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Phd Thesis Maiti Lommel.Pdf PhD-FSTC-2015-60 The Faculty of Sciences, Technology and Communication DISSERTATION Defence held on 04/12/2015 in Luxembourg to obtain the degree of DOCTEUR DE L’UNIVERSITÉ DU LUXEMBOURG EN BIOLOGIE by MAITI LOMMEL Born on 9 January 1988 in Luxembourg (Luxembourg) ANALYSIS OF SIGNAL TRANSDUCTION PATHWAYS LINKING L-PLASTIN SER5 PHOSPHORYLATION TO BREAST CANCER CELL INVASION Dissertation defence committee Dr. Elisabeth Schaffner-Reckinger, Dissertation Supervisor University of Luxembourg Dr. Serge Haan, Dissertation Co-supervisor and Vice Chairman Professor, University of Luxembourg Dr. Thomas Sauter, Chairman Professor, University of Luxembourg Dr. Christophe Ampe Professor, University of Ghent Dr. Guy Berchem Medical Doctor, Luxembourg Institute of Health Analysis of signal transduction pathways linking L-plastin Ser5 phosphorylation to breast cancer cell invasion A dissertation by Maiti Lommel Life Sciences Research Unit, University of Luxembourg Doctoral School in Systems and Molecular Biomedicine Dissertation defence committee: Supervisor: Dr. Elisabeth Schaffner-Reckinger University of Luxembourg, Life Sciences Research Unit, Cytoskeleton and Cell Plasticity Group Co-supervisor: Dr. Serge Haan Professor, University of Luxembourg, Life Sciences Research Unit, Molecular Disease Mechanisms Group Chairman: Dr. Thomas Sauter Professor, University of Luxembourg, Life Sciences Research Unit, Systems Biology Group Members: Dr. Christophe Ampe Professor, University of Ghent, Department of Biochemistry, Molecular Cell Biology and of the Actin Cytoskeleton Group Dr. Guy Berchem Medical Doctor, Luxembourg Institute of Health, Department of Oncology, Laboratory of Experimental Hemato-Oncology Acknowledgements First and foremost I would like to express my deep gratitude to my supervisor, Dr. Elisabeth Schaffner-Reckinger, who has supported me throughout my thesis with a lot of energy and a great enthusiasm for the subject matter. Your guidance and encouragement largely contributed to the completion of my dissertation and I would also like to thank you for your openness and informality which made it a pleasure working with you. I also thank my co-supervisor, Prof. Dr. Serge Haan, for always being available for giving advice and support and I thank Dr. Panuwat Trairatphisan and Prof. Dr. Thomas Sauter for their great collaboration on the modelling part of my dissertation. I also express my gratitude to our collaborators at Ghent University in the groups of Prof. Dr. Christophe Ampe and Prof. Dr. Kris Gevaert for receiving me for a research stay. Special thanks go to Dr. Laurent Vallar from the Luxembourg Institute of Health and his team members (Arnaud Muller, Tony Kaoma, François Bernardin and Petr Nazarov) for their help with the microarray experiments. I am grateful to Christina Laurini, Karoline Gäbler and Alexandre Baron for their meaningful contribution to my experiments and to Monique Wiesinger, Andreas Girod and Sébastien Plançon for their guidance in the P2 lab, in the microscopy and in the flow cytometry facilities respectively. Thanks also go to Nicolas Beaume and Aurélien Ginolhac for their help with statistical data analysis. Big thanks also go to all current and alumni lab members for giving outstanding advice and for creating an enjoyable working atmosphere. I also wish to thank my colleagues from the LSRU for helping out with material and for providing a very pleasant working environment. Thanks also go to Prof. Dr. Evelyne Friederich as an initiator of my research project. I would like to thank Prof. Dr. Christophe Ampe, Dr. Guy Berchem, Prof. Dr. Thomas Sauter, Prof. Dr. Serge Haan and Dr. Elisabeth Schaffner-Reckinger for accepting to be members of my Dissertation Defence Committee. A special thanks also goes to the members of my Dissertation Supervisory Committee, Dr. Thomas Clément and Prof. Dr. Serge Haan, for having accompanied my PhD project. I wish to express my thankfulness to the FNR for financing my thesis. Last but not least, I wish to express my deep gratitude to my family and my friends for their love and support and to my partner, Pit Ullmann, for constructive proofreading of my thesis and for his love and encouragement. Table of contents Abbreviations ......................................................................................................................... 1 Summary…… .......................................................................................................................... 5 Chapter 1 Introduction ....................................................................................................... 6 1.1 The actin cytoskeleton ........................................................................................ 6 1.1.1 Generalities .............................................................................................................. 6 1.1.2 Actin polymerisation ................................................................................................ 7 1.1.3 Actin‐binding proteins (ABPs) ................................................................................... 9 1.1.4 Formation of higher order actin structures .............................................................. 9 1.1.5 Function of higher‐order actin structures .............................................................. 11 1.1.6 Actin cytoskeleton and cancer ............................................................................... 14 1.2 The actin-bundling protein L-plastin .................................................................. 17 1.2.1 L‐plastin structure................................................................................................... 17 1.2.2 L‐plastin regulation and function ........................................................................... 18 1.2.3 L‐plastin phosphorylation and protein phosphorylation in general ...................... 21 1.2.4 L‐plastin interaction partners ................................................................................. 27 1.2.5 L‐plastin and cancer ................................................................................................ 28 1.3 Deregulation of signalling pathways in cancer .................................................. 32 1.3.1 ERK/MAPK pathway activation and activity ........................................................... 33 1.3.2 EGFR signalling in breast cancer ............................................................................. 37 Chapter 2 Aims and outline of the thesis ....................................................................... 41 Chapter 3 Materials and methods ................................................................................... 43 3.1 Cell culture ........................................................................................................ 43 3.2 Antibodies and reagents ................................................................................... 43 3.3 Treatment of cells with pharmacological agents ............................................... 44 3.4 Plasmid constructs ............................................................................................ 44 3.5 Transfections and siRNA knockdown ............................................................... 45 3.6 Immunofluorescence ........................................................................................ 45 3.7 Microarrays ....................................................................................................... 46 3.8 Kinase screening .............................................................................................. 47 3.9 In vitro kinase assays of full-length recombinant L-plastin ............................... 48 3.10 In vitro kinase assay of recombinant glutathione S-transferase (GST)-tagged L-plastin-deltaABD2 ................................................................................................. 48 3.11 Immunoblotting ................................................................................................ 48 3.12 Two-dimensional (2-D) gel electrophoresis ..................................................... 49 3.13 Invasion and migration assays ........................................................................ 50 3.14 Modelling ......................................................................................................... 50 3.15 GFP-Trap ........................................................................................................ 51 3.16 Mass spectrometry .......................................................................................... 51 Chapter 4 Results ............................................................................................................. 53 4.1 Characterisation of four breast cancer cell lines ............................................... 53 4.1.1 Epithelial and mesenchymal signature ................................................................... 53 4.1.2 Epidermal growth factor receptor (EGFR) .............................................................. 59 4.1.3 Migration and invasion capacities .......................................................................... 59 4.1.4 L‐plastin Ser5 phosphorylation levels ..................................................................... 61 4.1.5 Arguments in favour of a breast cancer origin of MDA‐MB‐435S .......................... 61 4.2 Signal transduction cascade leading to L-plastin Ser5 phosphorylation ........... 63 4.2.1 Analysis of protein kinases previously
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