EXPRESSION ANALYSIS AND FUNCTIONAL SIGNIFICANCE OF CHONDROITIN SULPHATE PROTEOGLYCANS AND HEPARAN SULPHATE PROTEOGLYCANS IN PROSTATE CANCER TENG HUI FANG, YVONNE (BSc, Hons) NATIONAL UNIVERSITY OF SINGAPORE A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF ANATOMY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements    ACKNOWLEDGEMENTS My PhD candidature would not have been easier, if not for the help, guidance and support from many mentors, family, friends and colleagues. My utmost gratitude goes to my mentor, Associate Professor George Yip Wai Cheong, for his unfailing guidance, patience and trust in me. Through him, I have learnt much, in terms of scientific skills and knowledge acquisition. In addition, I have benefitted from his many interesting stories that have also made my candidature a very enjoyable one indeed. The most important lesson that I have learnt from Dr George Yip would be the importance of thinking critically, a skill that is not only important in the scientific arena, but also one that applies to our everyday life. I would also like to thank my co-supervisor, Associate Professor Chia Sing Joo, for his support and advice. His comments have always added an interesting clinical perspective to the type of scientific research that I have been working on during my candidature. My deepest appreciation goes to Professor Bay Boon Huat for his timely advice and support. Professor Bay never fails to think for our welfare and has always shown genuine concern for us in all aspects of our life. He has taught us the importance of forming great relationships, one that supersedes any material wealth and status. Associate Professor Tan Puay Hoon has been a source of inspiration to me. Being one of the first female professors that I have personally met and known, she has demonstrated that with passion, hard work and determination, women can be leaders in their respective fields as well. I am grateful to her as she always takes time to help Page | i     Acknowledgements    and support me in my work despite a busy schedule; her boundless energy is amazing and can be infectious. I would also like to thank Dr Aye Aye Thike for spending much time to score the immunostained slides with me and for freely sharing her knowledge and expertise. To Dr Chong Kian Tai, thank you for your help in liasing with the collection of the prostate glands for the microarray study. Thank you for always trying so hard to ensure that the prostatic chips that we collect are ‘as fresh as possible’. This project would not have been possible without the excellent technical expertise offered by Ms Sii Lang Hiong (my immunohistochemical staining teacher), Ms Cheok Poh Yian and Ms Tan Yen. Thank you all for helping us cut all the prostate tissue sections and for compiling the clinicopathological data. Also to Mrs Yong Eng Siang, thank you for making the Cell and Developmental Biology Laboratory such a safe and clean place to work in. I also deeply appreciate the nice treats you have given me and the conversations that we had which makes my candidature at the Anatomy Department so much more enjoyable and memorable. Thank you, Mrs Ng Geok Lan, for your help and troubleshooting for problems that I had encountered in the Histology Laboratory. Also to Mr Poon Junwei, thank you for your help and expertise in the Tissue Culture Laboratory. My apologies to all of you if I had been messy or broken the rules at times! Many thanks to all my friends and colleagues whom I have worked with, either previously or presently: to Mr Gilbert Khor, my basic cell culture teacher and the first person to help orientate me around the laboratory when I first joined; all the Laboratory officers/Research Assistants, Ms Lee Xiao Hwee, Ms Choo Siew Hua, Ms Grace Leong, Ms Sim Wey Cheng, Ms Wendy Sim and Mr Nick Low, for Page | ii     Acknowledgements    helping keep things in order and in constant supply so that I can work without worries; my seniors, Dr Guo Chunhua and Dr Koo Chuay Yeng, for their help and advice; my fellow friends, Dr Omid Iravani, Ms Cao Shoufeng, Ms Chua Peijou, Mr Lo Soo Ling, Ms Sen Yin Ping, Mr Matthew Tan, Mr Koo Kah Chun, Ms Therese Kwan, Mr Alvin Low, Mr Jeffrey Lim, Mr Ong Han Kee, Mr Ho Soo Keng and Mr Ethan Khoo– it has really been fun and enjoyable learning together and working with all of you! I would also like to express my gratitude to all staff and students of the Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, for all the help, advices and stories you have willingly shared with me. I would also like to thank the National University of Singapore for giving me the Graduate Research Scholarship, without which, all my work would not have been made possible. Most importantly, I would like to thank my family for their unfailing support through the years. I dedicate this work to them for their love and patience all this while. Page | iii     Table of contents    TABLE OF CONTENTS Acknowledgements i Table of contents iv Summary xi List of Tables xiii List of Figures xvii List of Abbreviations xx List of Publications xxiii Chapter Introduction 1.1 Prostate gland and prostate cancer 1.1.1 The anatomy of the prostate gland 1.1.2 Functions of the prostate gland 1.1.3 The histology and histopathology of the prostate gland 1.1.3.1 Normal prostate histology 1.1.3.2 Histopathology of benign prostatic hyperplasia 1.1.3.3 Histopathology of prostatic intraepithelial neoplasia 1.1.3.4 Histopathology of prostate adenocarcinoma 1.1.4 Clinical symptoms and diagnosis of prostate cancer 1.1.5 Gleason grading of prostate cancer 1.1.6 Prognostic markers for prostate cancer 1.1.7 Treatment 10 1.1.8 Current challenges in prostate cancer 12 1.1.9 Genome-wide expression profiling of prostate cancer 12 Glycosaminoglycans (GAG) and proteoglycans (PG) – an 14 1.2 introduction 1.2.1 Structure 14 1.2.2 Biosynthesis of chondroitin sulphate/dermatan sulphate 19 and heparan sulphate 1.2.3 Enzymatic remodelling of chondroitin/dermatan sulphate 25 and heparan sulphate chains Page | iv     Table of contents    1.2.4 Functions of chondroitin/dermatan sulphate and heparan 26 sulphate and/or proteoglycans in cancer 1.2.4.1 Heparan sulphate in cancer biology 27 1.2.4.2 Chondroitin sulphate in cancer biology 31 1.3 Objectives of project 33 Chapter Materials and Methods 35 2.1 Genome-wide expression profiling of prostate 35 adenocarcinoma tissues 2.1.1 Collection of prostate adenocarcinoma tissues 35 2.1.2 Haematoxylin and eosin staining of prostate tissues 35 2.1.3 RNA isolation, yield and quality from prostate tissues 35 2.1.4 Microarray analysis 36 2.1.5 Analysis of microarray data 37 2.1.6 cDNA synthesis and quantitative real-time polymerase 37 chain reaction (qPCR) 2.2 Expression analysis of chondroitin sulphate and heparan 44 sulphate in prostate adenocarcinoma tissues using immunohistochemistry 2.2.1 Clinical samples and patient data collection 44 2.2.2 Immunohistochemical staining 44 2.2.3 Immunohistochemical evaluation 46 2.2.4 Statistical analysis 46 2.3 In vitro cell culture 47 2.3.1 Cell lines 47 2.3.2 Storage of cells 47 2.3.3 Antibodies used 48 2.3.4 RNA extraction, cDNA synthesis, real-time PCR of 48 prostate tumour cell lines 2.3.5 Silencing of prostate cell lines 54 2.3.6 Western blot 55 2.3.7 GAG extraction and quantification 56 2.3.8 Proliferation assay 56 Page | v     Table of contents    2.3.9 Migration assay 57 2.3.10 Invasion assay 57 2.3.11 Adhesion assay 58 2.3.12 Cell cycle assay 59 2.3.13 Cell apoptosis assay 59 2.3.14 Flow cytometry 59 2.3.15 Microarray analysis 60 Chapter Expression Profiling of Prostate Adenocarcinoma Tissues 62 3.1 Selection of tissues to be used for study 62 3.2 RNA quality of tissue samples used for Affymetrix GeneChip 62 microarrays 3.3 Quality control of microarray data 69 3.4 Differential expression of genes in prostate adenocarcinoma 71 3.5 Hierarchical clustering of microarray data 83 3.6 Principal component analysis (PCA) of microarray expression 83 data 3.7 Quantitative real-time polymerase chain reaction (qPCR) 86 verification of microarray results 3.8 Discussion 90 3.8.1 Aberrant expression of genes in prostate adenocarcinoma 90 3.8.2 Prostate cancer as a result of development gone wrong? 92 3.8.3 3.8.2.1 Dysregulation of Wnt signalling pathway 95 3.8.2.2 Dysregulation of homeobox genes 97 Prostate cancer – sustaining growth and motility while 98 evading death signals 3.8.4 Prostate cancer as a result of aberration of metabolic 98 processes? 3.8.5 Aberrant expression of GAGs and PGs genes in prostate 100 cancer 3.8.6 Conclusions 106 Page | vi     Table of contents    Chapter Expression and Functional Analysis of Chondroitin 107 Sulphate in Prostate Cancer 4.1 Expression analysis of chondroitin sulphate in prostate cancer 107 4.1.1 Clinical and demographic data 107 4.1.2 Expression analysis of non-sulphated chondroitin in 111 prostate adenocarcinoma 4.1.2.1 Localization of non-sulphated chondroitin in 111 prostatic tissues 4.1.2.2 Comparison of non-sulphated chondroitin expression 111 in benign prostatic and adenocarcinoma tissues 4.1.2.3 Comparison of non-sulphated chondroitin expression 117 in different grades of prostate adenocarcinoma tissues 4.1.3 Expression analysis of chondroitin-4-sulphate in prostate 120 adenocarcinoma 4.1.3.1 Localization of chondroitin-4-sulphate in prostate 120 tissues 4.1.3.2 Comparison of chondroitin-4-sulphate expression in 123 benign prostatic and adenocarcinoma tissues 4.1.3.3 Comparison of chondroitin-4-sulphate expression in 126 different grades of prostate adenocarcinoma tissues 4.1.4 Expression analysis of chondroitin-6-sulphate in prostate 129 adenocarcinoma. 4.1.4.1 Localization of chondroitin-6-sulphate in prostate 129 tissues 4.1.4.2 Comparison of chondroitin-6-sulphate expression in 129 benign prostatic and adenocarcinoma tissues 4.1.4.3 Comparison of chondroitin-6-sulphate expression in 134 different grades of prostate adenocarcinoma tissues 4.1.5 Expression analysis of chondroitin-2,6-sulphate in 137 prostate adenocarcinoma. 4.1.5.1 Localization of chondroitin-2,6-sulphate in prostate 137 tissues Page | vii     Table of contents    4.1.5.2 Comparison of chondroitin-2,6-sulphate expression 140 in benign prostatic and adenocarcinoma tissues 4.1.5.3 Comparison of chondroitin-2,6-sulphate expression 144 in different grades of prostate adenocarcinoma tissues 4.1.6 Comparisons of chondroitin sulphate staining amongst 147 each subtype 4.1.7 Association of chondroitin sulphate staining with 150 clinicopathological parameters 4.2 Gene transcript analysis of chondroitin-sulphate biosynthesis 157 and editing genes in prostate cancer cell lines 4.3 Functional analysis of CSGalNAcT-1 in prostate cancer cell, 161 PC-3 4.3.1 CSGalNAcT-1 is effectively silenced in PC-3 – gene 161 transcript and protein levels 4.3.2 Reduction of CS/DS chains after CSGalNAcT-1 siRNA 161 4.3.3 CSGalNAcT-1 silencing inhibited PC-3 proliferation 163 4.3.4 Apoptotic analysis of CSGalNAcT-1 silenced PC-3 cells 163 4.3.5 Cell cycle analysis of CSGalNAcT-1 silenced PC-3 cells 163 4.3.6 CSGalNAcT-1 silencing impeded PC-3 invasion 164 4.3.7 CSGalNAcT-1 silencing has no effect on PC-3 adhesion 164 to collagen type I 4.3.8 Microarray analysis of CSGalNAcT-1 silenced PC-3 cells 166 4.3.8.1 Quality control of microarray data 167 4.3.8.2 Differential expression of genes in CSGalNAcT-1 167 silenced PC-3 4.3.9 Cell cycle and apoptotic proteins were downregulated in 169 CSGalNAcT-1 silenced PC-3 cells 4.4 4.4.1 Discussion 177 Increased chondroitin sulphate expression and their potential 177 use as a biomarker of prostate cancer progression 4.4.2 Unraveling the functional significance of CSGalNAcT-1 179 upregulation in prostate cancer Page | viii     Table of contents    Chapter Expression and Functional Analysis of Heparan Sulphate 189 in Prostate Cancer 5.1 5.1.1 Expression analysis of heparan sulphate in prostate cancer 189 Localization of heparan sulphate in BPH and 189 adenocarcinoma tissues 5.1.2 Comparison of heparan sulphate expression in benign 189 prostatic and adenocarcinoma tissues 5.1.3 Comparison of heparan sulphate expression in different 190 grades of prostate adenocarcinoma tissues 5.1.4 Association of heparan sulphate staining with 197 clinicopathological parameters 5.2 Gene transcript analysis of heparan sulphate biosynthesis 206 genes in prostate cancer cell lines 5.3 Functional Analysis of HS3ST3A1 in prostate cancer 210 5.3.1 HS3ST3A1 is effectively silenced in RWPE-1 210 5.3.2 HS3ST3A1 silencing inhibited RWPE-1 proliferation 210 5.3.3 HS3ST3A1 silencing inhibited RWPE-1 migration 211 5.3.4 HS3ST3A1 silencing did not affect RWPE-1 adhesion to 211 collagen type I 5.4 Functional Analysis of HS6ST2 in prostate cancer 213 5.4.1 HS6ST2 is effectively silenced in RWPE-1 213 5.4.2 HS6ST2 silencing prevented the proliferation of RWPE-1 213 cells. 5.4.2 HS6ST2 silencing reduced the migratory ability of 214 RWPE-1 cells. 5.4.3 HS6ST2 silencing did not affect adhesion of RWPE-1 214 cells to collagen type I 5.5 5.5.1 Discussion Utility of 10E4-specific heparan sulphate as a prostate 216 216 cancer biomarker 5.5.2 HS3ST3A1 and HS6ST2 are not tumour suppressors in 217 prostate cancer. Page | ix     Appendix II   Section 3A Association of chondroitin-6-sulphate staining intensity with all clinicopathological parameters Stained tissue component Clinicopathological parameter Adenocarcinoma Total Weighted Average Intensity 8ng/ml 20 100.0% Total 41 20 95.2% 19 95.0% 4.8% 5.0% 1.000 Lymphovascular invasion Present 16 100.0% Absent 106 100.0% Total 122 16 100.0% 92 86.8% .0% 14 13.2% 0.211 Page | 306     Appendix II   Perineural invasion Seen 77 93.9% 36 80.0% 6.1% 20.0% 0.034 Circumferential resection margin Involved 32 30 100.0% 93.8% Uninvolved 40 39 100.0% 97.5% Total 72 6.3% 2.5% 0.581 Not seen Total Apex margin Involved Uninvolved Total Proximal margin Involved Uninvolved Total Clinical stage T1a to T1c T3a to T3b Total Lobe occurence Both Lobes Singles Lobe Total 82 100.0% 45 100.0% 127 34 100.0% 38 100.0% 72 31 91.2% 38 100.0% 8.8% .0% 0.100 100.0% 64 100.0% 73 88.9% 62 96.9% 11.1% 3.1% 0.330 34 100.0% 100.0% 42 32 94.1% 100.0% 5.9% .0% 1.000 61 100.0% 10 100.0% 71 59 96.7% 90.0% 3.3% 10.0% 0.370 32 94.1% 37 97.4% 5.9% 2.6% 0.599 Maximum tumour dimension [...]... Mean values of chondroitin- 2,6 -sulphate staining in prostate 143 adenocarcinoma samples versus paired BPH samples Table 4.13 Mean values of chondroitin- 2,6 -sulphate staining in different 146 grades of prostate adenocarcinoma tissues Table 4.14 Comparisons of chondroitin sulphate staining amongst each 148 subtype in the adenocarcinoma cells Table 4.15 Comparisons of chondroitin sulphate staining amongst... non-sulphated chondroitin in benign 114 hyperplastic prostate tissues and prostate adenocarcinoma tissues Table 4.3 Mean values of non-sulphated chondroitin staining in prostate 116 adenocarcinoma samples versus paired BPH samples Table 4.4 Mean values of non-sulphated chondroitin staining in different 119 grades of prostate adenocarcinoma tissues Table 4.5 Expression analysis of chondroitin- 4 -sulphate in benign... Comparisons of non-sulphated chondroitin expression in 118 different grades of prostate adenocarcinoma tissues Figure 4.4 Chondroitin- 4 -sulphate expression in BPH tissue and 121 prostate adenocarcinoma sections Figure 4.5 Comparisons of chondroitin- 4 -sulphate expression in 124 prostate adenocarcinoma samples against matched BPH samples Figure 4.6 Comparisons of chondroitin- 4 -sulphate expression in 127 different... tissues and prostate adenocarcinoma tissues Table 4.6 Mean values of chondroitin- 4 -sulphate staining in prostate 125 adenocarcinoma samples versus paired BPH samples Table 4.7 Mean values of chondroitin- 4 -sulphate staining in different 128 grades of prostate adenocarcinoma tissues Table 4.8 Expression analysis of chondroitin- 6 -sulphate in benign 131 hyperplastic prostate tissues and prostate adenocarcinoma... 4.9 Mean values of chondroitin- 6 -sulphate staining in prostate 133 adenocarcinoma samples versus paired BPH samples Page | xiv     List of tables    Table 4.10 Mean values of chondroitin- 6 -sulphate staining in different 136 grades of prostate adenocarcinoma tissues Table 4.11 Expression analysis of chondroitin- 2,6 -sulphate in benign 139 hyperplastic prostate tissues and prostate adenocarcinoma tissues... of prostate adenocarcinoma tissues Figure 4.7 Chondroitin- 6 -sulphate expression in BPH tissue and 130 Page | xvii     List of figures   prostate adenocarcinoma sections Figure 4.8 Comparisons of chondroitin- 6 -sulphate expression in 132 prostate adenocarcinoma samples against paired BPH samples Figure 4.9 Comparisons of chondroitin- 6 -sulphate expression in 135 different grades of prostate adenocarcinoma... 4.10 Chondroitin- 2,6 -sulphate expression in BPH and prostate 138 adenocarcinoma tissues Figure 4.11 Comparisons of chondroitin- 2,6 -sulphate expression in 142 prostate adenocarcinoma samples against paired BPH samples Figure 4.12 Comparisons of chondroitin- 2,6 -sulphate expression in 145 different grades of prostate adenocarcinoma tissues, Figure 4.13 CSGalNAcT-1 is effectively silenced and CS/DS 162 expression. .. and treatment plans for each patient In addition, understanding the complexities of this disease at a molecular level may aid in the identification and subsequent development of potential therapeutics Glycosaminoglycans and proteoglycans have been found to be important regulators of cellular growth and metastasis In this study, the expression of chondroitin sulphate (CS) and heparan sulphate (HS) in. .. George and Tan Puay Hoon (2006) Involvement of sulphated proteoglycans in the modulation of prostate cancer cells behaviour in Proceedings of the Singapore General Hospital 15th Annual Scientific Meeting, Singapore 4 Teng Hui-Fang Yvonne, Tan Puay-Hoon and Yip Wai-Cheong George (2007) Expression of chondroitin in prostate tumours and their association with several clinicopathological parameters of poor... proliferation of the prostatic ducts and acini PIN can be classified into low grade PIN (LGPIN) and high grade PIN (HGPIN), based on the cytologic characteristics of the cell [Petersen et al, 2009] Histologic features of LGPIN include intact basal cell layer, enlarged nuclei of variable sizes, normal or slightly increased chromatin content and presence of small or inconspicuous nucleoli In HGPIN, there . and heparan sulphate chains 25 Table of contents  Page|v  1.2.4 Functions of chondroitin/ dermatan sulphate and heparan sulphate and/ or proteoglycans in cancer 26 1.2.4.1 Heparan sulphate. Association of chondroitin sulphate staining with clinicopathological parameters 150 4.2 Gene transcript analysis of chondroitin- sulphate biosynthesis and editing genes in prostate cancer cell lines. upregulation in prostate cancer 179 Table of contents  Page|ix  Chapter 5 Expression and Functional Analysis of Heparan Sulphate in Prostate Cancer 189 5.1 Expression analysis of heparan sulphate