Aus dem Nationalen Zentrum für Strahlenforschung in der Onkologie - OncoRay Arbeitsgruppe: Molekulare und Zelluläre Strahlenbiologie Gruppenleiter: Herr Prof. Dr. med. habil. Nils Cordes The intermediate filament synemin promotes non- homologous end joining in an ATM-dependent manner D i s s e r t a t i o n s s c h r i f t zur Erlangung des akademischen Grades Doktor der Biomedizin Doctor rerum medicinalium (Dr. rer. medic.) Vorgelegt der Medizinischen Fakultät Carl Gustav Carus der Technischen Universität Dresden Sara Sofia Deville, M.Sc. born in Santa Fe, Argentina Dresden 2019 1. Gutachter: Prof. Dr. med. habil. Nils Cordes 2. Gutachter: Prof. Dr. rer. nat. Georg Breier Tag der mündlichen Prüfung: 27th January 2020 Vorsitzender der Promotionskommission: (Prof. Dr. med. Axel Roers) Contents Contents Contents ................................................................................................................................. I Abbreviations ........................................................................................................................ V 1 Introduction ..................................................................................................................... 1 2 Background .................................................................................................................... 3 2.1 Extracellular matrix, adhesion mediated radiation resistance and 3D cell culture model ................................................................................................................................ 3 2.2 Integrins and focal adhesion proteins (FAPs) .......................................................... 5 2.3 Intermediate filaments (IFs) ..................................................................................... 6 2.3.1 Cytoskeleton components ................................................................................ 6 2.3.2 Structure and assembly .................................................................................... 7 2.3.3 Family classification ......................................................................................... 7 2.4 Synemin, IV intermediate filament ........................................................................... 9 2.4.1 Structure .......................................................................................................... 9 2.4.2 Physiological role of synemin ..........................................................................10 2.4.3 Synemin in cancer ...........................................................................................11 2.5 Head and Neck squamous cell carcinomas (HNSSC) ............................................12 2.5.1 Common therapeutical approaches .................................................................13 2.5.2 Risk factors .....................................................................................................13 2.6 Effects of ionizing radiation on cellular level ...........................................................14 2.7 DNA damage response ..........................................................................................16 2.8 DNA double strand break repair mechanisms ........................................................17 2.8.1 Non-homologous end joining (NHEJ) ..............................................................19 2.8.2 Homologous recombination (HR) ....................................................................19 2.8.3 Alternative end-joining (Alt-EJ) ........................................................................20 I Contents 3 Hypothesis and Aims .....................................................................................................21 4 Materials and Methods ..................................................................................................23 4.1 Materials ................................................................................................................23 4.1.1 Devices ...........................................................................................................23 4.1.2 Further materials .............................................................................................25 4.1.3 esiRNA and siRNA ..........................................................................................26 4.1.4 Inhibitors and chemotherapeutic agents ..........................................................26 4.1.5 Plasmids .........................................................................................................26 4.1.6 Primers............................................................................................................28 4.1.7 Polymerases, Restriction enzymes and ligases ...............................................28 4.1.8 Bacterial culture ..............................................................................................28 4.1.9 Protein and DNA ladders .................................................................................29 4.1.10 Method kits ......................................................................................................29 4.1.11 Primary antibodies ..........................................................................................30 4.1.12 Secondary antibodies ......................................................................................31 4.1.13 Solutions for cell biological applications...........................................................32 4.1.14 Solutions for protein-biochemical and molecular-biological applications ..........33 4.1.15 Solutions for immunofluorescence ...................................................................35 4.1.16 Further solutions and chemicals ......................................................................36 4.1.17 PC programs ...................................................................................................36 4.2 Methods .................................................................................................................37 4.2.1 Cell culture ......................................................................................................37 4.2.2 Cell freezing and thawing ................................................................................37 4.2.3 siRNA knockdown ...........................................................................................38 4.2.4 Inhibitor treatment and chemotherapy .............................................................38 4.2.5 Radiation exposure .........................................................................................39 4.2.6 3D Colony formation assay .............................................................................39 4.2.7 3D high-throughput RNAi-based screening .....................................................40 4.2.8 Screening development ...................................................................................40 4.2.9 3D high-throughput screen using esiRNA (3D HTP-RNAi-S) ...........................42 II Contents 4.2.10 Immunofluorescence staining ..........................................................................42 4.2.11 Foci assay .......................................................................................................43 4.2.12 Total protein extracts, SDS-PAGE and Western Blotting .................................44 4.2.13 DRGFP and EJ5GFP-based chromosomal break reporter assay ....................46 4.2.14 Cell Cycle analysis ..........................................................................................48 4.2.15 Kinome analysis ..............................................................................................48 4.2.16 Immunoprecipitation ........................................................................................49 4.2.17 Chromatin fractionation ...................................................................................49 4.2.18 Expression constructs, site-directed mutagenesis and transfection of plasmids . ........................................................................................................................50 4.2.19 2D-Proximity Ligation Assay ............................................................................53 4.2.20 Statistics ..........................................................................................................53 5 Results ..........................................................................................................................54 5.1 3D high-throughput RNAi-based screening ............................................................54 5.1.1 Uncovering novel radioresistance-related focal adhesion proteins ..................54 5.1.2 Synemin modulates radiation sensitivity and DNA double strand break repair in HNSCC. ........................................................................................................................58 5.1.3 Analysis of synemin dynamics after irradiation ................................................63 5.2 The role of synemin in DNA repair mechanisms in HNSCCs ..................................66 5.2.1 The effect of synemin knockdown on NHEJ, HR and Alt-EJ in HNSCCs .........66 5.2.2 The effect of synemin knockdown on DNA repair kinetics in NHEJ and cell cycle ........................................................................................................................68 5.3 Synemin signaling in head and neck squamous cell carcinoma (HNSCC) ..............72 5.3.1 Regulation of kinase