CHARACTERIZATION, EPIGENETIC DRUG EFFECT, AND GENE DELIVERY TO BREAST CANCER CELLS A Dissertation Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of Requirements for the Degree Doctor of Philosophy Shan Lu December, 2015 CHARACTERIZATION, EPIGENETIC DRUG EFFECT, AND GENE DELIVERY TO BREAST CANCER CELLS Shan Lu Dissertation Approved: Accepted: Advisor Department Chair Dr. Vinod Labhasetwar Dr. Stephen Weeks Committee Chair Dean of the College Dr. Coleen Pugh Dr. John Green Committee Member Dean of Graduate School Dr. Abraham Joy Dr. Chand Midha Committee Member Dr. Ali Dhinojwala Committee Member Dr. Anand Ramamurthi Committee Member Dr. Peter Niewiarowski ii ABSTRACT Cancer relapse is strongly associated with the presence of cancer stem cells (CSCs), which drive the development of metastasis and drug resistance. In human breast cancer, CSCs are identified by the CD44+/CD24- phenotype and characterized by drug resistance, high tumorigenicity and metastatic potential. In this study, I found that MCF-7/Adr cells that are breast cancer cells resistant to doxorubicin (Dox) uniformly displayed CSC surface markers, possessed CSC proteins, formed in vitro mammospheres, yet retained low migratory rate. They were also able to self-renew and differentiate under floating culture condition and are responsive to epigenetic drug treatment. High degree of DNA methylation (modifications of the cytosine residues of DNA) and histone deacetylation are major epigenetic landmarks of CSCs. In this work, I showed that MCF-7/Adr cells are sensitive to histone deacetylation inhibitor suberoylanilide hydroxamic acid (SAHA). Through RNA-sequencing technology, I also found that decitabine (DAC) and SAHA similarly affected a large number of the examined pathways, including drug and nanoparticle cellular uptake and transport, lipid metabolism, carcinogenesis and nuclear transport pathways. However, DAC significantly down-regulated several drug metabolism and drug resistant genes whereas SAHA showed no effect on them. iii siRNA therapy is the only approved genomic drug up to date; and is under clinical trials for cancer treatment. Cationic polymers are effective in complexing nucleotide through electrostatic interactions, forming polyplexes. Therefore, I further investigated the ability of a polyethyleneimine (PEI)-based cationic polymer, polyethylene glycol (Peg) modified L-arginine oligo (alkylaminosiloxane)-graft PEI ((P(SiDAAr)5Peg3), in delivering siRNA. The delivery system enabled effective siRNA delivery in vitro. In vivo in xenograft tumor model, polyplex showed insignificant reduction in bioluminanance signal following single-dose intravenous administration than other control groups but the data were not statistically significant, suggesting that further dose optimization of polyplex required to achieve a greater effect. In a drug resistant cell line, the delivery system also enabled anti-ABCB1 siRNA to effectively knockdown its target ABCB1 protein and enhance the Dox uptake as well as its therapeutic efficacy. Furthermore, I conducted DNA-siRNA co-delivery using P(SiDAAr)5Peg3, and obtained significantly enhanced transfection levels for both DNA and siRNA using the co-delivery approach. In summary, this study identified stem-like characteristics in drug resistant cell line MCF-7/Adr, showed that epigenetic drugs DAC and SAHA are effective in targeting dug resistant cells, investigated the transcriptomic effects of DAC and SAHA through RNA sequencing, and developed a siRNA and DNA-co-siRNA delivery system that effectively transfected both metastatic and drug resistant breast cancer cell lines in vitro. iv DEDICATION This dissertation is dedicated to my parents Li Wang and James Halpert, and my grandparents Guimin Wang and Fengxian Yang, for their support and unending encouragement. -Shan Lu v ACKNOWLEDGEMENTS I would like to express my gratitude to all those who have helped and supported me in my graduate work. First and foremost, I would like to thank my advisor Dr. Vinod Labhasetwar for his generosity in providing his lab for me to conduct my doctoral study and research. Furthermore, thank you especially for providing me the opportunity to conduct research in my area of interest, the freedom to independently construct my project, tackle scientific issues, formulate hypothesis and test them. Thank you for guiding me, helping me to revise manuscript drafts and providing strategic guidance. I appreciate your patience, knowledge and best practices you shared with me through the 5 years of your mentorship. Your investment in my professional development was instrumental and I would not have been where I am without you. Dr. Pugh Coleen, thank you for serving as my committee chair and for your endless support and guidance. I thank you for the times you invited me to your house for thanksgiving dinner even after I had left Akron, and the times you took me out for dinner and shared your life experience with me. I appreciate your sincerity, openness, and kindness. I will always remember the eggs I got from Mad. They were delicious. It’s an honor to have you as my committee chair. I also want to express my sincerest appreciation to my committee: Dr. Abraham Joy, Dr. Ali Dhinojwala, Dr. Anand Ramamurthi and Dr. Peter Niewiarowski. You are the vi most wonderful committee members I have ever known. Your willingness to help me with any issues, your patience and encouragement has helped me significantly throughout my graduate career. It’s a true privilege to have you all on my committee, and I’m grateful for all your contributions from the deepest part of my heart. I thank the entire Labhasetwar lab, past and present, for all their generous sharing of expertise and support in completing my graduate studies. They are the most supportive, kind and pleasant people I’ve ever worked with. I want to thank Melinda Stees, Samantha Cramer, Dr. Yue Gao, Dr. Youzhi Kuang, Dr. Vijay Raghavan, and Dr. Jun Yang. I also thank my former labmates: Dr. Chiranjeevi Peetla, Dr. Sivakumar Vijayraghavalu, Dr. Isaac M. Adjei, Dr. Viola Morris, Dr. Susan Foy, Dr. Blanka Sharma, and Dr. Jaffer Hayder for their help and feedback with various experiments. I also thank the cores and the staff at the Cleveland Clinic Foundation, specifically, I thank Mei Yin, Dr. Judith A. Drazba, Eric Diskin and Sage O'Bryant, for their assistance with my experiments. I appreciate the Biology Department and the Integrated Bioscience Program at the University of Akron for providing a supportive environment as well as the financial support and the opportunity afforded to carry out my graduate studies. I deeply value the knowledge I gained from the Department of Polymer Science faculty and staff members. vii Last but not the least, I want to thank my parents, James Halpert and Li Wang, and grandparents, Guimin Wang and Fengxian Yang, and other family members for supporting me through this significant time of my life. Mom, thank you for always being there for me whenever I needed you. Thank you for providing valuable insights, suggestions, and honest opinions on all the matters we discussed. I would not be who I am without you. Jim, it is a privilege to have you in my family, and thank you for your encouragement through text messages, emails, e-cards which have supported me through the good and bad times. I also thank all my friends, Yu Pei, Yan Xing, Jinjin Ma, Zhaosheng Gao, and others for sharing the precious moments of life with me. This study was funded by grant R01 CA149359 (to VL) from the National Cancer Institute of the National Institutes of Health. viii TABLE OF CONTENTS Page LIST OF FIGURES..........................................................................................................xv LIST OF SCHEMES......................................................................................................xviii LIST OF TABLES.........................................................................................................xix CHAPTER I. INTRODUCTION............................................................................................................1 1.1 Breast Cancer...........................................................................................................1 1.1.1 Clinical Hurdles for Breast Cancer Treatment................................................2 1.1.2 Drug Resistance..............................................................................................2 1.1.3 Metastasis........................................................................................................3 1.2 Mechanisms of Cancer Progression.........................................................................4 1.2.1 Genetic Mutations...........................................................................................4 1.2.2 Epigenetic Alterations.....................................................................................5 1.2.3 Epithelial-Mesenchymal Transition (EMT) and Cancer Stem Cells (CSCs).....................................................................................................................6 1.3 Treatment Options for Breast Cancer......................................................................7 1.3.1 Hormone Therapy...........................................................................................7 1.3.2 Chemotherapy.................................................................................................8 1.3.3 Epigenetic Drug Therapy................................................................................8