Epithelial mesenchymal transition in breast cancer progression

EPITHELIAL-MESENCHYMAL TRANSITION IN BREAST CANCER PROGRESSION KELLY LIM SWEE YING B Sc (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PATHOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2009 ACKNOWLEDGEMENTS I wish to express my heartfelt gratitude and sincere appreciation to my mentor and project supervisor, Dr Robert Hewitt, for his constant guidance and vital support throughout my graduate programme His appreciation for science and lateral thinking has challenged me to grow in the way I view and analyse scientific data Always seeing the best in me, he never accepted less than my best efforts I would also like to acknowledge and extend my gratitude to: Prof Jean Paul Thiery, one of the leading experts on the topic of epithelialmesenchymal transition, for taking time despite his busy schedule to critique my data and offer words of encouragement, which are invaluable to me Dr Rajeev Singh, for scoring the immunohistochemical stains Prof Thomas Putti, for setting aside time for me to consult him on difficult cases with regards to immunohistochemistry Dr Lim Yaw Chyn and members of her lab, especially Chi Kuen and Joe Thuan, for sharing their expertise in immunohistochemistry techniques with me My colleagues in Special Histology Lab and NUH-NUS Tissue Repository, for being helpful and supportive whenever the need arose My beloved husband, David, for being my pillar of strength and constant source of joy, comfort and motivation Lastly, my utmost thanks to God, who makes all things possible   i TABLE OF CONTENTS i Acknowledgements Summary vii List of Tables ix List of Figures x Abbreviations xii Introduction 1 1.1 Breast Cancer 1.1.1 Incidence of Breast Cancer 1.1.2 Etiology of Breast Cancer 1.1.3 Progression of Breast Cancer 1.2 Epithelial-Mesenchymal Transition (EMT) 1.2.1 EMT in Normal Development 11 1.2.2 EMT in Breast Cancer 12 1.2.3 Recent Findings Linking EMT with Basal-like Phenotype in Breast 15 Cancer and Cancer Stem Cells 1.2.4 Reconciling the EMT Paradox 18 1.2.5 Clinical Significance of Understanding EMT in Cancer 21 23 1.3 Techniques Employed 1.3.1 Laser Capture Microdissection (LCM) 23 1.3.2 RNA Amplification 24   ii Objectives 25 Materials and Methods 26 3.1 Materials 26 3.2 Methods 28 3.2.1 Clinical Specimens 28 3.2.2 Laser Capture Microdissection (LCM) 30 3.2.2.1 OCT Embeddation of Frozen Tissues for Sectioning 30 3.2.2.2 Hematoxylin Staining 30 3.2.2.3 Performing LCM 30 31 3.2.3 RNA Extraction 3.2.3.1 Minimisation of RNase and Genomic DNA Contamination 31 3.2.3.2 Isolation of Total RNA 31 3.2.3.3 Quantification and Quality Assessment of Purified Total 32 RNA from LCM Tissues 3.2.4 Amplification of mRNA from Purified Total RNA 33 3.2.4.1 Reverse Transcription to Synthesise First Strand cDNA 33 3.2.4.2 Second Strand cDNA Synthesis 34 3.2.4.3 cDNA Purification 34 3.2.4.4 In Vitro Transcription (IVT) to Synthesise aRNA 35 3.2.4.5 aRNA purification 35 3.2.4.6 Second Round Synthesis of First Strand cDNA 36 3.2.4.7 Second Round Synthesis of Second Strand cDNA 36   iii 3.2.5 Generation of Biotinylated cRNA for Hybridisation with Illumina® 37 Beadchip Arrays 3.2.5.1 cDNA Purification 37 3.2.5.2 IVT to Synthesise cRNA 38 3.2.5.3 Biotinylated cRNA purification 38 3.2.5.4 Quantification and Quality Assessment of Purified 39 Biotinylated cRNA 3.2.6 Illumina® Beadchip 39 3.2.6.1 Hybridisation of Samples onto Beadchips 39 3.2.6.2 Washing and Staining of Beadchips 40 3.2.6.3 Scanning and Data Acquisition from Beadchips using 41 Illumina® BeadStudio 3.2.7 Validation by Quantitative Real-time PCR (QRT-PCR) 42 3.2.7.1 cDNA Synthesis 42 3.2.7.2 QRT-PCR using ABI TaqMan® Gene Expression Assays 43 3.2.7.3 Analysis of QRT-PCR Results 44 3.2.8 Immunohistochemistry (IHC) 45 3.2.8.1 Deparaffinisation and Rehydration of Tissue Sections 45 3.2.8.2 Antigen Retrieval and Blocking of Endogenous Peroxidase 45 Activity 3.2.8.3 Immunostaining and Visualisation 46 3.2.8.4 Assessment of Staining and Scoring 48 3.2.8.5 Statistical Analysis 48   iv 49 Results and Discussion 4.1 LCM Results 49 4.2 Quantity and Quality of RNA Extracted from LCM 51 4.3 Gene Expression Results 53 4.3.1 Overview of Transcriptomic Changes at Different Phases of 53 Progression 4.3.2 Functional Grouping of Differentially Expressed Genes 55 4.3.3 Expression of EMT-related Genes at Different Phases of Progression 58 4.4 Validation of Microarray Data 79 4.5 Further Validation by Immunohistochemistry 82 4.5.1 N-cadherin 82 4.5.2 SPARC 94 4.5.3 Keratin 14 104 4.5.4 Maspin 115 126 General Discussion 5.1 Significance of Technique Employed 126 5.2 Chronology of EMT Events in Breast Cancer Progression 129 5.3 Networks of Molecules Involved 129 5.4 Future Work 132 133 Conclusion   v Bibliography 134 Appendices 147 8.1 Appendix A: General Chemicals 147 8.2 Appendix B: Buffers and Solutions 148   vi SUMMARY In recent years, the epithelial-mesenchymal transition (EMT), in which epithelial cells lose their epithelial characteristics and gain mesenchymal features, has emerged as a process crucial in cancer progression Specifically, EMT induces a phenotypic, molecular and behavioural change in carcinoma cells, permitting them to invade and metastasise In our study on breast cancer tissue using microarray analysis validated by quantitative realtime polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC), we attempted to investigate genes that are differentially expressed in breast cancer progression, and determine at which point EMT-related genes become up- or downregulated Overall, 65 EMT-related genes were identified; 36 at the normal to ductal carcinoma in situ (DCIS) transition and 29 at the DCIS to invasive ductal carcinoma (IDC) transition The results of our study lead us to propose that EMT is a multi-step process, as opposed to an all-or-nothing event Instead, changes in gene and protein expression related to EMT can and occur as early as the normal to DCIS transition, but further accumulate as the cancer progresses from DCIS to IDC, eventually culminating in phenotypic changes sufficient for cellular invasion and metastasis Beginning with subtle losses of epithelial markers such as claudins and cytokeratins in DCIS, followed by gain of mesenchymal markers such as N-cadherin, cadherin-11 and SPARC in IDC, tumour cells continually undergo EMT Other noteworthy EMT-related genes that were identified to be upregulated included genes involved in cell adhesion (lumican, decorin, versican), proteolysis (matrix metalloproteases, cathepsins), regulation   vii of transcription (twist, HOXB7, TCF4) and in signal transduction (PDGF receptor-β, cycloxygenase-1, S100A4 and BMP4) EMT-related genes that were downregulated included genes also involving cell adhesion (p120ctn, laminin-β-3, plakophilin 1), proteolysis (maspin), regulation of transcription (EGR1, EGR2) and in signal transduction (frizzled-related protein, PI3-kinase regulatory subunit and TGF-β receptor II) Following up our findings, we performed IHC on four candidate proteins and found that keratin 14 and maspin were significantly downregulated at the normal to DCIS transition (53% and 33% of cases, respectively), while N-cadherin and SPARC were significantly upregulated at the DCIS to IDC transition (27% and 73% of cases, respectively) Additionally, in terms of technique and approach, we were able to successfully combine laser capture microdissection, RNA amplification and microarray technologies to generate epithelial specific gene expression profiles of premalignant (normal mammary epithelia), pre-invasive (DCIS) and invasive (IDC) stages of breast cancer from discrete subpopulations of cells within histologically complex tumour specimens This approach is useful because it eliminates confounding factors such as tumour stroma and interpatient variability, making the results from the expression profiling more meaningful Already, much work has been done to show that EMT is associated with poorer prognosis and decreased survival Therefore, it is imperative that the chronology of EMT events and their trigger mechanism are elucidated because identification of new EMT inhibitors could hold the promise of novel cancer treatment options   viii LIST OF TABLES Table EMT Markers Table Clinical characteristics of cases used for IHC studies 29 Table ABI TaqMan® Gene Expression Assays and Endogenous Control 44 Table Details of HIER pre-treatment and primary antibodies used for IHC 47 Table EMT-related Genes Differentially Expressed at the Normal to DCIS transition 61 Table EMT-related Genes Differentially Expressed at the DCIS to IDC transition 68 Table Observed frequency of N-cadherin staining intensities in 30 cases of breast carcinoma 83 Table Correlation between N-cadherin positivity and clinicopathological variables in IDC of the breast 84 Table Observed frequency of SPARC staining intensities in 30 cases of breast carcinoma 95 Table 10 Observed frequency of SPARC staining intensities in the surrounding stromal cells in 30 cases of breast carcinoma 95 Table 11 Correlation between SPARC positivity and clinicopathological variables in invasive ductal carcinoma of the breast 96 Table 12 Observed frequency of keratin 14 staining intensities in 30 cases of breast carcinoma 105 Table 13 Correlation between keratin 14 positivity and clinicopathological variables in invasive ductal carcinoma of the breast 106 Table 14 Observed frequency of maspin staining intensities in 30 cases of breast carcinoma 116 Table 15 Correlation between maspin positivity and clinicopathological variables in invasive ductal carcinoma of the breast 117   ix Bibliography BIBLIOGRAPHY Garcia M, Jemal A, Ward EM, Center MM, Hao Y, Siegel RL, Thun MJ (2007) Global Cancer Facts & Figures American Cancer Society 1: 1-52 Seow A, Koh WP, Chia KS, Shi LM, Lee HP, Shanmugaratnam K (2004) Trends in Cancer Incidence in Singapore 1968 - 2002 Singapore Cancer Registry 6: 1-181 Hulka BS, 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protease inhibitor inhibits epithelial growth factor-induced epithelial-mesenchymal transition of esophageal carcinoma cells Cancer 115: 36-48 140 Liu T, Pemberton PA, Robertson AD (1999) Three-state unfolding and selfassociation of maspin, a tumor-suppressing serpin J Biol Chem 274: 2962829632      146 Appendices APPENDICES 8.1 APPENDIX A: GENERAL CHEMICALS First Base Phosphate Buffered Saline, pH 7.4 0.5 M EDTA, pH 8.0 J T Baker Tris-Base Merck Xylene Ethanol Gills III Hematoxylin Canada Balsam Sigma Sodium Azide Sinopharm Chemical Reagent Co Bovine Serum Albumin (BSA)   147 Appendices 8.2 APPENDIX B: BUFFERS AND SOLUTIONS Tris-EDTA Buffer, pH 9.0 (10X concentration) 6.05 g Tris-Base 10 ml EDTA, 0.5 M, pH 8.0 2.5 ml Tween-20 PBS, 0.1% BSA, 0.01% Sodium Azide 0.05 g sodium azide 0.5 g bovine serum albumin 50 ml PBS 3% H2O2-PBS ml 30% H2O2 ml PBS   148 ... the breast 99 Figure 25 Keratin 14 immunostaining in normal breast and skin 107 Figure 26 Keratin 14 immunostaining in DCIS of the breast 108 Figure 27 Keratin 14 immunostaining in IDC of the breast. .. keratin 14 overexpression in IDC 110 Figure 29 Maspin immunostaining in normal breast epithelia 118 Figure 30 Maspin immunostaining in DCIS of the breast 119 Figure 31 Maspin immunostaining in. .. xii Introduction 1 1.1 Breast Cancer 1.1.1 Incidence of Breast Cancer 1.1.2 Etiology of Breast Cancer 1.1.3 Progression of Breast Cancer 1.2 Epithelial- Mesenchymal Transition (EMT) 1.2.1 EMT in