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Understanding the Etiology of Inflammatory Breast UNDERSTANDING THE ETIOLOGY OF INFLAMMATORY BREAST CANCER by Lauren M. Shuman A thesis submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Master of Science in Biological Sciences Spring 2015 © 2015 Lauren M. Shuman All Rights Reserved ProQuest Number: 1596894 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. ProQuest 1596894 Published by ProQuest LLC (2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, MI 48106 - 1346 UNDERSTANDING THE ETIOLOGY OF INFLAMMATORY BREAST CANCER by Lauren M. Shuman Approved: ____________________________________________________ Kenneth L. van Golen, Ph.D. Professor in charge of thesis on behalf of the Advisory Committee Approved: ____________________________________________________ Robin W. Morgan, Ph.D. Chair of the Department of Biological Sciences Approved: ____________________________________________________ George H. Watson, Ph.D. Dean of the College of Arts and Sciences Approved: ____________________________________________________ James G. Richards, Ph.D. Vice Provost for Graduate and Professional Education ACKNOWLEDGMENTS First and foremost, I would like to thank Dr. Kenneth van Golen for a list of things, but namely for the opportunity to work in this lab that gave me my first experience in cancer research. When I first spoke with Dr. van Golen (or KvG, as I like to call him), I was just excited that I had landed a lab in cancer research, regardless of the type of cancer. Little did I know that I stumbled upon a research project that would impact me emotionally just as much as it did educationally. I've always loved science, but the loss of my grandfather to a rare form of bladder cancer is what directed me to cancer research amidst the abundance of scientific career options. As I learned more about inflammatory breast cancer, I realized I was in the right place. These women get little help simply because IBC is uncommon. As a result, many of them do not survive, a consequence my family knows all too well. I feel honored to have had a hand in helping these women. For this, I thank Dr. van Golen for the opportunity and for showing me first hand that there are people passionate about fighting for the lesser known, but highly fatal, diseases. I also thank him for his advice, support, encouragement, enthusiasm, and confidence, especially since I worry about everything. I thank my committee members Dr. Randy Duncan and Dr. Michelle Parent. They both have helped me with new ideas, staying focused, and most importantly to me, they and Dr. van Golen have overwhelmingly shown their iii support and confidence in me as a scientist. Starting graduate school, I had never thought that I would have a team of such advocating mentors behind me and my work. I have grown more confident in myself because of them. I thank Dr. Melinda Duncan for her advice and support with the challenges graduate school presented for me. I will always remember her advice and use it to keep going. I also thank Dr. Robin Morgan for offering her time and help to push me forward and share with me experience that motivates me to continue to grow as a researcher. I also owe thanks to all of the former lab members who have taught me so much and brought me from "how-do-I-do-this" to "I-got-this", namely Erica Dashner, Madhura Joglekar, and Matt Weiztman, as well as those who have always offered their time and help in the lab, Chris Andrews and Manal Moalwi. I certainly would not be where I am without my family. Thank you to my parents for their continuing support as I establish myself and my career. You have always been role models of hard work, selflessness, and understanding. I've always felt like I could do anything I wanted and that's what has made this possible. I thank my boyfriend, Mark, for being there for me, going out of your way to help, and somehow dealing with my rollercoaster of emotions behind the scenes. I don't know how you put up with me, but I (and the rest of the world) thank you for keeping me sane. With this, I am happy to present my hard work and research. iv TABLE OF CONTENTS LIST OF TABLES ............................................................................................ vii LIST OF FIGURES ......................................................................................... viii ABSTRACT ......................................................................................................xi Chapter 1 INTRODUCTION ................................................................................... 1 1.1 Breast Anatomy and Physiology .................................................... 1 1.2 Breast Cancer ................................................................................ 3 1.3 Inflammatory Breast Cancer .......................................................... 8 1.4 Infectious Agents and Cancer: Evidence for the Possible Involvement of an Infectious Agent in Inflammatory Breast Cancer ......................................................................................... 11 1.5 Toll-like Receptor 4 Expression and Polymorphisms ................... 15 2 EXPERIMENTAL METHODS .............................................................. 20 2.1 Cell Culture .................................................................................. 20 2.2 Immunohistochemical (IHC) Staining........................................... 21 2.3 Polymerase Chain Reactions (PCRs) .......................................... 22 2.4 Creating a TLR4 Knockout Cell Line Using TALENs ................... 25 2.5 Bacterial Culture from SUM149 Cells .......................................... 27 3 RESULTS ............................................................................................ 28 3.1 Toll-like Receptor 4 Expression ................................................... 28 3.2 Toll-like Receptor 4 Polymorphism Screening ............................. 30 3.3 16s rDNA and MMTV PCR Screening of Cell Lines .................... 33 3.4 Bacterial Culture from the IBC SUM149 Cell Line ....................... 36 4 DISCUSSION ...................................................................................... 38 4.1 Toll-like Receptor 4 Expression and Polymorphisms in Inflammatory Breast Cancer ........................................................ 38 4.2 Infectious Agents in Inflammatory Breast Cancer ........................ 43 REFERENCES ............................................................................................... 48 v Appendix A PCR CONTROL TEMPLATES ............................................................ 56 A.1 TLR4 D299G T399I polymorphic positive control template.......... 56 A.2 HBV Internal control template ...................................................... 56 A.3 MMTV positive control template .................................................. 57 B PERMISSIONS .................................................................................... 58 C IRBS AND CONSENT FORMS ........................................................... 65 vi LIST OF TABLES Table 1.1. Breast cancer stages categorized using the TNM system. Adapted from cancerstaging.org. 7 ................................................. 5 Table 1.2. Distribution of TLR4 polymorphic haplotypes and incidence of IBC in African, Caucasian, and Asian populations . Adapted from "TLR4 polymorphisms, infectious disease, and evolutionary pressure during migration of modern humans." 65 ................................................................................... 19 vii LIST OF FIGURES Figure 1.1. A diagram of the fully developed female breast. After puberty, the female breast contains a network of lobes, lobules, and ducts for the production of milk upon pregnancy. Credit: For the National Cancer Institute © 2011 Terese Winslow LLC, U.S. Govt. has certain rights. Reprinted from www.cancer.gov with permission. 4 ................................................................................... 2 Figure 1.2. Cross-section illustrating the basic cellular structure of breast tissue. Lobes are composed of luminal epithelial cells, either lobular or ductal, surrounded by basal cells of the basement membrane and myoepithelial cells that facilitate the ejection of milk from the lobules to the nipple. Reprinted from "Myoepithelial cells: good fences make good neighbors" with permission. 3 ................................................................................... 3 Figure 1.3. An illustration of the common presentation of a breast tumor. Breast cancer typically develops as a hard, painless lump in the breast. Adapted from http://www.abc.net.au/health/. 6 ...................................................... 4 Figure 1.4. Clinical symptoms of inflammatory breast cancer. Classic symptoms of IBC include redness, swelling, and dimpling of the breast. The severity of symptoms may vary from patient to patient. Left image adapted from http://www2.mdanderson.org/ cancerwise/. 27 Right image reprinted from "Inflammatory Breast Cancer: The Disease, the Biology, the Treatment."
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