The Pattern of Expression of Receptor Tyrosine Kinases (RTK) EphB1, EphB2 and EphB4 in Colorectal Cancer Rashid Saeed Alrahbi Submitted for total fulfilment of the requirements of the degree of Master Department of Surgery North West Academic Centre (NWAC) University of Melbourne 2015 I Abstract Colorectal cancer (CRC) is a global health problem. It is the second leading cause of cancer related death worldwide and in Australia. Around 25% of CRC will experience recurrences postoperatively in the absence of lymph node metastasis and liver metastasis. We need to develop better diagnostic tools and more effective therapeutics. This will come through a thorough understanding of the molecular events in CRC progression. Erythropoietin producing hepatoma (Eph) receptors are the largest family of the receptor tyrosine kinases (RTK). They are divided into two subfamilies, A and B, Together with their cognate ligands, they play integral role in different body organs during embryo development and in adulthood. EphB receptors are considered as key coordinator of proliferation and migration of the intestinal stem cells (ISCs). They are Wnt signalling target genes. They are distributed in a complimentary pattern with their ligands, ephrinB1 and ephrinB2, along the intestinal crypt axis. Adenomatous polyposis coli gene mutation is considered as the initiating step in CRC development. It leads to Wnt signalling mutation and adenoma- carcinoma progression. Despite activation of Wnt signalling pathway, EphB receptors are inactivated during adenoma-carcinoma progression EphB1 and EphB2 receptors function as tumour suppressors in CRC, whereas for the EphB4 some studies found that EphB4 receptor is underexpressed whilst others found it overexpressed with the progression of the disease. It has also been suggested EphB4 expression is up-regulated as EphB2 expression is extinguished. Using immunohistochemistry we investigated pattern of expression of the three receptors together, EphB1, EphB2 and EphB4, across three stages of CRC, SII, SIII, SIV, in lymph node metastasis, and in liver metastasis, in a total population of 148 CRC patients. We found that: The three receptors are co-ordinately under-expressed in the primary CRC. The receptors are further under-expressed in the liver metastasis. II The EphB1 receptor is under-expressed in most undifferentiated CRC and in liver metastasis. The EphB4 receptors follow the same down-regulation pattern as the other two in both primary tumours and metastases There is no evidence that EphB2 –negative tumours express EphB4 The importance of this study of EphB receptors expression pattern in CRC and in liver metastasis is that it gives a general picture about the status of the three receptors together across different stages of CRC, in lymph node metastasis, and in liver metastasis of the same patients. Understanding the pattern of expression of three receptors together in CRC, highlights the significance of these receptors in CRC, and in long term revolutionizes current anticancer drug treatment of the disease. III Declaration This is to certify that: 1. The thesis comprises only my original work towards the Master except where indicated in the preface. 2. Due acknowledgment has been made in the text to all other material used. 3. This thesis is less than 40000 words in length, exclusive of tables, bibliographies and appendices. IV Preface Percentage contribution of the Author to total work for each data chapter is outlined below: Chapter 2 95% Chapter 3 95% Chapter 4 95% Chapter 5 100% Rashid Alrahbi V Acknowledgement Prophet Mohammed, peace be upon him, said, “ The one who does not acknowledge people, verily, he does not acknowledge God”. Foremost, I would like to give my sincere acknowledgement to all the people who were involved in this work. My deepest gratitude should be given to my supervisors: Dr. Paul Senior and Professor Steven Chan. I thank Paul for his genuine and persistent guidance throughout my master study. His enthusiasm and talented scientific mind has strengthened my research skills and sharpened my definite goal in this project. I would also like to express my special thankfulness to Professor Chan, without him this study does not exist today. I especially would like to thank him for his support during the most difficult moment I have experienced during this study. I am proud that he accepted to be my supervisor. I would also like to give my heartfelt appreciation to the pathologists: Dr. Chris Dow and Dr. Saurabh Prakash, from Western Health, for their volunteer to examine specimens under the microscope. They did not hesitate to provide me with the essential required skills to look at specimens microscopically. At the same level, I would like to thank Victorian Cancer BioBank (VCB) staff, particularly Ms. Leanne Taylor, VCB manager at Royal Melbourne Hospital, for her unremitting support to provide me with the required tissues for the project. It is hard to remember names as I have come across many people during my study, nevertheless, I will never forget Ms. Jill McGregor, personal assistant of Professor Chan, for her kindest help and support throughout my study. Last but not least, I have to deliver my deepest gratitude to my family. During this study, I passed with the most difficult experience in my life; I lost my father, the first and most influential teacher in my life, nevertheless, the great support and empathy I received from my family made it possible to complete this thesis. Thank God Almighty for granting me such great family. VI Publications VII Contents Page Chapter One 1 Introduction 1 1. Eph/ephrin signalling: a structural view 4 1.1 Introduction 4 1.2 Structures of Eph receptors 6 1.3 Structures of ephrin ligands 9 1.4 Structural differences between EphA and EphB receptors, ephrinA and ephrinB ligands, and the structural differences between EphA/ephrinA and EphB/ephrinB complexes 10 2. Eph-ephrin Bidirectional Signalling 11 2.1 Forward signaling 15 2.2 Reverse signaling 17 2.2.1 SH2/PDZ-dependent reverse signaling 19 2.3 Cross talk with other signaling system 19 2.4 Processing of Eph-ephrin complexes 22 3. Role of Eph/ephrin in the colon 23 3.1 Physiology of the colon 23 3.2 Histology of the colon 24 3.3 Intestinal stem cells (ISC) 25 3.4 Wnt signalling in normal cells 26 3.5 Eph/ephrin signalling is involved in maintaining intestinal integrity and homeostasis 28 VIII 4. Role of Eph/ephrins in cancer: tumorigenesis, deregulated cell positioning during tumor spreading, invasion and metastasis versus tumor suppression 32 5. Colorectal Cancer (CRC) 36 5.1 Pathogenesis of CRC 36 5.1.1 Environmental factors 36 a) Diet 36 b) Obesity 36 c) Alcohol consumption 36 5.1.2 CRC genetic factors 37 a) Familial Adenomatous Polyposis (FAP) 37 b) Hereditary Non Polyposis CRC (HNPCC) 38 c) Sporadic CRC 38 d) Adenoma-carcinoma sequence 39 e) Epigenetic mechanisms of CRC 40 5.2 Role of Eph/ephrin in CRC and some of their clinical implications in the cancer management 41 5.2.1 The role of EphAs in CRC 41 5.2.2 The role of EphBs and ephrinBs in CRC 43 a) EphB1 expression in CRC 43 b) EphB2 expression in CRC 43 c) EphB3 expression in CRC 49 d) EphB4 expression in CRC 49 e) EphB6 expression in CRC 51 f) EphrinB ligands expression in CRC 51 Concluding remarks 52 IX Chapter Two 54 Introduction 54 Research Hypotheses 55 Aims of the project 56 Ethics approval and ethics consideration 56 Inclusion and exclusion criteria 56 Design of the study 57 Overview 57 Immunohistochemistry 57 Common IHC methods 59 A. Avidin-Biotin Complex (ABC) based method 59 B. Polymer-Based Method 61 Antigen Retrieval (AR) Methods 62 A) Heat-induced epitope retrieval (HIER) methods 62 a) High pressure AR 63 b) Water bath heating 63 c) Hot plate heating: 64 B) Enzyme-induced epitope retrieval (EIER) method 64 Proteolytic Pre-treatment 64 Antibodies and their applications in IHC 65 IgG 65 IgM 66 Polyclonal Antibodies versus Monoclonal Antibodies 66 X Antibody-related factors which can affect the staining outcome 67 Antibody Titre 67 Antibody Dilution 67 Antibody Incubation Time 68 Antibody Incubation Temperature 68 Materials and Methods 68 Materials 68 Development of staining protocol for each receptor 70 Number of factors affect the sensitivity and specificity of IHC 70 EphB1 receptor 71 EphB1 receptor ABC based staining protocol 72 EphB2 receptor 73 EphB2 receptor staining protocol development using ABC method 73 EphB2 receptor Polymer based staining protocol 79 EphB4 receptor 80 EphB4 receptor Polymer based staining protocol 86 Technique 86 A) Overview 86 B) Procedure 89 Manual Staining of EphB2 and EphB4 Receptors 93 Clinicopathological Data Collection 95 XI A) Data Collection 95 B) Scoring System 98 a) The intensity of the receptor staining 98 b) The percentage of the positive cells stained 99 c) Degree of differentiation of the tumour 99 Statistical Analysis 100 Constituents of the solutions used in the staining protocol and their manufacturers 102 Chapter Three 104 Results 104 Introduction 104 EphB1, EphB2 and EphB4 receptors are positively expressed in the base of the intestinal crypt cells of the normal colonic epithelium and adenomatous tissues- adjacent to tumour tissues 107 EphB1 receptor expression in primary CRC and in liver metastasis 108 EphB1 receptor expression in CRC and liver m etastasis in relation to clinicopathological parameters 111 EphB2 receptor expression in primary CRC and in liver metastasis: first experiment 113 EphB2 receptor expression in primary CRC and in liver metastasis: second experiment