Role of Cyclooxygenase-2 and Fatty Acid Synthase in Breast Cancer Development
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ROLE OF CYCLOOXYGENASE-2 AND FATTY ACID SYNTHASE IN BREAST CANCER DEVELOPMENT by Suying Lu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Nutritional sciences University of Toronto © Copyright by Suying Lu 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-39890-6 Our file Notre reference ISBN: 978-0-494-39890-6 NOTICE: AVIS: The author has granted a non L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Canada ROLE OF CYCLOOXYGENASE-2 AND FATTY ACID SYNTHASE IN BREAST CANCER DEVELOPMENT Doctor of Philosophy, 2008 Suying Lu Graduate Department of Nutritional Sciences University of Toronto Abstract Cyclooxygenase-2 (COX-2) and fatty acid synthase (FAS) are over-expressed in breast cancer. Inhibitors of COX-2 and FAS decrease proliferation and induce apoptosis in breast cancer cells. A previous study showed that the COX-2 inhibitor celecoxib not only inhibited rat mammary carcinogenesis, but also decreased fat deposition in rats fed a high fat diet, suggesting that celecoxib may affect lipid metabolism. We demonstrated that celecoxib suppresses FAS expression, and decreases fat accumulation by down-regulating c-Jun N- terminal kinase 1 (JNK1) in rats fed a high fat diet rich in n-6 polyunsaturated fatty acids (PUFAs). This finding suggests that not only inhibition of COX-2 but also down-regulation of FAS contribute to the chemopreventive effect of celecoxib. This observation led us to investigate the roles of both COX-2 and FAS in breast cancer development. We showed that COX-2 over-expression in breast epithelial cells inhibits proliferation, detachment-induced apoptosis and differentiation, and causes epithelial to mesenchymal transition (EMT). This finding suggests that COX-2 over-expression disturbs the homeostatic status of mammary epithelial cells, leading to partial transformation of these cells, and predisposing the mammary gland to tumorigenesis. Malignant transformation of breast epithelial cells is associated with elevated expression of FAS. We showed that inhibition of FAS by triclosan suppresses rat mammary tumorigenesis induced by N-methyl-N-nitrosourea (MNU). This finding suggests that FAS is a promising molecular target for breast cancer prevention. In cancers, de novo fatty ii acid synthesis is functionally linked to cell proliferation by providing fatty acids for the biosynthesis of cellular membranes, but the molecular link between these two processes was not known. We demonstrated that Spl coordinately regulates the expression of FAS, a key enzyme of fatty acid synthesis, and CDC25A, a key cell cycle protein, in estrogen-sensitive breast cancer cells. Thus, Spl is a molecular link between de novo lipogenesis and proliferation in these cells. Overall, findings of this thesis contribute to our knowledge relating to how COX- 2 and FAS act to facilitate breast cancer development, and provide evidence that these enzymes could be potential targets for breast cancer prevention. in Acknowledgements Writing this part of the thesis is a humbling experience, which reminds me that many people have helped me to achieve this work. Without their help, this thesis would be impossible. First of all, I would like to thank my supervisor, Dr. Michael Archer, for accepting me into his lab, for his guidance and support over the years and for providing an excellent environment to explore my love of science. I would also like to thank the other members of my advisory committee, Dr. Young-In Kim and Dr. Dittakavi Sarma, for their time, guidance and support. My special thanks go out to Dr. Wendy Ward and Dr. Kelley Meckling, who served as my external examiners and provided many helpful comments. I learned a great deal from many other colleagues, past and present, either through practical help with lab work, advice on data interpretation, or suggestions for seminar presentations. This alphabetical list is undoubtedly incomplete, and I apologize for those I have forgotten: Jian Min Chen, Robin Duncan, Kafi Ealey, Ahmed El-Sohemy, James Korkola, Dominic Lau, George Saati, Runlan Song, Geoffrey Wood, Wanli Xuan, Guo Yu and Yonghong Zhu. I am very grateful to my husband, Xiaodong and my son, Alex for their understanding, patience, support and love. Permissions to include published papers in this thesis were kindly granted by COPYRIGHT.COM (Chapter 2 and Chapter 4) and JOHN WILEY & SONS INC (Chapter 3). Funding for this work was provided by the Natural Sciences and Engineering Research Council (NSERC) and Canadian Breast Cancer Research Alliance (CBCRA). iv Table of Contents Page Chapter One - Introduction and Literature review 1 1.1 Introduction 2 1.2Cyclooxygenase-2(COX-2) 5 1.2.1 The COX pathway 5 1.2.2 Functions of prostanoids 5 1.2.3 Nonsteroidal Anti-inflammatory Drugs (NSAIDs) 6 1.2.4 COX-2 and breast cancer 7 1.2.5 COX-2-independent mechanisms for NSAIDs 15 1.3 Fatty acid synthase (FAS) 17 1.3.1 The fatty acid synthesis pathway 17 1.3.2 Inhibitors of the fatty acid synthesis pathway 17 1.3.3 FAS in normal tissues 19 1.3.4 FAS and breast cancer 20 1.4 Hypothesis and organization of thesis 28 v Chapter Two - Celecoxib decreases FAS Expression via Down-regulation of JNK1 30 2.1 Abstract 31 2.2 Introduction 31 2.3 Materials and methods 33 2.4 Results 37 2.5 Discussion 46 Chapter Three - COX-2 Over-expression Causes Partial Transformation in MCF-10F Human Breast Epithelial Cells 53 3.1 Abstract 54 3.2 Introduction 54 3.3 Materials and methods 56 3.4 Results 59 3.5 Discussion 64 Chapter Four - FAS is a Potential Target for the Chemoprevention of Breast cancer 71 4.1 Abstract 72 4.2 Introduction 72 4.3 Materials and methods 73 4.4 Results 75 4.5 Discussion 79 vi Chapter Five - Coordinate Regulation of FAS and CDC25A Phosphatase in Human Breast Cancer Cells by Spl: a Molecular Link Between de novo Lipogenesis and Cell Proliferation 83 5.1 Abstract 84 5.2 Introduction 85 5.3 Materials and methods 86 5.4 Results 90 5.5 Discussion 96 Chapter Six - General Discussion 103 6.1 Overview 104 6.2 Future directions 110 References 113 vii List of Abbreviations AA arachidonic acid ACC acetyl-coenzyme A carboxylase ACL ATP citrate lyase ACO acyl-CoA oxidase ChREBP carbohydrate response element-binding protein COX cyclooxygenase COXIBs a family of COX-2 inhibitors CPTI carnitine palmitoyl transferase-I CREB cyclic AMP response element binding protein DCIS ductal carcinoma in situ DMBA 7,12-dimethylbenz[a]anthracene E2 17p-estradiol EMT epithelial to mesenchymal transition ER estrogen receptor Erkl/2 extracellular signal-regulated kinase 1/2 FAS fatty acid synthase FFA free fatty acids GAPDH glyceraldehyde phosphate dehydrogenase y-GT y-glutamyl transferase HSL hormone sensitive lipase IDPs intraductal proliferations JNK c-Jun N-terminal kinase viii LA linoleic acid LPL lipoprotein lipase LXR liver X receptor MAPK mitogen-activated protein kinase MI myocardial infarction MMTV mouse mammary tumor virus MNU N-methyl-N-nitrosourea NF-KB nuclear factor-kappaB NSAIDs nonsteroidal anti-inflammatory drugs PCNA proliferating cell nuclear antigen PDK1 3-phosphoinositide-dependent protein kinase-1 PGs prostaglandins PhIP 2-amino-1 -methyl-6-phenylimidazopyridine PI3K phosphoinositide 3-kinase PKA protein kinase A PKB protein kinase B PPAR peroxisome proliferator-activated receptor PR progesterone receptorreceptor PUFAs polyunsaturated fatty acids RXR retinoid X receptor SREBP sterol response element binding protein TXA2 thromboxane A2 USF upstream stimulatory factor VEGF vascular endothelial growth factor x List of Tables Page Table 2.1 Body, liver and intra-abdominal adipose tissue weights and liver triglyceride levels in rats fed a high fat diet with or without 1500 ppm celecoxib for 15 weeks 39 Table 2.2 Serum levels of y-glutamyl transferase (y-GT), insulin, triglycerides and free fatty acids (FFA) in rats fed a high fat diet with or without 1500 ppm celecoxib for 15 weeks 40 XI List of Figures Page Figure 1.1. COX signaling pathway and associated biologic activities 8 Figure 1.2. Fatty acid synthesis pathway 18 Figure 1.3. The as-acting elements and trans-acting factors involved in the regulation of transcription of the FAS gene 21 Figure 2.1.