Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast Cancer …€¦ · Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast Cancer Stem Cells In Vitro and In

Research ArticleSee related commentary by Rao, p. 760

Dietary Chemopreventative Benzyl Isothiocyanate InhibitsBreast Cancer Stem Cells In Vitro and In Vivo

Su-Hyeong Kim, Anuradha Sehrawat, and Shivendra V. Singh

AbstractA small subset of mammary tumor-initiating cells (also known as breast cancer stem cells; bCSC),

characterized by expression of different markers [CD44high/CD24low/epithelial-specific antigen (ESA)þ],

aldehyde dehydrogenase-1 (ALDH1) activity, and ability to form mammospheres under ultra-low attach-

ment culture conditions, are suspected to evade conventional therapies leading to disease recurrence.

Elimination of both therapy-sensitive epithelial tumor cells and therapy-resistant bCSC is therefore

necessary for prevention of breast cancer. We have shown previously that a nontoxic small-molecule

constituent of edible cruciferous vegetables (benzyl isothiocyanate; BITC) inhibits mammary cancer

development in mouse mammary tumor virus-neu (MMTV-neu) transgenic mice by causing epithelial

tumor cell apoptosis. The present study shows efficacy of BITC against bCSC in vitro and in vivo. Mammo-

sphere formation frequency andCD44high/CD24low/ESAþ and/or ALDH1þpopulations in culturedMCF-7

(estrogen receptor–positive) and SUM159 (triple-negative) human breast cancer cells were decreased

significantly in the presence of plasma achievable concentrations of BITC. BITC administration in the diet

(3 mmol BITC/g diet for 29 weeks) resulted in a marked decrease in bCSCs in the MMTV-neu mice tumors

in vivo. Overexpression of full-length Ron as well as its truncated form (sfRon), but not urokinase-type

plasminogen activator receptor, conferred near complete protection against BITC-mediated inhibition of

bCSCs inMCF-7 cells. The BITC treatment downregulated protein levels of Ron and sfRon in cultured breast

cancer cells and in tumor xenografts. Ron overexpression resulted in upregulation of bCSC-associated genes

Oct-4, SOX-2, andNanog. In conclusion, the present study indicates that BITC treatment eliminates bCSCs

in vitro and in vivo. Cancer Prev Res; 6(8); 782–90. �2013 AACR.

IntroductionBreast cancer remains a major public health concern for

womenworldwide accounting formore than 40,000 deathseach year in the United States alone despite tremendousadvances toward targeted therapies (1, 2). Preventive strat-egies, as exemplified by selective estrogen receptor (ER)modulators (e.g., tamoxifen and raloxifene) and aromataseinhibitors (e.g., exemestane) for breast cancer risk reduction(3–5), are necessary to alleviate disease-related cost, mor-bidity, and mortality associated with this disease. Unfortu-nately, the currently available preventive options are inef-

fective against ER-negative breast cancers as well as a subsetof women with ER-positive cancer (3–5). Unwanted sideeffects associated with the long-term use of selective ERmodulators and aromatase inhibitors further underscoresthe need for novel cancer preventive options that are notonly safe but can also target ER-positive as well as ER-negative breast cancers.

Bioactive small molecules from edible plants remainattractive for prevention of breast and other cancers dueto their favorable safety profile (6, 7). Benzyl isothiocya-nate (BITC) is one such naturally occurring phytochemicalin cruciferous vegetables (e.g., garden cress) with in vivopreventive efficacy in experimental animals (8, 9). For ex-ample, BITC treatment before the carcinogen challengeinhibited polycyclic aromatic hydrocarbon–induced ma-mmary cancer in rats (8). Previous work from our ownlaboratory has revealed that BITC administration in thediet confers significant protection against ER-negativebreast cancer development in mouse mammary tumorvirus-neu (MMTV-neu) transgenic mice without any signsof overt toxicity (9). The growth of human breast cancercells (MDA-MB-231) implanted in athymic mice is retard-ed markedly after treatment with BITC (10).

BITC is structurally quite simplebut elicits a complex set ofsignaling events leading to apoptotic as well as autophagic

Authors' Affiliation: Department of Pharmacology & Chemical Biology,and University of Pittsburgh Cancer Institute, University of PittsburghSchool of Medicine, Pittsburgh, Pennsylvania

Note:Supplementary data for this article are available atCancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

S.H. Kim and A. Sehrawat contributed equally to this work.

Corresponding Author: Shivendra V. Singh, University of PittsburghCancer Institute, 2.32A Hillman Cancer Center Research Pavilion, 5117Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-7828; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-13-0100

�2013 American Association for Cancer Research.

CancerPreventionResearch

Cancer Prev Res; 6(8) August 2013782

Research. on June 14, 2020. © 2013 American Association for Cancercancerpreventionresearch.aacrjournals.org Downloaded from

Published OnlineFirst May 9, 2013; DOI: 10.1158/1940-6207.CAPR-13-0100

http://cancerpreventionresearch.aacrjournals.org/

cell death in breast cancer cells (11–14). The molecularcircuitry of BITC-induced cell death involves inhibition ofcomplex III of the mitochondrial respiration and ensuingproduction of reactive oxygen species (11–13). Remarkably,a normal humanmammary epithelial cell line (MCF-10A) issignificantly more resistant to apoptotic and autophagic celldeath as well as production of reactive oxygen species result-ing from BITC exposure compared with cancerousmamma-ry cells potentially contributing to its safety profile (11–13).The BITC-induced apoptosis and autophagy are not con-fined to cellular models as these effects are observed inepithelial mammary tumor cells in vivo (9, 13).The current paradigm insists on elimination of both

therapy-sensitive epithelial tumor cells and therapy-insen-sitive breast cancer stem cells (bCSC) for prevention andtreatment of this disease (15, 16). The bCSCs are not onlyintrinsically resistant to therapies but also have the ability toself-renew and enrich after treatment leading to tumorrecurrence (15–17). Evidence continues to accumulate toindicate that bCSCs share biochemical features of epithe-lial–mesenchymal transition (EMT), a process implicated indevelopment of aggressive breast cancer (18–20). We haveshown previously that the BITC-mediated inhibition ofbreast cancer xenograft growth in vivo is associated withbiochemical features of EMT inhibition characterized byinduction of adherens junction protein E-cadherin andsuppression of mesenchymal marker vimentin (21, 22).Because of coalition between bCSCs and EMT, it was onlylogical to test whether BITC inhibits bCSCs. The presentstudy addresses this question using relevant in vitro and invivo models of breast cancer.

Materials and MethodsEthical issues concerning use and care of laboratoryanimalsThe use of mice and their care for the studies described

herein were in accordance with the University of PittsburghInstitutional Animal Care and Use Committee guidelines(protocol #12080818).

Reagents and cell linesBITC (purity > 98%) was purchased from LKT laborato-

ries. Reagents for cell culture including medium, FBS, andantibiotics were purchased from Invitrogen-Life Technolo-gies. Sources of the antibodies were as follows: anti-Ron,which recognizes both full-length Ron (pro-Ron), Ronb(active form), and short form (sfRon): Santa Cruz Biotech-nology, anti-N-cadherin: Cell Signaling Technology; anti-E-cadherin: BD Biosciences, and anti-vimentin and anti-actin:Sigma-Aldrich. The MCF-7 and MDA-MB-231 cells werepurchased from the American Type Culture Collection andauthenticated before the work. The cells were last authen-ticated in February 2012. The SUM159 cell line was pur-chased from Asterand and authenticated by the provider.TheMCF-7 andMDA-MB-468 cells stably transfected with aplasmid encoding for urokinase-type plasminogen activatorreceptor (uPAR) were generously provided by Dr. Steven LGonias (University of California, San Diego, CA; ref. 23).

The wild-type and Ron and sfRon overexpressing MCF-7cells were generously provided by Dr. Alana L. Welm (Uni-versity of Utah, Salt LakeCity, UT; ref. 24). Each cell linewasmaintained at 37�C in an atmosphere of 95% air and 5%CO2 according the recommendations of the providers.

Mammosphere formation assayMammosphere formation assay was conducted as

described by Li and colleagues (25). Single cells were platedin ultra-low attachment plates (Corning) at a density 500 to5,000 cells/well in serum-free mammary epithelium basalmedium (Lonza) supplemented with 1% penicillin/strep-tomycin, B27 (1:50, Invitrogen-Life Technologies), 5 mg/mLinsulin, 1 mg/mL hydrocortisone (Sigma), 20 ng/mL EGF(R&D Systems), 20 ng/mL basic fibroblast growth factor(Stem Cell), and 2-mercaptoethanol. Indicated concentra-tions of BITCwere added to themedia for primarymammo-sphere assay. Stock solution of BITC was prepared indimethyl sulfoxide (DMSO), andanequal volumeofDMSO(final concentration <0.15%) was added to controls. After 5days, the primary mammospheres were harvested, dissoci-atedwith trypsin, and thenpassed through a40-mmstrainer.Single cellswere then replated inultra-lowattachmentplatesfor second (5 days) and third-generation (7 days) mammo-sphere formation assays without further treatment withBITC or DMSO. The mammospheres were counted underan inverted microscope.

Flow cytometric analysis of aldehyde dehydrogenase1–positive (ALDH1þ) cells

The flow cytometric quantitation of ALDH1þ cells wascarried out by following the manufacturer’s instructions(Stem Cell). Briefly, cells were suspended in assay buffercontaining an ALDH1 substrate (bodipy-aminoacetalde-hyde) and incubated at 37�C for 30 minutes. As a positivecontrol, half of the sample was transferred to a tube con-taining the ALDH1 inhibitor diethylaminobenzaldehyde(DEAB). Before flow cytometric analysis, cells were resus-pended in assay buffer andmixed with 1 mg/mL propidiumiodide.

Analysis of CD44high/CD24low/epithelial-specificantigen-positive (ESAþ) population

Cellswere trypsinized,washedwithPBS twice, and stainedwith 20 mL of anti-ESA [fluorescein isothiocyanate (FITC)-conjugated, BDBiosciences], anti-CD24 (PE-conjugated, BDBiosciences), and anti-CD44 (APC-conjugated, BD bios-ciences) antibodies. Cells were incubated in dark for 30minutes at room temperature followed by washing withPBS. The cells were analyzed usingCyAnADPAnalyzer fromBeckman Coulter.

In vivo experimentThree- to 5-week-old female MMTV-neu transgenic mice

[homozygous; strain FVB/N-Tg(MMTV-neu) 202Mul/J]were purchased from Jackson Laboratories and acclimatedfor 1 week before start of the experiment. The mice wererandomized into 2 groups with 10 mice in each group. The

BITC Inhibits bCSC In Vitro and In Vivo

www.aacrjournals.org Cancer Prev Res; 6(8) August 2013 783




control mice were placed on basal AIN-76A diet, whereasthe experimental group of mice was fed AIN-76A dietsupplemented with 3 mmol BITC/g diet (447.6 mg BITC/kg diet). The dietwas replaced every 3 to 4days at the timeofcage change. Diet consumption and body weights wererecorded once weekly. Each mouse in every group was alsomonitored on alternate days for any other signs of distresssuch as impaired movement, unusual posture, indigestion,and areas of redness or swelling. Mice with tumors weresacrificed after 29 weeks of treatment. Tumor tissue wasdissected and washed with PBS and digested in Dulbeccos’modified Eagle medium (DMEM) supplemented with 300U/mL collagenase and 100 U/mL hyaluronidase for 3 to 4hours at 37�C. Cells were resuspended in Hank’s balancedsalt solution (HBSS) supplemented with 2% FBS andammonium chloride. The resultant suspension was resus-pended in 0.25% Trypsin-EDTA and 5 mg/mL dispase, 0.1mg/mLDNase1 in HBSS and filtered through 40 mm strain-er. The cells were used for ALDH1 assay or mammosphereformation assay. Mammosphere formation from tumorsamples was conducted as described by Liao and colleagues(26). Single cells were plated in ultra-low attachment platein DMEM/F12 supplemented with 2% FBS, 10 mmol/LHEPES, 5% bovine serum albumin, 1% penicillin/strepto-mycin, 10 mg/mL insulin, 20 ng/mL EGF, 20 ng/mL basicfibroblast growth factor, B27, 10 mg/mL heparin, and 2-mercaptoethanol for 7 days.

Western blottingCell lysates and xenograft supernatants were prepared as

described previously (27, 28). Western blotting was con-ducted as described previously (27, 28).

Real-time quantitative PCRTotal RNA from DMSO-treated control and BITC-treated

cells was isolated using RNeasy kit (Qiagen). First-strandcDNA was synthesized using Superscript Reverse Transcrip-tase (Invitrogen-Life Technologies)witholigo (dT)20primer.Primers were as follows: Ron: forward 50-AGCCCACGCT-CAGTGTCTAT-30, reverse 50-GGGCACTAGGATCATCTGT-CA-30; Oct-4: forward 50-GTGGAGAGCAACTCCGATG-30,reverse 50-TGCTCCAGCTTCTCCTTCTC-30; Nanog, forward50-ATTCAGGACAGCCCTGATTCTTC-30, reverse 50-TTTTT-GCGACACTCTTCTCTGC-30; and SOX-2, forward 50-CGAG-TGGAAACTTTTGTCGGA-30, reverse 50-TGTGCAGCGCTC-GCAG-30. The quantitative PCR (qPCR) was done using 2�SYBR Green master mix (Applied Biosystems-Life Technol-ogies) with 95�C (60 seconds), 55�C annealing (60 secondsfor Ron), 60�C annealing (60 seconds for Oct-4,Nanog, andSOX-2), and 72�C (60 seconds) for 40 cycles. Relative geneexpression was calculated using the method described byLivak and Schmittgen (29).

Quantitation of macrophage-stimulating proteinA commercially available kit fromR&D Systemswas used

for quantitative measurement of macrophage-stimulatingprotein (MSP) in cell lysates. Manufacturer’s instructionswere followed for quantitation of the MSP levels.

Statistical analysisTwo-tailedunpairedStudent t testwithWelch correctionor

one-way ANOVA followed by Dunnetts adjustment (fordose–response effects) or Bonferroni test (for multiple com-parisons) were used to determine statistical significance ofdifference in measured variables. Results are expressed asmean� SD.Differencewas considered significant atP< 0.05.

ResultsBITC inhibited self-renewal of bCSC

Initially, we used MCF-7 (ER-positive) and SUM159(triple-negative) human breast cancer cells to determinethe effect of BITC on bCSCs. The BITC concentrations usedin these experiments were (0.5–1 mmol/L) lower than pro-apoptotic doses (11, 12). Figure 1A exemplifies mammo-spheres resulting after 5 days of treatment of MCF-7 andSUM159 cells with DMSO (control) or the indicated con-centration of BITC. First-generation mammosphere fre-quency was decreased significantly and dose dependentlyin the presence of BITC in both cell lines when comparedwith DMSO-treated control (Fig. 1B). The mammospheresfrom the first generation were disaggregated and the result-ing single cells were replatedwithout further treatment withDMSO (control) or BITC. Second- (after 5 days) and third-generation (after 7 days) mammosphere frequency wassignificantly lower in BITC-treated MCF-7/SUM159 cellscompared with control (Fig. 1B). Inhibitory effect of BITCon bCSCs was confirmed by flow cytometric analysis ofALDH1 activity (Fig. 1C). As can be seen in SupplementaryFig. S1, the ALDH1 activity was reduced in the presence ofBITC in a dose-dependent manner. For example, the BITCtreatment (1 mmol/L, 72 hours) caused an approximate46% and 42% decrease in ALDH1 activity in MCF-7 andSUM159 cells, respectively, compared with DMSO-treatedcontrol (Fig. 1C). Consistent with these observations, per-centage of CD44high/CD24low/ESAþ fraction was lower inBITC-treated MCF-7 and SUM159 cell cultures comparedwith control (Fig. 1D). Collectively, these results indicatedin vitro inhibition of bCSCs in the presence of BITC.

Dietary BITC administration decreased bCSCs inMMTV-neu mice tumors in vivo

We next proceeded to determine the in vivo efficacy ofBITC against bCSCs using MMTV-neumouse model. Ratio-nale for the use of thismodel in the present study was basedon the following considerations: (i) we have shown previ-ously that dietary administration of BITC (3 mmol/g diet)prevents mammary cancer development in MMTV-neumouse model (9) and (ii) increased self-renewal and rep-licative potential of bCSCs has been reported in this model(30).We used the same protocol of BITC treatment (3 mmolBITC/g diet for 29 weeks) to determine the effect on bCSCs.After 29 weeks of BITC administration, the tumor incidencein the control and BITC treatment groups was 40% and20%, respectively. Tumors from the control mice (n ¼ 4)and those treated with BITC (n ¼ 2) were disaggregatedand single-cell suspensions were used for mammosphereformation assay (Fig. 2A). At each cell seeding density, the

Kim et al.

Cancer Prev Res; 6(8) August 2013 Cancer Prevention Research784




number of mammospheres from BITC-treated tumors wasmarkedly lower compared with those from control tumorsbut the difference did not reach statistical significance dueto an outlier in the control group and small sample size inthe BITC treatment arm (Fig. 2B). Nevertheless, consistentwith these results, the ALDH1 activity was about 83% lowerin the tumors from BITC-treated mice compared withcontrol mice (P ¼ 0.07 by two-tailed unpaired Student ttestwithWelch’s correction; Fig. 2C). These results providedin vivo evidence for BITC-mediated inhibition of bCSCs.

uPARwas dispensable for BITC-mediated inhibition ofbCSCsStudies have shown that overexpression of uPAR alone is

sufficient to drive both EMT and stemness in human breastcancer cells (23, 31). Recent work from our own laboratory

has revealed transcriptional repression of uPAR in BITC-treated breast cancer cells (22). We therefore raised thequestion of whether inhibition of bCSCs upon treatmentwith BITC was due to uPAR suppression. Overexpression ofuPAR (overexpression of uPAR was confirmed, results notshown) increased mammosphere formation frequency inMCF-7 cells by about 1.5-fold. Mammosphere formationfrequencywas decreased in a dose-dependentmanner upontreatment with BITC in MCF-7 cells stably transfected withthe empty vector as well as uPAR plasmid. However, whenthe results were normalized against corresponding DMSO-treated control, the percentage of inhibition in mammo-sphere formation frequency upon treatment with BITC wascomparable for MCF-7 cells and MCF-7/uPAR cells (Fig.3A). These results were confirmed by analysis of ALDH1activity (Fig. 3B). Consistent with results in MCF-7 cells

Figure 1. A, representative imagesdepicting MCF-7 and SUM159primary (first -generation)mammospheres after 5 days oftreatment with DMSO or theindicated concentration of BITC(100�magnification; scalebar, 100mm). B, quantitation of first- (after5 days), second- (after 5 days), andthird-generation (after 7 days)mammospheres. The bar graphshows the frequency ofmammosphere formationnormalized to DMSO-treatedcontrol (mean � SD, n ¼ 3). C,effect of BITC treatment onALDH1þpopulation. Thebar graphshows the percentage of ALDH1þpopulation relative to DMSO-treated control in MCF-7 andSUM159 cells (mean � SD, n ¼ 3).D, effect of BITC treatment onCD44high/CD24low/ESAþpopulation relative to DMSO-treated control (mean� SD, n¼ 3).�, significantly different (P < 0.05)compared with DMSO-treatedcontrol by one-way ANOVA withDunnett adjustment. Comparableresults were observed in twoindependent experiments.Representative data from one suchexperiment are shown.






(Fig. 3A), overexpression of uPAR failed to confer any pro-tection against BITC-mediated inhibition ofmammosphereformation (Fig. 3C) or ALDH1 activity (results not shown)in MDA-MB-468 cells. Collectively, these results indicatedthat uPAR suppression was dispensable for BITC-mediatedinhibition of bCSCs at least in breast cancer cells used in thepresent study.

BITC downregulated Ron in breast cancer cellsThe Ron tyrosine receptor kinase and/or its 55 kDa N-

terminally truncated form (sfRon) has been implicatedin EMT induction as well as metastasis of breast cancer(32–34). Initially, we designed experiments to determinewhether BITC treatment affected Ron and/or sfRon proteinlevels. Exposure of MDA-MB-231 cells, which express sub-stantial amount of Ron and sfRon proteins, to BITC resulted

in a dose- and time-dependent decline in protein levels ofpro-Ron and its active form (Ronb) and truncated form(sfRon; Fig. 4A). The BITC-mediated suppression of Ronprotein was accompanied by downregulation of its mRNA(Fig. 4B, top). BITC treatment also caused a significantdecrease in protein levels of Ron ligand MSP (Fig. 4B,bottom). Moreover, BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo (10) was associated withdownregulation of Ron and sfRon protein levels (Fig. 4C).

Figure 2. A, representative mammosphere images from tumors of MMTV-neu mice placed on basal (control) diet or 3 mmol BITC/g diet (100�magnification; scale bar- 100 mm). B, bar graph shows the number ofmammosphere from tumors of control and BITC-treated MMTV-neumice. Results shown aremean�SD (n¼ 4 for control and n¼ 2 for BITC-treated). C, bar graph shows the percentage of ALDH1þ population intumors from control and BITC-treated MMTV-neu mice. Results shownaremean�SD (n¼ 4 for control group and n¼ 2 for BITC-treated group).Statistical significance was determined by two-tailed unpaired Studentt test with Welch correction.

Figure 3. A, frequency of mammosphere formation (normalized torespective DMSO-treated control) from MCF-7 cells stably transfectedwith empty vector (MCF-7) or the same vector encoding for uPAR (MCF-7/uPAR). Results shown are mean � SD (n ¼ 3). B, representative flowhistograms showing ALDH1þ cell population in MCF-7 cells stablytransfectedwith empty vector or the samevector encoding for uPARafter72-hour treatment with DMSO or 1 mmol/L BITC. C, bar graph shows thefrequency of mammosphere formation (normalized to respective DMSO-treated control) from MDA-MB-468 cells stably transfected with emptyvector (MDA-MB-468) or the same vector encoding for uPAR (MDA-MB-468/uPAR). Results shown are mean� SD (n¼ 3). Each experiment wasrepeated at least twice and the results were similar. �, significantlydifferent compared with corresponding DMSO-treated control by one-way ANOVA with Bonferroni adjustment. There was no differencebetween empty vector–transfected cells and uPAR-overexpressing cellsat any dose.

Kim et al.





We used the MCF-7 cells for functional studies to deter-mine the role of Ron and sfRon in BITC-mediated inhibitionof EMT and bCSCs. Protein levels of exogenously expressedRon and sfRon were decreased in the presence of BITC (Fig.4D). Overexpression of sfRon, but not full-length Ron, trig-gered EMT as evidenced by suppression of E-cadherin andinduction of mesenchymal markers N-cadherin and vimen-tin (Fig. 4D). BITC treatment caused induction of E-cadherinin control and Ron-overexpressing MCF-7 cells but not incells with stable overexpression of sfRon (Fig. 4D). Theseresults indicated that overexpression of sfRon attenuatedBITC-mediated induction of epithelial marker E-cadherin.

OverexpressionofRonand sfRonconferredprotectionagainst BITC-mediated inhibition of bCSCsThe inhibition of ALDH1þ fraction resulting from BITC

exposure was nearly completely abolished by overexpres-sion of Ron as well as sfRon (Fig. 5A). The mammospheresfrom Ron- and sfRon-overexpressing MCF-7 cells were rel-

atively larger in size than those observed from parentalMCF-7 cells (Fig. 5B). In agreement with ALDH1 activitydata, BITC-mediated inhibition of mammosphere forma-tion (Fig. 5C) and CD44high/CD24low population (Fig. 5D)was significantly attenuated by overexpression of Ron andsfRon. Moreover, the Ron- and/or sfRon-overexpressingMCF-7 cells exhibited a significant increase in mRNA levelsof bCSC-associated genesOct-4, SOX-2, andNanog (Fig. 6).These results indicated that BITC-mediated inhibition ofbCSCs was attenuated by overexpression of Ron and sfRonat least in MCF-7 cells.

DiscussionWe have shown previously that mammary cancer pre-

vention by BITC in MMTV-neu mice is associated withtumor cell apoptosis. The present study building uponthese observations shows, for the first time, that BITCtreatment inhibits self-renewal of bCSC as evidenced bymammosphere formation, ALDH1 activity, and cell surface

Figure 4. A, Western blotting forpro-Ron, Ronb, and sfRon proteinsusing lysates from MDA-MB-231cells after 6-, 12-, and 24-hourtreatment with DMSO (control) orthe indicated concentrations ofBITC. B, quantitation ofRonmRNAlevel (top) by qPCR in MDA-MB-231 cells after 8- or 24-hourtreatment with DMSO (control) orBITC (2.5 or 5mmol/L). Quantitationof MSP (bottom) in lysates fromMDA-MB-231 cells after 12- or 24-hour treatment with DMSO(control) or the indicatedconcentrations of BITC. Resultsshown are mean � SD (n ¼ 3).�, significantly different (P < 0.05)compared with DMSO-treatedcontrol by one-way ANOVA withDunnett test. C, Western blottingfor Ron and sfRon using MDA-MB-231 xenograft supernatants fromcontrol and BITC-treated athymicmice (n¼ 5 for control and n¼ 3 forBITC-treated). D, immunoblottingfor Ron, sfRon, E-cadherin, N-cadherin, and vimentin usinglysates from parental MCF-7 cellsor those transduced tooverexpress full-length Ron orsfRon after 24-hour treatment withDMSO or the indicatedconcentrations of BITC. Similarresults were obtained fromreplicate experiments.






expression of cancer stem cell markers CD44 and ESA. Ofnote, BITC-mediated in vitro inhibitionof bCSC is observedat concentrations (0.5–1 mmol/L) that are not very cytotoxicbut within the plasma achievable levels (35). We also pro-vide evidence that BITC administration eliminates bCSCs invivo. On the basis of these observations, it is reasonable topropose that elimination of both epithelial tumor cells viaapoptosis and autophagy induction and bCSCs likely con-tributes to breast cancer prevention by BITC.

Overexpression of uPAR alone is sufficient to drive EMT(MDA-MB-468) and/or stemness (MCF-7 and MDA-MB-468) in breast cancer cells (23, 31). The uPAR and its liganduPA constitute an integral component of the extracellularmatrix proteolysis, cell–extracellular matrix interaction, aswell as cell signaling involving receptor tyrosine kinases (36,37). High uPA/uPAR level has been shown to be an adverseprognostic indicator for different cancers including breasttumors (38). Downregulation of constitutive uPA in cul-tured HT29 human colon cancer cells and suppression ofhepatocyte growth factor–stimulated secretion of uPA inMDA-MB-231 cells upon treatment with BITC has beenreported previously (39, 40). We have also shown recentlythat BITC treatment causes transcriptional repression ofuPAR protein in several breast cancer cells, but this systemis dispensable for BITC-mediated inhibition of EMT (22).The present study reveals that overexpression of uPAR failsto confer any protection against BITC-mediated inhibitionof bCSCs in MCF-7 or MDA-MB-468 cells. However, it ispossible that suppression of uPA/uPAR system by BITCcontributes to its inhibitory effect on breast cancer cellproliferation and possibly metastasis. Additional work isneeded to experimentally test this possibility.

Ron (recepteur d’origine nantais) tyrosine kinase expres-sion is very low in normal human breast epithelium, but itsoverexpression has been observed in about 50% of primarybreast cancers (41). Moreover, increased expression of theRon receptor correlates with more aggressive phenotype innode-negative breast tumors (42). Mammary-specific over-expression of wild-type Ron as well as constitutively activeRon induces highly metastatic breast tumors in mice (34).Studies have also indicated that Ron receptor tyrosine kinase

Figure 6. Quantitation ofOct-4,SOX-2, andNanogmRNA levels by qPCRin MCF-7 cells or those stably overexpressing Ron or sfRon. Resultsshown are mean � SD (n ¼ 3).

A

B

C

D

Figure 5. A, percentage of ALDH1þ population normalized to respectiveDMSO-treated control in MCF-7 cells or those overexpressing Ron orsfRon after 72-hour treatment with DMSO or BITC. Results shown aremean � SD (n ¼ 6). B, representative mammosphere images (100�magnification; scale bar, 100 mm) for MCF-7 cells and those transduced tooverexpress Ron and sfRon. C, bar graph shows the frequency ofmammosphere formation normalized to respective DMSO-treated controlin MCF-7 cells or those overexpressing Ron or sfRon after 4 days oftreatmentwithDMSOorBITC.Results shownaremean�SD (n¼6).D,bargraph shows the percentage of CD44high/CD24low population normalizedto respective DMSO-treated control in parental MCF-7 cells or thoseoverexpressingRon or sfRon after 3 daysof treatmentwithDMSOorBITC.Results shown are mean � SD (n ¼ 6). Significantly different (P < 0.05)compared with (�) respective DMSO-treated control and (#) between BITCtreated MCF-7 cells and BITC-treated Ron or sfRon overexpressing cellsby one-wayANOVA followedbyBonferronimultiple comparison test. Eachexperiment was carried out twice in triplicate and combined data fromboth experiments are shown (A, C, and D).

Kim et al.





can promote cell spreading and survival in breast cancer cellsindependent of its only known ligand MSP (43). Activationof Ron by hepatocyte growth factor–like protein has beenshown to confer resistance to tamoxifen in breast cancer celllines (44). The present study shows, for the first time, thatoverexpression of full-length Ron and sfRon induces stem-ness inMCF-7 cells that is accompanied by increased expres-sion of stem cell–associated genesOct-4, SOX-2, andNanog.We also found that BITC treatment inhibits protein and/ormRNA levels of full-length Ron receptor tyrosine kinase andits 55-kDa N-terminally truncated form (sfRon). Moreover,overexpression of full-length Ron and sfRon confers nearcomplete protection against BITC-mediated inhibition ofbCSC in MCF-7 cells. On the other hand, overexpression ofsfRon, but not the full-length Ron, is sufficient to attenuateBITC-mediated induction of E-cadherin.The novel findings of the present study are: (i) overexpres-

sionof full-lengthRonaswell as its truncated form(sfRon) issufficient to drive stemness in MCF-7 cells; (ii) BITC treat-ment inhibits bCSCs in vitro and in vivo; and (iii) BITC-mediated inhibition of bCSC is nearly fully abolished byoverexpression of Ron and sfRon at least in MCF-7 cells. Inconclusion, the present study identified Ron receptor tyro-sine kinase as a critical target of bCSC inhibition by BITC.

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

Authors' ContributionsConception and design: S.-H. Kim, A. Sehrawat, S.V. SinghDevelopment of methodology: S.-H. KimAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): S.-H. Kim, A. Sehrawat, S.V. SinghAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): S.-H. Kim, A. Sehrawat, S.V. SinghWriting, review, and/or revision of the manuscript: S.-H. Kim,A. Sehrawat, S.V. SinghStudy supervision: S.V. Singh

AcknowledgmentsThe authors thank Julie A. Arlotti for helping with the animal studies.

Grant SupportThis work was supported by the grant 2 RO1 CA129347-06 awarded by

the National Cancer Institute. This research used the animal facility and theflow cytometry facility supported in part by a grant from theNational CancerInstitute at the NIH (P30 CA047904).

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 March 18, 2013; revised April 23, 2013; accepted May 1, 2013;published OnlineFirst May 9, 2013.

References1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer

J Clin 2012;62:10–29.2. Higgins MJ, Baselga J. Breast cancer in 2010: novel targets and

therapies for a personalized approach. Nat Rev Clin Oncol 2011;8:65–6.

3. Fisher B, Costantino JP, Wickerham DL, Redmond CK, Kavanah M,Cronin WM, et al. Tamoxifen for prevention of breast cancer: report ofthe National Surgical Adjuvant Breast and Bowel project P-1 Study.J Natl Cancer Inst 1998;90:1371–88.

4. Cauley JA, Norton L, Lippman ME, Eckert S, Krueger KA, Purdie DW,et al. Continued breast cancer risk reduction in postmenopausalwomen treated with raloxifene: 4-year results from the MORE trial:multiple outcomes of raloxifene evaluation. Breast Cancer Res Treat2001;65:125–34.

5. Goss PE, Ingle JN, Ales-Martinez JE, Cheung AM, Chlebowski RT,Wactawski-Wende J, et al. Exemestane for breast-cancer preventionin postmenopausal women. N Eng J Med 2011;364:2381–91.

6. Surh YJ. Cancer chemoprevention with dietary phytochemicals. NatRev Cancer 2003;3:768–80.

7. Stan SD, Kar S, Stoner GD, Singh SV. Bioactive food components andcancer risk reduction. J Cell Biochem 2008;104:339–56.

8. Wattenberg LW. Inhibition of carcinogenic effects of polycyclic hydro-carbons by benzyl isothiocyanate and related compounds. J NatlCancer Inst 1977;58:395–8.

9. Warin R,ChambersWH, Potter DM, SinghSV. Prevention ofmammarycarcinogenesis in MMTV-neu mice by cruciferous vegetable constit-uent benzyl isothiocyanate. Cancer Res 2009;69:9473–80.

10. Warin R, Xiao D, Arlotti JA, Bommareddy A, Singh SV. Inhibition ofhuman breast cancer xenograft growth by cruciferous vegetable con-stituent benzyl isothiocyanate. Mol Carcinogenesis 2010;49:500–7.

11. XiaoD, Vogel V, SinghSV. Benzyl isothiocyanate-induced apoptosis inhuman breast cancer cells is initiated by reactive oxygen species andregulated by Bax and Bak. Mol Cancer Ther 2006;5:2931–45.

12. Xiao D, Powolny AA, Singh SV. Benzyl isothiocyanate targets mito-chondrial respiratory chain to trigger reactive oxygen species-depen-dent apoptosis in human breast cancer cells. J Biol Chem 2008;283:30151–63.

13. Xiao D, Bommareddy A, Kim SH, Sehrawat A, Hahm ER, Singh SV.Benzyl isothiocyanate causes FoxO1-mediated autophagic death inhuman breast cancer cells. PLoS One 2012;7:e32597.

14. Antony ML, Kim SH, Singh SV. Critical role of p53 upregulatedmodulator of apoptosis in benzyl isothiocyanate-induced apoptoticcell death. PLoS One 2012;7:e32267.

15. Oliveira LR, Jeffrey SS, Rebeiro-Silva A. Stem cells in human breastcancer. Histol Histopathol 2010;25:371–85.

16. Velasco-Vel�azquez MA, Homsi N, De La Fuente M, Pestell RG. Breastcancer stem cells. Int J Biochem Cell Biol 2012;44:573–7.

17. O'Brien CS, Farnie G, Howell SJ, Clarke RB. Breast cancer stem cellsand their role in resistance to endocrine therapy. Horm Cancer 2011;2:91–103.

18. RaimondiC,GradiloneA,NasoG, Vincenzi B, PetraccaA,NicolazzoC,et al. Epithelial-mesenchymal transition and stemness features incirculating tumor cells from breast cancer patients. Breast CancerRes Treat 2011;130:449–55.

19. Dave B, Mittal V, Tan NM, Chang JC. Epithelial-mesenchymal transi-tion, cancer stem cells, and treatment resistance. Breast Cancer Res2012;14:202.

20. Velasco-Vel�azquezMA, Popov VM, Lisanti MP, Pestell RG. The role ofbreast cancer stem cells in metastasis and therapeutic implications.Am J Pathol 2011;179:2–11.

21. Sehrawat A, Singh SV. Benzyl isothiocyanate inhibits epithelial-mes-enchymal transition in cultured and xenografted human breast cancercells. Cancer Prev Res 2011;4:1107–17.

22. Sehrawat A, Kim SH, Vogt A, Singh SV. Suppression of FOXQ1 inbenzyl isothiocyanate-mediated inhibition of epithelial-mesenchymaltransition in human breast cancer cells. Carcinogenesis 2013;34:864–73.

23. Jo M, Eastman BM, Webb DL, Stoletov K, Klemke R, Gonias SL. Cellsignaling by urokinase-type plasminogen activator receptor inducesstem cell-like properties in breast cancer cells. Cancer Res 2010;70:8948–58.

24. Liu X, Zhao L, Derose YS, Lin YC, BieniaszM, EyobH, et al. Short-formRon promotes spontaneous breast cancer metastasis through inter-action with phosphoinositide 3-kinase. Genes Cancer 2011;2:753–62.






25. Li Y, Zhang T, Korkaya H, Liu S, Lee HF, Newman B, et al. Sulforaph-ane, a dietary component of broccoli/broccoli sprouts, inhibits breastcancer stem cells. Clin Cancer Res 2010;16:2580–90.

26. Liao MJ, Zhang CC, Zhou B, Zimonjic DB, Mani SA, Kaba M, et al.Enrichment of a population ofmammary gland cells that formmammo-sphere and have in vivo repopulating activity. Cancer Res 2007;67:8131–8.

27. Xiao D, Srivastava SK, Lew KL, Zeng Y, Hershberger P, Johnson CS,et al. Allyl isothiocyanate, a constituent of cruciferous vegetables,inhibits proliferation of human prostate cancer cells by causing G2/Marrest and inducing apoptosis. Carcinogenesis 2003;24:891–7.

28. Powolny AA, Bommareddy A, Hahm ER, Normolle DP, Beumer JH,Nelson JB, et al. Chemopreventative potential of the cruciferousvegetable constituent phenethyl isothiocyanate in a mouse model ofprostate cancer. J Natl Cancer Inst 2011;103:571–84.

29. Livak KJ, Schmittgen TD. Analysis of relative gene expression datausing real-time quantitative PCR and the 2�DDC

T method. Methods2001;25:402–8.

30. Cicalese A, Bonizzi G, Pasi CE, Faretta M, Ronzoni S, Giulini B, et al.The tumor suppressor p53 regulates polarity of self-renewing divisionsin mammary stem cells. Cell 2009;138:1083–95.

31. Lester RD, Jo M, Montel V, Takimoto S, Gonias SL. uPAR inducesepithelial-mesenchymal transition in hypoxic breast cancer cells. JCellBiol 2007;178:425–36.

32. Bardella C, Costa B, Maggiora P, Patane' S, Olivero M, Ranzani GN,et al. Truncated RON tyrosine kinase drives tumor cell progression andabrogates cell-cell adhesion through E-cadherin transcriptionalrepression. Cancer Res 2004;64:5154–61.

33. Kretschmann KL, Eyob H, Buys SS, Welm AL. The macrophagestimulating protein/Ron pathway as a potential therapeutic target toimpede multiple mechanisms involved in breast cancer progression.Curr Drug Targets 2010;11:1157–68.

34. Zinser GM, LeonisMA, Toney K, Pathrose P, ThobeM, Kader SA, et al.Mammary-specific Ron receptor overexpression induces highly met-

astatic mammary tumors associated with beta-catenin activation.Cancer Res 2006;66:11967–74.

35. Boreddy SR, Pramanik KC, Srivastava SK. Pancreatic tumor suppres-sion by benzyl isothiocyanate is associated with inhibition of PI3K/AKT/FOXO pathway. Clin Cancer Res 2011;17:1784–95.

36. Hildenbrand R, Allgayer H, Marx A, Stroebel P. Modulators of theurokinase-type plasminogen activation system for cancer. Expert OpinInvestig Drugs 2010;19:641–52.

37. Jo M, Thomas KS, O'Donnell DM, Gonias SL. Epidermal growthfactor receptor-dependent and -independent cell-signaling path-ways originating from the urokinase receptor. J Biol Chem 2003;278:1642–6.

38. Kwaan HC, McMahon B. The role of plasminogen-plasmin system incancer. Cancer Treat Res 2009;148:43–66.

39. Lai KC, Huang AC, Hsu SC, Kuo CL, Yang JS, Wu SH, et al. Benzylisothiocyanate (BITC) inhibits migration and invasion of human coloncancer HT29 cells by inhibiting matrix metalloproteinase-2/-9 andurokinase plasminogen (uPA) through PKC and MAPK signaling path-way. J Agric Food Chem 2010;58:2935–42.

40. Kim EJ, Eom SJ, Hong JE, Lee JY, Choi MS, Park JH. Benzyl isothio-cyanate inhibits basal and hepatocyte growth factor-stimulatedmigra-tion of breast cancer cells. Mol Cell Biochem 2012;359:431–40.

41. MaggioraP,MarchioS,StellaMC,GiaiM,BelfioreA,DeBortoliM, et al.Overexpression of the RON gene in human breast carcinoma. Onco-gene 1998;16:2927–33.

42. Lee WY, Chen HH, Chow NH, Su WC, Lin PW, Guo HR. Prognosticsignificance of co-expression of RON and MET receptors in node-negative breast cancer patients. Clin Cancer Res 2005;11:2222–8.

43. Feres KJ, Ischenko I, Hayman MJ. The RON receptor tyrosine kinasepromotes MSP-independent cell spreading and survival in breastepithelial cells. Oncogene 2009;28:279–88.

44. McClaine RJ, Marshall AM,Wagh PK, Waltz SE. Ron receptor tyrosinekinase activation confers resistance to tamoxifen in breast cancer celllines. Neoplasia 2010;12:650–8.

Kim et al.





2013;6:782-790. Published OnlineFirst May 9, 2013.Cancer Prev Res Su-Hyeong Kim, Anuradha Sehrawat and Shivendra V. Singh

In Vivo and In VitroCancer Stem Cells Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast

Updated version

10.1158/1940-6207.CAPR-13-0100doi:

Access the most recent version of this article at:

Material

Supplementary

2

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2013/08/06/1940-6207.CAPR-13-0100.DC1http://cancerpreventionresearch.aacrjournals.org/content/suppl/2013/05/09/1940-6207.CAPR-13-0100.DCAccess the most recent supplemental material at:

Cited articles

http://cancerpreventionresearch.aacrjournals.org/content/6/8/782.full#ref-list-1

This article cites 44 articles, 13 of which you can access for free at:

Citing articles

http://cancerpreventionresearch.aacrjournals.org/content/6/8/782.full#related-urls

This article has been cited by 4 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://cancerpreventionresearch.aacrjournals.org/content/6/8/782To request permission to re-use all or part of this article, use this link



http://cancerpreventionresearch.aacrjournals.org/lookup/doi/10.1158/1940-6207.CAPR-13-0100

http://cancerpreventionresearch.aacrjournals.org/content/suppl/2013/05/09/1940-6207.CAPR-13-0100.DC1 http://cancerpreventionresearch.aacrjournals.org/content/suppl/2013/08/06/1940-6207.CAPR-13-0100.DC2




http://cancerpreventionresearch.aacrjournals.org/content/6/8/782.full#ref-list-1

http://cancerpreventionresearch.aacrjournals.org/content/6/8/782.full#related-urls

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

mailto:[email protected]

http://cancerpreventionresearch.aacrjournals.org/content/6/8/782


Documents

Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast Cancer …€¦ · Dietary Chemopreventative Benzyl Isothiocyanate Inhibits Breast Cancer Stem Cells In Vitro and In