Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2012 Identifying factors that conctribute to Phenotypic heterogeneity in melanoma progression Widmer, Daniel Simon Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-73667 Dissertation Originally published at: Widmer, Daniel Simon. Identifying factors that conctribute to Phenotypic heterogeneity in melanoma progression. 2012, University of Zurich, Faculty of Medicine. Eidgenössische Technische Hochschule Zürich Swiss Federal Institute of Technology Zurich Identifying factors that conctribute to Phenotypic heterogeneity in melanoma progression Daniel Simon Widmer 2012 Diss ETH No. 20537 DISS. ETH NO. 20537 IDENTIFYING FACTORS THAT CONTRIBUTE TO PHENOTYPIC HETEROGENEITY IN MELANOMA PROGRESSION A dissertation submitted to ETH ZURICH for the degree of Doctor of Sciences presented by Daniel Simon Widmer Master of Science UZH University of Zurich born on February 26th 1982 citizen of Gränichen AG accepted on the recommendation of Professor Sabine Werner, examinor Professor Reinhard Dummer, co-examinor Professor Michael Detmar, co-examinor 2012 Contents 1. ZUSAMMENFASSUNG...................................................................................................... 7 2. SUMMARY ................................................................................................................... 11 3. INTRODUCTION ............................................................................................................ 15 3.1 Melanoma ................................................................................................................. 15 3.1.1 Epidemiology ....................................................................................................... 15 3.1.2 Definition ............................................................................................................. 15 3.1.3 Risk factors.......................................................................................................... 16 3.1.4 Staging / Prognosis ............................................................................................. 16 3.1.5 Subtypes ............................................................................................................. 17 3.1.6 Familial predispositions ....................................................................................... 18 3.1.7 Somatic mutations............................................................................................... 18 3.1.8 Treatment options ............................................................................................... 19 3.1.9 Resistance........................................................................................................... 20 3.2 Models of melanoma progression ......................................................................... 22 3.2.1 Cancer Stem Cell model ..................................................................................... 22 3.2.2 Clonal evolution model ........................................................................................ 24 3.2.3 Phenotype switching model ................................................................................ 26 3.3 Hypoxia ..................................................................................................................... 28 3.3.1 The role of hypoxia in cancer .............................................................................. 30 3.4 Aim of the thesis ...................................................................................................... 33 3.5 References ................................................................................................................ 35 4. MATERIAL AND METHODS ............................................................................................. 45 4.1 Chemicals and consumables ................................................................................. 45 4.1.1 Transfection agents ............................................................................................ 46 4.1.2 Enzymes .............................................................................................................. 46 4.1.3 Cell isolation and culture reagents ..................................................................... 46 4.1.4 Growth factors, cytokines and drugs .................................................................. 47 4.1.5 Antibiotics ............................................................................................................ 47 4.1.6 Kits ....................................................................................................................... 47 4.1.7 Arrays and plates ................................................................................................ 48 4.1.8 Standard buffers .................................................................................................. 48 4.1.9 Lentiviral particles ............................................................................................... 50 4.1.10 Oligonucleotides................................................................................................ 50 4.1.11 Antibodies ......................................................................................................... 52 4.1.12 Custom RT2 Profiler PCR Array (CAH10586E-6; Qiagen) .............................. 54 4.2 Devices ..................................................................................................................... 57 4.3 Methods .................................................................................................................... 58 4.3.1 DNA methods ...................................................................................................... 58 4.3.2 RNA methods ...................................................................................................... 60 4.3.3 Protein methods .................................................................................................. 61 4.3.4 Cell culture .......................................................................................................... 64 4.3.5 Histology ............................................................................................................. 65 4.3.6 Phenotypic characterization of melanoma cells ................................................. 66 4.3.7 Hypoxia ............................................................................................................... 67 4.3.8 DNA Microarray analysis .................................................................................... 68 5. RESULTS .................................................................................................................... 73 5.1 Systematic classification of melanoma cells by phenotype-specific gene expression mapping. ..................................................................................................... 73 5.1.1 Summary ............................................................................................................. 74 5.1.2 Significance ......................................................................................................... 74 5.1.3 Introduction ......................................................................................................... 75 5.1.4 Results ................................................................................................................ 77 5.1.5 Discussion ........................................................................................................... 86 5.1.6 Acknowledgements ............................................................................................. 90 5.1.7 References .......................................................................................................... 91 5.2 Hypoxia contributes to melanoma heterogeneity by triggering phenotype switching. ....................................................................................................................... 95 5.2.1 Abstract ............................................................................................................... 96 5.2.2 Introduction ......................................................................................................... 97 5.2.3 Results .............................................................................................................. 100 5.2.4 Discussion ......................................................................................................... 120 5.2.5 Acknowledgements ........................................................................................... 125 5.2.6 Supplementary data .......................................................................................... 126 5.2.7 References ........................................................................................................ 130 5.3 Engineering melanoma progression in a humanized environment in vivo ... 137 5.4 Differential LEF1 and TCF4 expression is involved in melanoma cell phenotype switching ................................................................................................... 149 5.5 A proliferative melanoma cell phenotype is responsive to RAF/MEK inhibition independent of BRAF mutation status ..................................................................... 163 5.6 Novel MITF targets