Vergleichende molekulare Charakterisierung Ctnnb1 - und Ha-ras -mutierter Mauslebertumoren Comparative Molecular Characterization of Ctnnb1 - and Ha-ras -mutated Mouse Liver Tumors Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Elif Bettina Unterberger aus Den Haag Tübingen 2013 Tag der mündlichen Prüfung: 13.5.2013 Dekan: Prof. Dr. Wolfgang Rosenstiel 1. Berichterstatter: Prof. Dr. Michael Schwarz 2. Berichterstatter: Prof. Dr. Klaus Schulze-Osthoff The presented thesis was prepared at the Institute of Experimental and Clinical Pharmacology and Toxicology, Department of Toxicology, at the Eberhard Karls Universität Tübingen between September 2009 and March 2013 under the supervision of Prof. Dr. Michael Schwarz. During this time period I overtook the supervision of a diploma student, Kathrin Knötzsch, who carried out mutation analysis of spontaneous mouse liver tumors. Parts of this work have been conducted in cooperation with external partners. mRNA expression and DNA methylation profiling, as well as characterization of Meg3/Gtl2 by immunohistochemistry and in situ hybridization was conducted at the Novartis Institutes for BioMedical Research in Basel, Switzerland. CXR Biosciences in Dundee, Scotland, carried out miRNA expression measurements, and reverse phase protein microarray analysis was conducted at NMI, Reutlingen, Germany. Pre-processing and statistical analysis of raw data gained from these experiments was done by Johannes Eichner, Clemens Wrzodek (University of Tübingen, Department of Computer Science, Cognitive Systems) and Raphaëlle Luisier (Department for Discovery and Investigative Safety, Novartis Institutes for BioMedical Research, Basel, Switzerland). It is not the mountain we conquer, but ourselves. Sir Edmund Hilary Acknowledgements First and foremost, I would like to thank my supervisor Prof. Michael Schwarz for giving me the opportunity to work on a fascinating and relevant topic, for his support and trust, and for generously sharing his vast knowledge about carcinogenesis. My sincere thanks go to: The IMI MARCAR consortium for financial support and for generating and sharing a large amount of the raw data analyzed in the course of this thesis. Johannes Eichner and Clemens Wrzodek for their tremendous help in bioinformatically analyzing the datasets and for never growing tired of patiently answering my innumberable questions. The members of the Novartis Institutes for BioMedical Research for their support and technical guidance in conducting immunohistochemical stainings and in situ hybridization. Elke Zabinsky for excellent technical support with immunohistochemical stainings. All members of the Department of Toxicology in Tübingen for creating a positive working atmosphere and for their constant support in all scientific, bureaucratic and personal matters. My loving thanks go to my parents, who have always supported and encouraged me in every possible way, for their love and trust and for making me regain perspective when times were rough. I thank Jörg, my angel, my best friend through thick and thin, for everything. Parts of this work have been presented: Lempiäinen H, Couttet P, Bolognani F, Müller A, Dubost V, Luisier R, Espinola Adel R, Vitry V, Unterberger EB , Thomson JP, Treindl F, Metzger U, Wrzodek C, Hahne F, Zollinger T, Brasa S, Kalteis M, Marcellin M, Giudicelli F, Braeuning A, Morawiec L, Zamurovic N, Längle U, Scheer N, Schübeler D, Goodman J, Chibout SD, Marlowe J, Theil D, Heard DJ, Grenet O, Zell A, Templin MF, Meehan RR, Wolf RC, Elcombe CR, Schwarz M, Moulin P, Terranova R, Moggs JG.: Identification of Dlk1-Dio3 imprinted gene cluster non-coding RNAs as novel candidate biomarkers for liver tumor promotion , Toxicological Sciences 131(2), 375-386, doi: 10.1093/toxsci/kfs303 Unterberger EB , Schwarz M: Biomarkers and molecular tumour classification for non- genotoxic carcinogenesis , ToxNet Baden-Württemberg Symposium, Konstanz, Germany, 2011 ( poster presentation ) Unterberger EB , Schwarz, M: Mechanisms of non-genotoxic carcinogenesis – novel insights from integrated genetic, transcriptomic and epigenomic profiling , 26 th GUM meeting (Society of mutational and environmental research), Mainz, Germany, 2012 (invited lecture) Unterberger EB , Schwarz M: Biomarkers and molecular tumour classification for non- genotoxic carcinogenesis , 78 th meeting of the German Society of Pharmacology and Toxicology, Dresden, Germany, 2012 ( oral presentation, awarded with the Sanofi- Aventis-Prize for best oral presentation) Zeller E, Kramer-Potapenja V, Unterberger EB , Braeuning A., Schwarz M: Long non- docing RNA Gtl2/Meg3 and its role in hepatic cancer, 79 th meeting of the German Society of Pharmacology and Toxicology, Halle an der Saale, Germany, 2013 (poster presentation) Table of contents 1. Introduction ..................................................................................................... 1 1.1 Physiology of the liver ..................................................................................... 1 1.2 Chemical carcinogenesis ................................................................................ 4 1.2.1 Canonical Wnt signaling pathway ............................................................ 5 1.2.2 Mitogen-activated protein kinase (MAPK) signaling pathway ................... 6 1.3 Epigenetic mechanisms in carcinogenesis ...................................................... 7 1.3.1 The Dlk1-Dio3 imprinted gene cluster ...................................................... 8 1.4 Aims and objectives ...................................................................................... 10 2. Material ......................................................................................................... 11 2.1 Chemicals and biochemicals ......................................................................... 11 2.2 Antibodies ..................................................................................................... 12 2.2.1 Immunohistochemistry ........................................................................... 12 2.2.2 Reverse phase protein microarray ......................................................... 13 2.2.3 Western Blot .......................................................................................... 17 2.3 Buffers and solutions: .................................................................................... 18 2.3.1 Electrophoresis ...................................................................................... 18 2.3.2 Isolation of genomic DNA ...................................................................... 19 2.3.3 Immunohistochemistry ........................................................................... 19 2.3.4 Western Blot .......................................................................................... 20 2.4 Primers ......................................................................................................... 20 2.4.1 RFLP analysis and sequencing ............................................................. 20 2.4.2 LightCycler PCR .................................................................................... 20 2.5 Laboratory equipment ................................................................................... 21 2.6 Expandable items and small devices ............................................................. 22 2.7 Software ........................................................................................................ 23 3. Methods ........................................................................................................ 24 3.1 Animal treatment and housing ....................................................................... 24 3.1.1 Tumor tissue from previous initiation-promotion experiments ................ 24 Table of contents Page I 3.1.2 PB-treatment of Connexin32-deficient mice ........................................... 24 3.1.3 Obtaining of spontaneous liver tumors ................................................... 25 3.1.4 Tumors from DEN-initiated mice after withdrawal of PB-treatment ......... 25 3.1.5 APC-knockout mice ............................................................................... 25 3.2 Mutation analysis .......................................................................................... 25 3.2.1 Isolation of genomic DNA ...................................................................... 25 3.2.2 Standard PCR ....................................................................................... 25 3.2.3 RFLP analysis ....................................................................................... 26 3.3 Quantitative Real-time PCR .......................................................................... 27 3.3.1 Isolation of RNA from frozen liver .......................................................... 27 3.3.2 Reverse transcription ............................................................................. 27 3.3.3 LightCycler PCR .................................................................................... 28 3.4 Global gene expression analysis ................................................................... 29 3.5 miRNA expression analysis ........................................................................... 29 3.6 Analysis of proteome and phosphoproteome ................................................