CHARACTERIZATION OF THE MECHANISMS BY WHICH CARBONIC ANHYDRASE IX FACILITATES TUMOUR GROWTH AND METASTASIS by Maria de Lourdes Vallejo Espi MSc, UNAM, 2010 BSc, BUAP, 2008 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2015 © Maria de Lourdes Vallejo Espi, 2015 ii Abstract The presence of hypoxic microenvironments in solid tumours is a marker of poor prognosis in numerous cancer types, including breast cancer; the second leading cause of cancer-‐related death. Hypoxia results in an adaptive response in tumour cells through the activation of the transcription factor Hypoxia Inducible Factor-‐1α (HIF-‐1α), which stimulates the expression of a large number of genes that contribute to tumour progression. One of the most prominently activated genes is Carbonic Anhydrase IX (CAIX), which facilitates the acidification of the extracellular space and cell invasion by producing protons. Moreover, it assists in keeping the intracellular space neutral through the generation of bicarbonate, which is shuttled into the cytoplasm by bicarbonate transporters, ultimately favouring cell survival. CAIX facilitates breast tumour growth and metastasis; however the exact mechanism remains unknown. The overexpression of CAIX in hypoxic solid tumours, its limited expression in normal tissue and the presence of an extracellular catalytic domain makes this protein an excellent therapeutic target. The intent of this thesis was to unveil the mechanisms by which CAIX facilitates tumour progression, and to characterize novel small molecule inhibitors and antibodies targeting CAIX. It was found that the intracellular (IC) domain of CAIX regulates its catalytic activity, which is required for cell survival, cell invasion and metastasis. The extracellular proteoglycan-‐like (PG-‐like) domain of CAIX does not regulate CAIX catalytic activity; however it does modulate cell migration, invasion and metastasis. I identified a role of CAIX in promoting tumour cell invasion through interaction with membrane-‐bound matrix metalloprotease-‐14 (MMP-‐14) and localization in invadopodia. The IC domain of CAIX mediates this interaction and CAIX enzymatic activity appears to regulate the ability of MMP-‐14 to degrade type I collagen during cell invasion. From the pool of anti-‐CAIX inhibitors and antibodies characterized in this thesis, the inhibitor U-‐104 was excellent at blocking CAIX enzymatic activity and has entered iii phase I clinical trials. Likewise, anti-‐CAIX antibody MM-‐26 blocked 50% of CAIX activity and induced cell death in vitro. The work described here provides new insight into the mechanism of CAIX-‐mediated tumour invasion and metastasis and has identified two new therapeutic strategies for targeting CAIX. iv Preface This thesis is presented in six chapters. All of the experiments described in Chapter 3 were designed by Dr. Shoukat Dedhar, Dr. Paul McDonald and myself and carried out by myself. The flow enrichment experiments described in Chapter 4 were performed by the Flow Cytometry Core Facility (FCCF) housed in the Terry Fox Laboratory of the B.C. Cancer Research Center (BCCRC). Dr. Shawn Chafe and Dr. Eiko Kawamura helped me with the set up of the Flow Cytometer for membrane-‐bound CAIX analysis and Cell Cycle analysis, respectively (Chapter 4, Figures 4.4 and 4.13). Dr. Shawn Chafe generated the 4T1 knockdown cell lines (4T1shCAIX, Chapter 4, Figure 4.2) that I utilized for the overexpression of WT huCAIX and mutant forms. Dr. Mykola Maydan generated the CAIX constructs (Chapter 4, Figure 4.1). Dr. Shawn Chafe also generated the CAIX knockdown in MDA-‐MB-‐231 LM2-‐4, BxPC-‐3 and Pk-‐8 cell lines (Chapter 5, Figure 5.1). Mass spectrometry experiments described in Chapter 4 were performed in Dr. Leonard Foster’s laboratory at the Centre for High-‐Throughput Biology (CHiBi) in UBC. Dr. Yuanmei Lou, Jordan Gillespie and Christina Ostlund performed the in vivo experiments described in Chapter 5. All animal studies and procedures were performed in accordance with protocols approved by the Institution Animal Care Committee at the BC Cancer Research Centre and University of British Columbia (Vancouver, BC, Canada) as per protocol number: A14-‐0058 and under the project’s title: “Targeting Carbonic Anhydrase IX and hypoxia for the diagnosis and treatment of aggressive breast cancer”. Dr. Kevin Bennewith and Nancy E. LePard carried out the clonogenic experiments from the spontaneous metastasis assay (Figure 5.1) and Dr. Shawn Chafe and myself performed the clonogenic experiments from the experimental metastasis assay (Figure 5.2). The real names of the novel antibodies developed by SignalChem Lifesciences Corporation and described in Chapter 3 have been hidden for patenting reasons and alternative names are used instead. v All other experiments were designed by Dr. Shoukat Dedhar and myself and carried out by me. vi Table of Contents Abstract ................................................................................................................................................................. ii Preface .................................................................................................................................................................. iv Table of Contents .............................................................................................................................................. vi List of Tables ........................................................................................................................................................ x List of Figures .................................................................................................................................................... xi List of Abbreviations ..................................................................................................................................... xiv Acknowledgements .................................................................................................................................... xviii Dedication ......................................................................................................................................................... xix Chapter 1. Introduction .................................................................................................................................. 1 1.1 Breast cancer .......................................................................................................................................... 1 1.1.1 Classification .................................................................................................................................. 1 1.1.2 Risk factors ...................................................................................................................................... 2 1.1.3 Epidemiology ................................................................................................................................. 2 1.1.4 Models for studying breast cancer ........................................................................................ 3 1.2 Pancreatic cancer .................................................................................................................................. 4 1.2.1 Classification .................................................................................................................................. 4 1.2.2 Epidemiology ................................................................................................................................. 4 1.2.3 Models for studying pancreatic cancer ............................................................................... 5 1.3 Hallmarks of cancer ............................................................................................................................. 5 1.3.1 Cell migration, invasion and metastasis ............................................................................. 6 1.3.1.1 EMT ............................................................................................................................................ 7 1.3.1.2 Different types of cell migration ................................................................................... 8 1.3.1.2.1 Collective migration ................................................................................................... 8 1.3.1.2.2 Individual migration .................................................................................................. 8 1.3.1.3 pH and migration .............................................................................................................. 13 1.3.1.4 The role of MMPs in tumour cell invasion ............................................................. 14 1.3.1.5 pH and invasion ................................................................................................................. 15 1.3.1.6 Invadopodia and tumour cell invasion .................................................................... 16 1.4 Hypoxia, HIF-‐1α and pH homeostasis ....................................................................................... 21 1.4.1 Hypoxia and the hypoxia-‐inducible factor-‐1α (HIF-‐1α) ........................................... 21 1.4.2 pH homeostasis in the tumour cell .................................................................................... 24 1.5 CAIX ......................................................................................................................................................... 27 1.5.1 CAIX structure ............................................................................................................................ 27 1.5.2 CAIX expression and tissue distribution ......................................................................... 30 1.5.3 CAIX function .............................................................................................................................. 31 1.5.4 Proposed role of each domain in the regulation of CAIX enzymatic activity .. 33 1.5.5 CAIX role in the regulation of intracellular pH ............................................................. 34 1.5.6 CAIX role in cell survival and proliferation .................................................................... 34 1.5.7 CAIX role in cell adhesion, migration and invasion .................................................... 35 1.5.8 CAIX and cell signalling ........................................................................................................... 36 1.5.9 CAIX binding partners ............................................................................................................. 36 1.5.10 CAIX role in tumour progression and metastasis ..................................................... 37 1.5.11 CAIX as a therapeutic target .............................................................................................. 38 vii 1.6 CAIX-‐specific small molecule inhibitors .................................................................................. 38 1.6.1 CAIX inhibitors currently available ................................................................................... 38 1.6.1.1 Previous characterization of U-‐104, GC-‐205 and SLC-‐148, -‐149 and -‐150 ................................................................................................................................................................. 40 1.7 Anti-‐CAIX monoclonal antibodies ............................................................................................... 41 1.7.1 Mechanisms of tumour cell killing by antibodies ........................................................ 41 1.7.2 Pros and cons of antibodies as cancer therapy ............................................................ 42 1.7.3 Anti-‐CAIX antibodies currently available ....................................................................... 43 1.7.4 Novel anti-‐CAIX antibodies ................................................................................................... 44 1.8 Objectives and hypothesis ............................................................................................................. 45 1.8.1. Determine the mechanism by which CAIX is facilitating tumour growth and metastasis ................................................................................................................................................ 45 1.8.2. Characterize novel CAIX small molecule inhibitors and antibodies .................. 46 1.8.3 Hypothesis .................................................................................................................................... 46 Chapter 2. Materials and Methods .......................................................................................................... 47 2.1 Cell culture, antibodies and reagents ........................................................................................ 47 2.2 Generation of stable cell lines ....................................................................................................... 48 2.3 Analysis of protein expression ..................................................................................................... 49 2.4 Analysis of CAIX membrane localization ................................................................................. 50 2.5 Immunofluorescence ........................................................................................................................ 50 2.6 CAIX activity assay ............................................................................................................................ 51 2.6.1 In vitro activity assay (recombinant CAIX) .................................................................... 51 2.6.2 CAIX “In-‐cell” activity assay ................................................................................................... 52 2.6.3 Calculation of extent of inhibition ...................................................................................... 52 2.7 Binding of FITC-‐CAI .......................................................................................................................... 53 2.8 Migration and invasion assays ..................................................................................................... 53 2.9 Cell cycle analysis .............................................................................................................................. 54 2.10 Growth curves .................................................................................................................................. 54 2.11 MTT assays ......................................................................................................................................... 55 2.12 Immunoaffinity purification followed by protein identification by mass spectrometry ............................................................................................................................................... 55 2.12.1 Immunoprecipitation using CNBr beads ...................................................................... 55 2.12.2 Immunoprecipitation using A/G sepharose beads .................................................. 56 2.13 Apoptosis assays ............................................................................................................................. 57 2.14 Statistical analysis ........................................................................................................................... 57 Chapter 3. Characterization of Novel CAIX Inhibitors and Antibodies ................................... 58 3.1 Synopsis ................................................................................................................................................. 58 3.2 Inhibitory capacities of small molecules U-‐104 and GC-‐205 .......................................... 60 3.3 Inhibitory capacities of U-‐104 derivatives SLC-‐148, SLC-‐149 and SLC-‐150 ............ 64 3.4 Specificity and function-‐blocking capacities of novel anti-‐CAIX polyclonal antibody 359-‐1 and its derivatives .................................................................................................... 70 3.5 Specificity and function-‐blocking capacities of novel anti-‐CAIX monoclonal antibodies 538-‐542 .................................................................................................................................. 81 3.6 Specificity and function-‐blocking capacities of novel anti-‐CAIX monoclonal antibody MM-‐26 ......................................................................................................................................... 90 3.7 Discussion ............................................................................................................................................. 98 viii 3.7.1 Novel CAIX small molecule inhibitors .............................................................................. 98 3.7.2 Novel CAIX antibodies ........................................................................................................... 100 3.7.2.1 KalGene Pharmaceuticals anti-‐CAIX antibodies ................................................ 100 3.7.2.2 Deeley-‐UVic anti-‐CAIX antibodies ........................................................................... 103 3.7.2.3 SignalChem Lifesciences anti-‐CAIX antibody ..................................................... 104 Chapter 4. Characterization of CAIX Mutants .................................................................................. 107 4.1 Synopsis ............................................................................................................................................... 107 4.2 Expression and localization of human CAIX mutants ...................................................... 108 4.3 Activity of human CAIX mutants ............................................................................................... 118 4.4 Cell survival and proliferation of human CAIX mutants ................................................. 124 4.5 Cell migration of human CAIX mutants .................................................................................. 131 4.6 Intracellular binding partners of human CAIX. ................................................................... 133 4.7 Discussion ........................................................................................................................................... 138 4.7.1 Expression and localization ................................................................................................ 138 4.7.2 Activity ......................................................................................................................................... 140 4.7.3 Cell survival and proliferation ........................................................................................... 142 4.7.4 Migration ..................................................................................................................................... 144 4.7.5 Identification of binding proteins by immunoaffinity purification followed by mass spectrometry. ........................................................................................................................... 145 Chapter 5. Hypoxia-‐induced Carbonic Anhydrase IX Regulates Invasion and Metastasis by Interacting with and Activating MMP-‐14 within Invadopodia .......................................... 148 5.1 Synopsis ............................................................................................................................................... 148 5.2 Introduction ....................................................................................................................................... 148 5.3 Materials and methods .................................................................................................................. 150 5.3.1 Cell culture and antibodies ................................................................................................. 150 5.3.2 Cloning of CAIX truncation and point mutants ........................................................... 151 5.3.3 Generation of stable cell lines ............................................................................................ 151 5.3.4 Analysis of protein expression .......................................................................................... 152 5.3.5 Invasion assays ......................................................................................................................... 152 5.3.6 Mouse tumour models .......................................................................................................... 153 5.3.7 Gelatin zymography ............................................................................................................... 154 5.3.8 Collagen degradation ............................................................................................................. 154 5.3.9 Invadopodia formation and imaging .............................................................................. 154 5.3.10 Co-‐immunoprecipitation ................................................................................................... 155 5.3.11 Statistical analysis ................................................................................................................ 156 5.4 Results .................................................................................................................................................. 156 5.4.1 CAIX is required for breast and pancreatic tumour cell invasion and metastasis .............................................................................................................................................. 156 5.4.2 The intracellular and proteoglycan-‐like domains of CAIX are required for tumour invasion and metastasis .................................................................................................. 158 5.4.3 CAIX regulates MMP-‐14-‐mediated type I collagen invasion and degradation .................................................................................................................................................................... 160 5.4.4 CAIX co-‐localizes with MMP-‐14 in mature invadopodia ........................................ 163 5.4.5 CAIX and MMP-‐14 interact through the CAIX intracellular domain ................. 166 5.5 Discussion ........................................................................................................................................... 170 Chapter 6. Conclusions and Future Directions ................................................................................ 175 ix 6.1 Novel CAIX antibodies and small molecules inhibitors ................................................... 175 6.2 Characterization of CAIX mutants ............................................................................................ 177 6.3 Role of CAIX in cell invasion ........................................................................................................ 181 References ....................................................................................................................................................... 184 x List of Tables Table 3.1 Inhibitory constants of CAIX inhibitors U-‐104 and GC-‐205 ................................... 60 Table 3.2 Inhibitory constants of CAIX inhibitors SLC148-‐150 ................................................ 64 Table 4.1 List of proteins identified by mass spectrometry as binding partners of human CAIX .................................................................................................................................................................. 136 xi List of Figures Figure 1.1 Epithelial-‐mesenchymal transition .................................................................................... 7 Figure 1.2 Different types of cell migration ....................................................................................... 10 Figure 1.3 The five-‐step model of individual cell migration and the role of pH ................ 12 Figure 1.4 The steps of invadopodia formation ............................................................................... 19 Figure 1.5 Intracellular pH regulation in the tumour cell ........................................................... 26 Figure 1.6 Representation of CAIX in the cell membrane ............................................................ 28 Figure 3.1 Function-‐blocking abilities of novel CAIX inhibitors U-‐104 and GC-‐205 ........ 63 Figure 3.2 Function-‐blocking abilities of novel CAIX inhibitors SLC-‐148, -‐149 and -‐150 ............................................................................................................................................................................... 66 Figure 3.3 Dose-‐response of U-‐104 and SLC-‐149 ........................................................................... 68 Figure 3.4 Percent inhibition of CAIX activity by U-‐104 and SLC-‐149 ................................... 69 Figure 3.5 Specificity of novel anti-‐CAIX antibodies 359-‐1 and 360-‐1 .................................. 72 Figure 3.6 Immunoprecipitation abilities of novel anti-‐CAIX antibody 359-‐1 ................... 74 Figure 3.7 Immunofluorescence staining abilities of novel anti-‐CAIX antibody 359-‐1 ....... ............................................................................................................................................................................... 76 Figure 3.8 Function-‐blocking capacity of novel anti-‐CAIX antibody 359-‐1 ......................... 78 Figure 3.9 Specificity of novel anti-‐CAIX antibodies 4H1-‐4L1 and 16H1 ............................. 80 Figure 3.10 Specificity of novel anti-‐CAIX antibodies 538-‐542 ................................................ 82 Figure 3.11 Immunofluorescence staining abilities of novel anti-‐CAIX antibodies 538-‐542 ....................................................................................................................................................................... 84 Figure 3.12 Specificity of antibodies 539 and 541 towards CAIX ............................................ 86 Figure 3.13 Specificity of antibodies 539 and 541 towards the catalytic domain of CAIX ............................................................................................................................................................................... 87 Figure 3.14 Immunoprecipitation abilities of novel anti-‐CAIX antibodies 539 and 541 .... ............................................................................................................................................................................... 88 Figure 3.15 Function-‐blocking abilities of novel anti-‐CAIX antibodies 538-‐542 .............. 90 Figure 3.16 Epitope determination of anti-‐CAIX antibodies MM-‐26 and MM-‐04 ............. 92 Figure 3.17 Function-‐blocking ability of novel anti-‐CAIX antibody MM-‐26 ........................ 94 xii Figure 3.18 In-‐cell CAIX function-‐blocking ability of novel anti-‐CAIX antibody MM-‐26 ..... ............................................................................................................................................................................... 96 Figure 3.19 Anti-‐CAIX antibody MM-‐26-‐mediated induction of cell death .......................... 98 Figure 4.1 Diagram of human CAIX mutants .................................................................................. 109 Figure 4.2 Knockdown of mouse CAIX in 4T1 cells ..................................................................... 110 Figure 4.3 Expression, dimerization and localization of human CAIX mutants .............. 113 Figure 4.4 Percent of human CAIX present at the cell membrane ........................................ 115 Figure 4.5 Co-‐staining of human CAIX and the ER ....................................................................... 117 Figure 4.6 Contribution of each domain to the activity of CAIX: in vitro assay ............... 119 Figure 4.7 Contribution of each domain to the activity of CAIX: In-‐cell assay ................. 121 Figure 4.8 CAIX activity in 4T1shNS and 4T1shCAIX cell lines: binding of FITC-‐CAI ........... ............................................................................................................................................................................ 122 Figure 4.9 Contribution of each domain to the activity of CAIX: binding of FITC-‐CAI ......... ............................................................................................................................................................................ 123 Figure 4.10 The intracellular domain of CAIX regulates cell survival ................................. 125 Figure 4.11 Cell viability and proliferation of 4T1 cells expressing human CAIX mutants ............................................................................................................................................................................ 127 Figure 4.12 Growth curves of 4T1 cells expressing human CAIX mutants ....................... 128 Figure 4.13 Cell cycle analysis of 4T1 cells expressing human CAIX mutants ................ 130 Figure 4.14 Contribution of each domain of CAIX to the regulation of cell migration ......... ............................................................................................................................................................................ 132 Figure 4.15 Immunoprecipitation of human CAIX from 4T1sh/WT huCAIX and 4T1sh/ΔIC cell lines .................................................................................................................................. 134 Figure 5.1 CAIX is required for cell invasion and metastasis .................................................. 157 Figure 5.2 Intracellular (IC) and proteoglycan-‐like (PG-‐like) domains of CAIX regulate cell invasion and metastasis ................................................................................................................. 159 Figure 5.3 CAIX regulates MMP-‐14-‐mediated degradation of type I collagen ................. 162 Figure 5.4 CAIX co-‐localizes with MMP-‐14 in the invadopodia of 4T1 cells .................... 164 Figure 5.5 CAIX co-‐localizes with MMP-‐14 in the invadopodia of MDA-‐MB-‐231 cells ........ ............................................................................................................................................................................ 165 Figure 5.6 CAIX interacts with MMP-‐14 through its intracellular (IC) domain .............. 167 xiii Figure 5.7 Residues S448, Y449 and T443 modulate the interaction of CAIX with MMP-‐14 ....................................................................................................................................................................... 169 Figure 5.8 Model for CAIX-‐mediated regulation of MMP-‐14 activity .................................. 173 xiv List of Abbreviations aa amino acid Ab antibody ADCC antibody-‐dependent cell-‐mediat
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Characterization of the mechanisms by which Carbonic Anhydrase IX facilitates tumour growth and metastasis Vallejo Espi, Maria de Lourdes 2015
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| Title | Characterization of the mechanisms by which Carbonic Anhydrase IX facilitates tumour growth and metastasis |
| Creator |
Vallejo Espi, Maria de Lourdes |
| Publisher | University of British Columbia |
| Date Issued | 2015 |
| Description | The presence of hypoxic microenvironments in solid tumours is a marker of poor prognosis in numerous cancer types, including breast cancer; the second leading cause of cancer-related death. Hypoxia results in an adaptive response in tumour cells through the activation of the transcription factor Hypoxia Inducible Factor-1α (HIF-1α), which stimulates the expression of a large number of genes that contribute to tumour progression. One of the most prominently activated genes is Carbonic Anhydrase IX (CAIX), which facilitates the acidification of the extracellular space and cell invasion by producing protons. Moreover, it assists in keeping the intracellular space neutral through the generation of bicarbonate, which is shuttled into the cytoplasm by bicarbonate transporters, ultimately favouring cell survival. CAIX facilitates breast tumour growth and metastasis; however the exact mechanism remains unknown. The overexpression of CAIX in hypoxic solid tumours, its limited expression in normal tissue and the presence of an extracellular catalytic domain makes this protein an excellent therapeutic target. The intent of this thesis was to unveil the mechanisms by which CAIX facilitates tumour progression, and to characterize novel small molecule inhibitors and antibodies targeting CAIX. It was found that the intracellular (IC) domain of CAIX regulates its catalytic activity, which is required for cell survival, cell invasion and metastasis. The extracellular proteoglycan-like (PG-like) domain of CAIX does not regulate CAIX catalytic activity; however it does modulate cell migration, invasion and metastasis. I identified a role of CAIX in promoting tumour cell invasion through interaction with membrane-bound matrix metalloprotease-14 (MMP-14) and localization in invadopodia. The IC domain of CAIX mediates this interaction and CAIX enzymatic activity appears to regulate the ability of MMP-14 to degrade type I collagen during cell invasion. From the pool of anti-CAIX inhibitors and antibodies characterized in this thesis, the inhibitor U-104 was excellent at blocking CAIX enzymatic activity and has entered phase I clinical trials. Likewise, anti-CAIX antibody MM-26 blocked 50% of CAIX activity and induced cell death in vitro. The work described here provides new insight into the mechanism of CAIX-mediated tumour invasion and metastasis and has identified two new therapeutic strategies for targeting CAIX. |
| Genre |
Thesis/Dissertation |
| Type |
Text |
| Language | eng |
| Date Available | 2015-04-15 |
| Provider | Vancouver : University of British Columbia Library |
| Rights | Attribution-NonCommercial-NoDerivs 2.5 Canada |
| DOI | 10.14288/1.0166209 |
| URI | http://hdl.handle.net/2429/52795 |
| Degree |
Doctor of Philosophy - PhD |
| Program |
Biochemistry and Molecular Biology |
| Affiliation |
Medicine, Faculty of Biochemistry and Molecular Biology, Department of |
| Degree Grantor | University of British Columbia |
| GraduationDate | 2015-05 |
| Campus |
UBCV |
| Scholarly Level | Graduate |
| Rights URI | http://creativecommons.org/licenses/by-nc-nd/2.5/ca/ |
| AggregatedSourceRepository | DSpace |
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