Mari Tinholt

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

BackgroundIncreased 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).MethodsStable 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.ResultsDownregulation 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.ConclusionsOur results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

BackgroundTissue 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.MethodsTFPIα, 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.ResultsThe 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.ConclusionGPI-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.

Increased coagulation activity and genetic polymorphisms in the F5, F10 and EPCR genes are associated with breast cancer: a case-control study.

BackgroundThe 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.Methods366 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.ResultsFour 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.ConclusionsA 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.

Tumor expression, plasma levels and genetic polymorphisms of the coagulation inhibitor TFPI are associated with clinicopathological parameters and survival in breast cancer, in contrast to the coagulation initiator TF

IntroductionHypercoagulability in malignancy increases the risk of thrombosis, but is also involved in cancer progression. Experimental studies suggest that tissue factor (TF) and tissue factor pathway inhibitor (TFPI) are involved in cancer biology as a tumor- promoter and suppressor, respectively, but the clinical significance is less clear. Here, we aimed to investigate the clinical relevance of TF and TFPI genetic and phenotypic diversity in breast cancer.MethodsThe relationship between tumor messenger RNA (mRNA) expression and plasma levels of TF and TFPI (α and β), tagging single nucleotide polymorphisms (tagSNPs) in F3 (TF) (n = 6) and TFPI (n = 18), and clinicopathological characteristics and molecular tumor subtypes were explored in 152 treatment naive breast cancer patients. The effect of tumor expressed TF and TFPIα and TFPIβ on survival was investigated in a merged breast cancer dataset of 1881 patients.ResultsProgesterone receptor negative patients had higher mRNA expression of total TFPI (α + β) (P = 0.021) and TFPIβ (P = 0.014) in tumors. TF mRNA expression was decreased in grade 3 tumors (P = 0.003). In plasma, total TFPI levels were decreased in patients with larger tumors (P = 0.013). SNP haplotypes of TFPI, but not TF, were associated with specific clinicopathological characteristics like tumor size (odds ratio (OR) 3.14, P = 0.004), triple negativity (OR 2.4, P = 0.004), lymph node spread (OR 3.34, P = 0.006), and basal-like (OR 2.3, P = 0.011) and luminal B (OR 3.5, P = 0.005) molecular tumor subtypes. Increased expression levels of TFPIα and TFPIβ in breast tumors were associated with better outcome in all tumor subtypes combined (P = 0.007 and P = 0.005) and in multiple subgroups, including lymph node positive subjects (P = 0.006 and P = 0.034).ConclusionsThis study indicates that genetic and phenotypic variation of both TFPIα and TFPIβ, more than TF, are markers of cancer progression. Together with the previously demonstrated tumor suppressor effects of TFPI, the beneficial effect of tumor expressed TFPI on survival, renders TFPI as a potential anticancer agent, and the clinical significance of TFPI in cancer deserves further investigation.

Effect of hypoxia on tissue factor pathway inhibitor expression in breast cancer.

Essentials A hypoxic microenvironment is a common feature of tumors that may influence activation of coagulation. MCF‐7 and SK‐BR‐3 breast cancer cells and breast cancer tissue samples were used. The results showed transcriptional repression of tissue factor pathway inhibitor expression in hypoxia. Hypoxia‐inducible factor 1α may be a target for the therapy of cancer‐related coagulation and thrombosis.

Syndecan-3 and TFPI Colocalize on the Surface of Endothelial-, Smooth Muscle-, and Cancer Cells

Background Tissue factor (TF) pathway inhibitor (TFPI) exists in two isoforms; TFPIα and TFPIβ. Both isoforms are cell surface attached mainly through glycosylphosphatidylinositol (GPI) anchors. TFPIα has also been proposed to bind other surface molecules, like glycosaminoglycans (GAGs). Cell surface TFPIβ has been shown to exert higher anticoagulant activity than TFPIα, suggesting alternative functions for TFPIα. Further characterization and search for novel TFPI binding partners is crucial to completely understand the biological functions of cell associated TFPI. Methods and Results Potential association of TFPI to heparan sulphate (HS) proteoglycans in the syndecan family were evaluated by knock down studies and flow cytometry analysis. Cell surface colocalization was assessed by confocal microscopy, and native PAGE or immunoprecipitation followed by Western blotting was used to test for protein interaction. Heparanase was used to enzymatically degrade cell surface HS GAGs. Anticoagulant potential was evaluated using a factor Xa (FXa) activity assay. Knock down of syndecan-3 in endothelial,- smooth muscle- and breast cancer cells reduced the TFPI surface levels by 20-50%, and an association of TFPIα to syndecan-3 on the cell surface was demonstrated. Western blotting indicated that TFPIα was found in complex with syndecan-3. The TFPI bound to syndecan-3 did not inhibit the FXa generation. Removal of HS GAGs did not release TFPI antigen from the cells. Conclusions We demonstrated an association between TFPIα and syndecan-3 in vascular cells and in cancer cells, which did not appear to depend on HS GAGs. No anticoagulant activity was detected for the TFPI associated with syndecan-3, which may indicate coagulation independent functions for this cell associated TFPI pool. This will, however, require further investigation.

Determinants of acquired activated protein C resistance and D-dimer in breast cancer

BACKGROUND We have previously reported acquired activated protein C (APC) resistance and elevated plasma D-dimer levels in breast cancer patients. Here, we aimed to identify phenotypic and genetic determinants that contribute to the acquired APC resistance and increased D-dimer levels in breast cancer. Healthy controls served as reference. We also addressed whether higher APC resistance or D-dimer levels could be potential markers of clinicopathological breast cancer characteristics. MATERIALS AND METHODS 358 breast cancer patients and 273 healthy controls were enrolled and hemostatic plasma parameters were determined; factor (F) V, FVIII, FIX, FX, fibrinogen, von Willebrand factor (VWF), normalized APC sensitivity ratio (n-APC-sr), protein C, protein S, antithrombin, tissue factor pathway inhibitor (TFPI), and D-dimer. Common single nucleotide polymorphisms were genotyped in coagulation-related genes in the breast cancer patients. RESULTS The phenotypic hemostatic factors explained 25% and 31% of the variability in acquired APC resistance and D-dimer levels, respectively, in the breast cancer patients. Fibrinogen (β=-0.35, P<0.001), protein C (β=0.28, P<0.001), and FIX (β=0.22, P=0.026) were identified as determinants of n-APC-sr (in FV Leiden non-carriers), whereas TFPI (β=0.28, P<0.001), antithrombin (β=-0.25, P<0.001), and FX (β=0.15, P=0.040) were the major determinants of D-dimer. Moreover, borderline higher APC resistance (>75th percentile) was found in patients with triple negative tumors (odds ratio (OR) 1.97, 95% CI 0.99-3.90). CONCLUSIONS This study reports phenotypic hemostatic parameters that determine acquired APC resistance and D-dimer levels in breast cancer patients. The explanatory power was modest, however, our findings are hypothesis generating and may contribute to further understand the background for cancer associated-coagulopathy and thrombosis.

Polymorphisms of the coagulation system and risk of cancer

Hypercoagulability is a frequently finding in patients with cancer, and is associated with an increased risk of venous thrombosis (VT). Cancer-associated VT is associated with poor prognosis and represents the leading non-cancer cause of death among these patients. Conversely, patients experiencing VT are at increased risk of subsequent cancer, suggesting an epidemiological bidirectional link between cancer and hemostasis, and indicating a role of the hemostatic system in cancer development. How the coagulation system relates to cancer etiology at the genetic level is largely unexplored. Data on the association of polymorphisms in genes involved in coagulation with cancer development is important to clarify the role of the coagulation system in cancer pathogenesis. Effects of coagulation-related gene polymorphisms on cancer risk may possibly be translated into novel treatment- and prevention strategies of cancer-associated thrombosis and the cancer itself. This article reviews the current knowledge of the relation between polymorphisms in genes involved in coagulation and cancer risk in solid tumors.

Subtype-specific clinical and prognostic relevance of tumor-expressed F5 and regulatory F5 variants in breast cancer: the CoCaV study

Essentials The role of coagulation factor V (encoded by F5) in cancer pathogenesis is unknown. The clinical significance of tumor‐expressed F5 was evaluated in breast cancer patient cohorts. F5 was expressed in human breast tumors, and the expression was higher than in normal tissue. High F5 expression was associated with aggressive tumors, but also with survival in breast cancer.