DISS. ETH NO. 17606 The new melanoma: A novel model for disease progression A dissertation submitted to SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZÜRICH for the degree of DOCTOR OF SCIENCES presented by Natalie Schlegel Master of Science, Otago University (New Zealand) born on January 20th 1976 citizen of Zürich (ZH) accepted on the recommendation of Professor Sabine Werner, examinor Professor Reinhard Dummer, co-examinor Professor Josef Jiricny, co-examinor 2008 22 Table of Contents Abstract ...................................................................................................................................... 6 Résumé....................................................................................................................................... 8 Abbreviations ........................................................................................................................... 10 1. Introduction ...................................................................................................................... 13 1.1 Definition ................................................................................................................. 14 1.2 Clinical features........................................................................................................ 14 1.3 Pathological features and staging............................................................................. 16 1.3.2 Clark’s level of invasion and Breslow’s thickness........................................... 16 1.3.3 TNM staging .................................................................................................... 17 1.4 Epidemiology ........................................................................................................... 18 1.5 Genes and pathways................................................................................................. 19 1.5.1 Genes involved in familial melanoma.............................................................. 19 1.5.2 PTEN................................................................................................................ 21 1.5.3 MAPK .............................................................................................................. 22 1.5.4 Wnt................................................................................................................... 23 1.5.5 Microphthalmia-associated transcription factor............................................... 24 1.6 The transforming growth factor-β super-family ...................................................... 25 1.6.1 Receptors and Smad proteins ........................................................................... 27 1.6.2 Transforming growth factor-β.......................................................................... 28 1.6.3 Activin.............................................................................................................. 32 1.7 Id proteins................................................................................................................. 34 1.7.1 Ids, cell cycle regulation and cancer ................................................................ 34 1.7.2 TGF-β and Id proteins...................................................................................... 35 1.7.3 Id proteins and melanoma ................................................................................ 36 1.8 Aim........................................................................................................................... 37 1.9 References ................................................................................................................ 38 2. Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature................................................................................................................ 49 2.1 Abstract .................................................................................................................... 52 2.2 Introduction .............................................................................................................. 52 2.3 Results ...................................................................................................................... 54 2.3.1 No correlation between BRAF/NRAS mutations and gene expression........... 54 2.3.2 Microarray analyses reveal three cohorts......................................................... 56 2.3.3 Two groups of co-regulated genes define the cohorts...................................... 57 2.3.4 The cohorts reflect differences in metastatic potential..................................... 60 2.3.5 In vitro tests support the link between cohort distribution and metastatic potential............................................................................................................ 61 2.3.6 Wnt signalling controls Motif 1 ....................................................................... 62 2.4 Discussion ................................................................................................................ 63 2.5 Material and Methods............................................................................................... 67 2.5.1 Cell Culture and Media .................................................................................... 67 2.5.2 Genotyping....................................................................................................... 68 2.5.3 Total RNA Extraction and Expression Profiling.............................................. 68 2.5.4 Microarray Data Analysis ................................................................................ 68 2.5.5 Growth Inhibition Assays................................................................................. 70 2.5.6 Motility Assays ................................................................................................ 70 2.5.7 Western Analyses and ELISA.......................................................................... 70 2.5.8 Immunohistochemistry..................................................................................... 71 3 2.6 References ................................................................................................................ 72 3. In vitro phenotype validation.. ......................................................................................... 79 3.1 Introduction .............................................................................................................. 80 3.1.1 Modulation of TGF-β signalling...................................................................... 80 3.1.2 Vasculogenic mimicry...................................................................................... 83 3.2 Results ...................................................................................................................... 84 3.2.1 Confirming TGF-β1 and activin A secretion ................................................... 84 3.2.2 Follistatin secretion does not correlate with activin secretion ......................... 84 3.2.3 Smad2 and Smad3 are activated across all cohorts.......................................... 85 3.2.4 Ski is not responsible for the differential TGF-β signalling ............................ 86 3.2.5 The activation of the MAPK pathways does not correlate with the TGF-β signature ........................................................................................................... 87 3.2.6 Identifying vasculogenic mimicry as a discriminating phenotype................... 88 3.2.7 Phenotype switching ........................................................................................ 89 3.3 Discussion ................................................................................................................ 90 3.4 Material and Methods............................................................................................... 94 3.4.1 Cell culture ....................................................................................................... 94 3.4.2 Preparation of condition media ........................................................................ 95 3.4.3 ELISA............................................................................................................... 95 3.4.4 Preparation of total cell protein extracts .......................................................... 95 3.4.5 Preparation of cytosolic and nuclear protein extracts ...................................... 95 3.4.6 Western blot analysis ....................................................................................... 96 3.5 References ................................................................................................................ 98 4. In vivo switching of human melanoma cells between proliferative and invasive states 101 4.1 Abstract .................................................................................................................. 104 4.2 Introduction ............................................................................................................ 104 4.3 Results .................................................................................................................... 106 4.3.1 Phenotypic assignment of cell lines ............................................................... 106 4.3.2 Mitf is a marker of proliferative phenotype ................................................... 107 4.3.3 Mitf expression reflects signature phenotype................................................