The role of the homeobox gene HOXD10 in Lung and Head & Neck carcinoma Fahad Mohammad I Hakami BSc, MSc A thesis submitted to the University of Sheffield in part fulfilment for the degree of Doctor of Philosophy The School of Clinical Dentistry University of Sheffield December 2013 This thesis is dedicated to the memory of my father, Mohammad Ibrhaim Aiafi Hakami. ACKNOWLEDGEMENTS First and foremost, I thank The Almighty Allah for blessing, protecting and guiding me throughout my personal and professional life. This thesis is the end of my journey in obtaining my PhD, and it would not end happily without the advice, motivation, patience and, most importantly, the friendship of my principal PhD supervisor, Dr Keith Hunter. Keith has always been available to guide me through all the challenges I faced during my work until I developed independent thinking. I am deeply grateful for his endless support and his believing in me. It has been an honour and a privilege working with Dr Hunter. I would also like to extend my appreciation to my co-supervisors Professor Penella Woll for introducing me to Keith and for her contributions during discussing the progress of my work, and Dr Daniel Lambert for all his insightful discussions regarding my work and for his generosity in answering my endless questions. I gratefully acknowledge Dr Lynn Bingle for teaching me all the IHC optimisation tricks, Mrs Prachi Stafford for all the technical help, Dr Paul Heath for his help in the microarray analysis and Mrs Brenka McCabe for keeping my drawer full with all items I needed for my work. A special thank you to Dr Martin Nicklin for all the invaluable molecular biology knowledge I gained before I started my PhD studies. Also, it is my pleasure to thank everyone in the School of Clinical Dentistry, particularly, in the Unit of Oral and Maxillofacial Pathology. Big thank you to my fellow colleagues, Mr Lav Darda, Mrs Nada AlHindi, Mrs Ibtisam Zargoun, Miss Emma Hinsley, Miss Tasnuva Kabir, Miss Genevieve Melling, Mrs Hanan Niaz, Miss Dalal Al-Otaibi, Miss Kate Naylor, Miss Hayley Lunn and Miss Abigaile Rice. I am very grateful to Dr Mohammad Abdelaal, Mr Mohammad Al-Shehri and my wise mentor, Dr Baraa AlHaj-Huessin. Without their help and opportunities they offered me at the beginning of my career, I would not have been able to complete my postgraduate studies. Also, a special thank goes to the movies expert, Fawaz Albloui, who made my stay at Sheffield more enjoyable. Last but not least, words are short to express my deep sense of gratitude towards my wife, Hana Mobarki. I am grateful for her understanding when I worked late for long days, standing by me through the good and bad times, and for taking good care of our daughter, Hala. I doubt that I will ever be able to fully convey my appreciation to her. The work described in this thesis has been supported by the National Guard Health Affairs, Jeddah, through the Royal Embassy of Saudi Arabia Cultural Bureau in London. i ABSTRACT Alterations in gene expression in head and neck squamous cell carcinoma (HNSCC) and non- small cell lung cancer (NSCLC) are variable and correlate with cancer site and stage. Previous microarray analysis comparing HNSCC to normal oral keratinocytes (NOKs) revealed changes in HOX gene expression, particularly HOXD10. Similar data were identified in NSCLC in other study when a comprehensive analysis system based on the quantitative real-time polymerase chain reaction (qPCR) was performed. Moreover, HOXD10 expression is altered in many cancers including breast, ovarian, prostate and gastric cancers. This study demonstrates a low HOXD10 mRNA and protein expression in normal cells, variable levels in precancerous cells, high expression in most primary tumours, but low in cells derived from lymph node metastases. Moreover, it was demonstrated that HOXD10 expression is reduced in HNSCC metastases relative to their paired primary tumours. Over-expression of HOXD10 decreased cell invasion but increased proliferation, adhesion and migration, with siRNA and shRNA knock-down causing reciprocal effects, indicating possible role of HOXD10 in promoting the development of the primary tumour. Microarray analysis using the Agilent Sureprint G3 Array in cells with stable over-expression or transient knockdown of HOXD10 demonstrated increases in expression of a number of genes known to promote cell proliferation and modulate epithelial-to-mesenchymal transition (EMT), in keeping with its suggested role as a suppressor of metastasis. This was supported further by the observation that HOXD10 acts as a negative regulator of a number of genes known to promote cell invasion. Gene ontology (GO) analysis has mapped many of the genes affected by HOXD10 manipulation to different pathways responsible for different biological processes, particularly cell proliferation and migration. The microarray analysis also identified a number of HOXD10-associating molecules. Validation by qPCR and filtering by in- silico promoter analysis and applying some selection criteria identified a number of HOXD10 putative targets, including Angiomotin p80 isoform (AMOT-p80) and miR146a. These were confirmed as direct targets of HOXD10 by dual luciferase reporter (DLR) assay in the wild-type and mutated HOXD10 binding site(s) on the promoter. Suppression of miR-146a by HOXD10 was found to induce the expression of genes previously reported to induce tumour growth and are targets of miR-146a. Manipulation of AMOT-p80 expression in HNSCC cells resulted in similar phenotypic changes to those seen with manipulation of HOXD10 expression. Indeed, AMOT-p80 is able to rescue the migratory and proliferative phenotype of knockdown of HOXD10. Thus, HOXD10 expression varies by stage of disease in HNSCC and produces differential effects, with high expression giving premalignant and cancer cells a probable proliferative and migratory advantage over normal cells while low expression may later support metastasis. ii TABLE OF CONTENTS Abstract ........................................................................................................................................................ii Table of contents ........................................................................................................................................iii List of Figures ........................................................................................................................................... vii List of Tables ...............................................................................................................................................x Abbreviations ............................................................................................................................................. xi Publications ............................................................................................................................................. xvi CHAPTER 1: INTRODUCTION ....................................................................................................................1 1.1 Cancer ..........................................................................................................................................2 1.2 Head and neck cancer .................................................................................................................2 1.2.1 Epidemiology and incidence ....................................................................................................6 1.2.2 Diagnosis and staging .............................................................................................................6 1.2.3 Aetiology and risk factors .........................................................................................................7 1.2.3.1 Smoking and alcohol .................................................................................................................... 7 1.2.3.2 Human papillomavirus (HBV) ....................................................................................................... 9 1.2.3.3 Other HNSCC aetiological factors .............................................................................................. 10 1.2.1 Genetic alterations and molecular markers .......................................................................... 12 1.2.1.1 Tumour protein p53 (TP53) ........................................................................................................ 13 1.2.1.2 Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) ...................................................................... 14 1.2.1.3 Epidermal growth factor receptor (EGFR) .................................................................................. 15 1.3 Lung cancer .............................................................................................................................. 17 1.3.1 Epidemiology ........................................................................................................................ 20 1.3.2 Diagnosis and staging .......................................................................................................... 20 1.3.3 Aetiology and risk factors ...................................................................................................... 23 1.3.3.1 Smoking ..................................................................................................................................... 23 1.3.3.2 Radon and Asbestos .................................................................................................................