A Monoclonal Antibody against Human Notch1 Ligand Binding ... · Recent studies have suggested presence of cancer stem cell (CSC) subpopula-tion in various canceroustissues including

Therapeutic Discovery

A Monoclonal Antibody against Human Notch1 Ligand–Binding Domain Depletes Subpopulation of Putative BreastCancer Stem–like Cells

Ankur Sharma, Anurag N. Paranjape, Annapoorni Rangarajan, and Rajan R. Dighe

AbstractOverexpression ofNotch receptors and ligands has been associatedwith various cancers anddevelopmental

disorders,makingNotch a potential therapeutic target.Here,we report characterization ofNotch1monoclonal

antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF)

repeats 11 to 15 inhibited binding of Jagged1 andDelta-like4 and consequently, signaling in a dose-dependent

manner, the antibodies against EGF repeats 11 to 12 being more effective than those against repeats 13 to 15.

These data emphasize the role of EGF repeats 11 to 12 in ligand binding. One of the mAbs, 602.101, which

specifically recognizes Notch1, inhibited ligand-dependent expression of downstream target genes of Notch

such as HES-1, HES-5, and HEY-L in the breast cancer cell line MDA-MB-231. The mAb also decreased cell

proliferation and induced apoptotic cell death. Furthermore, exposure to this antibody reduced CD44Hi/

CD24Low subpopulation in MDA-MB-231 cells, suggesting a decrease in the cancer stem–like cell subpopu-

lation. This was confirmed by showing that exposure to the antibody decreased the primary, secondary, and

tertiary mammosphere formation efficiency of the cells. Interestingly, effect of the antibody on the putative

stem-like cells appeared tobe irreversible, because themammosphere-forming efficiency couldnot be salvaged

even after antibody removal during the secondary sphere formation. The antibody alsomodulated expression

of genes associated with stemness and epithelial–mesenchymal transition. Thus, targeting individual Notch

receptors by specific mAbs is a potential therapeutic strategy to reduce the potential breast cancer stem–like

cell subpopulation. Mol Cancer Ther; 11(1); 77–86. �2011 AACR.

Introduction

Notch receptors are single-pass transmembrane recep-tors that are involved in normal cell growth, differentia-tion, and death in multicellular organisms, in a context-dependent manner (1). The N-terminal part of Notchextracellular domain (N-ECD) consists of a series of epi-dermal growth factor (EGF)-like repeats (ELR) that arerequired for ligand binding (2). Notch signaling relies onthe ability of membrane-bound ligands of Delta/Serrate/Lag-2 (DSL) family to bring about conformational changesin the negative regulatory region (NRR) of the receptor,followed by a series of proteolytic events within thetransmembrane region, catalyzed by ADAM/TACEmetalloproteases and g-secretase (3–6). These proteolytic

events release Notch intracellular domain (N-ICD) intothe nucleus, which then associates with the DNA-bindingproteins to assemble a transcription complex that in turnactivates the downstream signaling cascade (7).

In addition, defects in Notch signaling lead to a largenumber of pathologic conditions (8). Both overexpressionand downregulation of Notch receptors and ligands havebeen implicated in human cancers (9–12). Many reportssuggest a strong correlation between coexpression ofNotch receptors and its ligands in the breast cancer path-ogenesis, which in turn leads to poor prognosis andsurvival in different cohorts (13–17). Recent studies havesuggested presence of cancer stem cell (CSC) subpopula-tion in various cancerous tissues including breast cancers(18). As in the case of normal stem cells, Notch signalingplays an important role in maintenance of CSCs. Activa-tion of Notch signaling results in elevated self-renewal, asshown by increased mammosphere formation, whereasinhibition of Notch signaling had the opposite effect (19).Inhibition of Notch signaling also affected the tumor-sphere-forming capacity of breast cancer cells (20). Thus,Notch signalingprovides apotential therapeutic target forbreast cancer treatment.

Different strategies are being developed to blockNotch signaling for therapeutic targeting (21–23),the most prominent being inhibition of proteolytic

Authors' Affiliation: Department of Molecular Reproduction Developmentand Genetics, Indian Institute of Science, Bangalore, India

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

Corresponding Author: Rajan R. Dighe, Department of Molecular Repro-duction Development and Genetics, Indian Institute of Science, Bangalore560012, India. Phone: 9180-2293-2660/3261; Fax: 9180-2360-0999;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0508

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on July 23, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst November 10, 2011; DOI: 10.1158/1535-7163.MCT-11-0508

http://mct.aacrjournals.org/

cleavages by g-secretase inhibitors (GSI; refs. 24, 25).However, GSIs being universal inhibitors of g-secretasesalso inhibit other signaling pathways (26) and havelimited therapeutic potential because of their seriousadverse effects (27, 28). Therefore, it is essential togenerate tools that can exclusively target Notch signal-ing pathway and specific antibodies are the ideal mole-cules. These antibodies could be against the ligands (29,30) or different domains of receptor that can distinguishbetween individual Notch receptors. The anti-ligandantibodies are likely to affect ligand binding to all Notchreceptors leading to pan-Notch inhibition and may notbe useful in pathologic situations precipitated by theparalogue-specific Notch signaling. This is particularlythe case for breast cancers, where overexpression ofNotch1 is correlated with poor prognosis whereas thatof Notch2 is associated with good clinical outcomes,thus highlighting the importance of individual receptor-specific inhibitors (14, 31).

Individual receptor-specific antibodies againstNotch1 and Notch3 NRR domains have been shownto stabilize the receptors in an autoinhibited state andare allosteric in nature (32–34). However, antibodiesspecific for the ligand-binding domain of the receptorcan serve dual functions of being the potential thera-peutic tools while providing insights into the mecha-nism of ligand binding and subsequent receptor acti-vation. Previous attempts of generating antibodiesagainst human Notch1 EGF repeats 1 to 36 suggestthat these antibodies can inhibit ligand-dependentNotch activation, but their precise epitopes have notbeen mapped and their mechanism of action needs tobe investigated (32). In present study, we report pro-duction and characterization of antibodies against EGFrepeats 11 to 15 of Notch1 and show that one of themonoclonal antibodies inhibits ligand binding andconsequently, signaling. We further show that thisantibody can inhibit cell proliferation and particularlytarget the putative CSCs by modulating expression ofgenes associated with stemness, as well as, the epithe-lial–mesenchymal transition (EMT) markers. This anti-body also inhibited proliferation and induced apopto-tic cell death in breast cancer cell lines.

Materials and Methods

Generation of stable cell linesThe stable cell lines overexpressing human Notch1

(hN1) and humanNotch2 (hN2) were generated by trans-fecting HEK293 cells with the respective cDNAs clonedinto pcDNA3.1Myc/His (Invitrogen). The Notch ligands,hJagged1, hJagged2, hDelta-like1, and hDelta-like4, wereexpressed as Fc fusion protein by cloning the respectivecDNAs into pFUSE-Fc1-IgG1 (InvivoGen) and purifiedfrom the culture supernatant by Protein-A affinity chro-matography (GE Healthcare). MCF-7 and MDA-MB-231cells were procured from American Type Culture Collec-tion. No further authentication was conducted.

Expression of Notch1 receptor fragmentsA fragment of Notch1 extracellular domain was

expressed as glutathione S-transferase (GST) fusion pro-tein by PCR amplifying the cDNA encoding EGF repeats11 to 15 (aminoacids 412–601) andcloning intopGEX-4-T1(GE Healthcare). The protein was purified from the sol-uble cell lysate using glutathione affinity chromatography(Supplementary Fig. S1A). The hN1 EGF repeats 1 to 12was expressed as Fc fusion protein (generously providedby Prof. Radtke, EPFL, Lausanne, Switzerland) and puri-fied using Protein-A affinity chromatography.

Polyclonal and monoclonal antibodiesPolyclonal antibodies were raised in the rabbits against

EGF repeats 11 to 15 using the immunization protocolswell established in the laboratory (35, 36). Mice wereimmunized with the same immunogen and monoclonalantibodies (mAb) were generated using protocols estab-lished for glycoprotein hormones (37, 38). Binding ofmAbs to EGF repeats 11 to 15 and GST was determinedusing standard ELISA protocol, and the clones reactingwith GST were eliminated by incubating the hybridomamediumwith the soluble GST (1mg/mL) and then deter-mining binding to EGF repeats 11 to 15. All interestingmAb-producing clones were further subcloned andmonoclonality established by isotyping the mAbs usingthe Isotyping Kit (Sigma-Aldrich) and determining thecross-reactivity with other Notch fragments. The detail ofantibody production is provided in the SupplementaryMethods.

Flow cytometric assayCells expressing Notch were harvested using Dulbec-

co’s PBS (DPBS)-EDTA, resuspended in DPBS containing2% FBS (Invitrogen; FBS/PBS), and incubated with theprimary antibody for 40 minutes at room temperature,followed by washing, resuspension, and incubation in0.1mLof FBS/PBS containing fluorescein isothiocyanate–conjugated antirabbit or mouse secondary antibodies for30 minutes at room temperature. The cells were washed,resuspended in DPBS, and analyzed using the BectonDickinson FACSCanto. The median values were calculat-ed using the Stat program of CellQuest by Becton Dick-inson. Flow cytometry–based ligand-binding assay wasconducted on ice as previously described (39).

Luciferase reporter assayTo determine effect of anti-Notch1 antibodies onNotch

signaling, functional assay for Notch signalingwas devel-oped. The cells expressing Notch1 receptor such as MCF-7, MDA-MB-231, or HEK293 cells overexpressing hN1 orhN2 were seeded (5� 104 cells per well) in a 24-well plate(Nunc) and transfectedwith 790 ng 12xCSL-Luc and 10 ngpGL3Basic or 800 ngpGL3 control alongwith 1 ngpRL-Tk(Promega) using Lipofectamine 2000 (Invitrogen) as perthe manufacturer’s instructions. Ligands were providedby precoating the wells with purified Jagged1 or Delta-like4 Fc (1 mg per well). The transfected cells were

Sharma et al.

Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics78




incubated with or without 10 mg/mL anti-Notch1 anti-bodies. Luciferase reporter activity was estimated after 36hours using the Dual Luciferase Assay Kit (Promega) anda TD-20 Luminometer (Turner Design) following themanufacturer’s protocol.

C2C12 differentiation assayThe C2C12 differentiation assay was conducted as

described previously (40). Briefly, C2C12mousemyoblastcells (5 � 104) were cultured in Dulbecco’s ModifiedEagle’s Media (DMEM) and 10% FBS and induced todifferentiate by replacing the original medium with thedifferentiatingmediumcontaining 2%horse serumwhichwas replenished every day. At the end of 4-day incuba-tion, cells were examined for expression of myosin heavychain by a confocal Zeiss LSM 510 META microscope.

Cell proliferation and apoptosis assayTo investigate effect of anti-Notch1 antibodies on pro-

liferation of cells endogenously expressing Notch1, MCF-7, and MDA-MB-231, the cells were seeded in a 96-wellplate (5� 103 cells perwell) for 4 hours and incubatedwithorwithout antibodies for 72hours.Cellswere subsequent-ly labeledwith bromodeoxyuridine (BrdUrd) for 12 hoursand its incorporation determined as per the protocolrecommended by the manufacturer (Calbiochem).

Collection and processing of primary breast cancertissuePrimary breast tumor tissue was obtained from the

Kidwai Memorial Hospital, Bangalore, India, as per theethical guidelines of the Institutional Review Board ofthe hospital and the Indian Institute of Science Bangalore,India. The tissue was aseptically minced and dissociatedenzymatically using 1 mg/mL Collagenase (Sigma-Aldrich) and 100 U/mL hyaluronidase (Calbiochem) at37�C for 16 hourswith constant rotation. Breast organoidswere separated by centrifugation, washed with PBS,and resuspended in DMEM-F12 with growth factors(10 mg/mL human EGF, 1 mg/mL hydrocortisone,10 mg/mL insulin, 20 ng/mL basic fibroblast growthfactor, 4 ng/mL heparin, and 1% B27), antibiotics andFungizone, and incubated at 37�C for 6 hours. The organ-oids were further dissociated with trypsin to yield singlecells which were filtered through a 70-mmBD cell strainerto remove clumps and to obtain a largely single-cellsuspension which was used for later experiments.

Mammosphere assayMCF-7, MDA-MB-231 (5 � 104 cells per well), and

the enzymatically dissociated single-cell suspensions(2.5 � 105 cells per well) of the primary breast cancer tis-sueswereseeded inaserum-freeDMEM-F12mediumwithgrowth factors in a semisolid medium containing methyl-cellulose as described earlier (19, 41). Effect of anti-Notch1antibodies on sphere-forming efficiency of these cells wasassessed by incubating the cells with or without anti-bodies (10 mg/mL) and culturing for 1 week, replenishing

the antibody every fourth day. The primary mammo-spheres were counted manually using ImageJ software,trypsinized, and allowed to form secondary and tertiaryspheres with or without antibodies. The same experimentwas also carriedout inpresenceof 5mmol/LDAPT(Sigma)used as a positive control for Notch inhibition.

Results

Characterization of soluble Notch ligandsThe biologic activities of purified soluble Notch ligands

(Jagged1/Delta-like4 Fc; Supplementary Fig. S1B) wereestablished by showing their binding to the full-lengthNotch1 receptor and also determining their ability to elicitresponse using the cell-based assays. The HEK293 hN1cells were suspended in DPBS (Caþ2, Mgþ2 free), incu-bated with purified Jagged1 and Delta-like4 dissolved inHanks’ balanced salt solution (HBSS) containing 1.26mmol/L CaCl2, and the ligand binding was determinedby flow cytometry. As shown in the Supplementary Fig.S2A, therewas specific ligand binding toNotch1 receptor.Furthermore, addition of 5 mmol/L EGTA to HBSSresulted in a significant decrease in ligand binding (Sup-plementary Fig. S2), suggesting that interactions of theseligands with Notch1 are also calcium dependent, as incase of Delta-like1 (39). As shown in the SupplementaryFig. S3A, there was a dose-dependent increase in bindingof Jagged1 and Delta-like4 to EGF repeats 11 to 15 proteinimmobilized on a plastic surface. Furthermore, preincu-bation of the ligandswith EGF repeats 11 to 15 or 1 to 12 Fcproteins abolished their subsequent binding to Notch1-expressing cells (data not shown), showing the functionalnature of the ligands as well as receptor fragments. Thiswas confirmed by showing that both ligands stimulated12xCSL Luc reporter activities in the HEK293 hN1 cells ina dose-dependentmanner (Supplementary Fig. S3B). Sim-ilarly, the precoated ligands led to inhibition of C2C12differentiation, as examined by the myosin heavy chainexpression in myotubes, using anti–myosin heavy chainantibody (Supplementary Fig. S4).

Characterization of anti-Notch1 antibodiesBinding of polyclonal antibody to EGF repeats 11 to 15

showed that it is a high titer antibody with considerablebinding to GST (Supplementary Fig. S5). Passing poly-clonal antibodies through GST-NHS-Sepharose removedall GST-specific antibodies but retained the antibodiesspecific for EGF repeats 11 to 15 (processed polyclonalantibodies). Furthermore, the processed polyclonal anti-bodies could specifically bind to HEK293 hN1 in the flowcytometric assay (data not shown).

A total of 35mAbs that recognized EGF repeats 11 to 15,but not GST, were selected for further analysis. Binding ofmAbs to EGF repeats 11 to 15 was determined by ELISA,and as shown in the Supplementary Table S1, severalmAbs showed varied binding to Notch receptor frag-ments. Partial epitope mapping was carried out by deter-mining binding of these mAbs to EGF repeats 1 to 12 Fc

Therapeutic Antibody Targeting of Notch1 in Breast CSCs

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 79




and EGF repeats 11 to 15 receptor fragments, followed byidentification of those mAbs that showed binding to boththe proteins, indicating that these antibodies recognizeEGF repeats 11 to 12 epitope shared by both proteins.Several mAbs did not recognize EGF repeats 1 to 12 Fcfragment, indicating that the epitopes recognized by theseantibodies reside in EGF repeats 13 to 15. Ability of allmAbs to recognize the full-lengthNotch1was establishedby flow cytometric assay, as shown in the SupplementaryTable S1.

Inhibition of ligand-dependent Notch signaling byanti-Notch1 antibodies

Having characterized both, Notch ligands and antibo-dies, effect of antibodies on ligand-receptor interactionswas investigated. The HEK293 hN1 cells were preincu-bated with polyclonal or monoclonal antibodies followedby incubation with either Jagged1 or Delta-like4 andbinding of the ligand to the cells was monitored by flowcytometry. As shown in the Fig. 1, Table 1, and theSupplementary Table S1, the antibodies specific for EGFrepeats 11 to 12 were more potent inhibitors of ligandbinding than the antibodies specific for EGF repeats 13 to15. Eight different mAbs were characterized for their

effects on ligand-stimulated Notch signaling in HEK293hN1 and hN2 cell lines. Ability of these antibodies toinhibit ligand-mediated receptor activation was in con-cordancewith their effect on ligand–receptor interactions.As shown in Table 1,mAbs specific to EGF repeats 11 to 12inhibited Jagged1- as well as Delta-like4–stimulatedreceptor activation. ThemAb 602.101was themost potentinhibitor of Jagged1/Delta-like4–stimulated Notch1 sig-naling but had no effect on the Notch2 signaling (Fig. 2A)clearly indicating the paralogue specificity of the mAb.Furthermore, this antibody showed a dose-dependentinhibition of Notch1 signaling (Fig. 2B) and proliferation(Fig. 2C), stimulated by both Jagged1 and Delta-like4.These results are in agreement with the previous studiessuggesting that EGF repeats 11 to 12 is the most criticaldomain for ligand binding (39, 42).

Inhibition of Notch signaling in breast cancer celllines by anti-Notch1 antibody

Several studies suggest that Notch receptor and ligandsare overexpressed in the breast cancer tissues comparedwith the normal breast epithelium (14, 27, 43, 44) and thereis a strong correlation between high expression of Notch1and Jagged1 and poor prognosis and survival (15).Because mAb 602.101 inhibited Jagged1 and Delta-like4activities in theNotch1-overexpressing cells, ability of thisantibody to inhibit Notch signaling in the cancer cell linesMCF-7 and MDA-MB-231 was investigated. As shown inthe Fig. 3A forMCF-7 and in Fig. 3B forMDA-MB-231, themAb 602.101was able to inhibit Notch signaling in a dose-dependent manner. MDA-MB-231 cells were cultured inpresence and absence of the antibody for 48 hours, and theendogenous transcript levels of Notch target genes, suchas HES-1, HES-5, and Hey-L, were determined by quan-titative real-time PCR (RT-PCR). As shown in Fig. 3C,mRNA levels of these 3 downstream genes weredecreased in presence of mAb, clearly indicating thatmAb 602.101 suppressed Notch signaling.

Inhibitory effect of anti-Notch1 antibody onproliferation and putative CSC subpopulation inbreast cancer cell lines

MCF-7 and MDA-MB-231 cells were incubated withmAb 602.101 or control IgG for 72 hours and BrdUrdincorporation into DNAwasmonitored. As shown in Fig.3D and E, the mAb inhibited cell proliferation in a dose-dependent manner. To investigate the effect of mAb onCSC population, the cells were grown in suspensionculture in presence or absence of the antibody andallowed to form mammospheres. The cells from cancercell lines, as well as, the primary breast cancer tissuesshowed significant decrease in mammosphere-formingefficiency in presence of mAb (Fig. 4A and D). The mAbalso inhibited secondary and tertiary sphere formation inbreast cancer cell lines (Fig. 4C). Interestingly, the cellstreated with mAb during the primary sphere formationstage could not form secondary spheres even after anti-body removal suggesting that treatment with this mAb

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Figure 1. Effect of anti-Notch1 antibodies on ligand binding. HEK293 hN1cells were preincubated with anti-Notch1 polyclonal (A) and monoclonal(B) antibodies, followed by incubation with the purified Jagged1/Delta-like4 Fc proteins. Ligand binding to Notch1 receptor was determined asdescribed in Table 1. The histograms represent observations from 3independent experiments. DLL4, Delta-like4; FITC, fluoresceinisothiocyanate; JAG1, Jagged1.

Sharma et al.





depletes the stem-like cell subpopulation (Fig. 4B), thuspreventing the recovery. This was further confirmed bythe observation that therewas a significant decrease in theCD44Hi/CD24Low subpopulation of breastCSCswhen theMDA-MB-231 cellswere exposed to the antibody (Fig. 5A)for 72 hours. Furthermore, the sphere-forming efficiencyof CD44Hi/CD24Low was significantly inhibited evenwhen these cells were grown in semisolid suspensionculture using methylcellulose along with the anti-Notch1mAb (Fig. 5B and Supplementary Fig. S6). Incubation ofMDA-MB-231 cells with mAb also increased Annexin V–positive cells clearly indicating that mAb induced apo-ptotic cell death of the cancer cells (Fig. 5C and Supple-mentary Fig. S7).

Modulatory effect of anti-Notch1 antibody on genesassociated with stemness and EMT in breast cancercell linesBecause mAb 602.101 inhibited mammosphere forma-

tion and reduced CD44HI/CD24Low population in breastcancer cell lines, effect of the antibody on expression ofstemness and EMTmarkers was next investigated. MDA-MB-231 cells were cultured in presence of mAb for 48hours and transcript levels of stemness (Bmi-1, Nanog,Sox-2, and Oct-4), epithelial (CK14, CK18, E-cadherin),

and EMT markers (Vimentin, N-cadherin, FN1, Fox-C2,Slug1, Zeb1, Zeb2, Snai1, andTwist1)were determined byquantitative RT-PCR analysis. As shown in Fig. 5D, all 4markers for the stemness were decreased in presence ofthe antibody. Furthermore, the genes associated withepithelial lineage were upregulated, whereas those asso-ciated with induction of EMT were downregulated(Fig. 5E).

Discussion

Antibodies in general have proved to be excellent toolstomap the ligand–receptor contact points andmechanismof receptor activation (38, 45). Recently, it was shown thatantibodies against Notch receptor EGF repeats inhibitligand–receptor interactions and signal transduction(32, 33). In the present study, antibody approach has beenused for in-depth analysis of Notch receptor–ligand inter-actions and potential therapeutic applications. SeveralmAbs were characterized for their effects on ligand bind-ing and consequent receptor activation. ThemAbs specificfor EGF repeats 11 to 12 appeared to be most effective ininhibiting Jagged1 and Delta-like4 binding to Notch1receptor and subsequent response confirming the impor-tance of EGF repeats 11 to 12 in theNotch ligand–receptor

Table 1. Characterization of Notch mAbs

Anti-Notch1 mAba

% binding inhi-bition Notch1

(n ¼ 3)b

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(n ¼ 3)b

% signalinginhibition

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% signalinginhibition

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Clone Epitope (EGF repeats) hJAG1 hDLL4 hJAG1 hDLL4 hJAG1 hDLL4 hJAG1 hDLL4 hNotch1 hNotch2

602.101 11–12 95 78 9 8 80 73 3 3 77 8602.110 11–12 81 71 39 35 63 59 34 32 68 29602.236 11–12 86 84 17 16 52 57 10 9 42 15602.109 13–15 53 54 8 7 23 24 3 3 28 7602.216 13–15 48 13 26 12 17 13 8 7 23 14602.335 13–15 52 55 7 8 17 18 8 7 27 14602.234 11–15 82 73 42 39 38 41 21 20 33 21602.244 11–15 73 51 8 7 27 23 7 7 26 14

aEpitope specificity of NotchmAbs:mAbswere incubatedwith either EGF repeats 11 to 15 or 1 to 12 adsorbed on a plastic surface andbinding was determined. The mAbs that recognized both receptor fragments were considered as EGF repeats 11 to 12 specific,whereas others were identified as EGF repeats 13 to 15–specific mAbs.bThe HEK293 hN1 or hN2 cells were incubatedwith the control IgG or mAbs (10 mg/mL) for 1 hour followed by washingwith DPBS andincubation with the saturating concentration of ligands (20 mg/mL) for 1 hour on ice. The cells were then incubated with antihuman Fcconjugated with fluorescein isothiocyanate antibody and binding was determined by flow cytometry. Ratio of median fluorescenceintensity in the presence of control IgG and Notch1 IgG was calculated for percent inhibition.cHEK293hN1or hN2cells transfectedwith 12xCSL-Luc reporter plasmidwere cultured for 36 hoursonplatesprecoatedwith 20mg/mLsoluble Jagged1/Delta-like4 Fc in the presence of 10 mg/mL control IgG or anti-Notch1 IgG, and the luciferase activities weredetermined by dual luciferase assay. The ratio of firefly luciferase to Renilla luciferase was calculated for normalization. Ratio ofnormalized values for the control IgG to anti-Notch1 IgG was calculated for determining percentage of inhibition.dHEK293hN1, hN2,or vector-alone cellswere culturedonJagged1Fcprecoatedplates in thepresenceof 10mg/mLcontrol IgGor anti-Notch1 mAb. BrdUrd incorporation was investigated by anti-BrdUrd–specific antibody in ELISA and percentage of inhibition wasdetermined compared with control IgG.






interactions. However, role of other EGF repeats in ligandbinding cannot be completely ruled out and antibodiesagainst other domains of Notch can modulate ligandbinding and signal transduction via a mechanism differ-ent from that of the EGF repeats 11 to 12 antibodies(Sharma and Dighe, manuscript in preparation). BecausemAb 602.101 inhibited binding of both Jagged1 andDelta-like4 equally, EGF repeats 11 to 12 could be the primarybinding site for all Notch ligands, as the same domain hasbeen shown to be the binding site for human Delta-like1(39). As in case ofDelta-like1, binding of both Jagged1 andDelta-like4 was increased in presence of calcium butdecreased significantly on chelation (Supplementary Fig.S2). Interestingly, mAb 602.101 binding was alsoincreased in the presence of calcium, indicating ability ofthismAb todetect conformational changes in the receptor,

thus unraveling the receptor activation process (Supple-mentary Fig. S8).

Overexpression of Notch receptors and ligands andconsequent increase in Notch activity has been reportedin number of cancers, particularly the breast cancer andtheir early precursors, linking upregulated Notch signal-ing to pathogenesis (15, 43, 46). Therefore, several attemptshave beenmade to inhibit Notch function by small-molec-ular inhibitors such as GSIs or short interfering RNA-mediated approaches. However, general inhibitors ofg-secretase have wide ranging effect, affecting at least 20different signaling pathways (47) and as discussed above,several adverse effects of such inhibitors havealreadybeenreported. Similarly, the short interfering RNA approach isalso impractical as a general therapeutic strategy. How-ever, domain-specific antibodies can be the magic bullets

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Figure 2. Specificity of anti-Notch1 antibodies. A,HEK293hN1or hN2cellswere cultured with Jagged1/Delta-like4 Fc in presence and absence ofanti-Notch1 mAbs, pAb, and control IgG (50 mg/mL), and the reporteractivity was determined as described in Table 1. B, the experimentdescribed in A was repeated with increasing concentrations of mAb602.101, showing the dose-dependent effect ofmAb onNotch1 signaling.C, HEK293 hN1 cells were cultured with Jagged1/Delta-like4 Fc asdescribed in A and B in presence of increasing concentration of mAb602.101 or control IgG for 72 hours followed by incubationwith BrdUrd fornext 12hours. Incorporation ofBrdUrd inDNAwas investigated using anti-BrdUrd–specific antibody in ELISA. Each experiment was carried out intriplicates and repeated 3 times. RLU, relative luciferase units.

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Figure 3. Effect of anti-Notch1 antibodies on Notch signaling andproliferation in breast cancer cell lines. Effect ofmAb602.101on thebasalNotch activity in the breast cancer cell lines was investigated bytransfecting MCF-7 (A) and MDA-MB-231 (B) cells with 12xCSL-Lucreporter plasmid in presence of increasing concentrations of mAb602.101 or control IgG and determining the reporter activities. C, MDA-MB-231 cells were cultured in presence of 10 mg/mL mAb 602.101 orcontrol IgG for 48 hours. The transcript levels of the downstream targetsof Notch such as HES1, HES5, and HEYL were determined byquantitative RT-PCR. The cell proliferation of MCF-7 (D) and MDA-MB-231 (E) in presence and absence of mAb was determined as describedin Fig. 2C. DMSO, dimethyl sulfoxide.

Sharma et al.





for targeting theNotch-associated pathobiology. Effective-ness of anti-NRR antibodies in targeting oncogenic Notchsignaling in T-acute lymphoblastic leukemia (T-ALL) celllines has already been reported (32, 34). Here, we showeffectiveness of the EGF repeats 11 to 12 specific mAbs inselectively affecting ligand-dependent Notch function inbreast cancer cell lines. The preliminary evidence alsosuggests that it can affect the primary breast tumor cells.Inhibition of Notch signaling leads to reduction in

mammosphere-forming capacity (19, 41, 43), hallmark ofputative stem-like cell subpopulation. The mAb 602.101inhibited cell proliferation and mammosphere formationup to 3 generations in cell lines, as well as, the primarytumor cells, suggesting a strong therapeutic prospect ofthis antibody. Furthermore, once treated with the anti-

body, the cells were unable to recover their stemness andcould not repopulate even in absence of the antibody,clearly indicating the long-term deleterious effects of theantibodyon theputativeCSCsubpopulation. InhibitionofNotch signaling alone has been shown to be not sufficientto inhibit neurosphere recovery and required combinato-rial therapy with a chemotherapeutic agent such as temo-zolomide (48). Recent study showed that GSI MRK-003treatment irreversibly affected sphere formation in theprimary breast tumor cells, but the effect was reversible inthe primary normal mammary epithelial cells suggestingdifferences in the sensitivities of the 2 cell populations(20). On the basis of these observations, it is tempting tospeculate that irreversible inhibition of sphere formationcaused by mAb 602.101 would be limited to the CSC

Figure 4. Effect of anti-Notch1antibody on the putative CSCpopulation. A, MCF-7 or MDA-MB-231 cells (5 � 104 cells) werecultured in semisolid medium(methylcellulose) in the presence of10 mg/mL mAb 602.101, control IgG,dimethyl sulfoxide (DMSO), andDAPT (5 mmol/L), and the number ofmammospheres formed wasdetermined; magnification, �10.B, MDA-MB-231 cells were firstcultured in semisolid medium inpresence of 10 mg/mL mAb 602.101for 1 week followed by trypticdigestion of spheres and culturedagain for secondary sphereformation in the absence of mAb. C,quantitation of mammosphere-forming efficiency of MCF-7 andMDA-MB-231 cells in presence orabsence of the antibody. D, breastcancer tissues from patients wereenzymatically digested, and thesingle-cell suspension wasincubated with 10 mg/mL mAb602.101 in semisolid medium for 1week, and mammosphere formationcapacity was determined;magnification, �10. E, quantificationof mammosphere-forming efficiencyof primary breast cancer cells inpresence or absence of mAb.

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subpopulation without any effect on the normal breaststem cells permanently. Furthermore, our mAb inhibitedthe chemotherapy- and radiotherapy-resistant CD44Hi/CD24Low subpopulation (49) and is potentially a strongtherapeutic tool to reduce these treatment-resistant cells.The antibody also induced apoptotic cell death of thecancer cells andmodulated expression of genes associatedwith stemness and EMT further highlighting therapeutic

potential of this antibody in targeting angiogenesis andmetastasis. It also altered the fate of breast cancer cellstoward myoepithelial lineage as suggested by increasedexpression of CK14 in these cells. The exposure to mAbhas probably initiated a differentiation program, leadingto reduction in stem cell population.

Antibodies are proving to be an extremely interestingtherapeutic strategy for cancer and a number of them

IgG controlA

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D

B77.8% 2.1% 0.2%

0.2%42.6%

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–3

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–2

–4

–6

–8

CD

44 (

FIT

C)

Figure 5. Effect of anti-Notch1 mAb602.101 on CD44Hi/CD24Low

subpopulation and apoptosis.MDA-MB-231 cells were treatedwith 10 mg/mL mAb or control IgG,DAPT (10 mmol/L), and DMSO for72 hours. A, expression of cellsurface markers CD44 and CD24was examined in flow cytometryusing specific antibodies. B,CD44Hi/CD24Low subpopulationwas flow-sorted from MDA-MB-231 cells and cultured in semisolidmedium using a limiting dilutionstrategy in presence of 10 mg/mLmAb 602.101 or control IgG. C,MDA-MB-231cellswere incubatedwith mAb or DAPT and with theirrespective controls for 48 hours,stained with Annexin V fluoresceinisothiocyanate, and analyzed byflow cytometry for apoptotic celldeath. MDA-MB-231 cells weretreated with mAb or DAPT(5 mmol/L) for 48 hours andquantitative RT-PCR was carriedout to determine the transcriptlevels of stemness (D) and EMT (E)markers using GAPDH asnormalizing control. Fold changewas calculated by normalizing thevalue of mAb treatment to controlIgG and DAPT treatment to DMSO.DMSO, dimethyl sulfoxide;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PE,phycoerythrin.

Sharma et al.





are in various stages of therapeutic development.Recent demonstration of the role of Notch1 in breastcancer metastasis to the brain (50) suggests that target-ing Notch as an effective therapeutic strategy and ourantibody appears to be a promising candidate. As dis-cussed above, different Notch receptors have differentfunctions and regulate cell fates differently. BecauseNotch1 and Notch2 have opposing effects on breastcancer progression, an antibody specific for Notch1 islikely to be more effective as a therapeutic tool com-pared with the GSIs. In addition to its therapeuticimportance, this antibody may prove to be a valuabletool in elucidating the molecular intricacies of Notchreceptor–ligand interactions.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Prof. Artavanis-Tsakonas (Harvard Medical School,Boston, MA) for Notch2, Jagged2, Delta-like1 cDNAs and Prof. ChrisBoshoff (UCL, London, UK) for Delta-like4 cDNA construct; Profs. UrbanLendahl (Karolinska Institutet, Stockholm, Sweden) and Freddy Radtke(EPFL, Lausanne, Switzerland) for generously sharing the 12XCSL-Lucreporter and hN1 EGF repeats 1-12 Fc cDNAs, respectively.

Grant Support

The in-house research support was provided by the Indian Institute ofScience, the University Grants Commission, and the Department of Sci-ence and Technology, Government of India, New Delhi, India. A. Sharmawas supported by fellowship from the Council of Scientific and IndustrialResearch, Government of India, New Delhi, India.

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 July 12, 2011; revisedNovember 2, 2011; acceptedNovember 4,2011; published OnlineFirst November 10, 2011.

References1. Artavanis-Tsakonas S, Muskavitch MAT. Notch: the past, the present,

and the future. Curr Top Dev Biol 2010;92:1–29.2. Rebay I, Fleming R, Fehon R, Cherbas L, Cherbas P, Artavanis-

Tsakonas S. Specific EGF repeats of Notch mediate interactions withDelta and Serrate: implications for Notch as amultifunctional receptor.Cell 1991;67:687–99.

3. Bray S.Notch signalling: a simple pathwaybecomes complex. Nat RevMol Cell Biol 2006;7:678–89.

4. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens J, et al. Anovel proteolytic cleavage involved in Notch signaling: the role of thedisintegrin-metalloprotease TACE. Mol Cell 2000;5:207–16.

5. Kopan R, Goate A. A common enzyme connects notch signaling andAlzheimer's disease. Genes Dev 2000;14:2799–806.

6. Kopan R, Ilagan M, Xenia G. The canonical Notch signaling pathway:unfolding the activation mechanism. Cell 2009;137:216–33.

7. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israel A.Signalling downstream of activated mammalian Notch. Nature 1995;377:355–8.

8. Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling in devel-opment and cancer. Endocr Rev 2007;28:339–63.

9. Allenspach EJ, Maillard I, Aster JC, Pear W. Notch signaling in cancer.Cancer Biol Ther 2007;1:466–76.

10. Ellisen L, Bird J, West D, Soreng A, Reynolds T, Smith S, et al. TAN-1,the human homolog of the Drosophila notch gene, is broken bychromosomal translocations in T lymphoblastic neoplasms. Cell 1991;66:649–61.

11. Radtke F, Raj K. The role of Notch in tumorigenesis: oncogene ortumour suppressor? Nat Rev Cancer 2003;3:756–67.

12. Weng A, Ferrando A, LeeW, Morris IV J, Silverman L, Sanchez-IrizarryC, et al. Activating mutations of NOTCH1 in human T cell acutelymphoblastic leukemia. Science 2004;306:269–71.

13. Reedijk M, Pinnaduwage D, Dickson BC, Mulligan AM, Zhang H, BullSB, et al. JAG1 expression is associated with a basal phenotype andrecurrence in lymph node-negative breast cancer. Breast Cancer ResTreat 2008;111:439–48.

14. Parr C, Watkins G, Jiang W. The possible correlation of Notch-1 andNotch-2 with clinical outcome and tumour clinicopathological para-meters in human breast cancer. Int J Mol Med 2004;14:779–86.

15. Reedijk M, Odorcic S, Chang L, Zhang H,Miller N, McCready DR, et al.High-level coexpression of JAG1 and NOTCH1 is observed in humanbreast cancer and is associated with poor overall survival. Cancer Res2005;65:8530–7.

16. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notchsignaling in human breast cancer. Cancer Res 2006;66:1517–25.

17. Dickson BC, Mulligan AM, Zhang H, Lockwood G, O'Malley FP, EganSE, et al. High-level JAG1 mRNA and protein predict poor outcome inbreast cancer. Mod Pathol 2007;20:685–93.

18. Dontu G, Liu S, Wicha MS. Stem cells in mammary development andcarcinogenesis. Stem Cell Rev 2005;1:207–13.

19. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM,WichaMS. Role of Notch signaling in cell-fate determination of humanmammary stem/progenitor cells. Breast Cancer Res 2004;6:R605–15.

20. Kondratyev M, Kreso A, Hallett RM, Girgis-Gabardo A, Barcelon ME,Ilieva D, et al. Gamma-secretase inhibitors target tumor-initiating cellsin a mouse model of ERBB2 breast cancer. Oncogene. 2011 Jun 13.[Epub ahead of print].

21. Nickoloff BJ, Osborne BA, Miele L. Notch signaling as a therapeutictarget in cancer: a new approach to the development of cell fatemodifying agents. Oncogene 2003;22:6598–608.

22. Miele L, Miao H, Nickoloff B. NOTCH signaling as a novel cancertherapeutic target. Curr Cancer Drug Targets 2006;6:313–23.

23. Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, et al. TargetingNotch signaling pathway to overcome drug resistance for cancertherapy. Biochim Biophys Acta 2010;1806:258–67.

24. Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM, et al. Notchpathway inhibition depletes stem-like cells and blocks engraftment inembryonal brain tumors. Cancer Res 2006;66:7445–52.

25. Lewis HD, Leveridge M, Strack PR, Haldon CD, O'Neil J, Kim H, et al.Apoptosis in T cell acute lymphoblastic leukemia cells after cell cyclearrest induced by pharmacological inhibition of notch signaling. ChemBiol 2007;14:209–19.

26. Beel A, Sanders C. Substrate specificity of g-secretase and otherintramembrane proteases. Cell Mol Life Sci 2008;65:1311–34.

27. Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L.Rational targeting of Notch signaling in cancer. Oncogene 2008;27:5124–31.

28. VanEsJH, VanGijnME,RiccioO, vandenBornM,VooijsM,BegthelH,et al. Notch/-secretase inhibition turns proliferative cells in intestinalcrypts and adenomas into goblet cells. Nature 2005;435:959–63.

29. Yan M, Plowman GD. Delta-like 4/Notch signaling and its therapeuticimplications. Clin Cancer Res 2007;13:7243–6.

30. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P,Gale NW, et al. Blockade of Dll4 inhibits tumour growth by promotingnon-productive angiogenesis. Nature 2006;444:1032–7.






31. O'Neill CF, Urs S, Cinelli C, Lincoln A, NadeauRJ, Le�onR, et al. Notch2signaling induces apoptosis and inhibits human MDA-MB-231 xeno-graft growth. Am J Pathol 2007;171:1023–36.

32. Aste-Am�ezagaM, ZhangN, Lineberger JE, Arnold BA, Toner TJ, GuM,et al. Characterization of Notch1 antibodies that inhibit signaling ofboth normal and mutated Notch1 receptors. PLoS One 2010;5:e9094.

33. Li K, Li Y, WuW, GordonW, Chang D, LuM, et al. Modulation of Notchsignaling by antibodies specific for the extracellular negative regula-tory region of NOTCH3. J Biol Chem 2008;283:8046–54.

34. WuY, Cain-HomC, Choy L, Hagenbeek TJ, de Leon GP, Chen Y, et al.Therapeutic antibody targeting of individual Notch receptors. Nature2010;464:1052–7.

35. Dighe R, Moudgal N. Use of [alpha]-and [beta]-subunit specific anti-bodies in studying interaction of hCG with Leydig cell receptors. ArchBiochem Biophys 1983;225:490–9.

36. Dighe RR, Murthy GS, Kurkalli BS, Moudgal NR. Conformation of the[alpha]-subunit of glycoprotein hormones: a study using polyclonaland monoclonal antibodies. Mol Cell Endocrinol 1990;72:63–70.

37. Dighe RR, Satyanarayana Murthy G, Raghuveer Moudgal N. Twosimple and rapid methods to detect monoclonal antibodies withidentical epitope specificities in a large population of monoclonalantibodies. J Immunol Methods 1990;131:229–36.

38. Gadkari RA, Sandhya S, Sowdhamini R, Dighe RR. The antigenbinding sites of various hCG monoclonal antibodies show homol-ogy to different domains of LH receptor. Mol Cell Endocrinol2007;260:23–32.

39. Cordle J, RedfieldZ C, Stacey M, van der Merwe P, Willis A, ChampionB, et al. Localization of the delta-like-1-binding site in human Notch-1and its modulation by calcium affinity. J Biol Chem 2008;283:11785–93.

40. Shawber C, Nofziger D, Hsieh J, Lindsell C, Bogler O, Hayward D, et al.Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 1996;122:3765–73.

41. Dey D, Saxena M, Paranjape AN, Krishnan V, Giraddi R, Kumar MV,et al. Phenotypic and functional characterization of human mammarystem/progenitor cells in long term culture. PLoS One 2009;4:e5329.

42. Ge C, Liu T, Hou X, Stanley P. In vivo consequences of deleting EGFrepeats 8–12 including the ligand binding domain of mouse Notch1.BMC Dev Biol 2008;8:48.

43. Mittal S, SubramanyamD,DeyD,KumarR,RangarajanA.Cooperationof Notch and Ras/MAPK signaling pathways in human breast carci-nogenesis. Mol Cancer 2009;8:128.

44. Zardawi SJ, Zardawi I, McNeil CM, Millar EKA, McLeod D, Morey AL,et al. High Notch1 protein expression is an early event in breast cancerdevelopment and is associated with the HER 2 molecular subtype.Histopathology 2010;56:286–96.

45. Agrawal G, Dighe R. Critical involvement of the hinge region of thefollicle-stimulating hormone receptor in the activation of the receptor. JBiol Chem 2009;284:2636–47.

46. Al-Hussaini H, Subramanyam D, Reedijk M, Sridhar SS. Notch sig-naling pathway as a therapeutic target in breast cancer. Mol CancerTher 2011;10:9–15.

47. Lleo A. Activity of gamma-secretase on substrates other than APP.Curr Top Med Chem 2008;8:9–16.

48. Gilbert CA, Daou M-C, Moser RP, Ross AH. g-secretase inhibitorsenhance temozolomide treatment of human gliomas by inhibitingneurosphere repopulation and xenograft recurrence. Cancer Res2010;70:6870–9.

49. Takebe N, Warren R, Ivy SP. Breast cancer growth and metastasis:interplay between cancer stem cells, embryonic signaling pathwaysand epithelial-to-mesenchymal transition. Breast Cancer Res 2011;13:211.

50. McGowan PM, Simedrea C, Ribot EJ, Foster PJ, Palmieri D, Allan AL,et al. Notch1 inhibition alters the CD44hi/CD24lo population andreduces the formation of brain metastases from breast cancer. MolCancer Res 2011;9:834–44.

Sharma et al.





2012;11:77-86. Published OnlineFirst November 10, 2011.Mol Cancer Ther Ankur Sharma, Anurag N. Paranjape, Annapoorni Rangarajan, et al. like Cells

−Domain Depletes Subpopulation of Putative Breast Cancer Stem Binding−A Monoclonal Antibody against Human Notch1 Ligand

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