Breast cancer stem cells (BCSC) are resistant to conventionalchemotherapy and radiotherapy, which may destroy tumormasses but not all BCSC that can mediate relapses. In the presentstudy, we showed that the level of Wnt/b-catenin signaling inBCSC is relatively higher than in bulk tumor cells, contributing toa relatively higher level of therapeutic resistance. We designed ahighly potent small-molecule inhibitor, CWP232228, whichantagonizes binding of b-catenin to T-cell factor (TCF) in thenucleus. Notably, although CWP232228 inhibited the growth ofboth BCSC and bulk tumor cells by inhibiting b-catenin–medi-
ated transcription, BCSC exhibited greater growth inhibition thanbulk tumor cells.We also documented evidence of greater insulin-like growth factor-I (IGF-I) expression by BCSC than by bulktumor cells and that CWP232228 attenuated IGF-I–mediatedBCSC functions. These results suggested that the inhibitory effectof CWP232228 on BCSC growth might be achieved through thedisruption of IGF-I activity. Taken together, our findings indicatethat CWP232228 offers a candidate therapeutic agent for breastcancer that preferentially targets BCSC as well as bulk tumor cells.Cancer Res; 75(8); 1691–702. �2015 AACR.
IntroductionBreast cancer is the most frequently diagnosed cancer among
women in theWestern world (1). Conventional therapies, such assurgery, chemotherapy, and radiotherapy, have limited effects onthe high rate of breast cancer recurrence (2). Recently, it has beensuggested that a subset of tumor cells, called cancer stem cells(CSC) or tumor-initiating cells (TIC), contribute to tumor growth,metastasis, and recurrence (3). Importantly, CSCs are resistant toconventional treatments, such as chemotherapy (4) and radiother-apy (5). CSCs have been identified in almost all cancer types,including colon cancer (6), leukemia (7), and breast cancer (8).Therefore, therapeutic strategies that selectively target breast cancerstemcells (BCSC)will ultimately improvebreast cancer treatments.
Wnt proteins are a large family of secreted, cysteine-rich mole-cules that play a critical role in the development of variousorganisms (9). The dysfunction of the Wnt/b-catenin signalingpathway has recently been implicated in several types of humancancers, including ovarian (10), colon (11), and breast cancers(12). Interestingly, accumulating evidence has revealed a criticalrole for Wnt/b-catenin signaling in CSCs (13). For example,mammary stem cells with high levels of Wnt/b-catenin signalingexhibit greater tumorigenic potential than their counterparts withlowWnt/b-catenin signaling (14). Therefore, these studies suggestthat Wnt/b-catenin signaling is a promising target for treatingbreast cancer through inhibiting BCSCs.
Functional Wnt signaling activities require an interactionbetween b-catenin and T-cell factor (TCF; ref. 15). The aberrantactivation or transcriptional activity of b-catenin has been asso-ciated with breast stem cell amplification and tumorigenesis in anumber of studies (16), suggesting that targeting the b-catenin/TCF protein–protein interaction, rather than other Wnt/b-cateninsignaling components, could effectively target BCSCs . However,as b-catenin is an intracellular signaling molecule with no dis-cernible enzymatic activity, this protein represents an "undrug-gable" target (17). Recent studies have demonstrated that smallmolecules, including both synthetic and natural compounds,inhibit Wnt/b-catenin signaling in various cancers through thedirect targeting of b-catenin. Although recently developed syn-thetic inhibitors targeting b-catenin, such as XAV939 (18) andIWP-2 (19), effectively inhibit the Wnt–b-catenin pathway underin vitro culture systems, the poor pharmacokinetic and pharma-codynamic (PK/PD) profiles of these molecules have preventedin vivo preclinical investigations. Therefore, the development ofinhibitors that target b-catenin and exhibit better in vivo PK/PDprofiles is needed. Herein, we designed a highly potent, selective
1Laboratory of Tumor Suppressor, Lee Gil Ya Cancer and DiabetesInstitute, Gachon University, Incheon, South Korea. 2Department ofMolecular Medicine, School of Medicine, Gachon University, IncheonSouth Korea. 3Division of Convergence Technology, Center for BreastCancer, Research Institute and Hospital, National Cancer Center 323,Ilsan-ro, Ilsandong-gu, Goyang-si Gyeonggi-do, South Korea. 4JWPharmaceutical, 2477 Nambusunhwan-ro, Seocho-gu, Seoul, SouthKorea. 5The Faculty of Liberal Arts, Jungwon University, Chungbuk,Republic of Korea.
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
G.-B. Jang and I.-S. Hong contributed equally to this article.
CorrespondingAuthor: Jeong-SeokNam, Laboratory of Tumor Suppressor, LeeGil Ya Cancer and Diabetes Institute, Gachon University, 7-45 Songdo-dong,Yeonsu-ku, Incheon 406-840, Korea. Phone: 82-32-899-6072; Fax: 82-32-899-6350; E-mail: [email protected]
small-molecule inhibitor, namely CWP232228 (U.S. Patent8,101,751 B2), which antagonizes the binding of b-catenin tothe TCF protein in the nucleus and specifically downregulates asubset of Wnt/b-catenin–responsive genes. In vitro and in vivostudies revealed that CWP232228 suppresses tumor formationand metastasis without toxicity through the inhibition of thegrowth of BCSCs and bulk tumor cells. The dysregulation ofinsulin-like growth factor-I (IGF-I) signaling in primary breastcancers has been associated with radioresistance and tumorrecurrence (20). Although IGF-I is important for the developmentof breast cancer, the role of this protein in BCSCs remains unclear.We also demonstrated, for the first time, the attenuation of IGF-I–mediated BCSC sphere formation mediated by CWP232228.Taken together, these results suggest that targeting b-catenin–mediated transcription using CWP232228 has significant thera-peutic potential for the treatment of breast cancer.
Materials and MethodsCell culture and reagents
The breast cancer cell lines 4T1 and 67NR were kindly providedby Dr. Wakefield, National Cancer Institute (Bethesda, MD).Human breast carcinoma cell lines MCF7, MDA-MB-435, andMDA-MB-231 were obtained from the ATCC. The human breastcarcinomacell lineHs578twas obtained from theKoreanCell LineBank (Seoul, Korea). Murine mammary cancer cell lines 4T1 and67NR (21) and human mammary carcinoma cell lines MDA-MB-435, MDA-MB-231, MCF-7, Hs578T (21) were cultured in DMEM(Invitrogen) supplemented with 10% FBS, 100 U/mL penicillin,and 100 U/mL streptomycin (Lonza) at 37�C and 5% CO2.CW232228 is designed by JW Pharmaceutical Corporation. Doc-etaxel (D-1000) and recombinant IGF-I (#250-19)were purchasedfrom LC Laboratory (Woburn, MA) and PeproTech, respectively.
Tumorsphere formationSingle cells were resuspended in serum-free DMEM (Invitrogen)
containing B27 (Invitrogen), 20 ng/mL EGF, 20 ng/mL basic fibro-blast growth factor (PeproTech), and 4 mg/mL heparin (Sigma-Aldrich). Primary tumorspheres were derived by plating 50,000 to200,000 single cells perwell into 6-well ultra-low attachment dishes(Corning). Secondary tumorspheres were plated at 50,000 cells perwell. Dishes were cultivated for 7 days to enumeration of spheres.Individual spheres�100 mm from each replicate well (n� 9 wells)were counted under an inverted microscope at �50 magnificationusing the Image-Pro Plus program (Media Cybernetics). The per-centage of cells capable of forming spheres, termed the "tumor-sphere formationefficiency,"was calculated as follows: [(number ofsphere formed/number of single cells plated) � 100].
Cell proliferation (cytotoxicity) assay4T1 and MDA-MB-435 cell lines were seeded in 96-well plates.
Cells were treated with increasing concentrations of CWP232228.After 48 hours of incubation, cell viability was assessed by cellcounting kit-8 (CCK-8) according to the manufacturer's instruc-tion. Thenumbers of viable cellsweremeasured at awavelength of450 nm using Versamax microplate reader.
Total RNA was extracted using TRIzol reagent (Invitrogen).RNA purity was verified by measuring 260/280 absorbance ratio.
The first strand of cDNA was synthesized with 2 mg of total RNAusing SuperScript II (Invitrogen), and one tenth of the cDNA wasused for each PCR mixture containing Express SYBR-Green qPCRSupermix (BioPrince). Real-time PCR was performed using aRotor-Gene Q (Qiagen). The reaction was subjected to 40-cycleamplification at 95�C for 20 seconds, at 60�C for 20 seconds, andat 72�C for 25 seconds. Relative mRNA expression of selectedgenes was normalized to HPRT and quantified using the DDCtmethod. The sequences of the PCR primers are listed in Supple-mentary Table S1. The stem cell PCR array (SABiosciences) andWnt signaling pathway PCR array (SABiosciences) were per-formed in triplicate according to the manufacturer's instructions.
Flow cytometryFACS analysis and cell sorting were performed using FACS
Calibur and FACS Aria machines (Becton Dickinson), respective-ly. FACS data were analyzed using Flowjo software (Tree Star).Antibodies to the following proteins were used: PE-conjugatedSca-1 (dilution 1/20), CD44 (dilution 1/40), CD24 (dilution1/40), CD61 (dilution 1/40), CD133 (dilution 1/40), and LEF1(dilution 1/40). The FACS gates were established by staining withisotype antibody or secondary antibody. APC-conjugated rabbitIgG antibodies (dilution, 1/500; Invitrogen) was used as thesecondary antibody to visualize LEF1 protein expression. TheAldefluor kit (Stem Cell Technologies) was used to isolate thepopulation with a high ALDH enzymatic activity. Cells werestained for ALDH using the Aldefluor reagent according to themanufacturer's instructions and analyzed on FACSCalibur. Asnegative control, for each sample of cell aliquot was treated with50 mmol/L DEAB, a specific ALDH inhibitor. Aldefluorpos cellswere quantified by calculating the percentage of total fluorescentcells compared with a control staining reaction. FACS Ariawas used to sort Aldefluor-stained cells into Aldefluorneg andAldefluorpos cell population.
Luciferase reporter assay4T1 cells were plated at a density of 2 � 104 cells per well in
48-well plates, and transfected using Genefectine transfectionreagent (Genetrone Biotech Co.) according to the manufac-turer's protocol. The TopFlash (Addgene; ref. 22), luciferasereporter (100 ng), and Renilla luciferase thymidine kinaseconstruct (Invitrogen; 50 ng) were used to determine luciferaseactivity. Luciferase activity was measured by a luminometer(Glomax, Promega), using a Dual-Luciferase assay kit (Pro-mega), according to the manufacturer's recommendations.Total value of reporter activity in each sample was normalizedto Renilla luciferase activity.
Protein isolation and Western blot analysisCells were lysed in RIPA buffer (20 mmol/L Tris-HCl, pH 7.5,
200 mmol/L NaCl, 1% Triton X-100, 1 mmol/L dithiothreitol)containing protease inhibitor cocktail (Roche). The concentrationof protein was measured with a Protein assay kit (Bio-Rad)following the manufacturer's protocol. Total protein was sub-jected to SDSPAGE and transferred to a polyvinylidene difluoridemembrane. The blot was probed with primary antibody; anti–b-catenin (Cell Signaling Technology). As a loading control,anti–b-actin antibody (Santa Cruz Biotechnology) was used.Subsequently, the blots were washed in TBST (10 mmol/L Tris-HCl, 50mmol/LNaCl, and 0.25%Tween-20) and incubatedwitha horseradish peroxidase–conjugated secondary antibody. The
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Cancer Res; 75(8) April 15, 2015 Cancer Research1692
presence of target proteins was detected using the enhancedchemiluminescence reagents (BioNote Inc.).
Immunofluorescent stainingThe use of fresh breast tumor specimens was approved by the
research ethic committees at the Korea National Cancer Center.Informed consent was obtained from all patients. Samples werefixedwith 4%paraformaldehyde forfluorescent staining. Sampleswere permeabilized with 0.3 mol/L glycine and 0.3% Triton X-100, and nonspecific binding was blocked with 2% normal swineserum (DAKO). Staining was performed as described previously(23), using the primary anti-ALDH1 (Abcam), and anti-LEF1(Cell Signaling Technology). Alexa Fluor 488–conjugated rabbitIgG (Molecular Probes) was used to visualize ALDH1 and LEF1.Samples were examined by fluorescence microscopy (Zeiss LSM510 Meta). The calculation of ALDH1 and LEF1 expression wasbased on green fluorescence area and density divided by cellnumber, as determined from the number of DAPI-stained nuclei,in three randomly selected fields for each specimen from a total ofthree independent experiments. For quantitation, an arbitrarythreshold was set to distinguish specific from background stain-ing, and this same threshold settingwas applied to all the samplesanalyzed.
Short hairpin RNAshRNA targeting mouse IGF-I and nontargeting RNA were
purchased from Sigma. For the efficient IGF-I shRNA transfection,reverse transfection was performed using Lipofectamine 2000(Invitrogen) according to the manufacturer's instructions. Wechose the IGF-I shRNA that is most effective in mRNA levels fromfive shRNA designed from the target sequence and determined byqRT-PCR and ELISA.
Animal studyAll mice were maintained according to Institutional Animal
Care and Use Committee-approved protocols of the Lee Gil YaCancer and Diabetes Institute. For titration experiments, anesthe-tized 7-week-old female Balb/c (OrientCharles River Technology)and NOD/SCID mice (Korea research institute of bioscience andbiotechnology) were inoculated with 5 � 104 4T1 cells and 5 �105 MDA-MB-435 cells into the mammary fat pad in 50 mLvolume (n ¼ 10 for each group), respectively. After inoculation,the mice were randomly assigned to treatment groups (i.p. atCWP232228 100 mg/kg in PBS) and the control group. For thecombination experiment, 7-week-old female BALB/c mice wereinoculated 4 � 104 4T1 into the mammary fat pad in 40 mLvolume (n¼ 10 for each group). After 10 days injection, the micewere then randomly divided into following four groups: (i) thecontrol group, (ii) the docetaxel (15 mg/kg, once a week) group,(iii) the CWP228 (100mg/kg, daily) group, (4) and the docetaxelþ CWP group. And it was monitored for 3 weeks.
Lung metastasis animal modelFor the metastasis and survival experiment, 9-week-old female
BALB/c mice were inoculated with 5 � 104 4T1-Luc cells into thetail vein in 0.1 mL volume. After inoculation, the mice wererandomly assigned to treatment groups (i.p. at CWP232228100 mg/kg in PBS) and the control group. Mice were euthanizedand lungs were collected on 3weeks, and fixed with 10% bufferedformalin. Metastasis incidence was assessed via in vivo biolumi-nescence measurement using an IVIS spectrum (Caliper Life
Sciences). For luciferase detection, 150 mg/mL D-luciferin(Caliper Life Sciences) in PBS was injected i.p. before imaging.Photometric measurement of metastasis was done by LivingImage software (V. 3.1.0; Caliper Life Sciences).
In vivo limiting dilution assayFor the limiting dilution experiment, primary tumors were
minced using scissors and incubated in DMEM (Invitrogen)mixed with collagenase/hyaluronidase (Stem cell Technologies)at 37�C for 15 to 20 minutes. Primary tumor-derived cells wereinoculated into the mammary fat pad of mice at varying celldensities ranging from 500 to 50,000 cells in a total volume of 50mL volume (n ¼ 8 for each group). 4T1 and MDA-MB-435 cellsinjectedmice were euthanized on 3 and 7weeks, respectively, andsecondary tumors were excised for analysis. The frequency of TICswas calculated using extreme limiting dilution assay (ELDA)webtool (http://bioinf.wehi.edu.au/software/elda). The volumeof the primary tumor was measured as previously described (24).
Statistical analysisAll the statistical data were analyzed by GraphPad Prism 5.0
(GraphPad Software) and evaluated by the two-tailed Student ttest. A P value of <0.05 was considered to indicate statisticalsignificance.
ResultsThe stem cell markers Oct4, Sca-1, and ALDH1 are enriched insphere-forming subpopulations
It has been suggested that three-dimensional (3D) spherecultures of tumor cells from various cancer types, including breast(25), colon (6), brain, and pancreatic (26) cancers, have enrichedcancer stem/progenitor cell populations. Recent studies havesuggested that the stem cell markers Oct4 (27), Sca-1 (28), andALDH1 (29) play important roles in maintaining the pluripo-tency of BCSCs. In the present study, we established a sphere-forming culture system to culture BCSCs as an in vitro model ofbreast cancer as previously described (30). To confirm whethersphere-forming subpopulations are enriched for stem cell–likeproperties under 3D culture conditions, we examined the expres-sion profiles of the stem cell markers Oct4, Sca-1, ALDH1, andSox2. Consistent with previous studies, the expression levels ofthese markers were higher in sphere-forming cells than in cells inmonolayers (Supplementary Figs. S1A–S1C and S2B–S2D). Wealso performed FACS analysis to quantitate the percentage of thetotal cell population that consisted of CD44þ/CD24� cells inboth the monolayer and sphere cultures. As expected, the per-centage of cells with this cell surface marker phenotype wasmarkedly higher in sphere-forming cells than in monolayer cells(Supplementary Figs. S1D and S2A). To determine whethersphere-forming subpopulations are enriched for stem cell–likeproperties under 3D culture conditions, we performed an ELDA.Monolayer and sphere-forming cells were harvested and trans-planted at limiting dilutions (from50,000 to 500 cells) intomice.The repopulating unit frequency of the basal population was 1 of1,591 for monolayer cultures and 1 of 425 for sphere cultures(Supplementary Table S2). Therefore, 4T1 sphere-forming sub-populations were compromised in their ability to repopulatefunctional BCSCs in a xenograft model, suggesting that thesecells exhibit the characteristics of BCSCs and can be used togenerate BCSCs in culture as an in vitro model for BCSC cultureto evaluate the efficacy of chemotherapeutic drugs.
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Enhanced expression of Wnt/b-catenin signaling-associatedgenes in putative BCSCs
Recently, accumulating evidence has illustrated a critical roleforWnt/b-catenin signaling in various CSCs (13). Thus, we used astem cell PCR array to identify potential therapeutic targets inBCSCs (Supplementary Fig. S7A). Consistent with previous stud-ies showing elevatedWnt/b-catenin signaling activity in CSCs, thesignaling array results revealed that Wnt/b-catenin signaling wasactivated to a greater extent in sphere-forming cells than inmonolayer cells (Supplementary Fig. S7B). To further assess thesignificance of Wnt/b-catenin signaling activity in BCSCs, weexamined quantitative changes in the gene expression regulatedthrough this pathway. The expression of downstream signalingcomponents of the Wnt–b-catenin signaling pathway, such asWnt1, PPAR-d, Dvl1, LEF1, Fzd1, TCF4, and b-catenin, was
markedly higher in sphere-forming cells than in monolayer cells(Fig. 1A).
Previous studies have demonstrated that aldehyde dehydroge-nase 1 (ALDH1) is a marker of both normal and malignanthuman mammary stem cells and a predictor of poor clinicaloutcomes (31), and lymphoid enhancer-binding factor-1 (LEF1),is a critical regulator ofWnt/b-catenin signaling (32). Therefore, toexamine the regulatory role of Wnt/b-catenin signaling in BCSCs,we analyzed the LEF1 expression levels in the ALDH-positivesubpopulation. The Aldefluor-positive subpopulation showed asignificantly higher level of LEF1 than the Aldefluor-negativesubpopulation in multiple breast cancer cell types (Fig. 1B),suggesting that ALDH1-positive BCSC subpopulations are highlyassociated with enhanced Wnt/b-catenin signaling activity. Toconfirm whether ALDH1-positive breast cancer cells represent the
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Figure 1.Expression profiles of Wnt/b-catenin signaling–related genes in BCSCs. A, the mRNA expression of Wnt1, TCF4, PPAR-d, Fzd1, b-catenin, LEF1, and Dvl1 in 4T1monolayer and sphere-forming cells was measured using real-time PCR. B, the percentages of LEF1-positive cells in both the ALDH-positive and ALDH-negativesubpopulations of various breast cancer cell types [murinemammary cancer cell lines 4T1 and 67NR and humanmammary carcinoma cell linesMDA-MB-435 (M435),MDA-MB-231 (M231), MCF-7, and Hs578T] were evaluated by flow cytometric analysis. C–E, the breast cancer cell lines 4T1 and MDA-MB-435 (C), premalignantMMTV-PyMT mammary tissues (kindly provided by Dr. Wakefield, National Cancer Institute, Bethesda, MD; D), and breast cancer tissues (kindly provided byDr. Lee, National Cancer Center, Korea; E) were costained with antibodies specific for ALDH1 and LEF1. The nuclear localization of LEF1 is dependent on thecytoplasmic expression of ADLH1. DAPI staining was used to label the nuclei within each field. The results are presented as the means� SD from three independentexperiments; � , P < 0.05; �� , P < 0.01; ��� , P < 0.001.
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LEF1-positive subpopulation, we investigated the coexpression ofthese markers in breast cancer cell lines (Fig. 1C), premalignantMMTV-PyMT mammary tissues (Fig. 1D and Supplementary Fig.S8A), and tissue samples from patients with breast cancer (Fig. 1Eand Supplementary Fig. S8B). As shown in Fig. 1C–E, we con-firmed that the ALDH1-positive populations mostly overlappedwith LEF1-positive subpopulations in both human and mousebreast cancer tissues. These results suggest that Wnt/b-cateninsignalingmight contribute to tumorigenic potential, representinga novel therapeutic target for eliminating BCSCs.
The b-catenin inhibitor CWP232228 suppresses BCSC growthand clonogenicity
Using a cell-based reporter system,we screened a diverse libraryof low molecular weight compounds for the inhibition of Wnt/b-catenin–mediated transcriptional activity. The results showedthat CWP232228 was the most effective Wnt/b-catenin inhibitor(Supplementary Fig. S9A). A PK/PD analysis in mice indicatedthat CWP232228, administered i.v. at a dose of 200 mg/kg,generated an exposure of the compound in the blood at aconcentration greater than 0.8 mg/mL for 7 hours (SupplementaryFig. S9B; Supplementary Table S3). Next, we further tested theefficacy and specificity of CWP232228 to inhibit Wnt/b-cateninsignaling in breast cancer cells transiently transfected with aluciferase reporter plasmid in the presence or absence of Wnt3a.In response to CWP232228 treatment, the transcriptional activityin breast cancer cells was significantly decreased in a dose-depen-dent manner (Fig. 2A). Using Western blot analysis (Fig. 2B) andimmunohistochemistry (Fig. 2C),we further investigatedwhetherCWP232228 was sufficient to inhibit key components of Wnt/b-catenin signaling. Consistently, CWP232228 treatment signif-icantly inhibited LEF1 expression in a dose-dependent manner.Approximate IC50 values were determined using a dose–responsecurve. In mouse (4T1) and human (MDA-MB-435) breast cancercell lines, the IC50 values were 2 and 0.8 mmol/L, respectively(Supplementary Fig. S10).
As a functional assay, we evaluated the effect of CWP232228onprimary and secondary sphere formation. Treatment withCWP232228 resulted in the disruption of primary sphere forma-tion of both 4T1 and MDA-MB-435 cells in a dose-dependentmanner (Fig. 2D). For the secondary sphere-forming assay, treatedprimary spheres were collected and dissociated into single cells.The cells from treated or untreated primary spheres were replatedon culture dishes without additional treatment. Interestingly, weobserved that in the presence of CWP232228, the cells derivedfrom primary spheres did not form secondary spheres as efficient-ly as the cells from untreated spheres (Fig. 2D). Moreover, weevaluated the effect of CWP232228 on the sphere formation ofprimary breast cancer cells obtained from four different patientsamples. Before performing in vitro experiments, the primarybreast cancer cells tested positive for cytokeratins 14 expressionand negative for vimentin expression (Supplementary Fig. S3Aand S3B). Treatment with CWP232228 disrupted the sphereformation of primary breast cancer cells in a dose-dependentmanner (Supplementary Fig. S4A–S4D). These findings suggestthat CWP232228 was sufficient to block subsequent secondarysphere formation from primary spheres in the absence of addi-tional treatment.
In breast carcinomas, cell populationswithhigh levels of ALDHactivity are enriched in tumorigenic stem/progenitor cells (33).Therefore,wehypothesized thatCWP232228might disrupt BCSC
sphere formationby regulating ALDHactivity. To test this hypoth-esis, we used FACS analysis to investigate the effect ofCWP232228 on ALDH activity. Indeed, the treatment of 4T1 andMDA-MB-435 cells with CWP232228 for 48 hours decreased thesize of theALDH-positive subpopulation (Fig. 2E). In this context,we examined the expression of BCSC markers in the presence orabsence of CWP232228. Consistent with our hypothesis, theexpression levels of these markers, including the phenotypesSca-1þ, CD133þ, CD61þ, CD44þ/CD24�, and side populations,were significantly lower after CWP232228 treatment (Supple-mentary Table S4).
CWP232228 targets chemoresistant BCSCsIt is important to compare CWP232228 with another well-
known small-molecule inhibitor (FH535) that targets the b-cate-nin/Tcf protein–protein interactions. Because deregulated cellproliferation is a hallmark of cancer cells, the antiproliferativeeffects of these two compounds were determined using the MTTassay. Overall, these data showed that CWP232228 more effec-tively suppressed the proliferative potential of MDA-MB-435 cellsand at much lower doses than the well-known small-moleculeinhibitor FH535 (Supplementary Fig. S5A). Normal humanfibroblast cell-based dose-dependent experiments showed nomarked signs of toxicity at the CWP232228 dose used in thisstudy (Supplementary Fig. S5B).
Recently, it has been suggested that BCSCs are resistant tomanyconventional therapeutic approaches, including chemotherapy(24) and radiotherapy (34). Thus, although traditional app-roaches might kill the majority of tumor cells, some of the BCSCsremain unaffected, surviving and generating new tumors. Toinvestigate the association between chemoresistance and Wnt/b-catenin signaling, we evaluated the available breast cancerdatasets using the Oncomine dataset repository (www.onco-mine.org). After specifically filtering for breast cancer datasetsshowing a response or nonresponse to conventional docetaxeltreatment, we observed significant correlations between chemore-sistance and the expression of negative (GSK3b) or positive(TCF4) regulators of Wnt/b-catenin signaling (Fig. 3A and B).Importantly, we observed that both the size of ALDH-positivepopulations and the sphere formation in 4T1 and MDA-MB-435cells increased in response to conventional docetaxel treatment.However, the docetaxel-enriched ALDH-positive populations(Fig. 3C and D) and sphere formation (Fig. 3E and F) weremarkedly reduced after CWP232228 treatment, suggesting thatCWP232228 targets BCSC populations are enriched in cellsresistant to conventional chemodrugs.
CWP232228 reduces tumor growth in a murine xenograftmodel
We further investigated the in vivo efficacy of CWP232228 ontumorigenesis using a mouse xenograft model. Importantly,CWP232228 treatment (100 mg/kg, administered i.p.) resultedin a significant reduction in tumor volume (Fig. 4A–C). Nosignificant changes in mortality (Supplementary Fig. S6A), bodyweight (Supplementary Fig. S6B), hematologic values (Supple-mentary Fig. S6C), and hemolytic potential (Supplementary Fig.S6D) were observed, indicating that CWP232228-associated tox-icity was minimal. No obvious clinical signs, including anorexia,salivation, diarrhea, vomiting, polyuria, anuria, and fecal changes,were observed. In addition, no significant differences in bodyweight were observed in mice inoculated with cancer cells
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(Supplementary Fig. S11). To determine whether and to whatextent CWP232228 treatment affects the proportion of BCSCs invivo, we performed FACS analysis to quantitate the percentage ofthe total cell population with ALDH activity in primary bulktumors with or without CWP232228 treatment. Indeed,CWP232228 treatment led to a smaller ALDH-positive subpop-ulation (Fig. 4D and E). The antitumor effects of CWP232228were further confirmed using serially regenerated secondary
tumor xenografts derived from primary tumor tissues withoutadditional treatment (Fig. 4F and G). Treatment withCWP232228 significantly reduced the incidence of secondarytumors, indicating that this molecule significantly impaired thetumor initiation potential of BCSCs. We further performed anELDA to evaluate the inhibitory effect of CWP232228 on tumor-igenesis. Following the isolation of cells from freshly digestedtumor tissues, we transplanted limiting dilutions (from 50,000 to
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Figure 2.Effect of CWP232228 on the growth and clonogenicity of BCSCs. b-Catenin–responsive TOPFlash luciferase assays revealed that CWP232228 inhibits recombinantWnt3a-inducedWnt/b-catenin signaling in mouse breast cancer cells (4T1). CWP232228 treatment strongly attenuatedWnt3a-induced TOPFlash activity (A). B and C,the inhibitory effect of CWP232228 on the expression of LEF1, a Wnt/b-catenin signaling target gene, was assessed in 4T1 cells through Western blot analysis (B)and immunocytochemistry (C). D, CWP232228 inhibited primary (with CWP232228 treatment) and second sphere formation (without additional CWP232228treatment) in both 4T1 and MDA-MB-435 cells. The sphere sizes greater than 100 mm were enumerated, and a representative image of a tumor sphere is shown.Data, an average of three independent experiments. TSFE, tumor sphere-forming efficiency. E, the treatment of 4T1 andMDA-MB-435 cellswith CWP232228 for 48hoursdecreased the percentage of ALDH-positive cells in the total cancer cell population. DAPI staining was used to label the nuclei within each field. b-Actin was used as aninternal control. The results are presented as the means � SD from three independent experiments; � , P < 0.05; �� , P < 0.01; ���, P < 0.001. MDA-MB-435, M435.
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500 cells) of the cell preparations into mice without additionalCWP232228 treatment. The repopulating unit frequency of thebasal population was 1 of 1,415 and 1 of 1 for controls and 1 of7,621 and 1 of 1,510 for CWP232228 treatment in 4T1 andMDA-MB-435 cells, respectively (Table 1). Therefore, CWP232228 didnot decrease the repopulation frequency of functional BCSCs in axenograft model.
CWP232228 targetsBCSCs, bulk tumors, andmetastatic tumorsAn ideal and completely curative breast cancer treatment targets
both BCSCs and bulk tumor cells to prevent recurrence. For eachxenotransplant, we observed the significant inhibition of tumorgrowth induced through CWP232228 treatment alone or incombination with docetaxel (Fig. 5A). Docetaxel treatment alonemoderately affected tumor growth (Fig. 5A). In bulk tumors, thepercentage of ALDH1-positive cells increased with conventionaldocetaxel treatment (Fig. 5B). However, these docetaxel-enriched
ALDH-positive populations were markedly reduced afterCWP232228 treatment (Fig. 5B). Moreover, we used metastatici.v. 4T1 cellmodels to investigate the effects ofCWP232228on themetastasis of breast cancer. We used cells stably expressing fireflyluciferase and whole-body bioluminescence to noninvasivelydetect i.v. injected xenografts. Lung metastasis was significantlylower in the CWP232228-treated groups than in the untreatedcontrol groups (Fig. 5C). An overall increase in the survival ofanimals treated with CWP232228 was also observed (Fig. 5D).These results indicated that CWP232228 treatment alone is effec-tive against conventional docetaxel-enriched BCSC populations,bulk tumors, and metastatic tumors in vivo.
Suppressive effects of CWP232228 on BCSCs are achievedthrough the disruption of IGF-I activity
We compared the expression of downstream components ofWnt/b-catenin signaling between sphere-forming cells and
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monolayer cells and between nontreated and drug-treatedspheres to identify potential downstream targets ofCWP232228 using a Wnt/b-catenin target PCR array. Wescreened differentially expressed genes associated with Wnt/b-catenin signaling. Two criteria for the selection of geneexpression differences were used: A significant t test andfold-change magnitude. Among the genes examined, the levelof IGF-I mRNA was significantly enhanced (�9-fold upregu-lated) in BCSCs (Fig. 6A). Interestingly, in both Wnt/b-cate-nin–targeting PCR array (Fig. 6B) and immunocytochemicalanalysis (Fig. 6C), CWP232228 treatment was correlated withdecreased IGF-I levels (�8-fold downregulated) under sphere-forming conditions. Previous studies demonstrated that theexpression of IGF-I in breast cancer tissues (35) and serumlevels of this protein in breast cancer patients (36) are signif-
icantly higher than those in healthy individuals. Therefore, it isreasonable to hypothesize that CWP232228 suppresses thegrowth of BCSCs and bulk tumors through the disruption ofIGF-I activity. Consistently, these results revealed that com-pared with control cells, IGF-I knockdown (Supplementary Fig.S12) led to smaller ALDH-positive subpopulations (Fig. 6D)and decreased BCSC sphere formation (Fig. 6E). The stimula-tory effects of IGF-I on BCSC sphere formation were success-fully attenuated after CWP232228 treatment (Fig. 6F). Tofurther evaluate the CWP232228-mediated inhibition ofIGF-I secretion, we performed an ELISA to quantitate the levelsof IGF-I in both monolayer and sphere cultures with or withoutCWP232228 treatment. Consistent with immunocytochemicalresults, CWP232228 treatment significantly decreased IGF-Isecretion under sphere-forming conditions (Supplementary
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Figure 4.The effects of CWP232228 ontumorigenesis in a murine xenograftmodel. A, schematic representation ofthe experimental protocol as describedinMaterials andMethods. Themicewereimplanted with 4T1 (5 � 104 cells/mouse) and MDA-MB-435 cells (5� 105
cells/mouse) through orthotopicinjection into the thoracic mammary fatpads. Tumor tissue was isolated frommice bearing 4T1 or MDA-MB-435 celltumors that had been treated withCWP232228 (100mg/kg, i.p.) or vehicle(PBS). B and C, tumor volumes weremeasured as described in Materials andMethods. D and E, the effects ofCWP232228 on the ALDH-positivesubpopulation as a proportion of thetotal cells in bulk tumors were assessedthrough flow cytometric analysis. 4T1andMDA-MB-435 xenografts frommicetreated with CWP232228 or vehiclewere dissociated into single-cellsuspensions and injected into themammary fat pads of mice in limitingdilutions (50,000, 10,000, 2,000, and500). Tumor formation was observedfor 4 weeks (4T1 cells) and 8 weeks(MDA-MB-435 cells) followinginoculation. F and G, the BCSCfrequency was calculated using theELDA. The results are presented as themeans � SD from three independentexperiments; � , P < 0.05; �� , P < 0.01;��� , P < 0.001.
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Cancer Res; 75(8) April 15, 2015 Cancer Research1698
Fig. S13). These results suggest that the suppressive effects ofCWP232228 on BCSCs are achieved through the disruption ofIGF-I activity.
DiscussionApproximately 30% to 50% of the patients diagnosed with
early-stage breast cancer are likely to progress to the metastaticstage, despite treatment with surgery and/or chemotherapy (37).Thus, the CSC concept has emerged as an important milestone in
the understanding of chemodrug resistance and cancer recurrence(38).On thebasis of their characteristics, targeting and eradicatingCSCs represent a potential strategy for significantly improvingclinical outcomes. Moreover, Fillmore and colleagues (39)revealed a 30-fold increase in the BCSC population in variousbreast cancer cell lines after conventional chemotherapy. Theavailable conventional therapeutic agents primarily eliminatethe bulk of a tumor mass but do not affect BCSCs (40). Thus,the identification anddevelopment of BCSC-targeting therapeuticagents is urgent.
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Figure 5.The effects of CWP232228 on BCSCs,bulk tumors, and metastatic tumors.A, the mice were implanted with 4T1cells (5 � 104 cells/mouse) throughorthotopic injection into themammary fat pads. Tumor tissue wasisolated from tumor-bearing micetreated with vehicle alone or withdocetaxel (15 mg/kg) andCWP232228 (100 mg/kg) aloneand in combination. Tumor volumeswere measured as described inMaterials and Methods(n ¼ 10). B, the ALDH-positivesubpopulation, as a proportion ofthe total cell population in bulktumors, was assessed throughimmunohistochemistry. C,monitoring tumor growth throughwhole-body bioluminescenceimaging, growing 4T1 cells expressingfirefly luciferase were i.v. injected intomice. At one day after cancer cellinjection, CWP232228 was i.v.administered (100 mg/kg bodyweight). The mice were subjected toweekly bioluminescence imaging.Representative images at week 2 areshown (n¼ 10). D, the survival rate of4T1 xenograft tumor-bearing micefollowing treatmentwith CWP232228(100 mg/kg body weight) or vehicle(PBS; n¼ 12). DAPI staining was usedto label the nuclei within each field.The results are presented as themeans � SD from three independentexperiments; �� , P < 0.01;��� , P < 0.001.
Table 1. ELDA to evaluate the tumor-forming potential
4T1 MDA-MB-435Estimated cell frequency Control CWP232228 P Control CWP232228 P
Cell number of inoculation, % (n)50,000 100% (8/8) 100% (8/8) — 100% (8/8) 100% (8/8) —
Estimated CSC Frequency by ELDA (95% CI) 1/1415 (1/2850-1/703) 1/7621 (1/15301-1/3796) 6.60E�04 1/1 (1/437-1/1) 1/1510 (1/3048-1/748) 3.70E�05
NOTE: 4T1 andMDA-MB-435 xenografts frommice treated with CWP232228 or vehicle were dissociated into single-cell suspensions and injected into themammaryfat pads ofmice in limiting dilutions (50,000; 10,000; 2,000; 500). Tumor formationwas observed for 4weeks (4T1 cells) and 8weeks (MDA-MB-435 cells) followinginoculation. BCSC frequency was calculated using ELDA.
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In recent years, a number of studies have suggested that thedysregulation of Wnt/b-catenin signaling occurs in human breastcancer (41). In this context, the high expression ofb-cateninmightbe an important clinical and pathologic feature of breast cancersand a predictor of poor overall survival (42). Mutations in the N-terminal domain of b-catenin have been observed in 92% ofpatients with metaplastic breast carcinoma (43). Consistent withthese findings, the results of the present study showed that thelevels of Wnt/b-catenin signaling activities observed in BCSCswere significantly higher than those of bulk cancer cells, althoughboth bulk tumor cells and BCSCs exhibit a basal level of Wnt/b-catenin signaling (Fig. 1). These results suggested that BCSCs aresensitive to therapeutic approaches targeted against Wnt/b-cate-nin. Theb-catenin–TCF interaction is required for functionalWnt/b-catenin signaling (15); therefore, the inhibition of Wnt/b-cate-nin signaling through the direct targeting of b-catenin is consid-ered an attractive therapeutic strategy. Despite academic pursuitand industrial investment, there is currently no small-moleculeinhibitor approved for human use. The majority of the inhibitorsdeveloped so far are in the preclinical or early clinical phase ofdevelopment: XAV939 (Novartis Pharmaceuticals) and JW55
(Tocris Bioscience) are in preclinical trials, and OMP-18R5(OncoMed Pharmaceuticals/Bayer), OMP-54F28 (OncoMedPharmaceuticals/Bayer), PRI-724 (Prism Pharma Co., Ltd/Eisai),and LGK974 (Novartis Pharmaceuticals) are in phase I/II trials. Ifthese chemical inhibitors prove to be both safe and effective fortreating human cancer, then these molecules will represent pow-erful tools to target chemotherapy-resistant CSCs that promotemetastasis.
Here, to inhibit the growth and/or self-renewal capacity ofBCSCs through the suppression of b-catenin–mediated signaling,we used the small-molecule inhibitor CWP232228, identified in ahigh-throughput screen. Follow-up analyses using this com-pound revealed a reduction in the expression of a Wnt/b-cateninluciferase reporter and the inhibition of the expressionof theWnt/b-catenin target gene LEF1 (Fig. 2A–C). Notably, whereasthe inhibition of b-catenin–mediated transcription throughCWP232228 had inhibitory effects on the growth of BCSCs andbulk tumor cells, BCSCs showed a markedly greater degree ofgrowth inhibition (Figs. 3–5). Moreover, CWP232228 treatmentwas sufficient to block subsequent secondary BCSC sphere for-mation in vitro (Fig. 2D) and secondary tumor development in the
Figure 6.Antagonistic interaction betweenWnt/b-catenin signaling and IGF-I inBCSC sphere formation. A and B, thedata from a PCR array of Wnt/b-catenin signaling targets arepresented as a heatmap ofdifferentially expressed genes in 4T1monolayer versus 4T1 sphere-formingcells (A) and in tumorspheres treatedwith CWP232228 (1 mmol/L) versustumorspheres treated with vehiclecontrol (B); decreased (green) orincreased (red) expression comparedwith the mean mRNA expression. TheIGF-I mRNA levels are presented asfold changes relative to controls. C,the inhibitory effect of CWP232228(1 mmol/L) on IGF-I expression in 4T1tumorspheres was assessed usingimmunocytochemistry. D and E, 4T1cells transfected with IGF-I shRNAversus cells treated with controlshRNA were evaluated for theALDH-positive subpopulation as aproportion of the total cells (D) andthe relative numbers of tumorsphere-forming units (E). F, the primarytumorspheres from 4T1 cells treatedwith vehicle alone orwith CWP232228(1 mmol/L) and IGF-I (100 ng/mL)alone or in combination wereevaluated for the relative numbers oftumorsphere-forming units. DAPIstaining was used to label the nucleiwithin each field. The results arepresented as the means � SD fromthree independent experiments;�, P < 0.05; �� , P < 0.01; ��� , P < 0.001.TSFE, tumor sphere-formingefficiency.
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Cancer Res; 75(8) April 15, 2015 Cancer Research1700
xenograft model (Table 1), without additional treatment. Thesefindings suggest that CWP232228 inhibits the initiation of tumordevelopment anddisrupts the physiologic requirements for BCSCmaintenance. The observation of markedly reduced ALDH-pos-itive BCSC populations in bulk tumors treated with CWP232228further supports this interpretation (Fig. 4D and E).
Breast cancer is likely to metastasize to multiple organs, pri-marily including the lungs, liver, and the brain. Therefore, weinvestigated the effect of CWP232228 on metastasis using breastcancer cell xenograft models. For this purpose, the mice were i.v.injectedwithmouse breast cancer cells expressing firefly luciferase(4T1-Luc), and the cells were noninvasively detected on the basisof bioluminescence. CWP232228 significantly reduced tumorbioluminescence in a fast-growing metastatic model of mousebreast cancer. This decrease in 4T1-Luc cells reflects both thenumber of metastatic cancer cells (data not shown), and thetumor volume as the bioluminescent signal was measured acrossthe whole body, integrating both optical parameters (Fig. 5C).Importantly, this efficacy was achieved without affecting primarytoxicity parameters, such as mortality, body weight, hematologicvalues, and hemolytic potential (Supplementary Fig. S6).
The aberrant activation or transcriptional activity of b-cateninhas been correlated with breast stem cell amplification and tumor-igenesis in a number of studies (16). Moreover, studies aiming totarget CSCs have primarily focusedondisrupting their self-renewalcapacity rather thandirectly causing toxic effects; these drugs could,therefore, be less toxic than conventional cytotoxic chemothera-peutic drugs, as reflected by their higher than anticipated IC50
values (44). This result suggests that theb-catenin–TCF interaction,rather thanotherWnt/b-catenin signaling components couldbeaneffective therapeutic target in BCSCs.
The IGF signaling pathway is one of the most important reg-ulators of the growth, migration, and invasion of various types ofcancer (45). Several in vitro experimental studies have providedsubstantial evidence of a role for IGF-I signaling in human breastcancer. The overexpression of the IGF-I receptor in the mousemammary gland results in rapid mammary tumorigenesis (46).Clinical studies also support the importance of IGF-I in breastcancer. IGF-I is detected at high levels in tissue specimens (35) andserum samples (36) from breast cancer patients compared withthose from healthy individuals. Moreover, the constitutive activ-ities of IGF-I in breast cancer cells are highly associated withradioresistance and tumor recurrence (20). These studies mighteventually reveal a role for IGF-I inbreastmalignancieswith respectto tumor growth,metastatic progression, and resistance to therapy.Although the IGF-I system is important in breast cancer develop-ment, themechanismsunderlying thepotential role of this proteinin BCSCs remain largely unknown. Cross-talk between IGF-I andWnt/b-catenin signaling was recently reported in colon cancer(47), oligodendroglial cells (48), and chondrocytes (49). There-fore, the interactions between these two signaling pathways inBCSCs are intriguing and need further investigation. In the presentstudy, we also provided the first evidence that IGF-I is expressed at
higher levels in BCSCs than in non-BCSCs. Furthermore, weobserved an attenuating effect of CWP232228 on IGF-I–mediatedfunctions in BCSC sphere formation and ALDH-positive BCSCpopulations (Fig. 6). The importance of IGF-I in the growth and/orself-renewal capacity of BCSCs is consistentwith previous observa-tions from breast cancer cell lines (50). These studies suggest thatIGF-I signaling is critical for the tumorigenicity andmaintenanceofBCSCs, and these cells could be selectively targeted to improveclinical outcomes through the inhibition of BCSCs.
In summary, these results provide the first demonstration thatthe small-molecule inhibitorCWP232228 inhibitsWnt/b-cateninsignaling and depletes BCSCs from bulk tumors. BCSC-depletedtumor cell populations showed diminished self-renewal capacityand decreased tumorigenicity. These findings suggest that theinhibition of Wnt/b-catenin signaling suppresses the growth andfunctionality of BCSCs, which might be key drivers of breastcancer metastasis and recurrence. To the best of our knowledge,the effect of CWP232228 on breast tumorigenesis has not previ-ously been assessed. Taken together, these results suggest that byantagonizing the binding of b-catenin to the TCF protein in thenucleus, b-catenin inhibitors, specifically CWP232228, might benovel BCSC-targeting agents for the treatment of breast cancer.
Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.
Authors' ContributionsConception and design:G.-B. Jang, I.-S. Hong, R.-J. Kim, J.H. Park, J.-U. Chung,H.-Y. Lee, J.-S. NamDevelopment of methodology: G.-B. Jang, R.-J. Kim, J.H. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G.-B. Jang, E.-S. Lee, J.H. Park, C.-H. YunAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G.-B. Jang, I.-S. Hong, R.-J. Kim, S.-Y. Lee, S.-J. Park,E.-S. Lee, J.H. Park, H.-Y. LeeWriting, review, and/or revision of the manuscript: G.-B. Jang, I.-S. Hong,J.H. Park, J.-U. Chung, H.-Y. Lee, J.-S. NamAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.-B. Jang, R.-J. Kim, S.-J. Park, E.-S. Lee,J.H. Park, J.-U. Chung, K.-J. LeeStudy supervision: G.-B. Jang, I.-S. Hong, J.H. Park, J.-S. NamOther (Technical or material support): J.H. Park
Grant SupportThis research was supported by a grant from the Korea Health Technology
R&DProject through the KoreaHealth Industry Development Institute (KHIDI)and the Ministry of Health and Welfare, Republic of Korea (grant number:HI11C1512). This work was also supported by the National Research Foun-dation of Korea (NRF) grant funded by the Korea government (MSIP) NRF-2013R1A2A2A01067703.
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 July 10, 2014; revised December 27, 2014; accepted January 28,2015; published OnlineFirst February 6, 2015.
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Cancer Res; 75(8) April 15, 2015 Cancer Research1702
2015;75:1691-1702. Published OnlineFirst February 6, 2015.Cancer Res Gyu-Beom Jang, In-Sun Hong, Ran-Ju Kim, et al. Inhibits the Growth of Breast Cancer Stem-like Cells
