title: Activity of plasma membrane V-ATPases is critical for the invasion of MDA-MB231 breast cancer cells.
abstract: The vacuolar (H(+))-ATPases (V-ATPases) are a family of ATP-driven proton pumps that couple ATP hydrolysis with translocation of protons across membranes. Previous studies have implicated V-ATPases in cancer cell invasion. It has been proposed that V-ATPases participate in invasion by localizing to the plasma membrane and causing acidification of the extracellular space. To test this hypothesis, we utilized two separate approaches to specifically inhibit plasma membrane V-ATPases. First, we stably transfected highly invasive MDA-MB231 cells with a V5-tagged construct of the membrane-embedded c subunit of the V-ATPase, allowing for extracellular expression of the V5 epitope. We evaluated the effect of addition of a monoclonal antibody directed against the V5 epitope on both V-ATPase-mediated proton translocation across the plasma membrane and invasion using an in vitro Matrigel assay. The addition of anti-V5 antibody resulted in acidification of the cytosol and a decrease in V-ATPase-dependent proton flux across the plasma membrane in transfected but not control (untransfected) cells. These results demonstrate that the anti-V5 antibody inhibits activity of plasma membrane V-ATPases in transfected cells. Addition of the anti-V5 antibody also inhibited in vitro invasion of transfected (but not untransfected) cells. Second, we utilized a biotin-conjugated form of the specific V-ATPase inhibitor bafilomycin. When bound to streptavidin, this compound cannot cross the plasma membrane. Addition of this compound to MDA-MB231 cells also inhibited in vitro invasion. These studies suggest that plasma membrane V-ATPases play an important role in invasion of breast cancer cells.
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine and the Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts 02111.
authors.forename: Kristina
authors.initials: K
authors.lastname: Cotter
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine and the Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts 02111.
authors.forename: Joseph
authors.initials: J
authors.lastname: Capecci
authors.affiliation: the Department of Cell Physiology and Molecular Biophysics, Texas Tech University, Lubbock, Texas 79430.
authors.forename: Souad
authors.initials: S
authors.lastname: Sennoune
authors.affiliation: the Department of Biology/Chemistry, Division of Animal Physiology, University of Osnabrück, 49069 Osnabrück, Germany, and.
authors.forename: Markus
authors.initials: M
authors.lastname: Huss
authors.affiliation: the Institute of Organic Chemistry, University of Tuebingen, 72076 Tuebingen, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Maier
authors.affiliation: the Department of Cell Physiology and Molecular Biophysics, Texas Tech University, Lubbock, Texas 79430.
authors.forename: Raul
authors.initials: R
authors.lastname: Martinez-Zaguilan
authors.affiliation: From the Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine and the Program in Cellular and Molecular Physiology, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts 02111, michael.forgac@tufts.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Forgac
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002465:Cell Movement; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport; D018942:Macrolides / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D021381:Protein Transport; D011522:Protons; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D018942:Macrolides; D011522:Protons; C057620:bafilomycin A; D025262:Vacuolar Proton-Translocating ATPases
keywords: Acidification; Breast Cancer; Invasion; Migration; Proton Transport; Vacuolar ATPase