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, Lambert J.1, Vanwynsberghe A.1, Gilles C. 1 1GIGA-Cancer, Laboratory of Tumor and Development Biology, 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 the activation of the coagulation system is a long-described correlate of malignancy. ○ We further collected clear cut data showing that EMT pathways induce the expression of TF in epithelial tumor cells, thereby providing EMT+ CTCs with enhanced pro-coagulant properties and promoting early metastasis. (Bourcy et al., Cancer Res., 2017). We propose here to identify EMT pathways that could regulate TF expression, and to 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 potential regulator of TF expression during EMT, and exploring the impact of these regulations on the metastatic spread. We pursued our aims 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 metastatic colonization. Results 4. Vimentin possibly interacts with TF 1. TF and vimentin are concomitantly induced during EMT Analyses by Proximity Ligation Assays suggest interactions between vimentin and TF in invasive human breast MDA-231 (vimentin+ and TF+). Similar A B Ctrl EGF Vimentin Tissue Factor GAPDH MDA-468 Ctrl EGF C MDA-468 MDA-468 Fold induction Mean number of dots by cells Vimentin Tissue Factor MCF-7 MDA-MB-231 MCF7 MDA-231 EGF- EGF+ PMC Ctrl PMC EGF Induction of EMT in MDA-468 cells by EGF increases TF expression and pro-coagulant properties. 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 induced to EMT by EGF. Similar results are obtained with EMT-inducible A549 lung tumor cells (TGF-b treatment). results were obtained for the EMT-inducible PMC-42-LA breast tumor cells treated or not with EGF. PMC-42-LA 5. Vimentin expression modulates in vitro coagulant properties 2. Vimentin contributes to TF regulation in EMT-inducible cell systems Visual Clotting Time (min) Ctrl Si1 15 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. + Tissue Factor/GAPDH MDA-468 EGF 0h - 4h 8h 12h 16h 20h 24h 48h Vim Si1 A B MDA-468 ctrl MDA-468 EGF Ctrl Si2 13 - - + + - - + + Vim siRNA Vimentin Vim Si1 65 Tissue Factor Vim Si2 21 GAPDH A 6. Vimentin expression promotes early metastasis Ctrl Si1 Vim Si1 Transfection of vimentin siRNA in MDA-231 reduces their early seeding properties after IV injection. (A) In vivo imaging of mice injected intravenously with MDA-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). B Transfection of a siRNA against vimentin reduces TF protein expression in MDA-468 cells. Analysis of TF expression by (A) RT-qPCR and (B) by western blotting. Similar results are obtained with others EMT+ cell lines. Lungs * p<0.05 3. Vimentin stabilizes TF mRNA 0.2 0.4 0.6 0.8 1 5 10 15 20 25 MDA-231 Transfection of vimentin siRNA accelerates TF mRNA decay after actinomycin D treatment. TF mRNA levels were measured by RT-PCR at different time points after actinomycin D treatment of MDA-231 cells transfected with a ctrl Si or a Vim Si. Human GAPDH/murin GAPDH Ctrl Si1 Vim Si1 * p=0,0187 Tissue Factor/GAPDH Ctrl Si1 Vim Si1 Time (h) Conclusion We have here demonstrated that the expression of TF and vimentin are concomitantly induced during EMT 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 collected in vivo results showing that EMT-positive cells silenced for vimentin, and injected intravenously in mice for 24 hours, have a diminished ability to accomplish early colonization of the 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