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Cancer enhances the risk of venous thromboembolism, but a hypercoagulant microenvironment also promotes cancer progression. Although anticoagulants have been suggested as a potential anticancer treatment, clinical studies on the effect of such modalities on cancer progression have not yet been successful for unknown reasons. In normal physiology, complex formation between the subendothelial-expressed tissue factor (TF) and the blood-borne liver-derived factor VII (FVII) results in induction of the extrinsic coagulation cascade and intracellular signaling via protease-activated receptors (PARs). In cancer, TF is overexpressed and linked to poor prognosis. Here, we report that increased levels of FVII are also observed in breast cancer specimens and are associated with tumor progression and metastasis to the liver. In breast cancer cell lines, tumor-expressed FVII drives changes reminiscent of epithelial-to-mesenchymal transition (EMT), tumor cell invasion, and expression of the prometastatic genes, SNAI2 and SOX9. In vivo, tumor-expressed FVII enhanced tumor growth and liver metastasis. Surprisingly, liver-derived FVII appeared to inhibit metastasis. Finally, tumor-expressed FVII-induced prometastatic gene expression independent of TF but required a functional endothelial protein C receptor, whereas recombinant activated FVII acting via the canonical TF:PAR2 pathway inhibited prometastatic gene expression. Here, we propose that tumor-expressed FVII and liver-derived FVII have opposing effects on EMT and metastasis.

This study was performed to determine whether murine alternatively spliced tissue factor (masTF) acts analogously to human alternatively spliced tissue factor (hasTF) in promoting neovascularization via integrin ligation. Immunohistochemical evaluation of a spontaneous murine pancreatic ductal adenocarcinoma model revealed increased levels of masTF and murine full-length tissue factor (mflTF) in tumor lesions compared with benign pancreas; furthermore, masTF colocalized with mflTF in spontaneous aortic plaques of Ldlr(-/-) mice, indicating that masTF is likely involved in atherogenesis and tumorigenesis. Recombinant masTF was used to perform in vitro and ex vivo studies examining its integrin-mediated biologic activity. Murine endothelial cells (ECs) rapidly adhered to masTF in a β3-dependent fashion. Using adult and embryonic murine ECs, masTF potentiated cell migration in transwell assays. Scratch assays were performed using murine and primary human ECs; the effects of masTF and hasTF were comparable in murine ECs, but in human ECs, the effects of hasTF were more pronounced. In aortic sprouting assays, the potency of masTF-triggered vessel growth was undistinguishable from that observed with hasTF. The proangiogenic effects of masTF were found to be Ccl2-mediated, yet independent of vascular endothelial growth factor. In murine ECs, masTF and hasTF upregulated genes involved in inflammatory responses; murine and human ECs stimulated with masTF and hasTF exhibited increased interaction with murine monocytic cells under orbital shear. We propose that masTF is a functional homolog of hasTF, exerting some of its key effects via β3 integrins. Our findings have implications for the development of murine models to examine the interplay between blood coagulation, atherosclerosis and cancer.

It is now widely recognized that a strong correlation exists between cancer and aberrant hemostasis. Patients with various types of cancers, including pancreatic, colorectal, and gastric cancer, often develop thrombosis, a phenomenon commonly referred to as Trousseau syndrome. Reciprocally, components from the coagulation cascade also influence cancer progression. The primary initiator of coagulation, the transmembrane receptor tissue factor (TF), has gained considerable attention as a determinant of tumor progression. On complex formation with its ligand, coagulation factor VIIa, TF influences protease-activated receptor-dependent tumor cell behavior, and regulates integrin function, which facilitate tumor angiogenesis both in vitro and in mouse models. Furthermore, evidence exists that an alternatively spliced isoform of TF also affects tumor growth and tumor angiogenesis. In patient material, TF expression and TF cytoplasmic domain phosphorylation correlate with disease outcome in many, but not in all, cancer subtypes, suggesting that TF-dependent signal transduction events are a potential target for therapeutic intervention in selected types of cancer. In this review, we summarize our current understanding of the role of TF in tumor growth and metastasis, and speculate on anticancer therapy by targeting TF.

Abstract 1135 Background. Aside from hemostatic maintenance, Tissue Factor (TF) also plays a major role in such pathophysiological processes as thrombogenesis and cancer progression. TF protein occurs naturally in two forms: full length TF (flTF), a well–studied integral membrane glycoprotein that serves as an obligatory enzymatic co-factor of the serine protease FVIIa, and alternatively spliced TF (asTF), which lacks a transmembrane domain and can be secreted. asTF has a unique 40 amino acid C-terminus and can be detected, alongside flTF, in organized arterial thrombi (Bogdanov et al, Nat Med 2003 Apr; 9(4):458-62). Following the discovery of human asTF, the murine form of asTF (masTF) was identified and characterized (Bogdanov et al, J Thromb Haemost. 2006 Jan;4(1):158-67). Like human asTF, masTF lacks a transmembrane domain due to the exclusion of exon 5 from the primary transcript during its splicing, possesses a unique 93 amino acid C-terminus, and exhibits minimal coagulant potential. Most recently, hasTF was discovered to induce cell adhesion and angiogenesis via integrin ligation, independent of FVIIa, PAR-2 cleavage and/or any other proteolytic events (van den Berg et al, Proc Natl Acad Sci U.S.A. 2009 Nov 17;106(46):19497-502). It has yet to be investigated whether masTF exhibits non-proteolytic biologic activity analogous to that of hasTF. As murine models comprise the preferred in vivo platform in preclinical cardiovascular and cancer research, it is highly warranted to ascertain these possible functional properties of masTF. In this study, we performed an initial set of experiments to address this issue. Results. N-terminally His-tagged recombinant masTF mature protein was generated in E. Coli, purified, and assessed by Coomassie staining; masTF's identity was successfully verified by western blotting. Analogously to hasTF, masTF induced adhesion of murine endothelial cells (bEnd.3) in a time-dependent fashion. A 15-fold increase over BSA (n = 3; p < 0.0001) was observed as early as 1 hour after the experiment's onset; at 4 hours, bEnd.3 cells exposed to masTF displayed a 37-fold increase over BSA (p < 0.0005). We noted that masTF also induced bEnd.3 cells to display characteristic endothelial morphology, whereas BSA did not. We subsequently used a wound healing assay to ascertain whether masTF promotes directional migration of bEnd.3 cells, and used VEGF (100 ng/mL) as a positive control to assess the degree of potency. Per wound, the area of complete closure generated by masTF (50 nM) was ∼4.9-fold greater than that of the vehicle (n = 3; p < 0.0001), an effect similar to that elicited by VEGF (area of closure ∼7-fold greater than that of the vehicle; p < 0.0001). Microarray analysis of masTF-treated bEnd.3 cells (Affymetrix Gene 1.0 ST platform) revealed upregulation of the genes encoding multiple CXC chemokines, with CXCL2, CXCL10, and CXCL1 topping the list (R = 1.42, 1.37, and 1.32, respectively). Notably, expression of the major adhesion molecule VCAM-1 was also upregulated (R = 1.3), suggesting that masTF may promote interactions between murine endothelial cells and leukocytes. Indeed, we found that stimulating bEnd.3 cells with 50 nM masTF caused J774A.1 cells (murine monocytes/macrophages) to adhere with a 75% increased affinity (n = 3; p = 0.0001 vs. vehicle) when exposed to orbital shear conditions. Depletion of masTF from the medium by Ni-charged beads and denaturation by heat eliminated increased monocyte adhesion, while addition of polymyxin B and non-charged beads had no effect, confirming that the observed biologic phenomena were elicited by the masTF protein. Conclusions. We report, for the first time, that murine asTF appears to possess non-proteolytic biologic properties analogous to those of human asTF, which indicates that the alternatively spliced TF may be a general cell agonist eliciting changes in gene expression via integrin ligation. We are currently investigating whether the endothelial surface molecules interacting with masTF, as well as the intracellular signaling pathways activated by masTF in endothelial cells, are analogous to those engaged by hasTF. Disclosures: No relevant conflicts of interest to declare.

Tissue factor (TF) is traditionally known as the initiator of blood coagulation, but TF also plays an important role in inflammatory processes. Considering the pivotal role of coagulation in inflammatory bowel disease, we assessed whether genetic ablation of TF limits experimental colitis. To this end, wild-type and TF-deficient (TFlow) mice were treated with 1.5% dextran sulfate sodium (DSS) for 7 d, and effects on disease severity, cytokine production and leukocyte recruitment were examined. Clinical and histological parameters showed that the severity of colitis was reduced in both heterozygous and homozygous TFlow mice compared with controls. Most notably, edema, granulocyte numbers at the site of inflammation and cytokine levels were reduced in TFlow mice. Although anticoagulant treatment with dalteparin of wild-type mice reduced local fibrin production and cytokine levels to a similar extent as in TFlow mice, it did not affect clinical and histological parameters of experimental colitis. Mechanistic studies revealed that IF expression did not influence the intrinsic capacity of granulocytes to migrate. Instead, TF enhanced granulocyte migration into the colon by inducing high levels of the granulocyte chemoattractant keratinocyte-derived chemokine (KC). Taken together, our data indicate that TF plays a detrimental role in experimental colitis by signal transduction-dependent KC production in colon epithelial cells, thereby provoking granulocyte influx with subsequent inflammation and organ damage. (C) 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org Online address: http://www.molmed.org doi: 10.2119/molmed.2011.00138

Abstract 343 Background: Alternatively spliced Tissue Factor (asTF) is a soluble form of Tissue Factor (TF) that lacks the transmembrane domain and exhibits low pro-coagulant activity compared to decrypted full-length TF (Bogdanov et al, Nat Med 2003). Human asTF was recently shown to ligate integrins α6β1 and αVβ3 on endothelial cell surfaces, which triggered neovascularization in vitro, ex vivo, and in vivo in fVIIa- and/or PAR-2 independent fashion (van den Berg et al., PNAS 2009). This principally novel, non-proteolytic biologic activity of asTF was found to activate multiple integrin-linked kinases in human and murine macrovascular endothelial cells (EC), including those comprising the PI3K/Akt pathway. Engagement of PI3K/Akt signaling is known to activate NFkB – a transcription factor involved in upregulation of multiple leukocyte adhesion molecules that play a major role in various disease states, most notably atherogenesis and tumorigenesis. In this study, we sought to determine whether human asTF acts as an agonist on microvascular EC – the endothelial sub-type most relevant to monocyte egress from the circulation. Results: Primary cardiac and retinal human microvascular endothelial cells (micro-EC) expressed α6, β1, and β3 mRNA. Both micro-EC sub-types rapidly adhered to the recombinant N-terminally His-tagged human asTF: a 20+ fold increase in the number of adherent micro-EC was observed as early as 30 min after the cells were added (n=3,100 ng asTF vs BSA, p Conclusions: We show for the first time that (i) human asTF ligates β1 integrin(s) on primary human micro-EC, (ii) this leads to transcriptional upregulation of CAMs, and (iii) this upregulation promotes adhesion and transendothelial migration of monocytic cells under physiologically relevant conditions. We note that the concentrations of asTF used here were in the range found in cervical cancer tissue (van den Berg et al., PNAS 2009). Our results expand the potential scope of asTF's contribution to atherogenesis and tumorigenesis. Evaluation of these findings in vivo is highly warranted as monocyte recruitment triggered by asTF may represent a novel nexus in atherosclerotic progression and/or tumor growth. Disclosures: No relevant conflicts of interest to declare.