SIPA1 As a Modulator of HGF/MET Induced Tumour Metastasis Via the Regulation of Tight Junction Based Cell to Cell Barrier
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SIPA1 as a modulator of HGF/MET induced tumour metastasis via the regulation of tight junction based cell to cell barrier By Chang Liu Cardiff China Medical Research Collaborative (CCMRC) School of Medicine, Cardiff University Cardiff April 2020 Thesis submitted to Cardiff University for the degree of Doctor of Philosophy Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed (candidate) Date 01/04/2020 STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of Ph.D. Signed (candidate) Date 01/04/2020 STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed (candidate) Date 01/04/2020 STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed (candidate) Date 01/04/2020 STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed (candidate) Date 01/04/2020 I Acknowledgements Firstly, I would like to thank my patient and gorgeous supervisors, Miss Rachel Hargest and Dr Tracey Martin, for your great support and guidance throughout my three-year study, your sincerely help made me become a better researcher. I would like to thank Professor Wen G. Jiang, and without you I couldn’t have the chance to study at Cardiff University and complete my project. Secondly, I am very grateful for all the current CCMRC members for their kindly help: Dr Lin Ye for your parental care and discussion on my career life; Dr Andrew Sanders for your delicate help at all times; Dr Yuxin Cui for your help on the my analysis of the online database; Fiona for v help on the IHC staining and all the efforts you did to make the lab working really enjoyable. And I also want to send my appreciation to the past CCMRC members: Juliet for the help in my application and enrolment; Dr Jun Cai for your discussion about science; Dr Moon Xie, my research role model; Dr Cathy Cai, the most delightful work partner. And I also would like to thank all the members in CCMRC who have been very nice, friendly and helpful all the time. Finally, I would like to express my heartfelt thanks to the most important people in my life, my parents, for your support and encouragement both in my academic career and my life. Thanks for my lifetime love, Dr Molly Xu, I can’t hold on for the three years without you on my back, I love you so much, my darling. And I would like to thank all my friends who helped and supported me. II Publications • C Liu, W G. Jiang, R Hargest, T A. Martin. 2019. SIPA1 is a modulator of HGF induced tumour metastasis via the regulation of tight junctions in lung adenocarcinoma cells. Annals of Oncology. Volume 30, Issue Supplement_5, mdz238.079. • C Liu, W G. Jiang, R Hargest, T A. Martin. 2019. SIPA1 in cancer and cancer metastasis. Molecular and Clinical Oncology. Accepted. • Z Sun, S Cai, C Liu, Y Cui, J Ji, W G. Jiang and L Ye. 2020. Increased expression of Gremlin1 promotes proliferation and epithelial mesenchymal transition in gastric cancer cells and correlates with poor prognosis of patients with gastric cancer. Cancer Genomics & Proteomics. 17: 49-60. • Z Sun, C Liu, W G. Jiang and L Ye. 2020. Deregulated bone morphogenetic proteins and their receptors are associated with disease progression of gastric cancer. Computational and Structural Biotechnology Journal. 18, 177-188 • J Liu, Z Zhao, Z Sun, C Liu, X Cheng, F Ruge, Y Yang, Wen G. Jiang and L Ye. 2020. Increased expression of Psoriasin is correlated with poor prognosis of bladder transitional cell carcinoma by promoting invasion and proliferation. Oncology reports. 43: 562-570. • Z Sun, S Cai, C Zabkiewicz, C Liu, L Ye. 2020. Bone morphogenetic proteins mediate crosstalk between cancer cells and the tumour microenvironment at primary tumours and metastases (Review). International Journal of Oncology. 56(6):1335-1351. doi: 10.3892/ijo.2020.5030. III Conference Attendance Oral presentation • Travelling Surgical Society of Great Britain and Ireland Annual Meeting, Cardiff, UK. In Sep. 2018. • Division of Cancer and Genetics PGR Day, University Hospital of Wales (UHW), Cardiff, UK. In Oct. 2018. • Surgical innovation - Royal Society of Medicine (RSM), King's College London, London, UK. In Sep. 2019. Poster presentation • European Society for Medical Oncology (ESMO) Congress 2019, Barcelona, Spain. In Sep. 2019. IV Abbreviations AF6 Afadin AIS Adenocarcinoma in situ AJs Adherens junctions Akt Protein kinase B ALK Anaplastic lymphoma kinase ANOVA Analysis of Variance APS Ammonium Persulfate AQP Aquaporin ASR Age-standardized rate ATCC American Tissue Culture Collection bp base pair BRAF B-Raf proto-oncogene Brd Bromodomain protein BSA Bovine Serum Albumin CaP Prostate cancer Cbl Casitas B-lineage lymphoma CCND Cyclin D CD Cluster of Differentiation cDNA Complementary DNA CDNA Circulation DNA CFC Change from control CIN85 Sh3 domain-containing kinase-binding protein 1 CLDN Claudin CSF-1 Colony-stimulating factor 1 CTCs Circulating tumour cells CTX Cholera toxin CXCL C-X-C motif ligand DAB Diaminobenzidine V DAPI 4’,6-diamidino-2-phenylindole del Deletion DEPC Diethylpyrocarbonate DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethylsuphoxide DNA Deoxyribonucleic acid DNMT DNA methyltransferases dNTPs Deoxynucleotide triphosphates E47 Transcription factor 3, TCF 3 ECIS Electric Cell-Substrate Impedance Sensing ECL Enhanced chemiluminescence ECM Extracellular Matrix EDTA Ethylenediaminetetracetic acid EGF Epidermal Growth Factor EGFR Epidermal Growth Factor Receptor eJM Extracellular juxtamembrane ELISA Enzyme-Linked Immunosorbent Assay EML4 Echinoderm microtubule-associated protein-like 4 EMT Epithelial to Mesenchymal Transition EP Etoposide and platinums ERK Extracellular Regulated Kinases ETS Environmental tobacco smoke EVOM Epithelial Volt/Ohm Meter FAK Focal Adhesion Kinase FBS Foetal bovine serum FITC Fluorescein isothiocyanate GAP GTPase activation Proteins GAPDH Glyceraldehyde 3-phosphate dehydrogenase GEF Guanine Nucleotide Exchange Factor VI GEO Gene Expression Omnibus GGA Golgi Associated Grb Proliferation factor receptor bound protein GRD Gap related domain GTP Guanosine Triphosphate HER2 Human epidermal growth factor receptor 2 HGF Hepatocyte Growth Factor HGFR Hepatocyte growth factor receptor HL Hairpin loop HRP Horseradish peroxidase IASLC International Association for The Study of Lung Cancer IF Immunofluorescent Ig Immunoglobulin IHC Immunohistochemical iJM Intracellular juxtamembrane ITGB1 Integrin beta 1 JAMs s Junctional Adhesion Molecule JNK Jun amino-terminal kinase kb Kilo base pairs KLF Kruppel like factor KRAS KRAS proto-oncogene LB Liquid Broth LCNEC Large cell neuroendocrine carcinoma LOX Lysyl oxidase LUSC Lung squamous cell carcinoma LUAD Lung adenocarcinoma LVSI Lymph vascular space invasion LZ Leucine zipper MAGI Membrane-associated guanylate kinase VII MAPK Mitogen-activated Protein Kinase MET MET Proto-oncogene MIA Minimally invasive adenocarcinoma MMP Matrix metalloproteinase mRNA Message RNA MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide NCCN National Comprehensive Cancer Network NGS Next Generation Sequencing technology NSCLC Non-small cell lung cancer nt Nucleotide ORF Open reading frame OS Overall survival OSCC Oral squamous cell carcinoma PBL Peripheral blood lymphocytes PBS Phosphate Buffer Saline PCP Paracellular Permeability PCR Polymerase Chain Reaction PD-1 Programmed death 1 PD-L1 Programmed death ligands 1 PD-L2 Programmed death ligands 2 PDZ PSD 95 Dig and ZO1 PEST Proline, glutamic acid, serine and threonine PFS Progression-free survival Phe Phenylalanine PI3K Phosphoinositide 3-Kinase PM Fine particular matter PTP1β Protein tyrosine phosphatase 1-beta qPCR Quantitative Polymerase Chain Reaction VIII Ran Ras related nuclear protein 1 Rap1 Ras proximate 1, Ras related protein 1 RET RET proto-oncogene RNA Ribonucleic Acid ROS1 ROS proto-oncogene 1 RPMI Roswell Park Memorial Institute rRNA Ribosomal RNA Rsr1 Ras related nuclear protein Rsr1 RT Reverse Transcriptase RTKs Tyrosine kinase receptors SCC Squamous cell carcinoma SCID Severe conbined immunodeficiency disease SCLC Small cell lung cancer SD Standard deviations SDS Sodium Dodecyl Sulphate SDS-PAGs Sodium dodecyl sulfate polyacrylamide gel electrophoresi SEM Standard Error of the Mean Ser Serine SF Scatter Factor SFM Serum Free Medium Sh2 Src homology 2 Sh3 Src homology 3 SIPA1 Signal induced proliferation-associated protein 1 SIPAL1 Signal induced proliferation-associated protein 1 like1 SNPs Single nucleotide polymorphisms SOCS suppressor of cytokine signalling SPH Serine protease homology Src Proto-oncogene tyrosine-protein kinase src STAT3 Signal transducer and activator of transcription-3 IX TAMP transmembrane adhesion membrane proteins TBE Tris-Boric-Acid TCDD 2,3,7,8-tetrachlorodibenzo-para-dioxin TEMED Tetramethylethylenediamine TER Transepithelial resistance TFs Transcription factors TGF-α Transforming growth factor-α TGF-β Transforming