University of Bradford eThesis This thesis is hosted in Bradford Scholars – The University of Bradford Open Access repository. Visit the repository for full metadata or to contact the repository team © University of Bradford. This work is licenced for reuse under a Creative Commons Licence. INVESTIGATION OF MECHANISMS OF DRUG RESISTANCE IN COLORECTAL CANCER: A PROTEOMIC AND PHARMACOLOGICAL STUDY USING NEWLY DEVELOPED DRUG-RESISTANT HUMAN CELL LINE SUBCLONES M. ORTEGA DURAN PhD 2017 Investigation of mechanisms of drug resistance in colorectal cancer: a proteomic and pharmacological study using newly developed drug-resistant human cell line subclones Mario Ortega Duran Submitted for the Degree of Doctor of Philosophy Faculty of Life Sciences University of Bradford 2017 Abstract Mario Ortega Duran Investigation of mechanisms of drug resistance in colorectal cancer: a proteomic and pharmacological study using newly developed drug- resistant human cell line subclones Keywords: Colorectal cancer, 5-Fluorouracil, Oxaliplatin, Irinotecan, Drug resistance, Proteomics, SILAC, chemotherapy, protein interactions, chemosensitivity assays Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer. i Acknowledgements I would like to express my deepest gratitude to my two Ph.D. supervisors: Dr. Steve Shnyder and Dr. Chris Sutton. Without their knowledge, support, guidance, and mentorship, I would never have become the scientist that I am today, and I will be grateful to them forever for that. I wish firstly to thank my main supervisor Dr. Steve Shnyder, for trusting me from the first moment. Thanks for his kindly welcome, and his weekly and constant supporting, patience, advising, and understanding of my professional and personal life. It was a pleasure to share all these issues during three years and a half with you. I am also thankful to Dr. Chris Sutton for his closeness, his support and for all the confidence placed in me. You have been always there whenever I needed advice to discuss any issue. Really, it has been a great pleasure to work with both of you, great professionals and wonderful people. I would like to thank my new in life-brother, Sadr-ul-shaheed for his invaluable spiritual and professional aid and the most important issue, he did always all of that with a smile on his face. I wish to offer my sincerest thanks to The University of Bradford and all ILSR scholarship and postgraduate program members (Dr. Anne Graham, Mrs. Shamim Haider) for their time and guidance. Thanks to all the staff and members at the Institute of Cancer Therapeutics for sharing three very pleasant years. Finally, I would like to express my deepest gratitude to all my beloved family. You have believed in me and offered your unconditional support. Thank you all. ii To my parents, you have been the mirror of my soul and the main source of all values I have received. Thank you for all you have done and especially for the love with which you have done it. You are the reference in my life. You have also been an example of love and constancy in the midst of difficulties. Thank you for the bountiful knowledge and experiences I gained with you, because is something that I will use during the rest of my life. You are the pride of my life, an example of fight, sacrifice, and perseverance. Thank you for showing me that all things in life must be done with love and at its necessary time. To my brother, I always feel you with me, as if you were by my side. Thank you for growing up with me. Thanks for taking part in so many adventures and new discoveries throughout our lives. Together we will share the dreams of the future. A special dedication to the soul of my beloved grandmother, all time invested in this work is the time that I will never share with any of my family members and friends anymore. Thanks to my homeland, my past history and all special people who entered our lives to enrich ourselves. Finally, this is a work is dedicated to all people around the world who are directly or indirectly affected by any form of cancer and which help us to learn more about life than death. To my family, I could never have been writing this today, without your love and support. Thank you. iii Table of Contents 1 General Introduction .................................................................................... 2 1.1 Colorectal Cancer ............................................................................................. 2 1.1.1 Colorectal Cancer Epidemiology ................................................................ 2 1.1.2 Stages of CRC ............................................................................................. 2 1.2 Treatments for CRC: Surgery, radiotherapy, and chemotherapy .............. 5 1.2.1 Surgery ........................................................................................................ 5 1.2.2 Radiotherapy ............................................................................................... 6 1.2.3 Chemotherapy ............................................................................................. 7 1.2.3.1 Common chemotherapeutics used in CRC ........................................ 10 1.2.3.1.1 5-FU................................................................................................ 10 1.2.3.1.2 OXA ............................................................................................... 11 1.2.3.1.3 IRI ................................................................................................... 11 1.3 A review of the known mechanisms of resistance in CRC chemotherapy 12 1.3.1 Known mechanisms of MDR .................................................................... 14 1.3.1.1 Mechanisms of MDR mediated by membrane transporters .............. 14 1.3.1.2 Mechanisms of MDR mediated by autophagy .................................. 17 1.3.1.3 Mechanisms of MDR mediated by polyploidy .................................. 17 1.3.1.4 Mechanisms of MDR mediated by cancer stem cells ........................ 17 1.4 Experimental approaches for identification of novel mechanisms of resistance in CRC ...................................................................................................... 18 1.4.1 Understanding of cancer resistance through genomics ............................. 18 1.4.2 Understanding genomics through proteomics .......................................... 20 1.4.3 Understanding of cancer resistance through proteomics .......................... 21 1.4.3.1 Mutational processes ......................................................................... 21 1.4.3.2 Transcriptional and Post-transcriptional processes ........................... 22 1.4.3.3 Protein alterations modelling chemotherapy resistance .................... 23 1.4.3.4 Proteomics approaches to identify mechanisms of resistance in cancer………….. ................................................................................................. 27 1.4.3.4.1 Proteomics separation approaches .................................................. 29 1.5 Mass spectrometry ......................................................................................... 30 1.5.1 Approaches in quantitative proteomics ..................................................... 33 iv 1.5.2 Multiple reaction monitoring (MRM) and Parallel reaction monitoring (PRM)…….............................................................................................................. 34 1.5.3 Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) .............. 35 1.5.4 Stable Isotope Labelling by Amino acids in Cell culture (SILAC) .......... 36 1.6 Aims & Objectives .......................................................................................... 41 2 Selection of CRC cell lines for use in resistance studies ................................ 43 2.1 Heterogeneity of CRC cell lines .................................................................... 43 2.2 Criteria for selection of a cell line for resistance studies ............................ 44 2.3 Material and Methods ..............................................................................