title: Nicastrin regulates breast cancer stem cell properties and tumor growth in vitro and in vivo.
abstract: Nicastrin (NCT) is a crucial component of the γ-secretase (GS) enzyme, which prompted investigations into its biological role in cancer. We have previously shown that nicastrin is overexpressed in breast cancer (BC), conferring worse overall survival in invasive, ERα negative patients. Here, we used 2D and 3D Matrigel, anchorage-independent growth conditions and a breast cancer xenograft mouse model to assess the impact of nicastrin on breast cancer stem cell (BCSC) propagation and invasion in vitro and tumor growth in vivo. Stable knockdown of nicastrin in HCC1806 breast cancer cells reduced cell invasion by 51.4 ± 1.7%, accompanied by a morphological change to a rounded cell phenotype and down-regulation of vimentin, Snail, Twist, MMP2, and MMP9. We observed a reduction of the pool of CD44(+)/CD24(-) and ALDH1 high breast cancer stem cells by threefold and twofold, respectively, and a reduction by 2.6-fold of the mammospheres formation. Nicastrin overexpression in nontransformed MCF10A cells caused an induction of epithelial to mesenchymal regulators, as well as a fivefold increased ALDH1 activity, a threefold enrichment for CD44(+)/CD24(-) stem cells, and a 3.2-fold enhanced mammosphere-forming capacity. Using the γ-sescretase inhibiton, Notch1/4 siRNA, and Akt inhibition, we show that nicastrin regulates breast cancer stem cells partly through Notch1 and the Akt pathway. Exploiting serial dilution transplantation of the HCC1806 cells expressing nicastrin and HCC1806 stably depleted of nicastrin, in vivo, we demonstrate that nicastrin inhibition may be relevant for the reduced tumorigenicity of breast cancer cells. These data could serve as a benchmark for development of nicastrin-targeted therapies in breast cancer.
authors.affiliation: Division of Surgery and Cancer, Department of Oncology, Imperial College London, Hammersmith Hospital Campus, London, United Kingdom. y.lombardo@imperial.ac.uk
authors.forename: Ylenia
authors.initials: Y
authors.lastname: Lombardo
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Filipović
authors.forename: Gemma
authors.initials: G
authors.lastname: Molyneux
authors.forename: Manikandan
authors.initials: M
authors.lastname: Periyasamy
authors.forename: Georgios
authors.initials: G
authors.lastname: Giamas
authors.forename: Yunhui
authors.initials: Y
authors.lastname: Hu
authors.forename: Pritesh S
authors.initials: PS
authors.lastname: Trivedi
authors.forename: Jayson
authors.initials: J
authors.lastname: Wang
authors.forename: Ernesto
authors.initials: E
authors.lastname: Yagüe
authors.forename: Loren
authors.initials: L
authors.lastname: Michel
authors.forename: R Charles
authors.initials: RC
authors.lastname: Coombes
mesh_terms: D053829:Amyloid Precursor Protein Secretases / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018929:Cell Culture Techniques; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics* / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D014183:Transplantation, Heterologous; D047368:Tumor Burden / Q000235:genetics
chemical_list: D008562:Membrane Glycoproteins; D051880:Receptors, Notch; C413604:nicastrin protein; D051057:Proto-Oncogene Proteins c-akt; D053829:Amyloid Precursor Protein Secretases