gms | German Medical Science

Objective: Malignant gliomas escape current treatment strategies by infiltrative growth and inevitable relapses of the tumor caused by residual tumor cells following resection. In gliomas, Tissue Factor (TF) expression is positively correlated with the grade of malignancy. Apart from its function in initiation of the coagulation cascade, TF has been recently discovered to act as a signaling protein leading to activation of MAP kinases. Our previous studies have demonstrated a role of the Tissue Factor/FVIIa pathway in glioma cell migration, invasion and proliferation. Here, we further dissect the role of PAR-2 and downstream ERK signaling for the highly invasive behavior of malignant glioma cells.

Methods: Expression levels of TF, FVII, PAR-2, phospho-ERK and total ERK were analyzed by Western blotting in glioma cell lines of oligodendroglial (Hs 683) and astrocytic (MZ-18) origin. Inhibition of TF/FVIIa/PAR-2 dependent signaling was achieved via a lentiviral-mediated knockdown of TF. For restoration of PAR-2 downstream signaling the PAR-2 selective agonistic peptide SLIGKV was used. Tumor cell migration and invasion were quantified by scratch migration assays and modified Boyden chamber assays, respectively.

Results: We previously demonstrated reduced migration and invasion in glioma cells with a stable knockdown of TF. Restoration of this pathway by addition of the selective PAR-2 agonist SLIGKV completely abrogated the effects of the TF knockdown and restored invasion and migration to levels comparable to those observed in control cells highly overexpressing TF. In contrast, addition of SLIGKV to control cells did not lead to any further increase in cell migration or invasion. Western blotting after SLIGKV treatment displayed a time-dependent increase of ERK1/2 phosphorylation. SLIGKV in the presence of the ERK pathway-specific inhibitor PD98059 could partly restore invasion, but not migration in TF knockdown cells. These data suggest that the pro-migratory, but not the pro-invasive effects of TF and PAR-2 are mainly mediated through activation of the ERK pathway.

Conclusions: The major findings of our study point out the crucial role of PAR-2 in TF-mediated signaling in glioma and therefore highlight PAR-2 as an interesting target for tumor therapy aimed at limiting invasion of gliomas of astrocytic and oligodendroglial origin. We were furthermore able to dissect the crucial role of ERK as the MAP kinase mediating the pro-migratory, but not the pro-invasive effects of TF and PAR-2 in glioma cells.