title: Drug uptake, lipid rafts, and vesicle trafficking modulate resistance to an anticancer lysophosphatidylcholine analogue in yeast.
abstract: The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.
authors: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: Álvaro
authors: Á
authors: Cuesta-Marbán
authors: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: Javier
authors: J
authors: Botet
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors: Ola
authors: O
authors: Czyz
authors: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: Luis M
authors: LM
authors: Cacharro
authors: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: Consuelo
authors: C
authors: Gajate
authors: Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas, Juan de la Cierva 3, E-28006 Madrid, Spain.
authors: Valentín
authors: V
authors: Hornillos
authors: Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas, Juan de la Cierva 3, E-28006 Madrid, Spain.
authors: Javier
authors: J
authors: Delgado
authors: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: Hui
authors: H
authors: Zhang
authors: Instituto de Química Orgánica General, Consejo Superior de Investigaciones Científicas, Juan de la Cierva 3, E-28006 Madrid, Spain.
authors: Francisco
authors: F
authors: Amat-Guerri
authors: Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Serrano 119, E-28006 Madrid, Spain.
authors: A Ulises
authors: AU
authors: Acuña
authors: Department of Pharmacology, Atlantic Research Centre, Dalhousie University, Halifax, Nova Scotia B3H 4R2, Canada.
authors: Christopher R
authors: CR
authors: McMaster
authors: Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain.
authors: José Luis
authors: JL
authors: Revuelta
authors: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada. Electronic address: vzarembe@ucalgary.ca.
authors: Vanina
authors: V
authors: Zaremberg
authors: Instituto de Biología Molecular y Celular del Cáncer, Centro de Investigación del Cáncer, Consejo Superior de Investigaciones Científicas-Universidad de Salamanca, Campus Miguel de Unamuno, E-37007 Salamanca, Spain. Electronic address: fmollin@usal.es.
authors: Faustino
authors: F
authors: Mollinedo
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm; D004354:Drug Screening Assays, Antitumor; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D055786:Gene Knockout Techniques; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D050296:Microbial Viability / Q000187:drug effects; D010742:Phospholipid Ethers / Q000378:metabolism / Q000494:pharmacology*; D021381:Protein Transport; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D010742:Phospholipid Ethers; D029701:Saccharomyces cerevisiae Proteins; C416672:VPS29 protein, S cerevisiae; C076106:VPS35 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C026659:edelfosine; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases