The IgLON Family in Ovarian Cancer: Functional Characterisation of LSAMP and NEGR1 in Comparison with the Tumour Suppressor OPCML Imperial College London, Department of Surgery and Cancer Thesis submitted by Sarah Christen for the degree of Doctor of Philosophy October 2012 1 PhD Thesis – Sarah Christen – Imperial College London Abstract I. Abstract Out of all gynaecological cancers, epithelial ovarian cancer (EOC), a mainly sporadically occurring disease affecting postmenopausal women, exhibits the highest fatality-to-case ratio. Further research is therefore urgently required to combat the disease. In previous studies, our group identified the IgLON protein OPCML to be an important, frequently epigenetically inactivated (>80%) tumour suppressor in EOC. On the molecular level, OPCML functions by directly interacting with and subsequently endocytotically downregulating a broad repertoire of receptor tyrosine kinases (RTKs). During this PhD project, the aim was to determine if LSAMP and NEGR1, two further IgLON family members, function in a similar manner. Bioinformatic approaches as well as in vitro experiments were used to address issues such as sequence similarity, colocalisation or interaction; furthermore, overexpression and knockdown clones were generated to determine the impact of IgLON presence/absence on cellular characteristics such as RTK expression, proliferation or migration. While LSAMP was found to potentially function in a similar manner as OPCML in the ovarian cancer cell line SKOV3 by reducing RTK expression, proliferation and colony formation, its functions in the ovarian epithelial surface cell line OSE-C2 were less clear and require further validation. For NEGR1, a striking downregulation of RTKs, including those which had been unaffected by OPCML in previous experiments – such as EGFR, HER3 or FGFR2 – was observed in transfected SKOV3 cells. Downregulation seemed to occur both via lysosomal and proteasomal pathways, and resulted in substantial reduction of cell proliferation and colony formation. Overall, these results suggest that the IgLON family could function as a broad regulator of RTK expression and function. Since IgLONs are extracellular proteins, they represent excellent subjects for drug development as no intracellular delivery or gene therapy is necessary. To our knowledge, this is the first report investigating the functional properties of LSAMP and NEGR1 in epithelial ovarian cancer. 2 PhD Thesis – Sarah Christen – Imperial College London Table of Contents II. Table of Contents I. Abstract .................................................................................................................... 2 II. Table of Contents ...................................................................................................... 3 III. List of Figures ............................................................................................................ 8 IV. List of Tables ........................................................................................................... 10 V. List of Frequently Used Abbreviations ..................................................................... 11 VI. Declaration of Originality ........................................................................................ 14 VII. Acknowledgements ............................................................................................. 15 Chapter 1: Introduction .................................................................................................. 19 1.1) Introduction............................................................................................................... 20 1.2) Cancer ........................................................................................................................ 20 1.2.1) Prevalence and Characteristics ........................................................................... 20 1.2.1.1) Classification ................................................................................................ 22 1.2.2) Causes and Prevention ....................................................................................... 23 1.2.2.1) Lifestyle Factors ........................................................................................... 23 1.2.2.2) Environmental Factors ................................................................................. 24 1.2.2.3) Infections ..................................................................................................... 25 1.2.2.4) Inherited Factors ......................................................................................... 26 1.2.3) Molecular Background ........................................................................................ 26 1.2.3.1) Oncogenes ................................................................................................... 27 1.2.3.2) Tumour Suppressor Genes .......................................................................... 27 1.2.3.3) Heredity of Oncogenes and Tumour Suppressor Genes ............................. 28 1.2.4) Development and Progression ........................................................................... 29 1.2.5) Treatment and Prognosis ................................................................................... 30 1.3) Ovarian Cancer .......................................................................................................... 32 1.3.1) Anatomy and Function of the Ovaries ................................................................ 32 1.3.2) Prevalence and Characteristics of Ovarian Cancer ............................................. 33 1.3.2.1) Classification ................................................................................................ 35 1.3.3) Causes and Prevention ....................................................................................... 37 1.3.4) Molecular Background ........................................................................................ 39 1.3.5) Development and Progression ........................................................................... 40 1.3.5.1) Origin of Epithelial Ovarian Cancer ............................................................. 41 1.3.5.2) Stages ........................................................................................................... 42 1.3.5.3) Grades .......................................................................................................... 43 1.3.6) Screening and Diagnosis ..................................................................................... 44 1.3.7) Treatment and Prognosis ................................................................................... 46 1.3.7.1) Surgery ......................................................................................................... 46 1.3.7.2) Chemotherapy ............................................................................................. 47 1.3.7.3) Alternative Approaches ............................................................................... 48 1.3.7.4) Relapse, Second-Line Treatment and Drug Resistance ............................... 49 1.3.7.5) Prognosis ..................................................................................................... 50 1.3.8) Outlook ............................................................................................................... 52 3 PhD Thesis – Sarah Christen – Imperial College London Table of Contents 1.4) IgLONs ....................................................................................................................... 53 1.4.1) Protein Structure ................................................................................................ 56 1.4.2) Expression ........................................................................................................... 58 1.4.3) Functions and Interactions ................................................................................. 60 1.4.4) Localisation in Lipid Rafts ................................................................................... 62 1.4.5) Pathological Implications .................................................................................... 63 1.4.5.1) IgCAMs and IgLONs in Cancer...................................................................... 64 1.5) Receptor tyrosine kinases (RTKs) .............................................................................. 69 1.5.1) Structure and Function ....................................................................................... 69 1.5.2) Regulation ........................................................................................................... 70 1.5.3) Selected RTK Families in Ovarian Cancer ........................................................... 72 1.5.3.1) RTK-targeting Drugs ..................................................................................... 73 1.6) The PhD Project ......................................................................................................... 75 1.6.1) Previous findings: OPCML and other IgLONs in EOC .......................................... 75 1.6.2) Hypothesis and Aims .......................................................................................... 81 Chapter 2: Materials and Methods.................................................................................. 82 2.1) Bioinformatics ..........................................................................................................