title: A monoclonal antibody against human Notch1 ligand-binding domain depletes subpopulation of putative breast cancer stem-like cells.
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 CD44(Hi)/CD24(Low) 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.
authors.affiliation: Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India.
authors.forename: Ankur
authors.initials: A
authors.lastname: Sharma
authors.forename: Anurag N
authors.initials: AN
authors.lastname: Paranjape
authors.forename: Annapoorni
authors.initials: A
authors.lastname: Rangarajan
authors.forename: Rajan R
authors.initials: RR
authors.lastname: Dighe
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000378:metabolism; D017209:Apoptosis; D001943:Breast Neoplasms / Q000188:drug therapy / Q000276:immunology*; D051927:CD24 Antigen / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004815:Epidermal Growth Factor / Q000276:immunology; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology / Q000378:metabolism; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D014411:Neoplastic Stem Cells / Q000276:immunology*; D051881:Receptor, Notch1 / Q000276:immunology* / Q000378:metabolism; D000072099:Serrate-Jagged Proteins; D015398:Signal Transduction
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D051927:CD24 Antigen; D002135:Calcium-Binding Proteins; C424126:DLL4 protein, human; D018960:Hyaluronan Receptors; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D000072099:Serrate-Jagged Proteins; D004815:Epidermal Growth Factor