title: Notch1 inhibition alters the CD44hi/CD24lo population and reduces the formation of brain metastases from breast cancer.
abstract: Brain metastasis from breast cancer is an increasingly important clinical problem. Here we assessed the role of CD44(hi)/CD24(lo) cells and pathways that regulate them, in an experimental model of brain metastasis. Notch signaling (mediated by γ-secretase) has been shown to contribute to maintenance of the cancer stem cell (CSC) phenotype. Cells sorted for a reduced stem-like phenotype had a reduced ability to form brain metastases compared with unsorted or CD44(hi)/CD24(lo) cells (P < 0.05; Kruskal-Wallis). To assess the effect of γ-secretase inhibition, cells were cultured with DAPT and the CD44/CD24 phenotypes quantified. 231-BR cells with a CD44(hi)/CD24(lo) phenotype was reduced by about 15% in cells treated with DAPT compared with DMSO-treated or untreated cells (P = 0.001, ANOVA). In vivo, mice treated with DAPT developed significantly fewer micro- and macrometastases compared with vehicle treated or untreated mice (P = 0.011, Kruskal-Wallis). Notch1 knockdown reduced the expression of CD44(hi)/CD24(lo) phenotype by about 20%. In vitro, Notch1 shRNA resulted in a reduction in cellular growth at 24, 48, and 72 hours time points (P = 0.033, P = 0.002, and P = 0.009, ANOVA) and about 60% reduction in Matrigel invasion was observed (P < 0.001, ANOVA). Cells transfected with shNotch1 formed significantly fewer macrometastases and micrometastases compared with scrambled shRNA or untransfected cells (P < 0.001; Kruskal-Wallis). These data suggest that the CSC phenotype contributes to the development of brain metastases from breast cancer, and this may arise in part from increased Notch activity.
authors.affiliation: Department of Medical Biophysics, University of Western Ontario, London, Ontario, Canada. patricia.mcgowan@ucd.ie
authors.forename: Patricia M
authors.initials: PM
authors.lastname: McGowan
authors.forename: Carmen
authors.initials: C
authors.lastname: Simedrea
authors.forename: Emeline J
authors.initials: EJ
authors.lastname: Ribot
authors.forename: Paula J
authors.initials: PJ
authors.lastname: Foster
authors.forename: Diane
authors.initials: D
authors.lastname: Palmieri
authors.forename: Patricia S
authors.initials: PS
authors.lastname: Steeg
authors.forename: Alison L
authors.initials: AL
authors.lastname: Allan
authors.forename: Ann F
authors.initials: AF
authors.lastname: Chambers
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001932:Brain Neoplasms / Q000378:metabolism / Q000556:secondary*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D051927:CD24 Antigen / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D051881:Receptor, Notch1 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D051927:CD24 Antigen; D018960:Hyaluronan Receptors; C497761:NOTCH1 protein, human; D051881:Receptor, Notch1; D053829:Amyloid Precursor Protein Secretases