ /C

D24–/lo

w

5x2 Gy

**

**

*

MCF7 cells Xenograft tumors

Matrigel invasion

0

1

2

3R

ela

tiv

e f

ille

d g

ap

(a

u)

wt C6 HER2

P = 0.086

P = 0.063

Gap filling

0

50

100

150

wt C6 HER2

Tu

mo

r s

ph

ere

s/d

ish

** **

Tumor sphere formation

0

0.1

0.2

0.3

0.4

0.5

0.6

Clo

no

ge

nic

su

rviv

al

** **

Clonogenic radiosensitivity

wt C6 HER2

0

0.5

1

1.5

2

2.5

3

0

0.5

1

1.5

2

2.5

3

C

Ap

op

toti

c c

ell

s

(%)

** **

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07**

**

Clo

no

ge

nic

su

rviv

al

wt C6 HER2 wt C6 HER2

Re

lati

ve

ga

p f

illi

ng

(a

u)

wt C6 HER2

** **

Apoptosis Clonogenic radiosensitivity Gap filling

0

20

40

60

80

100

120

140

160

In

va

siv

e c

ell

s/t

ran

sw

ell

wt C6 HER2

**

*

wt C6 HER2

HER2

ββ-Actin

HER2

β-Actin

IR (Gy) 0 1 2 4

MCF7 MDA-MB-231

Figure 1. HER2 protein expression causes the aggressive growth in breast cancer cells with HER2�/low status. A, HER2 protein expression was dose-dependently induced by IR in MDA-MB-231 cells and in radioresistant MCF7/C6 (C6) cells (8, 20). MDA-MB-231 were exposed to increased doses ofirradiation (0–4 Gy). Twenty-four hours postirradiation, cells were collected and tested for the expression of HER2. For MCF7/C6 cells, parental MCF7cells (wt) and MCF7 cells stably transfected with HER2 gene (HER2) were used as negative and positive controls, respectively. B, evaluation of cellaggressiveness.Matrigel invasion assay: cellswere cultured inMatrigel and cellsmigrating through the transwell were counted to reflect cell's aggressiveness(meanþSE; n¼ 6; �,P < 0.05; ��,P < 0.01; depicted images of invaded cells are illustrated in Supplementary Fig. S1C). The gap-filling capacitywas calculatedas the ratio of filled gaps at 96 hours postscraping compared with the filled gap at 0 hour (additional data are shown in Supplementary Fig. S1A).The tumor sphere formationwas evaluated as the number of tumor spheres formed per dish (meanþSE; n¼ 6; ��,P < 0.01; images of tumor sphere formationare illustrated in Supplementary Fig. S1D). Clonogenic survival was evaluated as the percentage of cells seeded that were able to form colonies 14 days afterirradiation (mean þ SE; n¼ 3; ��, P < 0.01). C, the radioresistant phenotype of MCF7/C6 (C6) cells was compared with MCF7/HER2 (HER2) and the parentalMCF7 cells after treatment with 5Gy IR bymeasuring apoptosis, clonogenic survival, and gap-filling rates (meanþSE; n¼ 3; ��,P < 0.01). D, quantification ofBCSCs with the feature of HER2þ/CD44þ/CD24�/low in the surviving fraction of MCF7 cells (left) or xenograft tumors (right) treated with 5 � 2 Gy IR. Cellsuspensions from control (sham) or irradiated cells or tumors were sorted by FACS with conjugated antibodies (APC for HER2, PE for CD44, and FITC forCD24; n ¼ 3 from separate sorting, �, P < 0.05; ��, P < 0.01; additional FACS data are shown in Supplementary Fig. S3 and Supplementary Table S1).

Duru et al.





(Fig. 1C) were detected in HER2 expressing MCF7/C6and MCF7/HER2 cells as compared with wt MCF7 cells.Inhibition of HER2 resulted in reduction of DYm, andHER2 inhibition followed by radiation further reducedDYm in both MCF7/C6 and MCF7/HER2 cells (Supple-mentary Fig. S2). Interestingly, contrary to a similargap-filling rate in all 3 cell lines without IR (Fig. 1B andSupplementary Fig. S1A), the gap-filling rates weremarkedly reduced in wt MCF7 cells but remained arelative high level in the MCF7/C6 and MCF7/HER2 cellsafter radiation (Fig. 1C and Supplementary Fig. S1B).To verify that the HER2-expressing BCSCs were enrich-ed by multiple IR, we analyzed the expression of HER2and BCSC markers in irradiated MCF7 cells and xenografttumors (Fig. 1D, Supplementary Fig. S3, and Supplemen-tary Table S2). IR was delivered by 5 fractionated doseswith 2 Gy each to cells or locally to tumor xenografts inmice. In both cases, CD44þ/CD24�/low, HER2þ/CD44þ,and HER2þ/CD24�/low populations were markedlyincreased. It should be noticed that about 1% of non-irradiated tumor cells were HER2þ/CD44þ, but this pop-ulation was increased about 30-fold after irradiation inxenograft tumors (Fig. 1D, right). The HER2þ/CD44þ/CD24�/low population was also significantly increased(95.8%) in the radioresistant MDA-MB231 cells (Supple-mentary Fig. S4; the FIR Clone 4 was derived from thetriple-negative wild-type MDA-MB231 cells after long-term irradiation; ref. 20). Similar to MCF7/C6, theincrease in the HER2þ/CD44þ/CD24�/low subpopulationwas due to the induction of HER2 expression in responseto irradiation. However, the CD44þ/CD24� cells per sedid not significantly change compared with the wt cells,probably due to the fact that MDA-MB-231 cells werefound to hold naturally a high level of BCSCs (29, 30).The high level of BCSCs may explain the inherited aggres-siveness phenotype of MDA-MB-231 compared withMCF7 cells. However, we believe that the overexpressionof HER2 will further increase their aggressiveness andresistance to radiation.Furthermore, we found that the increased population

of HER2þ/CD44þ cells was further enhanced in the irradi-ated xenograft tumors (Fig. 1D, right) compared with irra-diated cells in culture (Fig. 1D, left), highlighting the pos-sible roleofother factors in thedevelopmentof radioresistantBCSCs in vivo Taken together, these data suggest that thetumor microenvironment under therapeutic stress condi-tionspromotes the survivalofHER2-expressingBCSCs.Theseresults reinforce our hypothesis that HER2 expression inBCSC is a predominant feature of radioresistance inHER2�/low cancer.

Identification of HER2þ/CD44þ/CD24�/low cellsTo investigate whether HER2 expression is a unique

marker of radioresistant BCSCs in HER2�/low breast cancercells, CD44þ/CD24�/low population was further sortedaccording to the level of HER2 expression (Fig. 2A and Band Supplementary Table S1). Of the total of 2.4 � 106

MCF7/C6 cells, 3.7 � 104 cells were identified as CD44þ

/CD24�/low (1.51%). Interestingly, only 9.84% of theCD44þ/CD24�/low population was detected to express ahigh level of HER2 (termed as HER2þ/CD44þ/CD24�/low

population) and 6.85% showed low expression (termed asHER2�/CD44þ/CD24�/low population; Fig. 2B and Sup-plementary Table S1). The difference in HER2 expressionbetween these 2 populations was confirmed by immuno-blotting (Fig. 2B, right). ALDH is established to play amajorrole in cell-death resistance and is associated with BCSCs’aggressiveness (31). We found that the ALDH activitywas about 3-fold elevated in HER2þ/CD44þ/CD24�/low

cells compared with their counterpart HER2�/CD44þ/CD24�/low population (Fig. 2C and D). These results pro-vide the evidence indicating that a fraction of HER2-expres-sing CD44þ/CD24�/low BCSCs is present in the survivingbreast cancer cells with status of HER2�/low.

Enhanced aggressiveness of HER2þ/CD44þ/CD24�/low

BCSCsTheHER2þ/CD44þ/CD24�/low BCSCsweremore aggres-

sive in migration compared with the HER2�/CD44þ/CD24�/low BCSCs under the condition of sham or 5 Gy IR(Fig. 3C and Supplementary Fig S4), indicating that HER2-expressing BCSCs aremore resistant to genotoxic stress. Theincreased radioresistance of HER2þ/CD44þ/CD24�/low

BCSCs was further supported by elevated DYm, reducedapoptosis, and increased clonogenic survival (Fig. 3A andB). In addition, the invasiveness and tumor sphere forma-tion (Fig. 3C) were increased approximately 5- and 4-fold,respectively, in HER2þ/CD44þ/CD24�/low cells as compar-ed with the HER2�/CD44þ/CD24�/low cells. To elucidatewhether HER2 expression endows BCSC a higher tumori-genic capacity, we measured tumor formation of HER2þ/CD44þ/CD24�/low versus HER2�/CD44þ/CD24�/low cellsusing NOD scid gamma/SCID mouse xenograft model.HER2 status, indeed, affects the tumor-initiating rate ofBCSCs. All the sites inoculated with HER2þ/CD44þ/CD24�/low cells (500 cells/injection, HER2þ) developed tu-mors (6/6) with an average volume of 92 mm3 at day 21;whereas no detectable tumors were recorded in all sites(0/6) with the same number of HER2�/CD44þ/CD24�/low

cells (HER2�) up to day 45 (Fig. 3C). To verify the directfunction of HER2 expression in radioresistance, siRNA-mediated HER2 inhibition was applied and the resultsshowed a reduction in cell invasiveness by approximately70% that was further reduced by HER2 inhibition com-binedwith 5Gy IR (Fig. 4A).Noticeably, the gap-filling rateswere also severely reduced by HER2 inhibition with orwithout combination with IR (Fig. 4B and SupplementaryFig. S5). In agreement, treatment with HER2 siRNA (Fig.4C) or Herceptin (Fig. 4D) reduced the clonogenicity ofHER2þ/CD44þ/CD24�/low BCSCs, which was furtherdecreased when combined with IR treatment.

Coexpression of HER2 and CD44 in recurrent breastcancer

To investigate whether the feature of HER2þ/CD44þ/CD24�/low is related to the aggressiveness of metastatic






breast cancers, we analyzed the expression of HER2,CD44, and CD24 in 40 randomly selected pathologicspecimens that are clinically diagnosed as primary, recur-rent, or unknown breast tumors by the pathology depart-ment at the Emory University School of Medicine. Ineach tumor, we correlated the degree of HER2 geneamplification assessed by FISH with the HER2 proteinexpression detected by immunohistochemistry. By FISHanalysis, 12 of 40 tumors were determined as HER2-amplified (30%), whereas HER2 protein was expressedin 33 of 40 tumors by IHC (82.5%; Fig. 5A), suggestingthat mechanisms other than gene amplification areresponsible for the enhanced expression of HER2 pro-tein. Interestingly, cells positively stained with HER2and CD44 antibody by IHC were significantly in-creased (11/13, 85%) in the recurrent tumor samples

compared with the level of the samples of primarytumors (12/21, 57%; Fig. 5B). Subsequently, 85%of the recurrent tumors were CD24� contrasted with48% in primary tumors (Supplementary Fig. S6C). Arepresentative picture of the tumors costained withHER2 and CD44 without CD24 expression are shownin Supplementary Fig. S6D. Taken together, these resultssupport that HER2 expressing BCSC are enriched inrecurrent breast tumors.

Proteomics of HER2þ/CD44þ/CD24�/low versusHER2�/CD44þ/CD24�/low cells

To identify the signaling network causing the aggressivegrowth of HER2-expressing BCSCs, we conducted proteo-mics experiments using two-dimensional difference gelelectrophoresis (2-D DIGE; Supplementary Fig. S7) and

CD44+/CD24-/low

A

B

CD44/CD24

HER2+HER2–

Global

HER2–

HER2

98.49% 1.51% 83.31% 6.85%

CD

44

HE

R2

HER2+/CD44+/CD24–/low

HER2–/CD44+/CD24–/low

Global-HER2–/CD44+/CD24–/low

Non-CD44+/CD24–/low

HER2–/CD44+/CD24–/low HER2+/CD44+/CD24–/low

CD44+/CD24–/low

0

50

100

150

200

250

300

350

HER2– HER2+

ALD

H-p

ositi

ve c

ells

%

DC

1.25%

30,000

20,000

10,000

0

SS

C

30,000

20,000

10,000

0

SS

C

100 101 102

BAAA103 100 101 102

BAAA103

3.81%

Figure 2. Characterization of theHER2þ/CD44þ/CD24�/low BCSCswithin MCF7/C6 cell lines. A,CD44þ/CD24�/low cells weresorted from 2.5 � 107 MCF/C6cells with CD44 and CD24antibodies (left), from which thecells were further sortedwithHER2antibody (right). The orangebox in the left indicatesthe CD44þ/CD24�/low fractionused for sorting HER2þ/CD44þ/CD24�/low (yellow box), HER2�/CD44þ/CD24�/low cells (redbox), and a global HER2�/CD44þ/CD24�/low population (blue boxwith a broader gate). B,percentages of CD44þ/CD24�/low

and HER2þ/CD44þ/CD24�/low

cells derived from MCF7/C6 cells(left, also shown in SupplementaryTable S2). Western blot analysisof HER2 and CD44 proteinexpression in sorted HER2þ/CD44þCD24/�/low and HER2�/CD44þ/CD24�/low cells (right). C, analysisof ALDH activity in HER2�/CD44þ/CD24�/low versus HER2þ/CD44þ/CD24�/low cells via flow cytometryusing ALDEFLUOR staining. Asnegative control, cells wereincubated with ALDH inhibitorDEAB. SSC, side scatter. D,quantification of the ALDHexpression HER2þ/CD44þ/CD24�/low compared with HER2�/CD44þ/CD24�/low BCSCs analysisin C.

Duru et al.





RP-HPLC tandemmass spectrometry (MS-MS; Supplemen-tary Fig. S7 and Supplementary Tables S3 and S4) thatrevealed a specific protein profile differentially regulated inHER2þ/CD44þ/CD24�/low as compared with the HER2�

/CD44þ/CD24�/low BCSCs. 2-D DIGE results highlighteddifferences between these 2 samples and additional prote-

omics using RP-HPLC/MS-MS to separate trypsin-digestedpeptides identified 6,537 peptides differentially expressedin HER2þ/CD44þ/CD24�/low versus HER2�/CD44þ/CD24�/low cells. Using peptide counts determining differ-ential proteins, 499 and 182 proteins were upregulatedrespectively in HER2þ/CD44þ/CD24�/low and HER2�

B

A

Clo

no

ge

nic

su

rviv

al

Tu

mo

r v

olu

me

(m

m3)

Re

lati

ve

fil

led

ga

p (

au

)

Gap filling

Mitochondrial membrane potential Apoptosis

Clonogenic

Radiosensitivity

C

Ma

mm

os

ph

ere

/d

ish

Inv

as

ive

ce

lls

/tra

ns

we

ll

Tumor sphere formation Matrigel invasion

Tumorigenicity

ΔΨm

sham 5 Gy

Ap

op

toti

c c

ells (

%)

12.00

10.00

8.00

6.00

4.00

2.00

0.00

3.0

2.5

2.0

1.5

1.0

0.5

0.0

HER2–

** **

** ****

****

***

HER2+

HER2–

4.54.03.53.02.52.01.51.00.50.0

350

300

250

200

150

100

50

0

250

200

150

100

50

0

120

100

80

60

40

20

0

4.03.53.02.52.01.51.00.50.0

0.2

0.18

0.16

0.14

0.12

0.1

0.08

0.06

0.04

0.02

0

HER2+

HER2–

HER2+

HER2–

HER2+

HER2–

HER2+

HER2–

HER2–

HER2–

HER2+

HER2+

HER2+

HER2–

HER2+

HER2–

HER2+

0 Gy 5 Gy 0 Gy 5 Gy

Figure 3. Enhanced radioresistance and aggressiveness in HER2þ/CD44þ/CD24�/low BCSCs. A, mitochondrial membrane potential (left) andapoptosis (right) were measured in HER2þ/CD44þ/CD24�/low and HER2�/CD44þ/CD24�/Low BCSCs treated with or without 5 Gy IR for 24 hours.Data represent meanþ SE of 3 independent experiments and statistical significance was evaluated as �, P < 0.05; ��, P < 0.01. B, enhancedradioresistance in HER2þ/CD44þ/CD24�/low cells treated with sham (0 Gy) or 5 Gy of IR is tested by gap-filling and clonogenic survival assays.The gap-filling rate (left) was calculated as the ratio of the filled gap at 96 hours compared with 0 hour. The clonogenic survival (right) ofHER2þ/CD44þ/CD24�/Low BCSCs was evaluated by counting the number of colony formation (more than 50 cells) 14th day after cells were treatedwith 5 Gy of IR (n ¼ 6 and ��, P < 0.01; additional data are shown in Supplementary Fig. S5). C, the aggressiveness of HER2�/CD44þ/CD24�/low

(HER2�) and HER2þ/CD44þ/CD24�/low (HER2þ) BCSCs was further analyzed by Matrigel invasion assay (left), tumor sphere formation (middle,n ¼ 6; ��, P < 0.01; scale bar, 50 mm), and in vivo tumorigenesis (right). Tumor numbers and volumes were measured in NOD/SCID mice2 weeks after the inoculation of 500 cells from each BCSC population in 2 opposite sites (HERþ, left and HER�, right) of the animal as depictedby arrows.






/CD44þ/CD24�/low cells (cutoff-fold change � 2). Thesepeptides and proteins were further grouped according totheir cellular functions and a representative list was pre-

sented according to functions of tumor metastasis, mTORsignaling, apoptosis, mitochondrial function, Supplemen-tary and redox balancing (Supplementary Table S3).

*

Invasiv

e c

ells/t

ran

sw

ell

0

50

100

150

200

250

300

350

400

1 2 3 4 5 6

HER2siRNA - - s s + +

5 Gy - + - + - +

** **

Scrambled siRNA HER2 siRNA

A

B

C

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

Clo

no

gen

ic s

urv

ival

HER2siRNA + - + - 5 Gy IR - - + +

+ - + - - - + +

HER2+/CD44+/CD24–/low

5 G

y

Sh

am

*

**

0

5

10

15

20

25

0 24 48 72 96 (h)

No siRNA Scrambled

HER2 siRNA

0

5

10

15

20

25

0 24 48 72 96 (h)0

10

20

30

40

50

60

70

Sham 5 Gy

Rela

tive f

ille

d g

ap

(au

) ** **

Rela

tive g

ap

(au

)

Sham 5 Gy

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

Herceptin 5 Gy

Clo

no

gen

ic s

urv

ival

**

**

D

Rela

tive g

ap

(au

)

No

siR

NA

HE

R2 s

iRN

A

Scra

mb

led

siR

NA

HER2

β-actin

Figure 4. Inhibition of HER2 blocks the aggressiveness and radioresistant phenotypes of HER2þ/CD44þ/CD24�/low BCSCs. A, left, depicted images ofMatrigel invasion assays of HER2þ/CD44þ/CD24�/low BCSCs at 72 hours after 5 Gy IR and treatment with scrambled or specific HER2 siRNA. Middle,the specific capacity of HER2 siRNA to reduce HER2 expression in HER2þ BCSCs. Right, quantification of the number of cells invading the membrane pertranswell. Data represent mean þ SE of n ¼ 6 and statistical analysis was conducted with �, P < 0.05; ��, P < 0.01. B, represents the gap-filling valuesdetermined at different time points after cell's injury (0–96 hours) in sham (0 Gy) and irradiated cells (5 Gy) and in cells treated with specific or scrambledsiRNA against HER2. Right, mean þ SE of the gap-filling rate calculated from n ¼ 6 independent experiments; ��, P < 0.01. C and D, the effect oftargeting HER2 signaling pathway on aggressiveness and radiosensitivity of BCSCs. HER2þ/CD44þ/CD24�/low were treated either with siRNA (C; n ¼ 3;�, P < 0.05; ��, P < 0.01) or with anti-HER2 antibody, Herceptin (D; 10 mg/mL for 5 days, n ¼ 3; ��, P < 0.01). The clonogenic sensitivity to IR was evaluatedfollowing sham or 5 Gy IR (mean þ SE; n ¼ 3).

Duru et al.





Cross-talk betweenHER2 and STAT3 inHER2þ/CD44þ/CD24�/low BCSCsAmong the key signaling elements and effectors involved

in tumor proliferation and metastasis found in our proteinexpression profiling (Supplementary Table S3), the expres-sions of STAT3 and Src were detected in HER2þ/CD44þ/CD24�/low but not in the HER2�/CD44þ/CD24�/low

BCSCs (Supplementary Table S3). Because HER2 is knownto activate STAT3 through both JAK2- and Src-dependentmanners (32), resulting in tumor aggressiveness, we con-firmed the coexpression of HER2 and STAT3 in HER2þ/CD44þ/CD24�/low but not in HER2�/CD44þ/CD24�/low

BCSCs by Western blotting (Fig. 6A), and immunofluores-cence and IHC analyses (Fig. 6C and D). Reporter geneanalysis showed increased activity of STAT3 promoter onlyin HER2 expressing BCSCs (Fig. 6B). As shown in Fig. 6D,the overexpression of HER2 (green membrane) in radio-resistant cancer cells is strongly associated with high levelsof STAT3 (red cytoplasmic); and STAT3 level is low incells that are not expressing HER2 protein. InterestinglyHER2þ-positive human specimen showed similar pattern

of coexpression of HER2 and STAT3. Taken together, thesedata suggest a possible strong connection between HER2and STAT3 signaling pathways. Furthermore, using theMetaCore Version 6.9 program, we created the HER2–STAT3 signaling network consisting of all effectors that aredirectly linked to both HER2 and STAT3. This networkrevealed c-Src kinase that was expressed in HER2�

/CD44þ/CD24�/low BCSCs (Supplementary Table S3) asone of the mediators in the HER2–STAT3 connection (Fig.6E). Interestingly, recently Korkaya and colleagues reportedthat STAT3 activation might account for Herceptin resis-tance in HERþ breast cancer cells due to the expression andsecretion of IL-6 (33). Thus, interleukin-6 (IL-6) could beone of the factors mediating the radioresistance of CSCs inHER�/low breast cancers. Further investigation of theHER2–STAT3 signaling network and other HER2-linked effectorsand pathwaysmay generate additionalmechanistic insightsto understand the aggressive phenotype of CSCs.

DiscussionIncreasing evidence supports the concept that the tumor

bulk is consisted of heterogeneous population and poten-tially contains tumor-initiating cells or CSCs (34, 35). Here,we identified HER2-expressing CD44þ/CD24�/low BCSCsfrom both in vivo and in vitro irradiated HER2�/low breastcancer cells and tumor samples from patients with breastcancer. Compared with the HER2-negative BCSCs, HER2þ/CD44þ/CD24�/low cells showed a more aggressive pheno-type and in vivo tumorigenesis with the enhanced resistanceto radiation. Cells coexpressingHER2 and CD44weremorefrequently detected in recurrent than the primary tumors inpatients with breast cancer. Our results further provide aunique group of proteins in HER2þ/CD44þ/CD24�/low

cells governing therapy outcomes including metastasis,mTOR signaling, apoptosis, mitochondrial function, redoxbalancing, DNA repair, and HER2–STAT3 prosurvival net-work. These results suggest that HER2-expressing BCSCsmay be effective targets to treat recurrent tumors.

Although CSCs isolated from many tumors show anincreased tumor-initiating ability and invasiveness, not allCSCs are able to reside and proliferate at sites of metastasis(36). These results raise an important question aboutwhether or not CSCs are themselves a heterogenic popula-tion. Using patient-derived xenograft tumors, different radi-ation sensitivitieswere detected in theCSCpopulation (37),supporting that not all BCSCs are radioresistant. HER2-positive breast cancer are resistant to an array of therapieswith a high risk of local relapse and recurrence (38) andblocking HER2 expression or using specific agents targetingHER2 signaling pathway (trastuzumab, lapatinib, andothers) improve the cancer control (16). However, thisbenefit is not only reported for the HER2þ tumors, but alsofor patients with HER2�/low status (39, 40), suggesting thatacquired resistance due to HER2 induction could developunder anticancer treatments. In fact, we have shown thatHER2 expression is inducible in HER2�/low breast cancercells via NF-kB–mediated HER2 promoter transactivation,

Tu

mo

r n

um

be

r

FISH

40

35

30

25

20

15

10

5

0 IHC

HER2

(Moderate)

HER2 (–)

HER2 (+)

12/21

11/13 4/6

HE

R2-C

D44

Co

ex

pre

ss

ion

(%

)

A

B

9080706050403020100

Primary tumor Recurrent tumor Tumor with

unknown

clinical status

Figure 5. Increased number of HER2þ/CD44þ cells in recurrent breastcancer. A, HER2 expression status was assessed in breast cancersamples thatwerediagnosed asprimary, recurrent, or unknown status byFISH and IHC. Blue, HER2 þ (IHC 2þ and 3þ) or amplified (FISH, HER2:centromere 17 signal ratio >2.2); green, HER2� (IHC 0) or nonamplified(FISH, HER2:centromere 17 signal ratio <1.8); red, HER2 equivocal(IHC 1þ and FISH, HER2:centromere 17 signal ratio¼ 1.8–2.2). B, data ofIHC analysis for the expression of HER2 and CD44 in tumors harvestedfrom patients with breast cancer. Positive cells are depicted with arrows.HER2�/CD44� tumor was assigned if no HER2þ/CD44þ cells weredetected. Positive tumors were assigned if at least 1 cluster of HER2þ/CD44þ was detected (additional staining results are shown inSupplementary Fig. S6).






A

HER2

STAT3

B

BCSCs: HER2+ HER2– BCSCs: HER2+ HER2–

Lu

cif

era

se a

cti

vit

y

0

20,000

40,000

60,000

80,000

100,000

120,000

HER2 X40 STAT3 X40

D

C

HER2+ cells

HER2– cells β-Actin

Red: STAT3

Green: HER2

E

General receptor

General kinase

Generic binding protein

Protein kinase

Generic phosphatase

Transcription factor

Positive effect

Negative effect

G-protein-coupled receptor

Receptor with enzymatic

activity

Figure 6. HER2 andSTAT3 cross-talk in HER2þ/CD44þ/CD24�/low BCSCs. A, HER2 and STAT3 protein expression levels were further determined byWesternblot analysis in theHER2þ/CD44þ/CD24�/low andHER2�/CD44þ/CD24�/low BCSCs. B, increased STAT3 transcriptional activity in HER2þ/CD44þ/CD24�/low

compared with HER2�/CD44þ/CD24�/low BCSCs. STAT3 luciferase reporters (52) were transfected in HER2þ/CD44þ/CD24�/low and HER2�/CD44þ/CD24�/low cells and luciferase activity was measured 24 hours after transfection (n ¼ 3; ��, P < 0.01). C, colocalization of HER2 and STAT3 expression inradioresistant cancer cells and (D) HER2þ human breast specimen. E, a putative HER2–STAT3 cross-talk signaling network for the aggressiveness of HER2þ/CD44þ/CD24�/lowBCSCs. For the interactionbetweenHER2andSTAT3 signals, a cluster of factors searched from thedatabaseofMetaCoreVersion 6.9wasidentified to interact with both STAT3 and HER2. This network causing a cross-talk between HER2 and STAT3 may play a critical role for the overallaggressiveness of the HER2 expressing BCSCs in breast tumors with HER2�/low status.

Duru et al.





and the induced HER2 activation is responsible for theresistance to radiation (20). Our present data suggest thatHER2-expressing BCSCs grow more aggressively thanHER2-negative BCSCs under genotoxic conditions. Thus,tumor cells surviving a course of anticancer radiotherapy arelikely enriched with more aggressive cancer cells expressingHER2.A dynamic repopulation process may be initiated in

tumors under chemo- and/or radiotherapy (5, 41, 42). Inagreement with reported BCSC-resistant phenotype, BCSCswere increased in the surviving fraction of HER2�/lowMCF7cells and xenograft tumors after exposure to multiple dosesof irradiation (Fig. 1D). Together with an increased fre-quency of HER2þ/CD44þ in the recurrent tumors (Fig. 5AandB), these results raised the possibility that breast tumorsoriginally diagnosed as HER2�/low status (mainly by HER2transcript enhancements),may be induced to express a highlevel of HER2 protein. Thus, the radioresistant fractionwithin the CD44þ/CD24�/low population could beenriched because of HER2 expression, an oncogene well-recognized in tumor resistance, metastasis, and tumorrelapse (10, 11, 43). In this regard, HER2 protein expressioncould be more enhanced, whereas HER2 gene copies mayremain constant. These results may explain the additionalcurative benefit gainedwith trastuzumabas a coadjuvant forpatients initially classified as HER2� candidates (39, 40). Itis highly possible that genotoxic agents that activate NF-kB,may result in a selection of BCSCs with the feature ofHER2þ/CD44þ/CD24�/low in the HER2�/low breast cancer.In agreement with the low population of CSCs (44), we

found that 1.5% to 2.0% of the radioresistant MCF7/C6cells contain the feature of CD44þ/CD24�/low in which,9.8% was detected as HER2þ/CD44þ/CD24�/low cells (Fig.2). The HER2þ/CD44þ/CD24�/low BCSCs showed 4- to 5-fold increase in cell invasion, tumor sphere formation withan enhanced in vivo tumorigenesis (Fig. 3C). Although thetumor-initiating capacity of CD44þ/CD24�/low cells isolat-ed fromboth primary patient tumors and immortalized celllines is well established, our data suggested that CD44þ/CD24�/low cells fail to grow tumors when HER2 is notexpressed (HER2þ/CD44þ/CD24�/low vs. HER2�/CD44þ/CD24�/low cells, Fig. 3C; ref. 18). Importantly,tumors with HER2 protein expression were obviouslyincreased in the recurrent breast cancer tissues thanthe primary lesions, and the feature of HER2þ/CD44þ/CD24�/low was more frequently detected in recurrenttumors as compared with the primary tumors (Fig. 5).Therefore, it is highly possible that a small fraction of BCSCswith elevated de novoHER2 expression in HER2�/low breastcancer has the survival advantage under genotoxic condi-tion, such as ionizing radiation or anticancer chemothera-py, and thus will allow repopulation after a course oftreatment. However, we could not exclude the possibilitythatHER2 transactivation is preferably induced in a specificfraction of BCSC population under genotoxic stress that isresponsible for tumor repopulation.It has been reported that CD44 and HER2 interaction

promotes human ovarian tumor (45). Clinically, although

CD44þ/CD24�/low BCSC phenotype has been linked tobasal-type breast cancer tumors, particularly in BRCA1-inherited cancers, this phenotype does not correlate withthe clinical outcome (46), suggesting that, besides thefeature of CD44þ/CD24�/low, specific prosurvival networksare associated with CSC resistance. ALDH, a key BCSCmarker, also present in only about 30% of tumors, furtherdivide the CD44þ/CD24�/low population into highlytumorigenic ALDHþ/CD44þ/CD24�/low that is capable ofgenerating tumors as low as 20 cells; whereas ALDH�/CD44þ/CD24�/low show reduced tumorigenesis (31).Unlike CD44þ/CD24�/low, the ALDH phenotype correlateswith clinical outcome, tumor grade, and the status of HER2and Ki67. Korkaya and colleagues reported that HER2 over-expression enhances the ALDH activity (18) andDiehn andcolleagues found that ALDHþ/HER2 cells are more tumor-igenic than ALDH� cells (6), further confirming the linkbetween ALDH activity, HER2 expression and CSCs. In thisstudy, we found that ALDH activity is elevated in the HER2-expresing CD44þ/CD24�/low cells (Fig. 2C and D), suggest-ing that this radioresistant subpopulation of BCSCs is ableto activate both HER2 and ALDH. Thus, ALDHþ/HERþ andtheir potential downstream effectors may provide morespecific sets of targets to treatmetastatic and recurrent breastcancer, which needs to be further investigated.

To identify key factors causing the resistant phenotype ofHER2þ/CD44þ/CD24�/low cells, our proteomics data fromLC/MS-MS revealed a comprehensive protein profile inHER2þ/CD44þ/CD24�/low versus HER2�/CD44þ/CD24�/low BCSCs (Supplementary Table S3). The strongprosurvival and antiapoptotic factors are believed to be themajor mechanism the resistance of CSCs to radio- andchemotherapy (47, 48). Although many of these proteinsare already known for cell survival, such as DNA repair(Supplementary Table S3; refs. 47, 49), angiogenesis (41),cell adhesion and proliferation (50), elements involved inmetastasis, mTOR signaling, mitochondrial ATP genera-tion, and redox balancing seem to be required for theenhanced aggressiveness inHER2-expressing BCSCs. A low-ered rate of apoptosis in CSCs (50) with enhanced mito-chondrial function and DNA repair ability contributes tothe BCSC survival. Among key proteins and effectorsdetected in the in the HER2þ/CD44þ/CD24�/low versusHER2�/CD44þ/CD24�/low BCSCs profile, we found thatSTAT3 was expressed in HER2þ/CD44þ/CD24�/low but notin HER2�/CD44þ/CD24�/low BCSCs (SupplementaryTable S3). Because HER2 is known to activate STAT3through both JAK2- and Src-dependent manners (32) andSTAT3 promotes the microRNA expression and chemore-sistance in CD44-activated head and neck cancer cells (51),our data on the coactivation of HER2 and STAT3 furtherillustrate a HER2–STAT3 signaling network related to theaggressiveness ofHER2-expressing BCSCs (Fig. 6D). Furtherinvestigation of theHER2–STAT3 network and otherHER2-linked pathways may generate additional mechanisticinsights to understand the development of aggressive CSCs.

In summary, these results have important implications asthey suggest that HER2-overexpressing BCSCs are causally






associated with the aggressive phenotype and radioresis-tance of HER2�/low breast cancer cells. The ability ofHER2þ/CD44þ/CD24�/low to display an enhanced aggres-siveness suggests that the coexpressionofHER2andCD44 isa unique feature of BCSCs in HER2�/low breast cancer. TheHER2-associated prosurvival signaling network includingthe STAT3 pathway could be enhanced because of a sub-stantial HER2 gene transactivation during or afteranticancer therapy. Therefore, further characterization ofHER2-initiated pathways in the therapy-resistant CSCs,especially the HER2–STAT3 cross-talk (Fig. 6E) identifiedin this study, may generate new targets for the control ofrecurrent and metastatic tumors.

Disclosure of Potential Conflicts of InterestM.S. Wicha has ownership interest (including patents) in Oncomed

Pharmaceuticals and is a consultant/advisory board member of OncomedPharmaceuticals and Verastem. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: N. Duru, D. Candas, B.A. Chromy, D.R. Spitz,J.J. LiDevelopmentofmethodology:N.Duru,M. Fan,D.Nantajit, B.A. Chromy,D.R. SpitzAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.):N.Duru, D. Nantajit, Y.Wen, K. Xiao, A. Eldridge,B.A. Chromy, S. Li, D.R. Spitz

Analysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): N. Duru, D. Candas, C. Menaa,H.-C. Liu, Y. Wen, A. Eldridge, B.A. Chromy, S. Li, D.R. Spitz, M.S. Wicha,J.J. LiWriting, review, and/or revision of the manuscript: N. Duru, M. Fan, D.Candas, C. Menaa, D. Nantajit, Y. Wen, A. Eldridge, B.A. Chromy, S. Li, D.R.Spitz, K.S. Lam, J.J. LiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases):M. Fan, D. Candas, A. Eldridge, B.A.Chromy, S. Li, D.R. Spitz, J.J. LiStudyusupervision: M. Fan, D. Candas, B.A. Chromy, D.R. Spitz, J.J. Li

AcknowledgmentsThe authors thank Drs. Terry D. Oberley at the University of Wiscon-

sin-Madison, William C. Dewey at the University of California SanFrancisco, and members of our laboratory for valuable suggestions anddiscussions; and Khatereh Motamedchaboki at the Proteomics Facilityat Sanford-Burnham Medical Research Institute for assistance in massspectrometry analysis.

Grant SupportThis work was supported byNIH grants CA133402, CA152313 (to J.J. Li),

and CA133114 (to D.R. Spitz) as well as the Department of Energy Office ofScience Grant DE-SC0001271 (to J.J. Li).

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

Received May 4, 2012; revised October 11, 2012; accepted October 14,2012; published OnlineFirst October 22, 2012.

References1. Dowsett M, Harper-Wynne C, Boeddinghaus I, Salter J, Hills M, Dixon

M, et al. HER-2 amplification impedes the antiproliferative effects ofhormone therapy in estrogen receptor-positive primary breast cancer.Cancer Res 2001;61:8452–8.

2. Massarweh S, Osborne CK, Jiang S, Wakeling AE, Rimawi M,Mohsin SK, et al. Mechanisms of tumor regression and resistanceto estrogen deprivation and fulvestrant in a model of estrogenreceptor-positive, HER-2/neu-positive breast cancer. Cancer Res2006;66:8266–73.

3. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.Prospective identification of tumorigenic breast cancer cells. ProcNatlAcad Sci U S A 2003;100:3983–8.

4. Wicha MS. Targeting breast cancer stem cells. Breast 2009;18(Suppl3):S56–8.

5. Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/CD44þ breast cancer-initiating cells to radiation. J Natl Cancer Inst2006;98:1777–85.

6. Diehn M, Cho RW, Lobo NA, Kalisky T, Dorie MJ, Kulp AN, et al.Association of reactive oxygen species levels and radioresistance incancer stem cells. Nature 2009;458:780–3.

7. GuoG,Wang T, GaoQ, Tamae D,Wong P, Chen T, et al. Expression ofErbB2 enhances radiation-induced NF-kappaB activation. Oncogene2004;23:535–45.

8. Li Z, Xia L, Lee ML, Khaletskiy A, Wang J, Wong JYC, et al. Effectorgenes altered in MCF-7 human breast cancer cells after exposure tofractionated ionizing radiation. Radiat Res 2001;155:543–53.

9. Olayioye MA. Update on HER-2 as a target for cancer therapy:intracellular signaling pathways of ErbB2/HER-2 and family members.Breast Cancer Res 2001;3:385–9.

10. Haffty BG, Brown F, Carter D, Flynn S. Evaluation of HER-2 neuoncoprotein expression as a prognostic indicator of local recurrencein conservatively treated breast cancer: a case-control study. IntJ Radiat Oncol Biol Phys 1996;35:751–7.

11. Holbro T, Beerli RR, Maurer F, Koziczak M, Barbas CF III, Hynes NE.The ErbB2/ErbB3 heterodimer functions as an oncogenic unit: ErbB2

requires ErbB3 to drive breast tumor cell proliferation. Proc Natl AcadSci U S A 2003;100:8933–8.

12. WangSC, Zhang L, Hortobagyi GN, HungMC. Targeting HER2: recentdevelopments and future directions for breast cancer patients. SeminOncol 2001;28:21–9.

13. Li YM, Pan Y, Wei Y, Cheng X, Zhou BP, Tan M, et al. Upregulation ofCXCR4 is essential for HER2-mediated tumor metastasis. Cancer Cell2004;6:459–69.

14. Arpino G,Wiechmann L, Osborne CK, Schiff R. Crosstalk between theestrogen receptor and theHER tyrosine kinase receptor family: molec-ular mechanism and clinical implications for endocrine therapy resis-tance. Endocr Rev 2008;29:217–33.

15. Massarweh S, Osborne CK, Creighton CJ, Qin L, Tsimelzon A,Huang S, et al. Tamoxifen resistance in breast tumors is drivenby growth factor receptor signaling with repression of classicestrogen receptor genomic function. Cancer Res 2008;68:826–33.

16. Jones KL, Buzdar AU. Evolving novel anti-HER2 strategies. LancetOncol 2009;10:1179–87.

17. Kurokawa H, Arteaga CL. Inhibition of erbB receptor (HER) tyrosinekinases as a strategy to abrogate antiestrogen resistance in humanbreast cancer. Clin Cancer Res 2001;7:4436s–42s.

18. Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulates themammary stem/progenitor cell population driving tumorigenesis andinvasion. Oncogene 2008;27:6120–30.

19. MagnificoA, Albano L, Campaner S, Delia D,Castiglioni F,Gasparini P,et al. Tumor-initiating cells of HER2-positive carcinoma cell linesexpress the highest oncoprotein levels and are sensitive to trastuzu-mab. Clin Cancer Res 2009;15:2010–21.

20. Cao N, Li S, Wang Z, Ahmed KM, Degnan ME, Fan M, et al. NF-kappaB-mediated HER2 overexpression in radiation-adaptive resis-tance. Radiat Res 2009;171:9–21.

21. Song G, Zhang Y, Wang L. MicroRNA-206 targets notch3, activatesapoptosis, and inhibits tumor cell migration and focus formation. J BiolChem 2009;284:31921–7.

Duru et al.





22. Dontu G, AbdallahWM, Foley JM, Jackson KW, ClarkeMF, KawamuraMJ, et al. In vitro propagation and transcriptional profiling of humanmammary stem/progenitor cells. Genes Dev 2003;17:1253–70.

23. Al-Hajj M, Clarke MF. Self-renewal and solid tumor stem cells. Onco-gene 2004;23:7274–82.

24. Clarke MF. A self-renewal assay for cancer stem cells. Cancer Che-mother Pharmacol 2005;56(Suppl 1):64–8.

25. Corzett TH, Fodor IK, Choi MW, Walsworth VL, Turteltaub KW,McCutchen-Maloney SL, et al. Statistical analysis of variation inthe human plasma proteome. J Biomed Biotechnol 2010;2010:258494.

26. Corzett TH, Fodor IK, Choi MW, Walsworth VL, Chromy BA, Tur-teltaub KW, et al. Statistical analysis of the experimental variationin the proteomic characterization of human plasma by two-dimen-sional difference gel electrophoresis. J Proteome Res 2006;5:2611–9.

27. Brill LM, Motamedchaboki K, Wu S, Wolf DA. Comprehensive prote-omic analysis of Schizosaccharomyces pombe by two-dimensionalHPLC-tandem mass spectrometry. Methods 2009;48:311–9.

28. Guo G, Yan-Sanders Y, Lyn-Cook BD, Wang T, Tamae D, Ogi J,et al. Manganese superoxide dismutase-mediated gene expressionin radiation-induced adaptive responses. Mol Cell Biol 2003;23:2362–78.

29. Jo M, Lester RD, Montel V, Eastman B, Takimoto S, Gonias SL.Reversibility of epithelial-mesenchymal transition (EMT) induced inbreast cancer cells by activation of urokinase receptor-dependent cellsignaling. J Biol Chem 2009;284:22825–33.

30. Yin H, Glass J. The phenotypic radiation resistance of CD44þ/CD24(-or low) breast cancer cells is mediated through the enhanced acti-vation of ATM signaling. PLoS ONE 2011;6:e24080.

31. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, BrownM, et al. ALDH1 is a marker of normal and malignant human mammarystem cells and a predictor of poor clinical outcome. Cell Stem Cell2007;1:555–67.

32. Ren Z, Schaefer TS. ErbB-2 activates Stat3 alpha in a Src- and JAK2-dependent manner. J Biol Chem 2002;277:38486–93.

33. Korkaya H, Kim GI, Davis A, Malik F, Henry NL, Ithimakin S, et al.Activation of an IL6 inflammatory loop mediates trastuzumab resis-tance in HER2þ breast cancer by expanding the cancer stem cellpopulation. Mol Cell 2012;47:570–84.

34. Al-Ejeh F, Smart CE, Morrison BJ, Chenevix-Trench G, Lopez JA,Lakhani SR, et al. Breast cancer stem cells: treatment resistance andtherapeutic opportunities. Carcinogenesis 2011;32:650–8.

35. Croker AK, Allan AL. Inhibition of aldehyde dehydrogenase (ALDH)activity reduces chemotherapy and radiation resistance of stem-likeALDH(hi)CD44 (þ) humanbreast cancer cells. Breast Cancer Res Treat2011;133:75–87.

36. Sheridan C, Kishimoto H, Fuchs RK, Mehrotra S, Bhat-Nakshatri P,Turner CH, et al. CD44þ/CD24- breast cancer cells exhibit enhancedinvasive properties: an early step necessary for metastasis. BreastCancer Res 2006;8:R59.

37. Zielske SP, Spalding AC, Wicha MS, Lawrence TS. Ablation of breastcancer stem cells with radiation. Transl Oncol 2011;4:227–33.

38. Gonzalez-Angulo AM, Stemke-Hale K, Palla SL, Carey M, Agarwal R,Meric-Berstam F, et al. Androgen receptor levels and association withPIK3CA mutations and prognosis in breast cancer. Clin Cancer Res2009;15:2472–8.

39. Perez EA, Reinholz MM, Hillman DW, Tenner KS, Schroeder MJ,Davidson NE, et al. HER2 and chromosome 17 effect on patientoutcome in the N9831 adjuvant trastuzumab trial. J Clin Oncol2010;28:4307–15.

40. Ardavanis A, Kountourakis P, Kyriakou F, Malliou S, Mantzaris I,Garoufali A, et al. Trastuzumab plus paclitaxel or docetaxel in HER-2-negative/HER-2 ECD-positive anthracycline- and taxane-refractoryadvanced breast cancer. Oncologist 2008;13:361–9.

41. BaoS,WuQ,McLendonRE,HaoY,ShiQ,HjelmelandAB, et al. Gliomastem cells promote radioresistance by preferential activation of theDNA damage response. Nature 2006;444:756–60.

42. Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models andconcepts. Annu Rev Med 2007;58:267–84.

43. Slamon DJ. Studies of the HER-2/neu proto-oncogene in humanbreast cancer. Cancer Invest 1990;8:253.

44. Al-Hajj M, Becker MW, Wicha M, Weissman I, Clarke MF. Thera-peutic implications of cancer stem cells. Curr Opin Genet Dev2004;14:43–7.

45. Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC. Interactionbetween the adhesion receptor, CD44, and the oncogene product,p185HER2, promotes human ovarian tumor cell activation. J BiolChem 1997;272:27913–8.

46. Honeth G, Bendahl PO, Ringner M, Saal LH, Gruvberger-Saal SK,Lovgren K, et al. The CD44þ/CD24- phenotype is enriched in basal-like breast tumors. Breast Cancer Res 2008;10:R53.

47. Ropolo M, Daga A, Griffero F, Foresta M, Casartelli G, Zunino A, et al.Comparative analysis of DNA repair in stem and nonstem glioma cellcultures. Mol Cancer Res 2009;7:383–92.

48. Johannessen TC, Bjerkvig R, TysnesBB. DNA repair and cancer stem-like cells–potential partners in glioma drug resistance? Cancer TreatRev 2008;34:558–67.

49. Frosina G. DNA repair in normal and cancer stem cells, with specialreference to the central nervous system. Curr Med Chem 2009;16:854–66.

50. Bjerkvig R, Johansson M, Miletic H, Niclou SP. Cancer stem cells andangiogenesis. Semin Cancer Biol 2009;19:279–84.

51. Bourguignon LY, Earle C, Wong G, Spevak CC, Krueger K. Stemcell marker (Nanog) and Stat-3 signaling promote MicroRNA-21expression and chemoresistance in hyaluronan/CD44-activatedhead and neck squamous cell carcinoma cells. Oncogene 2012;31:149–60.

52. Yu CR, Wang L, Khaletskiy A, Farrar WL, Larner A, Colburn NH, et al.STAT3 activation is required for interleukin-6 induced transformation intumor-promotion sensitive mouse skin epithelial cells. Oncogene2002;21:3949–60.






2012;18:6634-6647. Published OnlineFirst October 22, 2012.Clin Cancer Res Nadire Duru, Ming Fan, Demet Candas, et al. Isolated from HER2-Negative Breast Cancer CellsHER2-Associated Radioresistance of Breast Cancer Stem Cells

Updated version

10.1158/1078-0432.CCR-12-1436doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2012/10/22/1078-0432.CCR-12-1436.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/18/24/6634.full#ref-list-1

This article cites 51 articles, 17 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/18/24/6634.full#related-urls

This article has been cited by 8 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/18/24/6634To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-12-1436

http://clincancerres.aacrjournals.org/content/suppl/2012/10/22/1078-0432.CCR-12-1436.DC1

http://clincancerres.aacrjournals.org/content/18/24/6634.full#ref-list-1

http://clincancerres.aacrjournals.org/content/18/24/6634.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/18/24/6634


her2-associated radioresistance of breast cancer stem ... · 6634 clin cancer res; 18(24) december...

Documents