Hollenberg, Morley D.Hyndman, Matthew Ericde Lima, Stacy Grace2021-01-282021-01-282020-01-24de Lima, S. G. (2021). The Role and Regulation of Proteinase Activated Receptors (PARs) in Urothelial Carcinoma (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.http://hdl.handle.net/1880/113033Introduction: Urothelial carcinoma (UC) is the most common type of bladder cancer in the North American population. UC is the most expensive cancer to treat per patient owing to the absence of noninvasive diagnostic and prognostic tests complemented by ineffective treatments and high recurrence rates necessitating long-term follow-up. One phenomenon that has been well demonstrated in UC is that the expression of proteinases and their inhibitors changes in the UC tumor microenvironment as the tissue progresses from healthy urothelium to low grade and then to high grade tumors. This information has not advanced clinical care likely due to the complexity of the proteolytic cascade in both healthy and tumor microenvironments. Proteinase activated receptors (PAR) are specific cell surface receptors that integrate complex proteinase signals into downstream cellular signaling pathways that have been shown to promote survival, proliferation, migration and invasion in carcinomas, but have yet to be explored in UC. Working hypothesis: UC cells produce proteinases that drive an oncogenic (pro-migratory and invasive) phenotype through proteinase activated receptor (PAR) 1 and 2 induced cell signaling which changes with grade, stage and prognosis of disease. Methods: The hypothesis was tested in 5 bladder and UC-derived cell lines of increasing aggressiveness (PC3-420-010, RT4, TCCSUP, 5637, and T24) with the postulation cell aggressiveness (i.e. migration and invasion capacity) would correlated with proteinase and PAR expression and signaling sensitivity. In vitro testing included: PAR expression (qPCR, IHC), PAR signaling (calcium and western blotting for MAP kinase), PAR cleavage (using a variety of published techniques using transfected PAR-indicator constructs in the cell of interest as well as “bystander” responding cells), proteinase expression (proteomics) and activity (fluorescent substrates, PAR-cleavage modeling) testing complemented by organotypic urothelium-fibroblast modeling, as well as immunohistochemical profiling of the expression and cleavage status of PAR1 in patient tumour samples. Results: PAR 1, 2 are expressed by most cell lines tested, but PAR 3, and 4 are not. The expression and sensitivity of these receptors was found to be lowest in healthy urothelium and low-grade papilloma-derived cell lines, and highest in UC cells with increased capacity to migrate and invade. All cell lines produce proteinases and inhibitors, but only high-grade-derived cells produced proteinases that could cleave PAR1 in an autocrine manner. Agonism of PAR1 or PAR2 had little effect on proliferation, but enhanced migration, and invasion in the cell lines that already had this capacity. Only PAR1 antagonism not PAR2 was able to inhibit migration and invasion. Interestingly, in an organotypic model there seemed to be little effect on cellular behaviour (i.e. proliferation, migration, invasion) but robust effects on phenotype with PAR1 targeting upregulating cellular markers associated with a basal phenotype. Patient-derived invasive UC tissue was found to express PAR1. Conclusions: UC-produced proteinases act through PAR1 and promote migration and invasion in UC cell lines in vitro but not in an organotypic model which supported PAR agonism in initiating cell differentiation. UC patient tissue was found to express PAR1 but more data will be necessary to understand the implications of this expression.engUniversity of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.PAR, proteinase, proteinase activated receptor, urinary bladder cancer, urothelial carcinomaBiologyBiology--CellGeneticsBiophysics--MedicalMedicine and SurgeryPathologyPharmacologyEngineering--Biomedicaldoctoral thesis10.11575/PRISM/38600