STUDIES OF IONIC MECHANISMS ASSOCIATED WITH HUMAN CANCERS Refika Mine GUZEL IMPERIAL COLLEGE LONDON DIVISION OF CELL AND MOLECULAR BIOLOGY Ph.D. Thesis This dissertation is the result of my own work unless otherwise acknowledged in the text or by reference(s). Signed: Refika Mine GUZEL Date: June, 2012 2 It's This Way I stand in the advancing light, my hands hungry, the world beautiful. My eyes can't get enough of the trees - they're so hopeful, so green. A sunny road runs through the mulberries, I'm at the window of the prison infirmary. I can't smell the medicines - carnations must be blooming nearby. It's this way: being captured is beside the point, the point is not to surrender. NAZIM HIKMET RAN (Translated by Randy Blasing and Mutlu Konuk) To my parents… 3 ABSTRACT The general aim of this thesis was to undertake a series of inter-related studies with a view to improving our understanding of the role of ion channel expression and its regulation in cancer cells with strong metastatic potential. The emphasis was on neonatal Nav1.5 (nNav1.5) subtype of voltage-gated sodium channel (VGSC). Chapter 1 (General Introduction) gives an account of the relevant literature and states the main aims of the studies. Chapter 2 details the Materials and Methods, ranging from quantitative molecular biology to in vitro assays of metastatic cell behaviour. Chapter 3 presents experiments on regulation of VGSCs by insulin and insulin-like growth factor1 (IGF1) in strongly metastatic human breast cancer (BCa) MDA-MB- 231 cells. The central strategy was to treat insulin and IGF1 as an integrated signalling system (”IIS”) and suppress it using pharmacological inhibitors and RNAi. Inhibiting IIS signalling suppressed metastatic cell behaviours (MCBs) and decreased nNav1.5 expression and activity. Chapter 4 describes studies on mRNA expression of a variety of cancer-associated ion channels (CAICs) in peripheral blood of normal human subjects with a view to laying the foundations for subsequent patient-based studies. The following 8 CAICs were studied: nNav1.5, VGSC-β1, VGSC-β1b, Kv1.3, Kv10.1, Kv11.1, KCa3.1 and TRPM8. Several differences were noted between healthy cases and cancer patients. In particular, nNav1.5 and Kv1.3 mRNA expressions were up- and down-regulated, respectively. Chapter 5 shows that the anti-diabetic drug Metformin suppressed Matrigel invasion and nNav1.5 mRNA expression in MDA-MB-231 cells. Chapter 6 involves studies on the strongly metastatic human colorectal cancer (CRCa) SW-620 cells, which were found to express nNav1.5 mRNA and protein. Silencing nNav1.5 expression had a significant 4 inhibitory effect on Matrigel invasion. The thesis ends with a General Discussion and Conclusion chapter, integrating the findings in the context of the field at large and pointing to future directions. 5 TABLE OF CONTENTS Abstract 4 Table of contents 6 List of figures 13 List of tables 17 Abbreviations 18 Chapter 1. General introduction 21 1.1 Pathophysiology of metastatic disease 22 1.1.1 Current problems in clinical management of BCa 22 1.1.2 Metastatic cascade 23 1.2 Cancer markers 26 1.2.1 Circulating tumour cells 27 1.3 Ion channels in cancer 29 1.3.1 Voltage-gated sodium channel expression in cancer 30 1.3.1.1 Structure-function of voltage-gated sodium channels 33 1.3.1.2 Expression of voltage-gated sodium channel in strongly metastatic cells 35 1.3.1.3 CELEX hypothesis 40 1.3.1.4 VGSC β subunits 42 1.3.1.5 Association of VGSC expression with mainstream cancer mechanisms: Regulatory mechanisms 45 1.4 Ion channel expression in blood mononuclear cells 45 1.4.1 Lymphocytes 46 1.4.2 Macrophages 48 1.4.3 Plasticity of ion channel expression in mononuclear cells 48 6 1.5 Insulin and insulin-like growth factor 1 system 50 1.5.1 IIS and cancer 54 1.5.2 IIS and VGSC 56 1.5.3 Metformin 57 1.5.3.1 Metformin and cancer 58 1.5.3.2 Metformin and ion channels 60 1.6 Aims, hypotheses and scope 61 Chapter 2. Materials and methods 64 2.1 Cell culture 65 2.1.1 Cell lines and basic maintenance 65 2.1.2 Pharmacological agents and treatments 65 2.1.3 Cellular toxicity 66 2.2 Functional assays 67 2.2.1 Measurement of cell number 67 2.2.2 Lateral motility 68 2.2.3 Transverse migration 70 2.2.4 Matrigel invasion 72 2.2.5 Crystal violet staining 72 2.3 Molecular biology 73 2.3.1 RNA extraction 73 2.3.2 cDNA synthesis 74 2.3.3 Conventional PCR 76 2.3.4 Quantitative real-time PCR 78 2.4 RNA interference 80 7 2.4.1 siRNA transfection 80 2.5 Western blotting 85 2.5.1 Protein extraction 85 2.5.2 Sodium dodecyl sulphate polyacrylamide gel electrophoresis 87 2.5.3 Antibodies 87 2.6 Immunocytochemistry 88 2.7 Immunohistochemistry 90 2.8 Protocols involving human blood 91 2.8.1 Collection of peripheral blood 91 2.8.2 Enrichment of mononuclear cells 92 2.8.3 RNA extraction 93 2.8.3.1 DNase treatment 93 2.8.4 cDNA synthesis 94 2.8.5 Quantitative real-time PCR 94 2.8.6 Generation of DNA standards 95 2.9 Electrophysiology 97 2.10 Data analysis 97 Chapter 3. (Results 1) Studies on the insulin – insulin-like growth factor system in MDA-MB-231 cells: Possible association with voltage-gated sodium channel 98 3.1 Introduction and rationale 99 3.2 Aims and scope 100 3.3 Results 101 3.3.1 Exploratory experiments 101 3.3.1.1 Expression of insulin and IGF1 receptors in MDA-MB-231 cells 101 8 3.3.1.2 Effects of serum deprivation 101 3.3.1.3 Functional effects of exogenous insulin in serum-free medium 106 3.3.1.4 Lack of functional effects of exogenous insulin in normal medium: “hyperinsulinemia” 106 3.3.1.5 Conclusion of exogenous insulin studies 110 3.3.2 Effects of AG1024 and OSI-906 112 3.3.2.1 Cell viability and cell number 112 3.3.2.2 Matrigel invasion 112 3.3.4 Effects of dual InsR-IGF1R siRNA 116 3.3.4.1 Control transfection experiments 116 3.3.4.2 Specificity of individual siRNAs 116 3.3.4.3 Dual siRNA transfections 118 3.3.4.4 Effects of dual-siRNA transfection on metastatic cell behaviours 123 3.3.4.5 Effects on nNav1.5 expression 123 3.4 Discussion 127 3.4.1 Effect of insulin on MCBs and the role of the cellular environment 129 3.4.2 Functional effects of inhibiting endogenous IIS 132 3.4.2.1 Lack of effect on cell growth 132 3.4.2.2 Effects on MCBs 133 3.4.3 IIS - VGSC (nNav1.5) interaction in control of MCBs 135 3.4.4 Conclusion 136 Chapter 4. (Results 2) Studies of cancer-associated ion channel expression in human blood 139 4.1 Introduction and rationale 140 4.2 Aims and scope 142 9 4.3 Results 142 4.3.1 Ethical aspects 142 4.3.2 Technical aspects 143 4.3.2.1 Validation of nNav1.5 primers 143 4.3.2.2 Primer efficiencies 143 4.3.2.3 Tests of PCR assay variability 146 4.3.3 Normal human blood 146 4.3.3.1 Initial sampling studies 146 4.3.3.2 CAIC mRNA expression patterns: Healthy controls 151 4.3.4 Blood from cancer patients: Comparison with healthy cases 151 4.4 Discussion 156 4.4.1 Technical aspects 156 4.4.2 Kv1.3 as a ‘control’ channel 157 4.4.3 Cancer cases: comparison with healthy controls 157 4.4.5 Circulating tumour cells 159 Chapter 5. (Results 3) Studies ionic mechanisms in relation to diabetes 161 5.1 Introduction and rationale 162 5.2 Aims and scope 163 5.3 Results 164 5.3.1 In vitro effects of metformin 164 5.3.1.1 Cellular viability and morphology 164 5.3.1.2 Cell number 166 5.3.1.3 nNav1.5 mRNA expression 166 5.3.1.4 Lateral motility 166 10 5.3.1.5 Matrigel invasion 169 5.3.2 Studies on human blood samples 169 5.4 Discussion 172 5.4.1 Effects of metformin on cancer cell invasive behaviour 175 5.4.2 Possible role of VGSC (nNav1.5) in the action of metformin 175 5.4.3 Changes in blood CAIC mRNA expression in T2D 176 Chapter 6. (Results 4) Voltage-gated sodium channel expression in human colorectal cancer 180 6.1 Introduction and rationale 181 6.2 Aims and scope 185 6.3 Results 185 6.3.1 nNav1.5 mRNA expression in SW620 cells 185 6.3.2 nNav1.5 mRNA expression in SW620 cells – PCRs of the ‘spliced’ region and sequencing results 187 6.3.3 Immunocytochemistry of VGSC expression in SW620 cells 191 6.3.4 Silencing nNav1.5 by RNAi 191 6.3.4.1 Effects on nNav1.5 expression 191 6.3.4.2 Effects on invasiveness of SW620 cells 198 6.3.5 nNav1.5 is expressed in CRCa biopsy tissue 202 6.4 Discussion 207 6.4.1 nNav1.5 RNA was expressed in SW620 cells 209 6.4.2 nNav1.5 protein was expressed in SW620 cells 210 6.4.3 siRNA transfection reduced nNav1.5 mRNA and functional protein expression in SW620 cells 210 11 6.4.4 siRNA knock-down of nNav1.5 suppressed invasiveness of SW620 cells 212 6.4.5 nNav1.5 protein is expressed in human CRCa biopsy tissues 212 Chapter 7.