REGULATION OF COLLAGENASES MATRIX METALLOPROTEINASES WITH CTCF/YB-1

(1)

REGULATION OF COLLAGENASES MATRIX METALLOPROTEINASES WITH CTCF/YB-1

TRANSCRIPTION FACTORS IN HUMAN MALIGNANT MELANOMA CANCER IN VITRO

BY

WISAM NABEEL IBRAHIM

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy

Kulliyyah of Allied Health Sciences International Islamic University Malaysia

September 2018

(2)

ii

ABSTRACT

The invasiveness of malignant melanoma is mainly attributed to the enzymatic destruction of the extracellular matrix and basement membrane components by a group of enzymes known as matrix metalloproteinases (MMPs). The expression of these proteinases is mainly regulated at the transcriptional level; therefore, high expression of MMPs is manly attributed to different transcription factors which enhance or inhibit the promoter activity of MMPs genes. Among these factors, YB-1 and CTCF proteins are transcription factors in which CTCF is mainly a tumour suppressor protein; while YB- 1 is an oncogenic factor and a prognostic indicator in a wide range of tumours that regulates most of the cancer processes such as proliferation, invasion and metastasis by regulating the expression of genes related to those processes. However; the expression of these transcription factors and their potential effect on the expression of collagenases MMPs in malignant melanoma cells are not yet confirmed. Therefore, this study was conducted to determine the expression of collagenases MMPs (MMP1, MMP8 and MMP13), YB-1 and CTCF transcription factors in A375 melanoma cancer cells. In addition, the stromal effect of normal skin fibroblasts on the expression of collagenases and proliferation in A375 cell was determined. The results of this experiment demonstrated an increase in the expression of YB-1, MMP8 and MMP13 in A375 cells.

Thereafter, this was followed by the establishment of YB-1 silenced strain of A375 cells using a silencing short hairpin RNA (shRNA) construct. The effect of YB-1 knockdown on the expression of collagenases MMPs was determined using reverse transcription PCR and Western blotting. In addition, the antiproliferative effect was determined using flow-cytometry, colorimetric MTT assay and cell counting; while the anti-invasive properties were determined using wound healing assay. The results of this experiment elucidated that YB-1 protein regulates the expression of MMP13, cell cycle progression, cell proliferation and cell migration of A375 cancer cells in vitro. Therefore, the direct interaction between YB-1 protein and the AP-1 promoter sequence of MMP13 was evaluated using Chromatin Immunoprecipitation assay (ChIP). The ChIP analysis has confirmed no interaction between YB-1 protein and the AP-1 promoter sequence.

Finally, these experiments demonstrated YB-1, MMP8 and MMP13 were highly expressed in the A375 cancer cells. The stromal cells were found to promote A375 cell proliferation and enhance the expression of MMP1. In addition, YB-1 silencing was significantly associated with reduced expression of MMP13 enzyme, reduction in cancer cell proliferation in a cell cycle specific manner and anti-invasive properties.

Therefore, YB-1, MMP13 and stromal cells are considered as promising elements that might help as a potential target in the treatment of melanoma tumour due to their roles in the processes of invasion, migration and proliferation. Further experiments are needed to demonstrate novel protein partners of YB-1 and novel binding sites within gene promoter region of MMP13 with determination of the involved signalling pathways.

(3)

iii

ثحبلا ةصلاخ

ناطرس نإ دلجلا

ينينلايملا وه

دحأ دشأ عاونأ ناطرس دلجلا

ةروطخ نم

ةيحان

،راشتنلاا اببسم

تلادعم ىلعأ

نم تايفولا نم

نم ىرخلأا عاونلأا نيب مارولاا

.ةثيبخلا

ىزعيو اذه

كولسلا يناطرسلا

ىلا للحتلا يميزنلاا

بحاصملا يف

تانوكم ءاشغلا

يدعاقلا جيسنلل

ةطساوب ةعومجم

نم تاميزنلاا فرعت

تاميزناب ميطحت

جيسنلا

ينيتوربلا وا

ما(ـلا ما .)سيب نم عونلا اذه

تاميزنلاا ةمهم

يف ديدعلا نم تايلمعلا

هيويحلا هيجولويسفلاو

روطتك نينجلا

مائتلاو

،حورجلا نكلو

تايلاعف هذه

تاميزنلاا

هطرفملا مهاست

يف نيوكت راشتناو

ديدعلا نم مارولاا رسلا

.ةيناط ىوتسم

زرف هذه

تاميزنلاا اهتيلاعفو

دمتعي ةروصب هيسيئر

ىلع لدعم خسنلا كلاذلو ،ينيجلا ناف

هذه

هساردلا فدهت

ىلا سايق ىوتسم نيوكت

تاميزنا ما(ـلا

ما يب

،) 1 ما(ـلا ما يب ) 8

ما(ـلاو ما

يب

،) 13 ةفاضلااب ىلا

لماوع خاسنتسلاا

ياو(ـلا يب

1 ) يس(ـلاو يت

يس

)فا يتلاو ربتعت نم تانيتوربلا هلاعفلا

يف مدقت مارولاا هيناطرسلا

نم للاخ ميظنت

خاسنتسلاا ينيجلا

يف ايلاخلا دقل .هيناطرسلا

تتبثا هساردلا هدايز

يف زارفا تاميزنا

ما(ـلا ما يب

،) 8 ما(ـلا ما يب ) 13 لماعو خاسنتسلاا

ياو(ـلا يب

1 ) كلاذلو تناك

هبرجتلا

هقحلالا يه

ءاشنا ايلاخ س ناطر ينينلايم عم

طيبثتلا مئادلا

لماعل خسنلا ينيجلا ياو(ـلا

يب 1 ) مادختساب هينقت

نيوكت ضامحا

هيوون هريصق هلمكم

ضامحلا ياو(ـلا

يب 1 )

هيوونلا ثيح

مت دكأتلا نم طيبثتلا اقحلا

سايقب ىوتسم

ضمحلا يوونلا

ىوتسمو

نيتورب ياو(ـلا

يب 1 .) ةفاضلااب ىلا

كلاذ مت هسارد ريثات

لا طيبثت ىلع ىوتسم نيوكتلا

ينيجلا ينيتوربلاو

تاميزنلال ةمطحملا

.ثلاثلا اضيأ

ةسارد تمت ريثات

طيبثتلا ىلع

هعرس رثاكت

ايلاخلا هيناطرسلا

اهتردقو ىلع

ماحتقلاا .يعضوملا

تتبثأ دقلو هذه

هساردلا نا

طيبثت ياو(ـلا يب

1 ) مئادلا ببسي صقن يف زارفا ميزنا ما(ـلا ما يب ) 13

بسيو ب ايجيردت يف

رثاكت ايلاخلا هيناطرسلا

ددحيو نم

اهتيلباق ىلع

ماحتقلاا كلاذلو

مت ءارجا هبرجت ىرخا مييقتل لعافتلا رشابملا

نيب هقطنملا همظنملا

يف نيج ميزنا ما(ـلا

ما يب يتلا ) 13 فرعت يا(ـلاب

يب 1 ) نيبو نيتورب ياو(ـلا

يب 1 ) نم للاخ مادختسا

ةينقت بيسرتلا يعانملا

نيتاموركلل يذلاو

تبثا دوجو هقطنملا

يف نوكملا ينيجلا

ايلاخلل نكلو

مدع هلعافت عم

يب 1 ) كلاذلو تتبثا

هذه هساردلا رودلا

يسيئرلا نيتوربل

ياو(ـلا يب

1 ) يف ميظنت زارفا

ميزنا ما(ـلا ما يب ) 13 يفو ميظنت

رثاكتلا راشتنلااو

ايلاخل ناطرس دلجلا

ينينلايملا .

كلاذلو ربتعي

يب

1 ) فده يجلاع مهم

دق يدؤي ىلإ جلاع ىضرم ناطرس

دلجلا

ينينلايملا

.

(4)

iv

APPROVAL PAGE

The thesis of Wisam Nabeel Ibrahim has been approved by the following:

_____________________________

Associate Prof. Dr Mohammad Syaiful Bahari bin Abdull Rasad Supervisor

____________________________

Associate Prof. Dr. Ridhwan Bin Abdul Wahab Co-Supervisor

________________________________________

Associate Prof. Dr. Muhammad Bin Ibrahim Co-Supervisor

__________________

Assistant Prof Dr.Mohd Arifin Kaderi External Examiner

__________________

Professor Shaharum Shamsuddin External Examiner

__________________

Professor Teh Lay Kek External Examiner

_____________________________

Associate Prof. Dr Jesni Shamsul Shaari Chairman

(5)

v

DECLARATION

I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Wisam Nabeel Ibrahim

Signature ... Date ...

(6)

vi

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

REGULATION OF COLLAGENASES MATRIX METALLOPROTEINASES AND ITS INTERACTING PARTNERS CTCF/YB-1 TRANSCRIPTION FACTORS IN HUMAN MALIGNANT MELANOMA CANCER IN VITRO

I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.

Affirmed by Wisam Nabeel Ibrahim

……..……….. ………..

Signature Date

(7)

vii

DEDICATION

“To my beloved parents for their support and to my beloved wife for her patience and support “

(8)

viii

ACKNOWLEDGEMENT

By the name of Allah, I praise him with gratitude for his guidance and his blessings which helped me to pass through all the difficulties and challenges to complete this thesis.

I wish to express my appreciation and thanks to my supervisor Associate Prof.

Dr Mohammad Syaiful Bahari for his valuable guidance, motivation and wisdom. I would like to thank him for his constructive comments and his time in evaluating this thesis, and for that, I will be forever grateful.

In addition, I wish to express my gratitude to Associate Professor Dr. Ridhwan Bin Abdul Wahab for his valuable encouragement and guidance in this study.

To Professor Dr. Ridzwan Bin Hashim and Associate Prof. Dr. Muhammad Bin Ibrahim thank you for your guidance and support in this study.

To the dedicated and supportive science officers and assistant science officers in Kulliyyah of Allied Health Sciences, thank you for your support in this project in providing guidance and appropriate laboratory setting to complete this project.

This project was supported by the Research Initiative grant Scheme 2015 (RIGS15-089-0089) and (FRGS 11-039-0188).

Finally, a special thanks to those who provided their time, effort and support for this project.

(9)

ix

CHAPTER TWO: LITERATURE REVIEW ... 8

2.1 Cancer biology ... 8

2.2 Malignant melanoma ... 10

2.2.1 Background ... 10

2.2.2 Molecular pathways in malignant melanoma ... 11

2.3 Matrix metalloproteinases ... 13

2.3.1 Introduction ... 13

2.3.2 Matrix metalloproteinases and cancer ... 17

2.3.2.1 Matrix metalloproteinases secretion within the tumour microenvironment ... 17

2.3.2.2 Functions of matrix metalloproteinases in cancer ... 18

2.3.3 Collagenases MMPs ... 22

2.3.3.1 Structure of collagenases ... 22

2.3.3.2 Action of collagenases ... 23

2.3.3.3 Collagenase-1 (MMP1) ... 25

2.3.3.4 Collagenase-2 (MMP8) ... 25

2.3.3.5 Collagenase-3 (MMP13) ... 26

2.3.4 Regulation of collagenases activity... 28

2.3.4.1 Transcriptional regulation of collagenases ... 28

2.3.4.2 Genetic polymorphisms ... 34

2.3.4.3 Epigenetic regulation ... 35

2.3.4.4 mRNA stabilization ... 36

2.3.4.5 Post-translational activation ... 36

(10)

x

2.3.4.6 Tissue inhibitors of matrix metalloproteinases (TIMPs) .. 36

2.3.4.7 Summary ... 37

2.4 CTCF protein ... 37

2.5 YB-1 protein ... 41

CHAPTER THREE: MATERIALS AND METHODS ... 45

3.1 Materials ... 45

3.1.1 Disposable items ... 45

3.1.2 Instruments and apparatus ... 45

3.1.3 Chemicals and reagents ... 45

3.2 General study design... 50

3.3 Methodology ... 52

3.3.1 Cell culture ... 52

3.3.1.1 Thawing procedure ... 52

3.3.1.2 Subculture procedure ... 53

3.3.1.3 Cryopreservation procedure ... 54

3.3.1.4 Coculture setting ... 54

3.3.2 Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR)... 55

3.3.2.1 RNA extraction ... 55

3.3.2.2 RNA quantity and purity ... 56

3.3.2.3 RNA integrity ... 57

3.3.2.4 cDNA synthesis from RNA samples ... 57

3.3.2.5 Running the RT-qPCR... 58

3.3.3 Western blotting ... 64

3.3.3.1 Preparation of protein lysate sample ... 64

3.3.3.2 Quantification of protein concentration ... 65

3.3.3.3 Protein sample preparation ... 66

3.3.3.4 SDS polyacrylamide gel electrophoresis ... 66

3.3.3.5 SDS polyacrylamide gel staining ... 68

3.3.3.6 Blotting protocol ... 69

3.3.4 Assessment of A375 cell proliferation ... 74

3.3.4.1 Serial trypan blue cell counting ... 74

3.3.4.2 MTT assay ... 76

3.3.4.3 Flow cytometry - cell cycle assay ... 77

3.3.5 Establishment of YB-1 knocked down strains of A375 cells ... 78

3.3.5.1 Preparation of LB broth liquid medium ... 78

3.3.5.2 Preparation of LB agar plates ... 78

3.3.5.3 Transformation of E. coli ... 79

3.3.5.4 Amplification and extraction of plasmid DNA ... 80

3.3.5.5 Plasmid quality and quantity ... 82

3.3.6 Transfection of A375 melanoma cells ... 82

3.3.6.1 Determination of puromycin kill curve ... 82

3.3.6.2 Plasmid transfection of A375 melanoma cells ... 83

3.3.6.3 Confirmation of YB-1 knockdown ... 87

3.3.7 Cell migration assay (Wound healing assay) ... 88

3.3.8 Chromatin Immunoprecipitation assay (ChIP) ... 88

3.3.8.1 The analysis of chromatin digestion ... 92

3.3.9 Statistical analysis ... 93

(11)

xi

CHAPTER FOUR: EXPRESSION OF COLLAGENASES MATRIX

METALLOPROTEINASES IN A375 MELANOMA CELLS ... 94

4.1 Introduction... 94

4.2 Literature review ... 95

4.2.1 Stromal cells in the tumor environment ... 95

4.2.2 Collagenases MMPs ... 96

4.2.3 YB-1 oncogenic factor and CTCF protein ... 96

4.3 Objective ... 98

4.4 Materials and methods ... 98

4.4.1 Study design ... 98

4.4.2 Cell culture conditions ... 100

4.4.3 Screening of MMPs expression using an RT-qPCR array ... 100

4.4.4 Expression of collagenases MMPs, YB-1 and CTCF in A375 melanoma cells ... 100

4.4.4.1 Coculture setting ... 102

4.4.4.2 Cell quantification ... 102

4.4.4.3 Determination of gene and protein expression levels ... 102

4.5 Results ... 103

4.5.1 Morphology ... 103

4.5.2 RNA extraction and RT-qPCR amplification ... 107

4.5.3 RT-qPCR array results ... 111

4.5.4 Coculture and A375 Cell Proliferation ... 114

4.5.5 RT-qPCR analysis ... 117

4.5.6 Protein expression analysis ... 121

4.5.6.1 Quantification of protein concentration ... 121

4.5.6.2 Western blotting analysis ... 123

4.6 Discussion ... 125

4.6.1 Western blotting ... 125

4.6.1.1 Internal control ... 125

4.6.1.2 Target proteins ... 126

4.6.2 RT-qPCR ... 127

4.6.2.1 Housekeeping genes ... 127

4.6.2.2 Target genes ... 128

4.6.3 A375 melanoma / CCD-1079 skin fibroblast coculture... 129

4.6.3.1 CCD-1079SK skin fibroblast cells as a model for stromal cells ... 129

4.6.3.2 Fibroblast cells enhanced the proliferation of A375 melanoma cells in vitro ... 130

4.6.3.3 Fibroblast cells enhanced MMP1 expression in A375 melanoma cells in vitro ... 131

4.6.4 Expression of collagenases MMPs in A375 melanoma cells ... 132

4.6.5 YB-1 protein is highly expressed in A375 Melanoma cells ... 134

4.6.6 CTCF expression in A375 cells ... 135

4.7 Conclusion ... 136

(12)

xii

CHAPTER FIVE: YB-1 KNOCKDOWN IN A375 MELANOMA CELL

LINE ... 138

5.2.2 RNA interference ... 139

5.2.3 YB-1 protein and the expression of collagenases MMPs ... 140

5.2.4 YB-1 protein and the cancer cell proliferation ... 141

5.2.5 YB-1 protein and the cell cycle in cancer cell ... 142

5.2.6 YB-1 protein and cancer cell migration ... 143

5.4.1 Study design ... 145

5.4.2 Cell culture and shRNA plasmid transfection... 147

5.4.3 Validation of YB-1 knockdown in silenced cell strains ... 147

5.4.4 A375 cell proliferation assay and cell cycle interference ... 147

5.4.5 Expression of collagenases MMPs ... 148

5.4.6 A375 cell migration assay ... 148

5.5 Results ... 148

5.5.1 Puromycin kill curve ... 148

5.5.2 Confirmation of successful plasmid transfection in A375 cells ... 150

5.5.3 Validation of YB-1 knockdown in A375 cells... 152

5.5.4 RNA extraction and RT-qPCR amplification ... 154

5.5.5 Effect of YB-1 silencing on the expression of collagenases in A375 cell line... 158

5.5.5.1 RT-qPCR ... 158

5.5.5.2 Western blotting analysis ... 160

5.5.6 Antiproliferative effect of YB-1 silencing in A375 cell line ... 162

5.5.7 Effect of YB-1 silencing on cancer cell cycle in A375 cell line ... 164

5.5.8 Effect of YB-1 silencing on A375 cell line migration assay in vitro ... 166

5.6.1 YB-1 silencing methodology ... 168

5.6.2 YB-1 protein and the expression of collagenases MMPs in A375 cancer cells ... 169

5.6.3 A375 cancer cell proliferation... 170

5.6.3.1 Proliferation assays ... 170

5.6.3.2 YB-1 protein and A375 cancer cell proliferation ... 171

5.6.4 YB-1 protein and A375 cancer cell migration ... 172

CHAPTER SIX: INTERACTION WITH AP-1 PROMOTER SEQUENCE OF MMP13 GENE ... 175

6.2.2 MMP13 gene promoter ... 176

(13)

xiii

6.2.3 AP-1 promoter site and the expression of MMP13 ... 177

6.3.2 Specific objective ... 178

6.5 Results ... 181

6.5.1 Micrococcal nuclease digestion of chromatin DNA ... 181

6.5.2 Chromatin immunoprecipitation assay ... 183

CHAPTER SEVEN: DISCUSSION AND CONCLUSION ... 189

7.2 Selection of Cell lines ... 189

7.3 Expression of MMPs, YB-1, CTCF and the stromal effect ... 190

7.4 YB-1 knock down with shRNA plasmid ... 195

7.5 YB-1 interaction with AP-1 sequence of MMP13 gene ... 196

7.6 Limitations ... 198

7.7 Recommendations... 198

Bibliography ... 199

Appendices ... 221

9.1 Appendix 1 Quality control data of YB-1 ... 221

9.2 Appendix 2 Quality control data of CTCF ... 222

9.3 Appendix 3 Quality control data of MMP1 ... 223

9.6 Appendix 6 Quality control data of TIMP1 ... 226

9.9 Appendix 9 Quality control data of α-tubulin ... 229

9.10 Appendix 10 Quality control data of RPL13A ... 230

9.11 Appendix 11 Quality control data of HPRT1 ... 231

9.12 Appendix 12 Quality control data of GAPDH ... 232

9.13 Appendix 13 Quality control data of β2-microglobulin ... 233

9.14 Appendix 14 Quality control data of β-actin ... 234

9.15 Appendix 15 Conference presentations and publications ... 235

9.15.1 Conferences ... 235

9.15.2 Publications ... 235

(14)

xiv

LIST OF TABLES

Table No. Page No.

Table 3.1 List of disposable items 46

Table 3.2 List of instruments and apparatus 46

Table 3.3 List of chemicals and reagents 47

Table 3.4 Amplicons sequences of the validated target and housekeeping genes used in qPCR (Bio-Rad, USA)

61

Table 3.5 Reagents specifications used for the preparation of SDS polyacrylamide gels

67

Table 3.6 Buffers and specifications used in Western blotting protocol

70

Table 3.7 List of monoclonal antibodies used in the Western blotting protocol

73

Table 4.1 List of genes included in the matrix and adhesion molecules 96 PCR array

101

(15)

xv

LIST OF FIGURES

Figure No. Page No.

Figure 2.1 Classification of matrix metalloproteinases 14 Figure 2.2 Roles of MMPS in cancer development and progression 16 Figure 2.3 Structure of collagenase matrix metalloproteinases

peptides

24

Figure 2.4 Structure of the promoter regions in collagenases genes 30

Figure 2.5 Domain structure of CTCF protein 38

Figure 2.6 Domain structure of YB-1 protein 42

Figure 3.1 The general study design 51

Figure 3.2 Extracellular Matrix and Adhesion Molecules Plate design

59

Figure 3.3 The designed customized 96 qPCR plate (Bio-Rad, USA) 62 Figure 3.4 Structure of retroviral pGFP-V-RS vector (OriGene,

USA)

85

Figure 4.1 Study design to determine collagenases expression in A375 melanoma cells

99

Figure 4.2 Morphology of A375 malignant melanoma cells 104 Figure 4.3 Morphology of CCD1079 skin fibroblasts cells 106 Figure 4.4 Denaturing agarose gel electrophoresis of RNA samples 108

Figure 4.5 RT- qPCR SYBR green amplification curve 110

Figure 4.6 Melting curve analysis of RT- qPCR amplicons 110 Figure 4.7 Relative mRNA expression of MMPs genes in A375 cell

line

112

Figure 4.8 Relative mRNA expression of TIMPs genes in A375 cell line

113

Figure 4.9 Microscopic growth and morphological changes of A375 melanoma cell line by the influence of fibroblasts cells

115

(16)

xvi

LIST OF FIGURES

Figure No. Page No.

Figure 4.10 Growth curve for A35 cells in monoculture & coculture 116 Figure 4.11 Relative mRNA expression analysis of target genes in

A375 melanoma cells and CCD-1079SK fibroblasts cells

118

Figure 4.12 Relative mRNA expression analysis of target genes of A375 melanoma cells and the coculture of A375 with CCD-1079SK fibroblasts cells

120

Figure 4.13 Standard curve of colorimetric Pierce BCA assay for calculation of protein concentration

122

Figure 4.14 Western blotting analysis of target genes in A375 melanoma cells, CCD1079SK fibroblasts cells and the coculture of both cell line

124

Figure 5.1 Flowchart of YB-1 shRNA silencing study 146

Figure 5.2 Puromycin kill curve observed in A375 melanoma cells 149 Figure 5.3 Validation of A375 cells transfection with pGFP-V-RS

vector

151

Figure 5.4 YB-1 protein expression analysis using Immune fluorescence staining

153

Figure 5.5 Denaturing agarose gel electrophoresis of RNA samples 155

Figure 5.6 RT-qPCR SYBR green amplification curve 157

Figure 5.7 Melting curve analysis of RT qPCR amplicons 157

Figure 5.8 RT-qPCR analyses of YB-1 knockdown 159

Figure 5.9 Western blotting and densitometry analysis 161

Figure 5.10 Anti-proliferative effects of YB-1 shRNA 163

Figure 5.11 Flow cytometry cell cycle analysis 165

Figure 5.12 Effect of YB-1 shRNA on cancer cell migration 167 Figure 5.13 Epithelial-mesenchymal transformation in A375

melanoma cells

167

(17)

xvii

LIST OF FIGURES

Figure No. Page No.

Figure 6.1 Workflow of chromatin immune precipitation assay 180 Figure 6.2 Agarose gel electrophoresis of digested chromatin

products from A375 cells

182

Figure 6.3 Chromatin immunoprecipitation assay 184

Figure 7.1 Changes in the expression of MMPs, TIMPs and transcription factors

192

Figure 7.2 The proposed mechanism involved in the stromal regulation of MMP1 expression

194

Figure 7.3 The proposed mechanism involved in the regulation of MMP13 expression, cell proliferation, and epithelial-

mesenchymal transition

197

(18)

xviii

LIST OF SYMBOLS

oC Degree Celsius

% Percentage

- Minus

bp Base pair

g Gram

mg Milligram

mM Millimolar

mL Millilitre

nm Nanometre

NaCl Sodium chloride

µg Microgram

µL Microliter

µM Micromolar

ng Nanogram

M Molar

V Volts

H2O Water

CO2 Carbon dioxide MgCl2 Magnesium chloride BSC Biosafety cabinet

(19)

xix

LIST OF ABBREVIATIONS

cDNA Complimentary DNA

DAPI 4',6-diamidino-2-phenylindole

DMEM Dulbecco's Modified Eagle Medium

DMSO Dimethyl sulfoxide

dNTP Deoxy nucleoside triphosphates

DNA Deoxyribonucleic acid

RNA Ribonucleic acid

EGF Epidermal growth factor

FGF Fibroblasts growth factor

PDGF Platelets derived growth factor

KDGF Keratinocyte growth factor

TGF-β Tumour growth factor -β

TNF-α Tumour necrosis factor -α

HGF Hepatocytes growth factor

VEGF Vascular endothelial growth factor

NGF Nerve growth factor

MAPK Mitogen activated phosphokinases

FITC Fluorescein isothiocyanate

IgG Immunoglobulin G

IgM Immunoglobulin M

MMPs Matrix Metalloproteinases

CTCF CCCTC-binding factor

(20)

xx

LIST OF ABBREVIATIONS

YB-1 Y-box binding protein

MMP13 Matrix Metalloproteinase 13

TIMPs Tissue inhibitors of matrix proteinases

mRNA Messenger ribonucleic acid

shRNA Short hairpin ribonucleic acid

PBS Phosphate buffer saline

PCR Polymerase chain reaction

RT-PCR Reverse transcriptase polymerase chain reaction

PFA Paraformaldehyde

RT-qPCR Quantitative reverse transcriptase polymerase chain reaction

TBE Tris/Borate/EDTA buffer

TAE Tris/Acetic acid/EDTA buffer

UV Ultraviolet

ACTB Actin Beta

B2M Beta-2-Microglobulin

GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase.

HPRT1 Hypoxanthine Phosphoribosyltransferase 1

RPL13A Ribosomal Protein L13a

TUBA1A Alpha Tubulin

Abs. Absorbance

(21)

1

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Malignant melanoma is the third most common malignant skin cancer and is the most serious cancer in terms of local invasiveness and mortality rate (Kumar, Abbas, Fausto,

& Aster, 2014). While the incidence rate of this malignancy is constantly increasing worldwide (Yamashita et al., 2013), malignant melanoma is considerably associated with poor prognosis, with an estimated 10-year survival rate of less than 10% (Hall &

Kudchadkar, 2014).

Cancer cells in general, possess a broad spectrum of migration and invasion mechanisms including the destruction of tissue basement membrane and extracellular matrix (ECM) components allowing the migration of cancer cells (Bourboulia &

Stetler-Stevenson, 2010). The matrix metalloproteinases (MMPs) group of enzymes play a major role in this destruction process that participates in the remodelling of the ECM to support the growth of tumour cells (Moro, Mauch, & Zigrino, 2014). These proteinases are involved in remodelling processes of normal tissue such as in morphogenesis, angiogenesis, tissue repair and embryonic development (Kessenbrock, Plaks, & Werb, 2010). The secretion of these enzymes is tightly controlled to regulate these normal functions; in which imbalance between the inhibition and activation of MMPs results in disturbances characterized by an excessive degradation of the ECM such as in osteoarthritis, rheumatoid arthritis & different types of malignancies (Frank et al., 2012; Hadler-Olsen, Winberg, & Uhlin-Hansen, 2013; Moro et al., 2014; Nissinen

& Kähäri, 2014). Within the tumour mass, MMPs are expressed by cancer cells and to a larger extent by the surrounding normal cells within the tumour environment known

(22)

2

as cancer stromal cells participating in the cleavage of ECM and basement membrane components (Hadler-Olsen et al., 2013; Hashimoto et al., 2016).

This family of enzymes is classified by their structure and substrate specificity into collagenases, gelatinases, stromelysins, and matrilysins (Gialeli, Theocharis, &

Karamanos, 2011a). Collagenases MMPs include collagenases-1 (MMP1), collagenase- 2 (MMP8) and collagenase-3 (MMP13) which are capable of initiating degradation of native fibrillary collagens (type I, II, III, V and IX) and obviously play a crucial role in degradation of collagenous extracellular matrix (ECM) in various tumours (Moro et al., 2014). Both MMP1 and MMP13 are associated with accelerated invasive processes by cancer cells and are either secreted by the cancer cells or the tumour surrounding stroma (Gialeli et al., 2011a; Kim et al., 2011; Martinet, Hirsch, Mulshine, & Vignaud, 2012).

Meanwhile, MMP8 is associated with some controversial effects within cancer microenvironment ranging from a promoter to an inhibitor in different malignancies (Schröpfer et al., 2010a; Thirkettle et al., 2013).

Generally, the expression of these collagenases is regulated at different levels and mechanisms; however, the most influential mechanism of control is at the transcriptional level at the noncoding promoter sequence via the specific binding of different transcription factors (Brown & Murray, 2015). The AP-1 DNA binding sites among the regulatory sites are present in the gene promoters of MMP1 and MMP13 gene and are mainly responsible for the transcriptional regulation of collagenases expression (Samuel, Beifuss, & Bernstein, 2007; Xia et al., 2015; Lu et al., 2016). The transcriptional regulation at this gene site is believed to participate actively in the progression of cancer cells by its binding with different oncogenic transcription factors expressed in cancer cells causing an increase in the expression of collagenases (Kessenbrock, Wang, & Werb, 2015).

(23)

3

CTCF and YB-1 transcription factors are among the many transcription factors that are involved with different regulatory roles in cancer cell development and progression. These factors are capable of specific binding with different gene promoter sequences and thereby can transcriptionally control the expression of different genes responsible for cancer cell proliferation and growth (H. Wang et al., 2015; Chen et al., 2016). CTCF is a well-defined tumour suppressor protein mainly regulating the epigenetic changes in cancer cells (Kemp et al., 2014). While, the YB-1 transcription factor is an oncogenic factor that is involved in stimulating different hallmarks of cancer cells (Lasham, Woolley, Dunn, & Braithwaite, 2013; Kemp et al., 2014).

Although both transcription factors might work in the opposite manner;

however, some studies reported their interaction with each other in a way that modifies their binding specificity with gene promoter sequences (Samuel et al., 2007; Wallace, 2007). Both factors were reported in the transcriptional regulation of MMPs through interaction with different promoter sequences including the AP-1 site (Abdull Rasad et al., 2008). However, the influence of these transcription factors on the expression of collagenases MMPs is not well elucidated in melanoma cells or other types of cancer cells. Therefore, this study would elucidate the possible mechanism regulating the expression of collagenases MMPs in A375 malignant melanoma cell line in vitro including YB-1, CTCF transcription factors and stromal cells in melanoma microenvironment in vitro.

1.2 SUMMARY OF THESIS

The aim of this study was to evaluate and discuss the expression of collagenases MMPs in malignant melanoma cancer cells and to determine novel mechanisms or transcription factors capable of regulating the expression of these enzymes.

(24)

4

In Chapter 1 and Chapter 2, all the theoretical background was provided to introduce the reader to cancer cells biology and to comprehensively describe the different regulatory aspects of the expression of MMPs from transcription to translation.

In Chapter 3, all the methods used in this study were described including the reagents, equipment and the basic protocols with their optimizations. The basic techniques of cell culture maintenance were fully described. In addition, the full description of reverse transcription polymerase chain reaction (RT-PCR) protocol and analysis were described in details. The Western blotting protocol with optimizations and the analysis were also fully described in this thesis. The description and explanation of plasmid transfection, validation, and maintenance of the cells was also described and finally, the protocol of chromatin immunoprecipitation (ChIP) assay was also described.

In Chapter 4, the expression of all MMPs and TIMPs were screened in the melanoma cancer cells followed by evaluating the expression of collagenases MMPs, YB-1 and CTCF proteins in melanoma cells and in the stromal- cancer cells model by the use of the human skin fibroblasts cells. The results were fully analysed and discussed in the Chapter which provided the preliminary data for the implementation of the next Chapter.

In Chapter 5, a stable strain of melanoma cells with constant knockdown of YB- 1 was established, validated and tested for its influence on the expression of collagenases MMPs, cancer cell proliferation and invasion in vitro.

In Chapter 6, the potential interaction of YB-1 and CTCF with the AP-1 promoter sequence of MMP13 was determined in ex vivo using ChIP assay to elucidate the possible protein – DNA interactions and their possible regulation of collagenases expression.

Kulliyyah of

REGULATION OF COLLAGENASES MATRIX METALLOPROTEINASES WITH CTCF/YB-1