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The Role of Mutant Phosphoinositide 3-Kinase P110α in Liver and Bile

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The Role of Mutant Phosphoinositide 3-Kinase P110α in Liver and Bile Fakultät für Medizin II. Medizinische Klinik und Polyklinik Klinikum rechts der Isar The role of mutant phosphoinositide 3-kinase p110 in liver and bile duct cancer Angelika Gertraud Kroemer Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doctor of Philosophy (Ph.D.) genehmigten Dissertation. Vorsitzende/r: Univ.-Prof. Dr. Jürgen Ruland Betreuer/in: apl. Prof. Dieter Karl Maximilian Saur Prüfer der Dissertation: 1. Priv.-Doz. Dr. Günter Schneider 2. apl. Prof. Dr. Klaus-Peter Janssen Die Dissertation wurde am 01.07.2015 bei der Fakultät für Medizin der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 27.08.2015 angenommen. Table of contents Table of contents .................................................................................................................... I List of tables ......................................................................................................................... IV List of figures ......................................................................................................................... V Abbreviations ....................................................................................................................... VI 1 Introduction .................................................................................................................... 1 1.1 Liver and biliary tract cancer .................................................................................... 1 1.1.1 Hepatocellular carcinoma (HCC) ...................................................................... 1 1.1.2 Cholangiocellular carcinoma (CCC) ................................................................. 3 1.1.3 Animal models for the study of liver and biliary tract cancer ............................. 6 1.2 Kirsten Rat Sarcoma viral oncogene homolog (KRAS) signaling ............................. 8 1.3 The phosphoinositide 3-kinase (PI3K) signaling pathway ........................................ 9 1.4 Aims of this work ....................................................................................................11 2 Materials .......................................................................................................................12 2.1 Technical equipment ..............................................................................................12 2.2 Disposables ...........................................................................................................13 2.3 Reagents and enzymes ..........................................................................................14 2.4 Kits .........................................................................................................................16 2.5 Plasmids and bacterial strains ................................................................................17 2.6 Primers...................................................................................................................17 2.7 Cell culture .............................................................................................................20 2.8 Histology ................................................................................................................21 2.9 Antibodies ..............................................................................................................22 2.10 Buffers and solutions ..............................................................................................23 3 Methods ........................................................................................................................25 3.1 Molecular biological techniques ..............................................................................25 3.1.1 Cloning of the targeting construct ....................................................................25 3.1.1.1 Generation and transformation of competent bacteria ..............................25 3.1.1.2 Isolation and cloning of plasmid DNA .......................................................25 3.1.1.3 Cloning strategy for the pRosa26-FSF-PIK3CAH1047R targeting vector ......27 3.1.2 Isolation of genomic DNA ................................................................................28 3.1.3 Polymerase chain reaction ..............................................................................28 3.1.4 Agarose gel electrophoresis ............................................................................33 3.2 Embryonic stem (ES) cell culture............................................................................33 I 3.2.1 Culture conditions, passaging and cryopreservation .......................................33 3.2.2 Transfection and selection of ES cells .............................................................34 3.3 Mouse experiments ................................................................................................35 3.3.1 Mouse strains ..................................................................................................35 3.3.2 Genotyping ......................................................................................................36 3.3.3 Serum collection and analysis .........................................................................37 3.3.4 Necropsy .........................................................................................................37 3.4 Histological techniques ...........................................................................................37 3.4.1 Tissue fixation, sectioning and documentation ................................................37 3.4.2 Hematoxylin and eosin staining on paraffin and frozen sections ......................38 3.4.3 Immunohistochemistry ....................................................................................38 3.4.4 Oil Red O staining on frozen sections .............................................................39 3.4.5 TOPRO®-3 staining on frozen sections ............................................................39 3.4.6 Immunofluorescence staining on frozen sections ............................................39 3.4.7 Senescence-associated -galactosidase staining on frozen sections ..............39 3.5 Statistical analysis ..................................................................................................40 4 Results ..........................................................................................................................41 4.1 Analysis of the role of PI3K signaling in liver tumorigenesis ...................................41 4.1.1 Liver specific expression of mutant p110 results in hepatomegaly due to lipid accumulation .................................................................................................................41 4.1.2 Signs of liver damage and increased cholesterol production in mice expressing mutant p110 in the liver ...............................................................................................44 4.1.3 Mutant p110 drives hepatocellular and intrahepatic cholangiocellular carcinogenesis ..............................................................................................................46 4.2 Analysis of molecular mechanisms involved in extrahepatic cholangiocellular carcinoma formation .........................................................................................................48 4.2.1 Targeting of the extrahepatic bile duct via Pdx1-Cre and characterization of the Pdx1-Cre;PIK3CAH1047R/+ mouse model .........................................................................48 4.2.2 The PIK3CA activating mutation is more relevant in extrahepatic bile duct carcinogenesis than the Kras activating mutation ..........................................................53 4.2.3 p53 loss favors pancreatic carcinogenesis ......................................................58 4.2.4 Disruption of cell cycle regulators is essential for tumor progression in the extrahepatic bile duct ....................................................................................................59 4.2.5 Generation and characterization of a new mouse line for Flp-dependent activation of the PIK3CA mutation .................................................................................65 5 Discussion .....................................................................................................................70 5.1 A new model for hepatic steatosis, NASH and liver cancer ....................................70 5.2 The first genetically engineered mouse model for carcinogenesis in the extrahepatic bile duct gives insight into relevant molecular pathways ...................................................77 II 5.3 A next-generation triple-recombination system .......................................................85 5.4 Outlook...................................................................................................................87 6 Summary .......................................................................................................................89 7 Zusammenfassung ........................................................................................................90 8 References ....................................................................................................................92 9 Acknowledgements ....................................................................................................
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