© 2016 Sumayah Abed Ahmed Al-Mahmood ALL RIGHTS RESERVED NANOTECHNOLOGY APPROACH FOR TARGETED TREATMENT OF TRIPLE NEGATIVE BREAST CANCER By SUMAYAH ABED AHMED AL-MAHMOOD A thesis submitted to the Graduate School-New Brunswick Rutgers, the State University of New Jersey In partial Fulfillment of the requirements For the degree of Master of Science Graduate Program in Pharmaceutical Sciences Written under the direction of Professor Tamara Minko And approved by ------------------------------------------------ -------------------------------------------------- -------------------------------------------------- New Brunswick, New Jersey May, 2016 ABSTRACT OF THE THESIS Nanotechnology Approach for Targeted Treatment of Triple Negative Breast Cancer By SUMAYAH ABED AHMED AL-MAHMOOD Thesis Director: Tamara Minko, PhD Breast cancer is one of the most devastating diseases worldwide. Triple negative breast cancer cells (TNBCs) are defined by the lack of progesterone receptor (PR), estrogen receptor (ER), and epidermal growth factor receptor 2 (EGFR2) expressions. TNBCs account for 10%- 20% of all breast carcinomas. The study is aimed at examining the efficacy of gefitinib and EGFR-targeted siRNA delivered by liposomes for treating triple negative breast cancer (TNBC). The experiments were carried out using two types of human breast cancer (BC) cell lines MCF-7 (estrogen positive BC, EPBC) and MDA- MB 231(TNBC). EGFR-targeted siRNA and gefitinib were delivered by cationic and neutral liposomes, respectively. A fluorescence microscope was used to study cellular internalization of labeled liposomes and siRNA. The expression of the targeted mRNA ii was performed using quantitative reverse transcription PCR. Finally, cytotoxicity of liposomal siRNA and gefitinib alone or in combination was measured using the modified MTT assay with appropriate controls. It was found that liposomes effectively delivered siRNA into both types of BC cells and suppressed the expression of targeted EGFR mRNA. However, formulations without gefitinib did not influence significantly on the viability of BC cells. Free drug demonstrated the ability to kill both types of cancer cells. Nevertheless, toxicity of gefitinib in TNBC was 2.5 times lower when compared with EPBC cells. The delivery of the drug by liposomes significantly enhanced its toxicity (1.2 and 2.5 times in EPBC and TNBC, respectively). The combination of liposomal siRNA and liposomal gefitinib demonstrated exceptionally high cytotoxicity when compared with the free drug (143 and 62 times higher in EPBC and TNBC, respectively). Suppression of EGFR mRNA effectively suppressed resistance of TNBC cells to gefitinib. The data obtained support the proposed approach and showed high potential of liposomal EGFR siRNA in combination with liposomal gefitinib in treatment of TNBC. iii DEDICATION To my wonderful parents Abid A. Al-Mahmood and Eehan A. Al-Sharefee To my lovely husband Jalal Nazar Abdulbaqi To my amazing kids Ibrahim, Mohammed, Abdullah To my brothers and sisters Asia, Aaeshah, Abdulrahman, Osama, Sara, Hamza To my amazing advisor Dr.Tamara Minko iv ACKNOWLEDGMENTS I would like to express my grateful and deepest appreciation to my Advisor Professor Tamara Minko for her continuous inspiration, mentoring and supporting me during my study. Also, I would like to thank my thesis committee members Professors Bozena Michniak-Kohnfor and Guofeng You for their generous time. I would like to thank my colleagues in Dr. Minko lab: Dr. Olga Garbuzenko, Dr. Natalia Pogrebnyak, Justin E. Sapiezynski and Andriy Kuzmov for their generous help and continuous discussions during my work in the lab. I would like to thank the office staff of the Department of Pharmaceutical Sciences at Rutgers University: Hui Pung, Marianne Shen, Fei Han and Sharana Taylor for always being helpful to complete my work. I am grateful to all the support from my parents and I hope that they become proud of me one day. Also, I would like to thank my brothers and sisters for their support during my life. Finally, this work cannot be completed without the unconditional love and support of my husband during my study. v TABLE OF CONTENTS ABSTRACT OF THE THESIS ................................................................................................... ii ACKNOWLEDGMENTS ............................................................................................................ v TABLE OF CONTENTS ............................................................................................................ vi LIST OF TABLES ........................................................................................................................ x LIST OF FIGURES ..................................................................................................................... xi 1 INTRODUCTION...................................................................................................................... 1 2 BACKGROUND AND SIGNIFICANCE ................................................................................ 2 2.1 Introduction ............................................................................................................... 2 2.2 Occurrence of metastasis breast cancer ..................................................................... 3 2.3 Predictive and prognostic factors of MBC ................................................................ 6 2.3.1 Axillary lymph nodal involvement ..................................................................... 6 2.3.2 Tumor size .......................................................................................................... 7 2.3.3 Estrogen receptor (ER) and progesterone receptor (PR) status .......................... 7 2.3.4 Circulating tumor cells (CTCs) .......................................................................... 8 2.3.5 Lymphovascular invasion (LVI) ........................................................................ 8 2.3.6 Age at diagnosis.................................................................................................. 9 2.3.7 Race and ethnicity .............................................................................................. 9 vi 2.3.8 Cathepsin D ........................................................................................................ 9 2.3.9 Angiogenesis Markers ...................................................................................... 10 2.3.10 Bone marrow micrometastasis ........................................................................ 10 2.3.11 Overexpression of the c-erb B-2 (HER2/neu) proto-oncogen ........................ 10 2.3.12 Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type I (PAI-1)............................................................................................................. 11 2.3.13 Mutation of p53 .............................................................................................. 11 2.3.14 Expression of topisomerase II-alpha (topo IIα) .............................................. 12 2.3.15 Proliferation markers ...................................................................................... 12 2.3.16 Gene expression profiling ............................................................................... 13 2.4 Models of breast cancer........................................................................................... 13 2.5 Treatments of metastasis breast cancer ................................................................... 15 2.5.1 Surgery ................................................................................................................. 16 2.5.2 Radiation .............................................................................................................. 16 2.5.3 Hormonal Therapy ............................................................................................... 17 2.5.3.1 Types of Hormonal Therapy .......................................................................... 17 2.5.3.2 Premenopausal Women with MBC ............................................................... 20 2.5.3.3 Postmenopausal Women with MBC.............................................................. 21 2.5.4 Chemotherapy ...................................................................................................... 21 vii 2.5.4.1. Common Chemotherapeutic Agents used in the Treatment of MBC ........... 22 2.5.5. Immune Therapy ................................................................................................. 26 2.5.6 Gene Therapy ....................................................................................................... 27 2.5.6.1 Targeting EGFR Family ................................................................................ 28 2.5.6.2 Targeting Metastasis and Invasion ................................................................ 34 2.5.6.3 Histone Deacetylase Inhibitors (HDACi) ...................................................... 35 2.5.6.4 Insulin-like Growth Factor Inhibitors (IGF-IR) ............................................ 35 2.5.6.5. Targeting Vascular Endothelial Growth Factor Family (VEGF) ................. 36 3 SPECIFIC AIMS ..................................................................................................................... 37 4 MATERIALS AND METHODS ............................................................................................ 37 4.1 Materials .................................................................................................................. 37 4.2 Cell Lines ................................................................................................................ 38 4.3 Liposome Preparation and Characterization ........................................................... 38 4.3.1 Liposome Preparation ....................................................................................... 38 4.3.2 Liposome Particle Size Measurement .............................................................. 40 4.3.3 Zeta Potential Measurement ............................................................................. 40 4.4 Cellular Internalization (Microscopic Technique) .................................................. 40 4.5 Cytotoxicity Assay .................................................................................................. 41 4.6 RNA Extraction ....................................................................................................... 41 viii 4.7 Gene Expression (Real-Time Quantitative Polymerase Chain Reaction) ............... 42 4.8 Statistical Analysis .................................................................................................. 42 5 RESULTS ................................................................................................................................. 43 5.1 Cellular Internalization of Liposomes ..................................................................... 43 5.2 EGFR mRNA Expression by Real-Time Quantitative Polymerase Chain Reaction (RT-QPCR) ................................................................................................................... 43 5.3 Cytotoxicity Assay .................................................................................................. 43 6 DISCUSSIONS ......................................................................................................................... 44 7 CONCLUSIONS ...................................................................................................................... 45 8 FUTURE DIRECTIONS ......................................................................................................... 45 9 ILLUSTRATIONS ................................................................................................................... 46 TABLES ........................................................................................................................ 46 FIGURES ...................................................................................................................... 48 ix