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Histone Deacetylase 6 Represents a Novel Drug Target in the Oncogenic Hedgehog Signaling Pathway

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Histone Deacetylase 6 Represents a Novel Drug Target in the Oncogenic Hedgehog Signaling Pathway Aus dem Institut für Molekularbiologie und Tumorforschung Geschäftsführender Direktor: Prof. Dr. Rolf Müller des Fachbereichs Medizin der Philipps-Universität Marburg Histone deacetylase 6 represents a novel drug target in the oncogenic Hedgehog signaling pathway Inaugural- Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften dem Fachbereich Medizin der Philipps-Universität Marburg vorgelegt von Dhanyamraju Pavan Kumar aus Secunderabad, Indien Marburg-2017 Angenommen vom Fachbereich Medizin der Philipps Universität Marburg am:3/11/2017 Gedruckt mit der Genehmigung des Fachbereichs Dekan: Prof. Dr. Helmut Schäfer Referent: PD. Dr. Matthias Lauth Korreferent: Prof. Dr. Elke Pogge von Strandmann Table of Contents 1. Summary……………………………………………………………………………...1 1. Zussamenfassung …………………………………………………………....……….2 2. Introduction………………………………………………………………………......3 2.1 The Hedgehog signalling pathway…………………………………………………3 2.2 The Mechanism of Hedgehog signal transduction…………………………………5 2.3 Ptch1 a tumor suppressor gene…………………………………………………....10 2.4 Primary cilium and Hedgehog signalling.………………….………………….….11 2.5 Histone deacetylases (HDACs)………………………………………………...…15 2.6 Classification of Histone deacetylases……………………………………………16 2.7 Histone deacetylase 6……………………………………………………….….…17 2.8 Regulation of Ciliogenesis by HDAC6…………………………………………...20 2.9 Hedgehog signalling and Cancer………………………………………………….22 2.10 Hedgehog signalling Type-I- Mutation driven-ligand independent………….….22 2.11 Hedgehog signalling Type-II-Autocrine-ligand dependent………………….….23 2.12 Hedgehog signalling Type-III-paracrine-ligand dependent………………….….24 2.13 Hedgehog signalling Type-IIIb-Reverse paracrine……………………………...24 2.14 Hedgehog signalling in Cancer stem cells……………………………………….25 2.15 Hedgehog signalling in Central Nervous System (CNS) tumours……...….…….27 2.16 Medulloblastoma (MB)....………………………………….…. ………….…….28 2.16.1 Classic medulloblastoma (CMB)………………………….........……….…….29 2.16.2 Desmoplastic or Nodular (D/N) medulloblastoma…………...………….…….29 2.16.3 Medulloblastoma with extensive nodularity (MBEN)….…………….…….….30 2.16.4 Large cell medulloblastoma…………………………………. ………….…….30 2.16.5 Anaplastic medulloblastoma………………………………… ……………….30 2.17 SHH-subtype medulloblastoma ………………………………….……….….….33 2.18 Drug resistance in medulloblastoma ……………………………….………. ….35 2.19 Mechanism of resistance to SMO inhibitors…………………………………….36 2.19.1 Resistance to SMO inhibitors due to mutation in SMO……………………….36 2.19.2 Resistance to SMO inhibitors via amplification of Gli2………………………37 2.19.3 Resistance to SMO inhibitors via upregulation of PI3K-AKT pathway…….…37 i 2.19.4 Resistance to SMO inhibitors via upregulation of atypical protein kinase PI3K-AKT pathway(aPKCɩ/λ) ……….……………………...……….……….38 2.19.5 p53......................................................................................................................40 2.19.6 Atoh1and Boc……………………………………………………………….…40 2.19.7 Survivin……………………………………………………………….…….…41 2.19.8 bFGF…………………………………………………………………….….…41 3. Clinical significance and purpose of this study……………………………………43 4. Materials and Methods………………………………………………………….….45 4.1 Materials………………………………………………………………...…….….45 4.1.1 Laboratory equipment and consumables……………………………………...45 4.1.2 Chemicals………………………………………………………………….….46 4.1.3 Cell lines………………………………………………………………………47 4.1.4 Mouse lines…………………………………………………………………...47 4.1.5 Primary antibodies ……………………………………………………............48 4.1.6 Secondary antibodies …………………………………………………………48 4.1.7 siRNA sequences targeting mouse genes…………………..............................48 4.1.8 qPCR primer sequences……………………………………………………….49 4.1.9 Buffers …………………………………………………………………….….50 4.1.10 Software………………………………………………………………….….51 4.2 Methods…………………………………………………………………………….52 4.2.1 Cell culture……………………………………………………………………52 4.2.2 Cryopreservation of cells…………………………………………….….….…52 4.2.3 Thawing of cells………………………………………………………………53 4.2.4 RNA isolation…………………………………………………………………53 4.2.5 cDNA synthesis……………………………………………………………….53 4.2.6 Quantitative real time PCR ………………………………………….….…….54 4.2.7 RNAi transfection…………………………………………………….……….55 4.2.8 Luciferase reporter assay………………………………………………………55 4.2.9 Osteogenic differentiation assay………………………………………………55 4.2.10 Immunofluorescence…………………………………………………………56 4.2.11 Immunohistochemistry ………………………………………………………56 4.2.12 Cilia formation assay…………………………………………………………57 ii 4.2.13 Cilia resorption assay…………………………………………………...……57 4.2.14 SDS PAGE and Immunoblotting ……………………………………………57 4.2.15 Microarray …………………………………………………………….….….58 4.2.16 Compound solubilisation ……………………………………………………58 4.2.17 Statistical analysis……………………………………………………………59 5. Results……………………………………………………………………………….60 5.1 In Murine Medulloblastoma HDAC6 is overexpressed ………………………….60 5.2 Active Hh signalling is blocked by pharmacological HDAC6 inhibitors……………………………………………………………………..…..61 5.3 Epistatic investigation of HDAC6 effects…………………………………….….64 5.4 Global investigation of HDAC6 blockade…………………………………….….67 5.5 HDAC6 and its dichotomous effect on Hh signaling……………….……………72 5.6 Inhibition of HDAC6 pharmacologically has repressive effects on growth of in vivo medulloblastoma………………………………………………72 5.7 Analysis of HDAC6 expression using publicly available datasets……………….75 6. Discussion …………………………………………………………………………...78 7. References………………………………………………………………………...…89 8. Appendix…………………………………………………………………………...145 8.1 Supplementary figures……………………………………………………….......145 8.1.1 Lack of significant cilia effects due to blockade of HDAC6 (Figure S1) ……145 8.1.2 Hdac6 siRNA sequences validation (Figure S2) ...……………………………146 8.1.3 Reduction in Hh signaling due to Hdac6-specific inhibition (Figure S3) ……147 8.1.4 Inhibition of HDAC6 and its dichotomy on Hh target gene expression (Figure S4) ………………………………………………………...……...…148 8.1.5 Inhibition of HDAC6 in vivo and its effect on Gli1 expression (Figure S5) …148 8.1.6 Alopecia phenotype in treated mice (Figure S6) ………………………….….149 8.2 Curriculum Vitae………………………………………………………………...150 8.3 List of academic teachers…………………………………...……………………151 8.4 Publications…………………………………………………...………………….152 8.5 Acknowledgements………………………………………………………………153 8.6 Declaration…………...…………………………………………………………..154 iii List of Figures Figure 1: In Medulloblastoma mouse model HDAC6 is overexpressed………………..60 Figure 2: Hh signaling is impaired by targeting endogenous HDAC6………………...63 Figure 3: HDAC6 epistatic analysis in the Hh cascade………………………………...65 Figure 4: Global gene expression analysis………………………………………….….68 Figure 5: Molecular connection between Hh signaling and HDAC6………………….71 Figure 6: In vivo effects of HDAC6 inhibition pharmacologically………….………...74 Figure 7: Analysis of HDAC6 expression using publicly available datasets………….76 Figure S1: Lack of significant cilia effects due to blockade of HDAC6…………......145 Figure S2: Hdac6 siRNA sequences validation………………………………………146 Figure S3: Reduction in Hh signaling due to Hdac6-specific inhibition………….….147 Figure S4: Inhibition of HDAC6 and its dichotomy on Hh target gene expression….148 Figure S5: Inhibition of HDAC6 in vivo and its effect on Gli1 expression……………148 Figure S6: Alopecia phenotype in treated mice…………………………………….…149 Figure A: The key components of mammalian Hedgehog pathway……………………10 Figure B: Structural and functional domains of HDACs………………………………16 Figure C: Functional domains of HDAC6……………….……………………….…….19 Figure D: Regulation of Ciliogenesis by HDAC6 ……………........……...…...……….22 Figure E: Diverse models of Hedgehog signaling pathway…………………………….27 Figure F: Histopathological classification of medulloblastoma…………….….………31 Figure G: Frequency of medulloblastoma molecular subtypes………………………...32 Figure H: Acquired resistance mechanisms to SMO inhibitors…………….….………39 Figure I: Dual role of HDAC6 in Hedgehog signaling…………….……………….….88 iv List of Abbreviations ABC ATP binding cassette AD Alzheimer’s disease AMC Academic medical centre (Amsterdam) AP Alkaline Phosphatase APS Ammonium persulfate ATO Arsenic trioxide AurA Aurora A BBS Bardet-Biedl syndrome BCC Basal Cell Carcinoma BDNF Brain-derived neurotrophic factor bFGF Basic fibroblast growth factor Boc Brother of Cdo Boi Brother of Ihog CALK Chlamydomonas aurora-like protein kinase cDNA Complementary DNA Cdo Cell adhesion molecule related/downregulated by oncogenes Cep70 Centrosomal protein of 70kDa CFTR Cystic fibrosis transmembrane conductance regulator CK1α Casein Kinase1α CMB Classic Medulloblastoma CML Chronic Myeloid Leukemia CMT Charcot Marie tooth disease CNS Central Nervous System CRD Cysteine-rich domain CREB cAMP response element binding protein CSC Cancer stem cells CTA Cancer testis antigens C-Terminus Carboxy Terminus v DAPI 4’6-Diamidino-2-Phenylindole DBD Dynein Binding Domain Dhh Desert Hedgehog Disp Dispatched DMEM Dulbecco’s Modified Eagles Medium DMSO Dimethyl sulfoxide DNA Deoxyribose Nucleic Acid DNMB Desmoplastic nodular medulloblastoma DPBS Distilled Phosphate Buffer Saline Dyrk1 Dual specificity tyrosine-phosphorylation- regulated kinase ECL Enhanced chemiluminescence EvC Ellis van Creveld Syndrome EMT Epithelial to mesenchymal transition FBS Fetal Bovine Serum FDA Food and Drug Administration GAGE G antigen GANT Gli antagonist Gas1 Growth arrest specific protein 1 GGS Gorlin-Goltz syndrome Gli Glioma associated oncogene Gli-A Activator form of Gli Gli FL Full length of Gli Gli-R Repressor form of Gli GNP Granule neuron precursors Gpr G-protein-coupled-receptor GSK3 Glycogen synthase kinase 3 HAT Histone Acetyltransferase HD Huntington’s disease HDAC Histone
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