A Monoclonal Antibody Against Human Notch1 Ligand– Binding Domain Depletes Subpopulation of Putative Breast Cancer Stem–Like Cells

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

Molecular Cancer Therapeutic Discovery Therapeutics

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

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

Abstract Overexpression of Notch receptors and ligands has been associated with various cancers and developmental disorders, making Notch a potential therapeutic target. Here, we report characterization of Notch1 monoclonal antibodies (mAb) with therapeutic potential. The mAbs generated against epidermal growth factor (EGF) repeats 11 to 15 inhibited binding of Jagged1 and Delta-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 subpopulation. 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 to be irreversible, because the mammosphere-forming efficiency could not be salvaged even after antibody removal during the secondary sphere formation. The antibody also modulated 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 events release Notch intracellular domain (N-ICD) into the nucleus, which then associates with the DNA-binding Notch receptors are single-pass transmembrane recep- proteins to assemble a transcription complex that in turn tors that are involved in normal cell growth, differentia- activates the downstream signaling cascade (7). tion, and death in multicellular organisms, in a context- In addition, defects in Notch signaling lead to a large dependent manner (1). The N-terminal part of Notch number of pathologic conditions (8). Both overexpression extracellular domain (N-ECD) consists of a series of epi- and downregulation of Notch receptors and ligands have dermal growth factor (EGF)-like repeats (ELR) that are been implicated in human cancers (9–12). Many reports required for ligand binding (2). Notch signaling relies on suggest a strong correlation between coexpression of the ability of membrane-bound ligands of Delta/Serrate/ Notch receptors and its ligands in the breast cancer path- Lag-2 (DSL) family to bring about conformational changes ogenesis, which in turn leads to poor prognosis and in the negative regulatory region (NRR) of the receptor, survival in different cohorts (13–17). Recent studies have followed by a series of proteolytic events within the suggested presence of cancer stem cell (CSC) subpopula- transmembrane region, catalyzed by ADAM/TACE tion in various cancerous tissues including breast cancers metalloproteases and g-secretase (3–6). These proteolytic (18). As in the case of normal stem cells, Notch signaling plays an important role in maintenance of CSCs. Activa- tion of Notch signaling results in elevated self-renewal, as Authors' Afﬁliation: Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India shown by increased mammosphere formation, whereas inhibition of Notch signaling had the opposite effect (19). Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). Inhibition of Notch signaling also affected the tumor- sphere-forming capacity of breast cancer cells (20). Thus, Corresponding Author: Rajan R. Dighe, Department of Molecular Repro- duction Development and Genetics, Indian Institute of Science, Bangalore Notch signaling provides a potential therapeutic target for 560012, India. Phone: 9180-2293-2660/3261; Fax: 9180-2360-0999; breast cancer treatment. E-mail: [email protected] Different strategies are being developed to block doi: 10.1158/1535-7163.MCT-11-0508 Notch signaling for therapeutic targeting (21–23), 2011 American Association for Cancer Research. the most prominent being inhibition of proteolytic

www.aacrjournals.org 77

Sharma et al.

cleavages by g-secretase inhibitors (GSI; refs. 24, 25). Expression of Notch1 receptor fragments However, GSIs being universal inhibitors of g-secretases A fragment of Notch1 extracellular domain was also inhibit other signaling pathways (26) and have expressed as glutathione S-transferase (GST) fusion pro- limited therapeutic potential because of their serious tein by PCR amplifying the cDNA encoding EGF repeats adverse effects (27, 28). Therefore, it is essential to 11 to 15 (amino acids 412–601) and cloning into pGEX-4-T1 generate tools that can exclusively target Notch signal- (GE Healthcare). The protein was purified from the sol- ing pathway and specific antibodies are the ideal mole- uble cell lysate using glutathione affinity chromatography cules. These antibodies could be against the ligands (29, (Supplementary Fig. S1A). The hN1 EGF repeats 1 to 12 30) or different domains of receptor that can distinguish was expressed as Fc fusion protein (generously provided between individual Notch receptors. The anti-ligand by Prof. Radtke, EPFL, Lausanne, Switzerland) and puri- antibodies are likely to affect ligand binding to all Notch fied using Protein-A affinity chromatography. receptors leading to pan-Notch inhibition and may not be useful in pathologic situations precipitated by the Polyclonal and monoclonal antibodies paralogue-specific Notch signaling. This is particularly Polyclonal antibodies were raised in the rabbits against the case for breast cancers, where overexpression of EGF repeats 11 to 15 using the immunization protocols Notch1 is correlated with poor prognosis whereas that well established in the laboratory (35, 36). Mice were of Notch2 is associated with good clinical outcomes, immunized with the same immunogen and monoclonal thus highlighting the importance of individual receptor- antibodies (mAb) were generated using protocols estab- specific inhibitors (14, 31). lished for glycoprotein hormones (37, 38). Binding of Individual receptor-specific antibodies against mAbs to EGF repeats 11 to 15 and GST was determined Notch1 and Notch3 NRR domains have been shown using standard ELISA protocol, and the clones reacting to stabilize the receptors in an autoinhibited state and with GST were eliminated by incubating the hybridoma are allosteric in nature (32–34). However, antibodies medium with the soluble GST (1 mg/mL) and then deter- specific for the ligand-binding domain of the receptor mining binding to EGF repeats 11 to 15. All interesting can serve dual functions of being the potential thera- mAb-producing clones were further subcloned and peutic tools while providing insights into the mecha- monoclonality established by isotyping the mAbs using nism of ligand binding and subsequent receptor acti- the Isotyping Kit (Sigma-Aldrich) and determining the vation. Previous attempts of generating antibodies cross-reactivity with other Notch fragments. The detail of against human Notch1 EGF repeats 1 to 36 suggest antibody production is provided in the Supplementary that these antibodies can inhibit ligand-dependent Methods. Notch activation, but their precise epitopes have not been mapped and their mechanism of action needs to Flow cytometric assay be investigated (32). In present study, we report pro- Cells expressing Notch were harvested using Dulbec- duction and characterization of antibodies against EGF co’s PBS (DPBS)-EDTA, resuspended in DPBS containing repeats11to15ofNotch1andshowthatoneofthe 2% FBS (Invitrogen; FBS/PBS), and incubated with the monoclonal antibodies inhibits ligand binding and primary antibody for 40 minutes at room temperature, consequently, signaling. We further show that this followed by washing, resuspension, and incubation in antibody can inhibit cell proliferation and particularly 0.1 mL of FBS/PBS containing fluorescein isothiocyanate– target the putative CSCs by modulating expression of conjugated antirabbit or mouse secondary antibodies for genes associated with stemness, as well as, the epithe- 30 minutes at room temperature. The cells were washed, lial–mesenchymal transition (EMT) markers. This anti- resuspended in DPBS, and analyzed using the Becton body also inhibited proliferation and induced apopto- Dickinson FACSCanto. The median values were calculat- tic cell death in breast cancer cell lines. ed using the Stat program of CellQuest by Becton Dick- inson. Flow cytometry–based ligand-binding assay was Materials and Methods conducted on ice as previously described (39).

Generation of stable cell lines Luciferase reporter assay The stable cell lines overexpressing human Notch1 To determine effect of anti-Notch1 antibodies on Notch (hN1) and human Notch2 (hN2) were generated by trans- signaling, functional assay for Notch signaling was devel- fecting HEK293 cells with the respective cDNAs cloned oped. The cells expressing Notch1 receptor such as MCF- into pcDNA3.1Myc/His (Invitrogen). The Notch ligands, 7, MDA-MB-231, or HEK293 cells overexpressing hN1 or hJagged1, hJagged2, hDelta-like1, and hDelta-like4, were hN2 were seeded (5 104 cells per well) in a 24-well plate expressed as Fc fusion protein by cloning the respective (Nunc) and transfected with 790 ng 12xCSL-Luc and 10 ng cDNAs into pFUSE-Fc1-IgG1 (InvivoGen) and purified pGL3Basic or 800 ng pGL3 control along with 1 ng pRL-Tk from the culture supernatant by Protein-A affinity chro- (Promega) using Lipofectamine 2000 (Invitrogen) as per matography (GE Healthcare). MCF-7 and MDA-MB-231 the manufacturer’s instructions. Ligands were provided cells were procured from American Type Culture Collec- by precoating the wells with purified Jagged1 or Delta- tion. No further authentication was conducted. like4 Fc (1 mg per well). The transfected cells were

78 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Therapeutic Antibody Targeting of Notch1 in Breast CSCs

incubated with or without 10 mg/mL anti-Notch1 anti- the antibody every fourth day. The primary mammo- bodies. Luciferase reporter activity was estimated after 36 spheres were counted manually using ImageJ software, hours using the Dual Luciferase Assay Kit (Promega) and trypsinized, and allowed to form secondary and tertiary a TD-20 Luminometer (Turner Design) following the spheres with or without antibodies. The same experiment manufacturer’s protocol. was also carried out in presence of 5 mmol/L DAPT (Sigma) used as a positive control for Notch inhibition. C2C12 differentiation assay The C2C12 differentiation assay was conducted as Results described previously (40). Briefly, C2C12 mouse myoblast cells (5 104) were cultured in Dulbecco’s Modified Characterization of soluble Notch ligands Eagle’s Media (DMEM) and 10% FBS and induced to The biologic activities of purified soluble Notch ligands differentiate by replacing the original medium with the (Jagged1/Delta-like4 Fc; Supplementary Fig. S1B) were differentiating medium containing 2% horse serum which established by showing their binding to the full-length was replenished every day. At the end of 4-day incuba- Notch1 receptor and also determining their ability to elicit tion, cells were examined for expression of myosin heavy response using the cell-based assays. The HEK293 hN1 þ þ chain by a confocal Zeiss LSM 510 META microscope. cells were suspended in DPBS (Ca 2,Mg 2 free), incubated with purified Jagged1 and Delta-like4 dissolved in Cell proliferation and apoptosis assay Hanks’ balanced salt solution (HBSS) containing 1.26 To investigate effect of anti-Notch1 antibodies on pro- mmol/L CaCl2, and the ligand binding was determined liferation of cells endogenously expressing Notch1, MCF- by flow cytometry. As shown in the Supplementary Fig. 7, and MDA-MB-231, the cells were seeded in a 96-well S2A, there was specific ligand binding to Notch1 receptor. plate (5 103 cells per well) for 4 hours and incubated with Furthermore, addition of 5 mmol/L EGTA to HBSS or without antibodies for 72 hours. Cells were subsequent- resulted in a significant decrease in ligand binding (Sup- ly labeled with bromodeoxyuridine (BrdUrd) for 12 hours plementary Fig. S2), suggesting that interactions of these and its incorporation determined as per the protocol ligands with Notch1 are also calcium dependent, as in recommended by the manufacturer (Calbiochem). case of Delta-like1 (39). As shown in the Supplementary Fig. S3A, there was a dose-dependent increase in binding Collection and processing of primary breast cancer of Jagged1 and Delta-like4 to EGF repeats 11 to 15 protein tissue immobilized on a plastic surface. Furthermore, preincu- Primary breast tumor tissue was obtained from the bation of the ligands with EGF repeats 11 to 15 or 1 to 12 Fc Kidwai Memorial Hospital, Bangalore, India, as per the proteins abolished their subsequent binding to Notch1- ethical guidelines of the Institutional Review Board of expressing cells (data not shown), showing the functional the hospital and the Indian Institute of Science Bangalore, nature of the ligands as well as receptor fragments. This India. The tissue was aseptically minced and dissociated was confirmed by showing that both ligands stimulated enzymatically using 1 mg/mL Collagenase (Sigma- 12xCSL Luc reporter activities in the HEK293 hN1 cells in Aldrich) and 100 U/mL hyaluronidase (Calbiochem) at a dose-dependent manner (Supplementary Fig. S3B). Sim- 37C for 16 hours with constant rotation. Breast organoids ilarly, the precoated ligands led to inhibition of C2C12 were separated by centrifugation, washed with PBS, differentiation, as examined by the myosin heavy chain and resuspended in DMEM-F12 with growth factors expression in myotubes, using anti–myosin heavy chain (10 mg/mL human EGF, 1 mg/mL hydrocortisone, antibody (Supplementary Fig. S4). 10 mg/mL insulin, 20 ng/mL basic fibroblast growth factor, 4 ng/mL heparin, and 1% B27), antibiotics and Characterization of anti-Notch1 antibodies Fungizone, and incubated at 37C for 6 hours. The organ- Binding of polyclonal antibody to EGF repeats 11 to 15 oids were further dissociated with trypsin to yield single showed that it is a high titer antibody with considerable cells which were filtered through a 70-mm BD cell strainer binding to GST (Supplementary Fig. S5). Passing poly- to remove clumps and to obtain a largely single-cell clonal antibodies through GST-NHS-Sepharose removed suspension which was used for later experiments. all GST-specific antibodies but retained the antibodies specific for EGF repeats 11 to 15 (processed polyclonal Mammosphere assay antibodies). Furthermore, the processed polyclonal anti- MCF-7, MDA-MB-231 (5 104 cells per well), and bodies could specifically bind to HEK293 hN1 in the flow the enzymatically dissociated single-cell suspensions cytometric assay (data not shown). (2.5 105 cells per well) of the primary breast cancer tis- A total of 35 mAbs that recognized EGF repeats 11 to 15, sues were seeded in a serum-freeDMEM-F12 medium with but not GST, were selected for further analysis. Binding of growth factors in a semisolid medium containing methyl- mAbs to EGF repeats 11 to 15 was determined by ELISA, cellulose as described earlier (19, 41). Effect of anti-Notch1 and as shown in the Supplementary Table S1, several antibodies on sphere-forming efficiency of these cells was mAbs showed varied binding to Notch receptor frag- assessed by incubating the cells with or without anti- ments. Partial epitope mapping was carried out by deter- bodies (10 mg/mL) and culturing for 1 week, replenishing mining binding of these mAbs to EGF repeats 1 to 12 Fc

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

Sharma et al.

and EGF repeats 11 to 15 receptor fragments, followed by effects on ligand-stimulated Notch signaling in HEK293 identification of those mAbs that showed binding to both hN1 and hN2 cell lines. Ability of these antibodies to the proteins, indicating that these antibodies recognize inhibit ligand-mediated receptor activation was in con- EGF repeats 11 to 12 epitope shared by both proteins. cordance with their effect on ligand–receptor interactions. Several mAbs did not recognize EGF repeats 1 to 12 Fc As shown in Table 1, mAbs specific to EGF repeats 11 to 12 fragment, indicating that the epitopes recognized by these inhibited Jagged1- as well as Delta-like4–stimulated antibodies reside in EGF repeats 13 to 15. Ability of all receptor activation. The mAb 602.101 was the most potent mAbs to recognize the full-length Notch1 was established inhibitor of Jagged1/Delta-like4–stimulated Notch1 sig- by flow cytometric assay, as shown in the Supplementary naling but had no effect on the Notch2 signaling (Fig. 2A) Table S1. clearly indicating the paralogue specificity of the mAb. Furthermore, this antibody showed a dose-dependent Inhibition of ligand-dependent Notch signaling by inhibition of Notch1 signaling (Fig. 2B) and proliferation anti-Notch1 antibodies (Fig. 2C), stimulated by both Jagged1 and Delta-like4. Having characterized both, Notch ligands and antibo- These results are in agreement with the previous studies dies, effect of antibodies on ligand-receptor interactions suggesting that EGF repeats 11 to 12 is the most critical was investigated. The HEK293 hN1 cells were preincu- domain for ligand binding (39, 42). bated with polyclonal or monoclonal antibodies followed by incubation with either Jagged1 or Delta-like4 and Inhibition of Notch signaling in breast cancer cell binding of the ligand to the cells was monitored by flow lines by anti-Notch1 antibody cytometry. As shown in the Fig. 1, Table 1, and the Several studies suggest that Notch receptor and ligands Supplementary Table S1, the antibodies specific for EGF are overexpressed in the breast cancer tissues compared repeats 11 to 12 were more potent inhibitors of ligand with the normal breast epithelium (14, 27, 43, 44) and there binding than the antibodies specific for EGF repeats 13 to is a strong correlation between high expression of Notch1 15. Eight different mAbs were characterized for their and Jagged1 and poor prognosis and survival (15). Because mAb 602.101 inhibited Jagged1 and Delta-like4 activities in the Notch1-overexpressing cells, ability of this A antibody to inhibit Notch signaling in the cancer cell lines MCF-7 and MDA-MB-231 was investigated. As shown in 1008060 1008060 Fc control Fc control the Fig. 3A for MCF-7 and in Fig. 3B for MDA-MB-231, the 11–15 pAb + DLL4 Fc mAb 602.101 was able to inhibit Notch signaling in a dose- 11–15 pAb + JAGI Fc DLL4 Fc dependent manner. MDA-MB-231 cells were cultured in JAGI Fc presence and absence of the antibody for 48 hours, and the endogenous transcript levels of Notch target genes, such Counts Counts 4020 4020 as HES-1, HES-5, and Hey-L, were determined by quantitative real-time PCR (RT-PCR). As shown in Fig. 3C, mRNA levels of these 3 downstream genes were decreased in presence of mAb, clearly indicating that 0 0 100 101 102 103 104 100 101 102 103 104 mAb 602.101 suppressed Notch signaling. B FITC-A FITC-A Inhibitory effect of anti-Notch1 antibody on 100 1008060 Fc control Fc control proliferation and putative CSC subpopulation in 602.101 mAb + DLL4 Fc

602.101 mAb + JAGI Fc 8060 breast cancer cell lines DLL4 Fc MCF-7 and MDA-MB-231 cells were incubated with mAb 602.101 or control IgG for 72 hours and BrdUrd JAGI Fc incorporation into DNA was monitored. As shown in Fig. Counts Counts 4020 4020 3D and E, the mAb inhibited cell proliferation in a dose- dependent manner. To investigate the effect of mAb on CSC population, the cells were grown in suspension

0 culture in presence or absence of the antibody and 0 100 101 102 103 104 100 101 102 103 104 allowed to form mammospheres. The cells from cancer FITC-A FITC-A cell lines, as well as, the primary breast cancer tissues showed significant decrease in mammosphere-forming Figure 1. Effect of anti-Notch1 antibodies on ligand binding. HEK293 hN1 efficiency in presence of mAb (Fig. 4A and D). The mAb cells were preincubated with anti-Notch1 polyclonal (A) and monoclonal also inhibited secondary and tertiary sphere formation in fi (B) antibodies, followed by incubation with the puri ed Jagged1/Delta- breast cancer cell lines (Fig. 4C). Interestingly, the cells like4 Fc proteins. Ligand binding to Notch1 receptor was determined as described in Table 1. The histograms represent observations from 3 treated with mAb during the primary sphere formation independent experiments. DLL4, Delta-like4; FITC, fluorescein stage could not form secondary spheres even after anti- isothiocyanate; JAG1, Jagged1. body removal suggesting that treatment with this mAb

80 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Therapeutic Antibody Targeting of Notch1 in Breast CSCs

Table 1. Characterization of Notch mAbs

% binding inhi- % binding inhi- % signaling % signaling bition Notch1 bition Notch2 inhibition inhibition % proliferation Anti-Notch1 mAba (n ¼ 3)b (n ¼ 3)b Notch1 (n ¼ 3)c Notch2 (n ¼ 3)c inhibition (n ¼ 3)d

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 8 602.110 11–12 81 71 39 35 63 59 34 32 68 29 602.236 11–12 86 84 17 16 52 57 10 9 42 15 602.109 13–15 53 54 8 7 23 24 3 3 28 7 602.216 13–15 48 13 26 12 17 13 8 7 23 14 602.335 13–15 52 55 7 8 17 18 8 7 27 14 602.234 11–15 82 73 42 39 38 41 21 20 33 21 602.244 11–15 73 51 8 7 27 23 7 7 26 14

aEpitope specificity of Notch mAbs: mAbs were incubated with either EGF repeats 11 to 15 or 1 to 12 adsorbed on a plastic surface and binding 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 incubated with the control IgG or mAbs (10 mg/mL) for 1 hour followed by washing with DPBS and incubation with the saturating concentration of ligands (20 mg/mL) for 1 hour on ice. The cells were then incubated with antihuman Fc conjugated with fluorescein isothiocyanate antibody and binding was determined by flow cytometry. Ratio of median fluorescence intensity in the presence of control IgG and Notch1 IgG was calculated for percent inhibition. cHEK293 hN1 or hN2 cells transfected with 12xCSL-Luc reporter plasmid were cultured for 36 hours on plates precoated with 20 mg/mL soluble Jagged1/Delta-like4 Fc in the presence of 10 mg/mL control IgG or anti-Notch1 IgG, and the luciferase activities were determined by dual luciferase assay. The ratio of firefly luciferase to Renilla luciferase was calculated for normalization. Ratio of normalized values for the control IgG to anti-Notch1 IgG was calculated for determining percentage of inhibition. dHEK293 hN1, hN2, or vector-alone cells were cultured on Jagged1 Fc precoated plates in the presence of 10 mg/mL control IgG or anti- Notch1 mAb. BrdUrd incorporation was investigated by anti-BrdUrd–specific antibody in ELISA and percentage of inhibition was determined compared with control IgG.

depletes the stem-like cell subpopulation (Fig. 4B), thus and EMT markers (Vimentin, N-cadherin, FN1, Fox-C2, preventing the recovery. This was further confirmed by Slug1, Zeb1, Zeb2, Snai1, and Twist1) were determined by the observation that there was a significant decrease in the quantitative RT-PCR analysis. As shown in Fig. 5D, all 4 CD44Hi/CD24Low subpopulation of breast CSCs when the markers for the stemness were decreased in presence of MDA-MB-231 cells were exposed to the antibody (Fig. 5A) the antibody. Furthermore, the genes associated with for 72 hours. Furthermore, the sphere-forming efficiency epithelial lineage were upregulated, whereas those asso- of CD44Hi/CD24Low was significantly inhibited even ciated with induction of EMT were downregulated when these cells were grown in semisolid suspension (Fig. 5E). culture using methylcellulose along with the anti-Notch1 mAb (Fig. 5B and Supplementary Fig. S6). Incubation of Discussion MDA-MB-231 cells with mAb also increased Annexin V– positive cells clearly indicating that mAb induced apo- Antibodies in general have proved to be excellent tools ptotic cell death of the cancer cells (Fig. 5C and Supple- to map the ligand–receptor contact points and mechanism mentary Fig. S7). of receptor activation (38, 45). Recently, it was shown that antibodies against Notch receptor EGF repeats inhibit Modulatory effect of anti-Notch1 antibody on genes ligand–receptor interactions and signal transduction associated with stemness and EMT in breast cancer (32, 33). In the present study, antibody approach has been cell lines used for in-depth analysis of Notch receptor–ligand inter- Because mAb 602.101 inhibited mammosphere forma- actions and potential therapeutic applications. Several tion and reduced CD44HI/CD24Low population in breast mAbs were characterized for their effects on ligand bind- cancer cell lines, effect of the antibody on expression of ing and consequent receptor activation. The mAbs specific stemness and EMT markers was next investigated. MDA- for EGF repeats 11 to 12 appeared to be most effective in MB-231 cells were cultured in presence of mAb for 48 inhibiting Jagged1 and Delta-like4 binding to Notch1 hours and transcript levels of stemness (Bmi-1, Nanog, receptor and subsequent response confirming the impor- Sox-2, and Oct-4), epithelial (CK14, CK18, E-cadherin), tance of EGF repeats 11 to 12 in the Notch ligand–receptor

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 81

Sharma et al.

A A B 100 100 100 hJAG1 Fc hDLL4 Fc 80 80 80 60 60

40 40 60

20 602.101 20 602.101

Relative luciferase activity luciferase Relative Control IgG activity luciferase Relative Control IgG 40 0 0 10–4 10–3 10–2 10–1 100 101 102 10–4 10–3 10–2 10–1 100 101 102

% Inhibition (RLU) Conc (µg/mL) Conc (µg/mL) 20 C Hes-1 Hes-5 Hey-L 0

0 N1N2 N1N2 N1N2 N1N2 N1N2 N1N2 N1N2 N1N2 N1N2 N1N2 –1

–2 101 109 110 216 234 236 244 335 pAb

Fold change Fold –3 Control IgG 602.101 IgG/control IgG BC –4 DAPT/DMSO 1.5 D E 0.6 60 1.5

450 1.0 40 0.4 1.0 450/595 450/595

hJAG1Fc + 602.101 0.5 20 hDLL4Fc + 602.101 hJAG1Fc + 602.101 0.2

Absorbance 0.5 hJAG1Fc + control IgG hDLL4Fc + 602.101 602.101 Absorbance 602.101 Absorbance hDLL4Fc + control IgG hJAG1Fc + control IgG Control IgG Control IgG

Relative luciferase activity luciferase Relative hDLL4Fc + control IgG 0 0.0 0.0 0.0 10–3 10–2 10–1 100 101 102 10–3 10–2 10–1 100 101 102 10–4 10–3 10–2 10–1 100 101 102 10–4 10–3 10–2 10–1 100 101 102 Conc (µg/mL) Conc (µg/mL) Conc (µg/mL) Conc (µg/mL)

Figure 2. Speciﬁcity of anti-Notch1 antibodies. A, HEK293 hN1 or hN2 cells Figure 3. Effect of anti-Notch1 antibodies on Notch signaling and were cultured with Jagged1/Delta-like4 Fc in presence and absence of proliferation in breast cancer cell lines. Effect of mAb 602.101 on the basal anti-Notch1 mAbs, pAb, and control IgG (50 mg/mL), and the reporter Notch activity in the breast cancer cell lines was investigated by activity was determined as described in Table 1. B, the experiment transfecting MCF-7 (A) and MDA-MB-231 (B) cells with 12xCSL-Luc described in A was repeated with increasing concentrations of mAb reporter plasmid in presence of increasing concentrations of mAb 602.101, showing the dose-dependent effect of mAb on Notch1 signaling. 602.101 or control IgG and determining the reporter activities. C, MDA- C, HEK293 hN1 cells were cultured with Jagged1/Delta-like4 Fc as MB-231 cells were cultured in presence of 10 mg/mL mAb 602.101 or described in A and B in presence of increasing concentration of mAb control IgG for 48 hours. The transcript levels of the downstream targets 602.101 or control IgG for 72 hours followed by incubation with BrdUrd for of Notch such as HES1, HES5, and HEYL were determined by next 12 hours. Incorporation of BrdUrd in DNA was investigated using anti- quantitative RT-PCR. The cell proliferation of MCF-7 (D) and MDA-MB- BrdUrd–speciﬁc antibody in ELISA. Each experiment was carried out in 231 (E) in presence and absence of mAb was determined as described triplicates and repeated 3 times. RLU, relative luciferase units. in Fig. 2C. DMSO, dimethyl sulfoxide.

interactions. However, role of other EGF repeats in ligand thus unraveling the receptor activation process (Supple- binding cannot be completely ruled out and antibodies mentary Fig. S8). against other domains of Notch can modulate ligand Overexpression of Notch receptors and ligands and binding and signal transduction via a mechanism differ- consequent increase in Notch activity has been reported ent from that of the EGF repeats 11 to 12 antibodies in number of cancers, particularly the breast cancer and (Sharma and Dighe, manuscript in preparation). Because their early precursors, linking upregulated Notch signal- mAb 602.101 inhibited binding of both Jagged1 and Delta- ing to pathogenesis (15, 43, 46). Therefore, several attempts like4 equally, EGF repeats 11 to 12 could be the primary have been made to inhibit Notch function by small-molec- binding site for all Notch ligands, as the same domain has ular inhibitors such as GSIs or short interfering RNA- been shown to be the binding site for human Delta-like1 mediated approaches. However, general inhibitors of (39). As in case of Delta-like1, binding of both Jagged1 and g-secretase have wide ranging effect, affecting at least 20 Delta-like4 was increased in presence of calcium but different signaling pathways (47) and as discussed above, decreased signiﬁcantly on chelation (Supplementary Fig. several adverse effects of such inhibitors have already been S2). Interestingly, mAb 602.101 binding was also reported. Similarly, the short interfering RNA approach is increased in the presence of calcium, indicating ability of also impractical as a general therapeutic strategy. How- this mAb to detect conformational changes in the receptor, ever, domain-speciﬁc antibodies can be the magic bullets

82 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Therapeutic Antibody Targeting of Notch1 in Breast CSCs

A Untreated IgG 602.101 DMSO DAPT MCF-7

Figure 4. Effect of anti-Notch1 antibody on the putative CSC population. A, MCF-7 or MDA- MB-231 cells (5 104 cells) were cultured in semisolid medium MDA-MB-231 (methylcellulose) in the presence of B 10 mg/mL mAb 602.101, control IgG, Control IgG–treated 602.101–treated Recovery of 602.101– dimethyl sulfoxide (DMSO), and primary spheres primary spheres treated spheres DAPT (5 mmol/L), and the number of mammospheres formed was determined; magnification, 10. B, MDA-MB-231 cells were first cultured in semisolid medium in presence of 10 mg/mL mAb 602.101 for 1 week followed by tryptic digestion of spheres and cultured again for secondary sphere C formation in the absence of mAb. C, 250 Primary Primary 600 Control IgG quantitation of mammosphere- Secondary DMSO Secondary forming efficiency of MCF-7 and 200 Control IgG Tertiary Tertiary MDA-MB-231 cells in presence or DMSO absence of the antibody. D, breast 150 400 cancer tissues from patients were 100 DAPT enzymatically digested, and the DAPT (5 µmol/L) 200 single-cell suspension was (5 µmol/L) incubated with 10 mg/mL mAb 50 mAb 602.101 mAb 602.101

602.101 in semisolid medium for 1 of MCF-7 sphere/20,000 cells No. 0 0 week, and mammosphere formation cells of MDA-MB-231sphere/20,000 No. capacity was determined; magnification, 10. E, quantification fi DE of mammosphere-forming ef ciency Patient 1 Patient 2 Patient 3 Patient 4 of primary breast cancer cells in presence or absence of mAb. 400 IgG 300 602.101 IgG 200 100 0 602.101 No. of mammosphere/50,000 cells No.

Patient 1 Patient 2 Patient 3 Patient 4

for targeting the Notch-associated pathobiology. Effective- body, the cells were unable to recover their stemness and ness of anti-NRR antibodies in targeting oncogenic Notch could not repopulate even in absence of the antibody, signaling in T-acute lymphoblastic leukemia (T-ALL) cell clearly indicating the long-term deleterious effects of the lines has already been reported (32, 34). Here, we show antibody on the putative CSC subpopulation. Inhibition of effectiveness of the EGF repeats 11 to 12 speciﬁc mAbs in Notch signaling alone has been shown to be not sufﬁcient selectively affecting ligand-dependent Notch function in to inhibit neurosphere recovery and required combinato- breast cancer cell lines. The preliminary evidence also rial therapy with a chemotherapeutic agent such as temo- suggests that it can affect the primary breast tumor cells. zolomide (48). Recent study showed that GSI MRK-003 Inhibition of Notch signaling leads to reduction in treatment irreversibly affected sphere formation in the mammosphere-forming capacity (19, 41, 43), hallmark of primary breast tumor cells, but the effect was reversible in putative stem-like cell subpopulation. The mAb 602.101 the primary normal mammary epithelial cells suggesting inhibited cell proliferation and mammosphere formation differences in the sensitivities of the 2 cell populations up to 3 generations in cell lines, as well as, the primary (20). On the basis of these observations, it is tempting to tumor cells, suggesting a strong therapeutic prospect of speculate that irreversible inhibition of sphere formation this antibody. Furthermore, once treated with the anti- caused by mAb 602.101 would be limited to the CSC

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 83

Sharma et al.

A B IgG control 602.101 IgG control 602.101 77.8% 2.1% 57% 0.2% cells 5 1 × 10

19.7% 0.4% 42.6% 0.2% cells 4 DMSO DAPT Figure 5. Effect of anti-Notch1 mAb 602.101 on CD44Hi/CD24Low

81.6% 0.3% 61.7% 0.3% 1 × 10 subpopulation and apoptosis. CD 44 (FITC) MDA-MB-231 cells were treated with 10 mg/mL mAb or control IgG, cells 3 DAPT (10 mmol/L), and DMSO for 17.9% 0.2% 37.9% 0.1% 72 hours. A, expression of cell 1 × 10 surface markers CD44 and CD24 was examined in flow cytometry fi CD 24 (PE) using speci c antibodies. B, CD44Hi/CD24Low subpopulation C D was flow-sorted from MDA-MB- 231 cells and cultured in semisolid Bmi-1 Nanog Sox-2 Oct-4 medium using a limiting dilution 0 strategy in presence of 10 mg/mL mAb 602.101 or control IgG. C, –1 MDA-MB-231 cells were incubated Control IgG DMSO with mAb or DAPT and with their –2 respective controls for 48 hours, stained with Annexin V fluorescein

Fold change Fold –3 isothiocyanate, and analyzed by 602.101 IgG/Control IgG ﬂow cytometry for apoptotic cell DAPT/DMSO –4 death. MDA-MB-231 cells were treated with mAb or DAPT (5 mmol/L) for 48 hours and 602.101 DAPT quantitative RT-PCR was carried out to determine the transcript levels of stemness (D) and EMT (E) E 4 markers using GAPDH as normalizing control. Fold change was calculated by normalizing the 2 value of mAb treatment to control IgG and DAPT treatment to DMSO. DMSO, dimethyl sulfoxide; Vimentin N-cadherin FN1 Fox-C2 Slug1 Zeb1 Zeb2 Snai1 Twist1 0 GAPDH, glyceraldehyde-3- phosphate dehydrogenase; PE, phycoerythrin.

–2 CK-14 CK-18 E-cadherin Fold change Fold –4

–6 602.101 IgG/Control IgG DAPT/DMSO –8

subpopulation without any effect on the normal breast potential of this antibody in targeting angiogenesis and stem cells permanently. Furthermore, our mAb inhibited metastasis. It also altered the fate of breast cancer cells the chemotherapy- and radiotherapy-resistant CD44Hi/ toward myoepithelial lineage as suggested by increased CD24Low subpopulation (49) and is potentially a strong expression of CK14 in these cells. The exposure to mAb therapeutic tool to reduce these treatment-resistant cells. has probably initiated a differentiation program, leading The antibody also induced apoptotic cell death of the to reduction in stem cell population. cancer cells and modulated expression of genes associated Antibodies are proving to be an extremely interesting with stemness and EMT further highlighting therapeutic therapeutic strategy for cancer and a number of them

84 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

Therapeutic Antibody Targeting of Notch1 in Breast CSCs

are in various stages of therapeutic development. Acknowledgments Recent demonstration of the role of Notch1 in breast cancer metastasis to the brain (50) suggests that target- The authors thank Prof. Artavanis-Tsakonas (Harvard Medical School, Boston, MA) for Notch2, Jagged2, Delta-like1 cDNAs and Prof. Chris ing Notch as an effective therapeutic strategy and our Boshoff (UCL, London, UK) for Delta-like4 cDNA construct; Profs. Urban antibody appears to be a promising candidate. As dis- Lendahl (Karolinska Institutet, Stockholm, Sweden) and Freddy Radtke (EPFL, Lausanne, Switzerland) for generously sharing the 12XCSL-Luc cussed above, different Notch receptors have different reporter and hN1 EGF repeats 1-12 Fc cDNAs, respectively. functions and regulate cell fates differently. Because Notch1 and Notch2 have opposing effects on breast cancer progression, an antibody specific for Notch1 is Grant Support likely to be more effective as a therapeutic tool com- The in-house research support was provided by the Indian Institute of pared with the GSIs. In addition to its therapeutic Science, the University Grants Commission, and the Department of Sci- importance, this antibody may prove to be a valuable ence and Technology, Government of India, New Delhi, India. A. Sharma was supported by fellowship from the Council of Scientific and Industrial tool in elucidating the molecular intricacies of Notch Research, Government of India, New Delhi, India. receptor–ligand interactions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conflicts of Interest Received July 12, 2011; revised November 2, 2011; accepted November 4, No potential conflicts of interest were disclosed. 2011; published OnlineFirst November 10, 2011.

References 1. Artavanis-Tsakonas S, Muskavitch MAT. Notch: the past, the present, 16. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch and the future. Curr Top Dev Biol 2010;92:1–29. signaling in human breast cancer. Cancer Res 2006;66:1517–25. 2. Rebay I, Fleming R, Fehon R, Cherbas L, Cherbas P, Artavanis- 17. Dickson BC, Mulligan AM, Zhang H, Lockwood G, O'Malley FP, Egan Tsakonas S. Speciﬁc EGF repeats of Notch mediate interactions with SE, et al. High-level JAG1 mRNA and protein predict poor outcome in Delta and Serrate: implications for Notch as a multifunctional receptor. breast cancer. Mod Pathol 2007;20:685–93. Cell 1991;67:687–99. 18. Dontu G, Liu S, Wicha MS. Stem cells in mammary development and 3. Bray S. Notch signalling: a simple pathway becomes complex. Nat Rev carcinogenesis. Stem Cell Rev 2005;1:207–13. Mol Cell Biol 2006;7:678–89. 19. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, 4. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens J, et al. A Wicha MS. Role of Notch signaling in cell-fate determination of human novel proteolytic cleavage involved in Notch signaling: the role of the mammary stem/progenitor cells. Breast Cancer Res 2004;6:R605–15. disintegrin-metalloprotease TACE. Mol Cell 2000;5:207–16. 20. Kondratyev M, Kreso A, Hallett RM, Girgis-Gabardo A, Barcelon ME, 5. Kopan R, Goate A. A common enzyme connects notch signaling and Ilieva D, et al. Gamma-secretase inhibitors target tumor-initiating cells Alzheimer's disease. Genes Dev 2000;14:2799–806. in a mouse model of ERBB2 breast cancer. Oncogene. 2011 Jun 13. 6. Kopan R, Ilagan M, Xenia G. The canonical Notch signaling pathway: [Epub ahead of print]. unfolding the activation mechanism. Cell 2009;137:216–33. 21. Nickoloff BJ, Osborne BA, Miele L. Notch signaling as a therapeutic 7. Jarriault S, Brou C, Logeat F, Schroeter EH, Kopan R, Israel A. target in cancer: a new approach to the development of cell fate Signalling downstream of activated mammalian Notch. Nature 1995; modifying agents. Oncogene 2003;22:6598–608. 377:355–8. 22. Miele L, Miao H, Nickoloff B. NOTCH signaling as a novel cancer 8. Bolos V, Grego-Bessa J, de la Pompa JL. Notch signaling in devel- therapeutic target. Curr Cancer Drug Targets 2006;6:313–23. opment and cancer. Endocr Rev 2007;28:339–63. 23. Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, et al. Targeting 9. Allenspach EJ, Maillard I, Aster JC, Pear W. Notch signaling in cancer. Notch signaling pathway to overcome drug resistance for cancer Cancer Biol Ther 2007;1:466–76. therapy. Biochim Biophys Acta 2010;1806:258–67. 10. Ellisen L, Bird J, West D, Soreng A, Reynolds T, Smith S, et al. TAN-1, 24. Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM, et al. Notch the human homolog of the Drosophila notch gene, is broken by pathway inhibition depletes stem-like cells and blocks engraftment in chromosomal translocations in T lymphoblastic neoplasms. Cell 1991; embryonal brain tumors. Cancer Res 2006;66:7445–52. 66:649–61. 25. Lewis HD, Leveridge M, Strack PR, Haldon CD, O'Neil J, Kim H, et al. 11. Radtke F, Raj K. The role of Notch in tumorigenesis: oncogene or Apoptosis in T cell acute lymphoblastic leukemia cells after cell cycle tumour suppressor? Nat Rev Cancer 2003;3:756–67. arrest induced by pharmacological inhibition of notch signaling. Chem 12. Weng A, Ferrando A, Lee W, Morris IV J, Silverman L, Sanchez-Irizarry Biol 2007;14:209–19. C, et al. Activating mutations of NOTCH1 in human T cell acute 26. Beel A, Sanders C. Substrate speciﬁcity of g-secretase and other lymphoblastic leukemia. Science 2004;306:269–71. intramembrane proteases. Cell Mol Life Sci 2008;65:1311–34. 13. Reedijk M, Pinnaduwage D, Dickson BC, Mulligan AM, Zhang H, Bull 27. Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. SB, et al. JAG1 expression is associated with a basal phenotype and Rational targeting of Notch signaling in cancer. Oncogene 2008; recurrence in lymph node-negative breast cancer. Breast Cancer Res 27:5124–31. Treat 2008;111:439–48. 28. Van Es JH, Van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H, 14. Parr C, Watkins G, Jiang W. The possible correlation of Notch-1 and et al. Notch/-secretase inhibition turns proliferative cells in intestinal Notch-2 with clinical outcome and tumour clinicopathological para- crypts and adenomas into goblet cells. Nature 2005;435:959–63. meters in human breast cancer. Int J Mol Med 2004;14:779–86. 29. Yan M, Plowman GD. Delta-like 4/Notch signaling and its therapeutic 15. Reedijk M, Odorcic S, Chang L, Zhang H, Miller N, McCready DR, et al. implications. Clin Cancer Res 2007;13:7243–6. High-level coexpression of JAG1 and NOTCH1 is observed in human 30. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, breast cancer and is associated with poor overall survival. Cancer Res Gale NW, et al. Blockade of Dll4 inhibits tumour growth by promoting 2005;65:8530–7. non-productive angiogenesis. Nature 2006;444:1032–7.

www.aacrjournals.org Mol Cancer Ther; 11(1) January 2012 85

Sharma et al.

31. O'Neill CF, Urs S, Cinelli C, Lincoln A, Nadeau RJ, Leon R, et al. Notch2 41. Dey D, Saxena M, Paranjape AN, Krishnan V, Giraddi R, Kumar MV, signaling induces apoptosis and inhibits human MDA-MB-231 xeno- et al. Phenotypic and functional characterization of human mammary graft growth. Am J Pathol 2007;171:1023–36. stem/progenitor cells in long term culture. PLoS One 2009;4:e5329. 32. Aste-Amezaga M, Zhang N, Lineberger JE, Arnold BA, Toner TJ, Gu M, 42. Ge C, Liu T, Hou X, Stanley P. In vivo consequences of deleting EGF et al. Characterization of Notch1 antibodies that inhibit signaling of repeats 8–12 including the ligand binding domain of mouse Notch1. both normal and mutated Notch1 receptors. PLoS One 2010;5:e9094. BMC Dev Biol 2008;8:48. 33. Li K, Li Y, Wu W, Gordon W, Chang D, Lu M, et al. Modulation of Notch 43. Mittal S, Subramanyam D, Dey D, Kumar R, Rangarajan A. Cooperation signaling by antibodies specific for the extracellular negative regula- of Notch and Ras/MAPK signaling pathways in human breast carci- tory region of NOTCH3. J Biol Chem 2008;283:8046–54. nogenesis. Mol Cancer 2009;8:128. 34. Wu Y, Cain-Hom C, Choy L, Hagenbeek TJ, de Leon GP, Chen Y, et al. 44. Zardawi SJ, Zardawi I, McNeil CM, Millar EKA, McLeod D, Morey AL, Therapeutic antibody targeting of individual Notch receptors. Nature et al. High Notch1 protein expression is an early event in breast cancer 2010;464:1052–7. development and is associated with the HER 2 molecular subtype. 35. Dighe R, Moudgal N. Use of [alpha]-and [beta]-subunit specific anti- Histopathology 2010;56:286–96. bodies in studying interaction of hCG with Leydig cell receptors. Arch 45. Agrawal G, Dighe R. Critical involvement of the hinge region of the Biochem Biophys 1983;225:490–9. follicle-stimulating hormone receptor in the activation of the receptor. J 36. Dighe RR, Murthy GS, Kurkalli BS, Moudgal NR. Conformation of the Biol Chem 2009;284:2636–47. [alpha]-subunit of glycoprotein hormones: a study using polyclonal 46. Al-Hussaini H, Subramanyam D, Reedijk M, Sridhar SS. Notch sig- and monoclonal antibodies. Mol Cell Endocrinol 1990;72:63–70. naling pathway as a therapeutic target in breast cancer. Mol Cancer 37. Dighe RR, Satyanarayana Murthy G, Raghuveer Moudgal N. Two Ther 2011;10:9–15. simple and rapid methods to detect monoclonal antibodies with 47. Lleo A. Activity of gamma-secretase on substrates other than APP. identical epitope specificities in a large population of monoclonal Curr Top Med Chem 2008;8:9–16. antibodies. J Immunol Methods 1990;131:229–36. 48. Gilbert CA, Daou M-C, Moser RP, Ross AH. g-secretase inhibitors 38. Gadkari RA, Sandhya S, Sowdhamini R, Dighe RR. The antigen enhance temozolomide treatment of human gliomas by inhibiting binding sites of various hCG monoclonal antibodies show homol- neurosphere repopulation and xenograft recurrence. Cancer Res ogy to different domains of LH receptor. Mol Cell Endocrinol 2010;70:6870–9. 2007;260:23–32. 49. Takebe N, Warren R, Ivy SP. Breast cancer growth and metastasis: 39. Cordle J, RedfieldZ C, Stacey M, van der Merwe P, Willis A, Champion interplay between cancer stem cells, embryonic signaling pathways B, et al. Localization of the delta-like-1-binding site in human Notch-1 and epithelial-to-mesenchymal transition. Breast Cancer Res 2011; and its modulation by calcium affinity. J Biol Chem 2008;283: 13:211. 11785–93. 50. McGowan PM, Simedrea C, Ribot EJ, Foster PJ, Palmieri D, Allan AL, 40. Shawber C, Nofziger D, Hsieh J, Lindsell C, Bogler O, Hayward D, et al. et al. Notch1 inhibition alters the CD44hi/CD24lo population and Notch signaling inhibits muscle cell differentiation through a CBF1- reduces the formation of brain metastases from breast cancer. Mol independent pathway. Development 1996;122:3765–73. Cancer Res 2011;9:834–44.

86 Mol Cancer Ther; 11(1) January 2012 Molecular Cancer Therapeutics

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

Ankur Sharma, Anurag N. Paranjape, Annapoorni Rangarajan, et al.

Mol Cancer Ther 2012;11:77-86. Published OnlineFirst November 10, 2011.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-11-0508

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2011/11/10/1535-7163.MCT-11-0508.DC1

Cited articles This article cites 48 articles, 13 of which you can access for free at: http://mct.aacrjournals.org/content/11/1/77.full#ref-list-1

Citing articles This article has been cited by 11 HighWire-hosted articles. Access the articles at: http://mct.aacrjournals.org/content/11/1/77.full#related-urls

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

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/11/1/77. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.