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.

Along with other proteins in the coagulation cascade, including tissue factor and thrombin, fibrinogen has been shown to promote tumor metastasis across numerous cancer types. However, the role of fibrin(ogen) in primary tumor growth is context dependent and not universally important. One cancer strongly dependent on fibrin(ogen) for primary tumor growth is colorectal cancer (CRC), but the mechanisms by which fibrinogen supports colon cancer growth are not well understood. To delineate the mechanism of fibrin(ogen)-supported tumor growth, we implanted C57BL/6-derived murine colon cancer cells (MC38) into the dorsal subcutis of syngeneic mice with specific deletions/alteration in fibrinogen or FXIII-deficiency. Complete fibrinogen deficiency significantly limited the growth of colon cancer cells. However, the imposition of FXIII-deficiency or specific mutations in fibrinogen that 1) prevent the formation of fibrin polymer (FibAEK), limit fibrin(ogen) binding to the integrin αMβ2 (Fibɣ390-396A), or remove the ɣ chain binding motif for the platelet integrin αIIbβ3 (FibɣΔ5) had no impact on colon cancer growth. To explore the mechanisms coupling fibrin(ogen) to colon cancer growth using a nonbiased approach, we performed RNA-Seq analyses of murine CRC tumors harvested from Fib+ and Fib- mice. We detected significant differences in the expression of 214 genes (128 downregulated and 86 upregulated) involved in cellular proliferation, survival, migration and metabolism. Notably, Stratifin (SFN), which encodes 14-3-3-σ, was one of the genes found to be highly upregulated in tumors from Fib- mice relative to Fib+ mice. 14-3-3-σ is a potent cell cycle regulator and it is also known to stabilize p53, which ultimately inhibits tumor growth. In a separate validation cohort, we observed significantly increased protein expression of 14-3-3-σ and its upstream and downstream targets (i.e., p53, p21 and p27) in tumors harvested from Fib- mice relative to controls. We also compared FAK activation, a key negative regulator of p53, in tumors from Fib+ and Fib- mice. FAK was inactive in tumors from Fib- mice, as indicated by lack of phosphorylation at tyrosine 397 (p-FAK Tyr397). Also, MDM2 was less phosphorylated at Ser166 in Fib- tumors, suggesting that p53 is not degraded by MDM2 in the absence of fibrinogen. Taken together, these data suggest that fibrin(ogen)-mediated downregulation of p53 and other targets via FAK activation and downregulation of 14-3-3-σ results in senescence of CRC cells. Consistent with this view, Ki67 positive nuclei were significantly less in tumor from Fib- mice relative to controls We also observed senescence-associated-β-galactosidase (SA-β-gal) activity in the tumors from Fib- mice, but not those from Fib+ animals. Furthermore, NMR-based metabolomics analyses demonstrated significantly less NAD+ and lactate levels in tumors from Fib- mice, which further confirms that fibrinogen deficiency hampers proliferation by inhibiting major metabolic pathways. In order to determine if fibrinogen-mediated support of CRC growth is tumor cell intrinsic, we compared MC38 growth in the 3D bioprinted matrices consisting of fibrin or albumin. MC38 cells showed a higher proliferation rate in the fibrinogen printed 3D environment compared to the albumin environment. These findings suggest that fibrin(ogen)-mediated engements of tumor cells activates FAK which inhibits p53 and its downstream targets including 14-3-3-σ and p21 which promote cellular proliferation and prevent senescence. Overall, these studies suggest that fibrin(ogen) is the important component of the CRC microenvironment and could be exploited for targeting and treating the CRC. Disclosures Whitlock: POSNA: Other: Research Committee; MTF Biologics Grant Program: Other: site co-investigator. Palumbo:Ionis Pharmaceuticals: Research Funding.