The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4+ and CD8+ T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect.

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<p>Pancreatic ductal adenocarcinoma (PDAC) is an incredibly lethal disease with a 5-year survival rate of less than 8 percent in the United States due to a lack of viable treatment options. The failures of chemo- and radiotherapies have been linked to the heterogeneous nature of the tumor microenvironment which forms a hypovascular, immunosuppressive and high coagulation activity tissue. Indeed, PDAC patients have one of the highest rates of thrombosis complications among all cancer types. The expression of two key coagulation factors, Tissue Factor (TF) and Protease Activated Receptor 1 (PAR-1), have been associated with poor patient prognosis and aggressive cancer progression. However, the molecular roles/mechanisms of TF and PAR-1 in PDAC progression are not known. To establish how clotting factors (PAR-1, TF) influence PDAC tumor progression, I utilized a genetically modified mouse model (KPC) where <i>KRas^G12D</i> and <i>TRP53^R172H</i> mutations were specifically introduced into mouse pancreas acinar cells to initiate PDAC progression. Multiple primary mouse PDAC cell lines were generated and characterized. TF and PAR-1 were highly expressed in primary KPC pancreatic lesions, in PDAC tumors, and in KPC-derived cell lines, an expression profile that is also observed in PDAC patient biopsies. In allograft studies, tumor growth and metastatic potential were significantly diminished by shRNA reduction of TF or PAR-1 in cancer cells or by genetic or pharmacological reduction of the coagulation zymogen prothrombin in mice. Notably, PAR-1 deleted KPC cells (KPC-Par-1^KO) failed to generate sizable tumors; a phenotype completely rescued by restoration of PAR-1 expression. To test the significance of targeting PAR-1 in a clinical setting, PAR-1 expression was withdrawn from established tumors to mimic a potential inhibitory effect of PAR-1 on solid PDAC tumors. Removal of PAR-1 from tumors (11 days post injection) yielded a diverse effect on tumor growth which can be categorized into (i) a decline in tumor growth; (ii) continued tumor growth; and (iii) stagnant tumor growth. Immunohistochemistry analysis of KPC2 shCon vs. shPar-1 subcutaneous allograft tumor samples revealed a massive immune cell infiltration in KPC2 shPAR-1 tumors when compared to KPC2 shCon control tumors. Accordingly, KPC-Par-1^KO cells failed to form tumors in immune-competent mice but displayed robust tumor growth in immune-compromised <i>NSG</i> mice, providing the first evidence of a PAR-1 mediated tumor immune evasion pathway operating in PDAC. </p> <p>Together, these results demonstrate that PDAC disease is driven by activation of the coagulation system through tumor cell-derived TF, circulating prothrombin, and tumor cell-derived PAR-1. These studies also highlight a novel mechanism by which thrombin/PAR-1-mediated tumor growth involves suppression of anti-tumor immunity in the tumor microenvironment. <b></b></p>

Le dogme voulant que les récepteurs couplés aux protéines G (GPCRs) activent des voies de signalisation seulement lorsqu’ils sont localisés à la membrane plasmatique, a récemment été remis en question. Des données récentes indiquent que certains GPCRs peuvent également induire une réponse intracellulaire à partir des compartiments intracellulaires dont le noyau. Les récepteurs activés par la protéase (PAR) sont des membres de la famille GPCR. Les PARs sont activés par le clivage de la partie N–terminale du récepteur ce qui permet au ligand attaché sur le récepteur de se lier à sa poche réceptrice. Quatre PARs ont été décrits : PAR1, PAR2, PAR3 et PAR4. PAR2 peut susciter des effets mitogéniques et participer aux processus comme l’angiogenèse et l'inflammation. Alors que beaucoup d'effets intracellulaires de PAR2 peuvent être expliqués lorsqu’il est localisé à la membrane plasmatique, une fonction intracrine de PAR2 a aussi été proposée. Pourtant les mécanismes par lesquels PAR2 peut provoquer l’expression de gènes ciblés sont toujours inconnus. Le but de notre étude était de vérifier l’existence d’une population nucléaire de PAR2. Nous avons également émis l’hypothèse que les voies activées par l’activation de PAR2 dépendent de sa localization cellulaire. En utilisant des techniques de microscopie confocale et de "Western Blot" nous avons démontré la présence d’une population nucléaire de PAR2. À la suite de la stimulation de PAR2, nous avons observé une augmentation de la translocation du récepteur de la membrane plasmatique au noyau. En utilisant la technique de "RT – PCR", nous avons observé des rôles différents de PAR2 à la surface de la cellule et du noyau dans l’initiation de l’expression des gènes. Afin d’identifier les mécanismes responsables de la translocation nucléaire de PAR2, nous avons évalué l’implication des membres de la famille de "Sorting Nexins (SNX)" dans la translocation nucléaire de PAR2. "Sorting Nexins" est un groupe de protéines avec des fonctions de transport bien établies. SNX1 et SNX2 ont été identifiés comme responsables du transfert de PAR1 vers les lysosomes. SNX11 n'a pas encore été étudié et nous avons émis l’hypothèse qu'il pourrait être un autre membre de la famille des SNX impliqué dans la signalisation de PAR2. Pour ce faire, nous avons développé des "knockdowns" stables pour SNX1, SNX2 et SNX11 dans les cellules HEK293. En utilisant les essais d’immunofluorescence, "Western Blot" et de cytométrie en flux, nous avons déterminé que tous les trois membres du groupe SNX sont des partenaires d'interaction de PAR2. Toutefois, seul SNX11 se co-localise avec son partenaire au noyau et est responsable de sa translocation nucléaire. Les expériences de "RT - PCR" sur les lignées de cellule de SNXs "knockdowns" ont démontré que la fonction de PAR2 nucléaire dépend surtout de SNX11; néanmoins SNX1 et SNX2 peuvent aussi l’influencer, suggérant qu'ils font aussi partie du réseau signalétique de PAR2. En conclusion, PAR2 est déplacé de la membrane plasmatique à la membrane nucléaire après sa stimulation avec un agoniste. La translocation nucléaire de PAR2 par un mécanisme impliquant SNX11, initie des effets intracellulaires différents de sa signalisation membranaire. Mots clés : récepteurs couplés à la protéine G, “Sorting Nexins”, récepteurs activés par la protéase, translocation nucléaire, membrane nucléaire, signal nucléaire. / During the recent years the existing statements that G – protein coupled receptors (GPCRs) are relaying signals only from the plasma membrane have been challenged. It has become clear that some GPCRs can also signal from intracellular compartments and the nucleus. The role and the function of these nuclear GPCRs are subject of intensive investigations. Protease - activated receptors (PAR) are members of the GPCR family. PARs are activated by the cleavage of the N – terminus of the receptor followed by binding of the tethered ligand on to the receptor. Four PARs have been described: PAR1, PAR2, PAR3 and PAR4. PAR2 can induce mitogenic effects and participate in processes such as angiogenesis and inflammation. While many of the intracellular effects of PAR2 can be explained with its plasma membrane signalling pathway, intracrine effects of PAR2 have also been proposed. However the mechanisms by which PAR2 can induce its target gene expressions are still unknown. The purpose of our study was to investigate whether a distinct nuclear population of PAR2 exists. We hypothesized that the roles of PAR2 at different cellular compartments are different since signalling pathways depend on subcellular context. Using confocal microscopy and Western blot techniques we were able to demonstrate the presence of a nuclear population of PAR2. Upon stimulation of the cell membrane PAR2, we observed significant translocation of the receptor from the plasma membrane to the nucleus. Using RT – PCR technique we detected diverse roles of cell surface and nuclear PAR2 on triggered gene expression. In the current study we have attempted to reveal the mechanisms responsible for PAR2 nuclear translocation. We tested the hypothesis that members of the Sorting Nexin (SNX) family are involved in PAR2 nuclear translocation. Sorting Nexins are a new, large group of proteins with well established cargo functions. SNX1 and SNX2 have been demonstrated to be responsible for lysosomal sorting of PAR1. SNX11 has not been studied yet, and we hypothesized that it may be another SNX involved in PAR2 signalling. We developed stable knockdowns for SNX1, SNX2 and SNX11 in HEK293 cells. Using immunofluorescence, Western Blot analysis and FACS assays, we determined that all three members of SNX group are interaction partners of PAR2. However only SNX11 co-localized with its partner in the nucleus and is responsible for its nuclear translocation. RT – PCR experiments on SNXs knockdowns cell lines demonstrated that PAR2 nucleus function is mostly dependent on SNX11; nevertheless SNX1 and SNX2 knockdowns can also attenuate it, suggesting that they are part of PAR2 signalling network. In conclusion, PAR2 is being translocated from the plasma membrane to the nuclear membrane after its stimulation with SLIGKV. PAR2 nucleus translocation triggers intracellular effects different from its cell membrane signalling. SNX11 is the major factor responsible for PAR2 nuclear sorting. Keywords: G – protein coupled receptors, Sorting Nexins, Protease - activated receptors, nuclear translocation, nuclear membrane, nuclear signalling

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