University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 2016 Role of Thrombin-Activatable Fibrinolysis Inhibitor in Breast Cancer Metastasis and Angiogenesis Zainab Ali Bazzi University of Windsor Follow this and additional works at: https://scholar.uwindsor.ca/etd Recommended Citation Bazzi, Zainab Ali, "Role of Thrombin-Activatable Fibrinolysis Inhibitor in Breast Cancer Metastasis and Angiogenesis" (2016). Electronic Theses and Dissertations. 5800. https://scholar.uwindsor.ca/etd/5800 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered. Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email ([email protected]) or by telephone at 519-253-3000ext. 3208. Role of Thrombin-Activatable Fibrinolysis Inhibitor in Breast Cancer Metastasis and Angiogenesis by Zainab A. Bazzi A Dissertation Submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Windsor Windsor, Ontario, Canada 2016 © 2016 Zainab A. Bazzi Role of Thrombin-Activatable Fibrinolysis Inhibitor in Breast Cancer Metastasis and Angiogenesis by Zainab Bazzi APPROVED BY: __________________________________________________ I. Podgorski, External Examiner Wayne State University __________________________________________________ Lisa Porter Department of Biological Sciences __________________________________________________ M. Koschinsky Department of Chemistry & Biochemistry __________________________________________________ S. Pandey Department of Chemistry & Biochemistry __________________________________________________ M. Boffa, Advisor Department of Chemistry & Biochemistry September 19, 2016 DECLARATION OF CO-AUTHORSHIP AND PREVIOUS PUBLICATION I hereby declare that this dissertation incorporates material that is result of joint research, as follows: This dissertation incorporates the outcome of joint research in collaboration with Jennifer Balun, Danielle Lanoue, Mouhanned El-Youssef, Rocco Romagnuolo, Janice Tubman, Dr. Lisa Porter and Dr. Cavallo-Medved under the supervision of Dr. Michael Boffa. In all cases, key ideas, primary contributions, experimental design, execution, data analysis, and interpretation were performed by the author (with exceptions listed below). Additionally, manuscripts were written initially by the author and revised and edited by Dr. Michael Boffa, Dr. Lisa Porter and Dr. Cavallo-Medved for Chapter 2, Dr. Michael Boffa, Jennifer Balun, Dr. Lisa Porter and Dr. Cavallo-Medved for Chapter 3 and revised and edited by Dr. Michael Boffa, for Chapter 4. Collaboration with Jennifer Balun is covered in Chapters 3 and 4 of this dissertation. For Chapter 3, Jennifer Balun performed experiments and data analysis for the following: cell invasion, tube formation, proteolysis and zymography experiments with PTCI and VEGF. For Chapter 4, Jennifer performed experiments and data analysis for the following: tube formation and proteolysis experiments with endothelial cells and co-culture experiments with endothelial cells and breast cancer cells. Collaboration with Danielle Lanoue is covered in Chapter 2 and Chapter 4 of this dissertation. For Chapter 2, Danielle Lanoue performed invasion and migration experiments with TAFI-CIIYQ. For Chapter 4, Danielle Lanoue assisted with molecular cloning of TM EGF3-6 mutants. Collaboration with Mouhanned El-Youssef is covered in Chapter 2 of this dissertation; this was through contribution of data analysis for proteolysis assays. Collaboration with Rocco Romagnuolo is covered in Chapter 2 of this dissertation; this was through intellectual contribution and experimental design for plasminogen activation assays. iii Collaboration with Janice Tubman is covered in Chapter 2 of this dissertation; this was through sample preparation. Collaboration with Dr. Lisa Porter is covered in Chapters 2, 3 and 4 of this dissertation; this was through intellectual contribution and joint funding. Collaboration with Dr. Cavallo-Medved is covered in Chapters 2, 3 and 4 of this dissertation; this was through intellectual contribution and joint funding. I am aware of the University of Windsor Senate Policy on Authorship and I certify that I have properly acknowledged the contribution of other researchers to my dissertation, and have obtained written permission from each of the co-author(s) to include the above material(s) in my dissertation. I certify that, with the above qualification, this dissertation, and the research to which it refers, is the product of my own work. This dissertation includes 3 original manuscripts that have been previously published, submitted, or are in preparation for publication in peer reviewed journals, as follows: Dissertation Publication Title and Full Citation Publication Chapter Status Chapter 2 Bazzi ZA, Lanoue D, El-Youssef M, Romagnuolo R, Published Tubman J, Cavallo-Medved D, Porter LA, Boffa MB. Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis. BMC Cancer. 2016 May 24;16(1):328. Chapter 3 Bazzi ZA, Balun, J, Cavallo-Medved D, Porter LA, Boffa Submitted MB. Activated thrombin-activatable fibrinolysis inhibitor attenuates the angiogenic potential of endothelial cells: Potential relevance to the breast tumour microenvironment. Clinical & Experimental Metastasis. CLIN-D-16-00098 iv Chapter 4 Bazzi ZA, Balun J, Lanoue D, Cavallo-Medved D, Porter In LA Boffa MB. Thrombomodulin as a cofactor for TAFI preparation and protein C activation: role in breast cancer metastatic behaviours and angiogenic potential. I certify that I have obtained a written permission from the copyright owner(s) to include the above published material(s) in my dissertation. I certify that the above material describes work completed during my registration as graduate student at the University of Windsor. I declare that, to the best of my knowledge, my dissertation does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my dissertation, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I have obtained a written permission from the copyright owner(s) to include such material(s) in my dissertation. I declare that this is a true copy of my dissertation, including any final revisions, as approved by my dissertation committee and the Graduate Studies office, and that this dissertation has not been submitted for a higher degree to any other University or Institution. v ABSTRACT Cancer metastasis is an important process in cancer progression. This process is facilitated by proteases that promote degradation of the extracellular matrix, cell migration and cell invasion. Angiogenesis is important to the metastatic process and also involves extracellular matrix degradation, endothelial cell migration and invasion. Proteases such as plasmin and matrix metalloproteinases are responsible for degradation of the extracellular matrix, facilitating cancer cell invasion and subsequent metastasis. These proteases are also important in promoting tumour angiogenesis. Therefore, developing methods to target these proteases may effectively inhibit cancer metastasis and tumour angiogenesis. TAFI is a plasma zymogen initially known for its role in attenuating fibrinolysis. TAFIa is formed through cleavage by thrombin, plasmin or, by thrombin in complex with thrombomodulin. TAFIa is a carboxypeptidase, cleaving carboxyl terminal lysine residues from plasminogen binding sites on cell surface receptors, which are important in accelerating plasminogen activation to plasmin. We have demonstrated that TAFIa is able to inhibit metastatic behaviours, including breast cancer cell invasion, migration and collagen degradation. We have shown that TAFIa inhibits these metastatic behaviours through attenuation of plasminogen activation to plasmin. Additionally, we have demonstrated that TAFIa also acts as an anti-angiogenic factor, inhibiting endothelial cell invasion, migration, tube formation and collagen degradation. We have also shown that these effects are mediated by the ability of TAFIa to inhibit plasminogen activation on the endothelial cell surface. Taken together these results indicate that TAFIa is an anti-metastatic and anti-angiogenic factor that may represent a novel therapeutic strategy to target cancer metastasis. vi DEDICATION To the loved ones I lost during my time as a graduate student, my grandmothers Fatima