Characterization of the Role of Cyclin D1 and Ral Gtpases in the Proliferation and Dissemination of Glioblastoma

Characterization of the Role of Cyclin D1 and Ral GTPases in the Proliferation and Dissemination of Glioblastoma Tània Cemeli Sánchez http://hdl.handle.net/10803/667907 Characterization of the Role of Cyclin D1 and Ral GTPases in the Proliferation and Dissemination of Glioblastoma està subjecte a una llicència de Reconeixement- NoComercial-CompartirIgual 4.0 No adaptada de Creative Commons Les publicacions incloses en la tesi no estan subjectes a aquesta llicència i es mantenen sota les condicions originals. (c) 2019, Tània Cemeli Sánchez TESI DOCTORAL Characterization of the Role of Cyclin D1 and Ral GTPases in the Proliferation and Disseminnation of Glioblastoma Tània Cemeli Sánchez Memòria presentada per optar al grau de Doctor per la Universitat de Lleida Programa de Doctorat en Salut Director/a Dr. Eloi Garí Marsol Dr. Francissco Ferrezuelo Muñoz Tutor/a Dr. Eloi Garí Marsol 2019 INDEX INDEX Abbreviations 1 Abstracts 5 English 7 Català 9 Castellano 11 Introduction 13 1 ‐ Astrocytic tumors 15 1.1 Classification..................................................................................................................... 18 1.2 Treatment ........................................................................................................................ 22 1.3 Origin and epidemiology .................................................................................................. 22 1.4 Invasive capacity of glioblastomas ................................................................................... 24 2 ‐ Cyclin D1 25 2.1 D‐type cyclins are essential .............................................................................................. 26 2.2 Cyclin D1 function ............................................................................................................ 26 2.3 Cyclin D1 structure and expression ................................................................................. 28 2.4 Cytoplasmic functions of cyclin D1 .................................................................................. 29 2.5 Cytoplasmic cyclin D1 and cell migration ........................................................................ 30 2.6 Cytoplasmic cyclin D1 and cell signaling .......................................................................... 31 2.7 Cyclin D1 and cancer: glioblastomas ............................................................................... 38 2.8 Cyclin D – CDK4/6 inhibition: Palbociclib ......................................................................... 40 3 ‐ Hypothesis 43 4 ‐ Objectives 45 Materials & Methods 47 1 ‐ Cell biology techniques 49 1.1 Cell cultures ...................................................................................................................... 49 1.2 Transfection ..................................................................................................................... 51 1.3 Virus production and infection ........................................................................................ 53 1.4 Cell proliferation .............................................................................................................. 54 1.5 Cell viability ...................................................................................................................... 55 1.6 Other cell biology methods .............................................................................................. 56 1.7 Tumorspheres .................................................................................................................. 57 1.8 In vitro pharmacological treatments ............................................................................... 58 2 ‐ Molecular biology techniques 59 2.1 Plasmid constructions ...................................................................................................... 59 3 ‐ Biochemical techniques 61 3.1 Antibodies ........................................................................................................................ 61 3.2 Protein extraction and Western Blot ............................................................................... 62 3.3 Ral activity assay .............................................................................................................. 64 3.4 YFP‐PBD ............................................................................................................................ 64 3.5 Peptide Array ................................................................................................................... 65 3.6 Proteome profiler ............................................................................................................ 65 4 ‐ Microscopy techniques 67 4.1 Immunohistochemistry & histology ................................................................................ 67 4.2 Immunofluorescence ....................................................................................................... 69 5 ‐ Experimental Animals 71 5.1 Previous considerations ................................................................................................... 71 5.2 SCID Mice ......................................................................................................................... 71 5.3 Intracranial injection ........................................................................................................ 72 6 ‐ Bioinformatic tools 73 6.1 ImageJ .............................................................................................................................. 73 6.2 Gliovis ............................................................................................................................... 73 6.3 Gene Ontology ................................................................................................................. 73 Results 75 Chapter 1 ‐ Cytoplasmic cyclin D1 regulates glioblastoma invasion 77 Retinoblastoma‐independent functions of cyclin D1 regulate the invasion efficiency of glioblastoma cells. .................................................................................................................. 77 Localization of the cyclin D1‐CDK4 activity in glioblastoma cells .......................................... 80 Cyclin D1 promotes invasion by human glioblastoma cells through the stimulation of Rac1, FAK and Ral A activities. ......................................................................................................... 83 Accumulation of cyclin D1 in the membrane promotes glioblastoma spread in vivo. ......... 87 Chapter 2 – Role of Ral GTPases in glioblastoma growth and dissemination 91 Study of the expression of Ral GTPases in primary human glioblastoma cells ..................... 91 Biological effects of the downregulation of Ral GTPases in primary glioblastoma cells ...... 92 Importance of Ral GTPases in glioblastoma growth and invasion ........................................ 94 Ral downregulation promotes senescence in primary glioblastoma cells ............................ 96 Ral GTPases as therapeutic targets to treat glioblastoma .................................................... 98 Effects of Ral GTPase inhibitors ........................................................................................... 100 Chapter 3 ‐ Signaling pathways activated by cytoplasmic cyclin D1 103 Cytoplasmic cyclin D1 is able to induce signaling pathways ............................................... 103 Signaling pathways activated by cyclin D1 in glioblastoma ................................................. 108 Discussion 111 Cytoplasmic cyclin D1 regulates glioblastoma dissemination 114 Role of Ral GTPases in glioblastoma growth and dissemination 117 Signaling pathways activated by citoplasmic cyclin D1 120 Conclusions 123 References 127 Annex 137 Publications 139 ABBREVIATIONS Abbreviations Aa – Amino acid ATP – Adenosine TriPhosphate BrdU – Bromodeoxyuridine BSA – Bovine Serum Albumin CAAX – Farnesylation site of K‐RAS protein Ccnd1 – Cyclin D1 CDK – Cyclin Dependent Kinase CEEA – Ethics Committee on Animal Experimentation CEI – Ethical Committee CKi – CDK inhibitor CMV – Citomegalovirus CNS – Central Nervous System CSCs – Cancer Stem Cells DMEM – Dulbecco’s modified Eagle’s Medium DMSO – Dimethyl Sulfoxide DTT – Dithiothreitol ECM – ExtraCellular Matrix EDTA – Ethylene diaminetetraacetic acid EGF – human Epidermal Growth Factor EGFR – human Epidermal Growth Factor Receptor EMT – Epithelial Mesenchymal Transition ERK – Extracellular signal‐Regulated Kinase FA – Focal Adhesion FAK – Focal Adhesion Kinase FBS – Fetal Bovine Serum FDA – Food and Drug Administration FGFR – Fibroblast Growth Factor Receptor 1 Abbreviations Fluc – Firefly Luciferase Gene GAP – GTPase Activating Protein GBM – Glioblastoma GDP – Guanosine Diphosphate GFP – Green Fluorescent Protein GSCs – Glioblastoma Stem Cells GSK‐3β – Glycogen Synthase Kinase 3 beta GTP – Guanine TriPhosphate H&E – Hematoxylin & Eosin HGF – Hepatocyte Growth Factor HRP – Horseradish peroxidase HUAV – Hospital Universitari Arnau de Vilanova IDH1 – Isocitrate Dehydrogenase 1 IF – Immunofluorescence IHC – ImmunoHistoChemistry iMEF – Immortalized Mouse Embryonic Fibroblast JNK – c‐JUN n‐terminal Kinase kDa – Kilodalton KO – Knock Out KOD1 – Cyclin D1 Knock Out MAPK – Mitogen‐Activated Protein Kinase MEF – Mouse Embryonic Fibroblast Mm – Mus musculus mTOR – Mammalian Target Of Rapamycin MTT – 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide NF1 – Neurofibromatosis type I NFкB –

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