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An Investigation Into the Function of WFDC2

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An Investigation Into the Function of WFDC2 An investigation into the function of WFDC2 Hannah Armes A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy School of Clinical Dentistry The University of Sheffield January 2019 (This page is left blank intentionally) i Acknowledgments First and foremost, I would like to thank my supervisor Dr Lynne Bingle for her endless advice, encouragement and kind words. I have learnt so much from her extensive knowledge and experience. Under Lynne’s supervision I have become a confident and independent researcher and for that I cannot thank her enough. I would also like to thank Professor Colin Bingle for his support throughout both my PhD and my undergraduate degree. His guidance and encouragement made me realise that a career in science was something achievable for me. I would like to give a huge thank you to the technical staff who helped me to make so much progress in my three years at the School of Clinical Dentistry: the wonderful Brenka McCabe and Jason Heath for teaching me molecular biology and microbiology techniques and to Hayley Stanhope for teaching me histological techniques and processing my tissue samples. I would also like to thank Jessica-Leigh Tallis for being an excellent SURE scheme student and for generating some brilliant immunohistochemistry analysis. Thank you to the Bingle group for their support and kind words over the years (Apoorva, Priyanka, Renata, Debora, Chloe, Zulaiha and Esra). A huge thank you to my friends in the 3rd floor PGR office for making me laugh constantly for 3 years, even when faced with lab work disappointments: Amy Harding, Sven Niklander, Marianne Satur and Alex Bolger. And finally, thank you to my lovely family and Chris, for their constant, unwavering love and encouragement. I could not have done this without you! ii Abstract Introduction Whey-acidic-protein (WAP) four-disulfide core domain protein 2 (WFDC2) is a small, secretory glycoprotein that is characterised by possession of two cysteine-rich protein domains. In healthy tissues, WFDC2 is most abundantly expressed in the trachea and oral cavity, yet its function at these sites is unknown. Structural similarities to other family members suggests that WFDC2 may play a role in host defence, thus antimicrobial and anti-protease functions have been hypothesised. WFDC2 expression is exacerbated in ovarian cancer and as a result it is used clinically as a serum biomarker. WFDC2 is also reported to be upregulated in other malignancies, including lung cancer, in addition to benign diseases such as cystic fibrosis and renal fibrosis. It is unclear whether WFDC2 mediates tumorigenic effects. The aim of this study was to determine the function of WFDC2, with the primary objective of understanding whether it is involved in tumour progression. Materials and methods Recombinant human and murine WFDC2 were synthesised by gene cloning and transfection into HEK293 cells. WFDC2 protein was purified from conditioned media and utilised for protease inhibition and bacteria assays. The N-glycosylation site of human WFDC2 was elucidated via site-directed mutagenesis and enzymatic cleavage. The WFDC2 expression of lung and oral cancer-derived cell lines was analysed by RT-qPCR, ELISA and Western blotting. An oral cancer cell line was utilised for CRISPR gene editing to silence endogenous WFDC2 gene expression. Successful gene silencing was confirmed via sequencing, RT-PCR, RT-qPCR, Western blotting and ELISA. Changes in cell behaviour between wild-type and CRISPR edited cells were analysed via standard in vitro cell behaviour assays. WT and heterozygous Wfdc2-knockout mice were studied via H&E staining and iii immunohistochemistry. Micro-CT scans of WT and homozygous knockout mice embryos at E14.5 and E18.5 were compared to elucidate differences in phenotype. Results Recombinant human and murine WFDC2 were unable to inhibit the growth of any of the bacterial strains tested, nor were they able to inhibit protease activity. CRISPR edited cells showed a significant reduction in their capacity to invade Matrigel-coated Transwells compared to controls. Homozygous Wfdc2-knockout mice died shortly after birth as a result of respiratory distress while heterozygous animals survived to adulthood and had no obvious histological phenotype. Micro-CT analysis showed that the trachea and bronchi of homozygote embryos were constricted at E14.5 and E18.5. Conclusions WFDC2 is not a host defence protein and is instead involved in development of the tracheal and bronchial lumina during embryogenesis. WFDC2 is involved in tumorigenesis by promoting cancer cell invasion. This suggests that WFDC2 is a prognostic biomarker and could represent an interesting therapeutic target. The role of WFDC2 in development requires further analysis. iv Contents Acknowledgements……………………………………………………………………….……..… ii Abstract……………………………………………………………………………………………... iii Contents……………………………………………………………………………………………... v List of figures……………………………………………………………………………...………. xii List of tables……………………………………………………………………………………… xvii List of abbreviations…………………………………………………………………………… xviii 1. Literature review……………………………………………………………………………….. 1 1.1. An introduction to WFDC2……………………………………………………………… 1 1.1.1. The WFDC2 gene………………………………………………………………….. 2 1.1.2. The WFDC2 splice variants……………………………………………………….. 2 1.1.3. The WFDC protein family………………………………………………………….. 4 1.1.4. The WFDC domain…………………………………………………………………. 5 1.1.5. Glycosylation………………………………………………………………..……… 7 1.1.6. The WFDC locus…………………………………………………………………… 8 1.2. WFDC2 expression……………………………………………………………………. 10 1.2.1. The expression profile of human WFDC2……………………………...……….. 10 1.2.2. Expression levels of WFDC2 splice variants…………………………………… 14 1.2.3. WFDC2 orthologues……………………………………………………………… 14 1.2.4. Rodent Wfdc2……………………………………………………………………... 15 1.2.5. Human WFDC proteins in secretions………………………………………….... 16 1.3. Activities of WFDC proteins…………………………………………………………… 19 1.3.1. The putative functions of human WFDC2………………………………………. 19 1.3.2. Antimicrobial activity of SLPI and elafin…………………………………………. 19 1.3.3. Putative antimicrobial activity of WFDC2……………………………………….. 24 1.3.4. Induction of expression of WFDC proteins……………………………………… 26 v 1.3.5. Anti-protease activity in the WFDC family………………………………………. 28 1.4. WFDC2 and cancer……………………………………………………………………. 31 1.4.1. WFDC2 as a biomarker for ovarian cancer……………………………………... 31 1.4.2. WFDC2 as a biomarker for other cancer types………………………………… 33 1.4.3. Amplification of the 20q13 locus…………………………………………………. 38 1.4.4. WFDC2 and metaplasia………………………………………………………….. 39 1.4.5. WFDC2 and cancer cell behaviours…………………………………………….. 39 1.5. WFDC2 and benign diseases………………………………………………………… 41 1.5.1. WFDC2 and benign lung diseases……………………………………………… 41 1.5.2. WFDC2 and renal fibrosis………………………………………………………... 43 1.6. Hypothesis and aims…………………………………………………...……………… 45 2. Materials and methods………………………………………………………………………. 46 2.1. Cell culture……………………………………………………………………………...… 46 2.1.1. Culture media……………………………………………………………………… 47 2.1.2. Collection of conditioned media…………………………………………………. 47 2.1.3. Harvesting cells………………………………………………………...…………. 47 2.2. DNA analysis…………………………………………………………………………...… 48 2.2.1. RNA extraction………………………………………………………………...….. 48 2.2.2. Reverse transcription………………………………………………………..…… 48 2.2.3. Polymerase chain reaction………………………………………………………. 48 2.2.4. DNA agarose gel electrophoresis……………………………………………….. 49 2.2.5. Quantitative polymerase chain reaction………………………………………… 49 2.3. Protein analysis…………………………………………………………………………... 50 2.3.1. Human and mouse samples……………………………………………………... 50 2.3.2. Antibodies………………………………………………………………………..... 51 2.3.3. BCA assay…………………………………………………………………………. 51 2.3.4. Western blotting…………………………………………………………………… 52 2.3.5. Densitometry…………………………………………………………...…………. 52 vi 2.3.6. SDS PAGE analysis………………………………………………………………. 53 2.3.7. Native PAGE analysis……………………..…………………….……………….. 53 2.3.8. Enzyme-linked immunosorbent assay……….…………………………………. 53 2.4. Synthesis of recombinant WFDC2…………………………………………...………… 53 2.4.1. Primer design and polymerase chain reaction………………………………… 53 2.4.2. TOPO TA subcloning……………………………………………….…………….. 54 2.4.3. Isolation of insert DNA……………………………………………………………. 55 2.4.4. Vector preparation………………………………………………………………… 55 2.4.5. Ligation…………………………………………………………………………….. 56 2.4.6. Transformation……………………………………………………………………. 56 2.4.7. Plasmid isolation ‘miniprep’……………………………………………………… 57 2.4.8. Plasmid isolation ‘maxiprep’……………………………………………………... 57 2.4.9. Confirmation of target insertion………………………………………………….. 57 2.4.10. Sequencing of clones…………………………………………………………….. 57 2.4.11. FuGENE transfection…………………………………………………………….. 57 2.4.12. Calcium phosphate transfection…………………………………………………. 58 2.4.13. Anti-FLAG M2 affinity gel purification…………………………………………… 59 2.4.14. Immunofluorescence microscopy……………………………………………….. 59 2.5. Glycosylation analysis…………………………………………………………………… 61 2.5.1. Site-directed mutagenesis……………………………………………………….. 61 2.5.2. PNGase F enzyme treatment……………………………………………………. 62 2.5.3. Sialidase enzyme treatment……………………………...……………………… 62 2.6. Protease inhibition assays………………………………………………………………. 62 2.6.1. Enzyme optimisation for protease inhibition assay……………………………. 62 2.6.2. Serine protease inhibition assay……………...…………………………………. 63 2.6.3. Zymography……………………………………………………………………….. 63 2.7. Bacteria assays………………………………………………………………………...… 64 2.7.1. Bacterial strains…………………………………………………………………… 64 vii 2.7.2. Bacterial culture…………………………………………………………………… 64 2.7.3. Colony
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