Effects of the activated coagulation factor X (FXa) and its protease-activated receptor-2 (PAR2) on colon cancer cell growth in vitro and in vivo
Authors: Meyer U , Polster S , Roennpagel V , Grammbauer S , Dombrowski F , Ritter C , Rauch B 
Published: November 2023

Abstract

Background
Protease-activated receptors are a family of four (PAR1-4) G-protein-coupled receptors with a unique mechanism of activation. Serine proteases such as the activated coagulation factor X (FXa) can activate PARs, selectively PAR2, by proteolytic cleavage unmasking its tethered ligand which induces consecutive receptor activation. PAR2 has been linked to various pathophysiological processes such as cardiovascular or inflammatory diseases. Recent studies indicate a key role of PAR2 in certain cancer types.

Purpose
In this study, we investigated the impact of FXa, PAR2 and of the direct, oral FXa-inhibitor Apixaban in colon cancer cell growth and migration in vitro and in vivo.

Methods
FXa and PAR2-dependent effects were investigated in various colon cancer cell lines such as human HCT116 or murine MC38 cells in vitro using different assays for cell proliferation and migration, Western blotting and PCR analyses. A subcutaneous tumor model was utilized to compare the impact of Apixaban (5 and 50 mg/kg body weight) in C57Bl/6 wild type (WT) versus PAR2-deficient mice (PAR2-KO). Tumor cells were allowed to grow for up to 21 days. Apixaban was applied orally by gavage. All animal studies were approved by the responsible animal welfare committee.

Results
Incubation with FXa at concentrations up to 30 nM stimulated growth and migration in both cultured human and murine cells in vitro. These effects were mimicked by peptide-stimulated activation of PAR2. In comparison, thrombin or selective peptide-induced activation of PAR1 did not induce a marked response. In addition, we observed an activation of mitogenic signaling pathways such as p44/42, p38 and AKT by FXa suggesting a direct response for colon cancer cells to coagulation factor signaling. In vivo, PAR2-deficiency resulted in a significantly reduced tumor growth compared to wild-type mice. In comparison, oral treatment with Apixaban both at 5 mg or 50 mg/kg body weight did not markedly affect tumor development in vivo. This effect was reflected by a reduced tumor weight in PAR2-deficient mice after excision of the tumor at the end of the study. In addition, a significantly lower rate of complications was seen in PAR2-deficient mice during the experiment. Interestingly, the spleen was larger in relation to body weight in PAR2-deficient mice independently from Apixaban treatment. We observed no obvious histological differences within the tumor tissue between the treatment groups or animal strains.

Conclusion
Deficiently of PAR2 resulted in reduced growth of colon cancer cells in vivo, while oral treatment with Apixaban did not significantly affect tumor growth. In vitro, FXa and selective activation of PAR2 did stimulate cell growth and migration indicating a potential direct growth promotion effect of PAR2 in colon cancer cells. The pharmacological targeting of PAR2 may represent a future therapeutic strategy in the treatment of colon cancer.