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Targeting the Hedgehog and PI3K/AKT/Mtor Signaling Pathways in Rhabdomyosarcoma

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Targeting the Hedgehog and PI3K/AKT/Mtor Signaling Pathways in Rhabdomyosarcoma Targeting the Hedgehog and PI3K/AKT/mTOR signaling pathways in rhabdomyosarcoma Doctoral Thesis In partial fulfillment of the requirements for the degree “Doctor rerum naturalium (Dr. rer. nat.)” in the Molecular Medicine Study Program at the Georg-August University Göttingen submitted by Natalie Geyer born in Darmstadt Göttingen 2018 Members of the Thesis Committee: Supervisor Prof. Dr. Heidi Hahn Institute of Human Genetics, University Medical Center Göttingen Second member of the thesis committee Prof. Dr. Matthias Dobbelstein Institute of Molecular Oncology, University Medical Center Göttingen Third member of the thesis committee Prof. Dr. Dieter Kube Department of Haematology and Oncology, University Medical Center Göttingen Date of Disputation: Affidavit Here I declare that my doctoral thesis entitled “Targeting the HH and PI3K/AKT/mTOR signaling pathways in rhabdomyosarcoma” has been written independently with no other sources and aids than quoted. Natalie Geyer Göttingen, May 2018 Table of Contents List of Figures ............................................................................................................ I List of Tables ............................................................................................................. II Abstract .................................................................................................................... III 1 Introduction ........................................................................................................ 1 1.1 Rhabdomyosarcoma (RMS) ................................................................................... 1 1.1.1 Current clinical trials for RMS ............................................................................... 2 1.2 The Hedgehog (HH) signaling cascade ................................................................ 3 1.2.1 Canonical HH signaling ........................................................................................ 3 1.2.2 Canonical HH signaling in cancer ........................................................................ 7 1.2.3 Non-canonical HH signaling ................................................................................. 8 1.2.4 HH signaling in RMS .......................................................................................... 10 1.3 Smoothened (SMO) inhibitors ............................................................................. 12 1.3.1 Cyclopamine, vismodegib, sonidegib and HhAntag ............................................ 12 1.3.2 Adverse effects .................................................................................................. 14 1.3.3 Resistance mechanisms .................................................................................... 14 1.3.1 SMO inhibitors in RMS ....................................................................................... 15 1.4 PI3K/AKT/mTOR signaling ................................................................................... 16 1.4.1 Inhibitors of PI3K/AKT/mTOR pathway .............................................................. 18 1.4.2 PI3K/AKT/mTOR signaling in RMS .................................................................... 20 2 Aim of the study ............................................................................................... 22 3 Material .............................................................................................................. 23 3.1 Technical equipment ............................................................................................ 23 3.2 Consumables ........................................................................................................ 24 3.3 Reagents and chemicals ...................................................................................... 25 3.3.1 Signaling pathway inhibitors ............................................................................... 27 3.3.2 Enzymes ............................................................................................................ 28 3.3.3 Kits and ready-to-use reagents .......................................................................... 28 3.4 Buffers and solutions .......................................................................................... 28 3.5 Media .................................................................................................................... 30 3.5.1 Media for prokaryotic cell cultivation .................................................................. 30 3.5.2 Media and reagents for eukaryotic cell cultivation .............................................. 31 3.6 Biological material ............................................................................................... 31 3.6.1 Bacterial strains ................................................................................................. 31 3.6.2 Eukaryotic cell lines and primary cells ............................................................... 31 3.7 Synthetic DNA oligonucleotides ......................................................................... 32 3.8 Plasmids ............................................................................................................... 33 3.9 Antibodies ............................................................................................................ 33 3.10 Software ............................................................................................................. 34 3.11 Databases........................................................................................................... 34 4 Methods ............................................................................................................. 35 4.1 Cell biology .......................................................................................................... 35 4.1.1 Cultivation of adherent cells ............................................................................... 35 4.1.2 Cryopreservation and thawing of eukaryotic cells .............................................. 35 4.1.3 Generation of SHH conditioned medium (SHH-CM) .......................................... 36 4.1.4 Isolation and cultivation of primary murine RMS cells ........................................ 36 4.1.5 Counting of living eukaryotic cells ...................................................................... 36 4.1.6 Proliferation assay ............................................................................................. 37 4.1.7 Cell viability assay ............................................................................................. 37 4.1.8 Apoptosis assay ................................................................................................ 37 4.1.9 Cell cycle analysis ............................................................................................. 38 4.1.10 Transfection of RMS cell lines ........................................................................... 39 4.1.11 Transfection of RD cells with a Gli reporter system ............................................ 39 4.1.12 Dual-luciferase assay ........................................................................................ 40 4.1.13 E.coli transformation .......................................................................................... 40 4.1 Protein chemistry ................................................................................................. 40 4.1.1 Protein isolation from cultured cells .................................................................... 40 4.1.2 Protein isolation from RMS tissue ...................................................................... 41 4.1.3 Bicinchoninic acid (BCA) assay .......................................................................... 41 4.1.4 Western Blot ...................................................................................................... 41 4.2 Molecular biology ................................................................................................. 42 4.2.1 Genomic DNA (gDNA) isolation from mouse tail biopsies .................................. 42 4.2.2 Total RNA isolation from cell culture .................................................................. 43 4.2.3 Total RNA isolation from RMS tissue ................................................................. 43 4.2.4 Photometric quantification of nucleic acids ......................................................... 44 4.2.5 Genotyping polymerase chain reaction (PCR) ................................................... 44 4.2.6 Agarose gel electrophoresis ............................................................................... 45 4.2.7 Quantitative Real-Time PCR (qRT PCR) ........................................................... 45 4.2.8 Plasmid DNA amplification, isolation and purification ......................................... 47 4.2.9 Restriction enzyme hydrolysis ............................................................................ 47 4.3 Histology and immunohistochemistry ................................................................ 48 4.3.1 Haematoxylin and eosin (HE) staining ............................................................... 48 4.3.2 Ki67 staining ...................................................................................................... 48 4.4 Animal experiments ............................................................................................. 49 4.4.1 Mouse lines .......................................................................................................
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