Tissue factor and tumor: Clinical and laboratory aspects

Clinica Chimica Acta 364 (2006) 12 – 21 www.elsevier.com/locate/clinchim

Invited critical review

Tissue factor and tumor: Clinical and laboratory aspects Yvonne Fo¨rster a, Axel Meye b, Sybille Albrecht c, Bernd Schwenzer a,* a

Institute of Biochemistry, Technical University Dresden, Bergstrabe 66 D-01069 Dresden, Germany b Department of Urology, Technical University Dresden, Dresden, Germany c Institute of Pathology, Technical University Dresden, Dresden, Germany Received 12 April 2005; received in revised form 13 May 2005; accepted 16 May 2005 Available online 2 September 2005

Abstract This review summarizes data demonstrating the role of TF in tumor development, metastasis and angiogenesis. TF is a transmembrane protein that is expressed constitutively in some kinds of extravascular cells and transiently in intravascular cells after stimulation with cytokines and growth factors. Originally TF was considered to have a function in the initiation of coagulation. In the last years it became evident that TF plays a role in physiological and pathological processes outside the hemostasis. Up-regulation of TF expression appears to be characteristic of tumor tissue. In a variety of human tumors it was shown by immunohistochemistry, that TF can be expressed in malignant cells as well as in tumor-infiltrating macrophages or endothelial cells. Such abnormal TF expression contributes to the angiogenic process by a shift in the balance between endogenous proangiogenic and antiangiogenic factors. Observations of a significant correlation between elevated TF expression with increased microvessel density and VEGF expression underline the TF involvement in tumor angiogenesis. Furthermore, TF expression influences also metastasis. The effect of TF on metastasis may result from its angiogenic effect, but also from the production of growth factors or adhesion proteins. D 2005 Elsevier B.V. All rights reserved. Keywords: Tissue factor; Tumor; Angiogenesis; Metastasis; Cancer diagnostic and therapy

Contents 1. 2. 3. 4.

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Introduction. . . . . . . . . . . . . . . . . . . . . . . Structure of TF gene, mRNA and protein . . . . . . . TF detection in plasma and tissues . . . . . . . . . . . TF and tumor. . . . . . . . . . . . . . . . . . . . . . 4.1. TF as a marker in oncology . . . . . . . . . . . 4.2. TF and metastasis . . . . . . . . . . . . . . . . 4.3. TF and tumor angiogenesis . . . . . . . . . . . Diagnostic and prognostic value of TF: limitations and TF as potential target for cancer therapies . . . . . . .

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Abbreviations: aa, Amino acids; bFGF, Basic fibroblast growth factor; CHO, Chinese hamster ovary; CD, Cytoplasmic domain; DIC, Disseminated intravascular coagulation; EGFR, Epidermal growth factor receptor; Egr-1, Early growth response gene-1; ELISA, Enzyme linked immunosorbent assay; ED, Extracellular domain; FVII, Factor VII; FX, Factor X; FXII, Factor XII; IF-a, Interferon-a; IF-g, Interferon-g; IL-8, Interleukin-8; IL-10, Interleukin-10; MD, Membrane spanning domain; MVD, Microvessel density; mTF, Monocyte tissue factor; NSCLC, Non-small cell lung cancer; PARs, Protease-activated receptors; PCA, Procoagulant activity; PDGF, Platelet-derived growth factor; RNAi, RNA interference; SCID, Severe combined immunodeficiency; siRNA, Small interfering RNA; TF, Human tissue factor; TSP-1, Thrombospondin-1; uTF, Urinary tissue factor; VEGF, Vascular endothelial growth factor. * Corresponding author. Tel.: +49 351 463 36447; fax: +49 351 463 35506. E-mail address: [email protected] (B. Schwenzer). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.05.018

Y. Fo¨rster et al. / Clinica Chimica Acta 364 (2006) 12 – 21

7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Human tissue factor (TF) is the cell surface receptor of factor VII (FVII) responsible for triggering blood coagulation [1,2]. Traditionally, TF is thought to initiate the extrinsic pathway of coagulation interacting with FVII to activate factor X (FX). FXa catalyses the conversion of prothrombin to thrombin resulting in the generation of fibrin clots from fibrinogen. The intrinsic pathway is initiated when factor XII (FXII) comes in contact with negatively charged surfaces resulting also in the generation of FXa [3,4]. However, the ‘‘cascade and waterfall hypothesis’’ does not accurately reflect hemostasis. Individuals deficient in one of the contact factors do not suffer bleeding problems, whereas patients with FVII deficiency bleed abnormally [3,5]. So, a revised theory of coagulation is evaluated involving linkages between the two pathways and incorporating the cell surface into the coagulation process [3,6 –8]. In the adult organism, TF is constitutively expressed in a variety of extravascular tissues. In addition, TF expression can be up-regulated transiently by growth factors and cytokines in intravascular cells as endothelial cells and monocytes [9 –12]. It is widely accepted that TF plays an essential role not only for coagulation but also outside the hemostasis. It seems to serve as a regulator of angiogenesis, tumor growth, and metastasis.

2. Structure of TF gene, mRNA and protein The human TF gene spans 12.4 kbp and is located on chromosomal region 1p21– 22 [13]. It is organized into six exons separated by five introns [14]. The first exon of the gene is encoding translation initiation by the 5V-region and the 32 residue leader sequence by the 3V-region. Exons two to five encode the extracellular domain (ED), while exon six provides the membrane spanning (MD) and cytoplasmic (CD) domains of the TF protein and a long 3V-untranslated region of the mRNA [13]. Transcription of the gene results in a 2.2/2.3 kb mRNA [15 –17]. Larger and less abundant species of TF mRNA were suggested to result from incomplete splicing of introns [16]. More than half of TF mRNA is 3V-non-coding sequence containing a highly AU-rich region [18]. The turnover of mRNA is normally very rapid (0.5 –1.5 h) and presumable related to this AU-rich destabilizing motifs [19]. The stability of TF mRNA in monocytes, endothelial cells and fibroblasts increases by inhibition of protein synthesis with cycloheximide, indicating that TF is in addition to a transcriptional regulation regulated also post-transcriptionally by mRNA degradation [20 – 23].

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18 18

The TF promoter contains five putative Sp1 sites, three Egr-1 sites that overlap with three Sp1 sites, two AP-1 sites and a NF-nB site. Functional studies indicate that Sp1 controls basal TF gene expression whereas c-Fos/c-Jun, c-Rel/ p65 and Egr-1 mediate inducible expression [24,25]. The TF protein consists of 295 amino acids from which 32 amino acids serve as a signal peptide for intracellular transport [15]. The mature peptide consists of 263 amino acids with an extracellular domain (residues 1 –219), a membrane spanning domain (residues 220 –242) and a cytoplasmic domain (residues 243– 263) [15 – 17]. The extracellular domain of TF has been classified as a member of cytokine receptor superfamily also including IF-a, IF-g and IL-10 receptors [26,27]. The crystal structure of the extracellular domain showed that this domain consists of two immunglobulin-like modules associated through a interdomain interface region [26,28], that contribute to the binding of FVIIa has been identified by mutagenesis [29,30]. The transmembrane domain is necessary for stabilization of the molecule [3], whereas the cytoplasmic domain plays a role in intracellular signaling as discussed later (Fig. 1). In addition to their role in fibrin formation, TF/FVIIacomplex as well as the downstream proteases FXa and thrombin initiate a cellular signal cascade by protease-activated receptors (PARs). PARs are members of a family of seven transmembrane domain surface receptors that mediate cell activation via G proteins [31]. The family consists of four members, PAR-1 to PAR-4 [32]. While thrombin activates PAR-1, -3 and -4 the TF/FVIIa complex activates PAR-2. Activation of PARs contributes to a variety of biological processes, including inflammation, angiogenesis, metastasis, and cell migration [8,32 – 34].

3. TF detection in plasma and tissues For immunochemical staining in tissues and measurement in body fluids numerous polyclonal and monoclonal antibodies were generated. A variety of these monoclonal antibodies was characterized and compared in a workshop [35], indicating that most of them are directed against two epitope areas of the TF extracellular domain. Quantitative measurement of TF in body fluids, culture supernatants or cell extracts became possible with the development of chromogenic assay [36], TF-RIA [37,38] and TF-ELISA [39 – 45]. Normal individuals have low levels of soluble tissue factor in the circulation [43,46]. The range detected by different ELISAs was from 108 pg/ml to 172 pg/ml [42 – 44,46 – 48]. TF can be released from cell surfaces by shedding of tissue factor-exposing microparticles from endothelial cells [49] and monocytes [50], which are capable of ex-

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Y. Fo¨rster et al. / Clinica Chimica Acta 364 (2006) 12 – 21

Fig. 1. Schematic illustration of TF gene, mRNA and protein. On the top the primary gene structure is presented. The following panels show the mRNA and the TF protein. The last panel shows the mature TF protein without the 32 amino acids (aa) leader sequence. ED extracellular domain (aa 1 – 219), MD membrane spanning domain (aa 220 – 242), CD cytoplasmic domain (aa 243 – 263).

pressing TF on their surface after stimulation. Platelets can internalize TF and release it after activation, but do not generate TF [51]. Plasma TF concentration has been reported to be increased in various diseases such as disseminated intravascular coagulation (DIC) [43,44,48], ischemic heart disease [52 – 54], antiphospholipid syndrome [55], liver-cirrhosis [56], in patients with chronic renal failure [47], in patients with microvascular complications of diabetes mellitus [57], and vasculitis [42], indicating that massive immunological reactions or tissue destruction increase TF release into the circulation. The procoagulant activity (PCA) of TF on cells or in biological fluids can be measured using clotting tests or chromogenic assays. Clotting tests determines the fibrin formation after addition of plasma to cells, whereas chromogenic assays can differentiate between the activity of FVII, FX or thrombin by specific peptides [58,59]. TF distribution in normal tissues has been investigated immunohistochemically with monoclonal [60] and polyclonal [61] antibodies in frozen sections. These studies confirmed the prominent expression of TF in tissues known to be rich in functional procoagulant TF activity, such as brain, lung and placenta. But the use of frozen tissue limited the ability to identify precisely the cells stained in these organs [61], whereas on microwaved paraffin sections a selective TF expression was detected [62]. The microtopologically prominent TF expression at biological boundaries gave rise to the

concept of a hemostatic envelope ready to activate coagulation when vascular integrity is disrupted [60].

4. TF and tumor 4.1. TF as a marker in oncology It is well known that cancer patients are at high risk for thrombosis. Increased TF expression was identified in many tumor types using immunohistochemistry such as glioma [63 – 65], breast cancer [66 – 72], lung cancer [73 – 76], colon cancer [77 –80], prostate cancer [81 – 83], pancreatic cancer [84,85], ovarian carcinoma [86], and hepatocellular carcinoma [87] on tumor cells and/or on tumor stroma cells. Tumor cells of epithelial origin often express more TF in comparison to nonepithelial malignant tumors [88]. This is not surprising because many normal epithelia express basal levels of TF. In some cases tumor cells express TF in the same way than the normal cells of this tissue. But more often malignant transformation results in alteration in antigen expression. That means that tumor cells originating from TFnegative cells express TF in the process of transformation or carcinoma cells lack TF, although the normal cells express TF. Expression of TF varies in the specimen within a tumor type, even among tumors of the same histological category. Even within one specimen tumor cells were heterogeneously stained [88]. Lung cancer cells derived from TF-positive

Y. Fo¨rster et al. / Clinica Chimica Acta 364 (2006) 12 – 21

bronchial epithelial cells were shown to express TF in the majority of the studies [73 – 76]. In contrast, in studies of mammary carcinomas the stromal cells (myofibroblasts, macrophages, in some studies also endothelial cells) were stained with anti-TF antibodies rather than tumor cells with high individual variety [66 – 72]. This coincides with the data that normal breast tissue express TF only rarely in stromal cell [88]. In addition to constitutively expressed TF, up-regulation of TF gene expression can be induced in malignant and normal host cells responding to inflammatory or tumorspecific signals. Therefore, the expression of TF on stroma cells (tumor-associated endothelial cell, tumor-infiltrating macrophages, fibroblasts) and tumor cells in the vicinity of host-tumor interface could be more interestingly for tumor growth and metastasis [68,69,89]. In pancreatic carcinoma TF expression occurred preferentially at the invasive front of the tumor [85]. Up-regulation of plasma TF concentration was also shown in breast cancer patients under chemo- and hormone therapy compared with normal controls [69]. In addition, the concentration of plasma TF was associated with tissue TF expression in both tumor and stroma cells [69]. The serum level of TF was elevated in patients with melanoma; however, it was not correlated with disease progression. These results suggest that TF was ubiquitously expressed in melanocytic cells and its expression was not correlated with disease progression and/or metastatic potency of melanoma cells [90]. Different studies showed higher levels of urinary tissue factor (uTF) in patients of bladder and prostate cancers in comparison to controls (patients with benign prostatic hyperplasia) [91,92], whereas others could discriminate between inflammatory and malignant disease only in bladder cancer [93]. Interestingly, both tumor entities have also increased monocyte TF (mTF) levels compared to controls, but no difference has been observed between malignant and benign inflammatory disease groups [93,94]. Furthermore, uTF and mTF level is also detectable in patients with breast and colorectal carcinomas [94,95]. Malignant and benign inflammatory groups, irrespective of the tumor entity, have higher uTF and mTF levels than controls. The increased mTF levels seem to be associated with higher tumor grade or stage and shorter survival. However, independent re-evaluation studies especially in prospective setting are needed. We conclude from these data that enhanced TF expression in tumors is not correlated to the TF level in plasma by normal shedding and destruction. But tumor related thrombotic disorders as well as massive tissue destruction after therapeutic treatment could cause abnormal enhancement of TF in the circulation. In our own study, the TF concentration in plasma or serum was not enhanced in patients with gastrointestinal tumors before surgery or chemotherapy (unpublished data), prostate and kidney tumor [96]. Only in patients with bladder tumor we found significantly increased TF levels compared with the

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healthy control group [96]. Further studies have to be performed with urologic patients to discriminate between bladder tumor and other bladder diseases. 4.2. TF and metastasis The expression of TF in tumor can induce activation of coagulation, fibrin formation and tumor stroma formation [97]. This can lead to encapsulation of tumor cells in a platelet- and fibrin-rich clot, and arrest in the microcirculation [98]. Local thrombin formation may facilitate arrest of tumor cells to the vessel wall by up-regulation of several adhesion molecules on the surface of endothelial cells and changes in the organization of cell –cell-junctions [98]. Different studies have recently focused on the relationship between TF expression and metastasis (Table 1). Sawada et al. [74] measured TF in a wide variety of non-small cell lung cancer (NSCLC) cell lines and found that human NSCLC cell lines derived from metastatic cells produced high levels of TF in vitro. Furthermore, they studied surgically resected specimens and found significantly stronger staining for TF in NSCLC specimens with metastasis than in those without [74], whereas Koomagi and Volm found no correlation to metastasis in lung cancer patients [73]. A strong correlation between TF expression and hepatic metastases, but not to lymphnode metastasis were recognized in colorectal carcinoma patients [78]. Another study aids the thesis that TF is related with the metastatic potential of colorectal cancer [77]. It was shown that TF expression in the primary tumor with simultaneous hepatic metastases was significantly higher than in primary tumors without metastases in the liver. Additionally, TF expression was significantly increased in metastatic cells of the hepatic metastases compared with the primary tumor cells [77]. Overexpression of TF by human melanoma cells lead to metastatic tumors in severe combined immunodeficiency (SCID) mice, whereas human melanoma cells expressing low levels of TF did not. A second experiment in this study showed that the cytoplasmic domain of TF is required for this effect [99]. Specific inhibition of TF function by antibodies result in insufficient adherence of the tumor cells in the target organ [100]. Transfection of Chinese hamster ovary (CHO) cells with cDNA of the TF gene enhances tumor cell metastasis [101]. Metastasis of CHO cells required the cytoplasmic domain but was also blocked by inhibitory antibodies to the binding sites of FVIIa. By the use of a number of transfected melanoma cell lines the role of the cytoplasmic domain and the extracellular domain of TF was investigated [102]. Alanine was substituted for each of the three serine residues in the cytoplasmic domain preventing phosphorylation. The four residues involved in the binding FVIIa in the extracellular domain were also substituted by alanine in another mutant removing procoagulant activity. The transfected cell lines were injected into SCID mice. The incidence of metastasis was decreased in all mutants compared to wild type. Both phosphorylation and the formation of the FVIIa/TF complex is necessary for full metastatic

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Table 1 Summary of published studies with described correlations between TF and parameters associated with tumor growth, metastasis and angiogenesis Invasive growth

Metastasis

Grade/stage

Negative prognosis

+ + +

+

+ + +

+

+ +

+ (+)

+ + +

+ +

+

+ + +

+

+ +

+

+ + + + + + + +

+ , lack of correlation.

It is widely accepted that tumors cannot grow beyond 2 to 3 mm in diameter without building new vasculature. The process of forming new blood vessels from pre-existing neighbouring vessels is called angiogenesis [103,104]. The switch to the angiogenic phenotype requires a tumor-induced imbalance between angiogenesis inhibitors and angiogenesis stimulators [105,106]. Angiogenesis can occur when the balance is changed towards an increased level of stimulators or a decreased level of inhibitors or both [104,106]. Examples for stimulators include vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8). Inhibitors of angiogenesis are thrombospondin-1 (TSP-1) and angiostatin, for example. Some of these angiogenesis-regulation factors are generated in the tumor and act locally, while others after being released into the circulation can act systemically [107,108]. The components and interactions of the angiogenic phenotype in tumor cells are summarized in Fig. 2. Different studies showed a correlation between TF and angiogenesis [73,79,109,110]. Zhang and collegues [109] transfected Meth-A sarcoma cells with cDNA constructs of TF. They found that tumor cells transfected to overexpress TF grew more rapidly, and established larger and more vascularized tumors than control or antisense transfectants in vivo. Further nuclear run on analysis showed higher transcription of VEGF and lower transcription of TSP-1 in cell lines transfected with sense construct. In NSCLC patients survival times were longer in patients with TF negative tumors. Further measurement of VEGF showed a significant corre-

tumor cell with multiple genetic alterations

cytokines? VEGF TF

VEGF, FGF, IL-8

cytokines

TSP-1↓ VEGF

stromal cell

VEGF , TF

inflammatory cell

vascular endothelium TF

local

hemostasis fibrinolysis

↓

4.3. TF and tumor angiogenesis

lation between TF and VEGF. They suggested therefore that TF may have prognostic relevance [73]. Another study with NSCLC found significantly higher TF mRNA expression in tumors that invaded blood vessels [111]. Primary tumor growth in mice was increased, when they were transfected with cells expressing a TF sense cDNA [112]. Further studies showed coherence between angiogenesis and expression of angiogenic factors. An apparent interaction between TF

Thrombin Plasmin

aaATIII

systemic

↓

function [102]. These data suggest that prometastatic function of TF is dependent on both procoagulant activity and the existence of the cytoplasmic domain of TF.

↓

+, significant correlation; (+), positive but not significant correlation;

+ +

↓

+

proliferation , adhesion , proteolysis , migration , metastasis

↓

19 55 191 213 7 19 100 67 79 67 73 66 34 44 23 55 113 58

Differentiation

↓ ↓

[76] [74] [73] [69] [67] [68] [79] [78] [77] [81] [83] [82] [65] [63] [64] [84] [85] [87]

VEGF

↓

Lung cancer Lung cancer Lung cancer Breast cancer Breast cancer Breast cancer Colon cancer Colon cancer Colon cancer Prostate cancer Prostate cancer Prostate cancer Glioma Glioma Glioma Pancreatic cancer Pancreatic cancer Hepatic cancer

MVD

↓

No. of patients

↓

Reference

↓

Tumor typ

Fig. 2. Components and interactions of the angiogenic phenotype in tumor cells (adopted and modified from [105]).

Y. Fo¨rster et al. / Clinica Chimica Acta 364 (2006) 12 – 21

expression and VEGF expression has been reported in different studies such as human breast cancer and human glioma [64,67]. However, the role of TF in angiogenesis that is discussed is controversial as well. In human melanoma cells some authors reported significant correlation between TF and VEGF [113,114] while others did not find any correlation [102]. Microvessel density (MVD) and VEGF are used as markers for tumor angiogenesis. A variety of studies investigated these markers by immunohistology (Table 1). In human hepatocellular carcinoma MVD was significantly higher in tumors with high immunoreactivity for TF than in tumors with low immunoreactivity for TF [87]. These results agree with the findings in other cancers [73,79,81,111,112,115]. NSCLC with low TF expression exhibited a low MVD [73]. A significant correlation between TF expression and MVD was also found in prostate carcinomas. Tumors with higher TF staining showed higher MVD [81]. In colorectal cancer TF positive tumors exhibited high MVD [79]. TF may contribute to angiogenesis by different pathways. In a clotting-dependent pathway activation of coagulation results in the generation of thrombin, subsequent activation of platelets, and deposition of fibrin. Platelets are a rich source of VEGF that is released by thrombin [116]. Additionally, VEGF stimulates endothelial cells to expose TF that promotes thrombin generation [64]. Thrombin forms an extracellular fibrin clot. This is an excellent scaffold to the formation of new blood vessels [110,116]. So, the tumor might ensure their own blood supply through TF dependent thrombin generation, local platelet activation, release of VEGF, and resultant subsequent angiogenesis [112]. The clotting-independent pathway is characterized primarily via PARs. Belting et al. [117] could demonstrate that PAR-2 signaling is controlled by the cytoplasmic domain of TF. The deficiency of the cytoplasmic domain as well as the phosphorylation of the CD resulted in angiogenesis. TFPAR-2 signaling was synergized with PDGF-BB, not with VEGF, bFGF or PDGF-AA in cytoplasmic domain-deficient mice aorta. PDGF-BB is synthesized from sprouting endothelial cell or released from activated platelets [117]. Phosphorylation initiates downstream signaling cascades that lead to transcriptional activation of angiogenic factors like VEGF [110]. Transfection of a TF cDNA vector containing a cytoplasmic domain deletion resulted in a significant reduction of VEGF expression in melanoma cells [113]. In contrast, genetic deletion of the CD of TF in mice resulted in enhanced angiogenesis [117]. These results show the complexity of pathways linking TF and coagulation dependent or independent angiogenesis.

5. Diagnostic and prognostic value of TF: limitations and perspectives Until today the diagnostic and prognostic value of TF is difficult to assess. Although up-regulation of TF is often de-

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tected on the surfaces of tumor-involved endothelial cells, inflammatory cells, and particularly on tumor cells themselves, a prediction of prognosis and survival is problematic. Table 1 shows an overview about the association between TF and clinicopathological parameters for different kinds of tumors. In gliomas [63,65], pancreatic carcinomas [85] and colorectal carcinomas [78,79] TF expression correlated with the histological grade of malignancy, in some tumor types TF detection correlated significantly with angiogenesis [64,73]. In several studies an association of TF expression level with the malignant phenotype in breast cancer was found [67,68]. However, in breast carcinoma a significant TF expression was detected also in cells of the tumor-associated stroma [66,68,70]. Furthermore, a correlation of high TF expression with a negative prognosis in NSCLC [73,74], colorectal cancer [77,78], pancreatic ductal adenocarcinoma [85], hepatocellular carcinoma [87] and breast cancer [69], was reported. TF expression was found predominantly in vascular endothelial cells and malignant tumor cells of glioma and breast carcinomas [64,67]. The level of TF expression correlated significantly with prognostic factors for prostate cancer as the serum level of prostate specific antigen and MVD [81,82]. Immunohistochemical analyses for biopsies from metastatic prostate cancer patients treated with withdrawal therapy showed that the TF content was not associated with histology, therapeutic response or the degree of metastasis [83]. However, patients with TF-positive biopsies had a shorter survival than those with TF-negative tumors. By multivariate analysis Akashi et al. [83] found that relative high TF content of tumor tissue represents a negative independent risk factor for the affected patients [70,90]. However, for some tumor types contradictory results have been described. In these studies no correlation between TF expression and disease prognosis was found. Our studies for tumor tissue extracts indicate that higher TF levels were observed most of non-malignant autologous kidney tissue specimens compared to renal cell carcinoma tissue by ELISA and quantitative PCR, respectively [96]. TF in tumor-free tissue specimens is probably derived from cells that are known to express TF also in normal healthy tissues, i.e. glomerules [60 –62], whereas tumor cells lost TF. Analyzing the TF protein content in pre-operatively collected serum samples of patients with bladder, renal and prostate cancer, we detected only in samples from bladder cancer patients in comparison to control samples from healthy probands of a significantly increased TF level. No causal association between TF levels in serum and TF content in tissue extracts for these three urologic cancer types was found. The relative high TF level in serum are alike the reported higher uTF levels found in patients with bladder tumors [91 – 93]. Taken together, the TF levels in serum and urine of patients with diagnosed bladder tumors represent an individual marker of disease.

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6. TF as potential target for cancer therapies TF seems to be an attractive target for directed cancer therapeutics since its expression is restricted to malignant (but not benign) tumor cells. TF was targeted either by nucleic acid inhibitors including antisense oligonucleotides, ribozymes and siRNA in vitro and in cell culture [85,118 – 122], immunoconjugates in mice [123,124], or antibodies [125]. These positive results from anticancer treatment studies targeted TF were supported also from studies inhibiting TF by antisense for prevention of renal ischemia-reperfusion injury of the kidney in a rat model [126,127]. Results from independent animal models for various tumor cell types clearly indicate the efficacy of anti-TF therapies in vivo [123 – 125,128,129]. Further, inhibition of TF expression can occur by disturbing the signals of regulation of angiogenesis triggered by induced oncogenic stimuli within a cancer cell clone. For example, data from clinical trials indicate that EGFR neutralizing antibody therapy (IMC-C225m Erbitux) suppresses also TF transcripts [130]. Also all-trans retinoic acid therapy in APL, a disease associated often with mild disseminated intravascular coagulation, down-regulates TF and VEGF [131]. Therefore, targeting of TF should be considered as combinational treatment schedules including i) oncogene-directed molecular therapies based on oligomeric nucleic acid inhibitors (e.g. AS-ODN, siRNA), ii) novel chemotherapies, and iii) antiangiogenic drugs.

7. Conclusion TF initiates the coagulation cascade by binding and activation of FVII. Outside the hemostasis TF plays an important role in tumor growth, metastasis, and angiogenesis. TF may support metastasis by a variety of processes like tumor angiogenesis, production of cytokines and growth factors or generation of adhesion molecules. An increasing number of studies showed the involvement of the protease activity of FVII via the proteolytic cleavage of PAR-2. Several studies demonstrate the involvement of the cytoplasmic domain of TF in these events. In addition, it is possible that the downstream coagulation factors FXa and thrombin influence also metastasis and angiogenesis. The question is how TF mediated signal transduction is translated into pathophysiological changes. TF can be detected in tissues using immuno- histochemistry. In plasma and serum TF is well quantifiable, however, additional studies are needed for an authentic and concluding evaluation of TF as a potential tumor marker and a useful diagnostic tool. Preclinical studies with TF-targeted cancer therapeutics are hopeful in the control of cancer growth and metastasis. Advances in antiangiogenic therapies are promising but continued studies of the regulation of TF are needed, as well as a detailed insight into tumor biology to develop new strategies of treatment of cancer.

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