University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School July 2017 Role of Amylase in Ovarian Cancer Mai Mohamed University of South Florida, [email protected] Follow this and additional works at: http://scholarcommons.usf.edu/etd Part of the Pathology Commons Scholar Commons Citation Mohamed, Mai, "Role of Amylase in Ovarian Cancer" (2017). Graduate Theses and Dissertations. http://scholarcommons.usf.edu/etd/6907 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Role of Amylase in Ovarian Cancer by Mai Mohamed A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Pathology and Cell Biology Morsani College of Medicine University of South Florida Major Professor: Patricia Kruk, Ph.D. Paula C. Bickford, Ph.D. Meera Nanjundan, Ph.D. Marzenna Wiranowska, Ph.D. Lauri Wright, Ph.D. Date of Approval: June 29, 2017 Keywords: ovarian cancer, amylase, computational analyses, glycocalyx, cellular invasion Copyright © 2017, Mai Mohamed Dedication This dissertation is dedicated to my parents, Ahmed and Fatma, who have always stressed the importance of education, and, throughout my education, have been my strongest source of encouragement and support. They always believed in me and I am eternally grateful to them. I would also like to thank my brothers, Mohamed and Hussien, and my sister, Mariam. I would also like to thank my husband, Ahmed. You have all been a bottomless source of strength, encouragement, and love throughout my Ph.D. Thank you. I love you. I would like to thank Dr. Kruk. Your continued support and patience are the reasons I was able to get his far. I would like to thank Stephanie Buttermore, my lab mate, for keeping me sane on my craziest days. Acknowledgements It is a pleasure to thank all of those who made this dissertation possible. I want to sincerely thank Dr. Patricia Kruk for accepting me into her lab and for her guidance, support, and patience over the years. The lessons and skills I learned from her during my time her lab will be invaluable in my future endeavors. I would like to thank my committee members, Dr. Paula C. Bickford, Dr. Meera Nanjundan, Dr. Marzenna Wiranowska, and Dr. Lauri Wright for their time and valuable insight on my dissertation research. I would also like to thank Dr. Eric Bennett for welcoming me into the program, as well as the rest of the Medical Sciences program and Pathology and Cell Biology departmental staff who have provided support for me during my time at the University of South Florida. Finally, thank you to all of the Kruk Lab members, past and present. Special thanks to my current fellow lab member: Stephanie Buttermore for her insight and for always making my time at the lab such a fun and entertaining experience. Finally, I would like to thank my family and friends for encouraging me over the last four years. It has been a long road that I would not have been able to travel on my own. Table of Contents List of Tables v List of Figures vi List of Abbreviations viii Abstract xiii Chapter 1 Overview of Ovarian Cancer Biomarkers 1 Ovarian Cancer 1 Literature-derived OC protein biomarkers 3 Cytokines and growth factors 5 Interleukin-6 (IL-6) 5 Interleukin-8 (IL-8) 5 Interleukin-10 (IL-10) 6 Prolactin (PRL) 6 Transforming growth factor beta 1 (TGF-β1) 6 Tumor necrosis factor alpha (TNFα) 7 Vascular endothelial growth factor (VEGF) 7 Structural and extracellular matrix proteins 8 Transmembrane proteins 8 Claudin-3 and -4 8 Mucin 1 (MUC1) 9 Mucin 4 (MUC4) 9 Mucin 16 (CA125) 9 Structural and matrix-related proteins 10 Collagen (COL1A1 and COL11A1) 10 Human plasminogen activator inhibitor-1 (PAI-1) 10 Kallikrein (KLK-10,-11) 11 Matrix metalloproteinases (MMP-2 and -9) 12 Osteopontin (SPP1) 12 Stress induced phosphoprotein 1 (STIP1) 13 Metabolic regulators 13 Apolipoprotein E (ApoE) 13 Fatty acid synthase (FASN) 13 Receptors 14 Folate receptor-α (FLOR1) 14 Proteins of unknown function in ovarian cancer 15 i Human epididymis protein 4 (HE4) 15 Mesothelin (MSLN) 15 Prostasin (PRSS8) 16 Shared Computational Characteristics 19 Characteristics of secreted proteins 19 Characteristics of stable proteins 20 Identifying additional key regulators of ovarian cancer 24 Rationale 31 Central Hypothesis 31 Aim 1 32 Aim 2 32 Aim 3 32 Chapter 2 Computational Analysis of Amylase Isozymes Contributing to Ovarian Cancer 34 Introduction 34 History of the amylase genes 34 Evolution and expression of amylase genes 35 Amylase gene regulation 37 Differentiating amylase isozymes 38 Known amylase characteristics 39 Amylase in cancer 40 Objectives 41 Materials and methods 41 Sequences used in computational characterization 41 Amylase isozyme homology 41 Protein biochemical properties databases 41 Protein disorder 42 Secondary structure prediction 42 Posttranslational modification 43 Transcription regulation by promoter analysis 43 Mutational analysis of the amylase isozymes 43 Clinical specimens 44 Western blot 44 Results 45 Amylase isozymes are highly homologous 45 Amylase isozymes have similar molecular weights and isoelectric points 45 Amylases are highly ordered proteins with one predicted protein binding site 45 Amylase isozymes are hydrophilic 50 Amylase is predicted to be glycosylated and phosphorylated 50 The amylase isozymes have common domains, and structural features 51 Amylase isozymes have unique binding regions for chloride, calcium and glucose 52 The amylase isozymes have similar secondary and tertiary structure profiles 54 Multiple regions of amylase are prone to aggregation 55 ii The amylase isozymes functionally interact with metabolic proteins 56 Amylase isozyme expression may be driven by differential promotor activation 59 AMY2B overexpression is the most likely to be associated with amplification mutations in cancer 60 Clinical validation confirms elevated levels of AMY2B protein in OC 67 Discussion 69 Chapter 3 Amylase Promotes Ovarian Cancer Cell Invasion In Vitro 79 Introduction 79 Materials and methods 80 Tissue culture 80 Transmission Electron microscope (TEM) 80 Quantitative PCR 81 Western blot 82 Amylase ELISA 82 Amylase activity assay 83 Amylase transfection 83 Invasion assay 84 Glycosaminoglycan (GAG)/proteoglycan quantitative assay 84 Immunogold staining 84 Statistical analysis 85 Results 85 Confirmation of yeast-free cultures 85 OC cells overexpress amylase in vitro 87 Amylase secreted by OC cells is metabolically active 91 Inhibiting amylase decreases OC invasion 93 The gylcocalyx of OC cells is thicker than the glycocalyx of IOSE cells 94 Inhibiting amylase increases GAG production 98 Discussion 99 Chapter 4 Regulation of amylase by spirulina 103 Introduction 103 Materials and methods 104 Tissue culture 104 Microarray 105 Quantitative PCR 105 Western blot 106 Amylase ELISA 106 Invasion assay 107 MTS assay 107 Statistical analysis 107 Results 108 Amylase is a downstream target of spirulina 108 Spirulina downregulates amylase mRNA expression in OC cell lines 108 Spirulina downregulates amylase protein expression in OC cell lines 109 iii Spirulina reduces amylase secretion by OC cell lines 112 Spirulina decreases the invasive capacity of OC cells 113 Spirulina decreased migration of OVCAR5 cells 113 Spirulina does not alter OC proliferation 115 Phycocyanin abrogates amylase RNA expression 116 Spirulina-induced inhibition of OC invasion is driven, in part, by Phycocyanin 117 Discussion 118 Chapter 5 Concluding Remarks 121 Chapter 6 References 126 Appendix I Potential novel regulators or biomarkers of OC – 683 proteins in total of non-redundant, secreted, ordered and aggregation-prone human proteins. 176 iv List of Tables Table 1.1 Functional and clinical characteristics of OC biomarkers 4 Table 1.2 Computational characteristics of OC biomarkers 17 Table 1.3 Proteins that interact with literature reported ovarian cancer biomarkers 28 Table 2.1 Biochemical properties of amylase determined by computational analyses 47 Table 2.2 Secondary structural and posttranslational modifications among amylase isozymes 51 Table 2.3 Proteins that functionally interact with amylase isozymes 58 Table 2.4 Distinguishing computational characteristics among amylase isozymes 72 Table 3.1 AMY1 and AMY2B are typically overexpressed in cancer cells 91 Table 4.1 Spirulina transcriptionally targets amylase 108 v List of Figures Figure 1.1 OC biomarkers have functional interactions 25 Figure 1.2 Summarial schematic identifying additional protein regulators of OC 26 Figure 1.3 Identification of proteins that interact with literature-derived OC biomarkers 30 Figure 2.1 Schematic of mammalian amylase gene distinction 36 Figure 2.2 Amylase proteins are highly homologous 46 Figure 2.3 Amylase isozymes are ordered proteins 48 Figure 2.4 Amylase isozymes are hydrophilic 49 Figure 2.5 Amylase isozymes share structural features 53 Figure 2.6 AMY2A has a unique 3D structure 54 Figure 2.7 Amylase isozymes have multiple aggregation prone regions 55 Figure 2.8 Amylase isozymes form functional interactions with other metabolic enzymes 57 Figure 2.9 AMY2B has unique potential transcription factors 61 Figure 2.10 Mutational events are highest in AMY2B 62 Figure 2.11 AMY2B is altered in most cancer types 63 Figure 2.12 AMY2B is the most expressed amylase isozyme in OC 66 Figure 2.13