1. 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.
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