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Contribution of HOXD10, HOXD11 and HOXD13 to Malignancy Of

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Contribution of HOXD10, HOXD11 and HOXD13 to Malignancy Of Technische Universität München Fakultät für Medizin Contribution of HOXD10, HOXD11 and HOXD13 to malignancy of Ewing sarcoma Laura Roth Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Medizin genehmigten Dissertation. Vorsitzender: Prof. Dr. Ernst J. Rummeny Prüfer der Dissertation: 1. Prof. Dr. Stefan Burdach 2. Prof. Dr. Uta Behrends Die Dissertation wurde am 23.05.2019 bei der Technischen Universität München eingereicht und durch die Fakultät für Medizin am 04.12.2019 angenommen. Table of contents Table of contents List of abbreviations ............................................................................................. 8 1. Introduction .................................................................................................. 14 1.1 Ewing sarcoma..................................................................................................... 14 1.2 Genes of the posterior HOXD locus ..................................................................... 19 1.2.1 General information on HOX genes .............................................................. 19 1.2.2 Current knowledge on HOXD10 ................................................................... 24 1.2.3 Current knowledge on HOXD11 ................................................................... 24 1.2.4 Current knowledge on HOXD13 ................................................................... 25 1.3 Aim of this study and overview of the experimental approach .............................. 26 2. Material ......................................................................................................... 28 2.1 List of manufacturers ............................................................................................ 28 2.2 General material ................................................................................................... 30 2.3 Instruments and equipment .................................................................................. 31 2.4 Chemical and biological reagents ......................................................................... 32 2.5 Commercial reagent kits ....................................................................................... 33 2.6 Media and solutions ............................................................................................. 34 2.7 Antibodies for immunofluorescence ...................................................................... 34 2.8 Small interfering RNAs ......................................................................................... 35 2.9 Oligonucleotides for retroviral gene transfer ......................................................... 35 2.10 Primers for PCR and qRT-PCR ............................................................................ 36 2.11 Gene expression assays for qRT-PCR ................................................................. 36 2.12 Human cell lines and mouse strain ....................................................................... 38 2.12.1 Human cell lines ........................................................................................... 38 2.12.2 Mouse strain ................................................................................................. 38 3. Methods ........................................................................................................ 39 3.1 Cell culture ........................................................................................................... 39 3.2 RNA isolation ....................................................................................................... 39 3.3 cDNA synthesis .................................................................................................... 40 3.4 Quantitative Real-Time PCR (qRT- PCR) ............................................................. 41 3.4.1 Standard qRT-PCR ...................................................................................... 41 3.4.2 Detection of EWS-FLI1 ................................................................................. 42 Table of contents 3.5 Transient RNA interference (RNAi) ...................................................................... 43 3.6 Retrovirus-mediated stable RNA interference ...................................................... 44 3.7 In vitro assays ..................................................................................................... 44 3.7.1 xCELLigence proliferation assay .................................................................. 44 3.7.2 Colony forming assay .................................................................................. 44 3.7.3 Invasion assay ............................................................................................. 45 3.7.4 Neuronal differentiation assay ...................................................................... 46 3.8 In vivo experiments.............................................................................................. 47 3.8.1 Investigation of local tumor growth ............................................................... 47 3.8.2 Investigation of invasive tumor growth ......................................................... 47 3.9 Immunohistochemistry of murine samples ........................................................... 48 3.10 Statistical analysis ............................................................................................... 48 4. Results .......................................................................................................... 49 4.1 Over-expression of posterior HOXD genes in ES ................................................ 49 4.2 Potential regulatory mechanisms of posterior HOXD genes in ES ....................... 51 4.2.1 No Regulation via EWS-FLI1 ....................................................................... 51 4.2.2 No Regulation via EZH2 .............................................................................. 52 4.2.3 Regulation via DKK2 .................................................................................... 53 4.3 Down-regulation of posterior HOXD genes via RNA interference ........................ 54 4.3.1 Transient RNA interference of single HOXD genes ...................................... 54 4.3.2 Triple HOXD gene knock down via transient RNA interference .................... 56 4.3.3 Constitutive HOXD gene knock down via RNA interference ......................... 57 4.4 Contribution of posterior HOXD genes to neuronal differentiation ability of ES cell lines ..................................................................................................................... 58 4.5 Influence of posterior HOXD genes on bone-associated genes and osteotropic tumor growth of ES .............................................................................................. 61 4.5.1 Suppressed expression of RUNX2 after triple HOXD knock down ............... 61 4.5.2 Influence of posterior HOXD genes on other bone-associated genes .......... 63 4.5.3 Contribution of posterior HOXD genes to osteolytic tumor growth in vivo ..... 67 4.6 Influence of HOXD10, HOXD11 and HOXD13 on ES growth and invasiveness .. 69 4.6.1 Inhibition of proliferation after HOXD10 and HOXD13 knock down in vitro ... 69 4.6.2 Reduction of colony formation after HOXD11 and HOXD13 knock down in vitro .......................................................................................................... 70 4.6.3 Decrease of invasiveness after HOXD10 and HOXD13 knock down in vitro 71 Table of contents 4.6.4 Suppressed expression of MMP1 after HOXD11, HOXD13 and triple HOXD knock down .................................................................................................. 73 4.6.5 Minor influence of posterior HOXD genes on other MMPs ............................ 75 4.6.6 Reduction of metastatic spread after HOXD11 and HOXD13 knock down in vivo ........................................................................................................... 77 5. Discussion.................................................................................................... 79 5.1 Over-expression of posterior HOXD genes in ES ................................................. 79 5.2 Regulatory mechanisms of posterior HOXD genes .............................................. 80 5.2.1 Absent regulation via EWS-FLI1 and EZH2 .................................................. 80 5.2.2 Regulation via DKK2 .................................................................................... 81 5.3 Diverse impact of posterior HOXD genes on neuronal differentiation ability of ES cell lines ............................................................................................................... 83 5.4 Role of HOXD10, HOXD11 and HOXD13 in ES pathology ................................... 85 5.4.1 Influence of posterior HOXD genes on bone-associated genes and osteotropic tumor growth .............................................................................. 85 5.4.1.1 Enhancement of osteotropic tumor growth mediated by RUNX2 ........... 86 5.4.1.2 Promotion of bone marrow invasiveness and osteolysis in tumor tissue in vivo ................................................................................................... 89 5.4.2 Influence of posterior HOXD genes on ES growth and invasiveness in vitro 91 5.4.3 MMP1 as posterior HOXD downstream target and key player
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