Genomic analysis of metallothionein expression in breast carcinogenesis

GENOMIC ANALYSIS OF METALLOTHIONEIN EXPRESSION IN BREAST CARCINOGENESIS Lai Yiyang [B.Sc.(Hons), NUS] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 I Acknowledgements My heartfelt gratitude goes especially to my supervisor, Professor Bay Boon Huat, Head of Department for Anatomy, for his guidance, mentoring and most importantly, his friendship. Under his stewardship, I have matured both as a researcher and as a person while his constant encouragement and support motivated me to complete this project successfully. His determination and dedication to research has become a role model for me to look up to and his resourcefulness has resulted in many of the fruitful collaborations with members of the scientific fraternity that has enriched and opened out new avenues for me to research on, thus making this learning journey extremely meaningful. Special thanks also go towards my co-supervisor, Associate Professor George Yip, for his patience in helping me resolve and overcome intriguing questions and experimental hiccups faced in my Doctor of Philosophy project. His wealth of knowledge and the challenges he throws at his students to exercise critical thinking has made this learning experience an enriching one. I also wish to thank Professor Ling Eng Ang, former Head of Department, and Associate Professor Samuel Tay, Deputy Head, for their continued support and stamp of approval that has made a lot of things possible during my candidature as a graduate student of the department. My great appreciation goes to Associate Professor Thomas Leung and his group, particularly, Ms Irene Lee, Ms Chia Shumei, Dr Ivan Tan and Mr Jeffery Yong for making my stay at the Institute of Molecular and Cell Biology a memorable one. My sincere gratitude to Associate Professor Tan Puay Hoon, Head of Pathology Department, and Dr Aye Aye Thike of Singapore General Hospital for the provision of breast cancer II tissue examined in study and consultancy advice on immunohistological evaluation conducted. I would take this opportunity to thank members from Professor Bay‟s and Associate Prosfessor Yip‟s groups, past and present, for their support and memorable friendships fostered during the last years. I would like to specially thank Drs Lim Daina, Koo Chuay Yeng and Guo Chun Hua and Yvonne Teng, for the technical assistance and advice rendered. I am also greatly indebted towards Ms Alice Zin and Esther Ng for their assistance in experimental procedures performed in this study. It has been a great pleasure working with Ms Jasmine Li, Mr Lo Soo Ling, Ms Yu Yingnan, Ms Grace Leung and Ms Chua Peijou. I also am extremely grateful to Ms Nicole Liu and Ms Jasmin Lim for coorganising many of the departmental events during my stay and the unforgettable lunches and afternoon tea sessions shared through the years. Special mention also goes to Mr Wong Yong Chiat for his companionship, technical assistance and advice rendered during our respective PhD candidatures. My sincere appreciation extends to Madam Bay Song Lin for her invaluable help to create many of the figures presented herein. My gratitude also goes out to all staff and members of Department of Anatomy, especially Mrs Yong Eng Siang, Mrs Ng Geok Lan, Ms Violet Teo, Ms Chan Yee Gek, Mrs Singh, Mr Kong Eng Chuan and Mr Poon Zhung Wei for their assistance to help resolve many non-scientific problems encoutered during my stay in the department. I would like to acknowledge the National University of Singapore for the provision of the Graduate Research Scholarship to pursue my PhD degree. III Finally, I am greatly indebted towards my family for their unfailing support and understanding during my course of study, particularly my wife who has been my pillar of support all these while. To her, I dedicate my thesis to. IV Table of Contents PAGE List of Publications i List of Figures ii List of Tables vi List of Abbreviations vii Summary Chapter . 1. Introduction . 1.1 Gross anatomy of the breast and its development . 1.2 Breast cancer . 1.2.1 Classification of breast disorders . 1.2.2 Non-invasive breast cancer . 1.2.3 Invasive ductal carcinoma . 1.2.4 Epidemiology of breast cancers 13 1.2.5 Risk factors of breast cancers . 14 1.2.6 Detection of breast cancer . 17 1.2.6.1 Mammography 17 1.2.6.2 Self and clinical examination . 17 1.2.6.3 Ultrasound . 18 1.2.6.4 Biopsy . 18 1.2.6.5 Positron emission tomography . 18 1.2.6.6 Magnetic resonance imaging 18 1.2.7 Treatments for breast cancers 19 1.2.7.1 Surgery . 19 1.2.7.2 Radiotherapy . 20 1.2.7.3 Chemotherapy . 20 1.2.7.4 Endocrine therapy 21 1.2.7.5 Biological therapy . 22 1.3 Metallothioneins 23 1.3.1 Biology of Metallothioneins (MTs) . 23 1.3.2 Structure of MT . 25 1.3.3 Metallothionein and cancers . 26 1.3.4 Expression of MT isoforms in breast tumours 27 1.3.5 Roles of MT isoforms in breast carcinogenesis . 29 1.3.6 Association of MT isoforms with pathological parameters and prognostication 33 1.4 Scope of Study . 34 V Chapter . 37 2. Materials and Methods . 38 2.1 Antibodies and Reagents 38 2.2 Cell Culture . 39 2.2.1 Maintenance of cell culture . 39 2.2.2 Cryopreservation of Cells 40 2.3 Manipulation of MT-2A gene expression 40 2.3.1 Down regulation of MT-2A gene using siRNA 40 2.3.2 Cloning of MT-2A and over-expression . 42 2.3.2.1 Transient over-expression of MT-2A 42 2.3.2.2 Stable transfection of MT-2A . 43 2.4 Silencing of FST gene 44 2.5 Quantitative Real-Time Polymerase Chain Reaction 45 2.5.1 Total RNA isolation . 45 2.5.2 First strand cDNA synthesis . 46 2.5.3 Real-time Ploymerase Chain Reaction . 47 2.5.4 Agarose gel electrophoreisis 49 2.5.5 Gene expression analysis of qRT-PCR data 50 2.6 Immunocytochemistry 50 2.7 Immunofluoroscence staining 51 2.8 Transmission Electron Microscopy . 52 2.9 Cell viability assay . 53 2.10 Growth curve analysis 54 2.11 Transwell migration assay . 54 2.12 Cell invasion assay 56 2.13 Genome wide expression analysis using GeneChip Microarray 57 2.13.1 Preparation of RNA starting material . 57 2.13.2 RNA quality check . 57 2.13.3 First strand cDNA synthesis . 58 2.13.4 Second-Strand cDNA Synthesis 59 2.13.5 Clean up of double stranded cDNA 60 2.13.6 Synthesis of Biotin-labelled cRNA 60 VI 2.13.7 Clean up of Biotin-Labelled cRNA . 61 2.13.8 Quantification of Biotin-labelled cRNA . 62 2.13.9 Fragmentation of cRNA 63 2.13.10 Target hybridization to Affymetrix GeneChip probe array 63 2.13.11 Washing and staining of probe array 64 2.13.12 Probe array scanning and quality assessment 65 2.13.13 GeneSpring-RMA Analysis . 67 2.13.14 GCRMA analysis . 67 2.13.15 PLIER analysis . 68 2.13.16 DAVID functional annotation . 68 2.14 Immuno-blot analysis . 69 2.14.1 Protein extraction . 69 2.14.2 Preparation of sodium dodecyl sulphate polyacrylamide gel 70 2.14.3 Sample preparation . 71 2.14.4 Protein separation on SDS-PAGE 71 2.14.5 Transfer of protein . 71 2.14.6 Western Blot . 72 2.14.7 Densitometric analysis of immuno-blot . 73 2.15 Statistical Analysis 74 2.16 Clinical patient samples 74 2.16.1 Tissue Scan Array 74 2.16.2 Immunohistochemical staining of invasive ductal carcinomas 74 2.16.3 Immunohistochemical staining of breast cancer tissues . 77 2.16.4 Quantitation of immunohistochemical staining 78 2.16.5 Statistical analysis 78 Chapter . 79 3. Results 80 3.1 Morphological features of breast cancer cells 80 3.2 Evaluation of MT isoforms in breast cancer cells 81 3.3 Immunocytochemistry of MT-1/2 expression in breast cancer cell lines . 83 3.4 Transient over-expression of MT-2A isoform in MCF-7 cells 85 3.5 Generation of a clone stably transfected MCF-7 cells over-expressing MT2A transgene 87 3.6 MT-2A over-expression increased MCF-7 cancer cell proliferation . 90 VII 3.7 Effects of MT-2A over-expression on cell motility . 93 3.8 Down regulation of MT-2A isoform via RNA interference . 95 3.9 MT-2A silenced cells show reduced protein expression via immunostaining . 98 3.10 MT-2A silencing decreases cell viability and proliferation . 100 3.11 Cell motility was affected in MCF-7 cells after MT-2A silencing . 102 3.12 Silencing of MT-2A gene induced the formation of cell-in-cell phenomenon . 105 3.13 Silencing MT-2A isoform did not dysregulate expression of autophagy related genes and ROCK . 107 3.14 Assessment of quality and integrity of RNA starting material for gene expression profiling . 110 3.15 GeneChip high density oligonucleotide microarray analysis and data mining . 112 3.16 Hierarchical clustering of microarray expression data . 115 3.17 Graphical summaries of cDNA microarray experiment . 117 3.18 Functional annotation of microarray expression data 119 3.19 Validation of microarray results by quantitative real-time PCR . 127 3.20 Effects of MT silencing in MT2AOE stable transfected cell line 131 3.21 MT-1/2 protein in MT over-expressing cells displayed concomitant down regulation after siMT2A_1 transfection . 133 3.22 Silencing of MT inhibited the growth advantage in MT2AOE cells 134 3.23 Assessment of cell motility in MT2AOE cells after MT silencing 136 3.24 Western blot analysis of FST protein expression after MT-2A manipulation . 137 3.25 Assessment of FST silencing in MCF-7 breast cancer cells 140 3.26 Silencing FST did not affect expression of MT and its associated genes . 141 VIII 3.27 Cell proliferation profile of MCF-7 was unaltered after FST silencing 142 3.28 Suppression of FST enhanced the migratory and invasive potential of breast cancer cells in vitro 144 3.29 FST silencing regulated gene expressions of bone morphogenetic protein and its receptor 146 3.30 Higher MT-2A mRNA expression is associated with the percentage of tumourigenic cells and cancer staging in breast cancer tissues 148 3.31 MT expression in breast cancer tissues . 150 3.32 Clinicopathological significance of MT expression in invasive ductal carcinoma . 153 Chapter . 156 4. Discussion . 157 4.1 MT expression enhances breast cancer cell proliferation . 159 4.2 MT expression potentiates cell motility and metastasis in breast cancers 169 4.3 Significance of cell-in-cell formation in adherent MCF-7 after MT-2A silencing 177 4.4 Clinico-pathological significance of MT expression in invasive ductal carcinoma . 180 Chapter . 185 5. Conclusion and future studies . 186 Chapter . 189 6. References 190 IX List of Publications Journals 1. Lai Y, Lim D, Tan PH, Leung T, Yip G, Bay BH. Silencing the Metallothionein 2A gene induces entosis in adherent MCF-7 breast cancer cells. Anat Rec (Hoboken) 2010 293: 1685-1691 2. Lai Y, Yip GW, Bay BH. Targeting metallothionein for prognosis and treatment of breast cancer. Recent Pat Anticancer Drug Discov (in press) 3. Yap X, Tan HY, Huang J, Lai Y, Yip GW, Tan PH, Bay BH. Over-expression of metallothionein predicts chemoresistance in breast cancer. J Pathol 2009 217: 563-570. Book Chapter 1. Lai Y, Yip GW, Tan PH, Kumar SD, Bay BH. Metallothionein and breast cancer. In: Zatta P, editor. Metallothioneins in biochemistry and pathology. New Jersey: World scientific; 2008. pp183-199. Meeting proceedings 1. Lai Y, Lim D, Yip GW, Tan PH, Bay BH. Silencing of the metallothionein gene induces entosis, a cell eats cell‟ phenomenon. In Proceedings of 18th Scientific Conference of Electron Microscopy Society of Malaysia, 15-17 December 2009, Selangor, Malaysia 2. Lai Y, Yip GW, Bay BH. Genomic analysis of breast cancer cells after Metallothionein-2A silencing. In Proceedings of the AACR 101st Annual Meeting. 17-21 April 2010, Washington DC, USA 3. Lai Y, Koo CY, Yip GW, Bay BH. Over-expression of Metallothionein-2A isoform increases the invasive potential of MCF-7 breast cancer cells in vitro. In Proceedings of International Anatomical Sciences and Cell Biology Conference, 26-29 May 2010, Singapore. 4. Hoe HM, Lai Y, Bay BH, Yip GW. 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J Environ Pathol Toxicol Oncol 19: 95-7 204 205 [...]... within the confines of their origins Commonly known as breast carcinoma in situ, two such forms of cancer exist in clinical setting, namely ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS) DCIS, believed to be the precursor of invasive breast cancers, contains a mass of rogue luminal epithelial cells proliferating into hollow of the milk ducts Cells of this pre-invasive form of breast. .. entry 26 3.1 Morphology of breast cancer cells 80 3.2 Quantitative expressions of MT-1 and MT-2 isoforms in various breast cancer cell lines 82 3.3 A typical melting curve analysis output 82 3.4 Expression profiles of functional MT isoforms in various lineages of breast cancer cells 83 3.5 Light micrographs of MT immunocytochemistry in breast cancer cells 84 3.6 Transfection of MCF-7 cells with PXJ40... Immunocytochemical analysis of MT-2A transient over -expression 87 3.8 Stable transfection of MT-2A isoform in MCF-7 88 3.9 Quantitative expressions of MT-1F and MT-1X isoforms in MT2AOE stable-transfected cells 89 Immunohistochemical profile of MT-1/2 staining in pIRES and MT2AOE stable transfected cell lines 90 Over -expression of MT-2A isoform enhanced cell proliferation in MCF-7 breast cancers 91... threatening disease Metallothioneins (MTs) belong to a superfamily of metal-binding proteins In human, four classes of MTs are represented by 10 function isoforms, viz MT-1A, 1B, 1E, 1F, 1G, 1H, 1X, 2A, 3 and 4 Averaging 7 kDa in size, a single mammalian polypeptide is capable of binding 7 divalent cations through the 20 cysteine residues present and forming thiolate clusters Inherently, MT binds to... Evaluation of MT-2A gene expression using cDNA from breast cancer patients 149 3.51 Categorization of MT intensity scoring in the nucleus 151 3.52 Categorization of MT intensity scoring in the cytoplasm 152 4.1 Postulated Pathways involving MT and its associated genes in the regulation of MCF-7 breast cancer cell proliferation 168 Schematic representation of the postulated pathways involving MT and... genes from microarray analysis 127 Validation of candidate genes from microarray analysis using cDNA from MT-2A over-expressing cells 129 Summary of fold changes of candidate genes as determined by real-time PCR 130 3.37 Evaluation of MT silencing with siMT2A_1 in MT2AOE cells 132 3.38 Immunohistological profile of MT-1/2 staining in MT2AOE cells after siMT2A_1 silencing 133 Reduction in cell proliferation... IBC Invasive breast cancers IDC Invasive ductal carcinoma IL1RAP Interleukin-1 receptor accessory protein IVT in vitro transcription JNK c-Jun N-terminal kinase Ki-67 Ki-67 nuclear antigen KLK12 Kallikrein-12 LCIS Lobular carcinoma in situ LEPC Luminal epithelial cells MAOB Monoamine oxidase B MAP1-LC3A Microtubule-associated proteins 1A/1B light chain 3A MAP1-LC3B Microtubule-associated proteins 1A/1B... 5-hydroxytryptamine Acetylcholinesterase American Joint Committee on Cancer Serine/Threonine protein kinase Alanine Ammonium persulphate Analysis of variance Autophagy related protein 5 Autophagy related protein 7 Autophagy related protein 12 Ataxia telangiectasia mutated Bcl-2 antagonist of cell death Restriction enzyme from Bacillus amyloliquefaciens Branched chain amino-acid transaminase 1, cytosolic... perturbed by FST silencing 141 3.45 Effect of FST silencing on growth of cancer cells in vitro 143 3.46 FST silencing did not affect MEK 5 protein expression 144 3.32 3.33 3.34 3.35 3.36 3.39 3.40 3.41 iv 3.47 FST silenced cells exhibited migratory behavior 145 3.48 FST silencing promoted invasiveness of MCF-7 cell in vitro 146 3.49 Gene expression profiles of BMPR-1B and BMP-7 in breast cancer cells... disease, enhancement of the predictive capability of clinical and pathological staging is necessary Hence, AJCC has recommended the grading of all invasive carcinomas to further delineate the subtypes that existed within invasive breast cancer category after staging (Thor, 2004) Histological grading estimates the extent of differentiation within the neoplastic establishments, looking out for microscopic . Metallothioneins 23 1.3.1 Biology of Metallothioneins (MTs) 23 1.3.2 Structure of MT 25 1.3.3 Metallothionein and cancers 26 1.3.4 Expression of MT isoforms in breast tumours 27 1.3.5 Roles of MT isoforms. Koo CY, Yip GW, Bay BH. Over -expression of Metallothionein- 2A isoform increases the invasive potential of MCF-7 breast cancer cells in vitro. In Proceedings of International Anatomical Sciences. Evaluation of MT isoforms in breast cancer cells 81 3.3 Immunocytochemistry of MT-1/2 expression in breast cancer cell lines 83 3.4 Transient over -expression of MT-2A isoform in MCF-7 cells