THE COMPLEX INTERACTIONS BETWEEN GENETICS AND ENVIRONMENT: DIET, INFLAMMATION AND INTESTINAL TUMORIGENESIS by STEPHANIE KAY DOERNER Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation advisors: Nathan A. Berger, M.D. Joseph H. Nadeau, Ph.D. Department of Genetics CASE WESTERN RESERVE UNIVERSITY January 2013 This thesis is dedicated to my brother, Nate. Thank you for everything. 1 TABLE OF CONTENTS Dedication 1 Table of Contents 2 List of Figures 7 List of Tables 10 List of Abbreviations 11 Acknowledgements 15 Abstract 18 CHAPTER 1 – Background and Significance 20 1.1 Summary of Research 21 1.2 Introduction to Colon Cancer 24 1.2.1 Biology of the Small Intestine 25 1.2.2 Biology of the Large Intestine 28 1.2.3 Regeneration of the Intestine 32 1.3 Immunity and Cancer 34 1.3.1 Intestinal Immunity 35 1.3.2 Lymphoid Cells of the Intestinal Mucosa 38 1.3.3 Immune System and Cancer 43 1.3.4 The Complement Component Cascade 44 1.3.5 Complement and Cancer 48 1.3.6 Prostaglandins and Cancer 49 1.4 Cancer of the Intestine 52 1.4.1 Types of Intestinal Polyps 52 1.4.2 Stages of Colon Cancer Development 56 2 1.5 Causes of Colon Cancer 61 1.5.1 Hereditary Forms of Colon Cancer 61 1.5.2 Rare Types of Colon Cancer 66 1.5.3 Inflammation Associated Colon Cancer 70 1.5.4 Sporadic Causes of Colon Cancer 72 1.5.5 The APC Mutation and Wnt Signaling 79 1.5.6 Environmental Causes of Colon Cancer 82 1.6 Understanding Digestion and Metabolism 83 1.6.1 Understanding the Process of Digestion 84 1.6.2 Regulation of Appetite and Metabolism 87 1.7 The Obesity Epidemic 90 1.7.1 Obesity-Related Diseases 91 1.7.2 Environmental Effects on Obesity 93 1.7.3 Inflammation and Obesity 94 1.8 Dietary Fat: The Good, the Bad and the Ugly 98 1.8.1 Saturated Fatty Acids 100 1.8.1.1 Butyric Acid 101 1.8.1.2 Lauric and Myristic Acids 103 1.8.1.3 Palmitic and Stearic Acids 104 1.8.2 Monounsaturated Fatty Acids 105 1.8.2.1 The Mediterranean Diet and Olive Oil 105 1.8.2.2 Oleic Acid 107 1.8.2.3 Oleic:Stearic Acid Ratio 107 1.8.3 Polyunsaturated Fatty Acids 108 1.8.3.1 Alpha-Linolenic Acid 109 3 1.8.3.2 Eicosapentaenoic and Docoshexaenoic Acids 109 1.8.3.3 Arachidonic Acid 112 1.8.3.4 Linoleic Acid 113 1.8.3.5 Conjugated Linoleic Acid 114 1.8.3.6 The Omega-3: Omega-6 Ratio 116 1.9 Using Mouse Models to Study Human Obesity and Colon Cancer 117 1.9.1 ApcMin/+ - A Mouse Model of Intestinal Neoplasia 119 1.9.2 Mouse Models of Polygenic Traits: Obesity 123 CHAPTER 2 – High Fat Diet Modulates Intestinal Polyp Formation, Separate from Diet-Induced Obesity 127 2.1 Introduction 128 2.2 Methods 131 2.2.1 Mice 131 2.2.2 Diets 132 2.2.3 Study Design 132 2.2.4 Metabolic Parameters and Cytokine Analysis 135 2.2.5 Quantitative RT-PCR and Protein Analysis 136 2.2.6 Statistical Analysis 136 2.3 Results 136 2.3.1 Constructing mouse models to study diet, separate from obesity 136 2.3.2 High fat diet decreases lifespan in ApcMin/+ 140 2.3.3 Contrasting CSS responses to HF diets 141 2.3.4 High dietary fat increases intestinal neoplasia 149 2.3.5 Neutrophils increased in strains fed high dietary fat 152 4 2.4 Discussion 154 CHAPTER 3 – Differential Effects of Dietary Fat on Inflammation and Cancer of the Intestine 158 3.1 Introduction 159 3.2 Methods 161 3.2.1 Mice 161 3.2.2 Diets 161 3.2.3 Study Design 162 3.2.4 Metabolic Parameters and Cytokine Analysis 164 3.2.5 Quantitative RT-PCR and Protein Analysis 164 3.2.6 Statistical Analysis 165 3.3 Results 165 3.3.1 Differential effect of different dietary fat sources on obesity and metabolic syndrome 165 3.3.2 Differential effects of fat on polyp burden of the small intestine 170 3.3.3 Contrasting responses to different fats on polyps of the colon 172 3.3.4 Early effects of different dietary fats on DIO and MetS 172 3.4.5 Early effects of different dietary fat sources on intestinal neoplasia 174 3.4.6 Effects of dietary fat sources on inflammation 180 3.4 Discussion 184 CHAPTER 4 – High Fat Diet-Induced Complement Signaling Contributes to Intestinal Adenoma Risk 188 4.1 Introduction 189 4.2 Methods 190 5 4.2.1 Mice 190 4.2.2 Diets 190 4.2.3 Study Design 191 4.2.4 Metabolic Parameters and Cytokines Analysis 192 4.2.5 Quantitative RT-PCR and Protein Analysis 192 4.2.6 Pharmacological Inhibition of C5aR 193 4.2.7 Fluorescence-Activated Cell Sorting (FACS) of Intestinal Immune Cells 193 4.2.8 Statistical Analysis 194 4.3 Results 194 4.3.1 A high fat diet induces inflammation in the circulation and intestine 194 4.3.2 Dietary mediation of complement induced inflammation 196 4.3.3 Inhibition of complement signaling attenuates intestinal neoplasia 200 4.4 Discussion 205 CHAPTER 5 – Summary of Conclusions and Future Directions 208 5.1 Summary of conclusions and evidence 209 5.2 Modulation of microbial profiles by dietary fat 215 5.2.1 Introduction 215 5.2.2 Questions and proposed experiments 217 5.2.2.1 Do microbial profiles differ in diets composed of specific fats? 217 5.2.2.2 Do microbial profiles associate with cancer severity in ApcMin/+ mice? 218 5.2.2.3 Can different dietary fats be used to change intestinal microbial profiles? 219 REFERENCES 220 6 LIST OF FIGURES CHAPTER 1 FIGURES Figure 1.1 – The structure of the small intestine 26 Figure 1.2 – The structure of the large intestine 29 Figure 1.3 – Immunity in the small and large intestine 37 Figure 1.4 – Differentiation of CD4+ T-cell subsets 40 Figure 1.5 – A model of innate and adaptive immunity during inflammation-induced cancer development 45 Figure 1.6 – Complement evasion by pathogens 47 Figure 1.7 – Synthesis of anti- and pro-inflammatory prostaglandins 51 Figure 1.8 – Stage 0 to Stage 2 of colon cancer progression 58 Figure 1.9 – Stage 3 of colon cancer progression 60 Figure 1.10 – Stage 4 of colon cancer progression 62 Figure 1.11 – The genetics of sporadic colon cancer 73 Figure 1.12 – Hallmarks of cancer 75 Figure 1.13 – Wnt signaling in health and disease 80 Figure 1.14 – Components of the digestive system 85 Figure 1.15 – Medical complications associated with obesity 92 Figure 1.16 – Adipose tissue, adipokines and inflammation 95 Figure 1.17 – Creation of the Chromosome Subsitution Strains (CSSs) 125 Figure 1.18 – CSSs after 100 days on LF and HF diets 126 CHAPTER 2 FIGURES Figure 2.1 – Construction of the CSS.ApcMin/+ strains 139 Figure 2.2 – Body weights during the duration of the diet study 142 7 Figure 2.3 – Survival curves for mice fed the coconut oil diets 143 Figure 2.4 – Blood cell counts in B6 and ApcMin/+ 144 Figure 2.5 – Growth weights from CSS.ApcMin/+ strains 145 Figure 2.6 – B6 and CSS body weight parameters 147 Figure 2.7 – B6 and CSS metabolic parameters 150 Figure 2.8 – High dietary fat increases polyp number and mass 151 Figure 2.9 – White blood cells in B6 and CSSs 153 CHAPTER 3 FIGURES Figure 3.1 – Growth curves for mice fed the coconut, corn and olive diets 166 Figure 3.2 – Body weight parameters in mice fed cocnut, corn or olive oil diets 168 Figure 3.3 – Specific dietary fat sources have differential effects on polyp number and mass 171 Figure 3.4 – Specific dietary fat sources have differential effects on colon polyp incidence 173 Figure 3.5 – Growth curves for mice fed coconut, corn or olive oil diets for 30 days 175 Figure 3.6 – Body weight parameters for mice fed coconut, corn or olive diets 176 Figure 3.7 – Metabolic parameters from mice fed coconut, corn or olive oils 177 Figure 3.8 – Specific dietary fat sources have differential effects on polyp number and mass after 30 days 178 Figure 3.9 – Expression analysis from B6 and ApcMin/+ fed the coconut diet 181 CHAPTER 4 FIGURES Figure 4.1 – Cytokines analysis in B6 and ApcMin/+ fed the coconut oil diets 195 8 Figure 4.2 – Total polyp numbers are decreased in A2.ApcMin/+ fed HF 197 Figure 4.3 – Complement C5a is elevated in mice fed a HF diet for 30 and 60 days 198 Figure 4.4 – Strains that carry A/J chromosome 2 are deficient in C5a 199 Figure 4.5 – Polyp numbers are reduced in mice deficient in C5a 201 Figure 4.6 – Complement C5a medicates diet-induced inflammation and intestinal neoplasia 204 9 LIST OF TABLES CHAPTER 1 TABLES Table 1.1 – Genetics of colon cancer 64 Table 1.2 – Mutations found in sporadic colon cancer 76 Table 1.3 – Effects of specific fatty acids on colon cancer 118 Table 1.4 – Known modifiers of ApcMin/+ 122 CHAPTER 2 TABLES Table 2.1 – Makers used in the creation of the CSS.ApcMin/+ strains 133 Table 2.2 – Composition of hydrogenated coconut oil diet 135 Table 2.3 – Primer sequences for quantitative RT-PCR reactions 137 Table 2.4 – Body weight and metabolic parameters after 60 days on the diet study 148 Table 2.5 – Summar of CSS.ApcMin/+ response to high saturated fat 156 CHAPTER 3 TABLES Table 3.1 – Composition of coconut, corn and olive oil diets 163 Table 3.2 – Body weight and metabolic parameters for mice fed coconut, corn or olive oil diets 169 Table 3.3 – Summary of dietary influences on B6 and ApcMin/+ 185 CHAPTER 4 TABLES 4.1 – Body weight and metabolic studies for complement studies 203 10 LIST OF ABBREVIATIONS AA Arachidonic Acid AAM Alternatively Activated Macrophage ACF Aberrant Crypt Foci AFAP Attenuated Familial Adenomatous Polyposis AgRP Agouti-Related Peptide AICR American Institute for Cancer Research ALA Alpha Linolenic Acid α-MSH Alpha Melanocyte-Stimulating Hormone AOM Azoxymethane APC Adenomatis Polyposis Coli B6 C57BL/6J BMI Body Mass Index CAM Classically Activated Macrophage CCK Cholecystokinin CCS Cronkhite-Canada Syndrome CD Crohn’s Disease CIMP CpG Island Methylator Phenotype CIN Chromosomal Instability CK1