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Binding RXFP1 in Brain Tumors

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Binding RXFP1 in Brain Tumors Functional role of C1Q-TNF related peptide 8 (CTRP8)- binding RXFP1 in brain tumors By THATCHAWAN THANASUPAWAT A Thesis submitted to the Faculty of Graduate Studies of The University of Manitoba in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY Department of Human Anatomy and Cell Science University of Manitoba Winnipeg, Manitoba Canada Copyright © 2017 by Thatchawan Thanasupawat TABLE OF CONTENTS ABSTRACT ....................................................................................................................... v ACKNOWLEDGEMENTS ........................................................................................... vii LIST OF TABLES ........................................................................................................... ix LIST OF FIGURES .......................................................................................................... x LIST OF ABBREVIATIONS ........................................................................................ xii LIST OF COPYRIGHTED MATERIALS ................................................................. xix CHAPTER 1: INTRODUCTION .................................................................................... 1 1.1 Relaxin family peptide receptors (RXFPs) ............................................................... 1 1.2 Relaxin family peptide receptor 1 (RXFP1) ............................................................. 1 1.2.1 Structural features of RXFP1 ............................................................................. 1 1.2.2 Ligands of RXFP1 .............................................................................................. 4 1.2.3 Signal transduction pathways of RXFP1 ............................................................ 5 1.2.4 Functional roles of the RLN2-RXFP1 system .................................................... 8 1.2.4.1 RLN2-RXFP1 in the reproductive system and breast .................................. 8 1.2.4.2 RLN2-RXFP1 in the central nervous system............................................. 10 1.2.4.3 RLN2-RXFP1 in the cardiovascular system .............................................. 11 1.2.4.4 RLN2-RXFP1 in extracellular matrix remodeling .................................... 12 1.2.4.5 RXFP1 and cancers .................................................................................... 13 1.3 Relaxin family peptide receptor 2 (RXFP2) and cancers ........................................ 17 1.4 P59 and P74 peptides .............................................................................................. 18 1.5 Tissue Distribution and Structure of CTRP Family Members ................................ 19 1.5.1 CTRP Members in Cancer ................................................................................ 23 1.5.2 CTRP8 is a Novel RXFP1 Ligand in Glioblastoma ......................................... 24 1.5.3 Summary and Prospective Goals ...................................................................... 26 1.6 Brain tumors ............................................................................................................ 26 1.6.1 WHO classification of brain tumors 2016 ........................................................ 27 1.6.2 Glioblastoma ..................................................................................................... 29 1.6.3 GB subtypes ...................................................................................................... 31 1.6.4 Glioblastoma invasiveness ............................................................................... 34 1.6.5 Glioblastoma chemoresistance ......................................................................... 35 1.7 DNA repair mechanisms ......................................................................................... 38 1.7.1 O6-methylguanine-DNA methyltransferase (MGMT) ..................................... 38 1.7.2 DNA mismatch repair (MMR) ......................................................................... 39 ii 1.7.3 Base excision repair (BER) .............................................................................. 40 1.8 The High Mobility Group (HMG) proteins............................................................. 41 1.9 High Mobility Group A (HMGA) ........................................................................... 42 1.9.1 HMGA2 functions in embryo development ..................................................... 43 1.9.2 HMGA2 in epithelial-mesenchymal transition (EMT) and stemness .............. 44 1.9.3 HMGA2 in cancer ............................................................................................ 46 1.9.4 The protective roles of HMGA2 ....................................................................... 49 1.9.5 HMGA2 as a novel therapeutic target .............................................................. 50 1.9.6 DNA minor groove binding agents .................................................................. 50 1.10 Thesis Overview and hypotheses .......................................................................... 52 CHAPTER 2: MATERIALS AND METHODS .......................................................... 54 2.1 Cell culture and primary human brain isolation ...................................................... 54 2.2 Human recombinant proteins .................................................................................. 55 2.3 Recombinant CTRP8 production ............................................................................ 55 2.4 Drugs and inhibitors ................................................................................................ 56 2.5 Motility assay .......................................................................................................... 57 2.6 RNA silencing ......................................................................................................... 58 2.7 RNA isolation, Reverse transcriptase polymerase chain reaction (RT-PCR) and Quantitative Real time PCR (qPCR) ............................................................................. 58 2.8 Cell viability assay (WST assay) ............................................................................ 63 2.9 Caspase 3/7 activity assay ....................................................................................... 64 2.10 Comet assay........................................................................................................... 64 2.11 Immunofluorescence ............................................................................................. 65 2.12 Immunohistochemistry .......................................................................................... 65 2.13 Western blot analysis ............................................................................................ 66 2.14 MPG molecular beacon activity assay .................................................................. 69 2.15 Measurement of apoptosis by flow cytometry ...................................................... 70 2.16 Colony formation assay ......................................................................................... 71 2.17 Statistical analysis ................................................................................................. 71 CHAPTER 3: RESULTS ............................................................................................... 72 Part I. Human CTRP8 is a new ligand of RXFP1 and induces migration of brain cancer cells ....................................................................................................................... 72 3.1 Expression of RXFP1 and CTRP8 in GB cell lines and human primary brain tumor cells................................................................................................................................ 72 iii 3.2 RXFP1 activation induced cell migration by PKC-dependent pathways ............... 76 3.3 Human recombinant Flag-tagged CTRP8 protein production ................................ 83 3.4 RXFP1 activation induces cell migration by a STAT3-dependent pathway .......... 87 Part II: The CTRP8-RXFP1 system protects GB against DNA damage and apoptosis........................................................................................................................... 91 3.5 RXFP1 activation protects GB cells from Temozolomide (TMZ) induced DNA damage .......................................................................................................................... 91 3.6 RXFP1 activation enhances cell survival ................................................................ 97 3.7 RXFP1 activation increases expression of anti-apoptotic proteins Bcl-2 and Bcl-XL ..................................................................................................................................... 100 3.8 The DNA protective function of RXFP1 involves members of the base excision repair (BER) pathway.................................................................................................. 101 Part III. High Mobility Group AT Hook 2 protein (HMGA2) increases temozolomide resistance in glioblastoma .................................................................... 108 3.9 HMGA2 is expressed in the nucleus of GB cells .................................................. 108 3.10 HMGA2 promoted TMZ chemoresistance ......................................................... 110 3.11 DNA minor groove binder agents show additive reduction in cell
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