Epigenetic Silencing of THY1 Tracks the EGFR Signaling in a … · Corresponding Author: Bianca Maria Veneziani, Department of Molecular Medicine and Medical Biotechnology, University

Signal Transduction

Epigenetic Silencing of THY1 Tracks theAcquisition of the Notch1–EGFR Signaling in aXenograft Model of CD44þ/CD24low/CD90þ

Myoepithelial CellsMicaela Montanari1,2, Maria Rita Carbone1, Luigi Coppola1, Mario Giuliano2, Grazia Arpino2,Rossella Lauria2, Agostina Nardone1,3, Felicia Leccia4, Meghana V. Trivedi3,5,Corrado Garbi1, Roberto Bianco2, Enrico V. Avvedimento1, Sabino De Placido2,6, andBianca Maria Veneziani1,6

Abstract

The surface glycoprotein THY is a marker of myoepithelialprecursor cells, which are basal cells with epithelial–mes-enchymal intermediate phenotype originating from theectoderm. Myoepithelial precursor cells are lost duringprogression from in situ to invasive carcinoma. To definethe functional role of Thy1-positive cells within the myoe-pithelial population, we tracked Thy1 expression in humanbreast cancer samples, isolated THY1-positive myoepithelialprogenitor cells (CD44þ/CD24low/CD90þ), and establishedlong-term cultures (parental cells). Parental cells were usedto generate a xenograft model to examine Thy1 expressionduring tumor formation. Post-transplantation cell cultureslost THY1 expression through methylation at the THY1locus and this is associated with an increase in EGFR and

NOTCH1 transcript levels. Thy1-low cells are sensitive to theEGFR/HER2 dual inhibitor lapatinib. High THY1 expressionis associated with poorer relapse-free survival in patientswith breast cancer. THY1 methylation may track the shift ofbipotent progenitors into differentiated cells. Thy1 is a goodcandidate biomarker in basal-like breast cancer.

Implications: Our findings provide evidence that THY1expression is lost in xenografts due to promoter methylation.Thy1-low cellswith increased EGFR andNotch1 expression areresponsive to target therapy. Because DNA methylation isoften altered in early cancer development, candidate methyl-ation markers may be exploited as biomarkers for basal-likebreast cancer.

IntroductionThe human breast gland is a ductal tree covered with a

monolayer of polarized epithelial cells whose basal surfacelies on contractile myoepithelial cells that are confined by thebasement membrane and surrounded by an interstitial stroma.Myoepithelial cells originate from the ectoderm and are basalcells, namely, cells in the basal position adjacent to the base-

ment membrane (1). Interest in basal cells was stimulated aftermolecular gene profiling divided breast cancer into five intrin-sic subtypes, one of which displays basal-like gene expression(2). Basal-like breast cancers are generally aggressive (3) andmost are triple-negative, that is, they test negative for estrogenreceptor (ER), progesterone receptor (PgR), and HER2 (HER;ref. 4). Treatment of patients with basal-like triple-negativebreast cancer (TNBC) is challenging because of the heteroge-neity of the disease and the absence of well-defined druggabletargets (5).

In breast cancer, myoepithelial cells are considered tumorsuppressors because they inhibit epithelial cell growth andinvasion (6), and because their oncosuppressive function dis-appears during progression from in situ to invasive carcinoma(7). Disappearance of the basement membrane and of themyoepithelial cell layer distinguishes invasive from in situcarcinomast (8), and the gene expression profiles of myoe-pithelial cells associated with in situ cancer are distinct fromthose in normal breast (9). The signals that initiate thesechanges are unknown, although it is recognized that tumor-associated fibroblasts and myofibroblasts counteract the tumorsuppressor function of myoepithelial cells by promotingtumorigenesis (10, 11) and cancer progression (11).

THY1 (also known as "CD90") is a surface glycoprotein of25–28 kDa (12) that is expressed on the cytoplasmic mem-brane of diverse cell types (13). The structural gene for human

1Department of Molecular Medicine and Medical Biotechnology, University ofNaples Federico II, Naples, Italy. 2Department of Clinical Medicine and Surgery,OncologyDivision, University of Naples Federico II, Naples, Italy. 3Lester and SueSmith Breast Center and Dan L. Duncan Comprehensive Cancer Center, andDepartment of Medicine, Baylor College of Medicine, Houston, Texas. 4CEINGE-Biotecnologie Avanzate, Naples, Italy. 5Department of Pharmacy Practice andTranslational Research, University of Houston College of Pharmacy, Houston,Texas. 6Oncotech, School of Medicine and Surgery, University of NaplesFederico II, Naples, Italy.

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

Corresponding Author: Bianca Maria Veneziani, Department of MolecularMedicine and Medical Biotechnology, University of Naples Federico II, Via S.Pansini 5, Napoli 80131, Italy. Phone: 39-081-7463758; Fax: 39-081-7463321;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0324

�2018 American Association for Cancer Research.

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THY1 lies on the long arm of chromosome 11 (11q23.3;ref. 14). Thy1 triggers a variety of cellular functions, namely,proliferation, differentiation, wound repair, and apoptosis. Inlung cancer, Thy1 differentiates between malignant pleuralmesothelioma and lung carcinoma (15). In melanoma, itcontributes to metastasis seeding by mediating the adhesionof melanoma cells to endothelial cells (16). Thy1 has beenassociated with tumor suppression in human ovarian cancer(17). It is also a cancer stem cell marker in esophageal cancer(18), high-grade gliomas (19), and hepatocarcinoma (20). Inbreast cancer, Thy1-expressing cells are undifferentiated cancerprogenitor/stem cells (21). Thy1 is upregulated in the epithe-lial-to-mesenchymal transition (EMT) core signature (22).

Moreover, it mediates the interactions of breast cancer stemcells with tumor-associated macrophages to maintain and rein-force the cancer stem cell state (23).

We previously demonstrated that sorted CD44þ/CD24low cellsdisplay differential expression of surface markers that identifyheterogeneous myoepithelial phenotypes (24). Among thesesurface markers, THY1/CD90 was commonly found highlyexpressed. In this study, we have investigated the relevance ofThy1 as a tracer biomarker of myoepithelial precursor cells also inrelation to receptor profile, in our model of sorted breast cancerstem/progenitor cells. We show that xenotransplantation ofCD44þ/CD24low/CD90þ myoepithelial cells in mouse reducesTHY1 expression through methylation of THY1 in conjunctionwith the acquisition of the Notch1–EGFR signaling.

Materials and MethodsCell lines and materials

Cell lines MCF-7, MDA-MB-231 (MDA), BT474, and MCF10Awere from ATCC. HER2-18 cells (25) were kindly provided byDr. R. Schiff. Cells were maintained in standard medium consist-ing of minimal essential Dulbecco/Ham F12 (1:1) (DMEM/F12;Sigma-Aldrich) supplemented with 2mmol/L glutamine (Sigma-Aldrich), 1% penicillin/streptomycin (Life Technologies), 15mmol/L HEPES (Sigma-Aldrich), and 5% FBS and kept at 37�Cin a humidified atmosphere of 5% CO2. Cell cultures wereroutinely checked for Mycoplasma with Hoechst 33258 (Sigma-Aldrich) staining; Mycoplasma-negative cell lines were usedfor experiments. Adherent and nonadherent 24-well ultra-low–

binding plates were used (Corning). FBS was purchased fromGibco (Invitrogen). The monoclonal anti-pancytokeratin andanti-vimentin antibodies were purchased from Sigma-Aldrich.Multicolor flow cytometry was performed with antihumanmAbsthat were conjugated with phycoerythrin (PE), fluorescein iso-thiocyanate (FITC), PE-Cy7 (PE-Cy7), or Alexa Fluor 647. Phy-coerythrin-conjugatedmAbs against CD10, CD29, CD49f, CD61,and FITC-conjugated mAbs against CD49b, CD90, CD227,CD324, and CD326 were from BD Biosciences and BD Pharmin-gen; PE-conjugated mAbs against CD133 were from MiltenyiBiotec; Alexa Fluor 647–conjugated mAbs against CD24 andPE-Cy7–conjugated mAbs against CD44 were from BioLegend.

Ethics and study designResidual breast cancer and paired normal specimens were

collected, after informed consent, from patients undergoingsurgery for breast cancer at the Azienda Ospedaliera Universi-taria Federico II (Naples, Italy). Nine patients with breast cancerwere recruited. Pathologic diagnosis was made based on the

histology of tumor specimens that had been examined byexperienced pathologists. Tumor histotype, size, grading, andmarkers including ERa were determined with standard proce-dures, and HER2 was determined with the Hercep Test TM(Dako). The receptor profile of human breast tumors, namely,ER status, PgR status, and HER2, are summarized in Supple-mentary Table S1. The breast cancer–intrinsic subtype wasdetermined using surrogate IHC definitions according toGoldhirsch and colleagues (26).

Sample collectionBreast cancer tissue (S#) and paired normal (N#) specimens

were collected using a biobanking standard operating procedureas reported previously (27). The samples were anonymouslyencoded to protect patient confidentiality and used according toprotocols approved by the Azienda Ospedaliera UniversitariaFederico II Ethics Committee (Ethical Committee ApprovalProtocol # 107/05). The primary objective of the approvedprotocol was to expand human breast cancer cells to characterizethe protein expression profile of in vitro cultured cells.

Primary cultures and breast cancer stem/progenitor cellsWithin 2 hours after surgery, fragmented aliquots of fresh

specimens were processed as reported previously (28). Briefly,the samples were extensively rinsed with PBS and suspended instandard culture media supplemented with 10% FBS. Afterthree cycles of differential centrifugation, cells were seededovernight in minimal DMEM/F12 medium (1:1; Sigma-Aldrich), supplemented with 2 mmol/L glutamine (Sigma-Aldrich), penicillin/streptomycin (100 mg/mL streptomycin,100 U/mL penicillin), 15 mmol/L HEPES (Sigma-Aldrich), and5% FBS. After exposure to trypsin (0.25% in 1 mmol/L EDTA;trypsin–EDTA solution, Invitrogen) for 2 minutes at 37�C, thefloating aggregates were transferred to 24-well plates and cul-tured in standard medium (DMEM/F12 þ 0.5% FBS) for 21–30days at 37�C in a humidified atmosphere of 5% CO2. Cells werecontinuously passaged with trypsin–EDTA until only the tumorepithelial cell population remained. The epithelial origin of thecells was confirmed by Western blot analysis with monoclonalanti-pancytokeratin antibody (Supplementary Fig. S1). Multi-ple vials of cells were cryopreserved. Frozen cells were thawed,allowed to adhere, and harvested within 15–20 days in stan-dard medium before sorting.

Flow cytometry and sortingFlow cytometry experiments were performed as reported pre-

viously (24). Briefly, samples and control cells, harvested atsubconfluence in 100-mm dishes, were dissociated by trypsin–EDTA, counted in a hemocytometer chamber, and 2 � 106 cells/sample were incubated for 5 minutes at room temperaturewith 50 mL of FBS. Cells were washed twice with PBS and stainedat 4�C for 20 minutes with the appropriate amount of thefluorescence-labeled mAb in PBS. After staining, all samples werewashed twice with PBS, centrifuged, and suspended in 0.5 mL ofFACS buffer (FACS Flow Sheat Fluid, BD Biosciences) for FACSanalysis. To exclude dead cells, immediately before FACS acqui-sition, cells were incubated at room temperature in the darkwith a vital dye (SytoxBlue, Invitrogen). We used a four-colorflow cytometric method tomeasure the expression of themarkersbased on a flow cytometry panel in which cells were stained withanti-CD24-Alexa Fluor 647 and anti-CD44-PE-Cy7 mAb, and

Epigenetic Silencing of Thy1 in CD44þ/CD24low/CD90þ Myoepithelial Cells

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with FITC-conjugated antibodies against CD90 (Thy1) andPE-conjugated anti CD133 (prominin 1; ref. 24). FluorescenceMinus One (FMO) control, was used as a negative control. Afterwashing twice with PBS/0.5% BSA, cells were pelleted, suspendedin 300 mL of PBS/0.5% BSA, and filtered through 50-mm filters.Cell analysis and sorting were performed with a FACSAriaflow cytometer and with the FACS Diva software (BectonDickinson). A total of 10,000 to 20,000 events were recordedand analyzed in each sample run. A three gating strategy wasadopted: first, to exclude dead cells and debris, cells were gatedon a two physical parameters dot plot measuring forward scatter(FSC) versus side scatter (SSC). Then, doublets were excludedby gating cells on FSC-Height versus FSC-Area dot plots, and,finally, SytoxBlue-negative cells were gated. The levels of expres-sion of surface markers were reported as percentage of positivecells in Count versus FITC- or PE-CD histograms. For cell sorting,the cells were suspended in PBS with 2% FBS and 0.5 mmol/LEDTA, sequentially labeled with a cocktail of mAb anti-CD24-Alexa Fluor 647and anti-CD44-PE-Cy7, mixed with magneticmicrobeads and separated using a magnet. The purity of sortedcells was evaluated by flow cytometry. Sorted CD44þ/CD24low

cells were cultured with standard medium for at least four/fivepassages before experiments. The steps of isolation of CD44þ/CD24low/CD90highmyoepithelial precursor cells (K#) are summa-rized in Supplementary Fig. S2. Cultures at the fourth to fifthpassage were used for the experiments.

ImmunofluorescenceFor immunofluorescence analysis, cells were plated on glass

coverslips, fixed, immunostained at 4�C overnight with rabbitpolyclonal anti-Thy1 antibodies (H-110; SC-9163; Lot #B2514; Santa Cruz Biotechnology, Inc.) and treated for 30minutes with goat anti-rabbit IgG secondary antibody taggedwith Alexa Fluor 594 (1/300; A-11012; Lot # 1420898; LifeTechnologies). Nuclei were stained for 30 minutes at roomtemperature with 1:1 (vol/vol) Hoechst 33258 (94403, Sigma-Aldrich)/DRAQ5 (ab108410; Abcam). Immunofluorescencewas visualized using a Zeiss LSM510 Meta argon/kryptonlaser-scanning confocal microscope.

Cumulative population doubling frequencyExperiments were performed in triplicate in 24-well plates

using 2.5 � 103 cells/well. The cells, routinely cultured in 100-mm dishes, were enzymatically detached, counted, and 2.5 �103 cells/well were seeded with standard medium. Cells weremaintained in a sterile environment, and at the times indicated inthe figures, they were trypsinized, counted in a hemocytometerchamber, and replated 1:2 in new wells. Cells viability wasassessed by Trypan bluewith paired triplicates. Lineage continuityfor Thy1 in 3D culture was assessed with immunofluorescence(representative at Supplementary Fig. S1). For 2D experiments,after trypsinization, the cells were plated overnight in 5% FBS,allowed to adhere, and then switched to 0.5% FBS. The prolifer-ation rate of the cells was measured by calculating the cumulativepopulationdoubling frequency (cpdf) in continuous culture froma known number of cells using the formula Ln(No/Nn)/Ln2,where Ln is the natural log andNo andNn are, respectively, initialand final cell numbers at each subcultivation. The sum of the cpdfof the subcultivation periods provides the cumulative final num-ber of total counts.

Semiquantitative multiplex RT-PCR analysis and real-timePCR analysis

Total RNAwas isolated fromsample and control cells, and frombreast tumor tissues using TRIzol Reagent (Invitrogen) accordingto the producer's instructions. Purity of RNA was checked bymeasuring the absorbance ratio at 260/280 nm in a BeckmanCoulter spectrophotometer (Beckman Coulter) with appropriatepurity values between 1.8 and 2.0. RNA was stored at �80�C inaliquots of 50 ng/L. The integrity of RNA was assessed on astandard 1% agarose/formaldehyde gel. The reverse transcriptionof 1.5 mg of total RNA was performed with the Super Script IIIReverse Transcriptase Kit (Invitrogen) according to the manufac-turer's instructions.

Multiplex PCR was performed in 50 mL reactions using thePTC-200 Peltier Thermal Cycler (Bio-Rad) and gene-specificsets of primers, including those for the internal standard b-actin.Agarose gel electrophoresis and staining with 0.3 mg/mL ofethidium bromide (Sigma) were carried out to assess templateproducts.

Real-time PCR amplifications were carried out on a Step OneReal-Time Thermocycler (Applied Biosystems) using the iTaqUniversal SYBR Green Supermix (Bio-Rad). Experiments wereperformed in triplicate for each data point, and the expressionof housekeeping b2-microglobulin gene (B2M, forward: 50-GCAGAA TTT GGA ATT CAT CCA AT-30; reverse: 50- CCG AGT GAAGAT CCC CTT TTT-30) was used for normalization.

Primer sequences are listed in Supplementary Table S2.

Nude mice cancer xenograftsThe tumorigenicity of sorted cells was assessed by injecting the

harvested cells into immunodeficient mice. Five-week-old femaleBALB/c athymic (null/null) mice (Charles River Laboratories)were maintained in accordance with the institutional guidelinesof theUniversity ofNaples AnimalCareCommitteewelfare policy(European Commission 86/609/EEC). All the animal experi-ments were approved by the Ethics Committee of the Universityof Naples Federico II Animal Care (ethical approval protocol# 83). Adherent harvested K197 cells were enzymatically disso-ciated, counted, diluted in PBS, mixed 1:1 with 200 mL Matrigel(CBP), and injected orthotopically in the fourthmammary fat padof triplicate mice, as reported previously (29). We injected 1 �102, 1 � 103, 1 � 104, 1 � 105, 1 � 106 of the K197 cells into thefourth mammary fat pad of immunodeficient mice. The experi-ment was performed twice. Tumor volume (cm3) was measuredwith calipers and calculated with the formula p/6 � largestdiameter � (smallest diameter)2. Within 8 weeks, the tumors(size: 1–3 cm3) were excised, digested with a trypsin/collagenasemixture, and plated for in vitro growth. The steps of isolation ofCD44þ/CD24low/CD90-Thy1-low cells (Topo9) are summarizedin Supplementary Fig. S3. The homogeneity of the cultures wasconfirmed with flow cytometry analysis of ten surface markers(Table 1). Cultures at their fourth/fifth passage were used for theexperiments.

5-Aza-20-Deoxycytidine treatmentTHY1-positive (K197) and THY1-low (Topo9) cells, 5 �

105cells/cell type, were seeded in 100-mm cell culturedishes (Corning) with standardmedium. After an initial 24 hoursof incubation, the cells were exposed to 5 mmol/L 5-aza-20-deoxycytidine (5-AZA-dC; Sigma) for 12, 24, 36, 48, 72, and96 hours. The medium was renewed every 24 hours. Control

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cultures lacking 5-AZA-dC treatment were incubated in the iden-tical culture condition. At the time indicated, the cells wereharvested for total RNA extraction.

Methylation-sensitive amplified polymorphismGenomic DNA was extracted with phenol/chloroform tech-

nique (30). For measurement of Thy promoter methylationstatus, 1 mg of DNA was digested overnight at 37 C with HpaIIor MspI (50 U/1mg DNA, Fermentas) restriction enzymes. DNAwas recovered by phenol/chloroform extraction and ethanolprecipitation, and resuspended in DNase/RNase free water.HpaII sensitivity was evaluated by amplifying a 276-bp frag-ment containing 4 CG upstream of the Thy promoter (position:�2328, �2297, �2259, and �2190 with respect to the firstATG). Methylation status was assessed by analyzing the effi-ciency of fragment amplification exposed to digestion (HpaII orMspI) on 2% agarose gel and quantified by real-time PCR.Amplification was performed on a PTC-200 Peltier ThermalCycler (Bio-Rad) through 40 PCR cycles using the followingtemperature profile: 95�C for 40 seconds, 61�C for 40 seconds,72�C for 1 minute, and one final elongation step at 72�C for10 minutes. All reactions were preceded by a primary denatur-ation step at 95�C for 5 minutes. Real-time PCR amplificationswere carried out on a Step One Real-Time thermocycler(Applied Biosystems) using the iTaq Universal SYBR GreenSupermix (Bio-Rad). Cycling conditions were: one cycle at 95�Cfor 5 minutes, followed by 40 cycles of 95�C for 15 seconds,61�C for 20 seconds, and 72�C for 30 seconds. Experimentswere performed in triplicate for each data point. The followingprimers were used for Thy-1 DNA amplifications: forward 50-CCAATGCGGGACCGCCTTCTCTTCC-3; reverse 50-GTCTTGCAT-GGGCGCCTGACGGCG-30.

Western blot analysisProtein preparations were obtained by lysing samples in 50

mmol/L Tris (pH 7.5), 150mmol/L NaCl, 1%Nonidet P40, 0.1%Triton, 1 mmol/L EDTA, 10 mg/mL aprotinin, and 100 mg/mL

phenylmethylsulfonylfluoride. Protein concentration was mea-sured by the Bio-Rad Protein Assay (Bio-Rad). Twenty-five–microgram aliquots were electrophoresed through 8%–15% SDSpolyacrylamide gels. After transfer onto nitrocellulose mem-branes (Hybond-C pure; Amersham Italia), the membrane wasstainedwith Ponceau S (Sigma) to evaluate the success of transfer,and to locate the molecular weight markers. Free protein-bindingsites were blocked with nonfat dry milk and Tween-20/TBSsolution. The membranes were washed, stained with specificprimary antibodies and then with secondary antisera, conjugatedwith horseradish peroxidase (1:3,000; Santa Cruz Biotechno-logy). Antibodies were: Ab anti-E-Cadherin (1:1,000, Cell Signal-ing Technology); Ab anti-Notch1 (C-20): sc-6014-R (1:200,Santa Cruz Biotechnology); Ab anti-Fibronectin (P1H11): sc-18825 (1:100, Santa Cruz Biotechnology); Ab anti-Fibronectin(EP5): sc-8422 (1:200, Santa Cruz Biotechnology); Ab anti-ERalfa (F-10):sc-8002 (1:200, Santa Cruz Biotechnology). Theluminescent signal was visualized with the ECL Western blot-ting Detection Reagent Kit (Amersham Italia) and quantified byscanning with a Discover Pharmacia scanner equipped with aSun Spark Classic Workstation. Expression levels were calcu-lated as the relative expression ratio compared with b-actin ortubulin using Image J (ImageJ.nih.gov).

Mammosphere formation assay and growth in soft agarCells were dissociated and seeded, 1,000 cells/well, in ultra-low

attachment 24-well plates (Corning) in DMEM/F12 plus 0.5%FCS medium, as reported previously (31). The medium wasrenewed twice weekly. Mammospheres were cultured for 15 daysand their diameter measured under an Axiovert 40 C invertedmicroscope (Zeiss) equipped with a Canon Powershot A640camera (Zeiss). Digital images were analyzed with AxioVisionsoftware (Zeiss). For colony growth in soft agar, cells were trypsi-nized, counted, and 104 cells/dishwere plated in 60-mm triplicatedisheswith 0.3%agar on a 0.5%agar (Type I, Sigma) bottom layerwith DMEM/F12 containing 0.5% FBS. Colonies, cultured for60 days, were counted in 10 fields per dish. The fields to be

Table 1. Surface marker percentage expression (%) and mean fluorescence intensity (MFI) in myoepithelial progenitors before (K197) and after transplantation(Topo9) and in cell lines

K197a Topo9a MCF7b MCF10Aa

% MFI % MFI % MFI % MFI

CD227-MUC1 0.0 49 0.6 41 90.2 605 42.6 227CD324-E-cadherin 0.5 45 0.8 52 6.0 90 3.3 148CD326-EpCAM 2.0 32 1.9 51 100.0 1377 72.1 425CD10-CALLA 8.4 65 15.0 20 18.7 8 82.4 388CD29-b1 Integrin 100.0 1474 100.0 1373 100.0 1300 100.0 3526CD49b-a2 Integrin 100.0 597 100.0 960 99.9 560 99.9 1996CD49f-a6 Integrin 100.0 519 100.0 554 23.8 66 100.0 6044CD61-b3 Integrin 60.0 11 33.0 18 0.4 6 25.2 121CD90-THY1 100.0 1065 11.0 76 0.2 45 58.6 208CD133-Prominin1 16.0 35 18.0 25 68.0 29 28.0 70

Percentage (%) Pearson R score P-value Significance at P < 0.001 CorrelationK197 vs Topo9 0.9818 1.5E-05 Yes Strong positiveK197 vs MCF7 0.3412 0.3688 No WeakK197 vs MCF10A 0.7603 0.0174 No Positive

MFI Pearson R score P Significance at P < 0.001 CorrelationK197 vs. Topo9 0.9659 9.7E-05 Yes Strong positiveK197 vs. MCF7 0.3263 0.3914 No WeakK197 vs. MCF10A 0.5006 0.1698 No Moderate Positive

NOTE: Numbers of positive cells are mean percentage of average of triplicates of three (a) or two (b) experiments; SD, not reported, was < 10%.






counted were ID-numbered fields on a 7 � 7 horizontal–verticaltransparency grid 60 mm in diameter. The same ID fields werecounted for all dishes. Results are reported asmean� SEMof threedifferent experiments performed in duplicate.

Cell viability assayCell viability experiments were performed as described previ-

ously (32). Cells were seeded at 2,500 cells/well, in 24-well plates,treated with the reported concentrations of lapatinib for 7 days,and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazoliumbromide (MTT) assay according to the manufac-turer's instructions (Sigma-Aldrich). The percentage of absor-bance of treated samples versus untreated is reported as a per-centage of viable cells/controls. Experiments were performedthree times; values represent means� SD from triplicate samplesfor each treatment.

Kaplan–Meier curvesKaplan–Meier curves were generated using the Kaplan–Meier

plot software and a public database of microarray datasets(probes: 213869_x_at (Thy1-CD90);211551_at (EGFR, ERBB,ERBB1; http://kmplot.com/analysis; ref. 33). Kaplan–Meier plotswere generated after averaging theprobes. For the analysis, eligiblepatients were divided according to the median expression value,and ERa-negative/HER2-negative cases were included. P valuewas determined by log-rank test.

Statistical analysisFlow cytometry, cell counting, sphere formation assay, and RT-

PCR experiments were carried out 2–3 times and found to bereproducible. Human tissue samples were not pooled; eachsample served as its own control. Values are presented as mean� SEM of multiple experiments, each experiment was performedat least in triplicate, or as mean � SD of triplicates when arepresentative experiment is shown. The statistical significancebetween two groups was determinedwith the Fisher exact test andmultiple group comparisons were made with ANOVA, asreported. Pearson's correlation coefficient was used to calculater. GraphPad software was used for all statistical analyses.

Ethical approvalAll procedures performed in studies involving human partici-

pants were in accordance with the ethical standards of the insti-tutional and/or national research committee and with the 1964Helsinki declaration and its later amendments or comparableethical standards.

Availability of data and materialsThe datasets used and/or analyzed during this study are avail-

able from the corresponding author on reasonable request. Thedatasets generated and/or analyzed during the Kaplan–Meiercurves are available in the [kmplot.com] repository (http://kmplot.com/analysis/).

ResultsCD44þ/CD24low/THY1þ myoepithelial progenitor cells

In our previous studies we profiled heterogeneous THY1-expressing myoepithelial phenotype in breast carcinomas. Toinvestigate the functional role of THY1-expressing cells in exper-imental models, we isolated breast cancer cells that displayed

features of stem/progenitor cells from fresh surgical breast tumortissues. Nine tumor specimens (S#) were chosen from our storedbreast cancer collection of fresh frozen tissues belonging tovarious molecular subclasses of breast tumors: S40, S43, S79,S88, S193 and S197 were luminal breast cancers; S66 was HER2-positive; and S77 and S90 were triple-negative (SupplementaryTable S1). Breast cancer stem cells are identified by one ormore ofthe following features: a CD44þ/CD24low phenotype, mammo-sphere formation in vitro, and ability to form new tumors whenxenografted into immunodeficient mice (34, 35). We establishednine primary cultures of breast cancer; to avoid fibroblasts, cellswere continuously passaged until only the tumor epithelial cellpopulation remained (Supplementary Fig. S1). Immunofluores-cence experiments with antibodies against Thy1/CD90 showed amixed population of THY-positive and THY1-negative cells in theprimary cultures with a percentage ranging from 27% to 58% ofThy1/CD90–stained cells versus nonstained cells (Fig. 1A). Fromeach culture, we sorted the CD44þ/CD24low cells. THY1/CD90expression assessed by immunofluorescence on cells harvested for10 days after sorting, showed expression of THY1 on 90%–100%of the sorted CD44þ/CD24low cells and on 98%–100% of cellsmaintained in culture for three months (Fig. 1A and B). To assessthe ability of the sorted cells to form spheres, we seeded 100 cells/well per each cell culture under nonadherent conditions andfound that all the nine cultures formed mammospheres in low-attachment plates (Supplementary Fig. S2). CD44/CD24 expres-sion on cultures of cells stabilized for 1–3months under adherentconditions (dot plots at Supplementary Fig. S2) was measured bycalculating the mean fluorescence intensity (MFI). The analysisconfirmed high expression of CD44 molecules per cell (1,153 �MFI� 7,967) and a low signal for CD24 (1�MFI� 72) in all thebreast cancer cell cultures. TheMFI for CD44/CD24 in BT474 (16/4,078), HER2-18 (123/250), MCF7 (15/79), MCF10A (914/27),and MDA-MB-231 (4,906/10) cell lines served as control (Fig.1C). Tomeasure the doubling frequency of cells dissociated frommammospheres in adherent (2D) and nonadherent conditions(3D), we enzymatically detached cells to obtain single-cell sus-pensions, seeded in ultra-low attachment or in adherent platesand counted the cells each month thereafter. After twomonths ofculture, growth andpropagationwas arrested in breast cancer cellsin low adhesion conditions, whereas the paired adherent culturegrewwith a doubling time ranging between 2 and 3 days (lowest)and 7 and 10 days (highest) across cell types. The cumulativepopulation doubling frequency (cpdf) at 120 days was 4.7� 105

in cell cultures with a high growth rate (K197), and 2.8 � 105 incell cultures with a low growth rate (K77). Themedian cpdf of thenine CD44þ/CD24low/Thy1þ cell cultures (K40, K43, K66, K77,K88, K79, K90, K193, and K197) at 120 days was 3.26 � 105 for2D cultures and 1.12� 105 for 3D cultures (Fig. 1D). In all cases,long-term culturing of Thy1-positive cells under nonadherentconditions delayed the proliferation. In fact, the mean doublingtime was 15–18 days during the initial two months, and wasarrested thereafter.

CD44þ/CD24low/THY1þ cells are basal cells with intermediateepithelial–mesenchymal phenotype

To evaluate whether Thy1 expression was stable in cells sortedfor stem features, we measured THY1messenger RNA (mRNA) instabilized cultures. As shown in Fig. 2A and B, THY1 was highlyexpressed in all nine THY1-positive cell cultures established fromthe CD44þ/CD24low population of primary culture, and low in

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MCF7, BT474, HER2/18, MCF10A and MDA-MB231 cells. Mes-senger RNA levels were consistent with the high expression of theprotein; indeed, the percentage of THY1/CD90 expression, mea-sured by immunophenotyping, ranged from 87.9% to 100%(Fig. 2C). To estimate the number of Thy1 molecules per cell,we calculated theMFI of Thy1/CD90 expression, and found that itwas low in all the cell lines BT474 (211), HER2-18 (189), MCF7(45), MCF10A (208), and MDA-MB-231 (81) (45 �MFI � 211)but high in all the nine cell cultures (1,065 � MFI � 8,117;Fig. 2D). THY1 expression per cell was significantly higher versusthe control cell lines (P ¼ 0.0078).

To investigate whether the CD44þ/CD24low/THY1þ popula-tions of sorted cells were tumorigenic, we injected the K197 cellsinto the fourth mammary fat pad of immunodeficient mice. Asfew as 1 � 104 CD44þ/CD24low/THY1þ cells generated tumors

with a calculated frequency of tumorigenic cells that resulted in 1/4,326 cells. Each of the dilutions that generated tumors showed alatency and a size when excised, at day 60, that correlated with thenumber of cells injected (R ¼ 0.949, P < 0.05). Indeed, the meantumor volume of triplicates was 950� 190mm3 per 1� 104 cellsinjected, 1,500 � 250 mm3 per 1 � 105 cells, and 2,800 �260 mm3 per 1 � 106 cells (Fig. 3A). The excised tumors (mea-suring 1–3 cm3) were minced and dissociated by enzymaticdigestion, and the cells derived were maintained in long-termculture. A signal for THY1 mRNA was detectable in carcinomaspecimens S197 (Fig. 3B, left) and absent in either normal N197tissue (Fig. 3B, middle) and xenotransplanted specimen Topo9(Fig. 3B, right). THY1expression levels were confirmed by real-time PCR (Fig. 3C). We next performed flow cytometry to inves-tigate whether the cell cultures derived from the transplanted

Figure 1.

Breast tissue specimens containTHY1-expressing cells. A, Percentageof THY1/CD90-positive cells asassessed by immunofluorescencein primary cultures, in CD44þ/CD24low cells cultured for 10 daysafter sorting and in long-term (3months) cultures. Numbers ofpositive cells were counted from 7–10representative fields of three slidesper cell culture (n ¼ 9 cultures). B,Immunofluorescence of THY1/CD90on two representative cell cultures(K43 and K197) at three months aftersorting; scale bar, 50 mm. C, Meanfluorescence intensity of CD24 (lightgray) and CD44 (dark gray) markersexpressed on the cell surface ofbreast cancer stem/progenitor cells(K40, K43, K66, K77, K88, K79, K90,K193, K197) and cell lines (BT474,HER2–18, MCF7, MCF10A, and MDA-MB231; log scale range: 1–10,000); theSE, not reported ongraph,was� 10%.For the analysis, cells were culturedfor 15–20 days in standard medium,then detached, counted, and 1–3 �106 cells were used. For each tube,20,000 events were recorded andanalyzed. D, Cumulative populationdoubling frequency (cpdf) of Thy1-positive cells. The median cpdf of thenine CD44þ/CD24low/THY1þ cellcultures (K40, K43, K66, K77, K88,K79, K90, K193, and K197 is plotted.Triplicate dishes of each cell culture,plated at 2.5 � 103 cells/well, werecultured on adherent dishes (lightgray) and as floating mammosphereson nonadherentwells (dark gray) andcounted in a hemocytometerchamber for the time indicated. Aftercounting, the cells, maintained in asterile environment, were replated 1:2in new wells. The cpdf was calculatedwith the formula Ln(No/Nn)/Ln2.Statistical analysis was done byx2 squares (P ¼ 0.069). Error barsindicate the SEM of triplicates.






tumors preserved the stem cell characteristics of the implantedcells. This analysis showed that, consistent with the implantedCD44þ/CD24low population, tumors generated by this popula-tion recapitulated the CD44þ/CD24low profile (Fig. 3D) and theMFI (Fig. 3E).Wemeasured themRNA expression to compare thereceptor profiles of the implanted cells (K197 cells) with those ofthe cells isolated and cultured from mouse tumor tissue (Topo9cells). Like the parental cells, transplanted cells expressed lowlevels of ERa, PgR, and HER2 (Fig. 3F), which indicates subtyperelationship with the implanted cells. Immunoblot for ER andHER2 (Supplementary Fig. S4) confirmed that both K197 andTopo9 cells do not express potentially functional levels of thesemarkers.

To further investigate the relationship between the preimplan-tation population (K197) and the population obtained from thecultivation in vitro of the CD44þ/CD24low cells from implantedtumors (Topo9), we profiled the phenotype by measuring thepercentage of expressing cells and the MFI of three epithelialmarkers (MUC1, E-cadherin, and EpCAM), and seven stem/mesenchymal markers (CALLA, b1integrin, a2 integrin, a6 integ-rin, b3 integrin, Thy1, andprominin 1). As reported in Table 1, thepercentage of expressing cells and the MFI of the markers of the

transplanted cells, except Thy1, overlapped that of the parentalcells. There was a strong positive correlation in percentage termsbetween the transplanted and the parental cells (P < 0.001), aweak correlation between the transplanted and theMCF7 cell line,and a possible, albeit not significant correlation between thetransplanted and the MCF10A cells. MFI data were in agreementwith the percentage data, and statistical analysis confirmed thehigh correlation between the transplanted and the parental cells(P<0.001). Like theparental cells, the transplanted cells preservedan a2b1 Integrin-CD44high/MUC1-EpCAM-CD24low phenotype,but THY1 expression was lost (Fig. 3G). To verify this finding,we measured THY1 mRNA expression in the cells obtained aftertransplantation, and found a consistent decrease of THY1mRNAin the cultures derived from the transplanted tumors versus thepreimplantation cells (Fig. 3H). qRT-PCR normalized to B2Mconfirmed these differences (P < 0.05; Fig. 3I).

Xenotransplantation silences Thy1 via promoter methylationThe cellular plasticity between the epithelial andmesenchymal

states is ascribed to epigenetic changes of cancer cells that result incellular heterogeneity (36). In metastatic breast cancer, the THY1gene is silenced bymethylation in those tumors that are hormone

Figure 2.

Thy1 expression in CD44þ/CD24low

breast cancer cells. A, RT-PCR of THY1mRNA (235 bp) expression of theK40,K43, K66, K77, K88, K79, K90, K193,and K197 myoepithelial progenitorcells. Cell lines MCF7, MCF10a, BT474,HER2/18, and MDA-MB-231 served asreferences. Standardization was onthe basis of b-actin cDNA levels. B,Densitometry RT-PCR for Thy1 inMCF7, MCF10a, BT474, HER2/18, andMDA-MB231, and in the K40, K43, K66,K77, K88, K79, K90, K193, and K197cells. C, Percentage of surface proteinexpression of THY1-CD90 in MCF7,MCF10a, BT474, HER2/18, andMDA-MB231, and in the myoepithelialprogenitor cells K40, K43, K66, K77,K88, K79, K90, K193, and K197. Thelevels of expression of surfacemarkerswere reported as percentage ofpositive cells in Count versus FITC- orPE-CD histograms. D, Proteinexpression of THY1-CD90 calculatedas mean fluorescence intensity (MFI)on breast cancer stem/progenitorcells (K40, K43, K66, K77, K88, K79,K90, K193, and K197) and cell lines(MCF7, BT474, HER2-18, MCF10A, andMDA-MB-231); (log scale range: 1–10,000) is plotted. The SEM oftriplicates, not reported on the graph,was � 10%. Statistical analysis ofdifferences of breast cancer stem/progenitor cells versus control cellswas done by one-way ANOVA(P ¼ 0.0078).

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Figure 3.

Thy1 expression in xenotransplanted cells. A, CD44þ/CD24low THY1-positive K197 cells were assayed for the ability to form tumors after injection into mice. Five-week-old female BALB/c athymic (null/null) mice were injected orthotopically in the fourth mammary fat pad, with K197 cells resuspended in Matrigel/PBS. Tumorvolume (mm3) was measured by calculating p/6� largest diameter� (smallest diameter)2, and correlated with the number of cells injected (R¼ 0.949, P < 0.05).Within 60 days, tumors were excised (250 mm3), digested with trypsin/collagenase mixture and mouse-derived tumor cells were harvested for in vitro growth(Topo9 cells). B, RT-PCR of THY1 mRNA (235 bp) expression in carcinoma specimens S197 (left), paired normal N197 (middle), and xenotransplanted specimenTopo9 (right) tissue. Standardization with b-actin cDNA levels. C, qRT-PCR of THY1 expression in carcinoma specimens S197, paired normal N197 andxenotransplanted specimen Topo9 tissue (S-Topo9). Experiments were performed in triplicate for each data point, and the expression of housekeeping b2-microglobulin gene (B2M) was used for normalization. D, Cells derived from xenotransplanted tumors recapitulate the CD44þ/CD24low profile of the grafted stem/progenitor cells. Dot plots of flow cytometry analysis of parental K197 and Topo9 cells. Cells were cultured for 15 days in standard medium, and 1 � 106 cells werestained for the flow cytometric analysis of CD44PE-Cy7A and CD24-Alexa Fluor 647. The expression of each antigen is represented on a frequency distributionhistogram (count vs. FITCor PE signal). The expression of the twomarkers is presented on abiparametric dot plot CD44-PE-Cy7 vs. CD24-AlexaFluor647 for each celltype. Vertical and horizontal markers delineate the quadrants used to identify the CD44/CD24 subsets and were set with the appropriate FMO control.E, The histogram reports the mean fluorescence intensity (MFI) of CD24 (light gray) and CD44 (dark gray) expressed on the cell surface of the indicated cells (logscale range: 1–10,000); SE, not reported on the graph, was � 10%. F, Topo9 cells, cultured from xenotransplanted tumors, are triple-negative, as the grafted K197THY1-positive cells. RT-PCR for mRNA of ERa (441 bp), PgR (121 bp) and HER2 (420 bp) of K197 and Topo9 cells. Standardization was based on b-actin cDNA levels.Protein expression determined withWestern blot analysis at Supplementary Fig. S4.G, Percentage of THY1/CD90 surface protein as determined by flow cytometrywith CD90-FITC. H, RT-PCR of THY1 mRNA (235 bp) expression in K197 (left), and xenotransplanted Topo9 (right) cells. Standardization with b-actin cDNAlevels. I, qRT-PCR of THY1 expression in MDA-MB231 cell line, in K197 and xenotransplanted Topo9 cells. Experimentswere performed in triplicate for each data point,and the expression of housekeeping b2-microglobulin gene (B2M) was used for normalization.






receptor–negative and basal-like (37). To determine whetherThy1 expression is relevant in breast cancer, we interrogatedpublic databases. Data mining of TCGA 450K DNA methyla-tion, at the human pan-cancer methylation database, MethHC(http://methhc.mbc.nctu.edu.tw/php/index.php), confirmed theenrichment of THY1 hypermethylation in human breast carci-noma compared with normal breast tissue (P � 0.005; Sup-plementary Fig. S5). To determine whether the expression ofThy1 in myoepithelial precursors is subject to epigenetic reg-ulation, we explored the possibility that methylation mightdetermine the disappearance of Thy1 when progenitors areimplanted in mice. Thus, we treated the Topo9 cells withmethylation inhibitors and found that THY1 expression wasrestored in a time-dependent fashion. Parental K197 cells andtransplanted Topo9 cells were cultured, for various times (Fig.4A), with the methylation inhibitor 5-aza-20-deoxycytidine (5-AZA-dC) and subjected to RT-PCR. THY1was not methylated inparental K197 cells (Fig. 4A, left blot) and highly methylated inuntreated Topo9 cells (Fig. 4A, right blot, lane 0). Treatmentwith the demethylation agent 5-AZA-dC time dependentlyrestored THY1 gene expression in Topo9 cells. The latter effectprogressively increased from 0 to 96 hours (Fig. 4A, right blot).

These data support the hypothesis that methylation contributesto THY1 silencing. To test this hypothesis, we analyzed theTHY1 promoter using the methylation-sensitive amplifiedpolymorphism (MSAP) technique. HpaII (CpG methylationinsensitive) digestion resulted in a 276-bp fragment inTHY1-low cells (Topo9), but not in THY1-positive (K197) orcontrol cells (C; Fig. 4B). At quantitative real-time PCR analysisofHpaII/MspI sensitivity, amplification rates (64% in THY1-lowcells vs. 0.54% and 0.18% in THY1-positive and control cells,respectively) were significantly correlated with methylationstatus (Fig. 4C). These results indicate that methylation occurson the THY1 loci in THY1-low Topo9 cells.

THY1-low cells activate the Notch1–EGFR programWe examined THY1-low cells for cellular functions (EMT-

marker expression, growth in 3D) that are critical when cells areallowed to adhere to a substrate (38, 39). The protein expressionprofile of the EMT markers CK18, CK19, CK5, vimentin, aSMA,and fibronectin (Fig. 5A) showed that THY1-low cells acquired apartial epithelial phenotype. In fact, these cells expressed CK18,CK19, and vimentin, reducedfibronectin, but they did not expressCK5 or aSMA.

Figure 4.

Methylation on the THY1 loci intransplanted cells. A, THY1-positive(K197) and THY1-low (Topo9) cellswere cultured, for the time indicated,with the methylation inhibitor 5-aza-20-deoxycytidine (5-AZA-dC) andqRT-PCR for THY1 (235 bp) wasperformed. Treatment with thedemethylation agent 5-AZA-dC did notaffect the methylation status of Thy1 inparental K197 THY1-positive cells (leftblot) whereas it restored theexpression of Thy1 mRNA in Topo9THY1-low cells (right blot). This effectprogressively increased from 0 to 96hours. Standardization with b-actin. B,Methylation analysis of the THY1promoter in K197 and Topo9 cells andcontrol cells. AfterHpaII/MspI digestion(blocked/unblocked by CpGmethylation, respectively), onlymethylated (undigested) genomicDNA produced a fragment. PCR-basedassay of HpaII/MspI sensitivity showeda 276-bp fragment only in lowexpressing/ hypermethylated samples(Thy1-low Topo9 cells) and not in highexpressing/hypomethylated controls(Thy-positive K197 cells and controlcells); no product is seen in MspIdigestions. C, Real-time analysis ofHpaII/MspI sensitivity. The differencesin amplification rates (64% in THY1-negative cells vs. 0.54% and 0.18% inTHY1-positive cells and control cells,respectively) relates to themethylation(mC) status. � indicates the differencebetween THY-low cells versusTHY1-positive cells and control cellswith P < 0.05; �� indicates thedifference between control cells versusTHY1-positive cells with P < 0.01.

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http://methhc.mbc.nctu.edu.tw/php/index.php


Figure 5.

THY1-methylated cells are Notch1-EGFR–expressing cells. A,Western blot analysis for EMT markers CK18, CK19, CK5, vimentin, and aSMA; THY1-low cells (Topo9)acquired CK18, CK19, and vimentin, and lost CK5 and aSMA. Standardization with tubulin. B, Representative phase-contrast microphotographs of culturesin 3D of mammospheres of THY1-positive (K197, left) and aggregates of THY1-low (Topo9, right) cells. Scale bar, 100 mm. C, Colony growth in soft agar ofTHY1-positive (K197) and THY1-low (Topo9) cells. Colonies were counted in 10 fields per dish on a horizontal/vertical grid. The mean results of two experiments oftriplicates dishes are plotted. D, RT-PCR for EGFR (348 bp) and NOTCH1 (520 bp) in K197 and Topo9 cells. Standardization with b-actin. E, Western blot analysisfor EGFR and cleaved NOTCH1 (NICD). Standardization with tubulin. F, RT-PCR for THY1 mRNA (235 bp) and EGFR (348 bp); G, and immunoblots for THY1and EGFR protein expression in K197 and Topo9 cells treated, 24 hours after seeding, without (�) and with (þ) 5 mmol/L 5-AZA-dC for 48 hours. Standardizationwith b-actin and tubulin, respectively. H, Lapatinib inhibited the proliferation of EGFR-expressing THY1-low (Topo9) cells. The effect of treatment on thesurvival of control cells (BT474, MCF7, and MDA-MB-231), parental THY1-positive (K197) and xenotransplanted THY1-low (Topo9) cells is expressed as percentageof viable cells over control. All cells were seeded at 2,500 cells/well, treated with lapatinib 0.1 mmol/L (gray) and 1 mmol/L (black) for seven days andanalyzed by the MTT assay. The percentage of absorbance of treated samples versus untreated samples is reported as a percentage of viable cells/controls.Values represent means � SD from triplicate samples for each treatment. Error bars indicate SD values. Asterisks indicate statistical significance, as determinedby two-tailed Fisher exact test (�� , two-sided P ¼ 0.0011). I, Real-time RT-PCR quantification of THY1, NOTCH1, and EGFR expression in six carcinoma tissuespaired with tissue used to obtain primary cultures of Thy1-positive cells; all samples were run in triplicate and normalized to B2M housekeeping.






To shed light on the growth features of THY1-low cells and toevaluate whether the cells exerted sphere-forming activity,we seeded the Topo9 cells under adherent and nonadherentconditions. In 2D cultures, THY1-low cells were smaller thanTHY1-positive cells and had and an epithelial-like morphology(Supplementary Fig. S3), the median cpdf was 6.3 � 105 at 120days, that was double that of THY1-positive cells (reported in Fig.1D). The 3D-harvested THY1-low cells in low adhesion formedaggregates in suspension instead of mammospheres (Fig. 5B). Tounderstand whether the loss of Thy1 affects growth on substrate,we performed soft-agar experiments. After seeding in semisolidmedium for 3 weeks, THY1-low Topo9 cell frequency was 4-foldhigher than Thy1-positive K197 cell (Fig. 5C), which confirmsthe greater propensity of THY1-low cells to proliferate on adhe-sion to a semisolid substrate. These observations prompted us toinvestigate on pathways involved in EMT.

To evaluate whether THY1-silencing in the grafted cellssignals a transition phenotype, we searched for signaling path-ways through which cells interact during development andtissue homeostasis. Molecular analysis had demonstrated thatboth parental K197, THY1-positive, and transplanted Topo9,THY1-low cells are triple-negative (Fig. 3F). Triple-negativebreast cancers generally express EGFR (40), which cross talkswith the Notch pathway in this setting (41). We measured thelevels of EGFR and NOTCH1 mRNA in THY1-positive andTHY1-low cells (Fig. 5D). At densitometry, EGFR mRNA wasthree times higher in Topo9 cells than in parental K197cells(grayscale: 0.62 vs. 1.93, respectively). Moreover, Topo9 cellsexpressed NOTCH1 mRNA, whereas K197 parental cells didnot (Fig. 5D). Immunoblots for EGFR and Notch1 (NICD)

confirmed that Topo9 cells express potentially functional levelsof these proteins (Fig. 5E). Further mRNA and protein analysisof Topo9 cells rescued after 48 hours with 5-AZA-dC showed aconcomitant reduction of EGFR levels together with Thy1induction (Fig. 5F-G).

Having identified activation of signaling pathways susceptibleto targeting, we investigated the ability of THY1-low cells torespond to tyrosine kinase inhibition. In gastric cancers, Thy1 isa cancer stem cell marker and trastuzumab (humanized anti-HER2 antibody) treatment of high tumorigenic gastric primarytumor models reduces the Thy1 population in the tumor massthereby suppressing tumor growth when combined with chemo-therapy (42). As HER2 has no ligand, antibodies against thisreceptor inhibit its activation by preventing heterodimerization(43) with other members of the HER2 family (44). Heterodimer-ization results in intrinsic kinase activation. We treated K197 andTopo9 cells, and BT474, MCF7 and MDA-MB231 cells withincreasing concentrations (0.7–2–5–10 mg/mL) of trastuzumaband measured cell viability with the MTT assay 4, 5, and 7 dayslater. Trastuzumab did not alter the viability of either the K197 orTopo9 cells (% of inhibition¼ 0). Because THY1-low Topo9 cellsexpress the EGFR (HER1), we tested the effect of lapatinib, areversible tyrosine kinase inhibitor that binds intracellularly andinhibits both the EGFR and HER2 activity. We found that lapa-tinib, in a concentration-dependentmanner, significantly reducedthe viability of THY1-low cells (Fig. 5H). Indeed, at concentra-tions of 0.1 mmol/L and 1 mmol/L, lapatinib reduced cell viabilityby 75% and 85%, respectively (P¼ 0.0011). These results suggestthat THY-low cell proliferation is sustained by tyrosine kinaseactivation.

Figure 6.

Low EGFR-high THY1 expression inhuman breast correlates with poorprognosis. A, Distribution of variousphenotypes with different ratio ofNotch1-EGFR/Thy1 expression in alarge collection of tumors derivedfrom cancer databases. TCGA RNA-sequencing data were extracted fromthe PCAWG website (https://www.ebi.ac.uk/gxa/) to compare combinedexpression of THY1, EGFR andNOTCH1in breast adenocarcinoma (IDC),normal-adjacent to breastadenocarcinoma, invasive lobularcarcinoma (ILC), and normal breast. Band C, Kaplan–Meier analysis ofrelapse-free survival on subjects withERa-negative HER2-negative breastcancer on the basis of THY1 (B) andEGFR (C). P value was determined bylog-rank test. The group with thelowest THY1 expression were morelikely to have a relapse-free survival.The group with highest EGFRexpression were more likely to have arelapse-free survival. Multivariateanalysis with ER (ESR1) and HER2(ERBB2) was performed. The plotswere generated using http://kmplot.com (probes: 213869_x_at (Thy1-CD90); 211551_at (EGFR, ERBB,ERBB1; ref. 33).

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https://www.ebi.ac.uk/gxa/


http://kmplot.com

http://kmplot.com


To determine whether Thy1 overepression might have clinicalrelevance in conjunction with EGFR and Notch1 expressionstatus, we analyzed our series of breast carcinoma and performedreal-time PCR experiments on the carcinoma tissue samplespaired with those used to establish primary cultures of THY1-positive cells. At qRT-PCR, we examined six tissue specimens andfound that THY1was highly expressed in breast carcinoma tissues(range 0.82–1.9), while transcript levels for EGFR and NOTCH1were very low (range 0.003–0.1; Fig. 5I).

We interrogated the Pancancer Analysis of Whole Genomes(PCAWG; https://www.ebi.ac.uk/gxa/) for the TCGA RNA-sequencing data, to compare combined expression of THY1,EGFR, and Notch1 in breast adenocarcinoma, normal-adjacentto breast adenocarcinoma, invasive lobular carcinoma, and nor-mal breast. THY1 was more highly expressed in invasive lobular(24FPKM) and invasive adenocarcinoma (18FKM) than in tissueadjacent to breast carcinoma (11FPKM) or normal breast(7FPKM). Interestingly, the levels of EGFR and NOTCH1 appearto be directly related each other, but inversely related to THY1.Indeed, EGFR in normal tissues (13 FPKM) was more than 3-foldhigher than in breast tumors (3/4 FPKM), as well NOTCH1 hadhigher levels (11 FPKM) in normal than in tumors (6/6 FPKM;Fig. 6A).

Furthermore, to situate our model within the context of abroad spectrum of "in vitro" cell lines, we interrogated Onco-mine (https://www.oncomine.org) for Neve collection of cul-tured cells (Supplementary Fig. S6). Also in this case, the resultssupport the notion of an inverse relationship between THY1and EGFR; as for Topo9 cells, the pattern with low THY1 andhigh EGFR is largely represented in most of the cell linesbroadly used to model breast cancer.

We then performed a Kaplan–Meier analysis to measurerelapse-free survival in subjects categorized ER- and HER2-nega-tive based on high/low Thy1 and EGFR expression. At the time ofanalysis, the median follow-up was 200 months (range, 0.1–200months). In this setting, high THY1 expression (THY1-high vsTHY1-low P ¼ 0.062) (Fig. 6B) as well as low EGFR expression(EGFR-high vs. EGFR-low P ¼ 0.061; Fig. 6C) identifies thosepatients with a worst relapse-free survival. Multivariate analysis ofESR1 andHER2 expression (stages I and II vs. stage III; HR, 2.815;95% confidence interval, 1.022–7.751; P ¼ 0.045) excludedinteraction of Thy1 and EGFR with ER and HER2, respectively.The analysis suggests that combined detection of Thy1 and/orEGFR expression might help to better identify subclasses ofpatients with breast cancer of basal origin.

DiscussionTumor cell heterogeneity, induced by a combination of genetic

and epigenetic events that lead to cancer cell plasticity, is one ofthe cancer features responsible for drug resistance and treatmentfailure (45). Gene expression profiling has revealed intertumorheterogeneity and identified five intrinsic breast cancer subtypes:luminal A and B (ERa- and/or PgR-positive and HER2-negative),HER2-enriched (HER2-positive), basal-like (which includes triplenegative) and normal-like, that differ in biologic, prognostic andpredictive features (2, 3, 46). In routine clinical practice, IHC isused to identify the molecular subclasses (26). Within the sub-classes, triple-negative (ER- and PgR- and HER2-negative), whichaccounts for 15%–20% of all invasive breast cancers, is the mostheterogeneous (47), has a higher risk of relapse and responds

poorly to targeted therapy (48). Eighty percent (80%) of triple-negative breast cancers harbor a signature that coincides with ahigh proportion of cells with the basal/myoepithelial phenotype(4). The "phenotypic" plasticity of cancer cells favors intratumorheterogeneity that, through the EMT, promotes the invasion anddissemination of cells, while the MET counterpart confers fitnessadvantage which enables cells to return to a highly proliferativestate andmediates tumor relapse atmetastatic sites (45). In tumorxenografts, the expansion of subclones with fitness advantages isascribed to cosegregating genomic factors, such as methylation,that, as determinants of fitness, lead to reproducible clonaldynamics (49). DNA methylation is a dynamic process thatcontributes to tumor heterogeneity (50). Because DNA methyl-ation is often altered in early cancer development, candidatemethylationmarkers may serve as prognostic or predictive factors(37). In this context, we investigated the relevance of Thy1 as abiomarker of myoepithelial progenitor to gain insight into therole of basal myoepithelial cells in breast cancer heterogeneity.

Here we isolated and harvested, from human breastcancer tissues, THY1-expressing cells with phenotypic andfunctional stem cell characteristics. When we transplantedthe (a2b1integrin-CD44)high (MUC1-EpCAM-CD24)low THY1-positivemyoepithelial progenitors in nudemice, Thy1 expressionwas lost. Recent studies on invasive breast cancer report that THY1is highly methylated in the group of HR�-Basal-like-p53mutant(37). To evaluate whether epigenetic changes modified Thy1expression, as occurs in patientswithmetastatic basal-like tumors,we treated the cells with methylation inhibitors and found thatthey time dependently restored THY1 mRNA expression. Thisresult suggested the emergence of epigenetic-induced transitingphenotypes. In agreement with data obtained with differentapproaches (7, 34, 51), in our model of human myoepithelialprogenitors, THY1-silenced cells displayed the alternative METphenotype, which resulted in a better propensity to proliferateand differentiate. This behavior, together with the finding thatTHY1-low cells, activated the Notch1-EGFR programmay explainthe rarity of tumors displaying myoepithelial features notwith-standing the ubiquity of myoepithelial cells in breast tissue. TheNotch signaling network is an evolutionarily conserved intercel-lular signaling pathway that regulates interactions between phys-ically adjacent cells. In mouse mammary cells, Notch activationincreases the proliferation potential of both bipotent and myoe-pithelial progenitor cells (52, 53). In normal breast, NOTCH1mRNA is expressed in luminal cells and its effect on lineagecommitment is irreversible (52). In breast cancer, Notch signalingpathways crosstalk with EGFR. Forced overexpression of Notch1by transfection increases EGFR expression (53), although inhibi-tion of EGFR or Notch signaling alone is not sufficient to suppresshuman breast cancer cell survival and proliferation (54). Our datasuggest that THY1methylation signals the acquisition of a cyclingepithelial phenotype (CK18-CK19-positive/CK5-aSMA-nega-tive) that activates the Notch1–EGFR pathway. Thy1 suppression,through methylation, at metastatic sites might be required forimplantation and growth of clones with fitness advantages.Intriguingly, examination on disease progression, of the SorlieBreast 2 dataset at Oncomine (https://www.oncomine.org) com-paring expression status of 107 breast carcinomas at primarysite versus 5 metastasis showed a decreased THY1 expression atmetastatic site (Supplementary Fig. S7). This observation mighthave clinical implications because high Thy1 expression mightidentify, within the heterogeneous basal-like (and TNBC)






https://www.oncomine.org

https://www.oncomine.org


subclass of breast cancer, a subset of primary tumors originatingfrom precursors/bipotent myoepithelial cells that have worstprognosis. If THY1is methylated and the loss of THY1 coincideswith the expression of EGFR, patients might have a better relapse-free survival. The tumors that we obtained after xenotransplan-tation have low levels of Thy1and high levels of EGFR andresemble those EGFR-overexpressing tumors with better progno-sis. Finally, because lapatinib inhibits the viability of THY1-lowcells, but not that of THY1-positive cells, the THY1-methylated/EGFR-expressing (THY�/EGFRþ) phenotype might define a sub-type, within the basal-like, that may benefit from tyrosine kinaseinhibition. Further studies are needed to determine the relativeimpact of these processes during cancer evolution.

ConclusionsIn our in vitro/in vivo model of stable basal breast cancer

progenitor cells in culture, Thy1 expression tracks the myoepithe-lial lineage. Quiescent myoepithelial progenitor cells are compo-nents of luminal and basal subtypes of breast cancer tissues, andcan be identified on the basis of their expression of Thy1. THY1-expressing myoepithelial progenitors are quiescent in 3D cultureand display a stable phenotypic profile of (a2b1integrin-CD44)high (MUC1-EpCAM-CD24)low that, in vivo, might beresponsible for attachment of cells to the extracellular matrix.Upon transplantation, THY1 expression is silenced by methyla-tion parallel to activation of Notch–EGFR signaling. This processmarks the emergence of differentiated clones, namely, CK18/CK19/vimentin–positive/CK5/aSMA–negative clones. The latterproliferate extensively, and, notably,might be arrested by tyrosinekinase inhibition. Collectively, our results suggest that THY1methylation may track the shift of THY1-positive bipotent pro-genitors into THY1-low differentiated cells. This behavior mayexplain the rarity of tumors with myoepithelial features, andconsidered to be of "basal" origin, such as some metaplasticcarcinomas, notwithstanding the ubiquity of myoepithelial cellsin breast tissue. An understanding of the dynamics of cellularstates in breast cancer evolution can lead to a more accurate

definition of the subtypes of breast cancer, and opens the wayto new therapeutic strategies.

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

Authors' ContributionsConception and design: G. Arpino, R. Lauria, S. De Placido, B.M. VenezianiDevelopment of methodology: M. Montanari, R. Lauria, B.M. VenezianiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):M. Montanari, M.R. Carbone, L. Coppola, G. Arpino,R. Lauria, A. Nardone, F. Leccia, C. Garbi, R. Bianco, B.M. VenezianiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):M.Montanari, G. Arpino, F. Leccia, E.V. Avvedimento,B.M. VenezianiWriting, review, and/or revision of themanuscript:M.Giuliano, G. Arpino, R.Lauria, M.V. Trivedi, R. Bianco, E.V. Avvedimento, S. De Placido, B.M. VenezianiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Montanari, M. Giuliano, B.M. VenezianiStudy supervision: R. Lauria, B.M. Veneziani

AcknowledgmentsBreast cancer tissue specimens were obtained from the Breast Cancer Tissue

Bank, developed under the auspices of the BIONCAM (Biobanca OncologicaCampania) Project and maintained by the CRPO (Centro Regionale Preven-zioneOncologica),University ofNaples "Federico II".We thank JeanAnnGilder(Scientific Communication srl) for editing the text. This work was supported byMinistero dell'Universita` e Ricerca, PRIN Grant 2015B7M39T (to S. DePlacidoand B.M. Veneziani), GrantMOVIE of the Rete delle Biotecnologie in Campania(to B.M. Veneziani), PON 03PE_00146_1 BIOBIOFAR (to R. Bianco, S. DePlacido, and B.M. Veneziani) M. Montanari is supported by a postdoctoralfellowship from POR CREME, M.R. Carbone is supported by a fellowship fromDottorato di Ricerca (PhD) in Medicina Molecolare e Biotecnologie Mediche,University Federico II of Naples, Italy.

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

Received June 21, 2017; revised September 13, 2017; accepted September 13,2018; published first September 21, 2018.

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2019;17:628-641. Published OnlineFirst September 21, 2018.Mol Cancer Res Micaela Montanari, Maria Rita Carbone, Luigi Coppola, et al. Myoepithelial Cells

+/CD90low/CD24+EGFR Signaling in a Xenograft Model of CD44− Tracks the Acquisition of the Notch1THY1Epigenetic Silencing of

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