**4. uPA/uPAR system**

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.

growth factor, exhibits further pleiotropic cellular effects.

10 Tumor Metastasis

potential in human tumors [73–75, 88, 89].

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

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

The physiologic role of fibrinolysis is dissolution of the fibrin clot and collagen degradation exerted by the action of plasmin. Generation of plasmin, the main enzyme in fibrinolysis, occurs upon activation of plasminogen by the tissue plasminogen activator (tPA) and the urokinase plasminogen activator (uPA).Therefore, function of the urokinase plasminogen activator (uPA) and its high-affinity cellular receptor (uPAR) is critical for fibrinolytic activities including targeted degradation of the basement matrix. Moreover, uPAR is motile within the cellular membrane, which allows its allocation at the cellular front of desired direction for proteolysis [100]. Under normal conditions, the process of active proteolysis is tightly control‐ led by the proteolytic systems.

In cancer, biology activation of uPA/uPAR system is a prerequisite for efficient focal proteol‐ ysis, adhesion, migration and enables penetrating tumor cells to invade and metastasize [101]. Extracellular matrix proteolysis acts at all stages of the metastatic cascade: detachment of tumor cells from primary site, intravasation, hematogeneous dissemination, extravasation, and metastases formation. These processes are executed by proteolytic enzymatic systems, including uPA/uPAR, matrix metalloproteinases, and cysteine proteinases, which interact synergistically and are responsible for the complex proteolytic activity of tumors [102].

The cellular receptor for the urokinase plasminogen activator (uPAR) is a key molecule for efficient pericellular proteolysis. Apart from potentiating proteolytic activity, the complex uPA/uPAR ignites series of intracellular signaling events associated with the processes of proliferation, adhesion, chemotaxis, migration, and angiogenesis. Tissue overexpression of uPA/uPAR is found in various human tumors—breast, prostate, GIT, and lung. It is associated with advanced disease and is independent adverse prognostic factor for survival [103]. Direct involvement of uPAR in processes of tumor biology characterizes it as a hallmark of the malignant invasive phenotype. Overexpression of uPAR cDNA in osteosarcoma cells increases its ability to penetrate the basal membrane [104]. Invasive potential of tumor cells in chorionallantois membrane of chicken embryos correlates with uPAR-associated proteolytic activity [105]. Expression of uPAR gene by tumor cells is required for vascular intravasation, whereas uPAR gene expression decreases invasive potential of transformed fibroblasts in vitro [106]. Experiments with anti-uPAR inhibitory antibodies demonstrate reduction of the matric proteolytic activity [107, 108]. Levels and activity of uPAR are regulated at the transcriptional level by oncogene-controlled promoter activation. uPAR promoter region contains binding motifs for several transcriptional factors that regulate cellular differentiation, migration, and apoptosis: specific protein 1 (SP1), activator protein 1 (AP1) and activator protein 2 (AP2) [109]. uPAR basal expression is regulated proximally from SP1 transcriptional starting point. In tumor models of colorectal cancer, constitutive, and induced uPAR expression is regulated by AP1 binding motif via MAPK and c-JUN NH2-terminal kinase (JNK) signaling [110]. AP2 binding motif is required for constitutive overexpression of uPAR promoter activity in invasive tumor cells after stimulation by the tumor promoter phorbol acetate.

The role of K-ras and SRC oncogenes in uPAR regulation has been identified. K-ras regulates uPAR mediated proteolysis by transcriptional binding of AP1 to the promoter motif. Down‐ regulation of promoter activity as in deletion of the AP1 binding activity has been observed in tumor clones with K-ras allelic deletion. The knockout effect is accompanied by significant reduction of uPAR expression and tumor-associated proteolysis [111]. Increased uPAR protein expression and laminin degradation parallel to c-SRC activation are observed in SW 480 cells transfected to constitutively overexpress s-SRC. Elevated uPAR expression is due to tran‐ scriptional activation secondary to increased binding of SP1 to the complementary promoter motif. This defines SP1 as distal factor of c-SRC-mediated regulation of uPAR [109]. Tissue overexpression of uPA/uPAR has been detected in many human tumors like breast, prostate, gastrointestinal, and lung cancers. Oncogenes responsible for enhancement of uPA/uPAR expression in malignant tissue include ras, jun, myc, fos, rel, and ets. uPAR expression can also be stimulated by the expression of TF and the epidermal growth factor receptor (EGFR) [112].


**Table 1.** Coagulation function vs. role in tumor biology of hemostatic factors.

Fibrinolytic system components can be identified as determinants of invasion in tumor biology and reflect the metastatic potential of tumors.
