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

<p>Abstract</p> <p>Background</p> <p>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).</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer.</p

Abstract 275: Hypoxic Conditions Regulate the Expression and Activity of Coagulation Factors Important in Atherothrombosis

Neovascularization is an important feature in advanced atherosclerosis. It is recognized by the formation of new blood vessels recruited from the adventitia that penetrate the plaque, presumably due to lack of oxygen (hypoxia) in the plaque core. These immature, leaky neovessels can cause intraplaque hemorrhages, and promote plaque vulnerability and rupture. Plaque rupture leads to activation of coagulation and the formation of a thrombus, which is the main contributor to acute manifestations, morbidity, and mortality in atherosclerotic disease. The extrinsic coagulation activator tissue factor (TF) and its inhibitor tissue factor pathway inhibitor (TFPI) have been detected in atherosclerotic lesions, and are expressed by macrophages, smooth muscle cells, and endothelial cells overlaying the plaques. We aimed to investigate how hypoxic conditions affected the endothelial expression and activity of coagulation factors important in the initiation of coagulation. Hypoxia was induced in primary human umbilical (HUVEC) and aortic (HAEC) endothelial cells with chemicals (DMOG/CoCl2) or 1% oxygen (O2) concentration, and mRNA and protein expressions were measured using qRT-PCR and ELISA, respectively. Microscopy of fluorescence labeled cells was used to visualize cell-associated TFPI. Cell-surface FXa activity was measured using a two-stage chromogenic substrate method. Results showed that hypoxia reduced the TFPI mRNA and protein levels in a dose-dependent manner. The effect was apparent on all the protein pools of TFPI; the secreted, the cell-surface associated, and the intracellularly stored, indicating that the regulation occurred at the transcriptional level. The TF mRNA expression was also affected in a dose-dependent way, where low doses of DMOG and CoCl2 resulted in an increased expression, while high dose of DMOG and 1% O2 reduced the TF mRNA levels. Activated endothelial cells showed a dose-dependent regulation of TF expression following hypoxic stimulation, which is identical to the regulation of the FXa activity in the cells. Our findings indicate that hypoxic conditions can influence the coagulant activity of endothelial cells, which may potentially promote clinical events and thus the severity of atherosclerotic disorders.</jats:p

Identification and evaluation of Pharmacological enhancers of the factor VII p.Q160R variant

Abstract Congenital factor (F) VII deficiency is caused by mutations in the F7 gene. The p.Q160R variant manifests with bleeding episodes due to reduced FVII activity and antigen in patient plasma, most likely caused by protein misfolding and intracellular retention. As current replacement therapy is expensive and requires frequent intravenous injections, there is an unmet need for new and less invasive therapeutic strategies. Drug repurposing allows for rapid, more cost-effective discovery and implementation of new treatments, and identification of pharmacological enhancers of FVII variant activity would be of clinical importance. High-throughput screening of > 1800 FDA-approved drugs identified the orally available histone deacetylase inhibitor abexinostat and the inhaled surfactant tyloxapol as enhancers of FVII p.Q160R variant activity. The positive hits were verified in an in vitro cell model transiently expressing wild type or variant FVII and ex vivo in patients’ plasma. Both drugs showed a dose-response effect on FVII antigen and activity levels in conditioned cell medium and on FVII activity in patients’ plasma. In conclusion, the efficacy of the FDA-approved drugs abexinostat and tyloxapol in enhancing FVII variant activity constitute a proof of principle for high-throughput identification of drugs that may be feasible for novel treatment of FVII deficiency