Epithelial–mesenchymal transitions (EMTs) generate hybrid phenotypes with an enhanced ability to adapt to diverse microenvironments encountered during the metastatic spread. Accordingly, EMTs play a crucial role in the biology of circulating tumor cells (CTCs) and contribute to their heterogeneity. Here, we review major EMT-driven properties that may help hybrid Epithelial/Mesenchymal CTCs to survive in the bloodstream and accomplish early phases of metastatic colonization. We then discuss how interrogating EMT in CTCs as a companion biomarker could help refine cancer patient management, further supporting the relevance of CTCs in personalized medicine.

Epithelial-mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3′-UTR-luciferase reporter vector assays implicated the 3′-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3′UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.

Previous work identified Tissue Factor (TF), a key activator of the coagulation cascade, as a gene induced in cellular contexts of Epithelial-Mesenchymal Transitions (EMTs), providing EMT+ Circulating Tumor Cells (CTCs) with coagulant properties that facilitate their metastatic seeding. Deciphering further molecular aspects of TF regulation in tumor cells, we report here that CD44 and TF coexpress in EMT contexts, and that CD44 acts as a regulator of TF expression supporting procoagulant properties and metastatic seeding. A transcriptional regulatory mechanism bridging CD44 to TF expression was further evidenced. Comparing different TF –promoter luciferase reporter constructs, we indeed found that the shortest -111 pb TF promoter fragment harboring three Specificity Protein 1 (Sp1) binding sites is still responsive to CD44 silencing. The observation that (i) mutation within Sp1 binding sites decreased the basal activity of the -111 pb TF promoter construct, (ii) CD44 silencing decreased Sp1 protein and mRNA levels and (iii) Sp1 silencing diminished TF expression further points to Sp1 as a key mediator linking CD44 to TF regulation. All together, these data thus report a transcriptional regulatory mechanism of TF expression by CD44 supporting procoagulant activity and metastatic competence of CTCs.

The aim here is to study the implication of the stem cell marker and fibrin receptor, CD44, in the regulation of tissue factor and its associated pro-coagulant and pro-metastatic properties in breast tumor cells. We examined this CD44/TF/coagulation axis in breast cancer EMT+ (MDA-MB-231, Hs578T), EMT- (MCF7, T47D) and EMT-inducible (EGF-treated MDA-MB-468, MDA-MB-468 harboring a doxycycline-inducible snail expression vector) cellular models. We used siRNA to inhibit CD44 expression and realized RT-qPCR, western blot, flow cytometry analyzes and promoter reporter assays to monitor regulations of our gene of interest. Furthermore, we realized in vitro clotting assays and TF activity assays to examine coagulant properties, and in vivo experimental metastasis assays to assess early metastatic potential. We first confirmed by WB, RT-qPCR and FACS analyzes that CD44 is over-expressed in our EMT+ and EMT-induced (EMTi) cellular models, concomitantly with TF expression. Using siRNAs, we showed that CD44 silencing in these same EMT+ and EMTi cellular models decreased both mRNA and protein levels of TF. Clotting assays and TF activity assays further showed that CD44 silencing decreased pro-coagulant properties of our EMT+ and EMTi cellular models in vitro and of their pro-metastatic properties in vivo. Examining further the molecular mechanisms of TF regulation by CD44, we employed truncated TF promoter reporter vectors to identify key transcription factors linking CD44 to TF transcription. These experiments revealed that a TF promoter region containing Sp1 binding sites is sufficient to mediate the diminution of TF promoter activity seen after CD44 silencing. Mutations in the Sp1 binding sites abrogated the activity of the TF reporter vector. Further pointing to Sp1 as a key transcription factor implicated in TF regulation by CD44, we observed that silencing CD44 inhibited Sp1 expression and that silencing Sp1 decreased TF mRNA levels. Our results thus demonstrate a key role of CD44 in regulating TF expression in EMT+ cancer cells, and point to Sp1 as a transcriptional mediator of this regulation. This modulation of TF expression by CD44 in EMT+ cells led to a concomitant regulation of pro-coagulant properties, and impacts metastatic potential. Citation Format: Amélie V. Villard, Anthony Genna, Christine Gilles. Regulation of tissue factor dependent procoagulant properties by CD44: Implication for metastasis of breast tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6336.