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Identification of Molecules Relevant for the Invasiveness of Fibrosarcomas and Melanomas

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Identification of Molecules Relevant for the Invasiveness of Fibrosarcomas and Melanomas Helsinki University Biomedical Dissertations No. 148 IDENTIFICATION OF MOLECULES RELEVANT FOR THE INVASIVENESS OF FIBROSARCOMAS AND MELANOMAS Pirjo Nummela Department of Pathology Haartman Institute Faculty of Medicine and Division of Biochemistry Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki Finland Academic dissertation To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences of the University of Helsinki in the Lecture Hall 2 of Haartman Institute (Haartmaninkatu 3, Helsinki), on 13.5.2011 at 12 o’clock noon. Helsinki 2011 Supervisor Docent Erkki Hölttä, M.D., Ph.D. Department of Pathology Haartman Institute University of Helsinki Thesis committee Docent Jouko Lohi, M.D., Ph.D. Department of Pathology Haartman Institute University of Helsinki and Pirjo Nikula-Ijäs, Ph.D. Division of Biochemistry Department of Biosciences University of Helsinki Reviewers Professor Veli-Matti Kähäri, M.D., Ph.D. Department of Dermatology University of Turku and Turku University Hospital and Docent Jouko Lohi, M.D., Ph.D. Opponent Professor Jyrki Heino, M.D., Ph.D. Department of Biochemistry and Food Chemistry University of Turku Custos Professor Kari Keinänen, Ph.D. Division of Biochemistry Department of Biosciences University of Helsinki ISBN 978-952-92-8821-2 (paperback) ISBN 978-952-10-6924-6 (PDF) ISSN 1457-8433 http://ethesis.helsinki.fi Helsinki University Print Helsinki 2011 To Juha, Joona, and Joel TABLE OF CONTENTS LIST OF ORIGINAL PUBLICATIONS ................................................................................... 7 ABSTRACT ............................................................................................................................... 8 ABBREVIATIONS ................................................................................................................. 10 INTRODUCTION ................................................................................................................... 12 REVIEW OF THE LITERATURE ......................................................................................... 14 1. CANCER DEVELOPMENT ........................................................................................... 14 2. TUMOR MICROENVIRONMENT ................................................................................ 16 2.1 Stromal cells and surrounding epithelia ..................................................................... 16 2.1.1 Keratinocytes ...................................................................................................... 17 2.1.2 Fibroblasts ........................................................................................................... 18 2.1.3 Endothelial cells .................................................................................................. 19 2.1.4 Inflammatory and immune cells ......................................................................... 19 2.2 Extracellular matrix molecules .................................................................................. 20 2.2.1 Collagens............................................................................................................. 22 2.2.2 Elastin ................................................................................................................. 23 2.2.3 Laminins ............................................................................................................. 24 2.2.4 Fibronectin .......................................................................................................... 24 2.2.5 Tenascins ............................................................................................................. 25 2.2.6 Transforming growth factor beta-induced .......................................................... 26 3. LOCAL INVASION ........................................................................................................ 27 3.1 Cell adhesion and adhesion receptors ........................................................................ 27 3.1.1 Cell-cell interactions ........................................................................................... 27 3.1.2 Cell-matrix interactions ...................................................................................... 28 3.2 Cell migration ............................................................................................................ 29 3.2.1 Actin polymerization .......................................................................................... 30 3.2.2 Acto-myosin contraction ..................................................................................... 31 3.3 Proteolysis of extracellular matrix ............................................................................. 31 3.3.1 Matrix metalloproteinases ................................................................................... 32 3.3.2 Serine proteases .................................................................................................. 34 3.3.3 Cysteine proteases ............................................................................................... 34 AIMS OF THE STUDY .......................................................................................................... 37 MATERIALS AND METHODS ............................................................................................. 38 1. CELL LINES AND TISSUE SPECIMENS (I-IV) ......................................................... 38 2. ANTIBODIES (I-IV) ....................................................................................................... 40 3. RNA ANALYSES (I-IV) ................................................................................................. 41 3.1 Microarray analyses (I-IV) ........................................................................................ 41 3.2 Northern blot analysis (I) ........................................................................................... 41 3.3 RT-PCR analysis (I-IV) ............................................................................................. 41 4. PROTEIN ANALYSES (I-IV) ........................................................................................ 43 4.1 Western blotting (I-IV) .............................................................................................. 43 4.2 Immunofluorescence staining (I-II, IV) ..................................................................... 43 4.3 Flow cytometry (II) .................................................................................................... 43 4.4 Immunoprecipitation (II) ........................................................................................... 43 4.5 Assay of cathepsin L activity (II) ............................................................................... 44 5. TRANSFECTION EXPERIMENTS (I-II, IV) ................................................................ 44 5.1 Tetracycline-inducible antisense-RNA expression vector (I) .................................... 44 5.2 siRNA oligonucleotides (I-II) .................................................................................... 44 5.3 shRNA expression vectors (II, IV) ............................................................................ 45 6. FUNCTIONAL ASSAYS (I-IV) ..................................................................................... 45 6.1 Adhesion assays (II, IV) ............................................................................................ 45 6.2 Analysis of cell growth (I-II, IV) ............................................................................... 46 6.3 Migration assays (I, III-IV) ........................................................................................ 46 6.4 Invasion assays (I-II, IV) ........................................................................................... 46 7. IMMUNOHISTOCHEMISTRY (II-IV) .......................................................................... 47 RESULTS AND DISCUSSION .............................................................................................. 48 1. MICROARRAY ANALYSES (I-IV) .............................................................................. 48 1.1 Incyte Genomics versus Affymetrix arrays (I-IV) ..................................................... 48 1.2 Mouse fibrosarcoma cells (I-II) ................................................................................. 51 1.3 Human melanoma cells (III-IV) ................................................................................. 52 1.4 Fibrosarcoma versus melanoma (I-IV) ...................................................................... 54 2. FUNCTIONAL CHARACTERIZATION (I-IV) ............................................................ 55 2.1 Thymosin β4 (I) ......................................................................................................... 55 2.1.1 Tβ4 is overexpressed in Amdc-s, Amdc-as, and E4 fibrosarcoma cells ............. 56 2.1.2 Tβ4 overexpression is related to the transformed phenotype ............................. 56 2.1.3 LAT-A, an inhibitor of Tβ4, affects the morphology and growth of Amdc-s and -as cells .................................................................................................... 57 2.1.4 LAT-A impairs the migration of Amdc-s, Amdc-as, and E4 cells ..................... 58 2.1.5 LAT-A blocks the invasion of Amdc-s and -as cells in Matrigel ......................
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