Opposite regulation by PI3K/Akt and MAPK/ERK pathways of tissue factor expression, cell-associated procoagulant activity and invasiveness in MDA-MB-231 cells

Abstract

Background: Tissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways.

Methods: A plasmid containing TF promoter -2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells.

Results: We show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment.

Conclusions: This study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

Figure 1

TF promoter activity in MDA-MB-231-TFluc cells treated by LY294002, wortmannin and PD98059. Panel a: MDA-MB-231-TFluc cells were treated by PD98059 at 10 μM for different time periods, then washed. Panels b-c: the cells were treated by LY294002 (b), wortmannin (c) and PD98059 (d) at indicated concentrations for 24 hrs. The activity of luciferase in the cells was measured. The data represent the means values of three independent experiments.

Figure 2

Expression levels of TF mRNA and TF protein in treated MDA-MB-231 cells. Panel a: The qPCR results of total TF mRNA levels in treated MDA-MB-231 cells. The cells were treated for 24 hr by the indicated agents at the indicated concentrations. qPCR was performed with primers Hs00175225_m1. The results were obtained from three independent experiments. Statistical significance (p<0.05) was found for all of the groups in comparison with the control group, except for the group of 5 μM PD98058. Panel b: The western blot of TF protein levels in PD98059-treated cells, showing a dose dependent increase in TF levels at 24 hrs. Panel c: The western blot of TF protein levels in the cells treated by LY294002 (10 μM) and wortmannin (0.1 μM) at 24 hrs. The data of the ratio were obtained with 3 repeated blots. * : p<0.05 in comparison with the controls.

Figure 3

TF promoter activity, TF mRNA and Akt phosphorilation after combined treatments. Panel a: MDA-MB-231-TFluc cells were treated by 10 μM of LY294002 and 0.1 μM of wortmannin, in the absence or the presence of 10 μM PD98059 for 24 hrs. The activity of luciferase was measured. The data was represented as means±SEM of three independent experiments. * : p<0.05. Panel b: qPCR results of TF mRNA (Hs00175225_m1) in the cells after the treatment for 24 hrs by 10 μM of LY294002, 10 μM of A6730, 0.1 μM of wortmannin, in the absence or the presence of 10 μM PD98059. The experiments were repeated for 3 times. ** : p<0.05. Panel c: MDA-MB-231 cells were treated with 0.1 μM of wortmannin, 10 μM of LY294002 in the presence or the absence of PD98059 at 10 μM or 50 μM for 24 hrs. Total protein was extracted for the determination of pAkt level by western blot using anti-Akt or anti-pAkt antibodies. The data were from three blots. * : p<0.05.

Figure 4

TF promoter activity, TF mRNA and Akt phosphorilation after combined treatments. Panela: MDA-MB-231-TFluc cells were treated by 0.1 μM of erlotinib in the absence or the presence of 10 μM PD98059 for 24 hrs. The cell activity of luciferase was measured. The data was from three independent experiments. Panel b: qPCR results of TF mRNA in the cells after the treatment for 24 hrs by 50 nM of cetuximab or 0.1 μM of erlotinib in the absence or the presence of 10 μM PD98059. The data were from 3 independent experiments. * : p<0.05. Panel c: The western blots of TF protein in the cells treated for 24 hrs by 10 μM PD98059, 10 μM of A6730, 50 nM of cetuximab and 0.1 μM of erlotinib. The data were from 3 blots. * : p<0.05 in comparison with control. Panel d: The western blots of TF protein in the cells treated for 24 hrs by 10 μM LY294002, 0.1 μM erlotinib and 10 μM A6730 in the presence or the absence of 10 μM PD98059. The data were from 3 blots. * : p<0.05.

Figure 5

Cell-associated procoagulant activity and invasion in MDA-MB-231 cells, flTF expression and asTF mRNA transcription in SKOV-3 and OVCAR-3 cells. Panel a: Cell procoagulant activity. MDA-MB-231 cells were treated with 10 μM of PD98059, 10 μM of LY294002, 0.1 μM of wortmannin for 24 hrs and washed. Then clotting time was measured with these cells after the addition of human blood plasma and calcium. The results represent 3 different experiments. * : p<0.05 between PD98059 group versus LY294002 and wortmannin groups. Panel b: Cell invassive capacity : the same cells as in the panel a were cultured in the invasion chambers for 22 hrs. The cells penetrated through the matrigel were counted in microscopy. The data were from 3 independent experiments. * : P<0.05 in comparison to the control cells. Panel c: Regulation of TF protein in SKOV-3 and OVCAR-3 cells. SKOV-3 and OVCAR-3 cells were treated 10 μM of PD98059 with or without A6730 and EGFR siRNA for 24 hrs. The protein was extracted from the washed cells and examined for TF expression by western blot. The data were from 3 repeated blot experiments. Significant difference was found between the PD98059 group versus all of the other groups within the same cells (p<0.05). Panel d: asTF levels in PD98059 treated cells. The cells were treated by 10 μM of PD98059 with or without 0.1 μM erlotinib 10 μM A6730 and EGFR siRNA for 24 hrs. qPCR data were from 3 dependent experiments. * : p<0.05 for PD98059 groups in comparison with all of the other groups in the same cell lines.