FUNCTIONAL ANALYSIS OF METALLOTHIONEIN-2A ISOFORM IN BREAST CANCER LIM DAINA [B.Appl.Sc. (Hons.), NUS] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN FACULTY OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGMENTS ACKNOWLEDGEMENTS I will like to thank the people that has been involved and contributed to this project. First and foremost, I will like to express my heartfelt gratitude to my supervisor and head of department, Prof Bay Boon Huat, for providing me an opportunity to pursue my PhD degree in the Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore (NUS). His dedicated guidance, support and encouragement have played an important part in making this project a successful one. Next, I will like to thank my co-supervisor, Dr. Yip George Wai Cheong, for his helpful suggestions and guidance in this project. I am grateful to my friends, Ms Koo Chuay Yeng for her encouragements, invaluable discussions and help on the analysis of the breast cancer clinicopathological data; Ms Alice Zin Mar Lwin for support and technical assistance during my pregnancy; Mr Lai Yiyang, Ms Yu Yingnan, Ms Yvonne Teng Huifang, Ms Grace Leong Shuxian, Ms Li Yinghui and all the present and former members from Prof Bay Boon Huat’s and Dr. Yip George Wai Cheong’s groups for their valuable discussion and friendship. My special thanks go to Dr. Liu Yang for his contribution on the structural modelling of the MT-2A protein mutant. I will like to express my appreciation to collaborators in the Department of Microbiology, Prof Sim Tiow Suan and Dr. Maurice Chan for the technical assistance rendered in the construction of the MT-2A overexpression vectors. I will like to thank the Department of Pathology, Singapore General Hospital for providing the breast cancer tissues sections and breast cancer TMAs. In particular, I am grateful to our collaborator, Assoc Prof Tan Puay Hoon (Department of Pathology, SGH) for the collaboration in this project and i ACKNOWLEDGEMENTS her guidance on the morphology of various breast pathology specimens; and Dr. Aye (Department of Pathology, SGH) for her guidance and verification on the MT immunostaining in breast TMAs. I will like to express my gratitude to the staff, with special thanks to Ms June Koh, in the core facilities of the teaching laboratory in the Department of Microbiology (NUS) for the use of the ABI7000 real-time PCR machine; Department of Pharmacology (NUS) for the use of Tecan2000 absorbance plate reader with special appreciation to Mr Wong Yong Jie (Department of Pharmacology, NUS) for his help. My apologies to those whom I have not mentioned by name I am indebted to them in many ways they have helped me. I will like to pay tribute to my husband, son and my immediate family members whose immense love and tremendous support has made this work possible. Last but not least, I will like to show my appreciation to National University of Singapore for providing me the research scholarship to pursue my PhD degree. ii TABLE OF CONTENTS TABLE OF CONTENTS ACKNOWLEDGEMENTS . i SUMMARY vi PUBLICATIONS ix LIST OF ABBREVIATIONS . x LIST OF FIGURES xiii LIST OF TABLES . xvi CHAPTER INTRODUCTION . 1.1 Breast anatomy and development 1.2. Breast cancer . 1.2.1. Classification of breast lesions . 1.2.1.1. Benign breast disorders .5 1.2.1.2. Non-invasive breast cancer .6 1.2.1.3. Invasive breast cancer .7 1.2.2. Epidemiology of breast cancer 11 1.2.3. Breast cancer symptoms and treatment 13 1.2.3.1. Surgery 13 1.2.3.2. Radiotherapy .14 1.2.3.3. Chemotherapy .17 1.2.3.4. Hormonal therapy 18 1.2.3.5. Targeted therapies 20 1.2.4. Risk factors of breast cancer 20 1.3. Cancer progression . 22 1.3.1. Cell proliferation . 22 1.3.2. Cell migration and invasion . 24 1.3.3. Cell death . 25 1.4. Biomarkers in breast cancer . 30 1.5. Metallothionein . 31 1.5.1 Biology of Metallothionein 31 1.5.2 Structure of MT 32 1.5.3. Protective effects of MT 34 1.5.3.1 Oxidative stress 34 1.5.3.2 Radiation 35 1.5.3.3 Infections 35 1.5.3.4 Alkylating agents 36 1.5.4. MT isoforms . 37 1.5.5. MT and cancer . 38 ii TABLE OF CONTENTS 1.6. Gene silencing . 39 1.6.1. Overview of gene silencing . 39 1.6.2. Mechanism of RNA interference 40 1.6.3. RNAi as a tool for gene analysis . 42 1.7. Scope of study 44 CHAPTER MATERIALS AND METHODS . 46 2.1. Antibodies and reagents 47 2.2. Cell culture 47 2.2.1. Maintenance of cell lines . 47 2.2.2. Cryopreservation of cells 48 2.3. Downregulation using siRNA transfection 49 2.3.1. Optimization of siRNA transfection 49 2.3.2. Silencing in MCF-7 breast cancer cells . 49 2.4. Cloning and overexpression of MT-2A 50 2.5. Quantitative real-time PCR . 51 2.5.1 Extraction of total RNA . 51 2.5.2 cDNA synthesis . 52 2.5.3 Quantitative real-time PCR 52 2.6. Automated Flow Cell Sorting (FACS) 54 2.7. Immunocytochemistry . 55 2.8. Growth curve analysis . 55 2.9. Cell proliferation assay . 56 2.10. Cell adhesion assay . 56 2.11. Cell cycle analysis 57 2.12. Cell migration assay 58 2.13. Invasion assay . 59 2.14. Superarray analysis . 60 2.15. Immunoblot analysis . 62 2.15.1. Protein extraction 62 2.15.2 Preparation of protein sample 62 2.15.3. Preparation of resolving and stacking gel . 63 2.15.4. SDS-PAGE . 64 2.15.5. Transfer of proteins . 64 iii TABLE OF CONTENTS 2.16. Electron microscopy 65 2.16.1 Transmission electron microscopy 65 2.16.2. Scanning electron microscopy 66 2.17. Scanning transmission ion micro-tomography (STIM) 66 2.18. Statistical analysis . 66 2.19. MT staining in invasive ductal breast carcinoma tissues 67 2.19.1. Patients and tissues . 67 2.19.2. Tissue microarrays 67 2.19.3. Clinicopathological parameters of patients of invasive ductal carcinomas . 68 2.19.4. Immunohistochemical staining 71 2.19.5. Scoring system . 71 2.19.6. Statistical analysis . 72 CHAPTER RESULTS . 73 3.1. Morphology of breast cell lines . 74 3.2. Expression of MT in breast cell lines . 76 3.3. Transfection in MCF-7 . 78 3.4. Down-regulation of MT-2A 81 3.5. Morphological changes after down-regulation of MT-2A . 85 3.6. Specificity of MT isoforms 88 3.6.1. Down-regulation of MT-1F . 88 3.7. Effect of down-regulation of MT-2A on cell proliferation 90 3.8. Effect of down-regulation of MT-2A on cell adhesion of breast cancer cells . 94 3.9. Effect of down-regulation of MT-2A on migration of breast cancer cells 95 3.10. Invasion of MCF-7 cells . 98 3.11. Cloning and expression of MT-2A plasmids . 98 3.12. Overexpression of MT-2A in MCF-7 cells 100 3.13. Structure of MT-2A and MT-2A’ . 102 3.14. Effect of overexpression of MT-2A on cell proliferation . 103 3.15. Differential effects of overexpression of MT-2A and MT-2A’ on cell migration 105 3.16. Differential expression of cancer related genes . 107 3.17. Differential expression of cell cycle related genes 111 3.18. Differential expression of cell cycle related proteins 116 iv TABLE OF CONTENTS 3.19. Differential expression of metastasis related proteins 119 3.21. Expression of MT in invasive ductal breast carcinoma tissues . 121 3.22. Association of MT cytoplasmic staining with clinicopathological parameters . 126 CHAPTER DISCUSSION . 137 4.1. General discussion 138 4.2. MT-2A and cell proliferation in breast cancer cells . 139 4.2.1. General overview of cell proliferation . 139 4.2.2. Role of MT-2A in cell proliferation 141 4.2.2.1. In vitro studies .141 4.2.2.2. Clinicopathological associations of MT with cell proliferation 147 4.3. MT-2A and breast cancer metastasis 151 4.3.1. General overview of cancer metastasis 151 4.3.2. Role of MT-2A in cancer metastasis 152 4.3.2.1. In vitro assay .152 4.3.2.2. Associations of MT-2A with lymph node metastasis in breast cancer patients .155 CHAPTER CONCLUSIONS AND FUTURE STUDIES 156 5.1. Conclusions . 157 5.2. Future studies 159 References 161 v SUMMARY SUMMARY Breast cancer is one of the most prevalent cancers in women worldwide. The rate of breast cancer has been increasing rapidly. Invasive ductal carcinoma makes up the bulk of all breast cancer cases. There has been a surge in the research on biomarkers in breast cancer for better evaluation and management of the disease. Metallothionein (MT) is a cysteine rich, low molecular weight protein that has a protective function against heavy metal detoxification, oxidative stress, radiation, infections and alkylating agents. MT has also been found to be associated with cell proliferation and apoptosis which has lead to the implication of MT in carcinogenesis. MT expression has been shown to be associated with a higher grade, more aggressive breast cancer and poorer prognosis. Despite so, the exact mechanism by which MT has had an impact on breast cancer has not been elucidated. Furthermore, there are ten functional isoforms in human, with MT-2A being the most abundant isoform in breast cancer. The exact roles of the individual MT isoforms in cancer progression have yet to be identified. This study aims to examine the functional roles of the most abundant MT isoform, MT-2A, in breast cancer progression. The results in this study showed that MT-2A has an effect on cell proliferation, cell death and metastasis in breast cancer. The data demonstrated that down-regulation of MT-2A in breast cancer cells led to an increase in cell death and a decrease in cell proliferation (with a rise in the sub G1 and G1 phase of the cell-cycle), while overexpression of MT-2A led to a significant increase in the G2/M phase of the cell cycle. Analysis of the expression of several cancer pathway-related and cell cyclerelated genes has shown significant altered regulation of several genes at both mRNA and protein level. MT-2A is postulated to affect the cell-cycle and cell proliferation via the ATM/Chk2/Cdc25a pathway. The gene expression results also indicated that another possible pathway by which MT-2A regulates vi SUMMARY cell proliferation is via the cyclin G1 or cyclin C/cdk8 pathway. Down-regulation of MT-2A also affected cell death as there was an apoptotic peak at the sub-G1 phase and SEM pictures had shown an increase in apoptotic bodies. Gene analysis revealed that this effect may be mediated via an increase in Bax or Cyclin G1 expression. In addition, it was also observed that cells, in which MT was downregulated, exhibited signs of entosis (a cell-eat-cell phenomenon) under TEM. The data has shown for the first time that MT-2A has an effect on metastasis. Down-regulation of MT-2A resulted in a significant decrease in the number of cells migrating through the transwell membrane while overexpression of MT-2A produced an opposite effect. It was shown that this phenomenon was specific to MT-2A isoform alone as down-regulation of MT-1F isoform was shown to have no significant effect on cell migration. From the gene expression analysis results, it is posited that MT-2A expression influences cell migration via differential regulation of the uPAR protein, through cMet. The uPA system is involved in metastasis and angiogenesis with uPAR being the primary modulator of the system. Moreover, cloning of the MT-2A gene has led to identification of a naturally occurring deletion mutant of the MT-2A gene, MT-2A’. The deletion mutant was found to have amino acids lacking in the amino terminus. Through reconstruction of MT-2A’ protein structure, the folding of the amino terminus was found to be affected. The deletion mutant was shown to have no difference to the native protein with regard to cell-cycle progression but was found to have a lesser potency in cell migration. Perhaps, cell migration via MT-2A mediation is dependent on the number of divalent ions MT-2A carries. MT expression was also examined in breast cancer tissues in a TMA format using general MT antibodies which recognises all MT isoforms. MT staining was observed to be present in both the cytoplasm and nucleus of the tumour cells. MT was documented to be expressed in the cytoplasm of vii SUMMARY the cancer cells in 122 cases (87.1 %), with only 18 cases (12.9 %) exhibiting no staining in the cytoplasm of the tumour cells. The percentage of MT staining in the nucleus was found to be relatively similar to the cytoplasm with 118 cases (84.3 %) showing positive MT staining and only 22 cases (15.7 %) showing absence of MT staining in the nucleus. 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Adv Immunol 2004;84: 131-179. 189 [...]... develop into invasive cancer 1.2.1.3 Invasive breast cancer Invasive ductal breast cancer is the most common form of invasive breast cancer accounting for most of the invasive breast cancer This occurs when the cancerous cells that originate from the ductal cells infiltrate into the surrounding normal tissues of the duct Other forms of invasive breast cancer include lobular breast cancer, which as the... 1.2.3 Breast cancer symptoms and treatment Breast cancer in its early stage is usually painless The typical first symptom of breast cancer is a lump in the breast (Bullough 1980) However 90 % of breast lumps are benign and include cysts and fibroadenoma Tell-tale signs of breast cancer other than presence of lumps include thickening in an area of the breast, change in the size or shape of the breast, ... (2006) Upregulation of metallothionein isoforms in keloid keratinocytes Int J Mol Med (17)2: 385-389 4 Lim D., Chan M., Yip G.W., Sim T.S., Bay B.H Metallothionein- 2A is involved in proliferation in breast cancer Manuscript in preparation, 2009 5 Lim D., Yip G.W., Tan P.H., Chan M., Sim T.S., Bay B.H Metallothionein- 2A has a positive effect on breast cancer metastasis Manuscript in preparation, 2009... carcinoma in situ These include cytological and histopathological features in the lesions Proliferative lesions with atypical hyperplasia result in a moderately elevated risk for breast cancer (Rosens 2008) 1.2.1.2 Non-invasive breast cancer Non-invasive breast cancer is also known as breast cancer in situ There are two forms of such breast cancer: ductal cancer in situ (DCIS) or lobular cancer in situ... females of 26 % (178,480 cases) in the United States (Jemal et al 2007) In the United States, there was a total of 394,891 cases of invasive breast cancer and 59,837 cases of in situ breast cancer from 1975 to 2003 in women 40 years old and above in the nine oldest SEER cancer registry areas of the U.S (Jemal, et 11 INTRODUCTION al 2007) The rising incidence is reflected by the increased use of mammography... Down-regulation of MT -2A resulted in cell cycle G1 arrest 93 Figure 27 Down-regulation of MT -2A using siMT2A_1 and siMT2A_2 resulted in an increased adhesiveness to collagen 1 matrix when compared to siNegative 94 Figure 28 Down-regulation of MT -2A using siMT2A_1 and siMT2A_2 resulted in an increased adhesiveness to fibronectin matrix when compared to siNegative 95 Figure 29 Down-regulation of. .. colorectal cancer, which accounts for less than half of that in breast cancer (14.4 % of the total cancer cases) Breast cancer remains as the highest ranking cancer in the three major ethnic groups The age standardized rates were highest in the Chinese female, followed by the Malay and Indian females Breast cancer has the highest age standardized mortality rate in Singaporean females (Seow, et al 2004) 12 INTRODUCTION... of Cancer related genes using Cancer Pathway Finder to examine the genes induced and repressed after siMT2A_1 and siMT2A_2 treatment 107 Table 7 Tabulation of CT values of 6 housekeeping genes in Cancer PathwayFinder Superarray for normalization of ΔCT values for the various gene of interest in siMT2A_1 treated samples with respect to siNegative treated samples 110 Table 8 Tabulation of. .. Treatment of MCF-7 cells with siMT -2A induced a reduction in c-met 120 Figure 43 Negative MT immunostaining in both the cytoplasm and nucleus of the carcinoma 123 Figure 44 Categorization of MT intensity scoring for the cytoplasm and nucleus 124 Figure 45 IRS of cytoplasmic MT-staining 125 xiv LIST OF FIGURES Figure 46 IRS of nuclear MT-staining 125 Figure 47 Bar charts showing... siMT2A _1 and siMT2A_2 on the MT -2A gene 81 Figure 15 Manipulation of MT -2A expression in MCF-7 cells by silencing the MT -2A gene with siRNA 82 Figure 16 Immunocytochemistry with primary monoclonal anti-MT E9 antibody and DAB staining after treatment with (A) siMT2A_1, (B) siMT2A_2 and (C) siNegative 83 Figure 17 Expression of MT isoforms in siMT2A treated (siMT2A_1 and siMT2A_2) . FUNCTIONAL ANALYSIS OF METALLOTHIONEIN- 2A ISOFORM IN BREAST CANCER LIM DAINA [B.Appl.Sc. (Hons.), NUS] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF. SUMMARY Breast cancer is one of the most prevalent cancers in women worldwide. The rate of breast cancer has been increasing rapidly. Invasive ductal carcinoma makes up the bulk of all breast cancer. impact on breast cancer has not been elucidated. Furthermore, there are ten functional isoforms in human, with MT -2A being the most abundant isoform in breast cancer. The exact roles of the individual