(RTK) Ephb1, Ephb2 and Ephb4 in Colorectal Cancer

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

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.

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.

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

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.

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

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

Antibody-related factors which can affect the staining outcome

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

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

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 117

EphB2 receptor expression in primary CRC and liver metastasis in relation to the clinicopathological parameters 119

EphB4 receptor expression in primary CRC and in liver metastasis 122

XII

Relationship between EphB4 expression in primary CRC and liver metastasis and the clinicopathological parameters 125

Do the EphB2 negative CRC cells become EphB4 positive with the progression of the disease? 128

The pattern of expression of the three receptors EphB1 EphB2 and EphB4 in CRC tissues 129

The pattern of expression of the three receptors EphB1, EphB2 and EphB4 in SIV CRC tissues and in liver metastasis of the same patients 131

Chapter Four 133

Introduction 133

EphB1, EphB2 and EphB4 Expression in CRC 134

EphB1, EphB2 and EphB4 expression and the presence of liver metastasis

137

EphB1 Receptor Expression in CRC 142

EphB2 Receptor Expression in CRC 147

EphB4 Receptor Expression in CRC 150

Do the EphB4 positive CRC cells turn to be EphB2 negative with the progression of the disease? 159

Other clinicopathological data in the study 161

Limitations of the study 162

Closing Remarks 164

Chapter Five 165

Closing Remarks 165

Future Directions 166 XIII

Appendix 1 168

Appendix 2 169

References 170

XIV

Tables

Chapter Two

Table 2.1: Total number of slides and the distribution of the slides based on the stage of the disease

Table 2.2: Fixation protocol of the project’s VCB tissues 70

Table 2.3: EphB2 receptor staining protocol development using ABC method

Table 2.4: EphB2 receptor staining protocol development using ABC method

Table 2.5: EphB2 receptor staining protocol development using ABC based modified approach, 76

Table 2.6: EphB2 receptor staining protocol development using ABC based modified approach, 77

Table 2.7: EphB2 receptor staining protocol development using polymer based method 78

Table 2.8: EphB4 receptor staining protocol development using ABC based method using water bath as a HIER method 80

Table 2.9: EphB4 receptor staining protocol development using ABC-based method using hotplate as a HIER method 81

Table 2.10: EphB4 receptor staining protocol development using ABC based method 81

Table 2.11: EphB4 receptor staining protocol development using ABC based method, 83

Table 2.12: EphB4 receptor ABC based staining protocol development using different HIER times and methods, 84

Table 2.13: Advantages and disadvantages of the IHC Select ® Manual Staining XV

System 89

Table 2.14: AR Buffers and HIER Methods and durations, 90

Table 2.15: Blocking Buffers and their durations 91

Table 2.16: Primary antibodies, their dilutions, diluents and incubation periods

Table 2.17: Secondary antibody 92

Table 2.18: Clinicopathological characters of the study population 97

Table 2.19: Example of a singly ordered 2 x 6 table 100

Table 2.20: Example of a doubly ordered 3 x 6 table 101

Chapter Three

Table 3.1: Clinicopatholigical parameters 105

Table 3.2: EphB1 receptor expression in primary CRC and in liver metastasis

108-109

Table 3.3: EphB1 receptor expression in CRC and liver metastasis in relation to clinicopathological parameters 111

Table 3.4: EphB2 receptor expression in primary CRC and in liver metastasis: first experiment 113-114

Table 3.5: EphB2 receptor expression in primary CRC and in liver metastasis: second experiment 117-118

Table 3.6: EphB2 receptor expression in primary CRC and liver metastasis in relation to the clinicopathological parameters 119

Table 3.7: EphB4 receptor expression in primary CRC and in liver metastasis

122-123

XVI

Table 3.8: Relationship between EphB4 expression in primary CRC and liver metastasis and the clinicopathological parameters 125

Table 3.9: Do the EphB2 negative CRC cells become EphB4 positive with the progression of the disease? 128

Table 3.10: The pattern of expression of the three receptors EphB1 EphB2 and EphB4 in CRC tissues 129

Table 3.11: The pattern of expression of the three receptors EphB1, EphB2 and EphB4 in SIV CRC tissues and in liver metastasis of the same patients

131

XVII

Figures

Chapter One

Fig 1.1: listing of classical and non-classical EphA/B ephrinA/B binding

Fig 1.2: structural domain of Eph receptors and their binding ephrins

Fig.1.3: interaction between Eph-expressing cell and ephrin-expressing cell

Fig 1.4: Potential effect of Ryk on Eph–ephrin responses 21

Fig 1.5: intestinal crypt of Leberkühn 25

Fig 1.6: Overview of Wnt signalling pathway 28

Fig 1.7: Reciprocal gradients of members of the EphB receptors and their ephrinB binding ligands along the intestinal crypt 30

Fig 1.8: Complimentary expression of the EphB receptors and their ligands along the crypt long axis 46

Fig 1.9: Compartmentalization phenomena induced by EphB-ephrinB interaction

Fig. 1.10: Tumour buds 48

Fig 1.11: The expression of the EphBs-ephrinBs in the normal colon, in adenoma and in different phenotypes of CRC 52

Chapter Two

Fig 2.1: IHC staining using ABC based method 60

Fig 2.2: Polymer-based IHC 62

Fig 2.3: Diagram showing the structure of IgG 65

Fig 2.4: The five subunits of mouse IgM linked by disulfide bridges 66 XVIII

Fig 2.5: EphB1 receptor staining protocol testing using VCB normal colon tissues,

Fig 2.6: EphB2 receptor IHC staining protocol testing using VCB normal colon tissues. The figure demonstrates the non-specific staining as one of the complications associated with ABC based method 73

Fig 2.7: EphB2 receptor staining protocol development 76

Fig 2.8: Colon basal crypts stained with anti-goat anti-EphB2 (AF467) using Polymer based method 78

Fig 2.9: CRC tissues stained with anti-goat anti-EphB2 (AF467) using Polymer based method 79

Fig 2.10: EphB4 receptor IHC staining protocol testing using anti-EphB4 Mouse monoclonal IgG (SC-130062) primary antibody 82

Fig 2.11: EphB4 receptor IHC staining protocol testing using rabbit polyclonal primary anti-EphB4 antibody (SC-5536) at 1:250 and overnight incubation

Fig 2.12: anti-EphB4 Invitrogen mouse monoclonal (37-1800) at 1:100 and overnight incubation mode x HIER for 30 min 84

Fig 2.13: anti-EphB4 Invitrogen mouse monoclonal (37-1800) at 1:200 and overnight incubation mode at RT x HIER for 50 min 85

Fig 2.14: anti-EphB4 Invitrogen mouse monoclonal (37-1800) following polymer based method with overnight incubation mode and HIER for 50 min

Fig 2.15: IHC Select Capillary Staining System 88

Fig 2.16: Different levels of the EphB receptor staining in CRC tissues

Fig 2.17: Degrees of differentiation of CRC 99

XIX

Chapter Three

Fig 3.1: percentage of cells stained for the receptor per tumour section, 106

Fig 3.2: IHC staining showing normal basal crypt distribution of (a) EphB1, (b) EphB2 and (c) EphB4 receptors. 108

Fig 3.3: Different levels of EphB1 receptor expression in CRC tissues, (a) no expression, (b) weak expression, (c) weak and moderate expression, and (d) high expression 110

Fig 3.4: EphB1 receptor expression in CRC liver metastasis showing weak and no expression of the receptor 110

Fig 3.5: Different levels of EphB2 receptor expression in CRC tissues, (a) no expression, (b) weak expression, (c) weak and moderate expression. 116

Fig 3.6: EphB2 receptor expression in CRC liver metastasis showing no expression of the receptor 116

Fig 3.7: Different levels of EphB4 receptor expression in CRC tissues, (a) no expression, (b) weak expression, and (c) moderate expression 125

Fig 3.8: EphB4 receptor expression in CRC liver metastasis showing (a) no expression of the receptor, and (b) weak expression of the receptor 125

Chapter Four

Fig. 4.1: The cancer cells change their morphology and gain mesenchymal cells characters inducing epithelial mesenchymal transition (EMT) 139

Fig. 4.2: The relationship between EphB receptors and E-cadherin in normal and tumour cells 140

Fig. 4.3: Eph receptors crosstalk with EMT-related signaling pathways 141

Fig 4.4: Moderately differentiated SIV CRC sections showing different levels of EphB1 expression 146

Fig 4.5: EphB1 receptor expression in CRC liver metastasis showing weak, moderate and high expression of the receptor in the same section 147

XXI

Abbreviations

CRC: Colorectal cancer

CEA: Carcinoembryonic Antigen

RTK: receptor tyrosine kinases

Eph: erythropoietin-producing hepatoma

Ephrin: Eph receptor interacting proteins

ISC: intestinal stem cell

LBD: ligand-binding domain,

CRD: cystine-rich domain

JM: juxtamembrane region

SAM: sterile-α-motif

RBD: receptor-binding domain

GPI: glycosyl-phosphatidylinositol domain

KD: tyrosine kinase domain

FGFR: fibroblast growth factor receptor

CNS: Central Nervous System

CST: corticospinal tract

ADAM: A-Disintegrin-And-Metalloproteinase

APC: Adenomatous Polyposis Coli

KO: Knock out mice

TNF: tumour necrosis factor

VEGF: vascular endothelial growth factor

FAP: familial adenomatous polyposis

HNPCC: hereditary non-polyposis CRC

SLAP: Src-like adaptor protein

qRT-PCR: quantitative Real Time- Poly Chain Reaction

AJCC: American Joint Cancer Committee staging system

XXII

WCHRE: Western Centre for Health, Research and Education

VCB: Victorian Cancer BioBank

IHC: immunohistochemistry

ABC: Avidin-biotin complex

DAB: 3,3'-diaminobenzidine substrate

FFPE: Formalin Fixed Paraffin Embedded

FS: frozen section

AR: Antigen Retrieval

Ig: immunoglobulin

HIER: heat-induced epitope retrieval

EIER: enzyme-induced epitope retrieval

HSB: Horse Serum Block

TBST: Tris-Buffered Saline and Tween 20

PBS: Phosphate-buffered saline

H2O2: Hydrogen Peroxide

TNM: Tumour-Node-Metastasis stage

DOD: Degree of Differentiation

ANOVA: Analysis of Variance test

POP: postoperative

CSC: colorectal cancer stem cells

HDAC: histone deacetylases

EMT: epithelial to mesenchymal transition

TIMP-1: tissue inhibitor of metalloproteinases-1

DFS: Disease Free Survival

OS: Overall Survival

XXIII

Chapter One

Introduction

Colorectal cancer (CRC) is a major health problem throughout the world. In Australia, it is the second leading cause of cancer-related death (Ngo, Williams et al. 2011). The incidence has risen dramatically in the 20th century and is still doing so due to the rapid evolution in the areas of agriculture, industry, environmental and life style changes (Chan and Giovannucci 2010). In 2010, there were 14,860 new cases of bowel cancer in Australia: 8,258 new cases in men and 6,602 new cases in women, accounting for 12.7 per cent of all new cancers (ACIM 2014). In 2014, about 16,980 Australians are expected to be diagnosed with bowel cancer and an estimated 19,960 are expected to be diagnosed in 2020 (AIHW 2012). Bowel cancer is more common in men than in women. In 2010, there were 73.7 cases per 100,000 men, compared with 51.1 cases per 100,000 women. Between 1982 and 2010, incidence rates for bowel cancer in men have increased from 66.7 cases per 100,000 men in 1982 to 73.7 cases per 100,000 men in 2010. The incidence rates for bowel cancer in women have virtually remained stable (52.0 cases per 100,000 women in 1982 and 51.5 cases per 100,000 women in 2010). The risk of developing bowel cancer increases with age. In 2010, the risk of developing bowel cancer before the age of 85 was 1 in 12 (ACIM 2014).

The five-year relative survival rate for bowel cancer has relatively increased in the recent years (AIHW 2012). This can be attributed to several factors. First, the surgical techniques of the bowel cancer have dramatically developed. For example, total mesorectal excision (TME) has become the gold standard technique in the treatment of rectal cancer and has been associated with decreased recurrence rate (Liang 2013). Second, the introduction of the adjuvant and neoadjuvant treatment modalities which has been associated with better surgical outcome and reduction in the recurrence rate in general (Rahbari 2013). Third, the proper screening program for CRC has been associated with the reduction in long term

mortality and early detection of tumour recurrences (Shaukat, Mongin et al. 2013).

One of the most challenging postoperative complications of CRC is the high recurrence rate after surgery (Goldberg 2006, Rodríguez-Moranta 2006). Approximately 25% of CRC patients without lymph node or distant metastasis develop recurrent disease after surgery (Kazuhiko Yoshimatsu 2014). The compromising feature of these recurrences is that most of them are not visible endolumenally by the conventional diagnostic tools (Patrick. E. Young 2014). In cancer, a biomarker refers to a substance or process that is indicative of the presence of cancer in the body. A biomarker might be either a molecule secreted by a tumor or it can be a specific response of the body to the presence of cancer (Mishra and Verma 2010). The most significant characteristic of predictive biomarkers of a cancer is that they can categorize high risk cancer patients who are likely to develop recurrences independent of the tumour stage (Wang 2004). Tumour markers should provide information useful for the management of cancer patients including early detection and risk stratification, prognosis, and the prediction of treatment responses (Grady and Pritchard 2014). Beside the physical examination and radiological investigation components of the CRC surveillance program, Carcinoembryonic Antigen (CEA) is also used as a blood-based predictive biomarker for the disease. Despite its high sensitivity and specificity to detect recurrences, about 40% of CRC patients experience recurrences without elevation of the CEA (Tsikitis 2014). In addition, the autopsies of patients dying of primary CRC have showed that there are significant discrepancies between the clinical stage of the primary disease at the time of resection and the actual stage of the disease in autopsy (Welch and Donaldson 1979), which indicates the presence of undetected micrometastasis at the time of resection of bowel cancer. These micrometastases are negatively correlated with the postoperative five-year survival rate of the disease. Molecular detection of these micrometastases can improve survival in CRC patients (Liefers, Cleton-Jansen et al. 1998). Therefore, it is essential to identify further biomarkers which can predict the relapse of the disease at earlier stages and hence improve the overall survival of the patients (Tsikitis 2014). 2

For three decades, since describing the basic molecular alterations during adenoma-carcinoma progression by Vogelstein (Vogelstein, Fearon et al. 1988), our understanding of the molecular biology of CRC has significantly evolved (Zoratto, Rossi et al. 2014). Currently, quantitative multigene expression assays of CRC are considered as diagnostic (Bhalla, Zulfiqar et al. 2013), prognostic (Bhalla, Zulfiqar et al. 2013, Sanz-García, Elez et al. 2014), reliable predictors (Bhalla, Zulfiqar et al. 2013) and therapeutic for the disease (Sanz-García, Elez et al. 2014). They are differentially expressed during the different stages of the disease. As the five-year survival of the CRC patients differs from early stages of that of the late stages, understanding the pattern of expression and early detection of these molecular changes can help to improve the overall survival of the affected individuals (Zoratto, Rossi et al. 2014). The erythropoietin-producing hepatoma (Eph) receptors and their binding ligands (ephrins) make up part of these molecules which are involved in CRC tumorigenesis (Genander 2012). They have diverse roles in CRC including suppression, progression and invasion (Genander and Frisén 2010). They are differentially expressed at different stages of CRC. Profound distortion of expression patterns correlates with altered tumour behaviour. Some of them are elevated with the advancement of the disease (Hafner, Schmitz et al. 2004). Recent genetic analyses have correlated expression of many Eph receptors in tumour tissues with disease stage, metastasis, recurrence and with patient survival (Brantley-Sieders 2012). Eph receptors and their ephrin binding ligands have been observed in many human cancers. They are strongly associated with CRC. Their involvement in the primary CRC is well documented not only in tumour progression, but also in its spread patterns (Herath and Boyd 2010). This study investigates the expression pattern of the subfamily EphB receptors: EphB1, EphB2 and EphB4 in different phenotypes of CRC. It also compares the expression pattern of these receptors in the primary tumor with that of the hepatic metastasis for the same patient. In the introduction, it will firstly review the biolphysiological structure of the Eph receptors and their binding ligands; the structural differences between the A- and B-subfamilies; the biochemistry of the Eph receptors and ephrins and the relationship with their various physiological

functions during embryogenesis and in adulthood, and the crosstalk between Eph/ephrins and other signalling pathways. Secondly, it will describe the normal histological structure of the colon; the biology of the intestinal stem cell (ISC) and its key regulator signalling pathways, and the essential role of the Eph/ephrins in the intestinal integrity and homeostasis. Then, it will describe the relationship between the Eph/ephrins and cancer in general and what is the role-played by the Eph/ephrins in different types of human cancer. The last section will be about CRC: the molecular background of the disease and the relationship between the two subclasses and CRC.

1. Eph/ephrin signalling: a structural view

1.1 Introduction

Eph are the largest subfamily of membrane-bound receptor tyrosine kinases (RTK). They include at least 16 distinct receptors and 9 distinct ligands. Both the Eph receptors and ephrin ligands are divided into 2 subgroups based on their structure and affinities for each other: the A and B subfamilies; ten EphAs (EphA1-EphA10) specifically interact with six ephrinAs (ephrinA1-A6) and six EphBs (EphB1-EphB6), (EphB5 is an avian protein and has no mammalian homologue (Richter, Murai et al. 2007)) interact with three ephrinBs (ephrinB1-B3) (fig 1.1a) (Noren and Pasquale 2004, Merlos- Suarez and Batlle 2008, Himanen 2012). However, a cross-class binding between the two subclasses has been reported; EphA4 can cross react with ephrinB2 and ephrinB3, EphB2 can cross react with ephrinA5 (Himanen 2004, Guo and Lesk 2013), while EphA2 can cross react with ephrinB1 (Paavilainen, Grandy et al. 2013) (fig 1.1b).

Fig 1.1: Listing of classical and non-classical EphA/B-ephrinA/B binding

Fig 1.1a: classical EphA/B-ephrinA/B binding ephrinA1 ephrinA2 ephrinA3 EphA1 - EphA10 ephrinA4 ephrinA5

ephrinA6

ephrinB1

EphB1 – EphB6 ephrinB2 ephrinB3

Fig 1.1b: non-classical EphA/B-ephrinA/B binding

ephrinB2 EphA4 ephrinB3

EphA2 ephrinB1

EphB2 ephrinA5

The first Eph receptor was cloned from a human erythropoietin-producing hepatocellular cell line (Héroult, Schaffner et al. 2006). Since its discovery more than two decades ago, the Eph families of the RTK and their binding ephrin ligands have been implicated in many physiological and pathological processes in many cell types and different organs. Understanding the biochemical structure as well as the mechanism of action of the Eph-ephrin complexes and their signaling networks is a corner stone in understanding the developmental processes, the physiology of adult organs, and the

pathogenesis of many diseases including cancer as well as the potential of drug development (Himanen and Nikolov 2003, Himanen 2012).

Eph-ephrin signaling has multiple biological activities, including widespread effects on the actin cytoskeleton, cell-substrate adhesion, intercellular junctions, cell shape, cell movement and tissue patterning (Pasquale 2008). They bind their opposite cell-bound ligands triggering bidirectional signalling and producing the various biological activities (Vearing and Lackmann 2005). In addition, effects on cell proliferation, differentiation and survival have also been proved (Pasquale 2008). Eph receptors and their ligands modulate cell proliferation in the physiological as well as pathological conditions. They exhibit their effect on cell proliferation through diverse mechanisms (Holmberg, Genander et al. 2006, Takahashi 2014).

1. Structures of Eph receptors

Eph receptors are composed of an extracellular N-terminal glycosylated ligand-binding domain (LBD), a cystine-rich domain (CRD) and two fibronectin-type III repeats. The intracellular part consists of a juxtamembrane region (JM), catalytic kinase domain, sterile-α-motif (SAM) domain and PSD95/Dlg/ZO1 binding site (PDZ-BS) domain. Specificity of the receptor to its ligand is mediated by the N-terminal LBD of the receptor (fig 1.2) (Himanen and Nikolov 2003).

Fig 1.2: structural domain of Eph receptors and their binding ephrins. The extracellular part of Eph receptor is the N-terminal: ligand-binding domain (LBD); cystine-rich domain consists of: EGF-like motif and two fibronectin (FN) III repeats. Transmembrane (TM) domain. Juxtramembrane (JM) domain. PDZ-binding site (PDZ-BS). Ephrin-A and B anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) domain for ephrinA and transmembrane (TM) region for ephrinB. Eph expressing-cell in contact with ephrin expressing-cell inducing bidirectional signaling (Miao and Wang 2009) (with permission).

The Eph-LBD structure is unique to the Eph family and shares no sequence or precise structural homology with other known proteins (Himanen 2003, Huan 2013). Each unbound Eph receptor-LBD forms a compact globular structure. It has β-strands, which are connected by loops of various lengths. There are three types of loops: one is well ordered loop (H-I) that packs against the concave surface of the β-strand sheets, while the other two loops (D-E and J-K) are less well-ordered and protrude from the middle of the other β-sheet. These loops play central role in ligand recognition and hence complex formation (Himanen 2012). The hallmark feature, which distinguishes between the two-subclass receptors, is the length of the H-I loop. In the EphA receptors, H-I loop four residues are shorter than the B- class molecules (Himanen, Goldgur et al. 2009). On the other hand, despite the sequence homology and the structural features of the LBD that are almost the same between Eph receptors of the same subclass, there are differences in the binding specificity as well as in the dynamic features of the LBD of these receptors. For example, there are structural and dynamic differences observed between the LBD of EphA5 and EphA4 receptors (Huan 2013). In the EphA5 LBD structure, all D-E and J-K loop residues form helical-like conformations over Ala 79-Ser 182 and Gly 189-Met 193 but

they form short antiparallel β-sheets in EphA4. Furthermore, EphA5 LBD D- E and J-K loops undergo, within 20 ns of ligand binding, conformational exchanges shortening the distance between them while this distance remains relatively large in EphA4 during the same period of time (Huan 2013). The structural differences in the LBD of the members of the same subclass explain the functional diversity in the cell response. EphA4 and EphA2 for instance, are members of the same subclass, however, stimulation of EphA4 forward signaling results in cell repulsion whereas EphA2 triggers cell adhesion due to structural differences in their LBD (Seiradake, Schaupp et al. 2013). In the four EphB receptors (EphB1- EphB4), the glycine in the outer lip of the active site is highly conserved in the LBD of the EphB 1, 2 and 4 but in EphB3 it is replaced with a cysteine, thus demonstrating LBD structural and binding differences between the members of the same subclass (Overman, Debreczeni et al. 2013).

The flexibility of the class specific loops observed in the Eph receptor LBD structure is another characteristic feature of the Eph receptors (Himanen 2012). For example, the overall structure of the EphA4 LBD is very similar to the other Eph receptors. But EphA4 LBD shows unusual flexibility of D–E, G- H and J–K loops which can be observed either in contact, pointing away from each other, or in the receptor/ligand complexes (Singla, Goldgur et al. 2010). This flexibility enables EphA4 to cross-class bind ephrinB2 and ephrinB3 ligands (Bowden, Aricescu et al. 2009, Guo and Lesk 2013) (fig 1.1b). However, despite this flexibility, EphA4 cannot bind ephrinB1; Met residue in ephrinB1 is smaller than Trp residue in ephrinB2, leading to loss of attraction between EphB4 and ephrinB1 (Guo and Lesk 2013).

There are subclass preferences in the interactions between receptors and their ligands based on the architectural arrangements of the receptors and their ligands; the interacting residues between the receptors and ligands as well as the flexibility of the receptor specific surface loops (Himanen 2012). For example, ephrinA1 is the preferred binding ligand for the EphA2 receptor. EphA2 functions as a tumor suppressor gene when it is activated by 8

ephrinA1. Glycosylation of ephrinA1 increases the affinity of the ligand to the receptor and hence helps to stabilize the EphA2/ephrinA1 tetramer which is observed in the EphA2/ephrinA1 but not in the EphA4/ephrinA1 complexes (Ferluga, Hantgan et al. 2013). Further, the high affinity of EphA4 receptor to the ephrinA5 ligand (Xu, Tzvetkova-Robev et al. 2013) and the high affinity of EphB4 receptor to the ephrinB2 among other ephrinBs (Chrencik, Brooun et al. 2006) are another examples to this phenomenon.

1.3 Structures of ephrin ligands

Unlike other RTKs, the membrane-bound ephrins can specifically bind and activate several Eph family members within the same subclass (Vearing and Lackmann 2005). They are characterized by the presence of a unique N-terminal receptor-binding domain (RBD). A-ephrins are attached to the cell via a glycosyl-phosphatidylinositol (GPI) domain and lack the transmembrane region and the cytoplasmic domain whereas B-ephrins have a transmembrane region and a short highly conserved cytoplasmic domain, which includes a C-terminal PDZ-binding site (fig 1.2) (Himanen and Nikolov 2003, Himanen 2012). The ligand ephrinA1 has uniquely an extra helix (E′), which is absent in the other ephrin structures. This extra structure enables ephrinA1 to accommodate different binding receptors at the same time (Himanen, Goldgur et al. 2009).

N-terminal-RBD of the ephrin ligands has a globular β-barrel structure with a Greek key folding topology, two beta sheets, three alpha helices and two stabilizing disulphide bonds. Receptor-binding G-H loop protrudes away from the rest of the molecule and has varying amount of conformational flexibility depending on the ligand type (Himanen 2003, Himanen 2012). The G-H loop in ephrinB1, for example, is more flexible and undergoes significant conformational rearrangements than that of ephrinB2 ligand (Nikolov, Li et al. 2005). Upon binding with Eph receptors, the overall ligand structure does not change except around the G-H loop where ephrin ligands have to undergo significant conformational rearrangements so that it can tightly bind their corresponding Eph receptors (Himanen 2003, Himanen 2012).

1.4 Structural differences between EphA and EphB receptors, ephrinA and ephrinB ligands, and the structural differences between EphA/ephrinA and EphB/ephrinB complexes

Although the unbound A-class receptor is structurally similar to that of B- class, there are some structural differences between the two subclasses. The main structural difference is the length of the H-I loop, which is already mentioned above. This loop is not part of the high affinity Eph/ephrin interaction in the A-subclass, whereas in the B-subclass, it is involved in the low-affinity interactions. However, the exact role of H-I loop in the formation of the Eph/ephrin cluster in not yet fully understood. The D-E and J-k loops also differ between the two subclasses. In the A-subclass, they prepare for Eph/ephrin binding in the preclustering phase by forming a ligand-binding channel, while in the EphB receptors, these two loops are involved in more open conformations (Himanen, Goldgur et al. 2009).

The overall structure of ephrinA ligand is similar to that of ephrinB ligand. However, conformational differences occur at several exposed loops especially in the region that lies between strands H and K. In this region, the A-subclass ephrins have a six-to-seven-residue deletion as compared to the B-subclass ephrins and, consequently, one of the two small helices (I and J) is absent in the structure of ephrinA5 (Nikolov, Li et al. 2007). Other structural differences between the A- and B-ephrins include the conformations of the A-B, C–D, E–F, and F–G loops which are proposed to participate in higher order clustering interactions (Nikolov, Li et al. 2007, Himanen, Goldgur et al. 2009). In addition, the N terminus of ephrin-A5 bends away from the core of the molecule as compared to ephrin-B2 (Nikolov, Li et al. 2007).

Although the A-class receptor-ligand complexes are structurally similar to that of B-class where the ligand/receptor interface centres around the G-H loop of the ephrin that is buried in a hydrophobic channel on the surface of the receptor, however, there are some structural differences between the two subclasses. A-class receptor/ephrin recognition is through a “lock-and- key” mechanism, whereas B-class receptor/ephrin recognition is through an “induced-fit” mechanism. A “lock-and-key” mechanism involves little 10

conformational rearrangements in the binding partner due to the pre-existing two relatively rigid molecular surfaces forming the hydrophobic channel, which are complementary to each other both in shape and in chemical nature, and this provides one explanation that A-class molecules interact with higher binding affinities than the B-class. In the B-class, Eph binding to ephrins involves the loops forming the side of the ephrin-binding channel rearrange on ligand binding and thus requires an extensive energy to generate the interaction surface between Eph receptors and their ligands (Himanen, Goldgur et al. 2009, Himanen 2012, Janes, Nievergall et al. 2012). Another explanation of the A-class high binding affinities is that in A- class, a salt bridge is formed between the Eph receptor and its binding ligand, which is not present in B-class (Himanen 2012). There are also differences in the Eph/ephrin binding molecules between the two subclasses. For example, Phe 114 from the G-H loop of ephrinA1 makes a close contact with Ala 190 from EphA2, whereas in the EphB/ephrinB complex, the molecules are polar in site (Asn 123 from ephrinB2 and Ser 194 from EphB2). Also, EphA2 Met 55 residue makes a contact with Ser 105 from ephrinA1, while Val 54 makes a contact with Thr 114 in ephrinB2 (Himanen 2012). However, in the subclass itself, for example EphB2 and EphB4, there are numerous differences exist between EphB2-specific binding peptide and EphB4-specific binding peptide. Although the direction of the peptide at the N-terminal end is almost the same in both peptides, the EphB4-peptide forms a 90 ͦ at the centre of the peptide but the EphB2 remains linear (Himanen 2012).

2. Eph-ephrin Bidirectional Signalling

Eph receptors and ligands interact together either in trans between opposite cell membranes, or in cis in the same cell membrane via the functional binding domains. The cis interaction does not mediate intracellular signaling like the trans interaction, but inhibits or fine-tunes the trans interaction (Yin, Yamashita et al. 2004, Egea and Klein 2007, Lisabeth, Falivelli et al. 2013). The trans communication between the cell membrane bound Eph receptors and their opposite cell membrane bound ligands initiates forward 11

signaling from the Eph receptor and reverse signaling from the ephrin ligand opposite each other (fig 1.2). The forward signalling and the reverse signalling are the components of a phenomenon known as the bidirectional signalling. The interacting Eph/ephrin complex regulates embryological tissuegenesis as well as adult tissue homeostasis through bidirectional signaling (Lisabeth, Falivelli et al. 2013), which is facilitated by the formation of adhesion molecules and gap junctions between adjacent cells to facilitate cell-cell communication (fig 1.2) (Arvanitis and Davy 2008). The signal is simultaneously transmitted in both Eph receptor-expressing cells and ligand expressing cells. The evidence shows that in normal conditions, bidirectional signaling resulting from Eph-ephrin communication works in balance to maintain body homeostasis. The class-A Eph-ephrin bidirectional signaling in the pancreatic β-cells, for example, contributes to maintain glucose homeostasis during fasting and feeding. EphA forward signaling inhibits insulin secretion during fasting, whereas ephrinA reverse signaling does the opposite during feeding. Class-B Eph-ephrin bidirectional signaling also exhibits the same effect in bone homeostasis. EphB forward signaling enhances osteoblasts, which are responsible for bone formation, while ephrinB inhibits osteoclasts, which are responsible for bone degradation (Himanen, Saha et al. 2007). However, deregulation of Eph-ephrin signaling can end with unfavourable conditions such as cancer. Eph-ephrin signaling has been involved in different body tumours with a diversity of complicated biological functions of Eph receptors and their membrane-bound ligands in the tumor cells (Gucciardo, Sugiyama et al. 2014).

Eph-ephrin bidirectional signalling triggers either cell segregation and contraction or cell-cell adhesion, spreading and intermingling during tissue patterning in embryonic development and during adult tissue homeostasis. The ultimate outcome of this interaction is determined by several factors: the cellular content of the receptor-expressing and ephrin-expressing cells, the degree of Eph/ephrin clustering, activation and phosphorylation, the composition of the Eph/ephrin complex, the predominance of one signal direction over the other, increasing flexibility of Eph receptors and the presence or absence of the cis interaction (Pasquale 2008, Janes, Nievergall et al. 2012, Gucciardo, Sugiyama et al. 2014). 12

Two conditions are needed to be fulfilled to initiate bidirectional signaling: receptor dimerization with their ligands to form hetero-tetrameric complexes and preclustering of ephrins into aggregates to induce Eph receptors phosphorylation and hence commence their biological responses (non- preclustering forms can act as Eph antagonists) (Egea and Klein 2007). Juxtamembrane (JM) domains are important for efficient receptor activation. In their unphosphorylated state, they repress Eph receptor phosphorylation, but once phosphorylated, they serve as docking sites for downstream signaling proteins (Himanen and Nikolov 2003). Agonistic and antagonistic peptides have been shown to bind the Eph receptor in the same way as the ephrin ligands. They are located in the same pocket as the G-H loop of the Eph receptor. They occupy large surface area of the receptor. They intersect at a conserved IIe residue that promotes the stability of the disulfide bridge (Himanen 2012). They have different mechanism of actions. In general, they target the Eph receptors and inhibit ephrin binding or tyrosine kinase activity. They show promise to deliver anticancer drugs in the future (Noberini, Lamberto et al. 2012). Furthermore, in cancer cells, ephrin-mediated cis interactions can attenuate and/or inhibit Eph receptor signaling induced by ephrin ligands in the opposite cells (Falivelli, Lisabeth et al. 2013).

The basic molecular structure of a functional bidirectional signalling is formed by 1:1 in which a single Eph receptor binds single ephrin ligand forming a hetero-dimer complex. Eph binds ephrin through insertion of an extended ephrin G-H loop into a preformed deep Eph channels. This complex joins a similar one in a ring like structure to form a hetero-tetramer 2:2 complex where each ephrin interacts with two Eph receptors and each Eph receptor with two ephrins to stabilize the complex (fig 1.3) (Himanen 2003, Janes, Nievergall et al. 2012).

Fig.1.3: interaction between Eph-expressing cell and ephrin-expressing cell. Adhesion between epithelial cells by the junction proteins. TJ, tight junction; AJ, adherens junction; GJ, gap junction; Des, desmosome; ECM, extracellular matrix; FA, focal adhesion; HD hemidesmosome. Eph/ephrin crosstalk with proteins in tight junctions, adherens junctions and gap junctions in epithelial cells. Crosstalk between Eph-ephrin and other receptors (Miao and Wang 2009) (with permission).

Further stabilization and lateral expansion are facilitated by receptor-receptor interactions between multiple domains of adjacent Eph receptors. It includes Eph-Eph homo-oligomerization via ECD subdomains through the conserved LBD and CRD residues in the form of LBD-LBD and CRD-CRD arrangements, through the transmembrane (TM) domain, and through Non- ECD SAM domains (Janes, Nievergall et al. 2012); Eph-Eph interaction between LBD and proximal FNIII (Xu, Tzvetkova-Robev et al. 2013), and cross-class activation between A- and B-subcalsses (Nikolov, Xu et al. 2013). Studies on glioblastoma cells observed that when the cells get activated by the EphA3-specific antibodies, EphB2 get phosphorylated (Nikolov, Xu et al. 2013). Interactions with other RTKs and non-RTKs contribute to clustering as well. These molecules act as helpers potentiating bidirectional signalling. For example, EphB2 and EphB4 involvement in SDF- 1-induced endothelial cell movement and assembly into cord like structure (Nikolov, Xu et al. 2013).

2.1 Forward signalling

Forward signalling is critical for cell morphology, cell-cell adhesion, cell shaping and cell migration (fig 1.2) (Pasquale 2008). During embryogenesis, Eph forward signalling is essential for the development of the integral parts of the central nervous system (CNS) including axon guidance of the anterior commissure, corpus callosum as well as for formation of the secondary palate. It has been shown that in vitro and in vivo mutation of EphB2 receptor can disrupt forward signalling resulting in a cleft palate phenotype (Bush and Soriano 2012). EphA4 forward signaling is essential for corticospinal tract (CST) formation and the central pattern generator (CPG) activity. EphA4 receptor mutation results in serious multiple CNS anomalies including defects in axon guidance of the anterior commissure, CST and spinal interneuron axons, and disrupts the CPG resulting in a rabbit-like hopping gait (Bush and Soriano 2012) and errors in CST pathfinding (Kullander and Klein 2002). Ephrin-B2/EphA4 forward signaling is required for regulation of radial migration of cortical neurons in the mouse. Deletion of the EphA4 gene in the embryonic cerebral cortex resulted in faster migration of cortical neurons, whereas knockdown or overexpression of ephrin-B2 did not alter the normal process of migration. These results suggest that EphA4- forward signaling is required for the precise control of cortical neuron migration (Hu, Li et al. 2014). EphB forward signalling is essential for the formation and maturation of the synapses as well as postsynaptic dendrites. They modulate N-methyl-ᴅ-aspartate (NMDA) receptors-dependent calcium influx in response to glutamate through Src tyrosine kinase phosphorylation. Further, EphB2 phosphorylates syndecan-2 which is essential for the postsynaptic dendrites formation in hippocampal neurons. However, EphB2- deficient mice have normal hippocampal synapses indicating that other receptors could contribute to synapse formation (Kullander and Klein 2002). EphB2 mediates cell sorting of Schwann cells thus can cause peripheral nerves regeneration upon axon transection. EphB2 activation in Schwann cells causes relocalization of N-cadherin mediated by the transcription factor Sox2 (Bush and Soriano 2012). EphB2 and EphB3 forward signalling regulates fluid distribution in the inner ear. They are attached to aquaporin-1

which is known fluid regulator domain that can be bound by PDZ-containing protein (Kullander and Klein 2002).

Transphsphorylation of Eph receptor by tyrosine kinase domain (KD) is an essential step required for forward signalling activation (Egea and Klein 2007). Before activation, Eph receptors are scattered on the cell surface and display minimal activity (Nikolov, Xu et al. 2013). Upon binding with membrane-bound ligands, autophosphorylation of cytoplasmic KD takes place leading to full activation of Eph receptor (Kullander and Klein 2002). EphB4 forward signaling, for example, is crucial for cardiomyocyte generation; the differentiation of embryonic stem (ES) cells into cardiomyocytes is impaired in EphB4-null ES cells. Receptor-KD-dependent EphB4-forward signaling plays a crucial role in the development of cardiac progenitor cells. While ectopic expression full-length EphB4 was able to restore the cardiac lineage development in EphB4-null ES cells, the truncated EphB4 that lack the intracellular domain of tyrosine KD and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular KD is essential for the development of cardiomyocytes (Chen, Bai et al. 2014). EphB1 and EphB2 forward signaling has a critical role in proper thalamocortical (TC) and corticothalamic (CT) axons interconnectivity through the ventral telencephalon (VTel). EphB1 and EphB2 are expressed in the developing caudal thalamus at a time when TC axons are turning from the ventral portion of the VTel toward the IacZ and developing cortical plate. Mutations within the conserved intracellular KD resulted in significant VTel axon guidance errors, including severe ventral misprojections (Robichaux, Chenaux et al. 2014).

Cytoskeleton reorganization is essential for cell shape change and movement (Miao and Wang 2012). Eph forward signalling regulates cell morphology and movement by rearranging the actin cytoskeleton through Rho GTPase-dependent and –independent pathways. Rho GTPase family

includes RhoA, Rac1 and Cdc42. RhoA is involved in the formation of focal adhesions as well as contractions of the actin cytoskeleton, whereas Rac1 and Cdc42 drive the formation of lamellipodia and filopodia. Ligand- dependent EphA receptor activation leads to activation of RhoA and/or inactivation of Rac1/Cdc42. Subsequently, this can result in repulsive effects of Eph forward signaling including cell retraction and inhibition of cell migration/invasiveness. In cancer cells, this can result in tumor suppression. In contrast, EphA2 can, independent of ligand activation, promote cell migration and invasion through activation of Rho GEF ephexin4. Eph forward signaling can also regulate cytoskeleton independent of the Rho GTPase family. This can be achieved through Cdk5 activation via Tyr15 phosphorylation and mTOR inactivation (Miao and Wang 2012, Lisabeth, Falivelli et al. 2013). On the other hand, Rho family of small GTPases plays an integral role in Eph forward signaling by activating specific Eph- associated effectors during Eph-mediated cell shaping. The activation state of Rho GTPase is regulated by guanine nucleotide exchange factors (GEFs) of the Dbl family such as ephexin, Vav and GTPase activating proteins (GAPs). They convert inactive GDP to active GTP. Eph receptors influence these conversions by regulating both GEFs and GAPs. The Rho GEF ephexin1 has specific attraction to EphA while Rho GEF Vav2 can bind both A- and B-subclasses. In the absence of ephrins, EphA-bound ephexin1 is a non-tyrosine kinase-phosphorylated that promotes axon outgrowth. When it is stimulated by ephrin, it becomes tyrosine-kinase-dependent promoting axon growth collapse (Egea and Klein 2007, Lisabeth, Falivelli et al. 2013).

2.2 Reverse signalling

Transmembrane ephrins reverse signalling is as important as the forward signalling. It is important for axon guidance, cell migration, midline fusion plasticity and synaptogenesis. Ephrins lack an enzymatic domain, and reverse signaling following Eph-ephrin binding, is initiated at cell membrane through recruitment of cytosolic proteins such as the Src family tyrosine kinases and this leads to the phosphorylation of the ephrin and thus enhancing reverse signalling (fig 1.2) (Lisabeth, Falivelli et al. 2013). EphrinAs can regulate cell adhesion by reverse signalling. This can take

place through several mechanisms. Mutation of ephrinA5 in mice, for example, can lead to decrease cell adhesion. Activation of ephrinA2 or ephrinA5 by EphA3 results in a beta-1-integrin-dependent increase in cell adhesion (Daar 2012). Also, ephrinA reverse signalling regulates haematopoietic stem cell adhesion and migration by phosphorylation of paxillin, which is a mediator of integrin signalling (Miao and Wang 2012). EphrinA5 reverse signaling in pancreatic β cells can stimulate Rac1 activity, which is necessary for insulin secretion after glucose stimulation (Lisabeth, Falivelli et al. 2013).

B-type ephrins initiate reverse signaling either through phosphorylation- dependent or phosphorylation–independent signaling pathways. Phosphorylation of ephrinBs can be either through in trans Eph-ephrin binding, in cis growth factor receptors or other cell surface molecules. For example, EphrinB1 is phosphorylated in response to binding the extracellular domain of its cognate EphB receptor, and this is accomplished through Src family kinases. Phosphorylation of ephrinB1 can also occur in cis by an active FGF receptor or by an interaction with claudins. Tubularization of the urethra and partitioning of the urinary and alimentary tract are induced by ephrinB2-mediated cell-cell adhesion. Mutation of ephrinB2 leads to severe hypospadias and incomplete cloacal septation. Closure of the ventral abdominal wall requires ephrinB reverse signalling but not the EphB forward signalling. The intercellular tight junctions are regulated by the ephrinB1 reverse signalling. Par-6, claudins, and connexin 43 are known structures involved in tight junctions, they undergo structural changes upon binding with ephrinB1 that lead to rearrangement of the actin cytoskeleton and thus enhancing the cell-cell contact. Tissue boundary formation has been linked to ephrinB1 reverse signalling. During the development of hindbrain compartments, ephrinB1 reverse signalling functions to restrict intermingling between even-numbered rhombomere cells and Eph receptor expressing odd-numbered rhombomere cells. EphrinB1 plays an integral role in cell- substrate adhesion via integrin. Integrin is a major receptor required for fibronectin matrix assembly. Mutations of ephrinB1 lead to congenital malformations such as craniofrontonasal syndrome (CFNS) due to failure of boundary formation between the coronal sutures, and cleft palate phenotype 18

due to failure of adhesion and epithelial-to-mesenchymal transition of the epithelial layer (Daar 2012).

2.2.1 SH2/PDZ-dependent reverse signalling

B-type ephrins have a carboxy terminal PDZ domain binding site, which is particularly important for the physiological functions of ephrin B. The C- terminal of ephrinB binds PDZ-domain containing proteins. Upon binding with EphB receptor, it activates PDZ-dependent reverse signalling. Point mutations of the C-terminal of ephrinB1 containing PDZ protein have resulted in complete agenesis of corpus callosum due to loss of axonal reception of a repulsive cue from EphB2 reflecting the importance of PDZ- mediated reverse signalling (Bush and Soriano 2012). An ephrinB interaction with PDZ-RGS3, a GTP exchange factor, regulates migration of cerebellar granule cells, and is critical for the maintenance of the neural progenitor cell state. Ligands ephrinB1 and ephrinB2 interact with syntenin through their C-terminal PDZ-binding and mediate presynaptic development. Dendritic spine morphogenesis is mediated by an interaction between ephrinB1and SH2/SH3-Grb4 and the G protein-coupled receptor kinase- interacting protein (GIT) (Daar 2012). Studies have also shown that PDZ- dependent ephrinB2 reverse signalling is important in vascular endothelial growth and sprouting of arteries. It is involved in internalization and phosphorylation of vascular endothelial growth factor receptor (VEGFR2 and VEGFR3). Defects in this system have resulted in lethal embryonic anomalies (Bush and Soriano 2012).

In conclusion, our knowledge has advanced regarding our understanding of the mechanisms of action and regulation of both ephrinA and ephrinB reverse signaling, however, further studies are required (Daar 2012).

2.3 Cross talk with other signaling system

In addition to the Eph-ephrin bidirectional signaling, Eph receptors and their ligands do communicate with a variety of other cell surface proteins (fig.3) (Egea and Klein 2007, Pasquale 2008). This cross-reaction with other receptors can widen the effect of Eph-ephrin signalling pathway. An example is fibroblast growth factor receptor (FGFR). FGFR is another family of RTK.

It has been reported that FGFR receptor plays dual roles with the Eph-ephrin signalling. Sometimes it inhibits Eph/ephrin pathway. For example, it inhibits ephrinB1-induced cell dissociation in blastomere by activating its phosphorylation. Eph-ephrin signalling helps retinal field development by allowing retinal progenitors to reach the eye field. FGFR antagonises this effect by inhibiting movements of the retinal progenitors. These two antagonising pathways work together to regulate access to the eye field by modulating cell movement. On the other hand, there is a direct cooperation between FGFR and Eph/ephrin signalling pathways in regulating several physiological functions. For example, the ectopic formation of posterior protrusions in Xenopus embryos is induced by overexpression of EphA4 as well as by activation of FGFR. The incidence of these structures increased when both proteins coinjected in the embryos, reflecting that these two RTK family members interplay with each other. In addition, FGFR and Eph/ephrin trans-phosphorylate each other during asymmetric cell division in Ciona embryos. Both systems act together to regulate extracellular kinase (Arvanitis and Davy 2008). The mutual relationship between EphA4 and FGFR1 also illustrates the agonistic relationship between both pathways. EphA4 promotes FGFR1 signalling in glioma cells. Ligand binding of EphA4 stimulates FGFR1 phosphorylation. In contrast, ephrinA5 negatively regulates EGFR signalling by promoting its ubiquitination through reverse and forward signaling by binding to EphA receptor (Miao and Wang 2012).

Ryk is atypical orphan RTK with a single-chain, single-pass transmembrane protein that possesses inactive tyrosine kinase domain (kinase-dead). Ryk binds cell through the C-terminal together with EphB2 and EphB3 receptors. It is implicated in cell positioning during craniofacial development, which is also mediated by the Eph-ephrin signalling pathway. Ryk-deficient mice have cleft palate and craniofacial defects similar to those with EphB2 and EphB3 double mutated mice suggesting that these receptors interact together during mice embryogenesis (Truitt 2011). Furthermore, Ryk is essential for the assembly of Eph-ephrin complexes resulting in the initiation of bidirectional signalling, which is the milestone to guide a normal craniofrontonasal development. In humans, craniofrontonasal syndrome 20

results from mutations in the ephrinB1 gene; this is located on the X chromosome. These mutations interfere with Eph binding capacity of the ligand. Since Ryk is associated with EphB2/EphB3, and since Ryk and double EphB2/EphB3 KO mice result in craniofacial defects, it is suggested that ephrinB1 interaction with EphB2 or EphB3 is mediated by the Ryk receptor (fig 1.4) (Truitt 2011).

Fig 1.4: Potential effect of Ryk on Eph–ephrin responses through its direct interaction with Eph receptors and stabilization of Eph–ephrin signaling complexes (Truitt 2011) (with permission).

Although the exact mechanism by which Ryk influences Eph/ephrin signaling is still not fully understood (Arvanitis and Davy 2008), Ryk interacts with PDZ domain-containing protein AF-6 (afadin), which is recruited by the Eph receptors. Ryk delivers AF-6 to the Eph receptors, where it is phosphorylated and then associates with the Eph receptor PDZ binding motif (Truitt 2011). On the other hand, earlier study had shown that AF-6 does not bind to Ryk. It also showed that Ryk is associated with EphB2 and EphB3 but is not phosphorylated by them (Trivier and Ganesan 2002). Ryk is a Wnt signalling receptor. It binds Wnt protein and participates in Wnt- regulated axon guidance (Keeble 2006, Truitt 2011). Wnt/Ryk pathway counterbalances Eph/ephrin pathway in controlling topographic mapping to ensure that axons project to their appropriate target in the brain. Eph/ephrin signalling has been involved in positioning retinotectal axons along the

medial-lateral axis whereas Wnt/Ryk pathway is involved in the lateral mapping of the retinotectal axons (Arvanitis and Davy 2008).

The chemokine CXCR4/SDF-1 receptor is involved in multiple cellular processes. The interaction between the Eph-ephrin signalling and the chemokine signalling is paradoxical; they either antagonise or agonise each other during cellular processes. Eph-ephrin activation, for example, inhibits SDF-1-induced chemotaxis of cerebellar granule cells. Eph-ephrin signalling downregulates chemokine induced cell migration, which is important for arteriole remodelling during placenta formation. In contrast, the two pathways cooperate together during morphogenesis of blood vessels. Activation of Eph-ephrin pathway is associated with stimulation of SDF-1- induced chemotaxis in endothelial cells (Arvanitis and Davy 2008).

2.4 Processing of Eph-ephrin complexes

Following Eph-ephrin binding, Eph-ephrin complexes are processed by several mechanisms. These mechanisms are particularly important for axon guidance during CNS development and for sorting of Eph-expressing cells from ephrin-expressing cells (Pasquale 2010). Examples of these mechanisms are: proteolytic cleavage by metalloproteases, endocytosis and dephosphorylation (Lisabeth, Falivelli et al. 2013)

Activation of metalloproteases such as Kuzbanian A-Disintegrin-And- Metalloproteinase (ADAM) 10 can switch Eph-ephrin mediated cell adhesion into cell repulsion. It is a cell membrane domain associated with Eph receptors and ephrin ligands. It is activated upon Eph-ephrin engagement leading to reposition of ADAM10 and activation of its N-terminal domain and subsequently proteolytic cleavage of the Eph-ephrin binding molecules (Egea and Klein 2007, Janes, Nievergall et al. 2012, Lisabeth, Falivelli et al. 2013, Nikolov, Xu et al. 2013) . On the other hand, ADAM19 stabilizes EphA4-ephrinA5 interactions at the neuromuscular junction independent of its proteolytic activity (Pasquale 2010).

Another mechanism of Eph-ephrin complex processing is endocytosis (Pitulescu 2010). It is a bilateral process that upon activation of Eph receptors, leads to the formation of intracellular vesicles containing Eph- 22

ephrin complexes in both cell populations. Trans-endocytosis terminates adhesion and enables cell retraction and repulsion (Zimmer, Palmer et al. 2003). Endocytosis requires actin cytoskeleton polymerization and activity of the small GTPase Rac1 (Pitulescu 2010). Tiam1 is a member of Rho family. Tiam1 activation is essential for EphA2 receptor endocytosis. Tiam1 activation leads to Rac1 activation. Inhibition of Tiam1 by RNA interference abrogates EphA2 endocytosis (Boissier Pomme 2013). Truncation of the Eph receptor and its binding ephrin ligand also inhibits bilateral internalization and thus enhances cell adhesion. It is commonly believed that endocytosed Eph ephrin complexes continue to signal after internalization (Pitulescu 2010, Daar 2012, Boissier Pomme 2013). Endocytosis of ephrinBs also helps regulating other receptor signalling pathways. PDZ- dependent activation of endothelial ephrinB2, for example, leads to VEGFR endocytosis (Daar 2012).

Eph forward signalling and ephrin reverse signalling both are inactivated by a phosphotyrosine-specific protein phosphatase (PTP). It dephosphorylates Eph receptors and ephrin ligands (Nikolov, Xu et al. 2013). Suppression of PTP, for example, shifted the dorsonasal axons anteriorly into a region of lower levels of ephrinAs indicating that PTP has a negative regulation effect on Eph forward signalling (Egea and Klein 2007). Overexpression of PTP induces EphA2-expressing epithelial cells transformation by decreasing EphA2 phosphorylation. In glioma cells, EphA/ephrinA interaction causes cell segregation, which is attenuated by PTP (Miao and Wang 2012).

3. Role of Eph/ephrin in the colon

3.1 Physiology of the colon

The large intestine is composed of the caecum, appendix, ascending colon, transverse colon, descending colon, sigmoid colon, rectum and anus. It is approximately 1.5-2 meters long. Functionally, it absorbs the remaining water and electrolytes from indigestible food matter; storing foods that were

not digested in the small intestine; and eliminating solid waste as faeces from the body (Ganong 2013).

3.2 Histology of the colon (Gartner. 2001)

The colonic epithelium mucosa is composed of four histological layers: mucosa, lamina propria, muscularis mucosa and subserosa.

The colonic mucosal layer is extensively embedded with crypts of Leberkühn (fig 1.5). The mucosa has a columnar epithelium composed of three differentiated cell types (enterocytes, enteroendocrine and goblet). The number of goblet cells increases along the colon from the proximal to the distal part of the colon. The absorptive cells are the most prominent type of the colonic mucosal cells and this correlates with the main physiological function of the colon, which is maintenance of the body fluid. Proliferative stem and precursor cells occupy the bottom two thirds of crypts, whereas differentiated cells constitute the surface epithelium and top third of crypts (Schoenwolf, Brauer et al. 2009). The intestinal epithelial cells are arranged in a characteristic way in which they become more differentiated as they migrate upwards along the crypt axis. Once they reach the lumen, they enter into apoptosis and then phagotysed. The epithelial lining of the crypts and of the mucosal surface is replaced every six to seven days due to the rapid mitotic activity of the progenitor cells. Proliferation occurs under the influence of growth factors and cytokines from the Wnt family that is produced by the underlying stromal cells (Leedham, Brittan et al. 2005).

Fig 1.5: intestinal crypt of Leberkühn, which represents the proliferative compartment of the intestinal epithelium. The lower two third consists of the proliferative precursor cells, which migrate upwards and give rise to the differentiated cells, which occupy the upper one third. The differentiated cells are specialized cells which expose different physiological functions (Wim de Lau 2007) (with permission).

The lamina propria of the colonic epithelium forms the centre revise of the crypts. It consists of loose highly vascularised connective tissue, a network of lymphoid cells and nodules which is very essential for immunoprotective mechanism of the epithelium. The muscularis mucosa is composed of inner circular layer and outer longitudinal layer of smooth muscles. These smooth muscles function in a coordinated way to propel forward the luminal content of the colon. They are controlled by the neuroendocrine system. The subserosa is composed of dense connective tissue, which is rich in lymphatic and vascular networks. It receives its innervations from the parasympathetic Meissner's plexus.

3.3 Intestinal stem cells (ISC)

Stem cells are undifferentiated self-renewing cells that can under appropriate conditions differentiate into specialized cells to give rise to different organs. They are the key cells, which are responsible for tissue homeostasis; they allow blood, bone, gametes, epithelial, nervous system, muscle, and other tissues to be replaced by fresh cells throughout life. Additional stem cells lie dormant, but can be activated at particular life cycle stages, or following 25

injury. These stem cells are confined within restricted tissue compartment known as “niches” which provide suitable microenvironment for stem cells to survive and proliferate (Sean J. Morrison 2008, Schepers and Clevers 2012).

In each colonic crypt, there are around six ISCs occupying the crypt base. These ISCs give rise to one ISC and one daughter cell. The daughter cell later on continues to divide into more differentiating cells. (van der Flier and Clevers 2009). The precursors are in a continuous proliferation, differentiation and maturation while moving upward into the epithelial surface where they are eventually expelled into the lumen whereas the stem cells skip this flow and remain at the bottom of the crypts. This is because the ISC are confined within the niche, which provides a biological microenvironment for the ISC in order to maintain their longevity (Ohlstein, Kai et al. 2004, Leedham, Brittan et al. 2005). In the intestinal crypts, this niche is formed by a fenestrated sheath of surrounding mesenchymal cells, which secrete growth factors and cytokines essential for regulating ISC behaviour. Within the niche, ISCs undergo asymmetric division producing one stem cell and one daughter cell per division or symmetric division producing either two ISCs or two daughter cells which go on to differentiate. The majority of divisions are thought to be asymmetric with retention of the template DNA strand within the ISC located in the niche (Leedham, Brittan et al. 2005).

3.4 Wnt signalling in normal cells

Wnt signaling was first discovered three decades ago in the murine mammary tumours (Nusse and Varmus 2012). It was found that these tumours were caused by murine mammary tumorus virus (MMTV) through the activation of the murine int-1 (integration1) gene. A Drosophila melanogaster Wingless (Wg) fly gene turned to be a homologue of the mammalian int-1, which was later on called Wnt1 as a combination of Wg and int-1 (Klaus and Birchmeier 2008, Polakis 2012). There are around 19 Wnt genes identified in humans (Kenneth C. Valkenburg 2011). Wnt signaling plays a major role in the growth of embryonic tissues as well as in the maintenance of adult tissues. It has an integral part in tissue specification, 26

cell-cell adhesion, cell to cell signaling and cell homeostasis (Logan and Nusse 2004). It was shown that loss of Wnt signaling in the intestinal crypts by genetic deletion of β-catenin or through inhibition of Wnt receptors by the Dikkopf-1 (DKK-1) proteins resulted in loss of the enteroendocrine cells and even complete absence of intestinal crypts in adult mouse (Haegebarth and Clevers 2009), impaired proliferation and breakdown of intestinal epithelium (Horvay and Abud 2013).

Under normal growth conditions, Adenomatous Polyposis Coli (APC) gene counteracts Wnt signaling in the cell by a multiprotein destruction complex (fig 1.6a), which promotes binding of β-catenin, and therefore triggering its degradation in the proteasomes. The central region of the 2843 amino acid APC protein is most directly implicated in Wnt signaling. It contains three 15- aa repeats (15R) and seven 20-aa repeats (20R) function in the binding and degradation of β-catenin (Aoki and Taketo 2007, Schneikert and Behrens 2007). Phosphorylation of this central part of APC promotes its affinity to β- catenin. The phosphorylation sites are contained in the N-terminal domain of β-catenin and are considered as potential mutations in tumorigenesis (Schneikert and Behrens 2007). Further, any mutation that destabilizes the aa-repeats in the APC gene will also interfere with the destruction complex (McCartney and Näthke 2008).

When cell receives Wnt signal (fig 1.6b), Wnt proteins bind to the transmembrane receptors frizzled family and to co-receptors low-density lipoprotein receptor-related protein (LRP-5 and LRP-6), which are essential for signal transmission. This leads to phosphorylation of LRP by GSK3β and CK1 and the recruitment of axin to the plasma membrane in a manner dependent on the cytoplasmic component Dishevelled (Dsh). This leads to degradation of axin. Stabilized β-catenin enters the nucleus where it associates with Tcf/Lef transcription factors to activate the transcription of Wnt target genes. The transcriptional force mediated by the β-catenin/Tcf complex is under continuous positive and negative regulation mediated by interacting factors (Sancho, Batlle et al. 2004, Schneikert and Behrens 2007).

Fig 1.6: Overview of Wnt signalling pathway (a) in the absence of Wnt signaling, APC/Axin/GSK3β destruction complex causes degradation of β-catenin. (b) In the presence of Wnt signallinig, Wnt interacts with its specific receptor complex containing a Frizzled family member and LRP5 and LRP6 triggering the formation of Dvl-Fzd complex, which counteracts the destruction complex. β-catenin form β-catenin/TCF complex in the nucleus and initiates the Wnt- target genes transcriptional process (Barker and Clevers 2006) (with permission).

Wnt inhibitory factors (WIFs) are found extracellularly (Aguilera, Fraga et al. 2006). The most potent WIF is Dikkopf (DKK). It inhibits Wnt signaling by binding to LRP5/6. This forces the LRP to enter the cell thus making it unavailable for transmembrane Wnt signaling. The DKK-1 gene is a target of β-catenin/TcF signaling pathway (Niida, Hiroko et al. 2004).

3.5 Eph/ephrin signalling is involved in maintaining intestinal integrity and homeostasis

Maintenance of the intestinal epithelium relies on a tightly regulated combination of cell division, differentiation, migration and apoptosis (Horvay and Abud 2013). Eph receptors of both subclasses and their corresponding ephrin ligands are directly involved in the maintenance of the intestinal integrity and homeostasis.

EphA receptors show differential expression in the intestinal epithelium. Genes encoding EphA1, EphA4 and EphA7 are highly expressed near the crypt base while EphA2, EphA5 and ephrinA1 are mostly encountered near the intestinal epithelial surface (Kosinski, Li et al. 2007, Miao and Wang 2009). EphA receptors and their ligand ephrinA1 have diverse contribution to the intestinal homeostasis (Miao and Wang 2009). EphA/ephrin-A signaling 28

differentially regulates cell–matrix and cell–cell adhesion. EphA2 forward signaling suppresses integrin function leading to cell deadhesion and cell rounding whereas ephrinA reverse signaling shows the opposite effect. Activation of EphA2 causes phosphorylation of claudin 4, leading to delayed assembly of claudin 4 to tight junctions. EphA/ephrin-A signaling regulates both Ca2+-dependent E-cadherin-based adherens junctions and Ca2+- independent nectin-based cell–cell adhesions. Nectins are linked to the actin cytoskeleton and are important for the formation of tight junctions in epithelial cells (Miao and Wang 2012).

EphB receptors and their associated ligands ephrinBs are expressed in an inverse gradient along the crypt long axis (Batlle, Henderson et al. 2002) (fig 1.7). The peak level of genes expressing EphB receptors, which are positively controlled by Wnt signaling, is in the ISC and their numbers gradually decline as they move towards the epithelial surface, whereas the genes expressing ligands ephrins, which are negatively controlled by Wnt signalling, are in a higher level near the intestinal lumen and their concentration gradually decreases downward the crypt axis (Genander 2012). For example, EphB1, EphB2, EphB3, EphB4 and EphB6 receptors are expressed at highest level in the intestinal crypt base (Vivian 2008). Among EphB receptors, EphB2 and EphB3, known sorting receptors, play an integral role in preventing the upward migration of the ISCs and in allocating cell population within the intestinal epithelium. Repulsive forces between EphB receptors and their binding ephrinB ligands position cells along the crypt axis. It has been found that β-catenin/TCF signaling positively controls EphB2 and EphB3 receptors while negatively controls their ligand ephrinB1 (Batlle, Henderson et al. 2002).

Fig 1.7: Reciprocal gradients of members of the EphB receptors and their ephrinB binding ligands along the intestinal crypt axis. EphB2 and EphB3 are highly expressed by the ISC at the crypt base and decrease gradually upwards. This correlates with Wnt pathway activity. Their cognate ligands ephrinB1 and ephrinB2 are highly expressed by the differentiated cells near the epithelial surface and decrease gradually downwards. Cells enter into apoptosis as they reach the epithelial surface (Holmberg 2006) (with permission).

This is consistent with the highest activity of Wnt target genes, which is at the crypt bottom and gradually decreases toward the top (van der Flier and Clevers 2009, Schepers and Clevers 2012). Also, cells in the lower third of the crypt contain nuclear bound β-Catenin while cells above this level contain membrane bound β-Catenin (Batlle, Henderson et al. 2002). In addition, genes downregulated by dominant-negative versions of β- catenin/TCF were physiologically expressed by the proliferative cells. By contrast, genes that were upregulated upon the inhibition of β-catenin/TCF were expressed in differentiated cells of the intestinal epithelium (Batlle, Henderson et al. 2002). Further, in double EphB2-EphB3-null mice, the unidirectional upward migration of the differentiating intestinal cells along the crypt axis was lost (Sancho, Batlle et al. 2003). EphB2 and EphB3 and their ligands ephrinB1 and ephrinB2 are expressed in a complementary pattern to form boundaries between proliferative and differentiated cells in the intestinal crypts. In EphB2 and EphB3 double mutant mice, proliferative cells were found mingling with the differentiated cells (Batlle, Henderson et al. 2002). A number of mechanisms have been attributed to the cell-cell segregation 30

and boundary formation phenomenon between proliferative and differentiated cells including: intercellular gap junction inhibition inducing cell segregation, EphB2 signalling-Mitogen-activated protein kinases (MAPK) activation resulting in decreased cell-matrix adhesion, and Wnt-pathway dependent RhoA activation resulting in actin contractility (Nievergall 2012).

The homeostasis of the intestinal epithelium is achieved through a balance between self-renewal and differentiation. Damaged or disrupted intestinal epithelium requires constant replacement by the ISC, which plays an essential role in the intestinal homeostasis (Amcheslavsky, Jiang et al. 2009). In addition, it has been found that ephrinB1 ligand and its associated receptor EphB1 and ephrinA1 and its binding receptor EphA2 are directly involved in the intestinal epithelial wound healing process. They can induce expression genes which are usually recruited in response to any epithelial disturbed homeostasis (inflammation, dehydration, breakdown) (Miao and Wang 2009). Ephrin-B signaling induces complex pathways, which are essential for rapid epithelial wound closure in the intestine. Such functions are essential for the protection of the host against microbial antigens and toxic factors abundantly present in the gut (Hafner, Meyer et al. 2005). In inflammatory bowel disease (IBD), data validated by real-time RT- PCR and immunohistochemistry (IHC), showed upregulation of ephrin-B2 in both perilesional and lesional intestinal epithelial cells of IBD patients, suggesting a role in epithelial homeostasis (Hafner, Meyer et al. 2005).

4. Role of Eph/ephrins in cancer: tumorigenesis, deregulated cell positioning during tumour spreading, invasion and metastasis versus tumour suppression

Considering their direct effect in cell adhesion, cell migration and cell positioning, it will not be surprising that many Eph receptors and ephrin ligands are also involved in tumour progression as well as tumour metastasis (Bogenrieder and Herlyn 2003). EphA2 overexpression, for example, is associated with more aggressive behaviour of several different tumours (Surawska, Ma et al. 2004). Some of these Eph/ephrins are considered as tumour suppressor genes whereas the others are considered as oncogenes (Genander and Frisen 2010). It is widely accepted that some of these Eph/ephrins can be considered as reliable prognostic indicators of the tumor survival rate (Fox and Kandpal 2004, Surawska, Ma et al. 2004). However, our current knowledge about the impact of Eph/ephrin signalling on tumour cell behaviour is incomplete (Miao and Wang 2012). More structural and biochemical studies are needed in order to understand more about the mechanisms of expressing these genes in tumours as well as in normal tissues, and the potential of developing anti-Eph/ephrin chemotherapeutic agents in the future (Miao and Wang 2012, Nikolov, Xu et al. 2013).

Eph receptors and ephrins have been isolated in many tumours. In most of the circumstances, their expression correlates with the degree of aggressiveness of the tumour (Surawska, Ma et al. 2004). Most of the CRC tissues express at least one of the Eph genes which are involved in the normal colon but at different level of expression (Herath, Spanevello et al. 2012). Microarray screening revealed that EphA3 receptor was identified as the third highly mutated gene in CRC (35% of tumours) after APC and K-Ras genes (Janes, Adikari et al. 2008, Herath and Boyd 2010). Other Eph receptors’ mutations have also been identified in CRC including EphA4, EphA7 and EphB6 (Janes, Adikari et al. 2008). In contrast, a series of studies have shown that particularly B-type receptors and their ephrinBs can function as either oncogenes or tumour suppressors in the same tumour depending on the tumour stage (Noren and Pasquale 2007, Genander, Halford et al. 2009). EphB4, for example, has both tumour-suppressing and 32

tumour promoting activities in breast cancer (Noren and Pasquale 2007). Its overexpression has been reported in breast cancer tissue. Nevertheless, tyrosine kinase phosphorylation of EphB4 with activated ephrinB2 has been found to inhibit breast cancer cell motility and invasion. Activation of ephrinB2 is associated with inhibition of the matrix metalloprotease MMP2 and increased activity of Abl/Arg/Crk signalling pathway thus inhibiting breast cancer cells invasion (Noren and Pasquale 2007).

Eph receptors and ephrins play an integral role in angiogenesis in embryos as well as in adults. EphrinA1, for example, induced by tumour necrosis factor-α (TNF-α) was the first vascular ephrin molecule identified in human umbilical venous endothelial cells (Melanie Heroult 2006). Ephrin-B2 is essential for mural-cell migration, spreading, and adhesion during blood- vessel-wall assembly (Foo, Turner et al. 2006). EphB4/ephrinB2 signalling plays a key role in angiogenesis. During development, EphB4 is essential for venous formation whereas ephrinB2 ligand for arterial formation. Both endothelial EphB4 receptor and ephrinB2 ligand display propulsive and repulsive functions during angiogenesis preventing intermingling of different endothelial cells (Mosch, Reissenweber et al. 2010). Overexpression of Eph receptors and their ligands in tumour is associated with tumour neo- angiogenesis. Angiogenic factors such as vascular endothelial growth factor (VEGF) and TNF-α in the tumour cells induce the expression and activation of ephrins in the tumour endothelial cells (Pasquale 2010). These in turn interact with the Eph receptors in the endothelial cells stimulating endothelial cell angiogenesis through the bidirectional signalling (Cheng, Brantley et al. 2002). Finally, Dll4/Notch and Ephrin-B2/EphB4 pathways play critical roles in tumour vessel development and maturation. Dll4/Notch pathway plays prominent role in angiogenesis. Dll4 is critical for embryonic vascular development and arterial specification and is markedly induced in murine and human tumour vessels. Combination targeting of Dll4/Notch and Ephrin- B2/EphB4 appears to prevent excessive vascular proliferation in the liver, which is seen in extended Dll4/Notch blockade alone. Therefore, this combination is a potential candidate for cancer therapy (Djokovic, Trindade et al. 2010).

Eph-ephrin signaling affects tumour cell migration and distant metastasis by interaction with cadherins (Cortina, Palomo-Ponce et al. 2007), by rearrangement of actin cytoskeleton (Egea and Klein 2007) and by deregulation of the metalloproteinase function (Solanas, Cortina et al. 2011). Elevated Eph receptor levels in tumour affect cell–cell attachment through interaction with cadherins. Loss of E-cadherin has been shown to affect the expression and cellular localization of several Eph receptors and ephrins (Orsulic and Kemler 2000). Furthermore, the integrity of epithelium is secured by tight junctions between the cells. E-cadherin is the molecule, which is responsible for cell-cell adhesion. It has been proven in mice that E- cadherin deletion leads to defects in cell to cell adhesion, cell differentiation and cell to matrix adhesion (Schneider, Dahlhoff et al. 2010). In CRC cells, EphB signaling couples cell contraction with cell-to-cell adhesion by relocalizing E-cadherin from cytoplasmic to transmembrane molecule resulting in packing of epithelial cells into tight clusters. Eph-ephrin interactions suppress CRC progression in its early stages by compartmentalizing tumor cells. Sorting and compartmentalized growth was severely impaired in E-cadherin knockdown cells, which intermingled and remained mostly individualized or in small clusters despite continuous EphB3–ephrinB1 interactions (Cortina, Palomo-Ponce et al. 2007). Solanas et al further investigated the mechanism by which Eph-ephrin signalling controls the compartmentalization of cells in epithelial tissues. They showed that EphB receptors interact with E-cadherin and with the metalloproteinase ADAM10 at sites of cell-cell adhesion and their activation induces shedding of E-cadherin by ADAM10 at interfaces with ephrin-B1- expressing cells. This process results in asymmetric localization of E- cadherin and, as a consequence, in differences in cell affinity between EphB-positive and ephrin-B-positive cells (Solanas and Batlle 2011). The RhoA small GTPase has a central role in the control of dynamic reorganization of the actin cytoskeleton required for cell migration and adhesion (Poliakov, Cotrina et al. 2004). RhoA small GTPase also controls Eph receptors forward signalling. Eph forward signalling regulates cell morphology by rearranging the actin cytoskeleton (Egea and Klein 2007). In tumour cells, Rho family small GTPase reorganizes actin cytoskeleton by

interaction with specific downstream effector, such as phosphatidyl inositide 3-kinase (PI3K). PI3K helps to establish cell polarity and to push the membrane forward in finger-like structures known as filopodia and in sheet- like structures known as lamellipodia. These structures generate the locomotive force in migrating cells and thus increase cell motility and tumor metastasis (Yamazaki, Kurisu et al. 2005). Inappropriate expression and regulation of Eph receptors also affect cell-matrix interaction by modulating integrin activity (Cheng, Brantley et al. 2002). In normal tissues, Eph receptors interact with the integrin enforcing cell to matrix adhesion (Poliakov, Cotrina et al. 2004). Integrins are cell surface receptors that bind to proteins in the extracellular matrix (ECM) and induce signal transduction pathways. Differential integrin expression affects cell adhesion, migration, survival, and differentiation, thus influencing cell motility and invasion and contributing to carcinogenesis (Surawska, Ma et al. 2004). For example, activation of EphA2 receptor in prostate carcinoma cells induces inhibition of integrins-mediated cell adhesion through rapid recruitment of the protein tyrosine phosphatase SHP2 and subsequent dephosphorylation and inactivation of focal adhesion kinase (FAK). This has resulted in converting non-invasive prostatic cancer cells into invasive pattern (Taddei, Parri et al. 2009). EphrinB1 reverse signalling has been reported to induce secretion of matrix metalloproteinase and promote invasion of glioma, pancreatic, gastric and leukemic cancer cells in vivo and in vitro (Pasquale 2010).

In contrast, Eph receptors and their ephrin ligands act as tumour suppressors (Chen, Zhuang et al. 2008). EphA2 KO mice, for example, show increased susceptibility to carcinogen-induced skin tumours and their invasive malignant transformation. EphA2 activation inhibited extracellular signal-regulated kinase ERK1/2 activities and reduced cell proliferation in wild-type mice. Interestingly, EphA2 was overexpressed in skin tumours compared with surrounding normal skin in wild-type mice, similar to the observations in human cancers. Together, these results make EphA2 an ideal molecule for antibody targeting, minimizing adverse effects in normal cells (Guo, Miao et al. 2006).

5. Colorectal Cancer (CRC)

5.1 Pathogenesis of CRC

The exact cause of CRC is unknown; however, there are multiple factors, which can predispose to the development of CRC. CRC development is a combination of environmental as well as genetic factors

6.1.1 Environmental factors d) Diet

Protein rich diet; red meat and saturated fatty food all are associated with increased risk of CRC. The exact cause is unknown but some theories said that red meat metabolism is associated with mitogens production. However, some studies negated such relationship. In contrast, low-fat diet is associated with decreased risk of CRC. The exact cause is not clear yet; nevertheless, the postulation that these protective foods are associated with methionine production, which improves DNA methylation, is largely supported (Chan and Giovannucci 2010). e) Obesity

Obesity and reduced physical activity are associated with increased risk of CRC. Obesity is associated with modifications in the level of adipocytokines and increased Insulin resistance leading to increased level of insulin and fatty acids. Cell exposure to high level of insulin and fatty acid interferes with cell signalling and produces high level of mitogens (Gunter and Leitzmann 2006, Bardou, Barkun et al. 2013). f) Alcohol consumption

The relationship between alcohol consumption and CRC is an area of ongoing controversy. In general, alcohol consumption increases CRC risk through reducing folate absorption, increasing acetaldehyde, suppressing tumor immune surveillance, delaying DNA repair, altering composition of bile acids, or inducing cytochrome p450 enzymes to activate hepatic carcinogens (Chan and Giovannucci 2010, DeVita 2011) (1).

6.1.2 CRC genetic factors

CRC develops as a result of multistep histological phenotypic changes in the epithelial lining of the large bowel. Initially, the environmental factors together with the genetic and epigenetic alterations in the lining epithelium result in the transformation from a normal colorectal epithelium into adenomas (Terry and Neugut 1998, Boutron-Ruault, Senesse et al. 2001). Over several years, adenomas gradually acquire further genetic changes and hence alteration in their phenotypes and become ultimately an invasive cancer. The multistep phenotypic changes of the CRC epithelium are caused by genetic alterations in the tumour suppressor genes as well as in the oncogenes. All the hereditary components of CRC are caused by the germline genetic mutations whereas 80% of the sporadic CRC are caused by these genetic alterations (Leslie, Carey et al. 2002, DeVita 2011, DeVita, Lawrence et al. 2011) (1).

Two inherited syndromes, familial adenomatous polyposis (FAP) and hereditary non-polyposis CRC (HNPCC) together account for about 5% of all CRC cases. They are caused by mutations in APC gene and DNA mismatch repair gene respectively (DeVita, Lawrence et al. 2011) (1).

a) Familial Adenomatous Polyposis (FAP)

The classical FAP is an autosomal dominant disorder that causes about 0.5% of all CRC. APC mutations almost invariably cause FAP phenotype. APC gene mutations are located between codons 1250 and 1464. Individuals develop hundreds of polyps by their teens and their lifetime risk of progression to CRC reaches 100%. The average age at which an individual develops colon cancer in classic familial adenomatous polyposis is 39 years. Extraintestinal manifestations include of the duodenal and gastric adenomas, congenital hypertrophy of the retinal pigment epithelium, jaw cysts, sebaceous cysts, and osteomata. The combination of polyposis, osteomas, fibromas and sebaceous cysts is termed Gardner's syndrome and less commonly, Turcot syndrome, if associated with brain tumours (Galiatsatos and Foulkes 2006, Rustgi 2007, Fearon 2011).

The attenuated FAP phenotype has fewer than 100 adenomas. The germline APC mutations are located in exons 1-4, exon 9 and distal to codon 1580 in exon 15 (Segditsas and Tomlinson 2006). b) Hereditary Non Polyposis CRC (HNPCC)

Hereditary non-polyposis syndrome (HNPCC), known as Lynch syndrome, is the most common type of hereditary CRC, contributes to approximately 5% of overall incidence. HNPCC patients have strong family history with multiple generations affected at early age, and usually develop tumours at proximal colon. Genetically, it is caused by mutations of the mismatch repair gene (MMR) leading to accumulation of mutations. HNPCC patients often carry germline mutation in one allele of any one of MMR genes, while the second allele would be targeted by mutation, deletion or promoter methylation of the CpG islands. MMR genes are tumor suppressor genes that any mutation in it would cause microsatellite instability (MSI), which is the hallmark of HNPCC patients. These patients are characterized by developing synchronous and metachronous CRCs, and the predisposition to extracolonic cancers in different organs (Umar, Boland et al. 2004, Fearon 2011). β-catenin mutations are largely observed in the context of the HNPCC (Segditsas and Tomlinson 2006). c) Sporadic CRC

The other type of CRC is the sporadic variant. Sporadic CRC cases usually do not have family history (de la Chapelle 2004). The majority (70-80%) of the CRC cases are sporadic. Genetic alterations of sporadic CRC are not in the germline level but are acquired by the cancer cells (Fearon 2011). These genetic alterations accumulate over the time. Together with the epigenetic alterations, they cause the phenotypic changes from adenoma to carcinoma.

Mutations of the APC gene have been identified in 70-80% of sporadic colorectal adenomas and carcinomas other than FAP. While germline half of the APC gene, somatic ׳mutations in FAP are confined to the 5 mutations accumulate in the central region of the frame, spanning codons 1286--1513 of exon 15 (Lüchtenborg, Weijenberg et al. 2004, Fearon 2011). These mutations lead to frameshifts or stop codons, that lack several 38

of the 20-amino acid repeats (20AAR) that interact with β-catenin and the serine alanine methionine proline (SAMP) interaction domains for axin/conductin. This leads to accumulation of β-catenin in the nucleus and nonantagonised transcription of Wnt target genes (Behrens 2005). APC somatic mutations are an early event in the development of adenomas. Later on, inactivation of both alleles takes place and results in the transformation from adenoma to invasive cancer (Fearon 2011).

β-catenin mutations are encountered in 10-15% of all sporadic CRC (Lugli, Zlobec et al. 2007). These either deletes the whole of exon 3 or target individual serine or threonine residues encoded by this exon. Serine/threonine are phosphorylated by the degradation complex, and hence causes β-catenin to escape from the destruction process (Segditsas and Tomlinson 2006). Studies have shown that nuclear β-catenin is a reliable prognostic marker for the progression of CRC. It is increasing during the transformation from adenoma to carcinoma. Its level is also increased in the presence of metastases (Lugli, Zlobec et al. 2007). Combination of β- catenin with other tumor markers increases its sensitivity as a tumor marker. It can detect most colorectal cancers and identify a subset of patients with poorer outcomes (Zhang, Ougolkov et al. 2003). However, other study showed that nuclear β–catenin expression in CRC is associated with a better prognosis (Chung, Provost et al. 2001).

Axin1 and Axin2 are important components of the β-catenin degradation complex. Axin1 mutations have been found in sporadic CRC at codons 395, 411 and 418 (GSK3β-binding domain) and codons 101, 103 and 122 whereas Axin2 mutations have been reported in MSI+ CRC (Segditsas and Tomlinson 2006). g) Adenoma-carcinoma sequence

The term adenoma-carcinoma sequence indicates sequential changes in the colonic epithelium from normal to dysplastic and then from dysplastic to carcinoma. The adenomatous polyps are considered as precancerous lesions as they contain dysplastic cells. Fortunately, only a small proportion of these polyps undergo malignant changes. Endoscopic resection of these

polyps prevents the occurrence of CRC. Both oncogenes and tumor suppressor genes are involved in adenoma-carcinoma sequence (Fearon 2011).

APC mutation is considered as the triggering point of the adenoma- carcinoma sequence. Subsequent accumulation of other mutations including the oncogenic mutations of K-RAS occurs early in the adenoma-carcinoma sequence. It helps in the tumourigenic progression of adenomas. The malignant transformation is often accompanied by the acquisition of the vital mutations of p53 at late stage. Inactivation of p53 most often occurs as a result of missense mutations in the DNA-binding domain and this leads to propagation of damaged DNA. It is considered as the guardian preventing damaged DNA from being processed (Leslie, Carey et al. 2002). e) Epigenetic mechanisms of CRC

Epigenetic changes can alter gene expression by affecting the promoter region without changing any DNA sequence. There are two major mechanisms attributed to epigenetic modulations, which include DNA hypermethylation and histone acetylation (Bandres, Agirre et al. 2009).

Methylation of CpG dinucleotides in the promoter region of genes is an important mechanism of gene regulation whereas aberrant hypermethylation of tumor suppressor genes plays a significant role in tumor development. It is associated with downregulation of tumor suppressor genes (Bandres, Agirre et al. 2009).

Histone modifications also play critical roles in epigenetic silencing. Histone proteins assemble into nucleosomes, which function as DNA packaging units as well as transcriptional regulators. Acetylation and/or methylation of histones lead to the formation of an open chromatin structure yielding a disfigured chromatin structure, which is incapable to function normally. Studies have shown that modifications of histone contribute to tumor suppressor gene silencing (Kondo, Shen et al. 2003).

5.2 Role of Eph/ephrin in CRC and some of their clinical implications in the cancer management

Both A- and B-subclasses of Eph receptors and their ligands ephrins are involved in CRC growth, progression and invasion. Some of these receptors are upregulated while the others are downregulated during the cancer progression (Herath and Boyd 2010).

5.2.1 The role of EphAs in CRC

EphA1 was cloned from a carcinoma cell line in a screen for new oncogenic tyrosine kinases (Pasquale 2010). It is widely expressed in different human tissues including the gut. Its role in CRC is still not fully understood. It has a biphasic pattern of expression in CRC (Herath and Boyd 2010). In a study conducted by Dong et al, EphA1 gene expression was downregulated in 37% and upregulated in 40% samples of CRC tested. Reduced expression was seen in a higher proportion of male patients, with poor differentiation and in lymph node metastasis. Overall, its underexpression correlates with invasion and metastasis (Dong, Wang et al. 2009). EphA1 has a CpG island encompassing 3′ and 5′ ends of the gene. These CpG islands are significantly hypermethylated in EphA1 low expressing CRC cells. Epigenetic silencing explains the low expression of EphA1 and low EphA1 correlates with poor prognosis of CRC. Thus, epigenetic silencing of EphA1 in CRC correlates with poor survival (Herath, Doecke et al. 2009, Pasquale 2010).

EphA2 is upregulated in many cancers and its expression has been linked to a poor clinical prognosis (Pasquale 2010). It is overexpressed in CRC cells (Bogan 2009). EphA2 has a prognostic role in CRC; high EphA2 receptor expression in CRC is associated with a worse outcome in patients treated with cetuximab-based therapy (Strimpakos 2013). It has been shown that E-cadherin regulates EphA2 in normal tissues by keeping cell-cell contact. Reduced expression of E-cadherin has been associated with aggressive clinicopathological phenotypes of CRC. Further, overexpression of EphA2 in CRC correlates with cancer progression. Thus, EphA2 and E-cadherin have an inverse relationship in CRC progression (Tetsuya Saito 2004). Furthermore, the activation of EphA2 by ephrinA1 can alleviate E-cadherin

mediated contact inhibition in tumor cells (Potla 2002). Src-like adaptor protein (SLAP) belongs to the subfamily of haematopoietic adaptors that inhibit intracellular signalling. In metastatic CRC cells, SRC phosphorylates EphA2, which is essential for the promotion of tumour cell growth and invasion. SLAP is implicated in the negative regulation of RTK. Thus, it has a tumor suppressing role in CRC via destabilization of the SRC substrate and receptor tyrosine kinase EphA2 (Naudin 2014). On the other hand, Kataoka and his team found that EphA2 is significantly overexpressed in early stages of CRC than late stages. They also found that there is a positive correlation between angiogenesis and EphA2-expressing tumor tissues supporting the hypothesis that EphA2 is important for tumor growth in stages I and II probably through their effect on microvessel induction (Kataoka, Igarashi et al. 2004).

EphA3 expression positively correlates with CRC progression, depth of invasion, lymph nodes metastasis and distant metastasis (Xi and Zhao 2011). EphA3 is considered as an independent reliable prognostic marker in CRC. Moreover, it is a potential therapeutic target in selected CRC cases. 5% of all CRC cell lines have a mutation in EphA3 gene (Bardelli, Parsons et al. 2003).

EphA4 is a reliable predictive gene of distant metastasis in CRC. EphA4 expression had been studied in surgical specimens of cancer tissue and adjacent normal mucosa obtained from 205 patients with untreated CRC. The relative expression levels of EphA4 mRNA in the specimens were measured by quantitative real-time, reverse-transcription polymerase chain reaction (RT-PCR). The relative expression level of EphA4 mRNA was higher in the presence than in the absence of liver metastasis. Overexpression of the EphA4 gene promotes liver metastasis in CRC. Overexpression of the EphA4 gene may thus be a useful predictor of liver metastasis in patients with CRC (Oshima, Akaike et al. 2008).

EphA7 is downregulated in CRC. Hypermethylation of 5´ CpG Island has been shown to inactivate EphA7 gene in CRC tissues in vivo and in vitro. Hypermethylation of EphA7 was more common in males than in females (Wang, Kataoka et al. 2005). In a study included 13 genes used for risk 42

stratification in stage II CRC patients (13-gene expression classifier), EphA7 was among these genes. It confirmed the independent prognostic value of the classifier (p≤0.004). The classifier was shown to be specific to stage II CRC and does not provide prognostic stratification of patients with stage III CRC (Agesen, Sveen et al. 2012). Hafner et al studied the expression of Eph receptors and ephrin ligands in different healthy and tumours tissues using RT-PCR and immunohistochemistry (IHC). They found that EphA8 was not expressed in any of the healthy tissues and was down-regulated in colon cancer (Hafner, Schmitz et al. 2004),

5.2.2 The role of EphBs and ephrinBs in CRC a) EphB1 expression in CRC

EphB1 is preferentially highly expressed in normal colon together with other EphB receptors. Loss of EphB1 expression in CRC occurs during transition from adenoma to carcinoma. Sheng et al have investigated the involvement of EphB1 in CRC. Underexpression of EphB1 in colorectal tumours is associated with poor differentiation and cancer cells with low level of EphB1 protein show more invasive power (Sheng, Wang et al. 2008). In contrast, Liu and his group examined the expression of EphB1 together with the other EphB receptors and ephrinB ligands in 11 colon carcinoma cell lines and 20 human colon carcinoma specimens including the adjacent uninvolved mucosa of the same patients. Although, the phenotype of the examined specimens was not mentioned, EphB1 was not expressed either in the colon cancer specimens or the adjacent normal mucosa in comparison to the other EphB receptors and their cognate ligands (Liu, Ahmad et al. 2002). b) EphB2 expression in CRC

EphB2 is expressed in several human gastrointestinal tumours including CRC (Lugli, Spichtin et al. 2005). Lugli’s group investigated the expression of EphB2 receptor across 138 human tumours including CRC using tissue microarray. They found that EphB2 is highly expressed in normal intestinal epithelium and colonic adenomas but only in ~ 33% of CRC. Loss of EphB2 was associated with unfavourable tumour phenotype, presence of vascular invasion and infiltrative tumour growth (Lugli, Spichtin et al. 2005). This is 43

in consistent with previous mouse model systems linking EphB2 loss of expression to defects in the compartmentalization and differentiation of colorectal epithelial cells (Batlle, Henderson et al. 2002). Further, loss of EphB2 expression showed a strong, inverse association with patient survival (P-value=0.0098). Survival was significantly different between negative, weakly positive and strongly positive EphB2 tumor tissues (Lugli, Spichtin et al. 2005).

EphB2 receptor is a Wnt-target gene. Therefore, activation of β-catenin/TCF complex in the nucleus induced by Wnt pathway mutations initiates the Wnt- target genes transcriptional process including EphB2 (Marc van de Wetering 2002). Overexpression of EphB2 in CRC patients is associated with prolonged survival (Batlle, Henderson et al. 2002, Jubb, Zhong et al. 2005, Lugli, Spichtin et al. 2005, Guo, Zhang et al. 2006, Oshima, Akaike et al. 2008, Kumar, Scehnet et al. 2009). In 2005, Jubb et al conducted a study about the power of EphB2 gene as an independent prognostic factor in CRC. They assessed the gene expression in different histological stages of the cancer and compared them with that of adenomas and liver metastasis using IHC. Although the gene expression was higher in the cancer (82%) than that of adenomas (77%), however, it was higher than that of liver metastasis (64%). Further, the pattern was homogeneous in adenomas whereas it was focal in the cancer. In addition, patients with high levels of EphB2 expression had a longer overall survival in CRC (P-value= 0.047) (Jubb, Zhong et al. 2005).

EphB2 is highly expressed in the early stages of CRC. With the advancement of the tumour, silencing of EphB2 receptors takes place resulting in downregulation of these tumour suppressor genes (Gregorieff, Pinto et al. 2005, Herath and Boyd 2010). A study was conducted about the expression of EphB2 in normal colon, colonic adenomas, primary CRC, positive lymph nodes and liver metastases. The study revealed that EphB2 underexpression correlates with invasion, metastasis and adverse patient outcome (Guo, Zhang et al. 2006). Another study conducted on APC mutant EphB2 null mice had similar conclusion (Batlle 2005). In vitro studies 44

showed that EphB2 overexpression is associated with inhibition of colon cancer invasion and migration and slows the growth of colon cancer cells in xenografts (Senior, Zhang et al. 2010).

EphB2 induces tumour suppression in CRC by induction of autophagy (Tanabe 2011). It has the same effect on breast cancer cells (Chukkapalli, Amessou et al. 2014). Autophagy, as the name indicates, is cellular self- degradation process characterized by the presence of abundant intracellular autophagic vacuoles termed autophagosomes. These vacuoles accumulate cellular proteins and organelles and present them to the lysosomal body for engulfing or recycling (Kim 2013) . Although its role in cancer is complex with controversy between tumor suppression versus tumour progression (Mathew 2007, Choi 2012), its role in CRC has been recently established as a tumor suppressor via generation of reactive oxygen species and activation of JNK in human colon cancer cells (Kim 2013). Sesamin, a major lipid- soluble lignan of sesame oil, is a natural chemopreventive through inhibition of the activation of NF-κB pathway; NF-κB is associated with carcinogenesis (Harikumar 2010). In the normal condition, EphB2 receptor tyrosine kinase gets activated upon receptor-ligand binding leading to forward signaling- dependent ISC proliferation (Holmberg 2006). In CRC, Sesamin induces autophagy by inhibition of tyrosine kinase phosphorylation of EphB2 receptor. Further, silencing of EphB2 receptor abrogates sesamin-induced autophagy (Tanabe 2011).

In normal colonic epithelium, EphB2 regulates proliferation of ISC in a kinase-dependent manner through Abl and cyclinD1 (cyclinD1 is an effector of EphB2 signalling), whereas migration of differentiated cells is mediated in a kinase-independent signaling cascade through PI3-kinase (Genander and Frisen 2010) (fig 1.8).

Fig 1.8: Complimentary expression of the EphB receptors and their ligands along the crypt long axis. The EphB receptors are responsible of proliferation and migration of the ISC whereas the ephrinB ligands are responsible for maturation of the progenitor cells into more function-specific form (a). EphB2 promotes ISC proliferation and migration through distinct pathways (b) (Genander and Frisen 2010) (with permission).

In adenoma and early stages of CRC (fig 1.9), EphB2 functions as a tumor suppressor gene by interacting with ephrinB ligands preventing tumor cell migration through forming compartments around them (Cortina, Palomo- Ponce et al. 2007).

Fig 1.9: Compartmentalization phenomena induced by EphB-ephrinB interaction isolating tumour cells (dashed line) from surrounding normal epithelium (arrowhead) (Cortina, Palomo-Ponce et al. 2007) (with permission).

During tumor progression, EphB2 inhibition is switched off and tumor cell proliferation and migration become independent. Genander and her team attributed this phenomenon to cyclinD1. During tumour progression, cyclinD1 becomes independent of EphB2 allowing continued proliferation and migration of the tumour cells (Genander, Halford et al. 2009).

Inactivation of EphB2 has been proven to cause rapid tumour growth. Alazzouzi and his team found for the first time that DNA hypermethylation of EphB2 promoter in CRC is the cause of EphB2 inactivation in the tumor. They analysed 60 microsatellite instable (MSI) and 41 microsatellite stable (MSS) CRC samples for DNA hypermethylation. DNA hypermethylation of CpG islands has been found in 54/101 (53%) of the cases. They concluded that DNA hypermethylation of EphB2 promoter in CRC is behind receptor inactivation in the tumor (Alazzouzi, Davalos et al. 2005). However, Fu et al have studied the potential mechanisms behind the downregulation of EphB2 in CRC. They found that DNA hypermethylation was not the actual cause behind EphB2 suppression in their series. Rather, they found that c-Rel acts as a transcriptional repressor of EphB2 causing downregulation of EphB2 in CRC. c-Rel is a member of NF-ᴋB family of transcription factors. NF-ᴋB family regulates gene transcription through co-activators and co-suppressors. c-Rel inhibits EphB2 transcription by direct binding to the negative regulatory element (NRE) in the EphB2 gene (Fu, Li et al. 2009). A second alternative to hypermethylation; is decreased histone levels in the EphB2 expressing cancer cells causing epigenetic silencing of the receptor (Ronsch 2011). Another mechanism of inhibition of EphB (EphB2 and EphB4) receptors expression in CRC is tissue hypoxia. Hypoxia inducible factor-1 (HIF-1) produced during hypoxia inhibits β-catenin from binding with TCF-4 thus inhibiting the transcription of the target genes including EphB receptors (Kaidi, Moorghen et al. 2007). In conclusion, there are distinct mechanisms that are claimed in the downregulation of EphB receptors in general

(Ronsch 2011).

Tumour budding is defined as detachment of tumour cells at the invasive front of CRC from the surrounding connective tissues and assembly into clusters of up to five cells to fight against the basement membrane in order to find their way to the surrounding lymphatic and blood vessels resulting in local and distant metastasis. It has been linked to the process of epithelial- mesenchymal transformation (EMT) where the epithelial cells silence the membranous E-cadherin and express fibronectin within the cytoplasm

acquiring a more mesenchymal phenotype. Tumour budding has positive correlation with the advancement of CRC and it is a predictor of lymphatic and blood vessels invasion. It is considered as a category IIB prognostic factor in CRC (A Lugli 2012) (fig 1.10).

Fig. 1.10: Tumour buds, defined as individual cells or small clusters (<5 cells) of tumour cells at the invasive front (arrows), (Mitrovic, Schaeffer et al. 2012) (with permission).

The first detailed description of tumor buds was by Gabbert et al who described tumour budding as a tumour within a tumour with well-developed glandular structures giving way to isolated tumor cells. Tumor budding is considered as an independent adverse prognostic factor in CRC that may allow for stratification of patients into risk categories irrespective to the TNM staging, and also potentially guide treatment decisions, especially in early stages of CRC without lymph nodes or distant metastasis. It has been found that SII CRC patients with tumour budding have worse outcome that SIII CRC without tumour budding. However, tumor budding has received little attention and the qualitative and quantitative integrity of tumour budding is yet to be further identified in order to make it more proactive histopathological prognostic tool (Mitrovic, Schaeffer et al. 2012). Karamitopoulou et al studied the relationship between the expression of several biomarkers including EphB2 receptor and the presence of high grade tumour budding in mismatch repair (MMR)-proficient CRC. The most accurate markers for predicting high-grade budding in MMR-proficient CRC was EphB2. Loss of EphB2/ Bcl-2 has been linked to the presence of high-

grad e tumour budding and subsequently more aggressive disease (Karamitopoulou, Lugli et al. 2010).

c) EphB3 expression in CRC

Over expression of EphB3 enhances cell-cell contact and suppresses tumour growth in CRC (Chiu, Chang et al. 2009). EphB3 suppresses tumor growth through promoting mesenchymal-epithelial transformation (MET) demonstrating the formation of tight junctions between the cells and thus restricting their motility. EphB3/ephrinB interaction potentiates junctional adhesion molecules E-cadherin, adherins and desmosomes. On the other hand, loss of EphB3 is associated with CRC invasiveness. Battle et al found that even single EphB3 null allele resulted in invasive phenotype of CRC in mice (Batlle 2005). Recently, Jagle et al identified a transcriptional enhancer located in the 5′-Flanking Region of EphB3 gene. It is controlled by Notch signalling and its dysfunction underlies EphB3 silencing. Downregulation of Notch signalling contributes to impaired enhancer activity (Sabine Jägle 2014).

d) EphB4 expression in CRC

Recent evidence shows a dual function of EphB4 receptor in CRC. While evidence of behaviour as a tumour promoter in CRC in some studies, a suppressor role has been suggested in others.

EphB4 is usually underexpressed or even lost in advanced stages of colorectal tumours (Batlle 2005, Dopeso, Mateo-Lozano et al. 2009). There is a significant correlation between the level of expression of EphB4 and tumour prognosis. Patients with low EphB4 tumour levels have significantly shorter survival than patients with high EphB4 level. Low level of EphB4 receptors can identify CRC patients that are at high risk of recurrence. Thus, EphB4 expression can be considered as prognostic marker in CRC

(Stephenson, Slomka et al. 2001, Davalos, Dopeso et al. 2006, Herath, Spanevello et al. 2012).

On the other hand, other studies revealed that EphB4 overexpression correlates with poor prognosis (Stephenson, Slomka et al. 2001, Liu, Ahmad et al. 2002, Herath and Boyd 2010). Kumar and his team made a comparison between the expression pattern of EphB2 and EphB4 in the same CRC patients. They found that while EphB2 expression was normal in healthy colon tissues, expression was lost as carcinoma progressed, whereas EphB4 was upregulated with the disease progression. They postulated that while EphB2 and EphB4 are Wnt signaling target genes, they are differentially expressed because they use different co-activators in the Wnt pathway. EphB2 uses p300 co-activator whereas EphB4 depends on CBP co-activator. With the disease progression Wnt signaling becomes more dependent on CBP at the expense of p300 resulting in upregulation of EphB4 and downregulation of EphB2. They also found that overexpression of EphB4 is associated with blocking of apoptosis resulting in prolonged survival of tumor cells (Kumar, Scehnet et al. 2009). Further, EphB4 expression in tumour cells favours the dissemination of metastatic cells by decreasing cell-cell contact and cell-matrix adhesion thus promoting more aggressive and invasive tumor phenotype (Noren, Lu et al. 2004).

One of the clinical applications of EphB4 expression in CRC is imaging of the cancer chasing the receptor expression in the malignant tissues. Positron Emission Tomography (PET) scan has become an essential diagnostic tool in cancer in general and in CRC in particular. It is a non- invasive imaging modality and helps to detect cancer at its early stages through the metabolic and the anatomical features of the cancer comparing to the other imaging modalities. Designed antibodies targeted against EphB4 expressing tumor cells labelled with radioactive materials have been used to detect CRC through PET scan using antibody-based imaging probes. Because these antibodies have high unmatched ability to selectively bind EphB4 receptor, and because EphB4 is over-expressed in CRC, these receptors can reliably be used as valid targets for CRC PET imaging (Liu 2013). However, the absolute tumor uptake of the antibodies, the

pharmacokinetics of these antibody-based probes and the clearance time of the antibody from the body need to be further improved in order to raise the sensitivity of the test. A similar study has been conducted targeting EphB4 receptor in CRC but using near-infrared fluorescence (NIRF)-dependent optical imaging rather than PET scan to detect the tumor. hAb47 antibody showed prominent uptake in vitro and in vivo and subsequently it can be used as an NIFR contrast agent for non-invasive imaging of EphB4- expressing CRC tissues (Li 2012).

Expression of EphB4–ephrin-B2 in tumour blood vessels has been well documented and EphB4 expressing tumours have higher blood vessel contents than EphB4-nonexpressing tumours. EphB4-ephrinB2 signalling mediates endothelial cells penetration during invasive angiogenesis and stabilizes newly formed blood vessels. VEGF upregulates ephrinB2 in endothelial cells. Endothelial ephrinB2 signalling mediates endothelial cell adhesion toward EphB4-expressing tumor cells by increasing cell-matrix adhesion (Noren, Lu et al. 2004). h) EphB6 expression in CRC

Loss of EphB6 gene in CRC correlates with poor prognosis. Samples for 124 CRC patients and samples of 57 adenoma cases were investigated for EphB6 expression using IHC method. The expression in cancerous tissues was less than that of the benign tissues. Loss of the receptor was associated with poorly differentiated phenotype (P-value<0.001) (Peng 2014).

f) EphrinB ligands expression in CRC

The ligand ephrinB2 is essential for angiogenesis in embryos as well as in adults. Loss of ephrin B2 results in fragile small vessels and the formation of microaneurysms (Foo, Turner et al. 2006). It is highly expressed in the colon (Batlle, Henderson et al. 2002). Ligand ephrinB2 is considered as tumor suppressor gene (Liu, Jung et al. 2004). Underexpression of ephrinB2 has been linked to cancer cell invasion and malignancy. It is also

involved in tumor angiogenesis. In contrast to the above mentioned, these newly formed blood vessels in cancer tissues are considered as non- functioning blood vessels; overexpression of ephrinB2 paradoxically results in new vessel formation but at the same time marked reduction of tumor growth (Liu, Jung et al. 2004).

Concluding remarks

Eph receptors and their binding ligands are largely involved in the primary CRC progression as well as in distant metastasis. They are considered as reliable prognostic markers (Stephenson, Slomka et al. 2001, Sheng, Wang et al. 2008). Most of the CRCs harbour at least one Eph receptor but at different level than the normal colon. EphB receptors and their ligands ephrinBs have higher contribution in CRC development than the A-subclass. With the exception to EphB4, all are considered as tumor suppressor genes in CRC (Fig 1.11).

High EphB1 EphB1 EphB1

EphB2 EphB2 EphB2 EphB3 EphB3 EphB3 EphB4 EphB4 EphB4 EphB6 EphB6 EphB6 ephrinB1 ephrinB1 ephrinB1 Low ephrinB2 ephrinB2 ephrinB2

Normal colon adenoma CRC I/II CRC III/IV

Fig 1.11: The expression of the EphBs-ephrinBs in the normal colon, in adenoma and in different phenotypes of CRC. With the exception to EphB4 gene, all are getting silenced with the progression of the disease.

However, to date, there is no single study in the literature, which has holistically investigated and compared the expression pattern of the EphB receptors and their ligands in the primary CRC with that in the hepatic metastasis of the same patients and correlating that with the influence on the patient survival. Three questions which could be answered in the future. First, can these molecules be considered as reliable anticancer agents relevant to the current chemotherapeutic agents or at least be conjugated with them? Although some of which are being investigated for anti-cancer therapy and showed significant results in the preclinical studies (Mao, Luis et al. 2004, Kertesz, Krasnoperov et al. 2006, Krasnoperov, Kumar et al. 2010, Tognolini 2014), however, because most of the EphB receptors and their ligands are downregulated in the most aggressive phenotype of the tumour, the value of this approach is currently of little utility. Moreover, the safety and efficacy of these agents in the human beings yet need to be elucidated and licensed to be used in clinical practice. Second, can they replace the current tumor predictive biomarkers of the CRC or at least be integrated with them to increase their predictive power? The clinical predictability of the EphB4 receptor, for instance, in the detection of the recurrence of the CRC patients treated with chemotherapy has been investigated and found it is a reliable predictive biomarker for CRC patients among other biomarkers (Guijarro- Munoz, Sanchez et al. 2013). However, a broader study, which would include other Eph receptors can confirm or negate the above assumption. Third, can these molecules replace the current conventional screening tools for the CRC disease or at least be integrated with them to enhance the screening power of these tools? Recent advances have greatly enhanced our understanding in the molecular and genetic basis of CRC. This improved understanding has enhanced the ability to provide information regarding etiology, prognosis, and even response to therapy for CRC. Nevertheless, significant research remains to be conducted to more finely characterize the underlying mechanism of CRC, allowing improved diagnosis of CRC (Daniel O. Herzig 2015).

Chapter Two

Introduction

CRC is one of the leading causes of mortality in Australia. Advances in the molecular basics of CRC have improved the management of CRC patients by integrating molecular biomarkers into the diagnostic checklist of the CRC. These advances have also unveiled other molecular aetiologies of CRC beside the current molecular pathways. However, the integrity of these molecular subgroups of CRC are not thoroughly studied, and further studies are needed to determine whether these molecular subclasses can be used as reliable and accurate indicators for the early detection of CRC (Grady and Markowitz 2014).

The unique RTK subfamily membrane-bound EphB receptors and ephrinBs interact together to organise structure and function of distinct epithelial cell compartments within the intestinal crypts. They are key regulators of intestinal homeostasis through governance of proliferation in the ISC and differentiation in the transient ambulatory cells. The deregulation of EphB and ephrinB expression in the intestinal epithelium is linked to malignant and disease status in the colon (Perez White and Getsios 2014). EphB receptors and their cognate ephrinB ligands are differentially expressed in CRC. The relationship between EphB receptors, their ephrinB binding ligands and CRC progression, invasion, metastasis and malignant neovascularisation has been unveiled in many occasions (Herath and Boyd 2010).

EphB1 is involved in the progression, invasion and metastasis of CRC. Reduced expression of EphB1 in CRC occurs in poorly differentiated phenotype. In vitro, cancer cells with a low level of EphB1 protein showed more invasive behaviour than EphB1-high expressing cancer cells (Sheng, Wang et al. 2008).

EphB2 is present at highest level in the ISCs and its expression gradually decreases in the progenitor cells as they migrate towards the lumen (Batlle, Henderson et al. 2002). Its down regulation is an independent prognostic factor for recurrence and death in CRC patients (Jubb, Zhong et al. 2005). 54

EphB2 is a tumour suppressor gene that abrogates a possible tumour growth advantage (Senior, Zhang et al. 2010). It works mutually with ephrinB1 and ephrinB2, which are secreted by the tumour stromal tissues to prevent migration of the tumour cells. Its loss and/or silence inhibit the repulsive forces imposed by the ephrinB1 and ephrinB2 and subsequently are associated with progression of the disease (Cortina, Palomo-Ponce et al. 2007). Hypermethylation of CpG islands in EphB2-expressing tumour cells (Alazzouzi, Davalos et al. 2005) and tissue hypoxia (Kaidi, Moorghen et al. 2007) have been linked to receptor inactivation in CRC.

Studies have given apparently contradictory results about EphB4 expression in CRC. While some studies have proven that EphB4 behaves as a tumour suppressor gene in CRC (Dopeso, Mateo-Lozano et al. 2009), the others showed that CRC cells preferentially express EphB4 over EphB2 and EphB4 promotes CRC metastasis (Kumar, Scehnet et al. 2009).

Although there have been several publications looking at the expression of the individual Eph receptors in CRC, none have looked at the expression patterns of the three EphB receptors and their ligands in the primary tumour and in the liver metastasis of the same patients.

Research Hypotheses

1. Loss of EphB1 in CRC only occurs in the most undifferentiated tumour tissues.

2. EphB2 downregulation is associated with EphB4 upregulation in CRC. EphB4 expression is only seen in EphB2 negative CRC tumour cells.

3. If EphB2 downregulation and EphB4 expression are associated with tumour progression, then liver metastasis would be expected to be EphB2 negative EphB4 positive cells, irrespective of the receptor status of the primary tumour.

Aims of the project

1. Assess the expression of EphB1, EphB2 and EphB4 transmembranous receptors in human primary colon tumors,

2. Correlate expression patterns with patient demographic and clinicopathological data,

3. Assess the expression pattern of EphB1, EphB2 and EphB4 in primary tumors and liver metastases from the same patient to determine whether receptor status changes during the metastatic process.

Ethics approval and ethics consideration

Ethics approval has been obtained from ethics committee in the Western Centre for Health, Research and Education (WCHRE) and from Victorian Cancer BioBank (VCB). Only de-identified patient data were obtained from the VCB. There was no direct contact with the participants. The project does not involve genetic testing and the outcomes are unlikely to affect individual patient management. All issues with informed consent and ethical recruitment of the patients have been dealt with by the VCB.

Inclusion and exclusion criteria

1. Colonic tumours from equal numbers of males and females aged 50- 70 years; the most common age of non hereditary CRC is 50 years and above and the incidence rate increases greatly with age, especially after 50 years old.

2. Stage II, III and IV CRC based on the American Joint Cancer Committee (AJCC) staging system,

3. Ideally the patient will not have undergone adjuvant therapy prior to resection,

4. In addition, a sub-cohort of patients for whom sections of primary tumour and hepatic metastatic lesions were banked was examined,

5. Patients with genetic predisposition to colon cancer (e.g. Lynch syndrome) were excluded, 56

6. Patients with predisposing conditions (e.g. Inflammatory Bowel Disease) were excluded,

Design of the study

Overview

In order to determine the expression of EphB1, EphB2 and EphB4 receptors in primary CRC and in liver metastasis, immunohistochemistry (IHC) was performed on the sections using antibodies directed against EphB1, EphB2 and EphB4 receptors (Senior, Zhang et al. 2010, Arik Drucker 2013).

IHC tissue reactivity was assessed using a combination of three scoring systems: intensity of staining, proportion of tumour cells staining throughout the tissue section, and degree of differentiation of the tumour. Data scoring was collected by the student conducting this project and by a qualified pathologist independently.

The statistical significance of the data was assessed and analysed using Linear Trend test and analysis of variance (ANOVA) test.

Immunohistochemistry

Immunohistochemistry (IHC), as the name indicates, is the identification of a certain antigen in a histological tissue section or cytological preparation via an antibody specific to the antigen. The localization of the primary antibody (and therefore the target antigen) is then visualized microscopically via an appropriate enzymatic or fluorescent detection system (Renshaw 2007).

In 1941, The Albert H. Coons was the first who attached a fluorescent dye to an antibody and used this antibody to localise its respective antigen in a tissue section (Coons, Creech et al. 1941). This was considered as the beginnings of IHC, which has developed remarkably since then. Currently, IHC is not only used for research but is increasingly employed for diagnosis and for the assessment of therapeutic biomarkers (Walker 2006). IHC has provided a revolutionary approach to histopathology in terms of analytical

and functional morphology. It has important applications in different body tissues in physiology as well as in the disease status. Its use in cancer has provided remarkable diagnostic, prognostic and predictive information relative to cancer status and biology. Due to the continuous advancements in tumour biology, and to allow for more understanding of the molecular changes in cancer development, progression and metastasis, there has been a constant effort to enhance the sensitivity of the IHC technique in molecular pathology to detect various tumour markers at different cellular levels (Dabbs 2013).

There are basically five types of specimens commonly used in IHC staining experiments (Renshaw 2007):

• Paraffin-embedded tissue sections, example is formalin fixed paraffin- embedded (FFPE),

• Frozen tissue sections (FS),

• Free-floating tissue sections,

• Cytological specimens as traditional smears,

• Cytological specimens as monolayer preparations.

Paraffin embedded tissues have become the tissues of choice for most clinical and research studies. Tissue blocks are fixed to a medium, which helps to preserve morphology, for example to formalin, and then processed to paraffin wax in order to give the tissue support during microtomy. Sections are cut at around 4µm thickness and floated out in a water bath before being picked up on to a glass microscope slide. The sections are then dried by keeping them in a 60°C oven for approximately 60 minutes in order to increase their adherence to the slide and to help to remove any creases obtained during microtomy or floating. Before immunochemical staining can occur, sections must be dewaxed and any necessary pre-treatments performed (Renshaw 2007). In this study, only FFPE sections were utilized to conduct the project.

Common IHC methods (Miller 2013):

The most common IHC methods used are avidin – biotin based method and polymer-based method.

A. Avidin-Biotin Complex (ABC) based method

In 1981, Hsu et al have defined the method of avidin-biotin-peroxidase complex which is abbreviated as ABC method (Hsu, Raine et al. 1981). The ABC based method takes advantage of the strong affinity of biotin for avidin, which has binding sites for 4 molecules of biotin. Basic steps in ABC staining method are as follows (fig 1.1):

1. Mounting sections on adhesive slides, deparaffinization in histoclear and rehydration in graded alcohols,

2. Performing antigen retrieval (AR) either enzyme-induced epitope retrieval (EIER), or heat-induced epitope retrieval (HIER),

3. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2),

4. Blocking with ImmunoHistoChemistry (IHC) blocking reagent to block non- specific binding,

5. Incubation with primary antibody,

6. Incubation with biotinylated secondary antibody,

7. Incubation with ABC,

8. Detection of the antigen-antibody immune complex by incubation with chromogen reagent such as DAB (3,3'-diaminobenzidine) substrate,

9. Counterstaining using haematoxylin,

10. Dehydration, coverslip, and view under the microscope.

Fig 2.1: IHC staining using ABC based method. Secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and ABC.

The main disadvantage of the ABC method is the artifactual staining, which interferes with the proper assessment of the target staining. Artifactual staining is precipitated by the reaction between the endogenous biotin and the avidin in the detection complex. The presence of endogenous biotin can be seen by omitting the primary antibody. Endogenous biotin artifact usually appears as granular cytoplasmic reactivity, and is particularly noticeable in cells that contain numerous mitochondria, such as liver cells and kidney proximal tubules (Miller 2013).

The use of HIER techniques (see section (A), antigen retrieval method), is associated with more artifactual staining due to the release of masked endogenous biotin and make them ready to be conjugated by avidin. Artifactual staining interferes with the interpretation of the actual staining as it has the same colour and subsequently, decreases the accuracy of the staining method (fig 2.6). For this reason, any laboratory employing HIER techniques should routinely include steps for blocking of endogenous biotin 60

in any immunostaining that is subjected to HIER. Steps taken to block endogenous biotin should be performed immediately after any HIER or even protease digestion steps and before the incubation with primary antibody (Miller 2013). These limitations are further exacerbated by the use of FS, in which levels of endogenous biotin are usually even higher than those encountered in paraffin-embedded specimens (Kumar 2009) (1). However, FS have been generally exempted from HIER methods because such sections are fragile and are easily destroyed by heating (Ino 2003).

Blocking endogenous biotin can be done with commercially available kits or by incubation with dilute egg white solution and dried milk solution (Igor B. Buchwalow 2010) (1).

B. Polymer-Based Method (Miller 2013)

Because of the limitations with the ABC based staining, polymer-based immunohistochemical methods have been introduced and are gaining popularity. Polymer-based staining system is an enzymatic, non-biotin, one- step detection kit that provides very high sensitivity with very low background staining in immunohistochemical applications. It contains bound secondary antibody and horseradish peroxidase detection complex enzymes, resulting in a simplified technique with fewer steps, as follows:

1. Mounting sections on adhesive slides, deparaffinization in histoclear and rehydration in graded alcohols,

2. Performing AR either enzyme-induced epitope retrieval (EIER), or heat- induced epitope retrieval (HIER),

3. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2),

4. Blocking with Ready-To-Use (RTU) blocking agent such as Horse Serum Block (HSB) block,

5. Incubation with primary antibody,

6. Incubate with RTU polymer complex (containing link antibody and enzyme detection complex) (fig 2.2),

7. Detection of the antigen-antibody immune complex by incubation with chromogen reagent such as DAB substrate,

8. Counterstain, coverslip, and view under the microscope.

Fig 2.2: Polymer-based IHC (such as the Vector Labs ImmPRESS™). After incubation with the primary antibody (a), RTU Impress reagent, which contains secondary antibody and conjugated horseradish peroxidase enzyme is added (b) followed by enzyme substrate (c)

Antigen Retrieval (AR) Methods (Kumar 2009) (2):

FFPE tissues require an AR step before immunohistochemical staining can proceed. While formalin fixation preserves tissue morphology, it results in the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of AR are heat-induced epitope retrieval (HIER) and enzymatic induced epitope retrieval (EIER). Both AR methods serve to break the methylene bridges and expose the antigenic sites in order to allow the antibodies to bind. Some antigens prefer enzymatic to heat mediated antigen retrieval and vice versa (Kumar 2009) (2). However, HIER was pioneered in the early 1990’s after it had become apparent that use of EIER alone to improve IHC staining was inadequate (Shi, Key et al. 1991). Therefore, throughout this study, only HIER method was performed.

A) Heat-induced epitope retrieval (HIER) methods

Wide application of the AR technique in pathology and other fields of morphology has demonstrated distinct enhancement of IHC staining on FFPE tissue sections. Several parameters govern the effect of any AR 62

solution used in IHC including: the temperature, the pH and the chemical composition of the solution and the exposure time. Heating is the most important factor for retrieval of antigens masked by formalin fixation. In general, higher temperature yields better results of the AR technique than lower temperature (Shi, Cote et al. 1997). However, for few antigens, higher temperature may induce a negative result of IHC staining. In this case, a lower-temperature heating treatment or a combining retrieval protocol (heat and enzyme digestion) may provide better results (Shi, Cote et al. 2001). The heating condition represents heating temperature and the time of heating, appearing to be a reverse correlation, the higher the temperature, the shorter the heating time (Shi, Cote et al. 2001). Another important factor is the pH of the AR solution. Antigens require heating in buffers of specific pH to obtain the strongest intensity of staining. Some of the antigens work more effectively in low pH whereas the others function better in high pH. Although the low-pH AR solution may yield the strongest intensity for some antibodies, weak false-positive nuclear staining may also be found in focal areas for some antibodies tested, giving rise to background staining (Shi, Cote et al. 1997). The third factor is the chemical composition of the AR solution. The chemical component of AR solution plays a role as a key factor that needs to be optimized for any given antibody. So far no single chemical has been identified that is best for AR (Shi, Cote et al. 1997).

a) High pressure AR

The temperature of AR can be increased to 120-125°C by using a pressure cooker or autoclave. The slides are usually put into the AR solution at room temperature then allowed to cool slowly back to room temperature after the procedure (Kumar 2009) (2).

b) Water bath heating

One of several advantages of the water bath heating method is the ready availability of water baths in most clinical laboratories. Temperature settings

just below the boiling point of water (95-99°C) are most commonly used (Kumar 2009) (2). c) Hot plate heating:

Temperature settings just below the boiling point of water (95-99°C) are most commonly used in this method. For both these method, the slides are usually put into the AR solution when it is at the required temperature then allowed to cool slowly back to room temperature (RT) after the procedure (Hayat 2002).

B) Enzyme-induced epitope retrieval (EIER) method

Proteolytic Pre-treatment

As with other pre-treatment methods, procedures for proteolytic pre- treatment vary due to laboratory-specific differences in formalin fixation. Proteolytic pre-treatment must be optimized according to the particular fixation process used in each laboratory. Examples of antigens most often treated with proteolytic enzymes include cytokeratins and immunoglobulins (Kumar 2009) (2).

Wide variety of proteases has been employed, including trypsin, proteinase K, pepsin, pronase, ficin, and others. The enzyme is usually dissolved in calcium chloride solution. Concentration of enzyme is usually 0.05–0.1%, depending on type of tissue and fixation. Incubation time within 5–30 min is commonly used. Incubation temperature is usually at 37°C (Igor B. Buchwalow 2010) (2).

The mechanism of action of the EIER is related to cleavage of the molecular cross-links by the proteolytic enzyme, liberating the antigens and preparing them for antibody binding (Igor B. Buchwalow 2010) (2).

Antibodies and their applications in IHC

An antibody or immunoglobulin (Ig) is a glycoprotein used by the immune system to identify and neutralize substances foreign to the body, such as bacteria and viruses and other infectious agents, known as antigens. Igs comprise five major classes: immunoglobulin g (IgG), IgA, IgM, IgD and IgE. Of the five classes of Igs, IgG and IgM are by far the most frequently utilized antibodies in IHC (Kumar 2009) (3).

IgG

The classical “Y” shape of the IgG molecule is composed of four polypeptide chains; two identical light chains and two identical heavy chains. The heavy (H) and light (L) chains are composed of variable (V) and constant (C) domains and are linked by inter- and intrachain disulfide bonds. Proteolytic digestion with papain yields two antigen-binding fragments (Fab) and one crystalline fragment (Fc), whereas digestion with pepsin yields one Fab fragment F(ab´)2. Fab, F(ab´)2 and Fc fragments are useful tools in immunoassays (Kumar 2009) (3) (fig 2.3).

Fig 2.3: Diagram showing the structure of IgG. The heavy (H) and light (L) chains are composed of variable (V) and constant (C) domains and are linked by inter- and intrachain disulfide bonds (|—◆—|). Proteolytic digestion with papain (• • • • •) yields two antigen-binding fragments (Fab) and one crystalline fragment (Fc), whereas digestion with pepsin (– – – –) yields one F(ab)2 fragment, (Kumar 2009) (with permission) 65

IgM

IgM is a pentamer consisting of five subunits. Each subunit is linked by a sulfhydryl-rich peptide, the J chain, and consists of two heavy chains μ and two light chains. The J-chains contribute to the integrity and stability of the pentamer. As with IgG, IgM subunits can be fragmented by enzymatic and reductive cleavage (fig 2.4).

Fig 2.4: (a) showing the five subunits of mouse IgM linked by disulfide bridges (|—◆—|) and the J chain to form a pentameric ring structure. Each subunit (b) comprises two mu heavy (H) chains and two light (L) chains each composed of constant (C) and variable (V) domains (Kumar 2009) (with permission).

Whereas IgG is the most abundant antibody in the hyperimmunized host, in the newly immunized animal, IgM is the first humoral antibody detectable, and whereas antibodies of class IgM have a relatively short half-life of only four to six days, IgG antibodies have a mean survival of approximately three weeks (Kumar 2009) (3).

Polyclonal Antibodies versus Monoclonal Antibodies

(Kumar 2009) (3).

Polyclonal antibodies are a heterogeneous mixture of antibodies directed against various epitopes of the same antigen. The antibodies are generated by different B-cell clones of the animal and as a consequence are immunochemically dissimilar. The antibodies in a polyclonal mixture can have slightly different specificities and affinities. Polyclonal antibodies are most frequently produced in rabbits but also made in other mammals. The

presence of antibodies to multiple epitopes can increase the chance for cross-reactivity with other proteins. The term “cross-reactivity” denotes an immunochemical activity that can occur either between an antibody and two or more antigens or vice versa, when an antigen reacts with several different antibodies.

Monoclonal antibodies are a homogeneous population of Ig directed against a single epitope. The antibodies are generated by a single B-cell clone from one animal and are therefore immunochemically similar. While the vast majority of monoclonal antibodies are produced in mice, a growing number of rabbit monoclonal antibodies are being manufactured. Monoclonal antibodies have the advantage of lot-to-lot consistency and lack the inherent variability and cross-reactivity of polyclonal antibodies.

Antibody-related factors which can affect the staining outcome (Kumar 2009) (4)

The staining quality of a processed tissue in IHC is governed by several factors among them are antibody titre, antibody dilutions, incubation time and incubation temperature. These variables are tightly connected together in their effect on the staining outcome.

Antibody Titre

Optimum antibody titre may be defined as the highest dilution of an antibody that results in maximum specific staining with the least amount of background under specific test conditions. This highest dilution is determined primarily by the absolute amount of specific antibodies present. An optimal antibody dilution is also governed by the intrinsic affinity of an antibody. A high-affinity antibody is likely to give more intense staining within the same incubation period than an antibody of low affinity.

Antibody Dilution

Accurate and consistent dilutions contribute directly to the quality of staining. Accurate dilutions are best determined by first selecting a fixed incubation time and then by making small volumes of a series of experimental dilutions. 67

It should be noted that in FFPE tissues, optimal dilutions of primary antibodies are not only signalled by a peak in staining intensity, but also by the presence of minimal background.

Antibody Incubation Time

There is an inverse relationship between incubation time and antibody titre: the higher the antibody titre, the shorter the incubation time required for optimal results. In practice however, it is important to first set a suitable incubation time before determining the optimal antibody dilution. For an antibody to react sufficiently strongly with the bound antigen in a short period of time, it must be of high affinity and concentration, as well as have the optimal pH. Primary antibody incubations with 24-hour duration allow for greater economy, because higher dilutions of the same may be used. Low affinity and/or low titre antibodies must be incubated for long periods in order to reach equilibrium. Consistent timing of this step is important. Inconsistent incubation times can cause variations in overall stain quality and intensity, and may lead to incorrect interpretation of results.

Antibody Incubation Temperature

Because antigen antibody reactions reach equilibrium more quickly at 37°C compared to room temperature, some workers prefer to incubate at the higher temperature. It is not known whether an increased temperature promotes the antigen antibody reaction selectively, rather than the various reactions that give rise to background. A temperature of 4°C is frequently used in combination with overnight or longer incubations. Slides incubated for extended periods, or at 37°C should be placed in a humidity chamber to prevent evaporation and drying of tissue sections.

Materials and Methods

Materials:

FFPE tumour sections were sectioned by VCB at 4 μm and mounted on normal slides (Grale Colourfrost HDS Microscope Slides- HD Scientific). Slides from 120 tumours were used; ideally, from equal number of men and women patients who have not undergone other therapy prior to surgery. 40 tumours comprise stage II CRC while 80 tumours were used for stage III CRC based on the AJCC staging system. In addition, 14 pairs of colon and liver tissues were selected from CRC patients who complicated with liver metastasis. Slides of adjacent normal colon tissue from 20 of the patients (7 slides from stage II and 13 slides from stage III) were used as negative staining controls (EphB1, EphB2 & EphB4 are present in normal colonic epithelium) (table 2.1). Haematoxylin and Eosin (H & E) slides from VCB were used by our pathologist to confirm degree of differentiation of each CRC section.

Table 2.1: Total number of slides and the distribution of the slides based on the stage of the disease:

Stage No of No of slides No of No of Total donors (6 slides per donors with negative number of donor) matched control slides of normal slides (6 the stage (negative slides per control) donor)

II 40 240 7 42 282

III 80 480 13 78 558

IV 14 12 (6 CRC + None None 168 6 liver metastases)

Total 134 20 slides 120 negative 1008 donors negative control slides tumour control slides

Development of staining protocol for each receptor

Number of factors affects the sensitivity and specificity of IHC

1) The fixation and processing protocol of the tissues. Fixation helps to preserve tissue morphology and hence to preserve the position of the antigens for the antibodies. However, many examples exist of situations that have led to incorrect interpretation of staining patterns, as a result of poor or inadequate fixation (Kumar 2009) (5). The following is the standard tissue fixation protocol that the VCB tissue samples are processed by:

Table 2.2: Fixation protocol of the project’s VCB tissues

Process Solution Time Retort

Fixation Formalin, 10% 120 min -

Fixation Alcoholic formalin 60 min -

Dehydrate Alcohol, 95% 60 min Vacuum

Dehydrate Alcohol, 95% 45 min -

Dehydrate Alcohol, absolute 45 min Vacuum

Dehydrate Alcohol, absolute 60 min -

Clearing Xylene 60 min - agent

Clearing Xylene 60 min Vacuum agent

Infiltrate Paraffin 30 min -

Infiltrate Paraffin 60 min -

Infiltrate Paraffin 90 min Vacuum

NB: this is a conventional, room temperature processing schedule. Total processing time is 12 hours.

2) The primary antibody: the characteristics of a good antibody are:

• High affinity to an antigen (affinity),

• High binding property (avidity),

• High concentration (titration).

However, these characteristics are not always available together in one antibody (Kumar 2009) (3). Therefore, during staining protocol development for each receptor in this study, several antibodies were tested for each receptor with different hosts, different dilutions, different diluents, different incubation times and different incubation modes.

3) Antigen retrieval method: different antibodies work differently with different antigen retrieval solutions, with different molarities and different pHs; different HIER methods with different times (Kumar 2009) (2). Therefore, as the case with the primary antibodies, several HIER methods with different antigen retrieval solutions, with different molarities and different pHs were tested against each primary antibody in order to achieve an optimised staining protocol. These are mentioned in details under each receptor.

Normal colonic sections sourced from the VCB were used for protocol validation. Each time, several sections are tested for each receptor based on the number of the antigen retrieval methods being used and the different primary antibody dilutions. Where they existed, protocols from the literature were tested. Once a protocol gave reliable and consistent tissue staining, next step, it was applied on the actual project tissues.

EphB1 receptor

An ABC based staining protocol for EphB1 receptor had been developed prior to this project. The staining protocol was tested against the VCB normal colon tissues before being commenced with the actual project’s tissues. The

tissues had shown normal uptake of the primary antibody mouse monoclonal antibody (5F10A4) (fig 2.5).

Fig 2.5: EphB1 receptor staining protocol testing using VCB normal colon tissues. The figure illustrates the normal microanatomy of EphB1 receptor at the base of the crypts, which decreases towards the lumen.

EphB1 receptor ABC based staining protocol

1. Deparaffinization in histoclear and rehydration in graded alcohols,

2. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2) for 10 min at RT,

3. HIER method: preheated 10mM Tris-HCl and 1mM EDTA on a hotplate for 10 min,

4. Blocking with IHC for 30 min at RT to block non-specific binding,

5. Incubation with primary antibody mouse polyclonal antibody (5F10A4) diluted in 1x TBST at 1:75 dilution for 2 hr at RT,

6. Incubation with biotinylated secondary antibody Vector biotinylated anti- mouse diluted in 1x TBST at 1:300 dilution for 15 min at RT,

7. Incubation with ABC complex for 15 min at RT,

8. Incubation with chromogen reagent DAB substrate for 15 min at RT,

9. Counterstaining using haematoxylin for 1 min at RT, 72

10. Dehydration, coverslip, and view under the microscope.

EphB2 receptor

EphB2 receptor staining protocol development using ABC method

Initially, an IHC staining protocol using ABC based method, which had been developed prior to this project, had been tested against VCB normal colon tissues. Goat polyclonal IgG (R&D-AF467) as primary antibody and donkey antigoat biotin conjugated (LS-C59905-1000) as secondary antibody were used. Unfortunately, it did not show a normal pattern of EphB2 receptor in the colon epithelium but rather it showed non-specific staining (fig 2.6).

Fig 2.6: EphB2 receptor IHC staining protocol testing using VCB normal colon tissues. The figure demonstrates the non-specific staining as one of the complications associated with ABC based method.

Then, the same proposed EphB2 staining protocol was tested against CRC tissues, which also contain adjacent normal mucosa. Several HIER methods with different antigen retrieval solutions and different pHs extracted from the literature relevant to EphB2 receptor were tried, but there was no actual staining of the basal crypts and tumour tissues together with non-specific staining (see examples in tables 2.3-2.4 & fig 2.7).

Table 2.3: EphB2 receptor staining protocol development using ABC method

Groups Group A Group B Group C Group D

AR 10mM Na 100mM EDTA pH 0.01M Tris-HCl pH 0.1M Tris-EDTA solution Citrate pH 6.0 8.0 9.5 and 0.001M pH 9.0 EDTA pH 8.0

HIER Hotplate @ Hotplate @ 95 ͦC Hotplate @ 95 ͦC X Hotplate @ 95 ͦC method 95 ͦC X 15 min X 15 min 15 min X 15 min

1ry ab AT1: 1:150, BT1: 1:150, CT1: 1:150, DT1: 1:150,

Goat BT2: 1:200, CT2: 1:200, DT2: 1:200, polyclonal IgG (R&D- AT2: 1:200, BT3: 1:400, CT3: 1:400, DT3: 1:400, AF467) AT3: 1:400, BC: control CC: control DC: control

AC: control

2ry ab 1:500 1:500 1:500 1:500

Donkey antigoat biotin conjugated (LS- C59905- 1000)

T: tumour tissues incubated with primary and secondary antibodies

C: positive control incubated with secondary antibody only

Table 2.4: EphB2 receptor staining protocol development using ABC method

Groups Group A Group B Group C Group D

AR 0.01M Na 0.02M Na 0.001M EDTA 0.01M Na solution Citrate pH 6.0 Citrate pH 6.0 pH 8.0 Citrate pH 6.0

HIER Autoclave fluids Autoclave fluids Autoclave Hotplate @ method cycle cycle fluids cycle 100 ͦC X 20 min

1ry AT: 1:100, BT: 1:100, CT: 1:100, DT: 1:100, antibody Goat AC: No 1ry, BC: No 1ry, CC: No 1ry, DC1: No polyclonal 1ry, IgG (R&D- AF467)

2ry 1:500 1:500 1:500 1:500 antibody Donkey antigoat biotin conjugated (LS- C59905- 1000)

T: tumour tissues incubated with primary and secondary antibodies

C: positive control incubated with secondary antibody only

The result was a) Non-specific staining of the tumour tissues, b) No staining of the tumour tissues.

Fig 2.7: EphB2 receptor staining protocol development. This figure demonstrates no actual staining of the tumour tissues (a) and non-specific staining (b).

Then, the staining protocol was tested against CRC tissues using two approaches; the conventional method in table 3, and a modified staining method, to find out the source of nonspecific staining.

Three slides were treated as follows (table 5)

Slides 1: received all the reagents as the conventional method.

Slide 2: was deprived of the secondary antibody only; to find out whether it might be the cause of the nonspecific staining, by comparing it with slide number 1 and 3.

Slide 3: was deprived of both, the secondary antibody and the ABC reagents, to find out whether these two reagents together might be the cause of the nonspecific staining, by comparing it with slide number 1 and 2.

Table 2.5: EphB2 receptor staining protocol development using ABC based modified approach

Slides Primary Secondary ABC DAB antibody antibody

Slide 1 Yes Yes Yes Yes

Slide 2 Yes No Yes Yes

Slide 3 Yes No No Yes

However, the result was non-specific staining of the cellular cytoplasm together with no actual staining of the tumour tissues in the slides of the conventional and modified staining methods.

Then, another approach was applied, to test whether secondary antibody, which was used before, is the cause of persistent nonspecific staining. Two types of controls were used; the first control was with no primary antibody as usual whereas the second control was deprived of primary and secondary antibodies (table 6). Both types of controls showed nonspecific staining.

Table 2.6: EphB2 receptor staining protocol development using ABC based modified approach

Groups Group A Group B Group C Group D

AR 0.01M Na 0.02 M Na 0.001M EDTA 0.01M Na solution Citrate pH Citrate pH 6.0 pH 8.0 Citrate pH 6.0 6.0

HIER Autoclave Autoclave Autoclave fluids Hotplate @ method fluids cycle fluids cycle cycle 100 ͦC X 20 min

1ry AT 1: 1:100, BT 1: 1:100, CT 1: 1:100, DT 1: 1:100, antibody AT 2: 1:200, BT 2: 1:200, CT 2: 1:200, DT 2: 1:200, Goat polyclonal AT 3: 1:400, BT 3: 1:400, CT 3: 1:400, DT 3: 1:400, IgG (R&D- AC1: no 1ry, BC1: no 1ry, CC1: no 1ry, DC1: no 1ry, AF467) AC2: no 1ry BC2: no 1ry & CC2: no 1ry & DC2: no 1ry & & no 2ry no 2ry no 2ry no 2ry

2ry 1:500 1:500 1:500 1:500 antibody

Donkey antigoat biotin conjugated (LS- C59905- 1000)

T: tumour tissues incubated with primary and secondary antibodies

C1: positive control incubated with secondary antibody only

C2: positive control incubated with ABC and DAB solutions only

Then, Polymer based method was tried as an alternative staining method. It was tested first against VCB normal colon tissues. Instead of the two-hour incubation time, two different overnight incubation temperatures were tried, at 4°C and at RT. Initially, incubation at 4°C was tried alone, which showed good uptake. In a separate occasion, 4°C incubation was compared with the RT. Both showed good uptake but the RT mode was better (table 2.7 & fig 2.8).

Table 2.7: EphB2 receptor staining protocol development using polymer based method

No of slides 4; 2 @ 4°C and 2 @ RT

AR solution Preheated 10mM Na Citrate pH 6.0

HIER Hotplate @ 100 °C x 20 min

1ry Antibody Anti-goat anti-EphB2 (AF 467) @ 1:100

2ry Antibody Ready-to-Use Impress Reagent

Fig 2.8: Colon basal crypts stained with anti-goat anti-EphB2 (AF467) using Polymer based method. Fig (a) is overnight incubation at 4°C whereas and fig. (b) is overnight incubation at RT. 78

Fig 2.9 is an example of EphB2 positive tumour.

Fig 2.9: CRC tissues stained with anti-goat anti-EphB2 (AF467) using Polymer based method.

The final protocol applied to all sections in the study was:

EphB2 receptor Polymer based staining protocol

1. Deparaffinization in histoclear and rehydration in graded alcohols,

2. HIER method: preheated 10mM Na Citrate pH 6.0 on a hotplate at 100°C for 20 min,

3. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2) for 15 min at RT,

4. Blocking with Ready-To-Use (RTU) 2.5% Horse Serum Block (HSB)

(MP-7405) for 1 hr at RT,

5. Overnight incubation with primary antibody anti-goat anti-EphB2 (AF467) at 1:100 at RT,

6. Incubation with RTU Anti-goat Impress reagent for 1 hr at RT,

7. Incubation with DAB substrate for 10 min at RT,

8. Counterstaining using haematoxylin for 1 min at RT,

9. Dehydration, coverslip, and view under the microscope.

EphB4 receptor

As the case with EphB2 receptor, proposed IHC staining protocol using ABC method was tried first to test for EphB4 receptor. Mouse monoclonal IgG (SC-130062) primary antibody was attempted first with several dilutions against several HIER methods with different antigen retrieval solutions and different pHs extracted from the literature relevant to EphB4 receptor. However, there was no actual staining of the basal crypts and tumour tissues together with non-specific staining (tables 2.8-2.10 & fig 2.10).

Table 2.8: EphB4 receptor staining protocol development using ABC based method using water bath as a HIER method

Groups Group A Group B Group C

AR solution 10mM Na 0.001M EDTA pH 10mM Citric Acid pH 6.0 Citrate pH 8.0 8.0

HIER Water bath @ Water bath @ Water bath @ 80°C X 10 method 80°C X 10 min 95°C X 10 min min

1ry AT1: 1:150, BT1: 1:150, CT1: 1:150, antibody AT2: 1:200, BT2: 1:200, CT2: 1:200, Mouse monoclonal AT3: 1:400, BT3: 1:400, CT3: 1:400, IgG (SC- AC: control BC: control CC: control 130062)

2ry 1:600 1:600 1:600 antibody

Vector biotinylated anti mouse

Table 2.9: EphB4 receptor staining protocol development using ABC- based method using hotplate as a HIER method

Groups Group A Group B Group C

Ag ret 10mM Na 0.001M EDTA pH 10mM Citric Acid pH 6.0 solution Citrate pH 8.0 8.0

HIER Hotplate @ Hotplate @ 95°C X Hotplate @ 95°C X 10 min method 95°C X 10 min 10 min

1ry AT1: 1:150, BT1: 1:150, CT1: 1:150, antibody AT2: 1:200, BT2: 1:200, CT2: 1:200, Mouse monoclonal AT3: 1:400, BT3: 1:400, CT3: 1:400, IgG (SC- AC: control BC: control CC: control 130062)

2ry 1:500 1:500 1:500 antibody

Vector biotinylated anti mouse

Table 2.10: EphB4 receptor staining protocol development using ABC based method

No of slides 3

AR solution Preheated 10mM Na Citrate pH 6.0

HIER method Hotplate @ 95°C X 10 min

1ry antibody mouse monoclonal IgG TB4 (1): 1:100 (SC- 130062) TB4 (2): 1:200

2ry antibody 1:500 Vector biotinylated antimouse

Fig 2.10: EphB4 receptor IHC staining protocol testing using anti-EphB4 Mouse monoclonal IgG (SC-130062) primary antibody. The figure demonstrates the non-specific staining as one of the complications associated with ABC based method.

Then, rabbit polyclonal primary anti-EphB4 antibody (SC-5536) at 1:250 and overnight incubation mode was tested against VCB normal colon sections and against CRC sections, following ABC staining method. Antigen retrieval buffer was preheated 10mM Na Citrate Buffer pH 6.0 and HIER method was hot plate for 15 min (>95°C). Goat pAb to rabbit (Biotin-ab97067) at 1:600 was used as a secondary antibody. However, the result was the same as before (fig 2.11).

Fig 2.11: EphB4 receptor IHC staining protocol testing using rabbit polyclonal primary anti-EphB4 antibody (SC- 5536) at 1:250 and overnight incubation. This figure demonstrates non-specific staining as one of the complications associated with ABC based method.

Then, anti-EphB4 Invitrogen mouse monoclonal primary antibody (37-1800) at three different dilutions, 1:100, 1:250 & 1:400, was tested against VCB CRC tissues containing adjacent normal colon tissues, following ABC staining method and overnight incubation mode at RT. Each primary antibody dilution was tested against three different HIER times, 10 min, 20 min and 30 min. Antigen retrieval buffer was preheated 10mM Na Citrate Buffer pH 6.0 and HIER method was hot plate. Vector biotinylated anti mouse at 1:500 was used as a secondary antibody (table 2.11).

Table 2.11: EphB4 receptor staining protocol development using ABC based method

HIER method Hotplate @ Hotplate @ 20 Hotplate @ 30 10 min min min

AR buffer Preheated 10mM Na Citrate pH 6.0

1ry Antibody Invitrogen mouse monoclonal @ 1:100, 1:250 &

1:400

Incubation mode Overnight incubation @ RT

2ry Antibody 1:500

The method showed promising results especially at 1:100 dilution with HIER method for 30 min (fig 2.12).

Fig 2.12: anti-EphB4 Invitrogen mouse monoclonal (37-1800) at 1:100 and overnight incubation mode x HIER for 30 min.

Therefore, the same primary antibody at 1:100 dilution was tested using higher hotplate times than the previous experiment; 40 min, 50 min, 60 min and 70 min, and also using autoclave fluid cycle (table 2.12). The best result was found with HIER method for 50 min as it showed good uptake of the primary antibody with less non-specific staining than the other HIER groups.

Table 2.12: EphB4 receptor ABC based staining protocol development using different HIER times and methods

HIER Hotplate @ Hotplate Hotplate Hotplate Autoclave method 40 min @ 50 min @ 60 min @ 70 min fluid cycle

Antigen 10mM Na Citrate pH 6.0 retrieval buffer

1ry Ab Invitrogen mouse monoclonal @ 1:100

Incubation Overnight incubation @ RT mode

Then, the same primary antibody at 1:100 dilution and hotplate HIER method for 50 min was assessed using ABC based method versus Polymer based staining method with overnight incubation at RT (fig 2.13). 84

Fig 2.13: anti-EphB4 Invitrogen mouse monoclonal (37-1800) at 1:200 and overnight incubation mode at RT x HIER for 50 min. Fig (a) ABC based method showed intense non-specific staining. Fig (b) polymer based method showed proper uptake of the anti-EphB4 antibody.

As it is shown in the figure, the polymer based staining method showed promising result whereas ABC based method showed non-specific staining.

Then, the Polymer based staining protocol was tested with the primary antibody at two lesser dilutions, 1:200 and 1:300.

Fig 2.14: anti-EphB4 Invitrogen mouse monoclonal (37-1800) following polymer based method with overnight incubation mode and HIER for 50 min. Figure (a) is at 1:200 dilution and is showing better uptake than figure (b) at 1:300 dilution.

The 1:200 dilution showed better uptake and less non-specific staining than the 1:300 dilution (fig 2.14). So, 1:200 concentration was selected as the dilution of choice for the anti-EphB4 Invitrogen mouse monoclonal antibody in this study.

EphB4 receptor Polymer based staining protocol

1. Deparaffinization in histoclear and rehydration in graded alcohols,

2. HIER method: preheated 10mM Na Citrate pH 6.0 on a hotplate at 100°C for 50 min,

3. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2) for 15 min at RT,

4. Blocking with homemade 2.5% Horse Serum Block (HSB) (MP-7402) for 1 hr at RT,

5. Overnight incubation with primary antibody anti-EphB4 Invitrogen mouse monoclonal antibody at 1:200 at RT,

6. Incubation with RTU Anti-mouse Impress reagent (37-1800) for 1 hr at RT,

7. Incubation with DAB substrate for 10 min at RT,

8. Counterstaining using haematoxylin for 1 min at RT,

9. Dehydration, coverslip, and view under the microscope.

Technique

Capillary staining of EphB1 and EphB2 receptors using IHC Select® Manual Staining System and Manual staining of EphB4 receptor

A) Overview

Once the staining protocols had been developed, we needed to stain a large number of slides consistently. The IHC Select® Manual Staining System allows up to 40 sections to be stained with up to 20 different antibodies at once. It reduces processing time and improves consistency. While the tissues are deparaffinized, the manual staining system is set up to be used

for tissue processing. It is a bench top instrument consisting of the following major components (Haase 2012 ) (fig 2.15):

1. Slide Holder: consists of an outer frame that holds 2 to 40 microscope slides. The slide holder is moved manually along the base platform, thus allowing the microscope slides to either be placed in a Reagent or on top of a wicking pad. The simple IHC procedure alternates moving the slide holder between a reagent and a wicking pad.

2. Base Platform: holds the nests in their proper positions.

3. Nests: are positioned onto the base platform to properly orient the slide holder, reagent trays, and wicking pads.

4. Reagent Trays: hold and separate each of the various IHC reagents. The tissues on the microscope slides are incubated in a particular IHC reagent, and then moved to a wicking pad.

5. Wicking Pads capture the reagents after they have been incubated with the tissue on the microscope slides.

6. Capillary Gap Slides, with Tissue Location.

The slides as supplied by VCB were not suitable to be used in a capillary staining system. Accordingly, all slides were treated in our laboratory by applying the Tough-Tags to each slide. Tough-Tags help to create a capillary gap between each pair of slides thus making them ready to be used in a capillary stainer.

Fig 2.15: IHC Select Capillary Staining System

Advantages and disadvantages of the IHC Select ® Manual Staining System (table 2.13)

Table 2.13:

Advantages Disadvantages

Allows staining of up to 40 slides at a Arrangement of the slides: time time consuming

Allows up to 20 different antibodies at Reagents may not be used in proper once order

Reduces processing time Any error can spoil the whole process

Improves consistency Incomplete solution drainage from slides can hamper quality of tissues staining

The slide holder is moved manually Slides not properly placed in the slide along the base platform: it allows holder moving the slides between reagents and a wicking pad

The reagent trays hold and separate each of the IHC reagents

Capillary Gap Slides, with Tissue Slides not properly placed rinsed Location the liquid is gently drawn between the glass slides

The Reagent Trays are numbered 1- 10 inside the bottom of the reagent wells. This orientation assists in inserting the reagent trays into the nests and in placing the reagents in the correct position

B) Procedure:

1. Deparaffinization and rehydration of the FFPE tissue sections were performed by putting the sections into a slide rack and then moved sequentially through the following baths: histoclear number 1 for 15 min; histoclear number 2 for 10 min; 100% Ethanol number 1 for 10 min; 100% Ethanol number 2 for 10 min; 70% Ethanol for 3-5 min; 30% Ethanol for 3-5 min and then to be rinsed in distilled water. The hot plate is switched on and is set to 300°C. 89

2. Pretreatment of the tissues: blocking of tissues in 3% H202 to paralyze endogenous peroxidase activity:

20mL of 30% H202 is added to 180mL of pure water and pour into a black slide box. Sections were Immersed in the 3% Hydrogen Peroxide for 10 min and then rinsed briefly in distilled water.

3. The AR buffer (table 2.14):

Table 2.14: AR Buffers and HIER Methods and durations:

Receptor EphB1 EphB2

AR Buffer 10mM Tris-HCl and 10mM Sodium Citrate 1mM EDTA

HIER Method Preheated in a Preheated in a microwave for 10 microwave for 10 min min then on a hot plate then on a hot plate

Duration 10 min 20 min

The hot AR solution is then placed on ice and left to cool down below 30ͦC. The slides are then briefly rinsed in pure water before being moved to the next step.

4. Stainer set up

Each pair of the slides is placed face to face and then inserted into the capillary stainer slide rack starting at A side for the slides to be incubated with the primary antibody and then B side which allocated for the control slides.

5. Blocking of non-specific tissue binding of the primary antibody (table 15):

Stain reservoirs are placed in the second staining position and 500µl of blocking buffer are added to all wells that correspond to slide pairs in the slide rack. The slides are dabbed on the wicking pad to wash out the

solution, placed in the wells with blocking buffer and incubated at room temperature for the proper duration. The primary antibody solution is prepared 10 min before end of incubation period with the blocking buffer.

Table 2.15: Blocking Buffers and their durations

Receptor EphB1 EphB2

Blocking Buffer IHC Ready-to-Use 2.5% Horse Serum Block (HSB) (MP-7405)

Duration 30min 1hr

6. Primary antibody (table 2.16)

Stain reservoirs are placed in the second staining position and 500µl of primary antibody are added to all wells that correspond to slide pairs in the slide rack. The slides are dabbed on the wicking pad to wash out the blocking buffer solution, placed in the wells with primary antibody and incubated at room temperature for the proper duration.

Table 2.16: Primary antibodies, their dilutions, diluents and incubation periods

Antigen EphB1 EphB2

Antibody Mouse polyclonal antibody Goat antibody R&D (AF467) (5F10A4)

Dilution 1:75 1:100

Diluent 1x TBST Ready-to-Use 2.5% HSB

Time 2hr at RT ON at RT

7. Washing: Fluid is removed from the slides using wicking pad. Slides are then washed in 1x TBST or 1x PBS and wicked frequently 5 times. The slides are then wicked to ensure all the fluid is removed.

8. Secondary antibody (Table 2.17)

Table 2.17: Secondary antibody

Antigen EphB1 EphB2

Antibody Vector biotinylated anti- Ready-to-Use ImmPRESS™ mouse reagent anti-goat Ig

(MP-7405)

Dilution 1:300 Ready-to-Use

Diluent 1x TBST Ready-to-Use

Time 15 min at RT 1 hr at RT

9. Washing

The same as in step 7.

10. Streptavidin-HRP complex (ABC)

Vector ABC reagent is only used for EphB1 receptor. 2 drops of reagent A and 2 drops of reagent B are mixed with 5 ml of PBS. Mixture is left at room temperature for 30 min before use. 300µl of the ABC mix are pipetted into each well of fresh reservoirs. The wicked slides are put into the wells and incubated for 15 min at room temperature.

11. Washing

The same as in step 7.

12. DAB Detection

The DAB solution containing chromogen reagent must be freshly made prior to use. Using the vector Labs DAB kit, 5ml of water is added to a 92

tube followed by 2 drops of the pH 7.5 buffer, 4 drops of DAB and 2 drops of H202. 300µl of the mixture are added into each well using fresh reservoir. Slides are placed in the wells and incubated for 10 min at room temperature.

13. Washing

The same as in step 7.

14. Counterstaining

300µl haematoxylin counterstain is added to each well of fresh reservoirs. The wicked slides are lowered into the wells and incubated for 1 min.

15. Washing

The slides are wicked and placed in water. Wicking and washing with water is repeated 4 times. Slides are wicked to ensure all the fluid is removed.

16. Dehydration & Mounting

The slides are carefully removed from the capillary stainer and placed in a slide rack in distilled water. Dehydration is performed as follows: 30% ethanol for 3minutes; 70% ethanol for 3min; 100% ethanol number 2 for 10min; 100% ethanol number 1 for 10min; Histoclear or xylene number 2 for 10min; Histoclear or xylene number 1 for more than 15min. DEPEX or Permount are used for mounting and then slides are covered with cover slip.

Manual Staining of EphB2 and EphB4 Receptors:

Because of the non-promising results associated with EphB2 receptor using capillary stainer, manual staining was used for repeated EphB2 receptor staining and for EphB4 receptor staining.

1. Deparaffinization in histoclear and rehydration in graded alcohols as for EphB1 and EphB2 receptors.

2. HIER method:

EphB2: Preheated 10mM Na Citrate pH 6.0 is placed on a hotplate at 100°C for 20 min.

EphB4: Preheated 10mM Na Citrate pH 6.0 is placed on a hotplate at 100°C for 50 min.

The hot AR solution is then placed on ice and left to cool down below 30ͦC. The slides are then briefly rinsed in pure water before being moved to the next step.

3. Quenching endogenous peroxidase using 3% hydrogen peroxide (H2O2) for 15 min at RT as for EphB1 and EphB2 receptors.

4. Blocking with

EphB2: RTU-HSB (MP-7405) for 1 hr at RT,

EphB4: homemade 2.5% Horse Serum Block (HSB) (MP-7402) for 1 hr at RT:

The slides are briefly rinsed in water after blocking with 3% H2O2. The slides are then placed in boxes with wet floor to reduce dryness. A Pap pen is used to draw a mark around tissues in the slide in order to prevent reagents from coming off the specimens. 2.5% HSB blocking agent is then dispensed to the specimens using a pipette at 100 µL for each section.

5. Overnight incubation with primary antibody:

EphB2: Goat antibody R&D (AF467) at 1:100 at RT

EphB4: anti-EphB4 Invitrogen mouse monoclonal antibody at 1:200 at RT:

The slides are cleaned of the blocking reagent. The primary antibody is then dispensed to the specimens using a pipette at 100 µL. The boxes are covered with lids and secured with cling film to reduce evaporation. 94

6. Incubation for 1 hr at RT:

EphB2: Ready-to-Use ImmPRESS™ reagent anti-goat Ig (MP-7405)

EphB4: RTU Anti-mouse ImmPRESS™ reagent (37-1800)

The slides are then cleaned of the primary antibody and washed in 1X PBS for 5 min. The slides are laid down in the boxes and Impress reagent is applied to the tissues using a pipette at 100 µL.

7. Incubation with DAB substrate for 10 min at RT:

The slides are then cleaned of the primary antibody and washed in 1X PBS for 5 min. The slides are laid down in the boxes and Impress reagent is applied to the tissues using a pipette at 100 µL.

The slides are then cleaned of the Impress reagent and washed two times in 1X PBS, each time for 5 min. The slides are then laid down in the boxes and DAB reagent is applied to the tissues using a pipette at 100 µL.

8. Counterstaining using haematoxylin for 1 min at RT:

The slides are then cleaned of the DAB reagent and rinsed briefly in water. The slides are laid down in the boxes and haematoxylin is added to the tissues using a pipette at 100 µL.

9. Dehydration, coverslip, and view under the microscope: as for EphB1 and EphB2 receptors.

Clinicopathological Data Collection

A) Data Collection

Data were collected in the two data collection sheets made for the project. The first one is for the pattern of expression of the receptor in the primary tumour whereas the second one is for the comparison between the pattern of the receptor expression in the primary CRC and of that in the hepatic metastasis. Each sheet was duplicated to be blindly filled by both the first

observer and the second observer (see examples in the appendix). The first observer was the student conducting this project while the second observer was a qualified pathologist. The pathologist’s findings were taken into consideration for any discrepancies between the two observers findings. Olympus microscope BX53 was used to visualise sections and score them. The clinicopathological data were received from the VCB. These data included age at surgery, gender, location of the tumour, depth of wall invasion, clinical stage (AJCC), lymph node metastases (LN), tumour differentiation, postoperative (POP) therapy, recurrence, tumour size and smoking status (table 2.18). The clinicopathological data were correlated with the expression pattern of EphB1, EphB2 and EphB4 receptors in the primary tumour and in the primary versus the hepatic metastasis.

Table 2.18: Clinicopathological characters of the study population

Parameters

Gender Male

Female

Age 50-55 yr

55-70 yr

Location Sigmoid and Rectum

Others (Right and transverse colon)

Depth of wall invasion Mucosa & SM

Subserosa and serosa

Stage (TNM) SII

SIII

SIV

LN mets Absent

Present

Tumor Differentiation Well

Moderate

Poor

POP Treatment No

Yes

Recurrence No

Yes

Tumour size <55mm

>55mm

Smoking No

Yes

B) Scoring System

The following scoring system was implemented throughout the study. It is divided into three categories. The first category is about the intensity of the receptor staining. The second category is about the percentage of the positive tumour cells stained whereas the third category is about the degree of the differentiation of the tumour. If there is a possibility of more than one score in the tested tumour section, strong 1+ expression and weak 2+ expression for instance, we usually take the most prominent score and omit the other to avoid any confusion.

a) The intensity of the receptor staining

0: receptor is not expressed (fig 2.16a)

1+: minimally expressed receptor (fig 2.16b)

2+: moderately expressed receptor (fig 2.16b)

3+: highly expressed receptor (fig 2.16c)

Fig 2.16: Different levels of the EphB receptor staining in CRC tissues. Fig(a) shows no staining of the EphB2 receptor (0); fig(b) shows weak staining of the EphB4 receptor (1+); fig(c) shows moderate staining of the EphB2 receptor (2+) and fig(d) shows high expression of the EphB4 receptor (3+).

b) The percentage of the positive cells stained (fig. 3.1)

0: no staining

1: 1-25% of cells stained,

2: 26–50% of cells stained,

3: > 50% of the tissue stained,

c) Degree of differentiation (DOD) of the tumour

Well differentiated: 95-100% glands present (fig 2.17a)

Mod differentiated: 50-95% glands present (fig 2.17b)

Poorly differentiated: < 50% glands present (fig 2.17c)

Fig 2.17: Degrees of differentiation of CRC; (a) demonstrates well differentiated CRC with good uptake of anti- EphB2 anti-goat primary antibody (AF467); (b) demonstrates moderately differentiated CRC with good uptake of anti-EphB2 anti-goat primary antibody (AF467) and (c) demonstrates poorly differentiated CRC with no uptake of anti-EphB2 anti-goat primary antibody (AF467).

Statistical Analysis

Descriptive statistics will be presented using mean and or median and Inter- Quartile Range (IQR), unless specified otherwise. Two variables were used to assess the expression of each receptor: the intensity of staining and the proportion of tumour cells stained. Each variable was first tested against the clinicopathological data. The two variables were then added together to get the total score of a receptor in each tumour. The total score was then tested against the clinicopathological data.

Categorical data generated with the total score and the clinicopathological variables were formatted in numerous “row x column” (r x c) contingency tables.

The approach to these analyses was by Exact (permutation) tests to make reliable inferences when the data may be small, sparse, heavily tied, or unbalanced and the validity of the corresponding large sample theory is in doubt (Ludbrook 2011, Agresti 2013). Tables in which the rows are “unordered” but the columns are “ordered” (singly ordered r x c tables); exact tests of independence were performed using Analysis of Variance (ANOVA).

Table 2.19: Example of a singly ordered 2 x 6 table

Score 0 2 3 4 5 6 Total Female 7 16 12 12 7 13 67 Male 14 20 17 8 7 13 79 Total 21 36 29 20 14 26 146

Tables in which both rows and columns are “ordered” (doubly ordered r x c tables), exact tests of independence were performed using Linear-by-linear tests (Agresti 2013).

100

Table 2.20: Example of a doubly ordered 3 x 6 table

DOD Score 0 2 3 4 5 6 0 2 9 5 1 4 11 1 12 24 23 16 9 11 2 8 4 1 2 2 4 Total 22 37 29 19 15 26

All tests were 2-sided and exact P-values less than 0.05 were deemed significant. Analyses were conducted using StatXact version 9.00 (Cytel Inc, Cambridge, Massachusetts, USA) and Stata® Software version 12.1 (College Station, Texas, USA).

101

Constituents of the solutions used in the staining protocol and their manufacturers

Stock Solutions Manufacturer & Constituents

High pH AR Buffer 10mM Tris-HCl and 1mM EDTA

Low pH AR Buffer 10mM Sodium Citrate pH 6.0

1X TBST 50mM Tris HCl, pH 7.4, 150mM NaCl, 0.1% Tween 20

H2O2 (Sigma Aldrich, Australia)

1:10 dilution of 30% H2O2

IHC 0.1% fish skin gelatine

10% Triton X100

10% Tween 20

In 1X PBS.

Homemade 2.5% HSB for each 100ml:

2.5% HS in 1xTBST stored at 4ͦC.

Primary Antibody (EphB1) (Santa Cruz Biotech. Inc, USA)

EphB1 (5F10A4) is a mouse monoclonal antibody raised against a recombinant protein corresponding to amino acids 19-133 of EphB1 of human origin 102

Primary Antibody (EphB2) (R&D Systems, Inc. USA)

EphB2 is produced in goats immunized with purified, NSO-derived, recombinant mouse EphB2 (mEphB2) extracellular domain. EphB2 specific IgG is purified by mouse EphB2 affinity chromatography.

Primary Antibody (EphB4) (Invitrogen Corporation, USA)

Invitrogen Mouse Monoclonal EphB4 antibody is highly purified from mouse ascites by protein A affinity

Vector biotinylated anti-mouse antibody (EphB1)

(Vector laboratories, USA)

Ready-to-Use ImmPRESS™ reagent anti-goat Ig (EphB2)

(Vector laboratories, USA)

Ready-to-Use ImmPRESS™ reagent anti-mouse Ig (EphB4)

(Vector laboratories, USA)

ABC kit (Vector laboratories, USA).

DAB substrate (Vector laboratories, USA)

Tween 20 (Sigma Aldrich, Australia)

103

Chapter Three

Results

Introduction (Table 3.1)

The total number of patients was 148; 78 (52.7%) were males and 70 (47.3%) were females. Most of the patients in the study were between 55 and 70 years (n=121) whereas only 27 patients were between 50 and 55 years. The mean patients age at the time of diagnosis was 61 years (range 50-70 years). Ninety two patients demonstrated lymph node metastasis (62.3%). Forty (27%) and eighty (54%) patients were diagnosed based on AJCC staging system as stage II and III, respectively, while 28 (19%) patients had stage IV due to the presence of liver metastatic disease. Anatomical classification of the tumours revealed that most of tumours were located in the left side colon (n=99) whereas the rest (n=49) were located in the right and transverse colon. The DOD of the sections was assessed by a qualified pathologist into well differentiated (n=32), moderately differentiated (n=95), and poorly differentiated (n=21) according to the World Health Organization system. The final histopathological workup of the surgical specimens showed that the majority of tumours had penetrated through the muscularis propria (n=105), followed by serosa and subserosa (n=35), but only few had infiltrated through the mucosa and submucosa (n=8). Postoperatively (POP), 64.1% (n=82) patients needed further treatment with chemoradiotherapy based on their histopathological report. Fifty seven patients developed recurrences after surgery. Most of the tumours were less than 55 mm in diameter (n=120), and 52% (n=66) patients had history of smoking.

104

Table 3.1: Clinicopathological parameters

Parameters Number

Gender Male 78

Female 70

Age 50-55 yr 27

55-70 yr 121

Location Sigmoid and Rectum 99

Others (Right and transverse 49 colon)

Depth of wall invasion Mucosa & SM 8

MP 105

Subserosa and serosa 35

Stage (TNM) SII 40

SIII 80

SIV 28 (14 1ry and 14 2ry)

LN mets Absent 56

Present 92

Tumor Differentiation Well 32

Moderate 95

Poor 21

POP Treatment No 66

Yes 82

Recurrence No 91

Yes 57

Tumour size <55mm 120

>55mm 28

Smoking No 82

Yes 66

105

Two variables were used to assess the expression of the three EphB receptors (EphB1, EphB2 and EphB4) in CRC tissues; the level of staining (negative=0, weak=1, moderate=2 and strong=3) (fig. 2.16), and the percentage of tumour cells stained in the section (0=negative, 1- 25%=low, 26-50%=moderate and >50%=high) (fig 3.1).

Fig 3.1: percentage of cells stained for the receptor per tumour section; a: 0=negative, b: 1-25%=low expression, c: 26-50%=moderate expression, and d: >50%=high expression.

106

During statistical analysis, the scores for the level of staining and the scores for the percentage of tumour cells stained were added together to form a third new variable indicated as EphB all; it has six scores (0, 2, 3, 4, 5, and 6). The three receptors were localized in the basolateral membrane of the epithelial cells.

Heterogeneous levels of staining were encountered in the sections of the same stage irrespective of the degree of differentiation of the tumours. Furthermore, same phenomenon was sometimes observed in tissues of the same section.

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

Strong staining for EphB1, EphB2 and EphB4 receptors was mainly seen in the normal colonic mucosa either of the normal specimens or of the adjacent “normal” colorectal mucosa from CRC specimens. Staining was most intense at the base of the crypts with the expression declining toward the luminal epithelium (fig 3.2). The normal sections were used to validate a staining protocol for each receptor before being applied to the project tissues. Also, normal mucosal slides were used as a positive control during each immunostaining run of the tumour tissues. The adenomatous tissues- adjacent to the tumour tissues also expressed the normal pattern of EphB1, EphB2 and EphB4 receptors.

107

Fig 3.2: IHC staining showing normal basal crypt distribution of (a) EphB1, (b) EphB2 and (c) EphB4 receptors.

Table 3.2: EphB1 receptor expression in primary CRC and in liver metastasis

A) Level of expression of EphB1 receptor per tumour section

EphB1

Score Number of Primary tumours (SII, SIII & SIV, n=134) Number of Secondary SII (40) SIII (80) SIV (14) tumours (n=14)

0 0 (0%) 19 (24%) 1 (7%) 2 (14%)

1 2 (5%) 45 (56%) 8 (57%) 6 (43%)

2 8 (20%) 14 (17.5%) 4 (29%) 6 (43%) 3 30 (75%) 2 (2.5%) 1 (7%) 0 (0%)

Total 40 80 14 14

0= no expression,

1= weak expression,

2= moderate expression, 108

3= strong expression.

B) Percentage of CRC cells with positive staining of EphB1 receptor per tumour section

EphB1

Score Number of Primary tumours (SII, SIII & SIV, n=134)) Number of Secondary SII (40) SIII (80) SIV (14) tumours (n=14)

0 0 (0%) 19 (24%) 1 (7%) 2 (14%)

1 1 (2.5%) 33 (41%) 6 (43%) 8 (58%)

2 5 (12.5%) 24 (30%) 7 (50%) 2 (14%)

3 34 (85%) 4 (5%) 0 (0%) 2 (14%)

Total 40 80 14 14

0= absent,

1= 1-25% tumour cells stained,

2= 26-50% tumour cells stained,

3= >50% tumour cells stained.

In general, 85% (114/134) of primary CRC and 86% (12/14) of metastatic CRC were positive (score 1) for EphB1 expression. In regards to the level

of expression of EphB1, ≥of primary CRC, 20 scored 0, 55 scored 1, 26 scored 2 and 33 scored 3 (fig 3.3). Among liver metastasis, 2 scored 0, 6 scored 1, 6 scored 2 and 0 scored 3 (table 3.2 (A) and fig 3.4). The score for percentage of CRC cells with positive staining of EphB1 protein showed 20 scored 0, 40 scored 1, 36 scored 2 and 38 scored 3. Of liver metastasis, 3 scored 0, 14 scored 1, 9 scored 2 and 2 scored 3 (table 3.2 (B)). Of 92 lymph node metastasis, in regards to the level of expression of EphB1 receptor, 79.3% (73/92) were positive for EphB1 expression; 19 scored 0, 54 scored 1, 17 scored 2 and 2 scored 3.

109

As it is shown in the table 3.2 (A), most of the SIV cases of the primary CRC (9/14 – 64%) and of the liver metastasis (8/14 – 58%), showed either no or weak expression of EphB1 receptor. This illustrates that loss or underexpression of EphB1 receptor is not only associated with poorly differentiated phenotype of the primary disease but is also associated with the presence of liver metastasis (see also table 3.10 and table 3.11).

Fig 3.3: Different levels of EphB1 receptor expression in CRC tissues, (a) no expression, (b) weak expression, (c) weak and moderate expression, and (d) high expression.

Fig 3.4: EphB1 receptor expression in CRC liver metastasis showing weak and no expression of the receptor 110

Table 3.3: EphB1 receptor expression in CRC and liver m etastasis in relation to clinicopathological parametersº

Parameters Number P-value

Gender Male 78 0.47

Female 70

Age 50-55 yr 27 0.45

55-70 yr 121

Location Sigmoid and Rectum 99 0.01*

Others (Right and 49 transverse colon)

Depth of wall Mucosa & SM 8 0.25 invasion MP 105

Subserosa and serosa 35

Stage (TNM) SII 40 <0.001*

SIII 80

SIV 28 (14 1ry and 14 2ry)

LN mets Absent 56 <0.001*

Present 92

Tumour Well 32 0.02* Differentiation Moderate 95

Poor 21

POP Treatment No 66 <0.001*

Yes 82

Recurrence No 91 0.08

Yes 57

Tumour size <55mm 120 0.62

>55mm 28

Smoking No 82 0.60

Yes 66

111

º Linear-by-linear and ANOVA tests were used for statistical analysis

* Significant result

POP: postoperative

The expression level of EphB1 receptor was significantly related to the DOD of the tumour. Loss of EphB1 receptor expression is more often seen in patients with poorly differentiated phenotype and in liver metastasis (P-value=0.02) (fig.3.4 and table 3.3). This is in line with the previous publication about EphB1 receptor (Sheng, Wang et al. 2008). EphB1 receptor expression loss has also a strong relationship with the lymph node metastasis (P- value<0.001), anatomical site of the tumour (P- value<0.01), TNM stage of the tumour (P-value<0.001), and POP treatment (P- value<0.001). In addition, EphB1 expression was also assessed against other clinicopathologic variables such as age, gender, recurrences, tumor size and smoking status. However, there was no significant relationship observed with these clinicopathologic variables.

112

Table 3.4: EphB2 receptor expression in primary CRC and in liver metastasis: first experiment

A) Level of expression of EphB2 receptor per tumour section

EphB2

Score Number of Primary tumours (SII, SIII & Number of SIV, n=134)) Secondary tumours (n=14) SII (40) SIII (80) SIV (14)

0 23(57.5%) 47 (58.7%) 8 (57%) 11 (79%) 1 13(32.5%) 32 (40%) 6 (43%) 2 (14%)

2 4 (10%) 1 (1.3%) 0 (0%) 1 (7%)

3 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Total 40 80 14 14

0= no expression,

1= weak expression,

2= moderate expression,

3= strong expression.

113

B) Percentage of CRC cells with positive staining of EphB2 receptor per tumour section

EphB2

Score Number of Primary tumours (SII, SIII & SIV, Number of n=134)) Secondary tumours SII (40) SIII (80) SIV (14) (n=14)

0 23(57.5%) 47 (58.7%) 8 (57.2%) 11 (79%)

1 13(32.5%) 29 (36.3%) 3 (21.4%) 2 (14%)

2 3 (7.5%) 3 (3.75%) 0 (0%) 1 (7%)

3 1 (2.5%) 1 (1.25%) 3 (21.4%) 0 (0%)

Total 40 80 14 14

0= absent, 1= 1-25% tumour cells stained,

2= 26-50% tumour cells stained,

3= >50% tumour cells stained.

In this experiment, capillary stainer was used to conduct the IHC staining similar to EphB1 receptor. EphB2 expression was variable across all samples. In contrast to the strong expression of EphB1, and to the strong expression of EphB2 receptor in the normal colonic mucosa, EphB2 expression in the CRC specimens was modest even in the early stages of the disease.

In this study, about 58.2% of the primary tumours (78/134) and 79% of the liver metastasis (11/14) showed no expression of the EphB2 receptor. This is in line with the published data where up to 60% of

114

the CRC cells tissues normally contain EphB2 receptor–negative cells despite evident nuclear β- catenin localization (Clevers and Batlle 2006). Only 42% (56/134) of the primary cancer and 21% (3/14) of the liver metastasis were positive (score 1) for

EphB2 receptor (table 3.4 (A)). Of the primary ≥tumour, in regards to the level of expression of the EphB2 receptor (table 3.4 (A)), 78 scored 0, 51 scored 1, 3 scored 2 and no one scored 3 (fig 3.5). Of the liver metastasis 11 scored 0, 2 scored 1, 1 scored 2 and no one scored 3 (fig. 3.6). In regards to the percentage of positively stained tumor cells of the primary CRC (table 3.4 (B)), 78 specimens scored 0, 45 scored 1, 6 scored 2 and 5 specimens scored 3. Of the liver metastasis, 11 specimens scored 0, 2 specimens scored 1, 1 specimen scored 2 and no specimen scored 3. Of 92 lymph nodes metastasis, in regards to the level of expression of the EphB2 receptor, 57 showed no expression, 34 showed weak uptake, 1 specimen showed moderate uptake and no one showed strong staining.

As it is shown in the table 3.4 (A), all the SIV cases of the primary CRC (14/14 – 100%), and most of the liver metastasis cases (13/14 - 93%), showed either no or weak expression of EphB2 receptor. This illustrates that loss or underexpression of EphB2 receptor is not only associated with poorly differentiated phenotype of the primary disease but also associated with the presence of liver metastasis (see also table 3.10 and table 3.11).

115

Fig 3.5: Different levels of EphB2 receptor expression in CRC tissues, (a) no expression, (b) weak expression, (c) weak and moderate expression

Fig 3.6: EphB2 receptor expression in CRC liver metastasis showing no expression of the receptor.

116

Table 3.5: EphB2 receptor expression in primary CRC and in liver metastasis: second experiment

A) Level of expression of EphB2 receptor per tumour section

EphB2

Score Number of Primary tumours (SII, SIII & SIV, Number of n=134)) Secondary tumours SII (40) SIII (80) SIV (14) (n=14)

0 32 (80%) 31 (39%) 7 (50%) 5 (36%)

1 7 (17.5%) 45 (56%) 7 (50%) 8 (57%)

2 1 (2.5%) 4 (5%) 0 (0%) 1 (7%)

3 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Total 40 80 14 14

0= no expression,

1= weak expression,

2= moderate expression,

3= strong expression.

117

B) Percentage of CRC cells with positive staining of EphB2 receptor per tumour section

EphB2

Score Number of Primary tumours (SII, SIII & Number of SIV, n=134)) Secondary

tumours

SII (40) SIII (80) SIV (14) (n=14)

0 32 (80%) 31 (39%) 8 (58%) 11 (79%)

1 8 (20%) 23 (29%) 3 (21%) 2 (14%)

2 0 (0%) 10 (12%) 0 (0%) 1 (7%)

3 0 (0%) 16 (20%) 3 (21%) 0 (0%)

Total 40 80 14 14

0= absent,

1= 1-25% tumour cells stained,

2= 26-50% tumour cells stained,

3= >50% tumour cells stained

Because of the non-significant result associated with the EphB2 receptor in the first run, another run was conducted using manual staining (chapter two). In this run, fewer cases of the primary (70/134 – 52%), and of the secondary (5/14 – 36%), showed no expression of the receptor in comparison to the first run. Also in this run, more cases of the primary (64/134 – 48%), and of the secondary (9/14 – 64.3%), were positive in regards to the level of expression of EphB2 receptor (table 3.5 (A)), in comparison to the

118

first run. Nevertheless, statistical analysis did not show any significant relationship between EphB2 expression in this run and the degree of differentiation (p-value = 0.2).

119

Table 3.6: EphB2 receptor expression in primary CRC and liver metastasis in relation to the clinicopathological parameters

Parameters Number P-value

Gender Male 78 0.84

Female 70

Age 50-55 yr 27 0.60

55-70 yr 121

Location Sigmoid and Rectum 99 0.81

Others (Right and transverse colon) 49

Depth of wall Mucosa & SM 8 0.70 invasion MP 105

Subserosa and serosa 35

Stage (TNM) SII 40 0.80

SIII 80

SIV 28 (14 1ry and 14 2ry)

LN mets Absent 56 0.05*

Present 92

Tumor Well 32 0.45 Differentiation Moderate 95

Poor 21

POP Treatment No 66 0.05*

Yes 82

Recurrence No 91 0.92

Yes 57

Tumour size <55mm 120 0.60

>55mm 28

Smoking No 82 0.48

Yes 66

* Significant result

120

Although EphB2 receptor is a well-known tumour suppressor (Cortina, Palomo- Ponce et al. 2007), in this series of patients, the expression of the EphB2 receptor did not show any significant relationship with the degree of the DOD of the tumours (p- value= 0.45) in comparison to EphB1 and EphB4 receptors. This finding, however, is consistent with the concept that EphB2 loss is associated with the most aggressive phenotype of CRC in comparison to the well-differentiated CRC. EphB2 gene identifies ISC-like tumour cells called colorectal cancer stem cells (CSCs), which are positioned within a cancer stem cell niche. These cells are aggressive in behaviour and play a central role in CRC relapse (Merlos-Suarez 2011). Similar to EphB1 receptor, EphB2 receptor expression showed almost significant relationship with LN metastasis and POP treatment (p-value = 0.05). However, EphB2 receptor did not show any significant relationship with the other parameters.

121

Table 3.7: EphB4 receptor expression in primary CRC and in liver metastasis

A) Level of expression of EphB4 receptor per tumour section

EphB4

Score Number of Primary tumours (SII, SIII & SIV, Number of n=134)) Secondary

tumours SII (40) SIII (80) SIV (14) (n=14)

0 18 (45%) 20 (25%) 4 (29%) 5 (36%)

1 16 (40%) 42 (52.5%) 10 (71%) 8 (57%)

2 6 (15%) 18 (22.5%) 0 (0%) 1 (7%)

3 0 (0%) 0 (0%) 0 (0%) 0 (0%)

Total 40 80 14 14

0= no expression,

1= weak expression,

2= moderate expression,

3= strong expression.

122

B) Percentage of CRC cells with positive staining of EphB4 receptor per tumour section

EphB4

Score Number of Primary tumours (SII, SIII & SIV, Number of n=134)) Secondary tumours SII (40) SIII (80) SIV (14) (n=14)

0 18 (45%) 20 (25%) 4 (29%) 5 (36%)

12 (30%) 13 (16%) 5 (35%) 6 (43%) 1 2 4 (10%) 24 (30%) 4 (29%) 2 (14%)

3 6 (15%) 23 (29%) 1 (7%) 1 (7%)

Total 40 80 14 14

0= absent,

1= 1-25% tumour cells stained,

2= 26-50% tumour cells stained,

3= >50% tumour cells stained.

In general, 69% (92/134) of primary CRC and 64.3% (9/14) of metastatic CRC were positive (score ≥ 1) for EphB4 expression (table 3.7 (A)). Of primary CRC, in regards to the level of expression of the receptor, 42 specimens showed no expression of the receptor, 68 showed weak staining, 24 showed moderate expression and none of the specimen showed strong expression of the receptor (table 3.7 (A) and fig. 3.7). Among liver metastasis, 5 specimens showed no expression of the receptor, 8 showed weak expression, only 1 specimen showed moderate expression and

123

none of the specimen showed strong expression of the receptor (table 3.7 (A) and fig. 3.8).

The score for percentage of primary CRC cells with positive staining of EphB4 protein showed 42 scored 0, 30 scored 1, 32 scored 2 and 30 scored 3 (table 3.7 (B)). Of liver metastasis, 5 scored 0, 6 scored 1, 2 scored 2 and 1 scored 3 (table 3.7 (B)). Of 92 lymph node metastasis (not shown in the table), 74% (68/92) were positive for EphB4 expression; 24 showed no expression, 50 showed weak expression, 18 scored showed moderate expression and none of the specimen showed high expression.

As it is shown in the table (3.7 (A)), all SIV cases (14/14 – 100%), and most of the liver metastasis (13/14 - 93%), showed either no or weak expression of EphB4 receptor. This illustrates that loss or underexpression of EphB4 receptor is not only associated with poorly differentiated phenotype of the primary disease but also associated with the presence of liver metastasis (see also table 3.10 and table 3.11).

124

Fig 3.7: Different levels of EphB4 receptor expression in CRC tissues, (a) no expression, (b) weak expression, and (c) moderate expression.

Fig 3.8: EphB4 receptor expression in CRC liver metastasis showing (a) no expression of the receptor, and (b) weak expression of the receptor.

125

Table 3.8: EphB4 receptor expression in primary CRC and liver metastasis in relation to the clinicopathological parameters

Parameters Number P-value

Gender Male 78 0.50

Female 70

Age 50-55 yr 27 0.40

55-70 yr 121

Location Sigmoid and Rectum 99 0.13

Others (Right and transverse colon) 49

Depth of wall Mucosa & SM 8 0.20 invasion MP 105

Subserosa and serosa 35

Stage (TNM) SII 40 0.80

SIII 80

SIV 28 (14 1ry and 14 2ry)

LN mets Absent 56 0.01*

Present 92

Tumor Well 32 Differentiation Moderate 95 0.04*

Poor 21

POP Treatment No 66 <0.001*

Yes 82

Recurrence No 91 0.71

Yes 57

Tumour size <55mm 120 0.32

>55mm 28

Smoking No 82 0.26

Yes 66

* Significant result

126

The expression level of EphB4 receptor was significantly related to the following parameter: DOD of the tumour. Loss of EphB4 receptor expression is more often seen in patients with poorly differentiated phenotype and in liver metastasis (P-value=0.04) (fig 3.7, fig 3.8 and table 3.8). This is in line with the previous publications which outlined the relationship between EphB4 receptor expression and CRC progression (Dopeso, Mateo-Lozano et al. 2009), and at the same time negates Kumar finding that EphB4 receptor functions as an oncogene in CRC (Kumar, Scehnet et al. 2009). EphB4 receptor expression loss has also a strong relationship with the lymph node metastasis (P-value=0.01), and POP treatment (P-value<0.001). It did not show significant relationship with the stage of the disease in comparison with the EphB1 receptor. In addition, EphB4 expression was also assessed against other clinicopathologic variables such as age, gender, and anatomical location of the tumour, depth of wall invasion, recurrences, tumor size and smoking status. However, there was no significant relationship observed with these clinicopathologic variables.

127

Table 3.9: Do the EphB2 negative CRC cells become EphB4 positive with the progression of the disease?

DOD SII SIII SIV 0 5 (50%) 5 (17%) 2 (50%)

1 3 (30%) 22 (76%) 2 (50%)

2 2 (20%) 2 (7%) 0 (0%)

Total 10/40 29/80 4/14

DOD: Degree of Differentiation

0: well-differentiated tumour,

1: moderately differentiated tumour,

2: poorly differentiated tumour.

Then, we investigated Kumar’s findings that, with the progression of the disease, CRC cells turn EphB2 receptor off, and at the same time turn EphB4 receptor on (Kumar, Scehnet et al. 2009). We picked up CRC cases, which did not show EphB2 expression (score = 0), but they are at the same time EphB4 positive (score 1), across the three stages, SII (10 cases), SIII (29≥ cases) and SIV (4 cases). Cases of the same stage are further subclassified based on their degree of differentiation, for examples, 10 cases of SII are divided as follows: 5 cases are well differentiated, 3 cases are moderately differentiated and 2 cases are poorly differentiated. All cases were compared against the degree of

128

differentiation (table 3.9). Although there is a high proportion of an EphB2-negative/EphB4-positive tumour in our cohort (total is 43/134 cases - 32%), our results did not support this notion (p-value = 0.54). However, it strongly supports the observation that expression of these receptors, EphB2 and EphB4, is coordinately silenced during CRC progression (Batlle 2005). It also means that with the progression of the disease, EphB4-positive tumour cells are not necessarily EphB2-negative. In general, this finding strengthens our observation that EphB4 receptor is underexpressed with the progression of the disease (table 3.8).

Table 3.10: The pattern of expression of the three receptors EphB1 EphB2 and EphB4 in CRC tissues

EphB1all, EphB2all, EphB4all (SII, SIII & SIV)

Score 0 2 3 4 5 6 Total

DOD

0 27 27 14 6 8 11 93

1 80 64 43 32 19 9 247

2 32 10 8 3 3 6 62

Total 139 101 65 41 30 26 402

129

Then, we studied the pattern of expression of the three receptors together in CRC tissues across the three stages, SII, SIII and SIV (134 tumours for each receptor, total is 402). For each single receptor, we added the level of expression to the percentage of staining of the tumour cells together, across the three stages, and indicated that as EphBall (EphB1all, EphB2all and EphB4all). Each EphBall has six scores (0, 2, 3, 4, 5, and 6). Each score is subclassified based on the DOD. Then, we added EphBalls together (EphB1all + EphB2all + EphB4all, 134 tumours per receptor, total is 402). Variables of the same score and same DOD are added together. For example, for score 0, we have a total of 139 tumours which are subclassified based on their DOD as follows: 27 tumours are well differentiated, 80 tumours are moderately differentiated and 32 tumours are poorly differentiated. And then, we compared the three EphBalls together against the DOD (table 3.10).

Our finding indicates that the three receptors together, EphB1, EphB2 and EphB4, are coordinately underexpressed with the progression of the disease (p- value = 0.02) and may function as tumour supressors, which is consistant with some of the functional data (Batlle 2005, Davalos, Dopeso et al. 2006, Dopeso, Mateo-Lozano et al. 2009).

130

Table 3.11: The pattern of expression of the three receptors EphB1, EphB2 and EphB4 in SIV CRC tissues and in liver metastasis of the same patients

SIV EphB1all, EphB2all, EphB4all (primary n=42 & liver metastasis n=42)

Score 0 2 3 4 5 6 Total

DOD

0 3 3 4 2 0 0 12

1 22 20 11 6 3 0 62

2 7 3 0 0 0 0 10

Total 32 26 15 8 3 0 84

Then, we conducted a comparison between the pattern of expression of the three receptors EphB1, EphB2 and EphB4 in SIV CRC patients with that in liver metastasis of the same patients. We followed the same style as what we did in table 3.10. For each single receptor, we added the level of expression to the percentage of staining of the tumour cells together, for stage IV CRC, and indicated that as EphBall (EphB1all, EphB2all and EphB4all). We did the same for liver metastasis tumours. Each EphBall has six scores (0, 2, 3, 4, 5, and 6). Each score is subclassified based on the DOD. EphBalls of the three receptors, EphB1all, EphB2all and EphB4all, in SIV CRC (14 tumours per receptor, total is 42), were added together. EphBalls of the three receptors in liver metastasis (14 tumours per receptor, total is 42), were

131

also added together. We then added SIV CRC tumours to liver metastasis tumours (42 tumours each, total is 84) and analysed them against DOD. Our finding indicates that the three receptors EphB1, EphB2 and EphB4, are also underexpressed in liver metastasis in comparison to SIV CRC tissues (p-value = 0.03).

This finding supports our observation that the three receptors are coordinately underexpressed in poorly differentiated CRC (table 3.10). It also emphasizes that underexpression of the three receptors EphB1, EphB2 and EphB4, is not only a sign of poorly differentiated CRC, but also a sign of the presence of liver metastasis. Furthermore, this evidence adds more power to our finding that with every receptor, the majority of SIV CRC and liver metastasis patients showed either weak or no expression of the receptor in their tissues (table 3.2, 3.4 and 3.7).

132

Chapter Four

Introduction

In analysing the results of this study, we were able to confirm two of the research hypotheses and refute one: we proved that EphB1 receptor underexpression is encountered in the most undifferentiated phenotype of CRC. We could also show that EphB1, EphB2 and EphB4 receptors are homogeneously underexpressed with the progression of the disease and in the presence of liver metastasis of the same patients. On the other hand, we rejected that EphB4 positive tumour cells tend to turn off EphB2 receptor expression, with the progression of the disease.

Our data revealed that EphB1 underexpression is not only associated with the presence of poorly differentiated CRC, and also that with the disease progression, EphB1 expression is reduced, being high in SII and SIII but low in SIV. For the first time in the literature, after studying EphB1 receptor expression in the liver metastatic tissues from the same patients of the primary CRC, we have evidenced that EphB1 underexpression is associated with the presence of distant metastasis such as lymph node metastasis and liver metastasis and this is supported by the notion that EphB1 underexpression is associated with invasion and distant metastasis. We could demonstrate, for the first time in the literature, that the three receptors, EphB1, EphB2 and EphB4, are homogeneously underexpressed during progression of the disease. We could also demonstrate that this underexpression in the primary cancer is imitated in liver metastasis of the same patients. We found that underexpression of these

133

three receptors is associated with the presence of lymph node metastasis and liver metastasis. The extent of EphB downregulation in CRC correlates with poor prognosis, cancer invasion and metastasis. We found that across the three stages of CRC, SII, SIII and SIV, EphB4 positive tumour cells are not necessarily be EphB2 negative cells.

EphB1, EphB2 and EphB4 Expression in CRC

Our data indicates that, with the progression of the disease, the three receptors, EphB1, EphB2 and EphB4, are consistently underexpressed across the three stages, SII, SIII and SIV. It is the first study, which investigates the pattern of expression of the three receptors together in CRC and compares it with the DOD of the disease. Hafner et al studied pattern of expression of several EphAs and EphBs receptors including EphB1, EphB2 and EphB4 in benign and in malignant tissues, however, it was more quantitative rather than being qualitative study. The study was not specifically about CRC as it included other organs such as lung and kidney. Even though, it included only 5 CRC specimens without further details about the clinicopathological data of the specimens. It did not compare the pattern of expression of the three receptors against the DOD of the disease and it did not include any liver metastasis tissues (Hafner, Schmitz et al. 2004). Functional studies showed that EphB2 and EphB3 receptors are key coordinators of ISCs proliferation and differentiating cells migration along the intestinal crypt axis. 50% of the mitogenic activity of the

134

ISCs is controlled by the EphB receptors. Knockout of EphB receptors is associated with loss of proliferation of the ISCs and mingling of the ISCs with the differentiating cells (Holmberg 2006). However, there is little evidence so far about the functional role of EphB1 and EphB4 receptors in the normal colon. EphB2 receptor controls ISCs proliferation and differentiating cells migration through two distinct pathways; proliferation is a kinase-dependent pathway through cyclin D1 domain whereas migration is a kinase-independent pathway through P13K domain (Genander and Frisén 2010). In 2007, Cortina et al systematically investigated the mechanism by which EphB receptors inhibit tumour progression. They looked specifically at EphB2, EphB3 and EphB4 receptors but not EphB1 receptor. Upon EphB-ephrinB interaction, E-cadherin is translocated from the cytoplasm to the basolateral membrane leading to coupling between cell-cell adhesion and cell-cell deadhesion. Cell-cell adhesion is between symmetrical cells and cell-cell deadhesion is between asymmetrical cells. Then, they assessed this observation in vitro. They found that stimulation of E-cadherin in a culture of EphB positive and ephrinB positive cells leads to clustering of the symmetrical cells into compartments and segregation between asymmetrical cells. In vivo, during intestinal tumorigenesis in APC deficient mice, EphB positive tumour cells appeared completely enclosed by the surrounding ephrinB positive normal epithelium restricting movements of tumour cells. This observation was further tested by knocking down ephrinB ligand in vivo. Compartmentalization phenomenon was abrogated resulting in acceleration of tumorigenesis in the colon of ephrinB deficient mice

135

(Cortina, Palomo-Ponce et al. 2007). As the tumour progresses, EphB receptors are silenced, kinase- dependent cyclin D1 domain becomes independent of EphB2 receptor and takes over cancer stem cells proliferation. Reduced EphB activity accelerates the progression of CRC, in the presence of mutated APC and Wnt/β-catenin genes (Genander, Halford et al. 2009). Cyclin D1 is Wnt-signaling transcription genes. Mutations in the APC gene are the initiating step in CRC development. Mutations of the Wnt-signaling genes result in the accumulation of β-catenin. Cells expressing mutant β-catenin produce high levels of cyclin D1. Increased transcription of cyclin D1 by β- catenin is likely to play an important role in the development of CRC (Tetsu and McCormick 1999). Downregulation of EphB receptors is linked to a poorer phenotype of CRC and poor phenotype of CRC is associated with poor prognosis. EphB2 receptor expression was evaluated as a survival factor among a group of 1,476 CRC patients. Survival data of only 1,414 patients were studied and assessed against the expression of EphB2 receptor. Significant variation in survival was observed in patients with different levels of EphB2 expression; negative expression was associated with the shortest survival whereas patients with strong expression had the longest survival rate (Lugli, Spichtin et al. 2005). The exact molecular mechanisms, which underlie the apparent loss of expression of EphB receptors upon progression of CRC, are largely unknown. Ronsch et al investigated the mechanisms behind downregulation of EphB2, EphB3 and EphB4 receptors in CRC tissues. They conducted their study on human cell lines, cells of normal colorectal mucosa, low- and high-grade

136

adenomas and CRC tissues. Overexpression of histone deacetylases (HDACs) has been linked to CRC progression. They concluded that functional class I and class III HDACs and absence of active histone marks contribute to epigenetic inactivation of EphB2, EphB3 and EphB4 receptors during CRC progression

(Ronsch 2011).

EphB1, EphB2 and EphB4 expression and the presence of liver metastasis

EphB1, EphB2 and EphB4 underexpression is associated with the presence of liver metastasis. There is a direct proportional relationship between tumour grade and the presence of distant metastasis; cells of high tumour grade are more invasive, have high turnover rate and associated with distant metastasis (DeVita 2011) (2). We could confirm a twofold relationship between expression of EphB1, EphB2 and EphB4 receptors, and the presence of liver metastasis. We found that underexpression of EphB1; EphB2 and EphB4 receptors can predict the presence of liver metastasis. Also, we found indirectly that underexpression of these three receptors is associated with the presence of high grade CRC. This is the first study in the literature, which established a clear relationship between EphB1, EphB2 & EphB4 receptor expressions and the presence of liver metastasis in CRC. Previous studies concentrated on single EphB receptors. Chen et al (Chen 2013), for instance,

137

analysed the differential gene expressions, which can be considered as markers for the presence of CRC- related liver metastasis. They showed that EphB2 gene is underexpressed in the presence of liver metastasis. Furthermore, in comparison to our study, Chen et al did not include any primary CRC tissues (Chen 2013). In a study conducted in 205 CRC patients who underwent surgery but without any adjuvant therapy, EphB2 underexpression was assessed against several clinicopathological factors including lymph node metastasis and liver metastasis. Oshima and his team found that EphB underexpression has a significant correlation with the presence of liver metastasis reduced. They concluded that reduced expression of the EphB2 genes might be a novel marker or predictor of liver metastasis (Oshima, Akaike et al. 2008). On the other hand, EphB2 and EphB4 receptor overexpression was reported to predict liver metastasis in cholangiocarcinoma. A study on 6 human cholangiocarcinoma cell lines showed that high EphB2 and EphB4 is a marker of the presence of distant metastasis (Khansaard, Techasen et al. 2014). Metastasis is often associated with several clinical and pathological characteristics. Among these are tumour size and lymph node involvement. The bigger the size of the tumour, the more is the chance of distant metastasis (DeVita 2011) (2). However, in our study, we could not establish a clear threshold between EphB1, EphB2 and EphB4 receptors expression, and the tumour size. In CRC, tumour cells undergo epithelial to mesenchymal transition (EMT), which is associated with several histological changes in the surrounding cellular and stromal environment (fig. 4.1).

138

Fig. 4.1: The cancer cells change their morphology and gain mesenchymal cells characters inducing epithelial mesenchymal transition (EMT). The invasive carcinoma stage involves epithelial cells losing their polarity and detaching from the basement membrane. The invasive carcinoma cells disseminate through blood and lymphatic vessels to distant organs (Kalluri and Weinberg 2009) (with permission).

In EMT, basement membrane becomes fragmented and cells lose their epithelial characteristics and gain mesenchymal properties including resistance to apoptosis and the gaining of stem cell properties. These cells can penetrate into lymph or blood vessels, allowing their passive migration to distant organs. The key changes in cells that undergo EMT are as follows: the loss of E-cadherin which is a prominent hallmark of

EMT causing disruption of adherins junctions and loss of cell polarity; the nuclear translocation of β-catenin leading to a more metastatic and invasive transcriptional program; gain of vimentin, a mesenchymal marker, and the upregulation of the Snail family of transcription factors (Wray, Shah et al. 2009). Eph receptors have been found to play an important role in many aspects of EMT. Some of the Eph receptors function as tumour suppressor genes inhibiting EMT, whereas other Eph receptors function as oncogenes promoting EMT. These paradoxical roles of Eph receptors in EMT are dependent on: the type of Eph receptor; the oncogene context within which the Eph receptor functions and the crosstalk of Eph receptors with EMT-related signal transduction

139

pathways such as those induced by Wnt, Ras/MAPK, Akt-mTOR, and Notch. For example, the tumour suppressor function exhibited by EphB2 and EphB3 can be explained in two ways. EphB might affect the redistribution of cellular E-cadherin to the plasma membrane, enhancing E-cadherin–mediated cell-cell adhesion through the Rac1 pathway, thus preventing invasion and distant metastasis. Inhibition of EphB receptors and E-cadherin during tumour progression abrogates cell-cell adhesion and helps tumour cells invasion (fig 4.2).

Fig. 4.2: The relationship between EphB receptors and E-cadherin in normal and tumour cells. The mutual regulation between ligand-dependent EphB function and E-cadherin helps to induce cell-cell adhesion. However, epithelial-mesenchymal transition (EMT) inhibits cell-cell adhesion and promotes tumour cell invasion and metastasis (Li, Chen et al. 2014) (with permission).

Furthermore, EphB-mediated compartmentalization restricts the spreading of EphB-expressing tumour cells into ephrinB1- positive regions. On the other hand, EphA2-expressing cells have been shown to acquire the EMT phenotype by up-regulating mesenchymal markers and down-regulating epithelial markers (Li, Chen et al. 2014) (fig. 4.3).

140

Fig. 4.3: Eph receptors crosstalk with EMT-related signaling pathways. Eph receptors initiate forward signaling to promote cancer progression via the MAPK signaling pathway in both ligand-dependent and ligand-independent manners. The Wnt/β-catenin signaling pathway promotes tumour progression by down-regulating EphB2. EphA2 promotes EMT through the Wnt/β-catenin signaling pathway (Li, Chen et al. 2014) (with permission).

The interactions between EphBs and ephrinBs play a role in mesenchymal stem cells (MSC)-mediated T-cell suppression by activating PI3Kinase, Src, Abl kinase, or JNK pathways and by modulating immunosuppressive factors secreted by MSC such as TGF-β1, and T-cell proliferation factors, including INF-g, TNF-a, IL-2, and IL-17. This has been proved as a mechanism in inhibition of cancer cell proliferation, motility and invasion (Nguyen, Arthur et al. 2013).

The presence of liver metastasis carries a poorer prognosis than the non-metastasizing primary CRC. The mortality rate associated with liver metastasis is higher than that of primary CRC (Dexiang, Li et al. 2012). As we first found that homogeneous underexpression of the three receptors, EphB1, EphB2 and EphB4, is associated with the most undifferentiated primary CRC, and subsequently with worse prognosis. And as we also found that underexpression of the three receptors is also associated with the presence of liver

141

metastasis and hence the worst prognosis in the disease. Then, this sheds the lights over the power of EphB receptors as reliable prognostic markers in the disease. A future study includes the three receptors together, and evaluates them against overall survival (OS) and disease free survival (DFS) can authenticate this notion. Furthermore, these findings may provide a novel anticancer therapeutic intervention, which can inhibit distant metastasis and hence improve overall survival.

EphB1 Receptor Expression in CRC

Our data showed that EphB1 underexpression was associated with the presence of poorly differentiated CRC. EphB1 expression was assessed across the three stages of CRC, SII, SIII and SIV, against the degree of differentiation of the disease. We found that the lowest expression of EphB1 receptor was encountered in the most undifferentiated phenotype of CRC. This was in consistent with the previously published in the literature. Sheng et al found that EphB1 receptor was underexpressed in poorly differentiated CRC (Sheng, Wang et al. 2008). They conducted their study using CRC cell lines as well as human CRC specimens with adjacent normal mucosa from 78 patients. Besides that, they also used 15 colorectal adenomatous tissues to compare between EphB1 expression in benign adenoma and in CRC. In their methodology, they used quantitative RT-PCR and IHC to detect the expression of EphB1 receptor in the CRC specimens. Our study extends the analysis of

142

EphB1 receptor expression in CRC to include liver metastasis specimens from the same patients of primary CRC. EphB1 underexpression has also been shown to be associated with other poorly differentiated cancers such as renal cell carcinoma (Shuigen Zhou 2014), gastric carcinoma (Wang, Dong et al. 2007), ovarian serous carcinoma (Haiyan Wang 2014), and of rare tumours such as cholangiocarcinoma (Yuan Y 2014). We found that, for the first time, EphB1 receptor was not only underexpressed in poorly differentiated CRC but also in liver metastasis of the same patients. This study demonstrated that EphB1 receptor underexpression was not only associated with poorly differentiated CRC, but was also associated with the presence of distant metastasis such as liver metastasis and lymph nodes metastasis. This was also in line with our findings that with the disease progression, EphB1 expression has inverse relationship with the TNM stage of the disease; high in SII and SIII but low in SIV. Sheng et al could not prove any significant relationship between EphB1 expression in CRC and TNM staging of the disease. As part of their study, Sheng et al investigated whether CpG island hypermethylation on the promoter region of EphB1 receptor underpins silencing of the EphB1 receptor in their CRC specimens. Although, their results were inconclusive, it has been shown in another study that CpG island hypermethylation causes EphB1 epigenetic silencing and underexpression (Kuang, Bai et al. 2010). In addition, somatic mutations in EphB1 contribute to cancer progression by disrupting cell-cell adhesion via RhoGTPase and Ras-MAPK signaling (Bonifaci, Gorski et al. 2010). In the normal colon and in the adenomatous tissues-adjacent to the tumour tissues,

143

we found that EphB1 receptor has the same distribution pattern as EphB2 and EphB3 receptors; it is localized to the base of the crypts and then decreased gradually towards the lumen (Batlle, Henderson et al. 2002). EphB2 and EphB3 receptors are Wnt signaling transcription genes. In the presence of active Wnt signaling, EphB2 and EphB3 receptors are directly involved in the proliferation of the ISC in the base of the crypts, and in the maturation, specialization and upward migration of the progenitor cells. Double EphB2-EphB3 knockout (KO) mice has resulted in loss of proliferation of the ISC, intermingling between ISC and the proliferating cells, and atrophy of the intestinal crypts (Batlle, Henderson et al. 2002). EphB1 receptor is also a Wnt transcription gene, and thus one may speculate that EphB1 has the same biological functions in the colon crypts as EphB2 and EphB3 receptors. Functional studies are required to prove this. Activation of Wnt signaling mutations are considered as essential key step in the initiation of CRC through adenoma-carcinoma progression. These mutations result in stabilization of β-catenin and constitutive transcription by the β-catenin/T cell factor-4 (Tcf-4) complex. EphB receptors are target genes of Wnt signaling pathway, however, they are silenced with the progression of CRC. Loss of EphB expression strongly correlates with degree of malignancy. They are highly expressed during early stages of CRC, but they are downregulated with the progression of the disease. Even in precancerous adenoma, EphB receptors function as adenoma suppressors. This is supported by the observation that there is a variation in EphB expression at different histopathological grades of adenomas; low grades adenomas show high

144

expression of EphB receptors, whereas high grade/dysplastic adenomas show less expression of EphB receptors (Batlle 2005). Although we do not have a direct evidence about the importance of EphB1 as a prognostic factor in CRC, however, it has been found that loss of expression of EphB1 protein in gastric carcinoma (Wang, Dong et al. 2006) and in ovary carcinoma (Wang, Wen et al. 2014) is associated with invasion and metastasis. This accumulating evidence indicates that EphB1 receptor expression has also a prognostic impact in CRC patients. Future studies about EphB1 expression in CRC as a reliable prognostic protein can support this notion.

Our data also showed, as a unique to this study, that expression of EphB1 receptor in CRC tissues had significant relationship with the anatomical location of the tumour as well as with the POP therapy. So far, there is no single study in the literature, which sheds the lights on the direct relationship between EphB1 expression and tumour location. Likewise, the significance of EphB1 underexpression on the outcome of treatment for CRC is still unknown, however, one may speculate that EphB1 underexpression can indirectly predict disease relapse, and subsequently, intensive adjuvant chemoradiotherapy may be tailored for these individuals in order to prevent disease recurrence and to improve overall survival. A dedicated future study, which can explore the relationship between EphB1 expression and the location of the tumour as well as the importance of EphB1 as a reliable predictor marker in CRC is highly

145

recommended.

Our data also showed that EphB1 expression was markedly varied among the CRC specimens. The three levels of EphB1 receptor expression (low, moderate and high) were encountered in the specimens of the same stage (fig 4.4).

Fig 4.4: Moderately differentiated SIV CRC sections showing different levels of EphB1 expression; (a): low expression, (b): moderate expression and (c): high expression.

Furthermore, sometimes within the same specimen, the three levels of the EphB1 receptor expression were seen (fig 4.5).

146

Fig 4.5: EphB1 receptor expression in CRC liver metastasis showing weak, moderate and high expression of the receptor in the same section.

In contrast, in the normal colonic tissues, in the normal adjacent tissues- adjacent to tumour tissues and in the adenomas-adjacent to tumour tissues, the level of expression of the receptor was exclusively homogeneous (fig 3.1a). This indicates that EphB1 receptor expression becomes heterogenous and possesses focal pattern during the transition from normal and adenomatous tissues to adenocarcinoma.

EphB2 Receptor Expression in CRC

Our data suggests that EphB2 receptor behaves as a tumour suppressor gene in CRC. We further studied the level of expression of the EphB2 receptor across the three stages of CRC, SII, SIII and SIV, and found that expression decreases as the tumour cells become less differentiated. Then, we investigated the receptor behaviour and level of expression in a group of CRC patients who developed liver metastasis. We could demonstrate that EphB2 receptor is further underexpressed in liver metastasis. We found that the majority of SIV population, who developed liver

147

metastasis, had either weak or no expression of EphB2 receptor in their primary and secondary tumours.

Guo et al studied the pattern of expression of EphB2 receptor at different types of tissues in CRC patients. They compared the expression pattern of EphB2 receptor in CRC tissues with adenomas. They also compared lymph nodes metastasis and liver metastasis with primary CRC tissues. They used 345 primary CRCs tissues, paired lymph node metastases from 98 patients and paired liver metastases from 82 patients. They used IHC and microarray to assess expression of EphB2 in the tissues. The liver metastases expressed a lower level of EphB2 compared with their primary cancers and lymph node metastases. They also compared EphB2 expression with the clinical outcome of the patients in the study. EphB2 underexpression was associated with adverse outcome of the prognosis. They found that EphB2 expression was independent factor predicting disease- free survival beside tumour stage (Guo, Zhang et al. 2006). This finding is supported by another study, which showed that EphB2 is a prognostic factor in CRC patients (Jubb, Zhong et al. 2005). Although our total study population was only 148 CRC cases including liver metastasis, however, our study, for the first time in the literature, investigated the pattern of expression of the three EphB receptors together EphB1, EphB2 and EphB4 in liver metastasis, whereas Guo et al’s study only investigated one EphB receptor.

EphB2 receptor functions as an ISC marker gene that can be used to identify CRC stem cells (CSCs) and predict disease relapse. Merlos et al developed intestinal crypt model formed of proliferating cells and

148

differentiating cells. They further divided both types of epithelial cells in their model, based on the EphB2 gene expression, into EphB2high, EphB2medium and EphB2low cells. EphB2high proliferating cells expressed around 5-fold higher levels of ISC-specific genes Lgr5 than EphB2med and EphB2low cells. In contrast, EphB2low differentiating cells corresponded mainly to crypt differentiating cells genes. EphB2 and Lgr5 genes can identify ISCs. Then, they tested EphB2 gene expression in CRC cells with different phenotypes. They found that the expression of EphB2-ISC gene Lgr5 was strongly associated with the most aggressive and metastatic CRCs compared with nonmetastatic colon cancer. In addition, the EphB2-ISC gene Lgr5 was associated with poorly differentiated tumours compared to more benign well-differentiated CRCs. Furthermore, they found that high expression of ISC- specific genes, EphB2 and Lgr5, predicts disease relapse in CRC patients than those with low expression. In contrast, differentiating cells was enriched in genes that considered non reliable to predict disease relapse over follow-up. This finding indicates that EphB2 and Lgr5 genes identify CSCs located at the bottom of the intestinal crypts, and predict cancer relapse. This is because CSCs have the potential to regenerate cancer after therapy (Merlos-Suarez, Barriga et al. 2011).

149

EphB4 Receptor Expression in CRC

Our data indicated that EphB4 receptor is underexpressed in the most unfavourable phenotype of CRC. In addition, we also found that EphB4 receptor is underexpressed in liver metastasis. This is supported by the previously reported descriptive studies about EphB4 expression in CRC. Davalos et al investigated EphB4 expression in CRC cell lines and in CRC samples using IHC and PCR in their study. They compared EphB4 expression with the survival outcome of their patients in the study. They found that EphB4 is lost with the progression of the disease. The expression of EphB4 was significantly lower in the lymph node metastases in comparison to the primary tumours. They also found that patients with low EphB4 tumour level had significantly shorter survival than patients with high EphB4 level. They concluded that low EphB4 tumour levels can be considered as independent prognostic marker in CRC patients. They also concluded that EphB4 is a reliable predictor tumour marker, which can predict disease recurrence in EphB4-low expression CRC patients who could therefore benefit from a more aggressive treatment of their disease. Davalos et al investigated the mechanism, which underpins decreased expression of EphB4 in CRC tissues. The results showed that aberrant hypermethylation of the EphB4 promoter occurs frequently in CRC and that this mechanism regulates EphB4 expression. EphB4 protein level was compared between samples with promoter hypermethylation with that with no promoter hypermethylation. EphB4 protein level was lower in the tumours with promoter hypermethylation than in the

150

tumours with no evidence of promoter hypermethylation (Davalos, Dopeso et al. 2006). This evidence is also potentiated by Kuang et al’s findings in acute lymphocytic leukaemia (ALL). Kuang and his colleagues investigated the mechanism of silencing of EphB4 in ALL and found that EphB4 acts as a tumour suppressor gene in ALL and promoter hypermethylation of EphB4 gene is the key mechanism of EphB4 gene silencing in ALL cells (Kuang, Bai et al. 2010). Dopeso et al also found in vivo in 2009 that EphB4 receptor is underexpressed in CRC with the proliferation of the disease. They used IHC and PCR in their methods to study EphB4 receptor expression in the large bowel tumours of APC deficient mice. To study the effect of EphB4 genes on the growth of the large bowel tumours of these mice, they used xenograft models of CRC cell lines with high and low levels of EphB4 genes. They found that large bowel tumours increased in size after injection of low levels of EphB4 genes into these mice in comparison to the control group. IHC staining showed decrease in the levels of expression of EphB4 receptors in these large bowel tumours, which increased in size. PCR also, showed decrease in the EphB4 mRNA gene levels in the tumour cells with high proliferation rate (Batlle 2005, Dopeso, Mateo-Lozano et al. 2009).

We found the EphB4 receptor is underexpressed in the poorly differentiated CRC; this is inconsistent with findings that EphB4 receptor is upregulated with the progression of CRC (Stephenson, Slomka et al. 2001, Kumar, Scehnet et al. 2009). Stephenson et al conducted a quantitative investigation of the level of

151

EphB4 expression in CRC samples using PCR and IHC in their methods. They used CRC and adjacent normal samples collected from 60 patients. They found that EphB4 receptor is overexpressed in CRC tissues in comparison to the adjacent normal colonic tissues. They concluded that EphB4 receptor plays a role in the progression of the disease (Stephenson, Slomka et al. 2001). Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody, which was approved as a first-line treatment for metastatic CRC in combination with chemotherapy. Using qRT- PCR arrays to measure EphB4 gene level in the responders and nonresponders to the treatment with bevacizumab, responders had lower EphB4 expression than the nonresponders indicating that EphB4 expression is useful in predicting response to treatment. Then, they assessed EphB4 expression against the median survival rate for high and low EphB4 expression groups. High EphB4 expression was associated with shorter overall survival in comparison to the low EphB4 expression group. The median survival time of patients with high EphB4 expression was 16 months, whereas the median survival time of patients with low EphB4 expression was 48 months. The influence of EphB4 on patient survival was independent of age, gender and histologic tumour grade. IHC on the CRC tissue samples used for qRT- PCR array analysis showed high expression of EphB4 in tissue sections with high EphB4 group as compared with specimens from patients in the low EphB4 group, in which EphB4 staining was faint or even absent (Guijarro-Munoz, Sanchez et al. 2013).

152

The functional effects of EphB4 gene in CRC cells, subjected to different biological conditions, were investigated by several researchers. Similar to the controversy in the descriptive studies, EphB4 has paradoxical functions in CRC cells. Dopeso et al found that inactive form of EphB4 gene, genetically modified in a cell line and then overexpressed in a xenograft nude mice containing large bowel tumours, had resulted in significantly larger tumours and shorter life span of these mice in comparison to the control group. Then, they investigated the effect of EphB4 gene on the migration of CRC cells by using a Matrigel invasion assay in a cell line. They found that in vitro reduced expression of EphB4 gene was associated with a remarkable increase in CRC tumour cells invasion, whereas increased expression of EphB4 gene was associated with decreased invasion of the tumour cells (Batlle 2005, Dopeso, Mateo-Lozano et al. 2009). Davalos et al found that forced expression of EphB4 in vitro resulted in a significant reduction in the number of CRC colonies suggesting that EphB4 acts as a novel tumour suppressor gene in CRC (Davalos, Dopeso et al. 2006). On the other hand, Kumar et al found that EphB4 gene is overexpressed with the progression of the CRC disease. However, they compared between EphB4 and EphB2 genes expression in CRC tissues. As a unique finding to their study, they observed that with the progression of the disease, CRC cells turn on EphB4 receptor and at the same time, they turn off EphB2 receptor. The same observation was also found in lymph node metastasis and in liver metastatic tissues of the same patients where all metastatic tissues were EphB4 positive but EphB2 negative. They further studied the biological mechanisms, which

153

underlie EphB4 overexpression and EphB2 underexpression in CRC cells. Both receptors are Wnt signaling transcriptional genes. Nevertheless, EphB4 gene expression is controlled by the coactivator CBP, whereas EphB2 expression relies on the coactivator p300. CBP stimulates proliferation and migration of tumour cells whereas p300 has the opposite effect. With the disease progression, tumour cells become more dependent on CBP pathway at the expense of the p300 pathway (Kumar, Scehnet et al. 2009). Although CBP is a known transcriptional co-activator that enhances tumour cell growth, and differentiation, however, CBP may function as a transcriptional co- repressor depending on its interaction with other factors in response to different cellular signals. For example, XIAP-associated factor 1 (XAF1) is a novel CBP-binding partner. Ectopic expression of XAF1 and CBP in a range of colon cancer cell lines suppressed cell growth. In addition, ectopic expression of CBP enhanced the suppressive effect of XAF1 on cell growth (Sun, Qiao et al. 2007). Furthermore, and in contrast to Kumar et al’s findings, CBP sometimes cooperates with p300 coactivator to restrict the growth of other tumours such as leukaemia (Iyer, Ozdag et al. 2004). Kumar and his colleagues conducted their study using fresh frozen tissues, which are known to be associated with loss of morphological integrity of the tissues including the antigen itself. This can subsequently compromise the study outcome. The use of paraffin-embedded tissues is therefore helpful in overcoming these problems (Buchwalow and Bocker 2010). Kumar and his team considered only the intensity of staining of the membranous receptors in their data scoring. In contrast, most of the published

154

articles in this context used percentage of positively stained tumour cells beside the intensity of staining in their data scoring to enhance their results (Sheng, Wang et al. 2008). Kumar et al used the American Joint Committee on Cancer (AJCC), but not the DOD of the disease, to compare receptor expression with the various tumour stages. In fact, DOD of the disease, and not the TNM staging, is the key reference which gives more precise assessment of the level of the EphB receptors expression against the different pathological grades of the disease (Davalos, Dopeso et al. 2006). Furthermore, the grading system is more accurate as a prognostic factor than the TNM staging system. Multivariate analysis has shown that histologic grading of tumour differentiation is a stage independent prognostic factor (Ueno, Kajiwara et al. 2012). Last but not least, Kumar et al partially attributed the differences between his results and Batlle study’s results about the expression of EphB receptors in CRC (Batlle 2005) to the potential biological difference in the CRC from the Netherlands and from the United States. Firstly, Whilst these biological differences observed in CRC are more attributed to the environmental influences and to the racial diversity (Virk, Gill et al. 2010), nevertheless, no existing evidence in the literature, so far, which links these biological differences in CRC or even racial differences literally with the expression of the EphB receptors, as the main reference in this issue is the DOD of the disease.

EphB4 is one of the RTKs, which has dual functions in

155

tumour cells. One explanation for this apparent paradoxical function is the activation of EphB4 by its cognate ligand ephrinB2. EphB4 promotes tumour cells proliferation in the absence of the cognate ligand ephrin-B2 whereas EphB4 acts as tumour suppressive in the presence of ephrin-B2. EphB4 enhances cell migration and invasion and ephrin-B2 reverses this effect. EphB4 overexpression protects cells from apoptosis but receptor activation by ephrin-B2 ligand induces apoptosis (Rutkowski, Mertens‐ Walker et al. 2012). One example is the dual function of EphB4 receptor in breast cancer. EphB4 is overexpressed in breast cancer and provides survival signal for breast cancer cells. EphB4 facilitates migration and invasion of tumour cells by increased production of matrix metalloproteases such as MMP-9 thus increases degradation of surrounding matrix proteins. EphB4 knockdown induces apoptosis through down-regulation of antiapoptotic proteins (Kumar, Singh et al. 2006). On the other hand, EphB4 receptor behaves as a tumour suppressor in breast cancer when stimulated by its ligand ephrin-B2. EphB4 suppresses breast cancer cells through Abl family tyrosine kinases and the Crk adaptor protein. Abl–Crk pathway inhibits breast cancer cell proliferation, invasion and metastasis through downregulation of MMP-2 (Noren, Foos et al. 2006).

In CRC stem cells, β-catenin/TCF complex controls expression of the EphB receptors and their binding ephrinB ligands through Wnt signaling pathway. Homogeneous expression of the EphB receptors is observed during early stages of CRC. However, the

156

majority of advanced CRC cells contain EphB receptor–negative cells despite active β-catenin/TCF signaling. This implies that other signaling pathways coincidentally control EphB receptors expression in CRC cells besides β-catenin/TCF signaling (van de Wetering, Sancho et al. 2002). ADAM10, for example, mediates shedding of E-cadherin at the EphB-positive and ephrin-B-positive contacts. ADAM10 redistributes E-cadherin at different sides of the cell resulting in increased adhesiveness between equivalent cells and decreased affinity between EphB-positive and ephrin- B-positive cells. ADAM10 deficient mice result in loss of segregation between EphB receptor positive cells and ephrinB positive cells. This compartmentalization phenomenon is observed during early stages of CRC in the presence of active ADAM10 signaling (Solanas, Cortina et al. 2011). In advanced stages of CRC, ADAM10 gets inhibited by the expression of tissue inhibitor of metalloproteinases-1 (TIMP-1). In CRC patients, (TIMP-1) is negatively correlated with their survival by enhancing liver metastasis (Kopitz, Gerg et al. 2007). Solanas et al findings and Kopitz et al findings together support the notion that EphB4 receptor is a tumour suppressor gene during early stages of CRC by forming compartments separating tumour cells from healthy epithelium. However, with the progression of the disease, inhibition of ADAM10 abrogates EphB-induced segregation phenomenon leading to a subset of tumour cells with unrestricted invasive capacity.

In contrast, EphB4 enhances tumour cells proliferation, mobilization and migration in different body tumours

157

through different biological mechanisms. For instance, EphB4 expression is associated with progression and aggressive behaviour of prostate cancer. EphB4 expression in prostate cancer is regulated by tumour suppressors such as p53 gene and by growth factors such as EGFR. Knockdown of EphB4 expression shows marked reduction in tumour growth both in vivo and in vitro. Downregulation of EphB4 expression in prostate cancer results in decreased migration and distant metastasis (Xia, Kumar et al. 2005). EphB4 is a valuable therapeutic target in ovarian cancer. EphB4- monoclonal bound with or without chemotherapeutic agent has resulted in significant increase in EphB4 positive tumour cells apoptosis and decrease in cancer cells migration and invasion. EphB4 targeting resulted in reduced tumour angiogenesis, proliferation, and increased tumour cell apoptosis, which likely occur through modulation of phosphoinositide 3-kinase P13K signaling (Spannuth, Mangala et al. 2010). EphB4 knockdown inhibits tumour cell migration and invasion in bladder cancer. Functional EphB4 is overexpressed in bladder cancer cell lines and tumour specimens. EphB4 expression in bladder cancer cells is regulated by EGFR signaling and p53 expression. EphB4 facilitates bladder cancer cell migration and invasion. EphB4 knockdown is associated with a predominant activation of caspase 8, or extrinsic apoptotic pathway. EphrinB2 is expressed in bladder cancer cell lines and tumour specimens (Xia, Kumar et al. 2006). EphB4 is overexpressed in papillary thyroid carcinoma (PTC) cells in comparison to normal thyroid tissues. EphB4 expression in PTC stimulates cell migration in a kinase- independent manner by inhibiting phosphorylation of focal adhesion molecules (FAK) and paxilin. Silencing

158

the expression of EphB4 inhibits metastasis of PTC cells in vivo by recruiting adhesion molecules (Xuqing, Lei et al. 2012). EphB4 promotes cell growth of glioma through activation of EGFR signaling. Ephb4 knockdown leads to inhibition of tumour cell growth (Chen, Liu et al. 2013). EphB4 promotes the formation of new blood vessels in ephrinB2-positive tumour endothelial cells tissue. Yet, functional quality of these newly formed blood vessels needs to be investigated. The EphB4 ectodomain also promotes endothelial cell migration, survival, and proliferation by decreasing cell–cell and cell–matrix adhesion (Noren, Lu et al. 2004).

Do the EphB4 positive CRC cells turn to be EphB2 negative with the progression of the disease?

Our data showed that EphB4 positive tumour cells are not always EphB2 negative. Different levels of evidence support this finding. First of all, both EphB2 and EphB4 receptors are target genes of Wnt/β- catenin/TCF4 signaling pathways. They are both positively controlled upon activation of β-catenin/TCF4 signaling pathway (Batlle 2005). Battle et al found in their series of CRC samples, that CRC cells, which did not express EphB2 receptors, were at the same time EphB4-negative cells. This indicates that these receptors are all orchestrated by the same mechanisms and are all underexpressed during CRC progression (Batlle 2005). In contrast, Kumar et al found that EphB4 expression in tumours was associated with EphB2 underexpression. However, this

159

observation can be scrutinised from different aspects. First of all, Kumar et all in their tumour samples, they found that EphB4 positive tumour cells turn off EphB2 from their cell membrane in only 12 liver metastasis and 5 CRC specimens (Kumar, Scehnet et al. 2009). Although it was statistically significant (p-value=0.02), however, it is difficult to make a definitive conclusion from small number of specimens. Secondly, as they found that EphB2 receptor expression was lower in the primary CRC tissues, then it is highly unlikely to be expressed in liver metastatic tissues irrespective of the status of EphB4 receptor. This is supported by the finding that more than 50% of EphB2 receptor are absent in the primary CRC tissues, which means that EphB2 absence is not affected by EphB4 expression in CRC. It also means that if EphB2 is absent in CRC, then it will be also absent in the metastatic tissues (Clevers and Batlle 2006). Thirdly, from statistical point of view, there are methodological issues in Kumar et al's analysis. "Excellent correlation” was claimed between immunoblotting and immunofluorescence for EphB4 and EphB2 receptors. Correlation analysis is not the appropriate method for comparison between two methods of measurement. They should have used methods of agreement analysis. Furthermore, all the r- values (correlation coefficients) cited were of negligible correlations irrespective of the p-values given. For example: r = 0.4 implies a coefficient of determination of 0.16; only 16% of the variation in the immunoblotting measurements may be accounted for by knowing the measurements from immunoflurescence technique. These methodological flaws throw doubt on their conclusion that there is preferential induction of EphB4 over EphB2 in CRC.

160

In conclusion, as Kumar made his observation based on a change in transcriptional activation, this could be assessed by looking in colon tumour tissues and using IHC for the activator proteins (p300 and CBP) to find out their level of expressions during different stages of CRC and in liver metastasis of the same patients, and then to correlate that with the level of expressions of EphB2 and EphB4 receptors.

Other clinicopathological data in the study

We also assessed the EphB receptors expression in CRC samples against other clinicopathological data. In our study, we found that the expression of EphB receptors has insignificant relationship with the following variables: gender, age, depth of wall invasion, recurrence of the disease, tumour size and smoking. While tumour markers profiling can identify CRC patients who are at higher risk of disease recurrence (Wang, Jatkoe et al. 2004), and while we could prove a significant relationship between EphB expression and the need for POP adjuvant therapy, yet, we could not prove such relationship between EphB receptors expression and the chance of CRC patients to develop recurrences. Although CRC is more prevalent in males than in females (Gao, Neutel et al. 2008), and its incidence increases with age (Okamoto, Shiratori et al. 2002), we could not find any relationship between EphB expression in CRC and the gender and age of the study population. Although the proportional relationship between depth of wall invasion and the presence of distant metastasis in CRC is significantly correlated (Kitajima, Fujimori et al. 2004), and although we could prove a significant relationship

161

between EphB receptors expression and TNM staging, and between EphB receptors expression and the presence of distant metastasis, however, we could not find any relationship between depth of wall invasion and EphB receptors expression in CRC samples. Smoking is considered as an adverse influence on CRC prognosis (Walter, Jansen et al. 2015), however, we could not establish any direct relationship between the effect of smoking and EphB receptors expression in CRC. The bigger the tumour size, the higher the chance to develop metastasis and recurrences (DeVita, Lawrence et al. 2011), nonetheless, we could not find any significant relationship between tumour size and the level of EphB receptors expression in CRC.

Limitations of the study

Although the present study has yielded some significant findings, its pattern is not without flaws. A number of caveats need to be noted regarding the present study. Disease-free survival (DFS) and overall survival (OS) were not included in the assessment of the EphB receptors expression against the clinicopathological data in the study, as most of the patients in the study are still under follow up. In cancer, DFS is the length of time after primary treatment for a cancer ends that the patient survives without any signs or symptoms of that cancer, whereas OS is the length of time from either the date of diagnosis or the start of treatment for the cancer, that patients diagnosed with the disease are still alive. In a clinical trial, measuring the DFS and OS is one way to see how well a new

162

treatment works. Cancer stage is the main determinant of OS and DFS (Andre, Boni et al. 2009). Guo et al confirmed, in a retrospective study, adverse relationship between EphB2 underexpression and OS and DFS of the CRC patients in their study (Guo, Zhang et al. 2006). A 5-year prospective study, which assesses the impact of EphB receptors (EphB1, EphB2 and EphB4) expression on DFS and OS in CRC patients with different phenotypes with and without adjuvant therapy can accurately evaluate the reliability of EphB receptors as independent prognostic markers for the disease. It is a single method study. IHC was the only method used throughout the study to assess the expression of EphB receptors in CRC samples. Adding another method, such as Western Blotting (WB), which can detect proteins post-electrophoresis, particularly proteins that are of low abundance (Kurien and Scofield 2006), The assessment of EphB receptors expression was done based on a retrospective study. Retrospective study has the potential of sampling bias causing some members of the population to be less likely or even inappropriately to be included in the study, resulting in a biased sample performed on statistical analysis. Selection bias can be minimized by carefully selecting cases in the study based on the inclusion and exclusion criteria (Hernan, Hernandez-Diaz et al. 2004). Last but not least, the study population was relatively small. Large study population gives more value to the study results than the small size population.

163

Closing Remarks

In summary, we could find answers to the proposed hypotheses in our study. EphB1 receptor underexpression is not only seen in the most undifferentiated CRC, but also in liver metastasis of the same patients. EphB receptors (EphB1, EphB2, and EphB4) are homogeneously underexpressed with progression of the disease and are further underexpressed in the presence of lymph nodes metastasis as well as liver metastasis of the same patients. EphB4 receptor is underexpressed in the poorly differentiated phenotype of the disease. With the progression of the disease, EphB4 positive tumour cells are not necessarily be EphB2 negative cells.

164

Chapter Five

Closing Remarks

CRC is a persistent health problem. It is the second leading cause of cancer related death worldwide and in Australia. Despite the improvements in the screening, diagnosis and management of the disease, the incidence rate is still rising due to the presence of undetectable micrometastasis at the time of the presentation. The current tumour markers such as carcinoembryonic embryonic antigen (CEA) have a false negative rate of up to 40%. Therefore, the necessity to find more tumour markers of high sensitivity and specificity that can detect the disease at its early stages is emphasized.

In CRC, EphB receptors have various biological effects. EphB1 and EphB2 receptors are considered as tumour suppressors whereas EphB4 receptor has contradictory effects; while some showed that EphB4 functions as a tumour suppressor, the others found the opposite.

We studied the pattern of expression of the EphB receptors (EphB1, EphB2 and EphB4) in a cohort of 120 patients with primary CRC and in 28 CRC patients who developed liver metastasis. We used IHC as the method of choice in our study; it is considered as the gold standard method in this type of research. We conducted the study on FFPE tissues.

165

We could show, for the first time in the literature, that the three receptors together, EphB1, EphB2 and EphB4, are homogeneously underexpressed in the same tumours with the progression of the disease. The same pattern was also seen in liver metastasis of the same patients. We also investigated EphB1 receptor expression in CRC and in liver metastasis of the same patients. We confirmed that EphB1 receptor is underexpressed in the most undifferentiated phenotype of the disease and we also found that EphB1 receptor is further underexpressed in liver metastasis of the same patients. In our series of patients, we found that EphB4 receptor is underexpressed with the progression of the disease. We rejected the hypothesis that EphB4 positive tumour cells turn off EphB2 receptor expression in order to become more aggressive and invasive cells.

Future Directions

This study is a quantitative study depending on a subjective estimation of EphB receptors expression in the CRC tissues. A future study, which includes the three receptors together, EphB1, EphB2 and EphB4, and assesses their functional role in different phenotypes of CRC, can add more value to the current study.

The cognate ligands, ephrinB1 and ephrinB2, are expressed in the stromal tissues surrounding the intestinal crypts, and are involved in mutual repulsive forces with the EphB receptors, to separate

166

proliferating cells from the differentiating cells along the intestinal crypts. A future study, which includes the EphB receptors and their cognate ligands, and compares their level of expression in the primary CRC with that of the liver metastasis of the same patients, would be of value.

Long term study, 5-year or 10-year study, which evaluates the relationship between EphB receptors expression and OS and DFS rates of CRC patients, can verify the importance of these receptors as prognostic tumour markers.

Last but not least, a holistic understanding of EphB receptors signaling in different cellular contexts during tumour progression in CRC may provide a novel anticancer therapeutic intervention, which can inhibit tumour progression and distant metastasis, and hence improve survival.

167

Appendix 1

A) Primary tumor

Slide Receptor Tumour Control Expression No Expression Weak Expression Moderate Expression High % of Deg No staining of Diff

168

Appendix 2

B) Primary tumour vs. hepatic metastasis

Slide Primary Tumour Hepatic Metastasis No Receptor T C Intensity of % of Deg Receptor T C Intensity % of Deg of staining staining of staining Diff of staining Diff

T: Tumour, C: Control

Intensity of staining:

1. no staining, 2. weak staining, 3. moderate staining, 4. high staining.

169

References:

ACIM, A. C. I. a. M. (2014). Bowel cancer for Australia Agesen TH, Sveen A, Merok MA, Lind GE, Nesbakken A, Skotheim RI and Lothe RA (2012). "ColoGuideEx: a robust gene classifier specific for stage II colorectal cancer prognosis." Gut 61(11): 1560-1567. Agresti, A. (2013). Categorical Data Analysis John Wiley & Sons. Aguilera O, Fraga MF, Ballestar E, Paz MF, Herranz M, Espada J, García JM, Muñoz A, Esteller M and González-Sancho JM. (2006). "Epigenetic inactivation of the Wnt antagonist DICKKOPF-1 (DKK-1) gene in human colorectal cancer." Oncogene 25(29): 4116-4121. AIHW (2012). Cancer in Australia: an overview. Alazzouzi H, Davalos V, Kokko A, Domingo E, Woerner SM, Wilson AJ, Konrad L, Laiho P, Espín E, Armengol M, Imai K, Yamamoto H, Mariadason JM, Gebert JF, Aaltonen LA, Schwartz SJr and Arango D. (2005). "Mechanisms of Inactivation of the Receptor Tyrosine Kinase EPHB2 in Colorectal Tumors." Cancer Research 65(22): 10170-10173. Amcheslavsky A, Jiang J and Ip YT. (2009). "Tissue Damage-Induced Intestinal Stem Cell Division in Drosophila." Cell Stem Cell 4(1): 49-61. André T, Boni C, Navarro M, Tabernero J, Hickish T, Topham C, Bonetti A, Clingan P, Bridgewater J, Rivera F and de Gramont A. (2009). "Improved overall survival with oxaliplatin, fluorouracil, and leucovorin as adjuvant treatment in stage II or III colon cancer in the MOSAIC trial." Journal of Clinical Oncology 27(19): 3109-3116. Aoki K and Taketo MM (2007). "Adenomatous polyposis coli (APC): a multi- functional tumor suppressor gene." Journal of Cell Science 120(19): 3327- 3335. Arvanitis D and Davy A. (2008). "Eph/ephrin signaling: networks." Genes & Development 22(4): 416-429. Bandres E, Agirre X, Bitarte N, Ramirez N, Zarate R, Roman-Gomez J, Prosper F and Garcia-Foncillas J. (2009). "Epigenetic regulation of microRNA expression in colorectal cancer." International Journal of Cancer 125(11): 2737-2743. Bardelli A, Parsons DW, Silliman N, Ptak J, Szabo S, Saha S, Markowitz S, Willson JK, Parmigiani G, Kinzler KW, Vogelstein B and Velculescu VE. (2003). "Mutational Analysis of the Tyrosine Kinome in Colorectal Cancers." Science 300(5621): 949. Bardou M, Barkun AN and Martel M. (2013). "Obesity and colorectal cancer." Gut 62(6): 933-947. Barker N and Clevers H (2006). "Mining the Wnt pathway for cancer therapeutics." Nature Reviews Drug Discovery 5(12): 997-1014. Batlle E, Bacani J, Begthel H, Jonkheer S, Gregorieff A, van de Born M, Malats N, Sancho E, Boon E, Pawson T, Gallinger S, Pals S and Clevers H. (2005). "EphB receptor activity suppresses colorectal cancer progression." Nature 435(7045): 1126-1130. Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, Meeldijk J, Robertson J, van de Wetering M, Pawson T and Clevers H (2002). "β-Catenin and TCF Mediate Cell Positioning in the Intestinal Epithelium by Controlling the Expression of EphB/EphrinB." Cell 111(2): 251-263.

170

Behrens J (2005). "The role of the Wnt signalling pathway in colorectal tumorigenesis." Biochemical Society Transactions 3(part 4): 672-675. Bhalla A, Zulfiqar M, Weindel M and Shidham VB (2013). "Molecular diagnostics in colorectal carcinoma." Clinics in Laboratory Medicine 33(4): 835-859. Bogan C, Chen J, O'Sullivan MG and Cormier RT (2009). "Loss of EphA2 receptor tyrosine kinase reduces Apcmin/+ tumorigenesis." International Journal of Cancer 124(6): 1366-1371. Bogenrieder T and Herlyn M (2003). "Axis of evil: molecular mechanisms of cancer metastasis." Oncogene 22(42): 6524-6536. Boissier P, Chen J and Huynh-Do U. (2013). "EphA2 Signaling Following Endocytosis: Role of Tiam1." Traffic 14(12): 1255-1271. Bonifaci N, Górski B, Masojć B, Wokołorczyk D, Jakubowska A, Dębniak T, Berenguer A, Serra Musach J, Brunet J, Dopazo J, Narod SA, Lubiński J, Lázaro C, Cybulski C and Pujana MA (2010). "Exploring the link between germline and somatic genetic alterations in breast carcinogenesis." PloS One 5(11): e14078. Boutron-Ruault MC, Senesse P, Méance S, Belghiti C and Faivre J. (2001). "Energy intake, body mass index, physical activity, and the colorectal adenoma-carcinoma sequence." Nutrition and Cancer-an International Journal 39(1): 50-57. Bowden TA, Aricescu AR, Nettleship JE, Siebold C, Rahman-Huq N, Owens RJ, Stuart DI and Jones EY. (2009). "Structural Plasticity of Eph Receptor A4 Facilitates Cross-Class Ephrin Signaling." Structure 17(10): 1386-1397. Brantley-Sieders, DM. (2012). "Clinical relevance of Ephs and ephrins in cancer: Lessons from breast, colorectal, and lung cancer profiling." Seminars in Cell & Developmental Biology 23(1): 102-108. Buchwalow IB and Bocker W (2010). IMMUNOHISTOCHEMISTRY: BASICS AND METHODS, Springer Science & Business Media. Bush JO and Soriano P (2012). "Eph/ephrin signaling: genetic, phosphoproteomic, and transcriptomic approaches." Seminars in Cell & Developmental Biology 23(1): 26-34. Chan AT and Giovannucci EL (2010). "Primary prevention of colorectal cancer." Gastroenterology 138(6): 2029-2043 e2010. Chen J, Zhuang G, Frieden L and Debinski W (2008). "Eph Receptors and Ephrins in Cancer: Common Themes and Controversies." Cancer Research 68(24): 10031-10033. Chen K, Bai H, Liu Y, Hoyle D, Shen WF Wu LQ and Wang ZZ (2015). "EphB4 forward-signaling regulates cardiac progenitor development in mouse ES cells." Journal of Cellular Biochemistry 116(3):467-75 Chen Q, Chen L, Zhao R, Yang XD, Imran K and Xing CG (2013). "Microarray analyses reveal liver metastasis-related genes in metastatic colorectal cancer cell model." Journal of Cancer Research and Clinical Oncology 139(7): 1169- 1178. Chen T, Liu X, Yi S, Zhang J, Ge J and Liu Z (2013). "EphB4 is overexpressed in gliomas and promotes the growth of glioma cells." Tumor Biology 34(1): 379-385. Cheng N, Brantley DM and Chen J (2002). "The ephrins and Eph receptors in angiogenesis." Cytokine & Growth Factor Reviews 13(1): 75-85.

171

Chiu ST, Chang KJ, Ting CH, Shen HC, Li H and Hsieh FJ (2009). "Over- expression of EphB3 enhances cell–cell contacts and suppresses tumour growth in HT-29 human colon cancer cells." Carcinogenesis 30(9): 1475-1486. Choi KS (2012). "Autophagy and cancer." Experimental and Molecular Medicine 44(2): 109-120 Chrencik JE, Brooun A, Kraus ML, Recht MI, Kolatkar AR, Han GW, Seifert JM, Widmer H, Auer M and Kuhn P (2006). "Structural and Biophysical Characterization of the EphB4·EphrinB2 Protein-Protein Interaction and Receptor Specificity." Journal of Biological Chemistry 281(38): 28185-28192. Chukkapalli S, A§messou M, Dilly AK, Dekhil H, Zhao J, Liu Q, Bejna A, Thomas RD, Bandyopadhyay S and Bismar TA (2014). "Role of the EphB2 receptor in autophagy, apoptosis and invasion in human breast cancer cells." Experimental cell research 320(2): 233-246. Chung GG, Provost E, Kielhorn EP, Charette LA, Smith BL and Rimm DL (2001). "Tissue Microarray Analysis of β-Catenin in Colorectal Cancer Shows Nuclear Phospho-β-catenin Is Associated with a Better Prognosis." Clinical Cancer Research 7(12): 4013-4020. Clevers H and Batlle E (2006). "EphB/EphrinB Receptors and Wnt Signaling in Colorectal Cancer." Cancer Research 66(1): 2-5. Coons AH, Creech HJ and Jones RN (1941). "Immunological properties of an antibody containing a fluorescent group." Experimental Biology and Medicine 47(2): 200-202. Cortina C, Palomo-Ponce S, Iglesias M, Fernandez-Masip JL, Vivancos A, Whissell G, Huma M, Peiro N, Gallego L, Jonkheer S, Davy A, Lloreta J, Sancho E and Batlle E (2007). "EphB-ephrin-B interactions suppress colorectal cancer progression by compartmentalizing tumor cells." Nature Genetics 39(11): 1376-1383. Daar IO (2012). "Non-SH2/PDZ reverse signaling by ephrins." Seminars in Cell & Developmental Biology 23(1): 65-74. Dabbs DJ (2013). DIAGNOSTIC IMMUNOHISTOCHEMISTRY, Ch 1, pages 1-2, Elsevier Health Sciences. Daniel O, Herzig DO and Tsikitis VL (2015). "Molecular markers for colon diagnosis, prognosis and targeted therapy." Journal of Surgical Oncology 111(1): 96–102. Davalos V, Dopeso H, Castaño J, Wilson AJ, Vilardell F, Romero-Gimenez J, Espín E, Armengol M, Capella G, Mariadason JM, Aaltonen LA, Schwartz S and Arango D (2006). "EphB4 and Survival of Colorectal Cancer Patients." Cancer Research 66(18): 8943-8948. de la Chapelle A (2004). "Genetic predisposition to colorectal cancer." Nature Reviews Cancer 4(10): 769-780. de Lau W, Barker N and Clevers H (2007). "WNT signaling in the normal intestine and colorectal cancer." Frontiers in Bioscience 12: 471-491. DeVita V, Lawrence S and Rosenberg A (2011). Cancer,Principle & Practice of Oncology, Ch.89, pages 1085-1086. Wolters Kluwer-Lippincott, Williams and Wilkins. DeVita V, Lawrence S and Rosenberg A (1) (2011). Cancer,Principle & Practice of Oncology, Ch 89, pages 1085-1086. Wolters Kluwer- Lippincott, Williams and Wilkins.

172

DeVita V, Lawrence S and Rosenberg A (2) (2011). Cancer,Principle & Practice of Oncology, Ch.10, pages 113-126. Wolters Kluwer- Lippincott, Williams and Wilkins. Dexiang Z., Li R, Ye W, Haifu W, Yunshi Z, Qinghai Y, Shenyong Z, Bo X, Li L, Xiangou P, Haohao L, Lechi Y, Tianshu L, Jia F, Xinyu Q and Jianmin X (2012). "Outcome of Patients with Colorectal Liver Metastasis: Analysis of 1,613 Consecutive Cases." Annals of Surgical Oncology 19(9): 2860-2868. Djokovic D, Trindade A, Gigante J, Badenes M, Silva L, Liu R, Li X, Gong M, Krasnoperov V and Gill PS (2010). "Combination of Dll4/Notch and Ephrin- B2/EphB4 targeted therapy is highly effective in disrupting tumor angiogenesis." BioMed Central Cancer 10(1): 641. Dong Y, Wang J, Sheng Z, Li G, Ma H, Wang X, Zhang R, Lu G, Hu Q, Sugimura H and Zhou X (2009). "Downregulation of EphA1 in colorectal carcinomas correlates with invasion and metastasis." Modern Pathology 22(1): 151-160. Dopeso H, Mateo-Lozano S, Mazzolini R, Rodrigues P, Lagares-Tena L, Ceron J, Romero J, Esteves M, Landolfi S, Hernández-Losa J, Castaño J, Wilson AJ, Ramon y Cajal S, Mariadason JM, Schwartz S and Arango D (2009). "The Receptor Tyrosine Kinase EPHB4 Has Tumor Suppressor Activities in Intestinal Tumorigenesis." Cancer Research 69(18): 7430-7438. Drucker A, Arnason T, Yan SR, Aljawad M, Thompson K and Huang WY. (2013). "Ephrin B2 Receptor and Microsatellite Status in Lymph Node– Positive Colon Cancer Survival." Translational Oncology 6 (5): 520–527. Egea J and Klein R (2007). "Bidirectional Eph-ephrin signaling during axon guidance." Trends in Cell Biology 17(5): 230-238. Falivelli, G., Lisabeth EM, Rubio de la Torre E, Perez-Tenorio G, Tosato G, Salvucci O and Pasquale EB (2013). "Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands." PloS One 8(11): e81445. Fearon ER (2011). "Molecular Genetics of Colorectal Cancer." Annual Review of Pathology: Mechanisms of Disease 6(6): 479-507. Ferluga, S, Hantgan R, Goldgur Y, Himanen JP, Nikolov DB and Debinski W (2013). "Biological and Structural Characterization of Glycosylation on Ephrin- A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase." Journal of Biological Chemistry 288(25): 18448-18457. Foo, SS, Turner CJ, Adams S, Compagni A, Aubyn D, Kogata N, Lindblom P, Shani M, Zicha D and Adams RH (2006). "Ephrin-B2 Controls Cell Motility and Adhesion during Blood-Vessel-Wall Assembly." Cell 124(1): 161-173. Fox BP and Kandpal RP (2004). "Invasiveness of breast carcinoma cells and transcript profile: Eph receptors and ephrin ligands as molecular markers of potential diagnostic and prognostic application." Biochemical and Biophysical Research Communications 318(4): 882-892. Fu T, Li P, Wang H, He Y, Luo D, Zhang A, Tong W, Zhang L, Liu B and Hu C (2009). "c-Rel is a transcriptional repressor of EPHB2 in colorectal cancer." The Journal of Pathology 219(1): 103-113. Galiatsatos P and Foulkes WD (2006). "Familial Adenomatous Polyposis." American Journal of Gastroenterology 101(2): 385-398. Ganong WF and Barrett KE (2013). Review of medical physiology, Ch 28, pages 475-478, McGraw-Hill Medical.

173

Gao RN, Neutel CI and Wai E (2008). "Gender differences in colorectal cancer incidence, mortality, hospitalizations and surgical procedures in Canada." Journal of public health 30(2): 194-201. Gartner L (2001). Colour Textbook of histology. 3rd edition. Elsevier Health Genander M (2012). "Eph and ephrins in epithelial stem cell niches and cancer." Cell Adhesion and Migration 6(2): 126-130. Genander M and Frisen J (2010). "Ephrins and Eph receptors in stem cells and cancer." Current Opinion in Cell Biology 22(5): 611-616. Genander M, Halford MM, Xu NJ, Eriksson M, Yu Z, Qiu Z, Martling A, Greicius G, Thakar S, Catchpole T, Chumley MJ, Zdunek S, Wang C, Holm T, Goff SP, Pettersson S, Pestell RG, Henkemeyer M and Frisén J. (2009). "Dissociation of EphB2 Signaling Pathways Mediating Progenitor Cell Proliferation and Tumor Suppression." Cell 139(4): 679-692. Goldberg, RM (2006). "Intensive Surveillance After Stage II or III Colorectal Cancer: Is It Worth It?" Journal of Clinical Oncology 24(3):330-331. Grady WM and Markowitz SD (2015). "The Molecular Pathogenesis of Colorectal Cancer and Its Potential Application to Colorectal Cancer Screening." Digestive diseases and sciences 60(3):762–772. Grady WM and Pritchard CC (2014). "Molecular alterations and biomarkers in colorectal cancer." Toxicologic pathology 42(1):124-139. Gregorieff A, Pinto D, Begthel H, Destrée O, Kielman M and Clevers H. (2005). "Expression Pattern of Wnt Signaling Components in the Adult Intestine." Gastroenterology 129(2): 626-638. Gucciardo E, Sugiyama N and Lehti K (2014). "Eph- and ephrin-dependent mechanisms in tumor and stem cell dynamics." Cellular and Molecular Life Sciences 71(19):3685-3710. Guijarro-Munoz I, Sanchez A, Martínez-Martínez E, García J, Salas C, Provencio M, Alvarez-Vallina L and Sanz L (2013). "Gene expression profiling identifies EphB4 as a potential predictive biomarker in colorectal cancer patients treated with bevacizumab." Medical Oncology 30(2):1-8. Gunter MJ and Leitzmann MF (2006). "Obesity and colorectal cancer: epidemiology, mechanisms and candidate genes." The Journal of Nutritional Biochemistry 17(3): 145-156. Guo DL, Zhang J, Yuen ST, Tsui WY, Chan AS, Ho C, Ji J, Leung SY and Chen X (2006). "Reduced expression of EphB2 that parallels invasion and metastasis in colorectal tumours." Carcinogenesis 27(3):454-464. Guo FY and Lesk AM (2013). "Sizes of interface residues account for cross- class binding affinity patterns in Eph receptor–ephrin families." Proteins: Structure, Function, and Bioinformatics 82(3):349-53. Guo H, Miao H, Gerber L, Singh J, Denning MF, Gilliam AC and Wang B (2006). "Disruption of EphA2 Receptor Tyrosine Kinase Leads to Increased Susceptibility to Carcinogenesis in Mouse Skin." Cancer Research 66(14): 7050-7058. Haase A. T. and Oldstone M. B. A. (2012) In Situ Hybridization. Pages 24-28. Springer-Verlag Haegebarth A and Clevers H (2009). "Wnt Signaling, Lgr5, and Stem Cells in the Intestine and Skin." The American Journal of Pathology 174(3): 715-721. Hafner C, Meyer S, Hagen I, Becker B, Roesch A, Landthaler M and Vogt T (2005). "Ephrin-B reverse signaling induces expression of wound healing

174

associated genes in IEC-6 intestinal epithelial cells." World Journal of Gastroenterology 11(29): 4511. Hafner C, Meyer S, Langmann T, Schmitz G, Bataille F, Hagen I, Becker B, Roesch A, Rogler G and Landthaler M (2005). "Ephrin-B2 is differentially expressed in the intestinal epithelium in Crohn's disease and contributes to accelerated epithelial wound healing in vitro." World Journal of Gastroenterology 11(26): 4024. Hafner C, Schmitz G, Meyer S, Bataille F, Hau P, Langmann T, Dietmaier W, Landthaler M and Vogt V (2004). "Differential Gene Expression of Eph Receptors and Ephrins in Benign Human Tissues and Cancers." Clinical Chemistry 50(3):490-499. Harikumar KB, Sung B, Tharakan ST, Pandey MK, Joy B, Guha S, Krishnan S and Aggarwal BB. (2010). "Sesamin Manifests Chemopreventive Effects through the Suppression of NF-κB–Regulated Cell Survival, Proliferation, Invasion, and Angiogenic Gene Products." Molecular Cancer Research 8(5): 751-761. Hayat MA (2002). Microscopy, Immunohistochemistry, and Antigen Retrieval Methods For Light and Electron Microscopy, Ch 8, page 175-176, Springer. Herath NI and Boyd AW (2010). "The role of Eph receptors and ephrin ligands in colorectal cancer." Interantional Journal of Cancer 126(9): 2003-2011. Herath NI, Doecke J, Spanevello MD, Leggett BA and Boyd AW (2009). "Epigenetic silencing of EphA1 expression in colorectal cancer is correlated with poor survival." British Journal of Cancer 100(7):1095-1102. Herath NI, Spanevello MD, Doecke JD, Smith FM, Pouponnot C and Boyd AW (2012). "Complex expression patterns of Eph receptor tyrosine kinases and their ephrin ligands in colorectal carcinogenesis." European Journal of Cancer 48(5):753-762. Hernan MA, Hernandez-Diaz S and Robins JM (2004). "A Structural Approach to Selection Bias." Epidemiology 15(5): 615-625. Heroult M, Schaffner F and Augustin HG (2006). "Eph receptor and ephrin ligand-mediated interactions during angiogenesis and tumor progression." Experimental Cell Research 312(5):642-650. Himanen JP, Chumley MiJ, Lackmann M, Li C, Barton WA, Jeffrey PD, Vearing C, Geleick D, Feldheim DA, Boyd AW, Henkemeyer M and Nikolov DB (2004). "Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling." Nature Neuroscience 7(5):501-509. Himanen JP and Nikolov DB (2003). "Eph receptors and ephrins." The International Journal of Biochemistry & Cell Biology 35(2):130-134. Himanen JP and Nikolov DB (2003). "Eph signaling: a structural view." Trends in Neurosciences 26(1):46-51. Himanen JP, Saha N and Nikolov DB (2007). "Cell–cell signaling via Eph receptors and ephrins." Current opinion in cell biology 19(5):534-542. Himanen JP (2012). "Ectodomain structures of Eph receptors." Seminars in Cell & Developmental Biology 23(1):35-42. Himanen JP., Goldgur Y, Miao H, Myshkin E, Guo H, Buck M, Nguyen M, Rajashankar KR, Wang B and Nikolov DB (2009). "Ligand recognition by A- class Eph receptors: crystal structures of the EphA2 ligand-binding domain and the EphA2/ephrin-A1 complex." European Molecular Biology Organization Reports 10(7): 722-728.

175

Holmberg J, Genander M, Halford MM, Annerén C, Sondell M, Chumley MJ, Silvany RE, Henkemeyer M and Frisén J (2006). "EphB Receptors Coordinate Migration and Proliferation in the Intestinal Stem Cell Niche." Cell 125(6): 1151-1163. Hime H and Abud H (2013). Transcriptional and Translational Regulation of Stem Cells. 786: 175-186. Springer Netherlands Hsu SM, Raine L and Fanger H (1981). "Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures." Journal of Histochemistry & Cytochemistry 29(4): 577-580. Hu Y, Li S, Jiang H, Li MT and Zhou JW (2014). "Ephrin-B2/EphA4 forward signaling is required for regulation of radial migration of cortical neurons in the mouse." Neuroscience Bulletin 30(3): 425-432. Huan X, Shi J, Lim L, Mitra S, Zhu W, Qin H, Pasquale EB and Song J (2013). "Unique Structure and Dynamics of the EphA5 Ligand Binding Domain Mediate Its Binding Specificity as Revealed by X-ray Crystallography, NMR and MD Simulations." PLoS ONE 8(9):e74040 Igor B and Buchwalow WB (2) (2010). Immunohistochemistry: Basics and Methods, Ch 6, Page 51, Springer. Igor B and Buchwalow WB (1) (2010). Immunohistochemistry: Basics and Methods, Ch 5 page 43, Springer. Ino H (2003). "Antigen Retrieval by Heating En Bloc for Pre-fixed Frozen Material." Journal of Histochemistry & Cytochemistry 51(8): 995-1003. Iyer NG, Ozdag H and Caldas C (2004). "p300/CBP and cancer." Oncogene 23(24): 4225-4231. Jagle S, Ronsch K, Timmec S, Andrlova H, Bertrand M, Jäger M, Proske, A Schrempp M, Yousaf A, Michoel T, Zeiser R, Werner M, Lassmann S and Hecht A (2014). "Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer." Proceedings of the National Academy of Sciences of The United States of America 111(13): 4886–4891. Janes PW, Adikari S and Lackmann M. (2008). "Eph/ephrin signalling and function in oncogenesis: lessons from embryonic development." Current Cancer Drug Targets 8(6): 473-479. Janes PW, Nievergall E and Lackmann M (2012). "Concepts and consequences of Eph receptor clustering." Seminars in Cell and Developmental Biology 23(1): 43-50. Jubb AM, Zhong F, Bheddah S, Grabsch HI, Frantz GD, Mueller W, Kavi V, Quirke P, Polakis P and Koeppen H (2005). "EphB2 is a Prognostic Factor in Colorectal Cancer." Clinical Cancer Research 11(14): 5181-5187. Kaidi A, Moorghen M, Williams AC and Paraskeva C (2007). "Is the downregulation of EphB2 receptor expression during colorectal tumorigenesis due to hypoxia?" Gut 56(11): 1637-1638. Kalluri R and Weinberg RA. (2009). "The basics of epithelial-mesenchymal transition." The Journal of clinical investigation 119(6): 1420. Karamitopoulou E, Lugli A, Panayiotides I, Karakitsos P, Peros G, Rallis G, Patsouris ES, Terracciano L and Zlobec I (2010). "Systematic assessment of protein phenotypes characterizing high-grade tumour budding in mismatch repair-proficient colorectal cancer." Histopathology 57(2): 233-243.

176

Kataoka H, Igarashi H, Kanamori M, Ihara M, Wang JD, Wang YJ, Li ZY, Shimamura T, Kobayashi T, Maruyama K, Nakamura T, Arai H, Kajimura M, Hanai H, Tanaka M and Sugimura H (2004). "Correlation of EPHA2 overexpression with high microvessel count in human primary colorectal cancer." Cancer Science 95(2): 136-141. Keeble TR and Cooper HM (2006). "Ryk: A novel Wnt receptor regulating axon pathfinding." The International Journal of Biochemistry & Cell Biology 38(12): 2011-2017. Kertesz N, Krasnoperov V, Reddy R, Leshanski L, Kumar SR, Zozulya S and Gill PS (2006). "The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth." Blood 107(6): 2330-2338. Khansaard W, Techasen A, Namwat N, Yongvanit P, Khuntikeo N, Puapairoj A and Loilome W (2014). "Increased EphB2 expression predicts cholangiocarcinoma metastasis." Tumor Biology 35(10): 10031-10041. Kim AD, Kang KA, Kim HS, Kim DH, Choi YH, Lee SJ, Kim HS and Hyun JW (2013). "A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells." Cell Death and Disease 1(4):e750. Kitajima K, Fujimori T, Fujii S, Takeda J, Ohkura Y, Kawamata H, Kumamoto T, Ishiguro S, Kato Y, Shimoda T, Iwashita A, Ajioka Y, Watanabe H, Watanabe T, Muto T and Nagasako K (2004). "Correlations between lymph node metastasis and depth of submucosal invasion in submucosal invasive colorectal carcinoma: a Japanese collaborative study." Journal of gastroenterology 39(6): 534-543. Klaus A and Birchmeier W. (2008). "Wnt signalling and its impact on development and cancer." Nature Reviews Cancer 8(5): 387-398. Kondo Y, Shen L and Issa JP (2003). "Critical role of histone methylation in tumor suppressor gene silencing in colorectal cancer." Molecular and Cellular Biology 23(1): 206-215. Kopitz C, Gerg M, Bandapalli OR, Ister D, Pennington CJ, Hauser S, Flechsig C, Krell HW, Antolovic D, Brew K, Nagase H, Stangl M, von Weyhern CW, Brücher BL, Brand K, Coussens LM, Edwards DR and Krüger A (2007). "Tissue inhibitor of metalloproteinases-1 promotes liver metastasis by induction of hepatocyte growth factor signaling." Cancer research 67(18): 8615-8623. Kosinski C, Li VS, Chan AS, Zhang J, Ho C, Tsui WY, Chan TL, Mifflin RC, Powell DW, Yuen ST, Leung SY and Chen X (2007). "Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors." Proceedings of the National Academy of Sciences of The United States of America 104(39): 15418-15423. Krasnoperov V, Kumar SR, Ley E, Li X, Scehnet J, Liu R, Zozulya S and Gill PS. (2010). "Novel EphB4 Monoclonal Antibodies Modulate Angiogenesis and Inhibit Tumor Growth." The American Journal of Pathology 176(4): 2029-2038. Kuang SQ, Bai H, Fang ZH, Lopez G, Yang H, Tong W, Wang ZZ and Garcia- Manero G (2010). "Aberrant DNA methylation and epigenetic inactivation of Eph receptor tyrosine kinases and ephrin ligands in acute lymphoblastic leukemia." Blood 115(12): 2412-2419. Kullander K and Klein R (2002). "Mechanisms and functions of eph and ephrin signalling." Nature Reviews Molecular Cell Biology 3(7): 475-486.

177

Kumar GL and Rudbeck L (1) (2009). Education Guide: Immunohistochemical Staining Methods: Pathology, Ch 17, page 116. Dako North America. Kumar GL and Rudbeck L (2) (2009). Education Guide: Immunohistochemical Staining Methods: Pathology, Ch 8, page 51-56. Dako North America. Kumar GL and Rudbeck L (3) (2009). Education Guide: Immunohistochemical Staining Methods: Pathology, Ch 1, page 1-9. Dako North America. Kumar GL and Rudbeck L (4) (2009). Education Guide: Immunohistochemical Staining Methods: Pathology, Ch 2, page 11-13. Dako North America. Kumar GL and Rudbeck L (5) (2009). Education Guide: Immunohistochemical Staining Methods: Pathology, Ch 5, pages 29-33, Dako North America. Kumar SR, Scehnet JS, Ley EJ, Singh J, Krasnoperov V, Liu R, Manchanda PK, Ladner RD, Hawes D, Weaver FA, Beart RW, Singh G, Nguyen C, Kahn M and Gill PS (2009). "Preferential Induction of EphB4 over EphB2 and Its Implication in Colorectal Cancer Progression." Cancer Research 69(9): 3736- 3745. Kumar SR, Singh J, Xia G, Krasnoperov V, Hassanieh L, Ley EJ, Scehnet J, Kumar NG, Hawes D and Press MF (2006). "Receptor tyrosine kinase EphB4 is a survival factor in breast cancer." The American journal of pathology 169(1): 279-293. Kurien BT and Scofield RH (2006). "Western blotting." Methods 38(4): 283- 293. Leedham SJ, Brittan M, McDonald SA and Wright NA (2005). "Intestinal stem cells." Journal of Cellular and Molecular Medicine 9(1): 11-24. Leslie A, Carey FA, Pratt NR and Steele RJ (2002). "The colorectal adenoma- carcinoma sequence." British Journal of Surgery 89(7): 845-860. Li D, Liu S, Liu R, Park R, Hughes L, Krasnoperov V, Gill, PS, Li Z, Shan H and Conti PS (2012). "Targeting the EphB4 Receptor for Cancer Diagnosis and Therapy Monitoring." Molecular Pharmaceutics 10(1): 329-336. Li RX, Chen ZH and Chen ZK (2014). "The role of EPH receptors in cancer- related epithelial-mesenchymal transition." Chinese journal of cancer 33(5): 231-40. Liang JT, Cheng JC, Huang KC, Lai HS and Sun CT (2013). "Comparison of tumor recurrence between laparoscopic total mesorectal excision with sphincter preservation and laparoscopic abdominoperineal resection for low rectal cancer." Surgical Endoscopy 27(9): 3452-3464. Liefers GJ, Cleton-Jansen AM, van de Velde CJ, Hermans J, van Krieken JH, Cornelisse CJ and Tollenaar RA (1998). "Micrometastases and survival in stage II colorectal cancer." The New England Journal of Medicine 339(4): 223-228. Lisabeth EM, Falivelli G and Pasquale EB (2013). "Eph Receptor Signaling and Ephrins." Cold Spring Harbor Perspectives in Biology 5(9): 1-20. Liu S, Li D, Park R, Liu R, Xia Z, Guo J, Krasnoperov V, Gill PS, Li Z, Shan H and Conti PS (2013). "PET Imaging of Colorectal and Breast Cancer by Targeting EphB4 Receptor with 64Cu-Labeled hAb47 and hAb131 Antibodies." Journal of Nuclear Medicine 54(7): 1094-1100. Liu W, Ahmad SA, Jung YD, Reinmuth N, Fan F, Bucana CD and Ellis LM (2002). "Coexpression of ephrin-Bs and their receptors in colon carcinoma." Cancer 94(4): 934-939.

178

Liu, W. Jung, YD, Ahmad SA, McCarty MF, Stoeltzing O, Reinmuth N, Fan F and Ellis LM (2004). "Effects of overexpression of ephrin-B2 on tumour growth in human colorectal cancer." British Journal of Cancer 90(8): 1620-1626. Logan CY and Nusse R (2004). "The Wnt signaling pathway in development and disease." Annual Review of Cell and Developmental Biology 20: 781-810. Luchtenborg M, Weijenberg MP, Roemen GM, de Bruïne AP, van den Brandt PA, Lentjes MH, Brink M, van Engeland M, Goldbohm RA and de Goeij AF (2004). "APC mutations in sporadic colorectal carcinomas from The Netherlands Cohort Study." Carcinogenesis 25(7): 1219-1226. Ludbrook J (2011). "Is there still a place for Pearson’s chi-squared test and Fisher’s exact test in surgical research?" Australian NewZeland Journal of Surgery 81(12): 923-926. Lugli A, Spichtin H, Maurer R, Mirlacher M, Kiefer J, Huusko P, Azorsa D, Terracciano L, Sauter G, Kallioniemi OP, Mousses S and Tornillo L (2005). "EphB2 Expression across 138 Human Tumor Types in a Tissue Microarray: High Levels of Expression in Gastrointestinal Cancers." Clinical Cancer Research 11(18): 6450-6458. Lugli A, Zlobec I, Minoo P, Baker K, Tornillo L, Terracciano L and Jass JR (2007). "Prognostic significance of the wnt signalling pathway molecules APC, β-catenin and E-cadherin in colorectal cancer—a tissue microarray-based analysis." Histopathology 50(4): 453-464. Luigi A, Karamitopoulou E and Zlobec L (2012). "Tumour budding: a promising parameter in colorectal cancer." British Journal of Cancer 106: 1713–1717. Mao W, Luis E, Ross S, Silva J, Tan C, Crowley C, Chui C, Franz G, Senter P, Koeppen H and Polakis P (2004). "EphB2 as a Therapeutic Antibody Drug Target for the Treatment of Colorectal Cancer." Cancer Research 64(3): 781- 788. Mathew R, Karantza-Wadsworth V and White E (2007) "Role of autophagy in cancer." Nature Reviews Cancer 7(12): 961-967. McCartney BM and Nathke IS (2008). "Cell regulation by the Apc protein: Apc as master regulator of epithelia." Current Opinion in Cell Biology 20(2):186- 193. Merlos-Suarez A, Barriga FM, Jung P, Iglesias M, Cespedes MV, Rossell D, Sevillano M, Hernando-Momblona X, da Silva-Diz V, Munoz P, Clevers H, Sancho E, Mangues R and Batlle E (2011). "The Intestinal Stem Cell Signature Identifies Colorectal Cancer Stem Cells and Predicts Disease Relapse." Cell Stem Cell 8(5): 511-524. Merlos-Suarez A and Batlle E (2008). "Eph-ephrin signalling in adult tissues and cancer." Current Opinion in Cell Biology 20(2): 194-200. Miao H and Wang B (2009). "Eph/ephrin signaling in epithelial development and homeostasis." The International Journal of Biochemistry and Cell Biology 41(4): 762-770. Miao H and Wang B (2012). "EphA receptor signaling—Complexity and emerging themes." Seminars in Cell & Developmental Biology 23(1): 16-25. Miller RT (2013). Critical Technical Aspects of Diagnostic Immunohistochemistry, pages 1-4 Propath-Texas. Mishra A and Verma M (2010). "Cancer biomarkers: are we ready for the prime time?" Cancers 2(1): 190-208.

179

Mitrovic B, Schaeffer DF, Riddell RH and Kirsch R (2012). "Tumor budding in colorectal carcinoma: time to take notice." Modern Pathology 25(10):1315- 1325. Morrison SJ and Spradling AC (2008). "Stem Cells and Niches: Mechanisms That Promote Stem Cell Maintenance throughout Life." Cell 132(4): 598–611. Mosch B, Reissenweber B, Neuber C and Pietzsch J (2010). "Eph Receptors and Ephrin Ligands: Important Players in Angiogenesis and Tumor Angiogenesis." Journal of Oncology 2010 (2010): 1-12. Naudin C, Sirvent A, Leroy C, Larive R, Simon V, Pannequin J, Bourgaux JF, Pierre J, Robert B, Hollande F and Roche S (2014). "SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2." Nature Communications 5(3159): 1-15 Ngo SN, Williams DB and Head RJ (2011). "Rosemary and Cancer Prevention: Preclinical Perspectives." Critical Reviews in Food Science and Nutrition 51(10): 946-954. Nguyen TM, Arthur A, Hayball JD and Gronthos S (2013). "EphB and Ephrin- B Interactions Mediate Human Mesenchymal Stem Cell Suppression of Activated T-Cells." Stem Cells and Development 22(20): 2751-2764. Nievergall E, Lackmann M and Janes PW (2012). "Eph-dependent cell-cell adhesion and segregation in development and cancer." Cellular and Molecular Life Sciences 69(11): 1813-1842. Niida A, Hiroko T, Kasai M, Furukawa Y, Nakamura Y, Suzuki Y, Sugano S and Akiyama T (2004). "DKK1, a negative regulator of Wnt signaling, is a target of the [beta]-catenin//TCF pathway." Oncogene 23(52): 8520-8526. Nikolov D, Li C, Lackmann M, Jeffrey P and Himanen J (2007). "Crystal structure of the human ephrin-A5 ectodomain." Protein Science 16(5): 996- 1000. Nikolov DB, Li C, Barton WA and Himanen JP (2005). "Crystal Structure of the Ephrin-B1 Ectodomain: Implications for Receptor Recognition and Signaling." Biochemistry 44(33): 10947-10953. Nikolov DB, Xu K and Himanen JP (2013). "Eph/ephrin recognition and the role of Eph/ephrin clusters in signaling initiation." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1834(10): 2160-2165. Noberini R, Lamberto I and Pasquale EB (2012). "Targeting Eph receptors with peptides and small molecules: Progress and challenges." Seminars in Cell & Developmental Biology 23(1): 51-57. Noren NK, Foos G, Hauser CA and Pasquale EB (2006). "The EphB4 receptor suppresses breast cancer cell tumorigenicity through an Abl-Crk pathway." Nature Cell Biology 8(8): 815-825. Noren NK, Lu M, Freeman AL , Koolpe M and Pasquale EB (2004). "Interplay between EphB4 on tumor cells and vascular ephrin-B2 regulates tumor growth." Proceedings of the National Academy of Sciences of the United States of America 101(15): 5583-5588. Noren NK and Pasquale EB (2004). "Eph receptor–ephrin bidirectional signals that target Ras and Rho proteins." Cellular Signalling 16(6): 655-666. Noren NK and Pasquale EB (2007). "Paradoxes of the EphB4 Receptor in Cancer." Cancer Research 67(9): 3994-3997.

180

Nusse R and Varmus H (2012). "Three decades of Wnts: a personal perspective on how a scientific field developed." European Molecular Biology Organization Journal 31(12): 2670-2684. Ohlstein B, Kai T, Decotto E and Spradling A (2004). "The stem cell niche: theme and variations." Current Opinion in Cell Biolology 16(6): 693-699. Okamoto M, Shiratori Y, Yamaji Y, Kato J, Ikenoue T, Togo G, Yoshida H, Kawabe T and Omata M (2002). "Relationship between age and site of colorectal cancer based on colonoscopy findings." Gastrointestinal endoscopy 55(4): 548-551. Orsulic S and Kemler R (2000). "Expression of Eph receptors and ephrins is differentially regulated by E-cadherin." Journal of Cell Science 113(10): 1793- 1802. Oshima T, Akaike M, Yoshihara K, Shiozawa M, Yamamoto N, Sato T, Akihito N, Nagano Y, Fujii S, Kunisaki C, Wada N, Rino Y, Tanaka K, Masuda M and Imada T (2008). "Overexpression of EphA4 gene and reduced expression of EphB2 gene correlates with liver metastasis in colorectal cancer." International Journal of Oncology 33(3): 573-577. Overman RC, Debreczeni JE, Truman CM, McAlister MS and Attwood TK (2013). "Biochemical and biophysical characterization of four EphB kinase domains reveals contrasting thermodynamic, kinetic and inhibition profiles." Bioscience reports 33(3): 427-437. Paavilainen S, Grandy D, Karelehto E, Chang E, Susi P, Erdjument-Bromage H, Nikolov D and Himanen J (2013). "High-level expression of a full-length Eph receptor." Protein Expression and Purification 92(1): 112-118. Pasquale EB (2008). "Eph-ephrin bidirectional signaling in physiology and disease." Cell 133(1): 38-52. Pasquale EB (2010). "Eph receptors and ephrins in cancer: bidirectional signalling and beyond." Nature Reviews Cancer 10(3): 165-180. Young PE, Womeldorph CM, Johnson EK, Maykel JA, Brucher B, Stojadinovic A, Avital I, Nissan A and Steele SR (2014). "Early Detection of Colorectal Cancer Recurrence in Patients Undergoing Surgery with Curative Intent: Current Status and Challenges." Journal of Cancer 5(4): 262-271. Peng L, Tu P, Wang X, Shi S, Zhou X and Wang J (2014). "Loss of EphB6 protein expression in human colorectal cancer correlates with poor prognosis." Journal of Molecular Histology 45(5): 555-563. Perez White BE and Getsios S (2014). "Eph receptor and ephrin function in breast, gut, and skin epithelia." Cell adhesion & migration 8(4): 327-338. Pitulescu ME and Adams RH (2010). "Eph/ephrin molecules—a hub for signaling and endocytosis." Genes & Development 24(22): 2480-2492. Polakis P (2012). "Wnt Signaling in Cancer." Cold Spring Harbor Perspectives in Biology 4(5):1-13 Poliakov A, Cotrina M and Wilkinson DG (2004). "Diverse Roles of Eph Receptors and Ephrins in the Regulation of Cell Migration and Tissue Assembly." Developmental Cell 7(4): 465-480. Potla L, Boghaert ER, Armellino D, Frost P and Damle NK (2002). "Reduced expression of EphrinA1 (EFNA1) inhibits three-dimensional growth of HT29 colon carcinoma cells." Cancer Letters 175(2): 187-195. Rahbari NN, Elbers H, Askoxylakis V, Motschall E, Bork U, Büchler MW, Weitz J and Koch M (2013). "Neoadjuvant Radiotherapy for Rectal Cancer:

181

Meta-analysis of Randomized Controlled Trials." Annals of Surgical Oncology 20(13): 4169-4182. Renshaw S (2007). Immunochemical staining techniques, , Chapter 4 page 45. Scion Publishing Ltd. Richter M, Murai KK, Bourgin C, Pak DT and Pasquale EB (2007). "The EphA4 receptor regulates neuronal morphology through SPAR-mediated inactivation of Rap GTPases." The Journal of Neuroscience 27(51):14205- 14215. Robichaux MA, Chenaux G, Ho HY, Soskis MJ, Dravis C, Kwan KY, Šestan N, Greenberg ME, Henkemeyer M and Cowan CW (2014). "EphB receptor forward signaling regulates area-specific reciprocal thalamic and cortical axon pathfinding." Proceedings of the National Academy of Sciences of The United States of America 111(6): 2188-2193. Rodríguez-Moranta F, Saló J, Arcusa À, Boadas J, Piñol V, Bessa X, Batiste- Alentorn E, Lacy AM, Delgado S, Maurel J, Piqué JM and Castells A (2006). "Postoperative Surveillance in Patients With Colorectal Cancer Who Have Undergone Curative Resection: A Prospective, Multicenter, Randomized, Controlled Trial." Journal of Clinical Oncology 24(3): 386-393. Ronsch K, Jäger M, Schöpflin A, Danciu M, Lassmann S and Hecht A (2011). "Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer." Epigenetics 6(5): 610-622. Rustgi AK (2007). "The genetics of hereditary colon cancer." Genes & Development 21(20): 2525-2538. Rutkowski R, Mertens‐ Walker I, Lisle JE, Herington AC and Stephenson SA (2012). "Evidence for a dual function of EphB4 as tumor promoter and suppressor regulated by the absence or presence of the ephrin‐ B2 ligand." International Journal of Cancer 131(5): E614-E624. Saito T, Masuda N, Miyazaki T, Kanoh K, Suzuki H, Shimura T, Asao T and Kuwano H (2004). "Expression of EphA2 and E-cadherin in colorectal cancer: correlation with cancer metastasis." Oncology Reports 11(3):605-11. Sancho E, Batlle E and Clevers H (2003). "Live and let die in the intestinal epithelium." Current Opinion in Cell Biology 15(6): 763-770. Sancho E, Batlle E and Clevers H (2004). "Signaling pathways in intestinal development and cancer." Annual Review of Cell and Developmental Biology 20: 695-723. Sanz-García E, Elez E, Macarulla T, Dienstmann R, Salazar R and Tabernero J (2014). "Prognosis and therapeutic implications for emerging colorectal cancer subtypes." Current Colorectal Cancer Reports 10(1): 55-61. Schepers A and Clevers H (2012). "Wnt signaling, stem cells, and cancer of the gastrointestinal tract." Cold Spring Harbor Perspectives in Biology 4(4): a007989. Schneider MR, Dahlhoff M, Horst D, Hirschi B, Trülzsch K, Müller-Höcker J, Vogelmann R, Allgäuer M, Gerhard M, Steininger S, Wolf E and Kolligs FT (2010). "A Key Role for E-cadherin in Intestinal Homeostasis and Paneth Cell Maturation." PLoS ONE 5(12): e14325. Schneikert J and Behrens J (2007). "The canonical Wnt signalling pathway and its APC partner in colon cancer development." Gut 56(3): 417-425. Schoenwolf GC, Bleyl SB, Brauer PR and Francis-West PH (2009). Larsen's Human Embryology, 4th edition, Churchill Livingstone Elsevier.

182

Segditsas S and Tomlinson I (2006). "Colorectal cancer and genetic alterations in the Wnt pathway." Oncogene 25(57): 7531-7537. Seiradake E, Schaupp A, del Toro Ruiz D, Kaufmann R, Mitakidis N, Harlos K, Aricescu AR, Klein R and Jones EY (2013). "Structurally encoded intraclass differences in EphA clusters drive distinct cell responses." Nature Structural and Molecular Biology 20(8): 958-964. Senior P, Zhang B and Chan SF (2010). "Loss of cell-surface receptor EphB2 is important for the growth, migration, and invasiveness of a colon cancer cell line." International Journal of Colorectal Disease 25(6): 687-694. Shaukat A, Mongin SJ, Geisser MS, Lederle FA, Bond JH, Mandel JS and Church TR (2013). "Long-Term Mortality after Screening for Colorectal Cancer." New England Journal of Medicine 369(12): 1106-1114. Sheng Z, Wang J, Dong Y, Ma H, Zhou H, Sugimura H, Lu G and Zhou X (2008). "EphB1 Is Underexpressed in Poorly Differentiated Colorectal Cancers." Pathobiology 75(5): 274-280. Shi SR, Cote RJ and Taylor CR (1997). "Antigen retrieval immunohistochemistry: past, present, and future." Journal of Histochemistry & Cytochemistry 45(3): 327-343. Shi SR, Cote RJ and Taylor CR (2001). "Antigen retrieval techniques Current perspectives." Journal of Histochemistry & Cytochemistry 49(8): 931-937. Shi SR, Key ME and Kalra KL (1991). "Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections." Journal of Histochemistry & Cytochemistry 39(6): 741-748. Singla N, Goldgur Y, Xu K, Paavilainen S, Nikolov DB and Himanen JP (2010). "Crystal structure of the ligand-binding domain of the promiscuous EphA4 receptor reveals two distinct conformations." Biochemical and Biophysical Research Communications 399(4): 555-559. Solanas G and Batlle E (2011). "Control of cell adhesion and compartmentalization in the intestinal epithelium." Experimental Cell Research 317(19): 2695-2701. Solanas G, Cortina C, Sevillano M and Batlle E (2011). "Cleavage of E- cadherin by ADAM10 mediates epithelial cell sorting downstream of EphB signalling." Nature Cell Biology 13(9): 1100-1107. Spannuth WA, Mangala LS, Stone RL, Carroll AR, Nishimura M, Shahzad MM, Lee SJ, Moreno-Smith M, Nick AM and Liu R, Jennings NB, Lin YG, Merritt WM, Coleman RL, Vivas-Mejia PE, Zhou Y, Krasnoperov V, Lopez-Berestein G, Gill PS and Sood AK (2010). "Converging evidence for efficacy from parallel EphB4-targeted approaches in ovarian carcinoma." Molecular cancer therapeutics 9(8): 2377-2388. Stephenson SA, Slomka S, Douglas EL, Hewett PJ and Hardingham JE (2001). "Receptor protein tyrosine kinase EphB4 is up-regulated in colon cancer." BioMed Central Molecular Biology 2(1): 1-9. Strimpakos A, Pentheroudakis G, Kotoula V, De Roock W, Kouvatseas G, Papakostas P, Makatsoris T, Papamichael D, Andreadou A, Sgouros J, Zizi- Sermpetzoglou A, Kominea A, Televantou D, Razis E, Galani E, Pectasides D, Tejpar S, Syrigos K and Fountzilas G (2013). "The Prognostic Role of Ephrin A2 and Endothelial Growth Factor Receptor Pathway Mediators in Patients

183

With Advanced Colorectal Cancer Treated With Cetuximab." Clinical Colorectal Cancer 12(4): 267-274.e262. Sun Y, Qiao L, Zou B, Xia HH, Gu Q, Ma J, Lin MC, Zhu Q, Zhu S, Dai Y and Wong BC (2007). "Interactions between XIAP associated factor 1 and a nuclear co-activator, CBP, in colon cancer cells." Digestion 77(2): 79-86. Surawska H, Ma PC and Salgia R (2004). "The role of ephrins and Eph receptors in cancer." Cytokine & Growth Factor Reviews 15(6): 419-433. Taddei ML, Parri M, Angelucci A, Onnis B, Bianchini F, Giannoni E, Raugei G, Calorini L, Rucci N, Teti A, Bologna M and Chiarugi P (2009). "Kinase- Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis." The American Journal of Pathology 174(4): 1492-1503. Takahashi Y, Itoh M, Nara N and Tohda S (2014). "Effect of EPH–Ephrin Signaling on the Growth of Human Leukemia Cells." Anticancer Research 34(6): 2913-2918. Tanabe H, Kuribayashi K, Tsuji N, Tanaka M, Kobayashi D and Watanabe N (2011). "Sesamin induces autophagy in colon cancer cells by reducing tyrosine phosphorylation of EphA1 and EphB2." International Journal of Oncology 39(1): 33-40. Terry MB and Neugut AI (1998). "Cigarette smoking and the colorectal adenoma-carcinoma sequence: a hypothesis to explain the paradox." American Journal of Epidemiology 147(10): 903-910. Tetsu O and McCormick F (1999). "[beta]-Catenin regulates expression of cyclin D1 in colon carcinoma cells." Nature 398(6726): 422-426. Tognolini M1, Incerti M, Pala D, Russo S, Castelli R, Hassan-Mohamed I, Giorgio C and Lodola A (2014). "Target Hopping as a Useful Tool for the Identification of Novel EphA2 Protein–Protein Antagonists." ChemMedChem 9(1): 67-72. Trivier E and Ganesan TS (2002). "RYK, a Catalytically Inactive Receptor Tyrosine Kinase, Associates with EphB2 and EphB3 but Does Not Interact with AF-6." The Journal of Biological Chemistry 277(25): 23037-23043. Truitt L and Freywald A (2011). "Dancing with the dead: Eph receptors and their kinase-null partners." Biochemistry and Cell Biology 89(2): 115-129. Tsikitis VL, Larson DW, Huebner M, Lohse CM and Thompson PA (2014). "Predictors of recurrence free survival for patients with stage II and III colon cancer." BioMed Central cancer 14(1): 336. Ueno H, Kajiwara Y, Shimazaki H, Shinto E, Hashiguchi Y, Nakanishi K, Maekawa K, Katsurada Y, Nakamura T, Mochizuki H, Yamamoto J and Hase K (2012). "New criteria for histologic grading of colorectal cancer." The American journal of surgical pathology 36(2): 193-201. Umar A, Boland CR, Terdiman JP, Syngal S, de la Chapelle A, Rüschoff J, Fishel R, Lindor NM, Burgart LJ, Hamelin R, Hamilton SR, Hiatt RA, Jass J, Lindblom A, Lynch HT, Peltomaki P, Ramsey SD, Rodriguez-Bigas MA, Vasen HF, Hawk ET, Barrett JC, Freedman AN and Srivastava S (2004). "Revised Bethesda Guidelines for hereditary nonpolyposis colorectal cancer (Lynch syndrome) and microsatellite instability." Journal of the National Cancer Institute 96(4): 261-268. Valkenburg KC, Graveel CR, Zylstra-Diegel CR, Zhong Z and Williams BO. (2011). "Wnt/β-catenin Signaling in Normal and Cancer Stem Cells." Cancers 3(2):2050-79

184

van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle E, Coudreuse D, Haramis AP, Tjon-Pon-Fong M, Moerer P, van den Born M, Soete G, Pals S, Eilers M, Medema R and Clevers H (2002). "The β-Catenin/TCF-4 Complex Imposes a Crypt Progenitor Phenotype on Colorectal Cancer Cells." Cell 111(2): 241-250. van der Flier LG and Clevers H (2009). "Stem cells, self-renewal, and differentiation in the intestinal epithelium." Annual Reviews Physiology 71: 241-260. Vauthey JN, Hoff PM, Audisio RA and Poston GJ (2009). Liver Metastases, Ch 2, pages: 15-24. Springer. Vearing CJ and Lackmann M (2005). "“Eph receptor signalling; dimerisation just isn't enough”." Growth Factors 23(1): 67-76. Virk R, Gill S, Yoshida E, Radley S and Salh B (2010). "Racial differences in the incidence of colorectal cancer." Canadian Journal of Gastroenterology 24(1): 47-51. Vivian LS (2008). "Profiling of gene expression changes in human colon crypt maturation and study of their dysregulation in tumourigenesis.". PhD thesis, University of Hong Kong Vogelstein B, Fearon ER, Hamilton SR, Kern SE, Preisinger AC, Leppert M, Nakamura Y, White R, Smits AM and Bos JL (1988). "Genetic Alterations during Colorectal-Tumor Development." New England Journal of Medicine 319(9): 525-532. Walker RA (2006). "Quantification of immunohistochemistry—issues concerning methods, utility and semiquantitative assessment I." Histopathology 49(4): 406-410. Walter V, Jansen L, Hoffmeister M, Ulrich A, Chang‐ Claude J and Brenner H (2015). "Smoking and survival of colorectal cancer patients: Population‐ based study from Germany." International Journal of Cancer. 137(6): 1433- 45. Wang H, Wen J, Wang H, Guo Q, Shi S, Shi Q, Zhou X, Liu Q, Lu G and Wang J (2014). "Loss of expression of EphB1 protein in serous carcinoma of ovary associated with metastasis and poor survival." International journal of clinical and experimental pathology 7(1): 313–321. Wang JD, Dong YC, Sheng Z, Ma HH, Li GL, Wang XL, Lu GM, Sugimura H, Jin J and Zhou X (2007). "Loss of Expression of EphB1 Protein in Gastric Carcinoma Associated with Invasion and Metastasis." Oncology 73(3-4): 238- 245. Wang J, Kataoka H, Suzuki M, Sato N, Nakamura R, Tao H, Maruyama K, Isogaki J, Kanaoka S, Ihara M, Tanaka M, Kanamori M, Nakamura T, Shinmura K and Sugimura H (2005). "Downregulation of EphA7 by hypermethylation in colorectal cancer." Oncogene 24(36): 5637-5647. Wang Y, Jatkoe T, Zhang Y, Mutch MG, Talantov D, Jiang J, McLeod HL and Atkins D (2004). "Gene Expression Profiles and Molecular Markers To Predict Recurrence of Dukes' B Colon Cancer." Journal of Clinical Oncology 22(9): 1564-1571. Welch, JP and Donaldson GA (1979). "The clinical correlation of an autopsy study of recurrent colorectal cancer." Annals of Surgery 189(4): 496-502. Xi HQ and Zhao P (2011). "Clinicopathological significance and prognostic value of EphA3 and CD133 expression in colorectal carcinoma." Journal of Clinical Pathology 64(6): 498-503.

185

Xia G, Kumar S, Stein JP, Singh J, Krasnoperov V, Zhu S, Hassanieh J, Smith D, Buscarini M, Broek D, Quinn DI, Weaver FA and Gill PS (2006). "EphB4 receptor tyrosine kinase is expressed in bladder cancer and provides signals for cell survival." Oncogene 25(5): 769-780. Xia G, Kumar SR, Masood R, Zhu S, Reddy R, Krasnoperov V, Quinn DI, Henshall SM, Sutherland RL, Pinski JK, Daneshmand S, Buscarini M, Stein JP, Zhong C, Broek D, Roy-Buman P and Gill PS (2005). "EphB4 expression and biological significance in prostate cancer." Cancer research 65(11): 4623- 4632. Xu K, Tzvetkova-Robev D , Xu Y, Goldgur Y, Chan YP, Himanen JP and Nikolov DB (2013). "Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5." Proceedings of the National Academy of Sciences of The United States of America 110(36): 14634-14639. Xuqing W, Lei C, Zhengfa M, Shengchun D, Xin F, Jianguo Q and Jianxin Z (2012). "EphB4 is overexpressed in papillary thyroid carcinoma and promotes the migration of papillary thyroid cancer cells." Tumor Biology 33(5): 1419- 1427. Yamazaki D, Kurisu S and Takenawa T (2005). "Regulation of cancer cell motility through actin reorganization." Cancer Science 96(7): 379-386. Yin Y, Yamashita Y, Noda H, Okafuji T, Go MJ and Tanaka H (2004). "EphA receptor tyrosine kinases interact with co-expressed ephrin-A ligands in cis." Neuroscience research 48(3): 285-295. Yoshimatsu K, Yokomizo H and Naritaka Y (2014). "Clinical impact of minimal cancer cell detection in various colorectal cancer specimens." World Journal of Gastroenterology 20(35): 12458-12461. Yuan Y, Yang ZL, Miao XY, Liu ZR, Li DQ, Zou Q, Li JH, Liang LF, Zeng GX and Chen SL (2014). "EphB1 and Ephrin-B, new potential biomarkers for squamous cell/adenosquamous carcinomas and adenocarcinomas of the gallbladder." Asian Pacific Journal of Cancer Prevention. 15(3): 1441-1446. Zhang B, Ougolkov A, Yamashita K, Takahashi Y, Mai M and Minamoto T (2003). "β-Cateninand ras Oncogenes Detect Most Human Colorectal Cancer." Clinical Cancer Research 9(8): 3073-3079. Zhou S, Wang L, Li G, Zhang Z and Wang J (2014). "Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas." International Journal of Clinical and Experimental Pathology 7(7): 4254– 4260. Zimmer M, Palmer A, Kohler J and Klein R (2003). "EphB-ephrinB bidirectional endocytosis terminates adhesion allowing contact mediated repulsion." Nature Cell Biology 5(10): 869-878. Zoratto F, Rossi L, Verrico M, Papa A, Basso E, Zullo A, Tomao L, Romiti A, Lo Russo G and Tomao S (2014). "Focus on genetic and epigenetic events of colorectal cancer pathogenesis: implications for molecular diagnosis." Tumor Biology: 1-12.

186

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Alrahbi, Rashid Saeed

Title: The pattern of expression of the receptor tyrosine kinases (RTK) EphB1, EphB2 and EphB4 in colorectal cancer

Date: 2016

Persistent Link: http://hdl.handle.net/11343/59635

File Description: The pattern of expression of the receptor tyrosine kinases (RTK) EphB1, EphB2 and EphB4 in colorectal cancer