Formation of Tissue Factor-Factor VIIa Complex and Generation of Factor Xa Induce Activation of the mTOR Pathway Which Regulates Migration of Human Breast Cancer Cells.

Tissue factor (TF) is a 47 kDa transmembrane glycoprotein that initiates blood coagulation when complexed with factor VIIa (FVIIa). TF is constitutively expressed in a variety of tumor cells and has been shown to have roles in cellular signaling and tumor progression. We showed previously that formation of TF-FVIIa-Factor Xa (FXa) complex induces cellular signaling in the Adr-MCF-7 cell line, a multidrug-resistant subline of the human breast cancer cell line, MCF-7, that leads to enhanced cell migration and inhibition of apoptosis. The Adr-MCF-7 cell line has high endogenous expression of TF and expression of protease-activated receptor 1 (PAR1) and protease-activated receptor 2 (PAR2). Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induces phosphorylation of p44/42 mitogen-activated protein kinase and protein kinase B. In the present study, we investigated the role of TF-FVIIa-mediated signaling in activation of the mammalian target of rapamycin (mTOR) pathway. Treatment of the Adr-MCF-7 cells with the combination of FVIIa (10 nM) and FX (150 nM) induced phosphorylation of mTOR and p70 S6 kinase (p70S6K) by 2 and 2.5 fold, respectively, compared with untreated cells. Phosphorylation of these proteins could be inhibited by pretreatment of the cells with either anti-TF antibody (TF85G9) or TAP, a specific FXa inhibitor. No increase in the basal level of phosphorylation of these proteins occurred with treatment of the cells with FVIIa (10 nM) alone. Moreover, phosphorylation of mTOR and p70S6K was probably mediated by PAR1 and/or PAR2 activation. Using a modified Boyden chamber chemotaxis assay, activation of the mTOR pathway with the combination of FVIIa (10 nM) and FX (150 nM) promoted migration of the Adr-MCF-7 cells. Inhibition of this pathway with the specific mTOR inhibitor, rapamycin, markedly decreased cell migration. Results from these studies suggest that TF-FVIIa-mediated signaling modulates mTOR pathway activation, which in turn regulates breast cancer cell migration.