Evaluation of Acid Ceramidase as Response Predictor and Therapeutic Target in Neoadjuvant Chemoradiotherapy for Rectal Cancer Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor of Medicine by David Lewis Bowden November 2018 Dedication To my family – you are my inspiration and motivation, thank you for all your love and support. x R A E J x i Declaration The work presented in this thesis was carried out in the Institute of Translational Medicine at the University of Liverpool and facilitated by the Madel research fellowship, supported by Health Education North West. The material contained within this thesis has not been, nor is currently being presented wholly, or in part, for any other degree or qualification. I declare that all the work presented in this thesis has been carried out by me except where indicated below: • Histopathological assessment of tumour regression grading and immunohistochemical expression of acid ceramidase was performed by Dr Michael Wall (FRCPath, Countess of Chester Hospital) • Tissue microarray construction and sectioning was performed by Mr Michael Neill (Liverpool Bio-Innovation Hub Biobank) David Bowden ii Acknowledgements I am indebted to everyone who has helped me with this research. Thank you. I would firstly like to thank my supervisors: Mr Dale Vimalachandran, Dr Neil Kitteringham, Dr Jason Parsons and Mr Paul Sutton. Your doors have always been open and your guidance and advice invaluable. Thank you particularly for your patience. One of the more laborious aspects of the work undertaken was in the assessment of histopathological tumour response and immunohistochemical expression of acid ceramidase on the tissue microarrays. I will be eternally grateful for the time and expertise provided by Dr Michael Wall. I am also grateful for the resourcefulness and assistance of Mrs Angela Kalaher and Mrs Julie Scanlon in identifying and retrieving the paraffin tissue blocks pertaining to the rectal cancer cases. Thank you to Mr Michael Neill for your effort in creating the tissue microarrays and thank you to Dr Helen Kalirai and Dr Theun van Veen for your expert assistance with the immunohistochemical staining. I would also like to thank Dr Katie Nickson for your welcoming approach and assistance with my weekly trip over the road for cell irradiation. I am indebted to the many other people in the Medical Research Council Centre for Drug Safety Science who have informally helped me acquire the necessary laboratory technique to produce this research, in particular Mr Jonathan Evans and Dr Charles Winiarski. I would finally like to thank the Bowel Disease Research Foundation for funding this project, and Health Education North West for the privileged opportunity to undertake this research with the Madel research fellowship. iii Contents Dedication i Declaration ii Acknowledgements iii Contents iv Abstract ix List of Figures x List of Tables xix Abbreviations xxi Chapter 1 – General Introduction 1 1.1 Colorectal Cancer 2 1.1.1 Epidemiology of Colorectal Cancer 2 1.1.2 Risk Factors for Colorectal Cancer 3 1.1.3 Genetics of Colorectal Cancer 4 1.1.4 Consensus Molecular Subtypes of Colorectal Cancer 10 1.1.5 NHS Bowel Cancer Screening Program (BCSP) 12 1.1.6 Diagnosis of Colorectal Cancer 13 1.1.7 Radiological Assessment of Colorectal Cancer 15 1.1.8 Staging of Colorectal Cancer 17 1.2 The Curative Management of Rectal Cancer 21 1.2.1 Early Rectal Cancer 21 1.2.2 Resectable Rectal Cancer 22 1.2.3 Radiotherapy in Rectal Cancer 23 1.2.4 Biological Effects of Radiation 24 1.2.5 Radiotherapy in Resectable Rectal Cancer 25 1.2.6 High Local Recurrence Risk Rectal Cancer 26 1.2.7 Low Rectal Cancer 27 1.2.8 Pelvic Radiotherapy Toxicity 28 1.2.9 Pharmacology of 5-fluorouracil 29 iv 1.2.10 Evaluation of Response to Chemoradiotherapy 32 1.2.11 Complete Response to Chemoradiotherapy 36 1.3 Biomarkers of Response to Chemoradiotherapy in Rectal Cancer 43 1.3.1 Gene Expression Analysis 43 1.3.2 miRNA Profiling 46 1.3.3 Protein Biomarkers 49 1.3.4 Conclusions and Future Direction 52 1.4 Acid Ceramidase 53 1.4.1 Precipitating Research 53 1.4.2 Biology and Pathophysiology of Acid Ceramidase 54 1.4.3 Acid Ceramidase in Apoptosis 56 1.4.4 Acid Ceramidase in Malignancy 57 1.4.5 Summary Statement and Further Research 62 1.5 Predicting and Modifying Response to Chemoradiotherapy in Rectal Cancer 64 1.5.1 Research Questions 64 1.5.2 Hypotheses 64 Chapter 2 – Validation of Acid Ceramidase as a Potential Predictive and/or 66 Prognostic Biomarker of Response to Chemoradiotherapy in Rectal Cancer 2.1 Introduction 67 2.1.1 Background 67 2.1.2 Hypotheses 68 2.1.3 Aims and Study Design 68 2.2 Methods 69 2.2.1 Ethical Approval 69 2.2.2 Assessment of Tumour Regression Grading 69 2.2.3 Tissue Microarray Construction 70 2.2.4 Immunohistochemical Staining 72 2.2.5 Acid Ceramidase Antibody Optimisation for Immunohistochemistry 73 2.2.6 Immunohistochemical Staining Analysis 76 2.2.7 Clinical and Pathological Data 77 v 2.2.8 Statistical Analysis 77 2.3 Results 78 2.3.1 Assessment of Tumour Regression Grading 78 2.3.2 Diagnostic Biopsy Tissue Microarrays 79 2.3.3 Cancer Site Tissue Microarrays 85 2.3.4 Normal Colon Tissue Microarrays 91 2.3.5 Quality Assessment of Tissue Microarray Staining 95 2.3.6 Patient Survival Analysis 96 2.4 Discussion 99 2.4.1 Summary of Aims 99 2.4.2 Summary of Results 99 2.4.3 Strengths and Limitations 101 2.4.4. Conclusion 106 Chapter 3 - Manipulation of Acid Ceramidase Expression and Activity in the 107 HCT116 Colorectal Cancer Cell Line 3.1 Introduction 108 3.1.1 Background 108 3.1.2 Hypotheses 111 3.1.3 Aims and Study Design 111 3.2 Methods 112 3.2.1 Culture of the HCT116 Colorectal Cancer Cell Line 112 3.2.2 Small Interfering RNA Transfection Targeting Acid Ceramidase 113 3.2.3 Reverse Transcription Polymerase Chain Reaction Detection of 114 Acid Ceramidase mRNA 3.2.4 Western Blotting for Acid Ceramidase 116 3.2.5 Acid Ceramidase Activity Assay 120 3.2.6 Statistical analysis 125 3.3 Results 126 3.3.1 Reverse Transcription Polymerase Chain Reaction Assessment 126 of ASAH1 mRNA Expression 3.3.2 Western Blotting for Acid Ceramidase 131 vi 3.3.3 Acid Ceramidase Activity Assay 139 3.4 Discussion 147 3.4.1 Summary of Aims 147 3.4.2 Summary of Results 147 3.4.3 Strengths and Limitations 149 3.4.4 Conclusion 151 Chapter 4 – Development of an in vitro Model of Acid Ceramidase 152 Dependent Sensitivity to Chemoradiotherapy in Rectal Cancer 4.1 Introduction 153 4.1.1 Background 153 4.1.2 Hypotheses 155 4.1.3 Aims and Study Design 155 4.2 Methods 157 4.2.1 MTS Assay 157 4.2.2 Clonogenic Model Development 158 4.2.3 Cell Counting and Plate Seeding 162 4.2.4 Colony Staining 164 4.2.5 Colony Counting 165 4.2.6 Cell Irradiation Delivery 166 4.2.7 Western Blotting for Thymidylate Synthetase 166 4.2.8 Post-Clonogenic Evaluation of Treatment Group Characteristics 167 4.2.9 Statistical Analysis 170 4.3 Results 171 4.3.1 MTS Assay – Chemotherapeutic Dose-Response 171 4.3.2 Western Blotting for Thymidylate Synthetase 175 4.3.3 Clonogenic Assay – Post-Irradiation / Acid Ceramidase Inhibition 177 4.3.4 Baseline Surviving Fraction 180 4.3.5 MTS Assay – Post-Irradiation / Acid Ceramidase Inhibition 184 4.3.6 Post-Transfection Proliferation 185 4.3.7 Post-Transfection Caspase 3/7 Activity 188 vii 4.4 Discussion 189 4.4.1 Summary of Aims 189 4.4.2 Summary of Results 189 4.4.3 Strengths and Limitations 191 4.4.4. Conclusion 194 Chapter 5 – Concluding Discussion 197 5.1 Background Summary 198 5.2 Original Aim 199 5.3 Initial Research Questions and Study Design 200 5.4 Summary of Results 201 5.4.1 Chapter 2 201 5.4.2 Chapter 3 201 5.4.3 Chapter 4 202 5.5 Study Limitations and Further Work 203 5.6 Review of Hypotheses 208 5.7 Conclusion 210 Bibliography 211 Appendix A Differentially Expressed Proteins Identified at Original Proteomic 237 Profiling Appendix B Clinical and Pathological Patient Data for Tissue Microarray 242 Analysis Appendix C Acid Ceramidase Expression from 10K Proteome in 49 Colorectal 244 Cancer Cell Lines Appendix D Supporting Publications, Presentations and Grants 245 viii Abstract Evaluation of acid ceramidase as response predictor and therapeutic target in neoadjuvant chemoradiotherapy for rectal cancer. Bowden D, Sutton P, Wall M, Parsons J, Kitteringham N, Vimalachandran D Introduction Colorectal cancer represents the second commonest cause of cancer-related mortality in the UK, with one-third of cases involving the rectum. Response to chemoradiotherapy (CRT) in rectal cancer varies from pathological complete response (pCR, with associated survival benefit) to disease progression. Predicting response is not currently possible but biomarkers to do this would facilitate personalised treatment, minimise morbidity from CRT and prevent delays in the systemic and local management of the disease in non-responders. Therapeutic targets may also be revealed to improve the efficacy of CRT, and further facilitate non- operative or rectal-preservation strategies. Initial proteomic profiling of rectal cancer has revealed differential expression of acid ceramidase (AC) between relative responders and non-responders to CRT. Aims 1) Validation of initial proteomic findings. 2) Confirmation of biological manipulation of AC in vitro.