In this study, TF expression was studied with pharmacological inhibitors and siRNA that suppress PI3K/Akt and MAPK/ERK pathways . Previous reports showed that these two pathways regulate both flTF and asTF transcription [15, 24, 25].
In agreement with other reports [7, 8], an essential role of PI3K/Akt in TF expression in MDA-MB-231 cells was found because treatment by either LY294002 or wortmannin decreased TF expression in a dose-dependent manner. Experiments using Akt siRNA gave the same results. This was demonstrated by a decrease in the reporter gene expression using MDA-MB-231 cells transfected with the plasmid PGL4-TFluc as well as by qPCR using the parental cells. The decrease in TF gene expression was well correlated with the decrease in flTF protein and with the decrease in the cell surface-associated TF activity as shown by plasma clotting assays. We further showed that treatment with LY294002 and wortmannin resulted in inhibition of the catalytic activity of PI3K and Akt phosphorylation by western blot. All these findings confirmed that PI3K/Akt phosphorylation plays a critical role in TF gene expression.
In contrast to Akt inhibitors, we found that treatment with the ERK inhibitor PD98059 surprisingly resulted in a remarkable increase in TF gene expression in a dose-and time-dependent manner. This finding was initially observed in MDA-MB-231-TFluc cells, and then confirmed by qPCR and western blot with their parental cells. The use of ERK siRNA further confirmed this observation. Therefore, Akt and ERK modulated TF expression in opposite ways.
To study the mechanisms involved, we blocked PI3K/Akt activation by LY294002, wortmannin, A6730 or Akt siRNA in PD98059-treated MDA-MB-231 cells. These experiments gave concomitant results showing that PD98059-induced TF expression was indeed inhibited at mRNA and protein levels by blocking the PI3K/Akt pathway, and in particular, the blockage was complete using Akt inhibitor A6730. These results emphasized the importance of the PI3K/Akt pathway in the control of TF expression.
In the literature, many studies reported the interaction of growth factor receptors with ERK and PI3K/Akt pathways and the crosstalk between ERK and PI3K/Akt pathways [26–31]. Gan et al. demonstrated that blockage of ERK activity enhanced EGF receptor activation and turnover, which in turn enhanced PI3K activation and Akt phosphorylation . For this reason, we explored the role of EGFR in the PD98059-induced TF up-regulation. Our results from qPCR and western blot experiments showed that the EGFR inhibitor erlotinib indeed suppressed PD98059-induced TF expression. We also observed that the inhibitory effect of erlotinib was much more noteworthy in PD98059-treated cells than in non-treated cells. The experiments using EGFR siRNA gave similar results. These results strongly suggest that the similar regulation described by Gan et al. occurred in MDA-MB-231 cells . In brief, the inhibition of ERK activity by PD98059 enhanced EGFR activity, which in turn up-regulated Akt activity, resulting in high levels of TF expression. Such a mechanism can explain how the blockage of ERK induced a high level of TF expression, and why blockage of the Akt pathway suppressed such an induction. The same profile of TF regulation was again observed in OVCAR-3 and SKOV-3 cells, suggesting a widespread mechanism. Our results do not exclude other signal interconnections and we believe that the full mechanism of TF regulation is likely more complicated and further study is needed. Our results contradict a previous report showing inhibition of TF expression by ERK inhibitor , however, the reason for this discrepancy is unclear.
As the inhibition of PI3K/Akt may reduce asTF mRNA in endothelial cells , we evaluated the asTF isoform in response to the addition of inhibitors of PI3K/Akt and MAPK/ERK. We observed in MDA-MB-231, SKOV-3 and OVCAR-3 cells that PD98059 up-regulated asTF. However, the inhibition of PD98059-enhanced asTF mRNA transcription by Akt inhibitors was observed only in MDA-MB-231. The results of the asTF mRNA levels in SKOV-3 and OVCAR-3 cells seem to suggest that asTF level could also be regulated independently from flTF expression . They indicate the complexity of the regulation of TF isoform transcription. Further investigation is needed to clarify these. Our observation in MDA-MB-231 also suggests that the increase in the membrane-associated flTF and in the secretion of asTF can occur concomitantly during malignant transformation. flTF is known to stimulate tumor progression via FVIIa and PAR2 and asTF has been shown to induce tumor angiogenesis by its binding to integrins . The level of asTF was found to be related to poor clinical prognostics . The secretion of asTF by cancer cells has been shown to be a complex process which is under the control of SR proteins in addition to TF promoter and miRNA regulation [15, 31], Further investigation can be expected to better understand the regulation of TF including its isoforms in detail. Our results do not exclude a distinct SR protein-mediated regulatory mechanism for asTF production which has been reported to be independent from transcriptional regulation for TF .
Our results support and underline the roles of Akt and EGFR in TF-related tumor growth and metastasis. We believe that targeting TF expression could potentially improve clinical cancer therapy by inhibiting tumor angiogenesis and metastasis as well as by controlling thrombotic complications [32, 33].