MOLECULAR INSIGHTS INTO THE EFFECTS OF CANCER AND CHEMOTHERAPY ON THE BRAIN Anna Kovalchuk Bachelor of Science, University of Lethbridge, 2014 Master of Science, University of Lethbridge, 2015 A Thesis Submitted to the School of Graduate Studies of the University of Lethbridge in Partial Fulfilment of the Requirements for the Degree DOCTOR OF PHILOSOPHY Department of Neuroscience University of Lethbridge LETHBRIDGE, ALBERTA, CANADA © Anna Kovalchuk, 2017 MOLECULAR INSIGHTS INTO THE EFFECTS OF CANCER AND CHEMOTHERAPY ON THE BRAIN Anna Kovalchuk Date of Defence: June 12, 2017 Dr. Bryan Kolb Professor Ph.D. Supervisor Dr. Robbin Gibb Assoc. Professor Ph.D. Co-Supervisor Dr. Aru Narendran Professor M.D./Ph.D. Thesis Examination Committee Member Dr. James Thomas Professor Ph.D. Thesis Examination Committee Member Dr. Claudia Gonzalez Assoc. Professor Ph.D. Internal External Examiner Dr. Quentin J Pittman External Examiner Professor Ph.D. Dr. David Euston Assoc. Professor Ph.D. Chair, Thesis Examination Committee Member ABSTRACT An array of central nervous system complications, neurological deficits, and cognitive impairments come about and persist as a result of cancer and cancer treatments. These conditions are known as ‘tumor brain’ and ‘chemo brain’, respectively, and affect many cancer survivors. We proposed that the mechanisms behind tumor and chemo brain were epigenetic and associated with aberrant global gene expression and metabolome deregulation. We used mouse TumorGraft models of breast, lung, and pancreatic cancer, and sarcoma to profile the epigenetic, transcriptome, small RNAome, and metabolome changes in chemo-treated tumor-bearing mice, as compared to untreated tumor-bearing mice, and to control mice. We noted that tumor presence alone caused oxidative stress and affected global gene and small RNA expression, DNA methylation, and metabolic activity in the brain. Chemotherapy affected similar domains, at times seeming to build on tumor effects. The observed molecular changes strongly resembled those associated with neurodegenerative diseases and brain aging. iii ACKNOWLEDGEMENTS First and foremost, I would like to express my deepest appreciation and sincere gratitude to Professor Bryan Kolb for the opportunity to work on this project, the mentorship, support, and guidance along the way – and for your confidence in me. The experience and knowledge that I have gained as part of your group has given me the confidence and skills to pursue a career in medical research. This thesis would not have been competed without your help and encouragement. Thank you for helping me grow as both a researcher and a person. Thank you also to my co-supervisor, Dr. Robbin Gibb, for your warm encouragement, support, and guidance. Thank you for always being there for me and for all your help and advice along the way. It was a great pleasure to be able to participate in other research going on in the lab. I would like to thank my committee members, Dr. James Thomas and Dr. Aru Narendran, for their mentorship, constant support, insightful discussions, and valuable advice throughout my PhD program. I also extend my gratitude to our valuable collaborators, Drs. David Sidransky, Amanda Katz and Maria Mancini from the Champions Oncology for providing the TumorGraft mouse models, and Drs. Igor Pogribny, Svitlana Shpyleva and Stepan Melnyk from the National Center for Toxicological Research for their continuing collaboration, valuable input, and expertise. I am very grateful to my friends and colleagues from the CCBN and Hepler Hall for their collaboration, support, encouragement, and friendship. My thanks go to Rocio Rodriguez Juarez, Naomi Cramer, Slava Ilnytsky, Andrey Golubov, Dongping Li, Bo Wang, Rommy Rodriguez Juarez, Rachel Stark, Allonna Harker, Serena Jenkins, and iv Candace Burke for being there for me, answering questions (even the silly ones!), and for being wonderful people. My heartfelt thanks and love to my family, my parents Drs. Olga and Igor Kovalchuk, my grand-parents Drs. Viktor and Valentina Titov, my aunt and uncle Dr. Lyuba Titova and Dr. Artur Teymurazyan (and their two boys, Alexander and Gregory), who have given me their support every step of the way. You always encouraged me to believe in myself and to strive for excellence in everything I do, and gave me your unconditional love. I only wish my late great-grandmother, Lyubov Reznikova, was here to celebrate the completion of this important chapter in my life, as I am sure it would have made her happy. My appreciation goes to my best friends, Coral Rajchgot, Cleo Lenox, and Jacqueline Walz, who have been there for me through the good and the bad times and never failed to make me laugh when I needed it the most. I am thankful to the Alberta Innovates – Health Solutions, Vanier Canada Graduate Scholarship program, and the University of Lethbridge for financial support. I am also thankful to the Department of Neuroscience and the CCBN for being a great hub for graduate research. v TABLE OF CONTENTS ABSTRACT iii TABLE OF CONTENTS vi LIST OF TABLES ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii CHAPTER 1: Introduction ............................................................................................... 15 Malignant tumors – Facts and figures ........................................................................... 16 Breast cancer ................................................................................................................. 18 Other cancer types ......................................................................................................... 19 Lung cancer ............................................................................................................... 19 Pancreatic cancer ....................................................................................................... 20 Sarcoma ..................................................................................................................... 20 Cancer chemotherapy .................................................................................................... 20 Cancer survivorship ....................................................................................................... 25 Side effects of cancer chemotherapy ............................................................................. 25 Neurotoxic chemotherapy side effects – chemo brain .................................................. 26 Cancer-associated cognitive change – the tumor brain ................................................. 29 Mechanism of chemo brain and tumor brain ................................................................ 29 Epigenetics .................................................................................................................... 31 DNA methylation ...................................................................................................... 31 Histone modifications ................................................................................................ 35 Small RNAs ............................................................................................................... 36 Metabolomics ................................................................................................................ 38 The hippocampus and prefrontal cortex – main chemo brain targets ........................... 38 Hippocampus ............................................................................................................. 38 Prefrontal cortex ........................................................................................................ 40 Study Rationale ............................................................................................................. 41 Theory and Hypotheses ..................................................................................................... 44 Main findings .................................................................................................................... 46 CHAPTER 2: Chemo brain or tumor brain - that is the question: the presence of extracranial tumors profoundly affects molecular processes in the prefrontal cortex of TumorGraft mice .............................................................................................................. 49 Abstract ......................................................................................................................... 50 Introduction ................................................................................................................... 51 Results ........................................................................................................................... 54 vi Breast cancer growth affects global gene expression in prefrontal cortex tissue of tumor-bearing mice.................................................................................................... 54 Oxidative damage in the PFC tissues of tumor-bearing mice ................................... 56 Global DNA methylation and level of DNA methyltransferases and methyl-CpG- binding protein MeCP2 in the PFC tissues of tumor-bearing mice ........................... 57 Effects of chemotherapy treatments on the molecular processes in the PFC tissues of tumor-bearing animals ............................................................................................... 58 Discussion ..................................................................................................................... 59 Outlook .........................................................................................................................