Morgane Bourcy

Soluble factors regulated by epithelial–mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment

Epithelial–mesenchymal transition (EMT) programmes provide cancer cells with invasive and survival capacities that might favour metastatic dissemination. Whilst signalling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumour cells and the tumour microenvironment remains elusive. We aimed to identify EMT‐regulated soluble factors that facilitate the recruitment of host cells in the tumour. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL‐8, IL‐6, sICAM‐1, PAI‐1 and GM‐CSF, and implicate the EMT‐transcription factor Snail as a regulator of this process. We further show that EMT‐derived soluble factors are pro‐angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT‐positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple‐negative breast cancers, tumours presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumours with little or no EMT. Taken together, our results show that EMT programmes trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favour cancer spread. Copyright

Tissue factor induced by epithelial-mesenchymal transition triggers a pro-coagulant state that drives metastasis of circulating tumor cells.

Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 4270-82. ©2016 AACR.

Epithelial‐Mesenchymal Plasticity and Circulating Tumor Cells: Travel Companions to Metastases

Epithelial–mesenchymal transitions (EMTs) associated with metastatic progression may contribute to the generation of hybrid phenotypes capable of plasticity. This cellular plasticity would provide tumor cells with an increased potential to adapt to the different microenvironments encountered during metastatic spread. Understanding how EMT may functionally equip circulating tumor cells (CTCs) with an enhanced competence to survive in the bloodstream and niche in the colonized organs has thus become a major cancer research axis. We summarize here clinical data with CTC endpoints involving EMT. We then review the work functionally linking EMT programs to CTC biology and deciphering molecular EMT‐driven mechanisms supporting their metastatic competence. Developmental Dynamics 247:432–450, 2018.

Abstract LB-147: EMT transcription factors induce tissue factor expression and pro-coagulant properties, supporting early metastasis of circulating tumor cells

The presence of circulating tumor cells (CTCs) expressing traits of epithelial-mesenchymal transition (EMT) in cancer patients is increasingly recognized. Recently, we identified a new mechanism by which EMT induces the expression of Tissue Factor (TF, a major cell-associated initiator of coagulation), triggering a local activation of the coagulation cascade that favors the early metastatic colonization of EMT-positive CTCs (Bourcy et al., Cancer Research, 2016). Strengthening the functional contribution of EMT to TF regulation and coagulation, we further accumulated evidence that EMT transcription factors regulate TF expression. Thus, silencing ZEB1 inhibited both EMT-associated TF expression and coagulant activity. EMT-positive cells also exhibited a higher survival/persistence in lungs of mice colonized after intravenous injection, which was diminished by silencing ZEB1. Inversely, triggering de novo expression of Snail in MDA-MB-468 increased TF, coagulant properties and early metastasis in mice. Using TF promoter reporter vectors (mutated at different potential ZEB1 and Snail transcription factors) and CHIP, we further identified a binding region for ZEB1 in the proximal TF promoter encompassing a MCAT box, suggesting that ZEB1 and YAP might interact to regulate TF expression. Ongoing experiments are being performed to verify this hypothesis. We also collected data suggesting that targeting this new EMT/TF axis with anti-EMT drugs may represent an important extension to global anticoagulant strategies against cancer. Citation Format: Morgane Bourcy, Nicolas Skrypek, Erik W. Thompson, Geert Berx, Brett Hollier, Myriam Polette, Christine Gilles. EMT transcription factors induce tissue factor expression and pro-coagulant properties, supporting early metastasis of circulating tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-147. doi:10.1158/1538-7445.AM2017-LB-147

Abstract 3954: Regulation of pro-angiogenic and pro-inflammatory cytokines in tumor cells: Implication of epithelial-to-mesenchymal transitions .

Increasing data suggest a contribution of epithelial-to-mesenchymal transitions in specific steps of the metastatic cascade, particularly in tumor invasion and intravasation, and in the release of circulating tumor cells (CTCs) in the blood stream. The completion of these steps largely relies on interactions disseminating tumor cells establish with host cells. Because cytokines are particularly implicated in tumor-host cross-talks, we have examined whether EMT programs could be involved in the regulation of pro-inflammatory and pro-angiogenic cytokines which could favor angiogenesis, intravasation and CTC release. To investigate EMT implication in cytokine regulation, we used two cell lines, MDA-MB-468 (a mammary adenocarcinoma cell line) and A549 (a lung carcinoma cell line). These cell lines were shown to undergo EMT-changes following EGF and TGF-β treatment respectively, characterized by an upregulation of vimentin, snail and slug, and a downregulation of E-cadherin. A cytokine array on cell supernatant showed that EMT induction in these cell lines is associated with the upregulation of a consistant panel of cytokines. We then confirmed that interleukin-8 (IL-8), interleukin-6 (IL-6) and Granulocyte Monocyte-Colony Stimulating Factor (GM-CSF) are upregulated during EMT in the two carcinoma cell lines by RT-qPCR and ELISA. Emphasizing a functional role of such regulations, conditioned medium from EMT-induced MDA-468 cells was shown to stimulate HUVEC cell migration in a boyden chamber assay. In order to investigate whether slug and snail EMT transcription factors are involved in the regulation of IL-8, IL-6 and GM-CSF, we used both siRNA and cDNA transfection approaches. Combined repression of snail and slug inhibited the EMT-associated upregulation of IL-8, IL-6 and GM-CSF, suggesting that snail and slug are necessary for this process. Ectopic expression of each transcription factor was not sufficient to induce cytokines expression. However, ectopic expression of a proteasome degradation-resistant mutant of snail was sufficient to induce expression of IL-6, IL-8 and GM-CSF, suggesting that the stabilization of snail is necessary to regulate the expression of these cytokines. We thus here demonstrate that EMT is associated with an overexpression of specific pro-angiogenic and pro-inflammatory cytokines. We further emphasize a role of snail and slug transcription factors in this regulatory process. These data suggest that EMT programs could directly contribute to the stimulation of angiogenesis and inflammatory cell recruitment in the tumor microenvironment. The impact of such regulations on angiogenesis and the metastatic spread is currently under investigation. Citation Format: Meggy Suarez-Carmona, Morgane Bourcy, Jean-Michel Foidart, Philippe Delvenne, Agnes Noel, Myriam Polette, Christine Gilles. Regulation of pro-angiogenic and pro-inflammatory cytokines in tumor cells: Implication of epithelial-to-mesenchymal transitions . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3954. doi:10.1158/1538-7445.AM2013-3954