MASTER of SCIENCE (2014) Hamilton, Ontario (Biochemistry and Biomedical Sciences)
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NICOTINIC ACETYLCHOLINE RECEPTORS IN BREAST CANCER IDENTIFICATION OF NICOTINIC ACETYLCHOLINE RECEPTORS REQUIRED FOR BREAST TUMOURIGENESIS BY NATALLIA KASMACHOVA, B.Sc. A Thesis Submitted to the School of Graduate Studies in Partial Fulfillment of the Requirements for the Degree Master of Science McMaster University ©Copyright by Natallia Kasmachova, September 2014 MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences McMaster University MASTER OF SCIENCE (2014) Hamilton, Ontario (Biochemistry and Biomedical Sciences) TITLE: Identification of Nicotinic Acetylcholine Receptors Required for Breast Tumorogenisis AUTHOR: Natallia Kasmachova, B.Sc. (ISEU) SUPERVISOR: Dr. John A. Hassell NUMBER OF PAGES: xi, 71 ii MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences ABSTRACT Breast cancer continues to be the most common diagnosed cancer among women, and radiation or chemotherapy generally fails to provide durable cure, especially in the context of advanced or metastatic disease. Tumours recurrence is believed to be driven by cancer stem cells, which resist anti-cancer therapy and survive to seed relapse after remission in breast cancer patients. Small molecules inhibitors of nicotinic acetylcholine receptors (nAChR) target cancer stem cells, however, the precise nAChR required for breast cancer stem cell activity is unknown. Hence, we propose to test the capacity of shRNAs that target each individual nAChR to inhibit breast cancer stem cell activity. Briefly, we performed a cancer stem cell based pooled lenti-vector shRNA screen, to identify receptors required for the propagation of breast cancer stem cell enriched cultures. Our results demonstrate that the suppression of multiple receptors can be detected and corresponding genes are essential for TIC viability and survival. We anticipate our approach will identify the relevant nAChR receptor required for breast cancer stem cell activity. Such receptors may represent useful drug targets for the development of anti- breast cancer stem cell therapeutics. iii MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences ACKNOWLEDGMENTS It is my pleasure to express my sincere gratitude to the people who contributed to my academic research career and emotional being. I am expressing my special appreciation and thanks to my thesis supervisor Dr. John A. Hassell who introduced me the world of cancer stem cells and whose immense knowledge, enthusiasm and support has helped to make my research project exciting. He has provided me an outstanding opportunity to gain skills and knowledge as a scientific researcher. Without his expertise, motivation and guidance this thesis would not have been possible. A special gratitude I give to my committee members Drs. Kristin Hope and Andre Bedard for their time and consideration to reading my reports and providing brilliant comments and suggestions. I am thankful for their incredible contribution to my scientific progress. I wish to thank Bonnie Bojovic who has been a tremendous mentor to me during this long two-year journey. Her professional expertise, patience, and sense of humour made everyday of my lab work easy-going. To Adele Girgis-Gabardo, being a tissue culture guru, she showed me all tricks and tips to maintain sphere-cultures. I am indebted to many lab members for their interest and involvement into my project. I am especially thankful to Robin Hallett for assisting me in so many different ways, to Jennifer Seager for her tremendous organizational skills. I am grateful to Anna Dvorkin-Gheva, who has become my genuine friend, for her warm personality, amazing mind, and outstanding knowledge of bioinformatics. iv MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences Many thanks to Biochemistry Department, especially to Lisa Kush, who kindly provided all necessary academic information, helped and supported me from the very first day of enrolment. Special thanks to my friend and my soul mate Alex for providing a supportive environment for me. I cannot overstate his involvement and contribution to my emotional well-being. I am thankful for your wise words, sincere attitude and empathic nature. Your adventurous personality motivated and taught me to think outside of the box. I will be grateful forever for your love and support. Lastly, and most importantly, my dearest thanks go to my beloved family, my parents and my sister Anna. I am expressing my appreciation to you for being with me throughout the entire process, for sharing stressful situations and happy moments, for playing as a team, for sacrifices that have been made on my behalf. Words can never explain how truly grateful and proud I am to have you three in my life. To you I dedicate this thesis. v MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences TABLE OF CONTENTS 1 INTORDUCTION 1.1 Breast cancer overview ………………………………...……………... 12 1.2 Breast cancer types…………………………………………………….. 12 1.3 Breast cancer treatments……………………………………………….. 14 1.4 Molecular classification of breast cancer………………………………. 15 1.5 History of CSC and breast CSCs………………………………………. 16 1.6 Acetylcholine receptors………………………………………………… 18 1.7 nAChRs and CSC……………………………………………………..... 21 1.8 Experimental Rationale………………………………………………… 22 2 EXPERIMENTAL PROCEDURES 2.1 Cell culture…………………………………………………………… 23 2.2 Lentivius Production………………………………………………… 24 2.3 Lentiviral Infection………………………………………………….. 26 2.4 gDNA extraction…………………………………………………….. 26 2.5 gDNA PCR amplification…………………………………………… 28 2.6 Illumina primer design and high-throughput DNA sequencing…….. 29 2.7 Statistical methods………………………………………………….. 30 2.7.1 Data visualization…………………………………………………… 30 2.7.2 Data filtering………………………………………………………… 30 2.7.3 Examining changes in shRNA………………………………………. 31 3 RESULTS 3.1 shRNA dropout screen revealed multiple nAChRs that are essential for breast CSC viability and proliferation, and required for breast tumourogenisis……. 32 vi MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences 4 DISCUSSION 4.1 Role of nAChR with antiproliferative effect…………………………. 49 4.2 Role of nAChR co-regulation in inhibition of breast tumourgenisis… 54 4.3 Role of nAChR that promote breast cancer stem cell proliferation…. 55 4.4 Screen limitations……………………………………………………. 55 4.5 Conclusions………………………………………………………….. 57 5 APPENDIX 6 REFERENCES vii MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences LIST OF FIGURES Figure 1. Heterogeneity of nAChR …………………………… 20 Figure 2. Dropout viability screen experimental plan………… 33 Figure 3. Percentage distribution of single shRNA within the control samples (p0A, p0B, p0C) and tumours (T1, T2, T3, T4, T7, T8)............................... 35 Figure 4. Removing low readings from p0 data by using EM (Expectation Maximalization) algorithm……………………………………………. 37 Figure 5. Hierarchical clustering of the control samples (p0A, p0B, p0C) and tumours (T1, T2, T3, T4, T7, T8).............................. 38 Figure 6. Principal Coordinate Analysis (PCoA) 2D plots indicating similarities/differences between the control samples (p0A, p0B, p0C) and tumours (T1, T2, T3, T4, T7, T8)……………... 39 Figure 7. shRNA targeting β-galactasidase and luciferase…… 40 Figure 8. shRNA that were statistically significant different in the tumour samples compared to the control……………………………………….. 42 viii MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences LIST OF TABLES Table 1. Number of reads of single shRNA, presented in the control samples (p0A, p0B, p0C) and tumours (T1, T2, T3, T4, T7, T8)………............. 46 Supplementary Table 1. shRNA plasmids sequences ……………………. 58 Supplementary Table 2. Titers of shRNA-encoding lentiviruses ………… 60 Supplementary Table 3. Reverse and forward Illumina fusion primer templates ………………………………… 61 Supplementary Table 4. shRNA sequences found to be not presented in the control and in the tumour samples……….. 63 Supplementary Table 5. Pair-wise comparison performed for 79 sequences……………………………………… 63 ix MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences LIST OF ABBREVIATIONS cAMP Cyclic Adenosine Monophosphate CK Cytokeratin CREB cAMP Response Element-Binding Protein CSC Cancer Stem Cell ER Estrogen Receptor FDR False Discovery Rate gDNA Genomic DNA GRB Growth Factor Receptor-Bound Protein HER2 Human Epidermal Growth Factor Receptor 2 MAPK Mitogen-Activated Protein Kinases MOI Multiplicity of Infection nAChR Nicotinic Acetylcholine Receptor PR Progesteron Receptor TIC Tumour Initiating Cell TM Transmembrane TRC The RNAi Consortium x MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences CONTRIBUTION BY OTHERS I would like to thank Jason Moffat from Donnelly Centre for Cellular and Biomolecular Research at University of Toronto for providing the TRC library shRNA-sequences library. I would also like to thank Christine King from Francombe Metagenomics Facility at McMaster University for sequencing the samples and generating the data, Fiona Whelan from Dr. Surrette laboratory at McMaster University for processing the sequences data. I would also like to thank the following Hassell lab members who contributed to the data presented herein: Robin Hallett for accurate planning of the experiment, Bonnie Bojovic for her assistance with gDNA preparation and Anna Dvorkin-Gheva for the analysis of the sequences data. xi MSc Thesis – N. Kasmachova; McMaster – Biochemistry and Biomedical Sciences 1 INTRODUCTION 1.1 Breast cancer overview