Tissue factor-factor VIIa complex triggers protease activated receptor 2-dependent growth factor release and migration in ovarian cancer.
Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC.



TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expressions were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists.



Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4-10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present.



Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation.
Departments of Medicine/Hematology-Oncology and Pathology, University of Wisconsin-Madison, Madison, WI 53792, United States.
Alice
A
Chanakira
Departments of Medicine/Hematology-Oncology and Pathology, University of Wisconsin-Madison, Madison, WI 53792, United States.
Pamela R
PR
Westmark
Biostatistics and Medical Informatics, UW Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI 53792, United States.
Irene M
IM
Ong
Departments of Medicine/Hematology-Oncology and Pathology, University of Wisconsin-Madison, Madison, WI 53792, United States. Electronic address: jps@medicine.wisc.edu.
John P
JP
Sheehan
D015153:Blotting, Western; D000077216:Carcinoma, Ovarian Epithelial; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D002633:Chemotaxis; D015942:Factor VIIa / Q000378:metabolism*; D005260:Female; D006801:Humans; D009361:Neoplasm Invasiveness; D009375:Neoplasms, Glandular and Epithelial / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010051:Ovarian Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000378:metabolism*; D044463:Receptor, PAR-1 / Q000235:genetics* / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000235:genetics* / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism; D013925:Thromboplastin / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D012333:RNA, Messenger; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D044462:Receptors, Proteinase-Activated; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D013925:Thromboplastin; D015942:Factor VIIa; D013917:Thrombin
Microparticles; Ovarian cancer; Protease activated receptor 2; Tissue factor-factor VIIa complex; Vascular endothelial growth factor