Abstract 149: TF-FVIIa activates VEGF-A secretion in ovarian epithelial cancer cell line via a PAR-2-dependent mechanism
Authors: Chanakira Alice , Sheehan John P. 
Published: May 2015

Abstract
Background and Objective: Venous thromboembolism (VTE) is intrinsic to the biology of EOC. Progression of malignancy is associated with increased tissue factor (TF) expression. Formation of the TF-factor VIIa (FVIIa) complex triggers local thrombin generation, with thrombin activating protease activated receptor-1 (PAR-1). The TF-FVIIa complex also triggers intracellular signaling via phosphorylation of the TF C-terminus and activation of PAR-2. Although PAR-2 analysis in ovarian cancer has been limited, PAR-2 activation in renal cell carcinoma is associated with MAPK signaling pathway activation and induction of growth factor release. We hypothesize that TF-FVIIa dependent activation of PAR-2 on EOC cells up-regulates expression of angiogenic mediators (VEGF-A) that modify the peritoneal microenvironment, enhancing vascular leakage and ascites formation.
Methods: TF antigen expression in EOC cell lines (CaOV-3, SKOV-3 & OvCAR-3) was determined by western blot (WB) analysis. TF-bearing microparticles were isolated from conditioned media by differential centrifugation (20,800 g) and TF coagulant activity was detected by factor X activation. VEGF-A levels in conditioned media were determined by ELISA. Proliferation of the SKOV-3 and OVCAR-3 cell lines in response to thrombin (PAR-1 agonist) was determined via direct cell count.
Results: WB analysis of EOC cell line lysates demonstrated PAR-2 expression (CaOV-3&gt;OvCAR-3&gt;&gt;SKOV-3), and exposure of SKOV-3 and OVCAR-3 cells to 50 nM FVIIa resulted in 3-fold induction of VEGF-A release over 48 hr relative to untreated cells (p&lt;0.05). TF-FVIIa complex was a more potent inducer of VEGF-A when compared to the PAR-2 peptide agonist (SKIGKL-NH2). Pre-incubation with the PAR-2 antagonist ENMD-1068 reduced VEGF-A to basal levels in the presence of TF-FVIIa; further supporting PAR-2 dependent induction of VEGF-A. Pan-expression of PAR-1 was observed in EOC cell lines by WB. Thrombin (0.1-1U/mL), an established PAR-1 agonist, induced dose-dependent proliferation of SKOV-3 and OVCAR-3 cell lines under serum free conditions (p&lt;0.001). Pre-incubation with the PAR-1 antagonist vorapaxar reduced proliferation to basal levels in the presence of thrombin.
Conclusion: TF expression and coagulant activity is up regulated in EOC. FVIIa triggers a PAR-2 dependent induction of VEGF-A release from EOC cell lines. Similarly, the PAR-1 agonist thrombin triggered a significant increase in EOC cell proliferation. These results suggest that interaction of EOC with the coagulation system in the tumor microenvironment may enhance both PAR-1 dependent cellular proliferation and PAR-2 dependent release of VEGF, a potent regulator of angiogenesis. Thus, PAR-1 and PAR-2 may represent pharmacologically relevant drug targets to block ovarian cancer progression via inhibition of growth and angiogenesis.
Citation Format: Alice Chanakira, John P. Sheehan. TF-FVIIa activates VEGF-A secretion in ovarian epithelial cancer cell line via a PAR-2-dependent mechanism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 149. doi:10.1158/1538-7445.AM2014-149