Thrombin-mediated proteolysis is a major determinant of metastasis, but is not universally important for primary tumor growth. Here, we report that colorectal adenocarcinoma represents one important exception whereby thrombin-mediated functions support both primary tumor growth and metastasis. In contrast with studies of multiple nongastrointestinal cancers, we found that the growth of primary tumors formed by murine and human colon cancer cells was reduced in mice by genetic or pharmacologic reduction of circulating prothrombin. Reduced prothrombin expression was associated with lower mitotic indices and invasion of surrounding tissue. Mechanistic investigations revealed that thrombin-driven colonic adenocarcinoma growth relied upon at least two targets of thrombin-mediated proteolysis, protease-activated receptor-1 (PAR-1) expressed by stromal cells and the extracellular matrix protein, fibrinogen. Colonic adenocarcinoma growth was reduced in PAR-1–deficient mice, implicating stromal cell-associated PAR-1 as one thrombin target important for tumor outgrowth. Furthermore, tumor growth was dramatically impeded in fibrinogen-deficient mice, offering the first direct evidence of a critical functional role for fibrinogen in malignant tumor growth. Tumors harvested from fibrinogen-deficient mice displayed a relative reduction in cell proliferative indices, as well as increased tumor necrosis and decreased tumor vascular density. Collectively, our findings established a functional role for thrombin and its targets PAR-1 and fibrinogen in the pathogenesis of colonic adenocarcinoma, supporting tumor growth as well as local invasion and metastasis.

The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. Based on evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/− mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacological inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, pre-cancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression.

Essentials Protease activated receptor-1 (PAR-1) has been proposed to drive cancer progression. Surprisingly, PAR-1 deletion accelerated tumor progression in two distinct experimental settings. PAR-1 deletion was shown to limit the apoptosis of transformed epithelial cells. Thrombin- and activated protein C-mediated PAR-1 activation have unique effects on tumor cell biology. SUMMARY: Background Multiple studies have implicated protease-activated receptor-1 (PAR-1), a G-protein-coupled receptor activated by proteolytic cleavage of its N-terminus, as one target coupling thrombin-mediated proteolysis to tumor progression. Objective To analyze the role of PAR-1 in the setting of two distinct spontaneously developing tumor models in mice. Methods We interbred PAR-1-deficient mice with Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice, which spontaneously develop prostate tumors, and adenomatous polyposis coli Min (APC ) mice, which spontaneously develop intestinal adenomas. Results Analyses of TRAMP mice with advanced disease (30 weeks) revealed that PAR-1 deficiency resulted in significantly larger and more aggressive prostate tumors. Prostates collected at an earlier time point (12 weeks of age) revealed that PAR-1 promotes apoptosis in transformed epithelia. In vitro analyses of TRAMP-derived cells revealed that activated protein C-mediated PAR-1 cleavage can induce tumor cell apoptosis, suggesting that tumor cell-intrinsic PAR-1 functions can limit tumor progression. Paralleling results in TRAMP mice, PAR-1-deficient APC mice developed three-fold more adenomas than PAR-1-expressing mice, and the adenomas that formed were significantly larger. Moreover, loss of PAR-1 expression was shown to limit apoptosis in transformed intestinal epithelial cells. Conclusions Together, these results demonstrate a previously unrecognized role for PAR-1 in impeding tumor progression in vivo. These results also offer a cautionary note suggesting that long-term PAR-1 inhibition could increase malignancy risk in some contexts.

Eradication of leukemia stem cells (LSCs) is the ultimate goal of treating acute myeloid leukemia (AML). We recently showed that the combined loss of Runx1/Cbfb inhibited the development of MLL-AF9-induced AML. However, c-Kit+/Gr-1− cells remained viable in Runx1/Cbfb-deleted cells, indicating that suppressing RUNX activity may not eradicate the most immature LSCs. In this study, we found upregulation of several hemostasis-related genes, including the thrombin-activatable receptor PAR-1 (protease-activated receptor-1), in Runx1/Cbfb-deleted MLL-AF9 cells. Similar to the effect of Runx1/Cbfb deletion, PAR-1 overexpression induced CDKN1A/p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1 deficiency also prevented leukemia development induced by a small number of MLL-AF9 leukemia stem cells (LSCs) in vivo. PAR-1 deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose-dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there were a large number of LSCs, while a small number of LSCs required PAR-1 for their efficient growth. Mechanistically, PAR-1 increased the adherence properties of MLL-AF9 cells and promoted their engraftment to bone marrow. Taken together, these data revealed a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML.

Background-Protease-activated receptor-1 (PAR-1) plays a major role in multiple disease processes, including colitis. Understanding the mechanisms coupling PAR-1 to disease pathogenesis is complicated by the fact that PAR-1 is broadly expressed across multiple cell types.Objective-Determine the specific contributions of PAR-1 expressed by macrophages and colonic enterocytes to infectious colitis.Methods-Mice carrying a conditional PAR-1 allele were generated and bred to mice expressing Cre recombinase in a myeloid-(PAR-1 ΔM ) or enterocyte-specific (PAR-1 ΔEPI ) fashion.

Background We previously showed that fibrinogen is a major determinant of the growth of a murine model of colorectal cancer (CRC). Objective Our aim was to define the mechanisms coupling fibrin(ogen) to CRC growth. Results CRC tumors transplanted into the dorsal subcutis of Fib− mice were less proliferative and demonstrated increased senescence relative to those grown in Fib+ mice. RNA‐seq analyses of Fib+ and Fib− tumors revealed 213 differentially regulated genes. One gene highly upregulated in tumors from Fib− mice was stratifin, encoding 14‐3‐3σ, a master regulator of proliferation/senescence. In a separate cohort, we observed significantly increased protein levels of 14‐3‐3σ and its upstream and downstream targets (i.e., p53 and p21) in tumors from Fib− mice. In vitro analyses demonstrated increased tumor cell proliferation in a fibrin printed three‐dimensional environment compared with controls, suggesting that fibrin(ogen) in the tumor microenvironment promotes tumor growth in this context via a tumor cell intrinsic mechanism. In vivo analyses showed diminished activation of focal adhesion kinase (FAK), a key negative regulator of p53, in Fib− tumors. Furthermore, nuclear magnetic resonance–based metabolomics demonstrated significantly reduced metabolic activity in tumors from Fib− relative to Fib+ mice. Together, these findings suggest that fibrin(ogen)‐mediated engagement of colon cancer cells activates FAK, which inhibits p53 and its downstream targets including 14‐3‐3σ and p21, thereby promoting cellular proliferation and preventing senescence. Conclusions These studies suggest that fibrin(ogen) is an important component of the colon cancer microenvironment and may be exploited as a potential therapeutic target.

Protease-activated receptor 1 (PAR1) is widely expressed in humans and mice, and is activated by a variety of proteases, including thrombin. Recently, we showed that PAR1 contributes to the innate immune response to viral infection. Mice with a global deficiency of PAR1 expressed lower levels of CXCL10 and had increased Coxsackievirus B3 (CVB3)-induced myocarditis compared with control mice. In this study, we determined the effect of cell type-specific deletion of PAR1 in cardiac myocytes (CMs) and cardiac fibroblasts (CFs) on CVB3-induced myocarditis. Mice lacking PAR1 in either CMs or CFs exhibited increased CVB3 genomes, inflammatory infiltrates, macrophages and inflammatory mediators in the heart and increased CVB3-induced myocarditis compared with wild-type controls. Interestingly, PAR1 enhanced poly I:C induction of CXCL10 in rat CFs but not in rat neonatal CMs. Importantly, activation of PAR1 reduced CVB3 replication in murine embryonic fibroblasts and murine embryonic cardiac myocytes. In addition, we showed that PAR1 reduced autophagy in murine embryonic fibroblasts and rat H9c2 cells, which may explain how PAR1 reduces CVB3 replication. These data suggest that PAR1 on CFs protects against CVB3-induced myocarditis by enhancing the anti-viral response whereas PAR1 on both CMs and fibroblasts inhibits viral replication.

Previous studies suggested that contact pathway factors drive thrombosis in mechanical circulation. We used a rabbit model of veno-arterial extracorporeal circulation (VA-ECMO) to evaluate the role of factors XI and XII in ECMO-associated thrombosis and organ damage. Factors XI and XII were depleted using established antisense oligonucleotides (ASO) prior to placement on a blood-primed VA-ECMO circuit. Decreasing FXII or FXI to <5% of baseline activity significantly prolonged ECMO circuit lifespan, limited the development of coagulopathy, and prevented fibrinogen consumption. Histological analyses suggested that FXII depletion mitigated interstitial pulmonary edema and hemorrhage whereas heparin and FXI depletion did not. Neither FXI nor FXII depletion were associated with significant hemorrhage in other organs. In vitro analyses showed that membrane oxygenator fibers (MOFs) alone are capable of driving significant thrombin generation in a FXII and FXI-dependent manner. MOFs also augment thrombin generation triggered by low (1 pM) or high (5 pM) tissue factor (TF) concentrations. However, only FXI elimination completely prevented the increase in thrombin generation driven by MOFs, suggesting MOFs augment thrombin-mediated FXI activation. Together, these results suggest that therapies targeting FXII or FXI limit thromboembolic complications associated with ECMO. Further studies are needed to determine the contexts where targeting FXI and FXII, either alone or in combination, would be most beneficial in ECMO. Further studies are also needed to determine the potential mechanisms coupling FXII to end organ damage in ECMO.