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Role of Magi Proteins in Invasiveness of Human

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Role of Magi Proteins in Invasiveness of Human ROLE OF MAGI PROTEINS IN INVASIVENESS OF HUMAN COLORECTAL CANCER CELLS Zubair Khanzada 1370322 Doctor of Medicine (MD) thesis Supervisors: Dr Tracey Martin, Miss Rachel Hargest, Prof Wen Jiang Cardiff China Medical Research Collaborative (CCMRC) Cardiff University. Declaration I hereby declare that this thesis has been composed by myself. I confirm that the work submitted is my own. The work has not been submitted for any other degree or professional qualification. I confirm that appropriate credit has been given within this thesis where reference has been made to the work of others. Zubair Khanzada 2 Acknowledgement My sincere thanks to Dr Martin, Miss Hargest, Prof Jiang and every single person at the Cardiff China Medical Research Collaborative (CCMRC), Cardiff University who has helped me to make it possible. My special gratitude to my parents, my wife and my two daughters, my brothers and my friends, for their constant support and encouragement. 3 Abstract Introduction: MAGI -1 plays an essential role in cancer metastasis. We aimed to study the expression of MAGI -1, -2 and -3 in colorectal cancer tissue with specific focus on the role of MAGI -1 in colorectal cancer metastasis, through its function in the maintenance of cell-to-cell tight junctions. Materials and Methods: MAGI -1, -2 and -3, expression was determined in colorectal cancer tissue by using Q-PCR and RT-PCR. MAGI -1 knockdowns were created in cancer cell lines by electroporation method and were confirmed on RT- PCR, morphology and immunofluorescence. The impact of aberrantly expressed MAGI -1 on adhesion, invasion and migration of colorectal cancer tissue was studied by using trans-epithelial resistance (TER) and electrical cell impedance (ECIS) assays. Results: Q-PCR was consistent with the reduced expression of MAGI-1, -2 and -3 in colorectal cancer tissue. Survival curves showed reduced survival in cohort with reduced MAGI expression. MAGI -2 lacked expression on RT-PCR. MAGI -1 KDs showed decrease trans-epithelial resistance in the KDs. Reduced electrical resistance across epithelial cell lines in MAGI -1 KDs and also reduced adhesion and increase in migratory function was observed in MAGI -1 KDs on Trans-epithelial resistance (TER) and electronic cell impedance sensing (ECIS) experiments respectively. Conclusion: Our experimental work suggests that MAGI-1 plays a vital role in maintaining the barrier function of tight-junctions in colorectal cancer tissue, which when knocked down, has a significant implication in colorectal cancer metastasis. 4 Contents 1.1. Introduction ....................................................................................................18 1.1.1. Risk factors ......................................................................................................19 1.1.2. Regional variations ..........................................................................................20 1.1.3. WHO action plan for cancer prevention and control .......................................21 1.1.4. Cancer incidence in United Kingdom ...............................................................22 1.1.5. Bowel Cancer...................................................................................................23 1.2. Inherited bowel cancer ....................................................................................25 1.4. Anatomy and physiology of the colon and rectum ..........................................29 1.5. Natural history of the disease .........................................................................35 1.6. Presentation ....................................................................................................35 1.7. National institute for health care and excellence (NICE) – suspected cancer referral pathway ..........................................................................................................36 1.8. UK department of health criteria for high risk and low risk colorectal cancer .37 1.9. Diagnostic investigations .................................................................................37 1.10. Staging of colorectal cancer .............................................................................39 1.11. Management of colorectal cancer ...................................................................41 1.11.1. Patients with resectable primary rectal cancer ...............................................42 1.11.2. Patients with high risk rectal cancer ................................................................42 1.11.3. Role of colonic stents in acute large bowel obstruction ..................................43 1.11.4. Patients with stage 1 colorectal cancer ...........................................................43 1.11.5. Surgical Resection of colorectal cancer............................................................44 1.11.6. Adjuvant chemotherapy in patients with colorectal cancer ............................45 1.11.7. Management of metastatic disease ................................................................46 1.11.8. Imaging hepatic and extra hepatic metastasis.................................................46 1.11.9. Ongoing care ...................................................................................................46 1.12. Mechanism of spread ......................................................................................47 5 1.12.1. Reduction in cell to cell adhesion ....................................................................47 1.12.2. Tumour cell motility ........................................................................................47 1.12.3. Angiogenesis ...................................................................................................47 1.12.4. Vascular invasion.............................................................................................48 1.12.5. Establishment of a new colony ........................................................................48 1.13. Pathophysiology of metastasis ........................................................................49 1.14. Tight junctions .................................................................................................51 1.14.1. Structure of TJs: ...............................................................................................52 1.14.2. Functions of TJs ...............................................................................................57 1.14.3. Tight junctions in colorectal cancer cells .........................................................58 1.15. MAGI proteins .................................................................................................58 1.15.1. MAGI -1 ...........................................................................................................60 1.15.2. MAGI -2 ...........................................................................................................62 1.15.3. MAGI -3 ...........................................................................................................63 1.15.4. MAGI proteins in colorectal cancer .................................................................64 1.15.5. MAGI associated proteins in colorectal cancer ................................................65 1.16. Hypothesis and aims of study ..........................................................................66 2.1.1. Materials .........................................................................................................69 2.1.2. Culture mediums, buffers and standard solutions ...........................................69 2.1.3. 0.05M EDTA .....................................................................................................69 2.1.4. Trypsin.............................................................................................................69 2.1.5. Balanced salt solution (BSS) ............................................................................69 2.1.6. Antibiotic solution ...........................................................................................70 2.1.7. 2.1.1.5. Normal culture medium......................................................................70 2.1.8. Selection culture medium................................................................................71 2.1.9. Maintenance culture medium .........................................................................71 2.1.10. DPC water........................................................................................................71 2.1.11. PCR water ........................................................................................................71 2.1.12. T5X Tris, Boric acid, ETDA (TBE) .......................................................................71 6 2.1.13. RNA-ase free water .........................................................................................72 2.1.14. Master mix ......................................................................................................72 2.1.15. RT mix .............................................................................................................72 2.1.16. DNA ladder ......................................................................................................72 2.1.17. Electrophoresis gel ..........................................................................................72 2.1.18. Syber safe ........................................................................................................72 2.1.19. TBS
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