Fibrinogen activates focal adhesion kinase (FAK) promoting colorectal adenocarcinoma growth.

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.

Cell type-specific mechanisms coupling protease-activated receptor-1 to infectious colitis pathogenesis.

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-1ΔM ) or enterocyte-specific (PAR-1ΔEPI ) fashion. Citrobacter rodentium colitis pathogenesis was analyzed in mice with global PAR-1 deletion (PAR-1-/- ) and cell type-specific deletions. RESULTS: Constitutive deletion of PAR-1 had no significant impact on weight loss, crypt hypertrophy, crypt abscess formation, or leukocyte infiltration in Citrobacter colitis. However, colonic shortening was significantly blunted in infected PAR-1-/- mice, and these animals exhibited decreased local levels of IL-1ß, IL-22, IL-6, and IL-17A. In contrast, infected PAR-1ΔM mice lost less weight and had fewer crypt abscesses relative to controls. PAR-1ΔM mice had diminished CD3+ T cell infiltration into colonic tissue, but macrophage and CD4+ T cell infiltration were similar to controls. Also contrasting results in global knockouts, PAR-1ΔM mice exhibited lower levels of IL-1ß, but not Th17-related cytokines (ie, IL-22, IL-6, IL-17A). Infected PAR-1ΔEPI mice exhibited increased crypt hypertrophy and crypt abscess formation, but local cytokine elaboration was similar to controls. CONCLUSIONS: These studies reveal complex, cell type-specific roles for PAR-1 in modulating the immune response to Citrobacter colitis that are not readily apparent in analyses limited to mice with global PAR-1 deficiency.