Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften der Fakultät für Biologie der Ludwig-Maximilians- Universität München The role of the transcription factor SOX2 in tumorigenesis and development of the stomach Vorgelegt von Katharina Antonia Hütz München 2013 2 „Wenn Du ein Schiff bauen willst, dann trommle nicht Männer zusammen um Holz zu beschaffen, Aufgaben zu vergeben und die Arbeit einzuteilen, sondern lehre die Männer die Sehnsucht nach dem weiten, endlosen Meer.“ Antoine de Saint-Exupery 3 4 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29. Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von Prof. Thomas Cremer von der Fakultät der Biologie vertreten. Eidesstattliche Versicherung Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet. München, ……………………………………………………. Katharina Hütz Dissertation eingereicht am 06. Juni 2013 1. Gutachter: Prof. Thomas Cremer 2. Gutachter: Prof. Elisabeth Weiss Mündliche Prüfung am 21. Oktober 2013 5 6 Meinen Eltern und meinen Schwestern 7 8 Table of Contents Table of Contents ................................................................................................................. 9 List of Abbreviations .......................................................................................................... 13 1. Introduction ................................................................................................................ 17 1.1. The stomach ...................................................................................................... 17 1.1.1. Development of the stomach ...................................................................... 17 1.1.2. Anatomy and function of the stomach ........................................................ 17 1.1.3. Gastric stem cells ......................................................................................... 19 1.2. Gastric cancer .................................................................................................... 24 1.2.1. Epidemiology of gastric cancer .................................................................... 24 1.2.2. Classification of gastric cancer ..................................................................... 24 1.2.3. Development of gastric cancer .................................................................... 25 1.2.4. Helicobacter pylori and gastric cancer ........................................................ 30 1.2.5. The Cancer stem cell theory ........................................................................ 33 1.3. The transcription factor SOX2 ............................................................................ 34 1.3.1. General characteristics of SOX (Sry box) proteins ....................................... 34 1.3.2. Classification and localization of SOX2 ........................................................ 35 1.3.3. SOX2 in pluripotency and development ...................................................... 36 1.3.4. SOX2 and cancer .......................................................................................... 38 1.4. Aims of the study .............................................................................................. 41 2. Methods ..................................................................................................................... 43 2.1. Molecular cloning .............................................................................................. 43 2.1.1. Cloning strategies ........................................................................................ 43 2.1.2 Restriction digestion of DNA ........................................................................ 44 2.1.3 Ligation ......................................................................................................... 44 2.1.4 Competent E. coli cells ................................................................................. 44 2.1.5 Transformation of DNA in bacteria .............................................................. 45 2.1.6 Preparation of plasmid DNA ........................................................................ 45 2.1.7 shRNA cloning .............................................................................................. 45 2.1.8 Agarose gel electrophoresis ........................................................................ 46 2.2 Cell culture ........................................................................................................ 46 2.2.1. General cell culture methods ...................................................................... 47 2.2.1.1. Cell counting ......................................................................................... 47 2.2.1.2. Freezing cells ........................................................................................ 47 9 2.2.1.3. Thawing and maintaining cells ............................................................. 47 2.2.2. Transfection of cells ..................................................................................... 48 2.2.2.1. Transient transfection by lipofection ................................................... 48 2.2.2.2. Transient transfection by electroporation ........................................... 49 2.2.2.3. Titration of antibiotics (Killing curve) ................................................... 49 2.2.2.4. Stable transfection of cells ................................................................... 49 2.2.2.5. Inducible cell clones .............................................................................. 50 2.2.3. shRNA mediated RNA interference.............................................................. 51 2.3. Biological assays ................................................................................................ 52 2.3.1. Luciferase reporter gene assay .................................................................... 52 2.3.2. Proliferation analysis .................................................................................... 53 2.3.3. Analysis of apoptosis .................................................................................... 53 2.3.4. Cell cycle analysis ......................................................................................... 54 2.3.5. Wound healing/migration ............................................................................ 55 2.3.6. Analysis of senescence associated -galactosidase (SA--gal) activity ....... 55 2.4. Immunofluorescence ......................................................................................... 56 2.5. Infection with Helicobacter pylori ...................................................................... 56 2.6. Sodium-dodecyl-sulfate- Polyacrylamide Gel Electorphoresis (SDS-PAGE) ........... 57 2.7. Western Blot ..................................................................................................... 58 2.8. RNA Analysis ..................................................................................................... 58 2.8.1. RNA Isolation ................................................................................................ 58 2.8.2. Reverse transcription ................................................................................... 59 2.8.3. Quantitative real-time polymerase chain reaction (qRT-PCR)..................... 60 2.8.4. RNA Microarray ............................................................................................ 61 2.9. In vivo studies .................................................................................................... 62 2.9.1. Genotyping transgenic mice ........................................................................ 62 2.9.2. Preparation of blastocystes ......................................................................... 64 2.9.3. Tamoxifen application .................................................................................. 65 2.9.4. Histology ....................................................................................................... 65 2.9.4.1. Organ isolation and embedding ........................................................... 65 2.9.4.2. Hematoxylin-Eosin (HE) staining ........................................................... 66 2.9.4.3. Immunohistochemistry ......................................................................... 66 2.9.5. Analysis of AZ-521 tumorgenicity in vivo ..................................................... 68 2.10. Materials ........................................................................................................... 68 2.10.1. Buffers and Solutions ............................................................................... 68 10 2.10.2. Chemicals and Kits.................................................................................... 76 2.10.3. Antibodies ................................................................................................ 80 2.10.4. Consumables ............................................................................................ 81 2.10.5. Instruments .............................................................................................. 82 3. Results ........................................................................................................................ 85 3.1. Role of SOX2 in gastric cancer ............................................................................ 85 3.1.1. SOX2 expression in gastric tumors and