**3. Tissue factor**

Besides its major role in hemostasis, TF has been identified as an important signaling receptor in cancer biology. Ample preclinical evidence has accumulated over the past decade, impli‐ cating TF as an important effector in the processes of tumor initiation, growth, angiogenesis, and metastasis. Moreover, this has led to the development of approaches exploring TF as a potential target for anticancer therapy [71]. TF is overexpressed in many types of human tumors, including breast cancer, pancreatic cancer, gastric cancer, prostate cancer, colorectal cancer, non-small-cell lung cancer, melanoma, leukemia, lymphoma, esophageal cancer, hepatocellular carcinoma, brain glioblastoma, but not in their normal tissue counterparts [1]. TF overexpression in cancer cells has been correlated with tumor progression and unfavorable prognostic indicators such as increased angiogenesis, advanced disease stage, and resistant phenotype [72–74]. Therefore, TF overexpression in situ could be considered a biomarker for solid tumors.

Enhanced TF expression in cancer has been reported to be an oncogenic-driven event. In colorectal cancer, activation of the K-ras oncogene and loss-of-function mutation of p53 result in constitutive activation of mitogen-activated protein kinase (MAPK) and PI3K pathways leading to increased TF expression [75]. In turn, inhibition of the PI3K and MAPK by restora‐ tion of the PTEN tumor suppressor gene in glioma cells downregulates TF expression dependent on EGFR amplification [76]. In medulloblastoma cell lines, TF expression has been shown to result from mutation in the c-Met oncogene and subsequent activation of Src kinases [77]. It has been observed that a certain subset of tumor cells, known as cancer stem cells, which constitutively express activated oncogenes and are capable of undergoing multilineage differentiation, are characterized by TF abundant phenotype [5]. Moreover, enhanced TF expression is observed during the processes of epithelial-to-mesenchymal-transition, whereby epithelial cells acquire a mesenchymal, more aggressive and motile phenotype [78]. This indicates that TF is possibly involved in maintaining cancer cell self-perpetuance.

There is a structure function dependency in TF mode of action. TF plays a role in cancer progression both by initiating tumor growth and by promoting efficient tumor cell dissemi‐ nation. Tumor-promoting activities of TF occur via non-hemostatic mechanism and can be attributed to the cytoplasmic domain signaling dependent mostly on the activation of the protease activated receptor 2 (PAR2). Prometastatic properties of TF can rather be coupled with its extracellular domain and the subsequent generation of thrombin, which, as a potent growth factor, exhibits further pleiotropic cellular effects.

TF-mediated signaling is critical for both physiological and pathological angiogenesis. TF deficiency in murine knockout experiments caused early embryonic lethality due to impaired vasculature development [79]. Zhang et al. demonstrated that tumors overexpressing TF become highly vascularized once implanted into mice and the observed growth induction could not be inhibited despite maximal anticoagulation [80]. It has been revealed that involve‐ ment of TF cytoplasmic domain in several transduction cascades accounts for the production by tumor cells of angiogenic cytokines and contributes to increased angiogenesis in a paracrine fashion [81]. Formation of the complex TF/VIIa leads to increase of intracellular Ca2+ and phosphorylation of serine residues on TF cytoplasmic tail. This triggers signaling via the Gprotein coupled membrane receptor PAR2 that activates MAPK and PI3K transduction cascades, resulting in increased gene expression of the angiogenic cytokines VEGF, VEGF-C, CXCL1, Il-8, and Cyr61 [82–84]. In addition to PAR2 signaling, the cytoplasmic domain of TF can be phosphorylated independently of f.VII ligand binding by protein kinase C (PKC) resulting in the transcriptional activation of VEGF, VEGFR, TGF, and suppressed expression of anti-angiogenic molecules such as thrombospondin [85, 86]. The relationship between TF and VEGF has been extensively studied and is manifested by reciprocal co-stimulation of their expression profiles. VEGF induces TF expression by orchestrating the binding of nuclear factors NFAT and AP-1 to the promoter region of TF gene [87]. Regulation of VEGF is in turn dependent on the TF cytoplasmic domain signaling, which is demonstrated by the finding that tumor cells transfected with truncated TF cDNA lacking the cytoplasmic domain fail to produce VEGF, but preserve the TF procoagulant function [86]. Experimental ex vivo and in vivo studies have further supported the TF-VEGF interrelationship by finding increased coexpression on tumor sections and their association with increased angiogenesis and malignant potential in human tumors [73–75, 88, 89].

TF enhances tumor growth via TF/VIIa/PAR2 signaling. This is evidenced by studies on cancer cell lines, where overexpression of TF by cancer cells conferred growth advantage compared to cell lines expressing low levels of TF [71, 86]. Delay in primary and metastatic tumor growth was observed after specific TF/VIIa inhibition with the anticoagulant rNAPc2 but not after inhibition of f.Xa [90]. Studies on selective targeting of different domains of the complex TF:VIIa revealed that PAR2 signaling and integrin ligation is sufficient for TF tumor-promoting properties [91].

Prometastatic properties of TF can be attributed to its extracellular domain, which is required for its major role in triggering coagulation. The extracellular mutant domain (TFmut) has markedly diminished function for activation of f.X, while full-length or cytoplasmic taildeleted TF retains its procoagulant activity [85, 92]. The expression of TF in tumors can induce downstream coagulation activation via the TF/VIIa/Xa pathway leading to fibrin and tumor stroma formation. Tumor cells become encapsulated in fibrin and platelet-rich thrombi, being protected from the host immune defense and arrest in the microcirculation. Local thrombin formation facilitates arrest of tumor cells to the vessel wall by upregulation of adhesion molecules and strengthening cell-to-cell junctions [93]. Fibrin matrix in the tumor stroma builds itself a multifunctional scaffold rich on growth factors such as platelet derived growth factor (PDGF), transforming growth factor (TGF), fibroblast growth factor (FGF), that is not only protective, but also promotes matrix-cell interactions necessary for neovascularization [94]. Thrombin, constitutively generated by the activated coagulation cascade in the tumor surrounding, mobilizes the adhesion molecules αIIbβ3-integrin, P-selectin, CD40 ligand, and enhances tumor cell interactions to platelets, endothelial cells and matrix [95–97]. Thrombin has also an important function in angiogenesis by inducing the activation of endothelialsecreted collagenase type IV, which degrades basement matrix proteins and collagen during neoangiogenesis [98]. In vivo experimental models of metastasis demonstrated dramatic increase of lung metastasis with thrombin-treated tumor cells compared with untreated cells [99].

In summary, TF-mediated effects either in a coagulation-independent mechanism via direct cytoplasmic domain signaling and TF/VIIa/PAR2 or dependent on the coagulation products induce diverse sets of cellular responses inherent to tumor cells including tumor growth, neoangiogenesis, cell migration, and metastasis. Taking into account the specific biologic role of TF in the malignant tissue, detecting circulating TF in cancer patients might be informative of active disease and ongoing processes of matrix reorganization, cell destruction, and neovascularization.
