Epithelial-to-Mesenchymal Transitions Circulating Tumor Cells Regulation of Tissue Factor by Epithelial-to-Mesenchymal Transitions: impacts for the metastatic progression   Francart M-E.1, Bourcy M.1, Suarez-Carmona M.1, Lambert J.1, Oury, C.3, Noël A. 1, Gilles C. 1 C.1 1GIGA-Cancer, Laboratory of Tumor and Development Biology, University of Liège, 2GIGA-Cardiovascular Sciences, Laboratory of Thrombosis and Hemostasis, University of Liège. Epithelial-to-Mesenchymal Transitions Circulating Tumor Cells Tissue Factor Metastasis ○ Epithelial-to-mesenchymal transitions (EMTs) generate tumor cells with higher invasive and metastatic properties. Increasing data support the involvement of EMT pathways in the liberation of circulating tumor cells (CTCs). ○ Enhanced expression of Tissue Factor (TF), a major cellular factor of coagulation, by aggressive epithelial tumor cells has been reported and activation of the coagulation system is a long-described correlate of malignancy. ○ We further collected clear cut data showing that EMT pathways induces the expression of Tissue Factor in epithelial tumor cells, thereby providing EMT+ CTCs with enhanced pro-coagulant properties and promoting early metastasis. (Bourcy et al., Cancer Res., accepted for publication). We propose here to identify EMT pathways that could regulate TF expression, and further investigate the impact of such regulation on pro-coagulant properties of CTCs and metastasis development. Objectives Our project aims at examining the contribution of vimentin as a molecular EMT actor potentially involved in EMT-modulated TF expression, and to further explore the impact of these regulations on the metastatic spread. We propose to pursue our aim along 2 specific objectives: Explore the molecular mechanisms by which vimentin could contribute to TF regulation. Examine the functional implication of such regulations on pro-coagulant activity in vitro and, in vivo, on survival in the blood stream and metastasis development. Results 4. Vimentin seems to interact with TF Induction of EMT in MDA-468 cells by EGF increases TF expression and pro-coagulant properties. 1. TF and vimentin are concomitantly induced during EMT B Ctrl EGF Vimentin Tissue Factor GAPDH MDA-MB-468 A C Analyses by Proximity Ligation Assays suggest interactions between vimentin and TF in invasive human breast MDA-MB-231 (vimentin+ and TF+). Similar MDA-MB-468 Mean number of dots by cells Fold induction Vimentin Tissue Factor MCF-7 MDA-MB-231 EGF- EGF+ Analyses by (A) RT-qPCR and (B) western blotting of vimentin and TF expression. (C) Visual clotting assay of whole blood incubated with MDA-468 cells treated or not with EGF. Similar results are obtained with TGF-β treated and non treated A549 cells. PMC-42-LA results were obtained for the inducible cell line PMC-42-LA treated or not with EGF. 2. Vimentin contributes to TF regulation in EMT-inducible cell systems 5. Vimentin expression modulates in vitro coagulant properties Transfection of vimentin siRNA in MDA-468 decreases their pro-coagulant activity. Analysis by visual clotting assay in whole blood of the coagulant properties of MDA-468 cells treated with EGF and transfected with 2 siRNA controls or 2 siRNA against vimentin. A MDA-MB-468 EGF B Si Ctrl1 Si Ctrl2 Si Ctrl1 Si Ctrl2 B Si Ctrl1 Si Ctrl2 Vim Si1 Vim Si2 MDA-MB-468 ctrl Vim Si1 Vim Si2 MDA-MB-468 EGF Vim Si1 Vim Si2 - - + + - - + + Vim siRNA TF/gapdh Vimentin Tissue Factor GAPDH A 6. Vimentin expression promotes early metastasis Si Ctrl1 Vim Si1 Transfection of vimentin siRNA in MDA-MB-231 reduces their early seeding properties after IV injection. (A) In vivo imaging of mice injected intravenously with MDA-MB-231 cells (transfected with a Ctrl si or a Vim si) 24h after injections, and of their collected lungs. (n=14). (B) RT-nested qPCR against human GAPDH performed on total RNA extracted from lungs (n=10). *p<0.05 B Transfection of siRNA against vimentin reduces the protein expression of TF in MDA-MB-468 cell line. Analysis of TF expression by (A) RT-qPCR and (B) by western blotting. Similar results are obtained with others EMT+ cells lines. Lungs 3. Vimentin sounds to stabilize TF mRNA (preliminary data) Analysis of TF mRNA stabilization by decay rates of TF mRNAs in MDA MB 231 cells transfected with a ctrl si or a Vim si. Transcription was blocked by actinomycin D (ACT-D) and TF mRNA levels were measured after the block by RT-PCR. The expression of TF mRNAs was normalized to the expression of GAPDH mRNA and set as 1 for 0h. Human Gapdh Si Ctrl1 Vim Si1 Conclusion We have here demonstrated that the expression of TF and vimentin are concomitantly induced during EMT processes together with high pro-coagulant properties. So far, our data support a role of vimentin in the regulation of TF at the mRNA and/or protein level. We are currently investigating the possibility that TF and vimentin could interact directly or through other intermediaries. In parallel, we have performed a first in vivo study showing that EMT-positive cells silenced for vimentin injected intravenously in mice for 24 hours have a diminished ability to seed in the colonized lungs. These data point towards a regulatory mechanism of TF by vimentin that could modulate the coagulant properties and early seeding ability of EMT+ CTCs. Contact : mefrancart@ulg.ac.be