EVALUATION OF THE ROLE OF EGFR, SOX2, AND PAX9 IN ORAL CARCINOGENESIS Timothy John Bates A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Northern Institute for Cancer Research Newcastle University August 2015 Acknowledgements I would like to acknowledge my three supervisors, Dr M. Robinson, Dr R. Kist, and Professor P. Sloan, without whom this project would not have been possible. Dr Kist and Dr Robinson shared the initial vision for this project and, in collaboration, designed and developed its overall structure. The project therefore arose from a unique collaboration between principle investigators with backgrounds in developmental oral biology and oral pathology. Professor Sloan’s experience and insight have proved invaluable to the analysis, interpretation, and presentation of our data. I would like to thank Miss A. Long and all members of the Research and Development Team, Department of Cellular Pathology, for their essential contribution to the processing, sectioning, and automated immunostaining of both human and mouse tissue during this project. Dr R. Mahsoub and Miss V. Rook contributed to the manual immunohistochemical staining of sections during this project, work which formed part of their Masters by Research projects. Mr M. Kennedy played a vital role in collecting clinical data for the group of early-stage oral squamous cell carcinoma patients, during a Master of Science project that he undertook at the University of Edinburgh. I would like to thank Miss M. Elias and all the team in the Dermatology Laboratory, Institute of Cellular Medicine. The generation of the tetracycline- inducible cell lines would not have been possible without the support of Martina and others in the Dermatology team: Carol, Keith, and Emma, to name just a few. It was a privilege to work with this group, whose commitment to and passion for science is an inspiration. Finally, I would like to thank my parents, Mr and Mrs C.G. Bates, for their patience, wisdom, and support, not only during this project, but throughout my career to date. I Table of Contents Chapter 1. General Introduction .................................................................. 1 1.1 Cancer ................................................................................................... 1 1.2 Oral cancer ............................................................................................ 1 1.2.1 Epidemiology ................................................................................... 1 1.2.2 Histological classification of oral cancer .......................................... 2 1.2.3 Tobacco and alcohol: major environmental risk factors for OSCC .. 3 1.2.4 High-risk human papillomavirus infection in oral cancer and oro- pharyngeal cancer ....................................................................................... 4 1.2.5 The contribution of other infective conditions to OSCC formation ... 5 1.2.6 Histopathological staging of OSCC ................................................. 5 1.2.7 Treatment modalities ....................................................................... 7 1.2.8 Clinical outcomes for patients with OSCC ....................................... 7 1.2.9 Second primary tumours and the phenomenon of ‘field cancerisation’ ............................................................................................... 8 1.2.10 Oral potentially malignant disorders ........................................... 10 1.2.11 Genetic and molecular alterations in oral cancer ....................... 11 1.3 Epidermal growth factor receptor ......................................................... 14 1.3.1 Structure and biological function ................................................... 14 1.3.2 EGFR as a cancer biomarker ........................................................ 14 1.3.3 EGFR in oral potentially malignant disorders ................................ 15 1.4 The SOX gene family ........................................................................... 16 1.4.1 SOX2 in development ................................................................... 16 II 1.4.2 SOX2 in cancer ............................................................................. 17 1.4.3 SOX2 in oral cancer ...................................................................... 18 1.5 The PAX gene family ........................................................................... 19 1.5.1 PAX9 in development and disease................................................ 20 1.5.2 A potential role for PAX9 in cancer................................................ 22 1.6 Animal models ..................................................................................... 23 1.6.1 Chemical induction of OSCC in animal models ............................. 24 1.6.2 4-Nitroquinoline 1-oxide (4-NQO) .................................................. 24 1.6.3 4-NQO method of action ............................................................... 25 1.6.4 Experimental protocols for 4-NQO induction of carcinogenesis in mouse models ........................................................................................... 26 1.6.5 The 4-NQO model and chemotherapy .......................................... 27 1.6.6 Genetic induction of OSCC in mouse models ............................... 28 1.6.7 Investigating a potential tumour-suppressor role of Pax9 in OSCC .. ...................................................................................................... 29 1.6.8 Normal lingual microanatomy of the Pax9-deficient mouse ........... 29 1.7 Oral keratinocyte cell lines ................................................................... 32 1.7.1 Primary culture of normal oral keratinocytes ................................. 32 1.7.2 OSCC cell lines ............................................................................. 33 1.8 Conclusion ........................................................................................... 34 1.9 Hypothesis ........................................................................................... 34 1.10 Aims ................................................................................................. 34 III Chapter 2. Materials and Methods ............................................................ 35 2.1 Human tissue samples: oral potentially malignant disorders ............... 35 2.1.1 Attendance at an OPMD clinic ...................................................... 35 2.1.2 Electronic database of the Cellular Pathology Department ........... 35 2.1.3 Histopathological specimens ......................................................... 36 2.1.4 Histological grading of epithelial dysplasia .................................... 36 2.1.5 Clinical outcomes .......................................................................... 36 2.1.6 Data collection and management .................................................. 37 2.2 Human tissue samples: early-stage OSCC .......................................... 38 2.2.1 Patients ......................................................................................... 38 2.2.2 Histopathological specimens ......................................................... 39 2.2.3 Data collection and management .................................................. 39 2.3 Human tissue samples: OSCC that transformed from OPMD ............. 39 2.4 Immunohistochemical staining and analysis ........................................ 40 2.4.1 Automated immunohistochemistry ................................................ 40 2.4.2 Manual immunohistochemistry protocol ........................................ 40 2.4.3 Digital image analysis of immunohistochemical staining ............... 43 2.5 In situ hybridisation for EGFR chromosome 7 ..................................... 45 2.5.1 Automated staining ........................................................................ 45 2.5.2 Scoring and interpretation of ISH-stained sections........................ 45 2.6 Mouse model of oral carcinogenesis ................................................... 47 2.6.1 Experimental outline ...................................................................... 47 2.6.2 Preparation of 4-NQO solutions .................................................... 48 IV 2.6.3 4-NQO administration .................................................................... 48 2.6.4 Health and safety measures during the handling of 4-NQO .......... 49 2.6.5 Cage cleaning and bedding changes ............................................ 49 2.6.6 Monitoring of mice during exposure to 4-NQO .............................. 49 2.6.7 Sacrifice for macroscopical and histological analysis .................... 50 2.6.8 Autopsy procedures ...................................................................... 50 2.6.9 Fixation and cut-up ........................................................................ 51 2.6.10 Processing, embedding, and sectioning .................................... 51 2.7 Cell Culture Techniques ....................................................................... 52 2.7.1 Maintenance of oral squamous cell carcinoma cell lines ............... 52 2.7.2 Subculture and cell number determination .................................... 52 2.7.3 Long-term cell storage ................................................................... 53 2.7.4 Retrieval of cells for culture ........................................................... 53 2.7.5 Mycoplasma testing ......................................................................