Background: Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231).Methods: Stable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion.Results: Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis.Conclusions: Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

The procoagulant state in cancer increases the thrombotic risk, but also supports tumor progression. To investigate the molecular mechanisms controlling cancer and hemostasis, we conducted a case-control study of genotypic and phenotypic variables of the tissue factor (TF) pathway of coagulation in breast cancer. 366 breast cancer patients and 307 controls were genotyped for SNPs (n = 41) in the F2, F3 (TF), F5, F7, F10, TFPI and EPCR genes, and assayed for plasma coagulation markers (thrombin generation, activated protein C (APC) resistance, D-dimer, antithrombin, protein C, protein S, and TF pathway inhibitor (TFPI)). Associations with breast cancer were evaluated using logistic regression to obtain odds ratios (ORs) and 95% confidence intervals (CIs), or the chi-square test. Four SNPs in F5 (rs12120605, rs6427202, rs9332542 and rs6427199), one in F10 (rs3093261), and one in EPCR (rs2069948) were associated with breast cancer. EPCR rs2069948 was associated with estrogen receptor (ER) and progesterone receptor (PR) positivity, while the SNPs in F5 appeared to follow hormone receptor negative and triple negative patients. The prothrombotic polymorphisms factor V Leiden (rs6025) and prothrombin G20210A (rs1799963) were not associated with breast cancer. High APC resistance was associated with breast cancer in both factor V Leiden non-carriers (OR 6.5, 95% CI 4.1-10.4) and carriers (OR 38.3, 95% CI 6.2-236.6). The thrombin parameters short lag times (OR 5.8, 95% CI 3.7-9.2), short times to peak thrombin (OR 7.1, 95% CI 4.4-11.3), and high thrombin peak (OR 6.1, 95% CI 3.9-9.5) predicted presence of breast cancer, and high D-dimer also associated with breast cancer (OR 2.0, 95% CI 1.3-3.3). Among the coagulation inhibitors, low levels of antithrombin associated with breast cancer (OR 5.7, 95% CI 3.6-9.0). The increased coagulability was not explained by the breast cancer associated SNPs, and was unaffected by ER, PR and triple negative status. A procoagulant phenotype was found in the breast cancer patients. Novel associations with SNPs in F5, F10 and EPCR to breast cancer susceptibility were demonstrated, and the SNPs in F5 were confined to hormone receptor negative and triple negative patients. The study supports the importance of developing new therapeutic strategies targeting coagulation processes in cancer.

Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA‐27a/b and microRNA‐494 through the estrogen receptor α. MicroRNA‐27a/b and microRNA‐494 are partly involved in estrogenic downregulation of TFPIα. Summary Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPIα. Methods Computational analysis of the TFPI 3′‐untranslated region (UTR) identified potential binding sites for miR‐19a/b, miR‐27a/b, miR‐494, and miR‐24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3′‐UTR by miR‐27a/b and miR‐494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17α‐ethinylestradiol (EE2) and fulvestrant on relative miR‐27a/b, miR‐494, and TFPI mRNA levels in MCF7 cells were determined by qRT‐PCR and secreted TFPIα protein by ELISA. Transient knockdown of ERα was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR‐19a, miR‐27a/b, miR‐494, and miR‐24 mRNA levels in MCF7 cells through ERα. miR‐27a/b and miR‐494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR‐27a/b and miR‐494 on TFPI mRNA. Impaired estrogen‐mediated downregulation of TFPI mRNA was detected in anti–miR‐27a/b and anti–miR‐494 transfected cells. Conclusions Our results provide evidence that miR‐27a/b and miR‐494 regulate TFPIα expression and suggest a possible role of these miRNAs in the estrogen‐mediated downregulation of TFPIα.

Background: Tissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIα and TFPIβ, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIα circulates in plasma. The functional importance of TFPIβ is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIα, TFPIβ, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells.Methods: TFPIα, TFPIβ, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay.Results: The expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIα and TFPIβ from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIα without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIα in the breast cancer cells.Conclusion: GPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIβ isoform.