Thesis for the degree of Philosophiae Doctor Membrane dynamics in cancer cells Simona Kavaliauskiene Faculty of Mathematics and Natural Sciences, University of Oslo, Norway Department of Molecular Cell Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Center for Cancer Biomedicine, Oslo University Hospital, Oslo, Norway © Simona Kavaliauskiene, 2016 Series of dissertations submitted to the Faculty of Mathematics and Natural Sciences, University of Oslo No. 1754 ISSN 1501-7710 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. Table of Contents ACKNOWLEDGEMENTS .................................................................................................. 1 ABBREVIATIONS AND LIPID NOMENCLATURE ........................................................ 3 LIST OF PUBLICATIONS ................................................................................................... 7 ABSTRACT .......................................................................................................................... 8 INTRODUCTION ................................................................................................................. 9 STRUCTURE AND ORGANIZATION OF CELLULAR MEMBRANES .................. 10 SPHINGOLIPIDS ........................................................................................................... 12 Sphingolipid biosynthesis ............................................................................................ 12 Sphingolipids and membrane organization ................................................................. 14 Glycosphingolipids ...................................................................................................... 15 Functions of glycosphingolipids .................................................................................. 17 Globotriaosylceramide (Gb3) ...................................................................................... 18 Glycosphingolipids and cancer .................................................................................... 20 GLUCOSE ANALOGUES AND THEIR CELLULAR EFFECTS ............................... 22 2-Deoxy-D-glucose (2DG) .......................................................................................... 23 2-Fluoro-2-deoxy-D-glucose (FDG) ........................................................................... 24 PROTEIN TOXINS ......................................................................................................... 25 Shiga toxins ................................................................................................................. 25 Factors regulating Shiga toxin binding and intracellular transport ............................. 28 Diphtheria toxin ........................................................................................................... 32 Ricin ............................................................................................................................ 33 Toxin application in research and clinic ...................................................................... 34 AIMS OF THE STUDY ...................................................................................................... 36 SUMMARY OF PUBLICATIONS .................................................................................... 37 Paper I. Cell density-induced changes in lipid composition and intracellular trafficking .................................................................................................................... 37 Paper II. Novel actions of 2-deoxy-D-glucose: protection against Shiga toxins and changes in cellular lipids ............................................................................................. 38 Paper III. Cellular effects of fluorodeoxyglucose: Global changes in the lipidome and alteration in intracellular transport .............................................................................. 39 DISCUSSION ..................................................................................................................... 42 Cell growth in culture: implications of cell density on the molecular level ............... 42 Different factors modulating Gb3 receptor function ................................................... 45 Shiga toxin transport modulation by lipids ................................................................. 47 Potential applications of 2DG and FDG in the clinic .................................................. 48 EXPERIMENTAL SYSTEMS ........................................................................................... 52 Cell lines ...................................................................................................................... 52 Cell-based toxin assays ............................................................................................... 52 Stx transport ................................................................................................................ 53 Fluorescence microscopy-based analysis of Stx transport .......................................... 53 SNAP-tag method for studying retrograde Stx transport ............................................ 55 Quantitative MS analysis of lipids .............................................................................. 57 CONCLUSIONS AND PERSPECTIVES .......................................................................... 58 REFERENCE LIST ............................................................................................................. 60 ENCLOSED PAPERS I-III ................................................................................................. 79 2 ACKNOWLEDGEMENTS Acknowledgements This work was carried out in Prof. Kirsten Sandvig’s laboratory at the Department of Molecular Cell Biology, Centre for Cancer Biomedicine, Institute for Cancer Research at the Oslo University Hospital from 2011 to 2016. The work was financed by South-Eastern Norway Regional Health Authority. I would like to express my gratitude to Kirsten Sandvig for giving me this wonderful opportunity to complete my Ph.D. under her supervision. Your support, advice and skilful guidance have been much appreciated. I am truly grateful for believing in me and for giving me freedom to feed my scientific curiosity. I would like to thank all the coauthors for excellent collaboration; without your help, ideas and critics this work would not have been possible. In particular, I would like to thank Tore Skotland for countless fruitful discussions, for exceptional attention to details and for being enthusiastic about new ideas. In addition, I want to express my gratitude to Kim Ekroos and his group for delivering excellent lipidomic data for all three papers. The Sandvig lab is truly a perfect place to carry out a Ph.D., and I would like to thank each and every member of the group along the side with the rest of the people at the Department of Molecular Cell Biology for providing a wonderful atmosphere and a great scientific environment for me during my time here. In particular, I want to express my gratitude to Anne Engen and the whole ‘crew’ of the cell lab, for providing me with the top quality cells and thus making the Ph.D. life much easier. In addition, I want to thank Anne Grethe Myrann for performing countless tox-assays, and for being the most persistent and patient Norwegian teacher I have ever had. I also want to express my special gratitude to Chema Bassols for being our computer ‘superman’ and giving absolutely everything for fixing any problem in the lab. I would like to thank Kotryna, Ieva, Audun, Nagham and Santosh for being my friends and for sharing experience of growing as personalities and as PhD students. Without you my time in the lab (and the lunch breaks) would have never been so joyful. I would also like to express my true gratitude to Anne Mari for being the best office mate I have ever had. 1 ACKNOWLEDGEMENTS Finally, I want to thank my mom for laying the first stones in my path to science. Thank you for seeing and encouraging my passion for knowledge. I also thank my best friend and husband Arturas for his fascinating ideas and thinking out-side the box. Thank you for always being with me and staying strong when I wanted to give up. And to my daughter Ruta, thank you for showing me the unbelievable joys of motherhood, you taught me things far more complicated and fascinating than I have ever anticipated. Simona Kavaliauskiene Oslo, May 2016 2 ABBREVIATIONS AND LIPID NOMENCLATURE Abbreviations and Lipid Nomenclature 2DG 2-deoxy-D-glucose Acc1 Acetyl-CoA carboxylase 1 ADP Adenosine diphosphate ALPS Amphipathic lipid packing sensor AMP Adenosine monophosphate ARF ADP-ribosylation factor BFA Brefeldin A BG Benzylguanine BiP Immunoglobulin binding protein (also called GRP78) CDase Ceramidase CerS Ceramide synthase CERT Ceramide transfer protein CoA Coenzyme A COG Conserved oligomeric Golgi COP I Coat protein I COP II Coat protein II cPLA2 Cytoplasmic phospholipase A2 DAGK Diacylglycerol-consuming kinase DRM Detergent resistant membrane EGF Epidermal growth factor EGFP Enhanced green fluorescent protein EGFR Epidermal growth factor receptor EHD Eps15 homology domain-containing ER Endoplasmic reticulum ERAD Endoplasmic reticulum associated degradation FA Fatty acid FAPP2 Four-phosphate