View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by ScholarBank@NUS PATHWAY DETERMINANTS OF 5-FLUOROURACIL SENSITIVITY TAN WEN LEE B.Sc. (Pharm)(Hons.), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PATHOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS First of all, I wish to thank Prof. Teh Ming for giving me an opportunity to study in the Department of Pathology. I would like to express my heartfelt gratitude to my supervisor – A/Prof Richie Soong for giving me this opportunity to undertake this project. I am indebted to him for his invaluable understanding, guidance and support throughout this course. I would also like to thank Prof. Limsoon Wong and Mr. Difeng Dong from School of Computing, National University of Singapore, and Ms. Marie Loh from Oncology Research Institute (Singapore) for their invaluable advice and inputs for the statistical analyses; Dr. Ross Soo from Department of Haematology Oncology, National University Hospital (Singapore) for his expert opinions in clinical research; Dr. Iduna Fichtner from Max Delbrück Centre for Molecular Medicine, Berlin-Buch, (Germany) for the provision of tumor xenograft materials; Dr. Robert Hewitt, Dr. Rajeev Singh, Dr. Chon Boon Eng, Ms. Almeda and Ms Wentong Yang from NUH-NUS Tissue Repository (Singapore) for the provision of human tissue samples and clinical information and, Ms. Cecilia Lee from Institute of Molecular and Cell Biology (Singapore) for the provision of human cancer cell lines. Lastly, I would like to thank my family members, my hubby, Eng Lee and all my lab colleagues from Translational Interface, Cancer Science Institute (Singapore) for their companionships and their help in one way or another. 1 CONTENTS List of Tables ..................................................................................................................... 3 List of Figures .................................................................................................................... 4 ABSTRACT ....................................................................................................................... 5 CHAPTER 1: LITERATURE REVIEW ....................................................................... 6 1.1 Colorectal Cancer ............................................................................................... 6 1.2 5-fluorouracil ...................................................................................................... 7 1.2.1 Transport ....................................................................................................... 9 1.2.2 Metabolism ................................................................................................. 10 1.2.3 Mechanism of Action .................................................................................. 14 1.3 Fluorouracil-based therapies ............................................................................. 15 1. 4 Molecular determinants of 5FU sensitivity ...................................................... 18 1. 4.1 Candidate gene approach ............................................................................ 18 1.4.2 Genome-wide approach .............................................................................. 24 1.5 Study approaches .............................................................................................. 30 1.5.1 Quantification of mRNA expression using real-time PCR ......................... 31 1.5.2 Regulation patterns of genes in response to 5FU treatment ....................... 35 1.5.3 RNA interference (RNAi) ........................................................................... 35 1.6 Scope of Study .................................................................................................. 38 CHAPTER 2: MATERIALS AND METHODS .......................................................... 41 2.1 Cell lines ........................................................................................................... 41 2.2 Cell sensitivity analysis .................................................................................... 41 2.3 Xenografts ......................................................................................................... 42 2.4 Primary Tumors ................................................................................................ 42 2.5 Gene expression analysis .................................................................................. 42 2.6 Small interfering RNA (siRNA) transfections .................................................. 49 2.7 Statistical analysis ............................................................................................. 49 CHAPTER 3: RESULTS ............................................................................................... 52 3.1 Correlation between gene expression and 5FU sensitivity ............................... 52 3.2 Identification of baseline gene expression associated with 5FU sensitivity ..... 60 3.3 Identification of post-treatment gene expression changes associated with 5FU sensitivity ...................................................................................................................... 65 3.4 Modulation of 5FU sensitivity by CTPS2 siRNA ............................................ 69 CHAPTER 4: DISCUSSION ......................................................................................... 73 CONCLUSION ............................................................................................................... 79 REFERENCES ................................................................................................................ 80 APPENDIX ...................................................................................................................... 94 PUBLICATIONS ............................................................................................................ 95 2 List of Tables Table 1: Overview of studies on gene/protein expression levels in relation to 5FU chemosensitivity. Table 2: Summary of genome-wide and proteomic studies to identify determinants for 5FU-based chemosensitivities. Table 3: List of 96 genes selected for LDA analysis. Table 4: Characterization of 5FU sensitivity: (A) IC50 values of 21 colorectal cancer cell lines; (B) % of tumor shrinkage in 14 xenografts treated by 5FU; (C) Recurrence data of 25 stage II or III colorectal cancer patients treated with standard 5FU and leucovorin therapy. Table 5: Associations (χ2 test) between 5FU sensitivity and clinicopathological parameters Table 6: Candidate genes with differential gene expression (p<0.05) identified by unsupervised clustering analysis for xenografts and primary tumors. Table 7: Differential expression of genes in xenograft and patient series (p<0.05). 3 List of Figures Figure 1: Chemical structure of uracil and 5-fluorouracil (5FU). Figure 2: 5FU metabolism. Figure 3: Pathways of fluoropyrimdine and folate metabolism and downstream events. Figure 4: Activation of DNA repair mechanisms and stress response in relation to 5FU effects. Figure 5: Diagram illustrating the concepts for the TaqMan probe assay. Figure 6: TaqMan® LDA. Figure 7: The RNA Interference pathway. Figure 8: Genes analyzed in this study for their roles in transport and metabolism of 5FU and its downstream events. Figure 9: Unsupervised hierarchical clustering of samples based on the expression of 96 genes for (A) cell lines, (B) xenografts and (C) primary tumors. Figure 10: Difference in expression levels of CTPS2 and NME4 between clusters in (A) xenografts and (B) patients. Figure 11: Patterns of gene expression at 0, 4, 8, 12, 24 and 48 hours after 10 μM 5FU treatment for (A) RKO and (B) SW837. Figure 12: CTPS2 and GAPDH mRNA expression levels in SW620 cells transfected with (A) CTPS2 siRNA and (B) GAPDH siRNA 48 and 120 hours after transfection. Growth curves of cells either treated or untreated with 10 μM 5FU after 48 hours of transfection of (C) CTPS2 siRNA, (D) GAPDH siRNA and (E) negative control siRNA. 4 LITERATURE REVIEW ABSTRACT Purpose: Large inter-patient variability in patient response to the chemotherapeutic agent, 5-Fluorouracil (5FU), makes markers for predicting 5FU response highly desirable. We describe here a pathway-based gene expression analysis of the relative roles of all known components of 5FU transport, metabolism, its downstream effects and co-factor (folate) metabolism in determining 5FU sensitivity in cell lines, xenografts and tumours.Experimental design: The expression of 96 rationally-selected genes was measured by low-density array real-time PCR in 21 colorectal cancer cell lines, 14 xenografts and 25 tumors. Samples were grouped by unsupervised hierarchical clustering and the groups tested for associations with respective sensitivity, response and patient survival data. Patterns of expression changes of the 96 genes in 5FU sensitive and resistant cells following a time course of 5FU treatment were also clustered to identify the most defining expression changes, and their component genes. siRNA knockdown was performed to verify the influence of the top candidate on 5FU sensitivity in-vitro. Results: 5FU sensitivity associated with clustered groups for xenografts and tumours, but not cell lines. CTPS2 and NME4 were the genes common to the significantly differentially regulated genes in xenografts and tumors. CTPS2 was also in the group of genes with the most distinct expression change following 5FU treatment in