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Combating Resistance to Epidermal Growth Factor Recpetor Inhibitors in Triple Negative Breast Cancer Julie Marie Madden Wayne State University

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Combating Resistance to Epidermal Growth Factor Recpetor Inhibitors in Triple Negative Breast Cancer Julie Marie Madden Wayne State University Wayne State University Wayne State University Dissertations 1-1-2014 Combating Resistance To Epidermal Growth Factor Recpetor Inhibitors In Triple Negative Breast Cancer Julie Marie Madden Wayne State University, Follow this and additional works at: http://digitalcommons.wayne.edu/oa_dissertations Part of the Cell Biology Commons, and the Oncology Commons Recommended Citation Madden, Julie Marie, "Combating Resistance To Epidermal Growth Factor Recpetor Inhibitors In Triple Negative Breast Cancer" (2014). Wayne State University Dissertations. Paper 1017. This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. COMBATING RESISTANCE TO EPIDERMAL GROWTH FACTOR RECEPTOR INHIBITORS IN TRIPLE NEGATIVE BREAST CANCER by JULIE M MADDEN DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2014 MAJOR: CANCER BIOLOGY Approved by: ______________________________________ Advisor Date ______________________________________ Co-Advisor Date ______________________________________ ______________________________________ ______________________________________ DEDICATION Vires, Fides, Motum Ducit This work is dedicated to my unwavering parents. They never questioned when I wanted to stay in school forever and always encouraged me to follow my dreams no matter where they took me. They were always there to offer support when I decided to fly halfway across the world to study (three times) or travel hundreds of miles to see Oasis in concert or watch Man Utd play. Your trials with cancer led me into this field and your strength through it all drove me to help others fight and survive. To my Dad who taught me the importance of education and that science can be cool, your guidance and willingness to help when I didn’t understand something made me want to get a PhD because I wanted to be like Daddy and be a scientist. To my Mother who is the most caring and thoughtful person on the planet, thank you for always listening to my ramblings about lab while proclaiming, “that word sounds familiar but I don’t know what it means” and inserting commas copiously into all my papers. Without you both I would never be where I am now as an individual or a scientist. ii ACKNOWLEDGEMENTS First and foremost I have to thank my mentor, Dr. Julie Boerner for being the best mentor anyone could ask for. You made my experience in lab wonderful and your advice and guidance professionally and personally successfully led me through this stage in my life and education. It was a joy coming into lab every day. I must thank my co-mentor, Dr. Mattingly, for always giving good advice and pushing me to do my best. Thank you for listening to my talks and presentations before I gave them and making suggestions on how to improve. To Kelly Mueller who put up with my endless tormenting for the past four years. Just like a big sister you took it like a champ and made my life in lab so easy, always helping me with a protocol or running my Westerns down to the cold room. To my siblings who had to deal with me their entire lives and my siblings in law who will have to deal with me for the rest of it. To my friends who were always there to vent after a long day or hard test and made life enjoyable. Thank you for study parties, shopping trips, and ice creams runs. To the following people who have influenced and entertained my life tremendously and will never even know it: Manchester United, who entertained me endlessly for the past 20 years and is the sole entity that has brought the most emotion out of me. Special note to Scholes, Giggs, and Sir Alex, best day of my life when I saw you play Barcelona and I might still cry publically if anyone mentions your retirements. William Shakespeare who taught me, “And though she be but little, she is fierce”(Shakespeare). Oscar Wilde for his endless wit, “Education is an admirable thing, but it is well to remember from time to time that nothing that is worth knowing can be taught” (Wilde, 1894). Oasis because how could I not include the band I have stood hours in line for to be in the front row and chat about leaving Beatles Monopoly in airport security bins with and provided the soundtrack to my education? “Cos all of the stars have faded away/ Just try not to worry/ You'll see them someday/ Take what you need/ And be on your way/ And stop crying your heart out” (Oasis, 1996). iii TABLE OF CONTENTS Dedication ....................................................................................................................... ii Acknowledgements ....................................................................................................... iii List of Tables ............................................................................................................... viii List of Figures ................................................................................................................ ix List of Abbreviations ..................................................................................................... xi CHAPTER 1: Introduction .............................................................................................. 1 1.1 Breast Cancer: A Review ..................................................................................... 1 1.1.1 Stage and Grading ........................................................................................... 3 1.1.2 Triple Negative Breast Cancer ......................................................................... 5 Table 2: TNBC Subtypes. ........................................................................................... 8 1.2 Epidermal Growth Factor Receptor .................................................................. 13 1.2.1 EGFR as a Target in Cancer .......................................................................... 16 1.2.2 Mechanisms of Resistance ............................................................................ 21 1.3 PI3K/AKT/mTOR .................................................................................................. 23 1.4 Protein Translation ............................................................................................. 27 1.4.1 eIF4 ................................................................................................................ 31 1.4.2 Ribosomal S6 Kinases ................................................................................... 34 1.5 STAT3 ................................................................................................................... 36 CHAPTER 2: Exploring Resistance Pathways to EGFR Inhibitors .......................... 43 2.1 Hypothesis and Specific Aims ........................................................................... 43 CHAPTER 3: Materials and Methods .......................................................................... 45 3.1 Human Breast Cancer Cell Lines and inhibitors .............................................. 45 3.2 Phospho Mass Spectrometry ............................................................................ 45 3.3. Ingenuity® Pathways Analysis ......................................................................... 46 iv 3.4 Cell Viability Assays ........................................................................................... 46 3.5 Cell Growth Analysis .......................................................................................... 47 3.6 Clonogenic Survival Assays .............................................................................. 47 3.7 Immunoblotting ................................................................................................... 48 3.8 siRNA silencing ................................................................................................... 48 3.9 Bicistronic luciferase Assay .............................................................................. 49 3.10 Stat3c plasmid ................................................................................................... 49 3.11 STAT3 DNA Binding ELISA .............................................................................. 50 CHAPTER 4: Identification of proteins remaining phosphorylated after gefitinib treatment in TNBC ................................................................... 52 4.1 Introduction ......................................................................................................... 52 4.2 Results ................................................................................................................. 53 4.2.1 Summary of proteins remaining phosphorylated in the presence of gefitinib . 53 4.2.2 mTOR signaling remains activated in the presence of EGFR inhibitors in TNBC ............................................................................................................... 54 4.3 Conclusions ........................................................................................................ 54 CHAPTER 5: EGFR and mTOR Inhibitor Synergy in TNBC ...................................... 58 5.1 Introduction ......................................................................................................... 58 5.2 Results ................................................................................................................. 58 5.2.1 Inhibiting
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