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Tissue factor and PAR signaling in tumor progression
Thrombosis Research (2007) 120 Suppl. 2, S7–S12
intl.elsevierhealth.com/journals/thre
Tissue factor and PAR signaling in tumor progression Wolfram Ruf* Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA
Abstract Tumor development depends on multiple reciprocal interactions of tumor cells with the host cell compartment. Tumor cells initiate TF-dependent crosstalks with the tumor microenvironment by releasing procoagulant microparticles, soluble cytokines and angiogenic growth factors. Conversely, the hemostatic system in the host compartment provides multiple circuits that regulate tumor growth and sustain angiogenesis. A combination of experimental models of spontaneous and transplanted tumor development and metastasis start to delineate the role of TF in tumor progression and identified potential therapeutic approaches to target the TF pathway. © 2007 Elsevier B.V. All rights reserved. Keywords: Tissue factor; Cancer; PAR; Angiogenesis
1. Two faces of TF in tumor biology Local and systemic coagulation activation is a hallmark of advanced cancer [1]. TF expressing tumor cells initiate the coagulation pathways within the tumor microenvironment and shedding of procoagulant activity is a key factor for the disseminated coagulation activation frequently detected in advanced cancer. Although upregulation of TF is well documented in cancer cells, the hypoxic tumor microenvironment induces TF expression by endothelial cells, tumor associated macrophages, or myofibroblasts [2,3]. Whether host cell TF contributes to the hypercoagulable state is incompletely understood. TF expression is correlated with tumor progression in several cancers [4]. Intriguingly, prolonged anticoagulant therapy for recurrent thromboembolism reduces cancer incidence [5]. Therefore, a detailed understanding of the diverse effects of the TF coagulation pathway in cancer remains an important area of research. Typically, TF exerts its biological activities by forming a catalytic enzyme complex with coagulation factor VIIa. The TF VIIa complex then triggers coagulation by binding and activating factor X, leading to thrombin-dependent fibrin deposition and platelet activation. Thrombin furthermore exhibits pleiotrophic cellular effects mediated through Gprotein-coupled, protease activated receptor (PAR) * Correspondence address: Wolfram Ruf. Tel.: +1 858 784 2748; fax: +1 858 784 8480. E-mail address: [email protected] (W. Ruf)
1 or 4 signaling [6]. The TF VIIa complex signals directly by cleaving PAR2, but not PAR1 [7,8]. We have recently shown that TF’s procoagulant activity can be switched off by protein disulfide isomerase-dependent pathways without impairing direct TF VIIa signaling [9]. The relevance of this regulatory pathway for tumor cell TF function has not been elucidated in detail. Nevertheless, these results show that TF can function independently of activating coagulation to initiate direct cell signaling. TF triggers two distinct signaling pathways that are partially linked. On the one hand, TF expression influences integrin function. TF regulates migration of non-cancerous epithelial cells specifically on laminin 5, a matrix for the integrin a3b1 and a6b1 [10]. This pathway is dependent on TF cytoplasmic domain signaling and extracellular interaction of the physiological ligand VIIa that induces TF cytoplasmic domain phosphorylation in a PAR2dependent manner [11]. The TF cytoplasmic domain is also upstream of MAP kinase p38, ERK1/2, and rac pathways [12,13] and synergizes with PDGF-induced chemotaxis [14], emphasizing the relevance of the TF cytoplasmic domain to pathways of cell motility and promigratory signaling. The second major direct TF VIIa signaling pathway is cleavage and activation of PAR2 [15]. TF VIIa PAR2 signaling promotes breast cancer migration dependent on the chemokine interleukin 8 (IL8) and regulation of the cofilin pathway [16 18]. It is of interest that a number of tumors in vivo and in vitro ectopically express VIIa. Ectopic synthesis
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W. Ruf / Thrombosis Research 120 Suppl. 2 (2007) S7–S12
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of VIIa is sufficient to induce tumor cell migration when exposed to gradients of substrate FX [19]. Although promigratory effects appear to be specific for direct TF, but not thrombin [20] signaling in certain tumor models, the role of TF-dependent motility pathways in vivo has not been conclusively established. In addition, TF VIIa signaling has antiapoptotic effects [21,22] and induces immune modulatory cytokines and angiogenic regulators [23 25]. These downstream targets may play an important role in shaping the tumor microenvironment. However, it has not been delineated in detail how direct tumor cell TF signaling influences tumor progression.
2. Experimental approaches to dissect tumor cell host interactions in vivo The essential role of the TF-initiated coagulation pathway in normal hemostasis and embryonic survival of mice has limited approaches using conventional knock-out technology for TF and other coagulation factors. This has particularly hindered studies on the role of TF in spontaneous tumor development using oncogene driven tumor models in the mouse. Models of spontaneous tumor development are highly suited to analyze early stage of tumorigenesis, including the angiogenic switch, recruitment of hematopoietic progenitor populations that prime and shape the tumor environment, and the escape from immune surveillance by the host immune system. Conditional knock-out of TF in tumor cells may be a feasible strategy to address specifically pathways of TFdependent tumor growth. Nevertheless, studies in transplanted tumor models have delineated in some detail how the TF coagulation pathway supports key steps in tumor progression. Two approaches have proven successful to dissect the respective contributions of tumor cells and host Tumor growth
Signaling
Coagulation
Angiogenesis
TF
TF–VIIa
Tumor cell
Host
TF
Hemostatic system Thrombin Platelets Fibrin
Metastasis Fig. 1. TF coagulation and signaling pathways established by in vivo models.
factors to tumor progression. Syngeneic tumor lines have been used in inbred mouse strains to address the role of host factors, such as PARs or fibrinogen [26 28] for angiogenesis and metastasis. Moreover, TFdeficient cell lines have been established from mouse embryos by oncogene transformation and used to address the specific role of tumor cell TF in immune competent hosts [29]. In addition to reconstitution experiments, species-specific antibodies to human proteins have clarified the role of tumor cell TF in hematogenous metastasis and tumor growth [9,30]. From these experiments, pathways emerged that are dependent on either coagulation-initiation or direct signaling by TF (Fig. 1).
3. Tumor cell TF interacts with the host hemostatic system to promote metastasis The prometastatic pathway downstream of TF [30] has been delineated in detail and is largely dependent on thrombin that through its various activities promotes tumor cell survival during the initial phase of homing to target organs (for reviews, see [4,31]). Experimental hematogenous metastasis models primarily analyze the end stage of TF-dependent homing to the lung, but no study addressed the specific roles of TF in spontaneous metastasis which requires tumor cell escape from the tissue and intravasation. In experimental metastasis, thrombin promotes tumor cell survival through direct signaling to the tumor cell and by encapsulating the arrested cells in a fibrin and platelet rich microthrombus that transiently shields early metastases from cytotoxic elimination by natural killer cells. The TF cytoplasmic domain also contributes to metastasis, but cytoplasmic domain deletion does not consistently decrease metastasis in all tumor types [29,32,33]. Differential expression of the integrin a3b1 which is regulated by TF and involved in metastatic tumor cell arrest [34] may explain the tumor cell specific effects of TF cytoplasmic domain signaling. Nevertheless, the central TF-dependent pathway in metastasis is to activate thrombin, fibrin and platelets for successful tumor cell implantation.
4. The TF PAR signaling pathway in the host compartment Studies in experimental metastasis have firmly established the concept that tumor cell TF can initiate crosstalks with the host hemostatic system to promote tumor progression. However, transplanted tumor growth studies in TF cytoplasmic domaindeleted (TFDCT ) mice uncovered a pro-angiogenic role of direct TF signaling in host cells [35]. In vitro aortic
W. Ruf / Thrombosis Research 120 Suppl. 2 (2007) S7–S12
ring endothelial cell sprouting assays demonstrated that upon TF cytoplasmic domain deletion, TF VIIa drives PAR2-dependent angiogenesis in the presence of growth factor signaling, in particular plateletderived growth factor (PDGF) BB. PAR2 deletion per se had little effect on angiogenesis, indicating that other pathogenic events are required to possibly modify the TF cytoplasmic domain. We have recently demonstrated that endotoxin-induced TF upregulation in macrophages is negatively regulated by the TF cytoplasmic domain [13]. Increased TF expression and consequently PAR2 signaling in relevant cell types of TFDCT mice may thus cause enhanced angiogenesis in this mouse strain. Further studies confirmed in vivo that the proangiogenic phenotype of TFDCT mice is dependent on PAR2 and growth factor signaling pathways [26]. In the hypoxia-driven angiogenesis model of oxygen induced retinopathy, TFDCT mice revascularized areas of central vaso-obliteration significantly faster than wild-type animals. Genetic deletion of PAR2, but not of PAR1, abolished the accelerated revascularization of TFDCT mice. In addition, blockade of tyrosine kinase receptor pathways with Gleevec reversed accelerated angiogenesis of TFDCT mice. This experimental model also allowed us to evaluate how pharmacological inhibitors interfere with the proangiogenic pathway of TF signaling in vivo. Inhibition of the TF VIIa complex, but not of Xa, abolished the enhanced angiogenesis phenotype of TFDCT mice. Thus, a potent coagulation inhibitor that blocks Xa and downstream thrombin generation as well as PAR1-deficiency had no effect on angiogenesis, indicating that TF PAR2 signaling is sufficient to drive angiogenesis independent of coagulation activation. The apparently normal angiogenic response of PAR1 / mice in the model of retinal neoangiogenesis was surprising, because PAR1 knock-out mice show partial embryonic lethality due to vascular failure [36] and thrombin-dependent PAR1 signaling stimulates angiogenesis in different angiogenesis models in vivo [37 39]. The tumor stroma is enriched in several cell types that support angiogenesis, such as reactive myofibroblasts that prominently express PAR1 and PAR2 in breast cancer [40] and myeloid lineage macrophages or dendritic cells that also express PARs [41 43]. To address the role of proangiogenic PAR signaling in the more complex tumor microenvironment, we further analyzed subcutaneous tumor growth of several syngeneic tumor lines. These experiments also did not uncover a role for host cell PAR1 or PAR2 in tumor expansion [26]. PAR1-deficient animals show no alterations in experimental metastasis assays [27], further indicating that thrombindependent PAR signaling is dispensable in the host cell compartment with the exception of platelets that
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are necessary for metastasis and known to enhance angiogenesis [44]. Although it is possible that local synthesis of proangiogenic factors by tumor cells compensated for the loss of host cell PAR1 activation, the proangiogenic phenotype of TFDCT mice is found in several independent models, demonstrating that direct TF PAR2 signaling on host cells is sufficient to enhance angiogenesis in vivo.
5. TF-signaling by tumor cells How TF expressed by tumor cells enhances primary tumor growth remains incompletely understood. Overexpression of TF in fibrosarcoma [45], pancreatic cancer cells [46] and melanoma cells [47] enhances tumor growth and conversely knock-down of TF in colon cancer cells [48] attenuates tumor expansion. Tumor growth in transplanted models is reduced by treatment with the thrombin inhibitor hirudin, indicating the possibility that TF-dependent tumor growth involves proangiogenic thrombin signaling, fibrin deposition or platelet activation [31]. However, normal growth of transplanted tumors in PAR1deficient mice [26] argues against a major role of thrombin PAR1 signaling in the host compartment. Alternatively, direct proangiogenic TF signaling is frequently discussed [45,47,48], but direct signaling of TF is difficult to separate from complex indirect effects of TF-dependent coagulation activation in the tumor microenvironment. Direct tumor cell TF signaling involves the activation of either PAR1 by the ternary TF VIIa Xa coagulation initiation complex or PAR2 that is cleaved by either TF VIIa or TF VIIa Xa. PAR1 is upregulated in human cancers and linked to invasion and oncogenic transformation [31,49 51]. Most epithelial cancers also express PAR2 that is an upstream activator of promigratory pathways [16 18]. In addition, breast cancer cell TF VIIa, but not thrombin signaling induces a broad array of chemokines and cytokines that may play immune-modulatory and proangiogenic roles [25]. Thus, direct TF-signaling may be responsible for increased angiogenesis observed upon TF upregulation in tumor cells [45,47,48]. Interestingly, one of the TF VIIa induced cytokines, CXCL1, is a component of the lung metastasis signature of aggressive breast cancer [52]. However, CXCL1 is also induced in endothelial cells by thrombin [53]. Whether tumor cell PAR signaling contributes to tumor progression may thus not only depend on the expression of PARs, but also on the availability of activating proteases in the tumor microenvironment. Ectopic synthesis of VIIa under hypoxic conditions [19] may contribute to the angiogenic switch in early stages of tumor development, whereas in vascularized
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W. Ruf / Thrombosis Research 120 Suppl. 2 (2007) S7–S12
tumors abundantly generated coagulation proteases and thrombin may override upstream coagulation signaling. However, the list of tumor cell synthesized proteases is constantly growing and PAR signaling may be triggered by a variety of redundant protease pathways in a tumor type specific manner. Nevertheless, blocking TF signaling on breast cancer and melanoma cells by a monoclonal antibody that does not inhibit coagulation was sufficient to attenuate tumor growth of transplanted aggressive human breast cancer cells in mice. These data provide initial evidence that direct tumor cell TF signaling is contributing to the establishment of a supportive tumor microenvironment for certain tumors in vivo.
6. Therapeutic implications of the targeting the TF pathway in cancer A prethrombotic state correlates with colorectal cancer incidence [54] and anticoagulant therapies delay cancer progression and tumor incidence [5, 55 58]. Additional preclinical research is required to fully understand the mechanisms by which the TF pathway contributes to tumor progression and to select appropriate approaches to inhibit relevant targets. While some of the therapeutic benefit of traditional anticoagulants likely resulted from the inhibition of thrombin, TF VIIa signaling of tumor or host cells may represent an escape mechanism for anti-thrombin therapy. Blockade of TF VIIa with specific inhibitors may circumvent this problem by providing a dual strategy that targets both coagulation-dependent and direct TF VIIa proteolytic signaling pathways. Consistently, cancer growth and angiogenesis is more potently inhibited by targeting TF directly with the nematode-derived inhibitor NAPc2 versus inhibition of downstream coagulation with FXa-specific inhibitors [26,59]. In addition, we find that selective inhibition of TF signaling is sufficient to suppress the growth of certain tumors. Because, aggressive anticoagulant therapy is problematic in cancer patients due to bleeding complications, selectively inhibition of TF signaling should be considered as an alternative approach to more safely treat tumor promoting activities of the TF pathway in the clinic.
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intl.elsevierhealth.com/journals/thre
Tissue factor and PAR signaling in tumor progression Wolfram Ruf* Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA
Abstract Tumor development depends on multiple reciprocal interactions of tumor cells with the host cell compartment. Tumor cells initiate TF-dependent crosstalks with the tumor microenvironment by releasing procoagulant microparticles, soluble cytokines and angiogenic growth factors. Conversely, the hemostatic system in the host compartment provides multiple circuits that regulate tumor growth and sustain angiogenesis. A combination of experimental models of spontaneous and transplanted tumor development and metastasis start to delineate the role of TF in tumor progression and identified potential therapeutic approaches to target the TF pathway. © 2007 Elsevier B.V. All rights reserved. Keywords: Tissue factor; Cancer; PAR; Angiogenesis
1. Two faces of TF in tumor biology Local and systemic coagulation activation is a hallmark of advanced cancer [1]. TF expressing tumor cells initiate the coagulation pathways within the tumor microenvironment and shedding of procoagulant activity is a key factor for the disseminated coagulation activation frequently detected in advanced cancer. Although upregulation of TF is well documented in cancer cells, the hypoxic tumor microenvironment induces TF expression by endothelial cells, tumor associated macrophages, or myofibroblasts [2,3]. Whether host cell TF contributes to the hypercoagulable state is incompletely understood. TF expression is correlated with tumor progression in several cancers [4]. Intriguingly, prolonged anticoagulant therapy for recurrent thromboembolism reduces cancer incidence [5]. Therefore, a detailed understanding of the diverse effects of the TF coagulation pathway in cancer remains an important area of research. Typically, TF exerts its biological activities by forming a catalytic enzyme complex with coagulation factor VIIa. The TF VIIa complex then triggers coagulation by binding and activating factor X, leading to thrombin-dependent fibrin deposition and platelet activation. Thrombin furthermore exhibits pleiotrophic cellular effects mediated through Gprotein-coupled, protease activated receptor (PAR) * Correspondence address: Wolfram Ruf. Tel.: +1 858 784 2748; fax: +1 858 784 8480. E-mail address: [email protected] (W. Ruf)
1 or 4 signaling [6]. The TF VIIa complex signals directly by cleaving PAR2, but not PAR1 [7,8]. We have recently shown that TF’s procoagulant activity can be switched off by protein disulfide isomerase-dependent pathways without impairing direct TF VIIa signaling [9]. The relevance of this regulatory pathway for tumor cell TF function has not been elucidated in detail. Nevertheless, these results show that TF can function independently of activating coagulation to initiate direct cell signaling. TF triggers two distinct signaling pathways that are partially linked. On the one hand, TF expression influences integrin function. TF regulates migration of non-cancerous epithelial cells specifically on laminin 5, a matrix for the integrin a3b1 and a6b1 [10]. This pathway is dependent on TF cytoplasmic domain signaling and extracellular interaction of the physiological ligand VIIa that induces TF cytoplasmic domain phosphorylation in a PAR2dependent manner [11]. The TF cytoplasmic domain is also upstream of MAP kinase p38, ERK1/2, and rac pathways [12,13] and synergizes with PDGF-induced chemotaxis [14], emphasizing the relevance of the TF cytoplasmic domain to pathways of cell motility and promigratory signaling. The second major direct TF VIIa signaling pathway is cleavage and activation of PAR2 [15]. TF VIIa PAR2 signaling promotes breast cancer migration dependent on the chemokine interleukin 8 (IL8) and regulation of the cofilin pathway [16 18]. It is of interest that a number of tumors in vivo and in vitro ectopically express VIIa. Ectopic synthesis
1590-8658/ $ – see front matter © 2007 Elsevier B.V. All rights reserved.
W. Ruf / Thrombosis Research 120 Suppl. 2 (2007) S7–S12
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of VIIa is sufficient to induce tumor cell migration when exposed to gradients of substrate FX [19]. Although promigratory effects appear to be specific for direct TF, but not thrombin [20] signaling in certain tumor models, the role of TF-dependent motility pathways in vivo has not been conclusively established. In addition, TF VIIa signaling has antiapoptotic effects [21,22] and induces immune modulatory cytokines and angiogenic regulators [23 25]. These downstream targets may play an important role in shaping the tumor microenvironment. However, it has not been delineated in detail how direct tumor cell TF signaling influences tumor progression.
2. Experimental approaches to dissect tumor cell host interactions in vivo The essential role of the TF-initiated coagulation pathway in normal hemostasis and embryonic survival of mice has limited approaches using conventional knock-out technology for TF and other coagulation factors. This has particularly hindered studies on the role of TF in spontaneous tumor development using oncogene driven tumor models in the mouse. Models of spontaneous tumor development are highly suited to analyze early stage of tumorigenesis, including the angiogenic switch, recruitment of hematopoietic progenitor populations that prime and shape the tumor environment, and the escape from immune surveillance by the host immune system. Conditional knock-out of TF in tumor cells may be a feasible strategy to address specifically pathways of TFdependent tumor growth. Nevertheless, studies in transplanted tumor models have delineated in some detail how the TF coagulation pathway supports key steps in tumor progression. Two approaches have proven successful to dissect the respective contributions of tumor cells and host Tumor growth
Signaling
Coagulation
Angiogenesis
TF
TF–VIIa
Tumor cell
Host
TF
Hemostatic system Thrombin Platelets Fibrin
Metastasis Fig. 1. TF coagulation and signaling pathways established by in vivo models.
factors to tumor progression. Syngeneic tumor lines have been used in inbred mouse strains to address the role of host factors, such as PARs or fibrinogen [26 28] for angiogenesis and metastasis. Moreover, TFdeficient cell lines have been established from mouse embryos by oncogene transformation and used to address the specific role of tumor cell TF in immune competent hosts [29]. In addition to reconstitution experiments, species-specific antibodies to human proteins have clarified the role of tumor cell TF in hematogenous metastasis and tumor growth [9,30]. From these experiments, pathways emerged that are dependent on either coagulation-initiation or direct signaling by TF (Fig. 1).
3. Tumor cell TF interacts with the host hemostatic system to promote metastasis The prometastatic pathway downstream of TF [30] has been delineated in detail and is largely dependent on thrombin that through its various activities promotes tumor cell survival during the initial phase of homing to target organs (for reviews, see [4,31]). Experimental hematogenous metastasis models primarily analyze the end stage of TF-dependent homing to the lung, but no study addressed the specific roles of TF in spontaneous metastasis which requires tumor cell escape from the tissue and intravasation. In experimental metastasis, thrombin promotes tumor cell survival through direct signaling to the tumor cell and by encapsulating the arrested cells in a fibrin and platelet rich microthrombus that transiently shields early metastases from cytotoxic elimination by natural killer cells. The TF cytoplasmic domain also contributes to metastasis, but cytoplasmic domain deletion does not consistently decrease metastasis in all tumor types [29,32,33]. Differential expression of the integrin a3b1 which is regulated by TF and involved in metastatic tumor cell arrest [34] may explain the tumor cell specific effects of TF cytoplasmic domain signaling. Nevertheless, the central TF-dependent pathway in metastasis is to activate thrombin, fibrin and platelets for successful tumor cell implantation.
4. The TF PAR signaling pathway in the host compartment Studies in experimental metastasis have firmly established the concept that tumor cell TF can initiate crosstalks with the host hemostatic system to promote tumor progression. However, transplanted tumor growth studies in TF cytoplasmic domaindeleted (TFDCT ) mice uncovered a pro-angiogenic role of direct TF signaling in host cells [35]. In vitro aortic
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ring endothelial cell sprouting assays demonstrated that upon TF cytoplasmic domain deletion, TF VIIa drives PAR2-dependent angiogenesis in the presence of growth factor signaling, in particular plateletderived growth factor (PDGF) BB. PAR2 deletion per se had little effect on angiogenesis, indicating that other pathogenic events are required to possibly modify the TF cytoplasmic domain. We have recently demonstrated that endotoxin-induced TF upregulation in macrophages is negatively regulated by the TF cytoplasmic domain [13]. Increased TF expression and consequently PAR2 signaling in relevant cell types of TFDCT mice may thus cause enhanced angiogenesis in this mouse strain. Further studies confirmed in vivo that the proangiogenic phenotype of TFDCT mice is dependent on PAR2 and growth factor signaling pathways [26]. In the hypoxia-driven angiogenesis model of oxygen induced retinopathy, TFDCT mice revascularized areas of central vaso-obliteration significantly faster than wild-type animals. Genetic deletion of PAR2, but not of PAR1, abolished the accelerated revascularization of TFDCT mice. In addition, blockade of tyrosine kinase receptor pathways with Gleevec reversed accelerated angiogenesis of TFDCT mice. This experimental model also allowed us to evaluate how pharmacological inhibitors interfere with the proangiogenic pathway of TF signaling in vivo. Inhibition of the TF VIIa complex, but not of Xa, abolished the enhanced angiogenesis phenotype of TFDCT mice. Thus, a potent coagulation inhibitor that blocks Xa and downstream thrombin generation as well as PAR1-deficiency had no effect on angiogenesis, indicating that TF PAR2 signaling is sufficient to drive angiogenesis independent of coagulation activation. The apparently normal angiogenic response of PAR1 / mice in the model of retinal neoangiogenesis was surprising, because PAR1 knock-out mice show partial embryonic lethality due to vascular failure [36] and thrombin-dependent PAR1 signaling stimulates angiogenesis in different angiogenesis models in vivo [37 39]. The tumor stroma is enriched in several cell types that support angiogenesis, such as reactive myofibroblasts that prominently express PAR1 and PAR2 in breast cancer [40] and myeloid lineage macrophages or dendritic cells that also express PARs [41 43]. To address the role of proangiogenic PAR signaling in the more complex tumor microenvironment, we further analyzed subcutaneous tumor growth of several syngeneic tumor lines. These experiments also did not uncover a role for host cell PAR1 or PAR2 in tumor expansion [26]. PAR1-deficient animals show no alterations in experimental metastasis assays [27], further indicating that thrombindependent PAR signaling is dispensable in the host cell compartment with the exception of platelets that
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are necessary for metastasis and known to enhance angiogenesis [44]. Although it is possible that local synthesis of proangiogenic factors by tumor cells compensated for the loss of host cell PAR1 activation, the proangiogenic phenotype of TFDCT mice is found in several independent models, demonstrating that direct TF PAR2 signaling on host cells is sufficient to enhance angiogenesis in vivo.
5. TF-signaling by tumor cells How TF expressed by tumor cells enhances primary tumor growth remains incompletely understood. Overexpression of TF in fibrosarcoma [45], pancreatic cancer cells [46] and melanoma cells [47] enhances tumor growth and conversely knock-down of TF in colon cancer cells [48] attenuates tumor expansion. Tumor growth in transplanted models is reduced by treatment with the thrombin inhibitor hirudin, indicating the possibility that TF-dependent tumor growth involves proangiogenic thrombin signaling, fibrin deposition or platelet activation [31]. However, normal growth of transplanted tumors in PAR1deficient mice [26] argues against a major role of thrombin PAR1 signaling in the host compartment. Alternatively, direct proangiogenic TF signaling is frequently discussed [45,47,48], but direct signaling of TF is difficult to separate from complex indirect effects of TF-dependent coagulation activation in the tumor microenvironment. Direct tumor cell TF signaling involves the activation of either PAR1 by the ternary TF VIIa Xa coagulation initiation complex or PAR2 that is cleaved by either TF VIIa or TF VIIa Xa. PAR1 is upregulated in human cancers and linked to invasion and oncogenic transformation [31,49 51]. Most epithelial cancers also express PAR2 that is an upstream activator of promigratory pathways [16 18]. In addition, breast cancer cell TF VIIa, but not thrombin signaling induces a broad array of chemokines and cytokines that may play immune-modulatory and proangiogenic roles [25]. Thus, direct TF-signaling may be responsible for increased angiogenesis observed upon TF upregulation in tumor cells [45,47,48]. Interestingly, one of the TF VIIa induced cytokines, CXCL1, is a component of the lung metastasis signature of aggressive breast cancer [52]. However, CXCL1 is also induced in endothelial cells by thrombin [53]. Whether tumor cell PAR signaling contributes to tumor progression may thus not only depend on the expression of PARs, but also on the availability of activating proteases in the tumor microenvironment. Ectopic synthesis of VIIa under hypoxic conditions [19] may contribute to the angiogenic switch in early stages of tumor development, whereas in vascularized
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tumors abundantly generated coagulation proteases and thrombin may override upstream coagulation signaling. However, the list of tumor cell synthesized proteases is constantly growing and PAR signaling may be triggered by a variety of redundant protease pathways in a tumor type specific manner. Nevertheless, blocking TF signaling on breast cancer and melanoma cells by a monoclonal antibody that does not inhibit coagulation was sufficient to attenuate tumor growth of transplanted aggressive human breast cancer cells in mice. These data provide initial evidence that direct tumor cell TF signaling is contributing to the establishment of a supportive tumor microenvironment for certain tumors in vivo.
6. Therapeutic implications of the targeting the TF pathway in cancer A prethrombotic state correlates with colorectal cancer incidence [54] and anticoagulant therapies delay cancer progression and tumor incidence [5, 55 58]. Additional preclinical research is required to fully understand the mechanisms by which the TF pathway contributes to tumor progression and to select appropriate approaches to inhibit relevant targets. While some of the therapeutic benefit of traditional anticoagulants likely resulted from the inhibition of thrombin, TF VIIa signaling of tumor or host cells may represent an escape mechanism for anti-thrombin therapy. Blockade of TF VIIa with specific inhibitors may circumvent this problem by providing a dual strategy that targets both coagulation-dependent and direct TF VIIa proteolytic signaling pathways. Consistently, cancer growth and angiogenesis is more potently inhibited by targeting TF directly with the nematode-derived inhibitor NAPc2 versus inhibition of downstream coagulation with FXa-specific inhibitors [26,59]. In addition, we find that selective inhibition of TF signaling is sufficient to suppress the growth of certain tumors. Because, aggressive anticoagulant therapy is problematic in cancer patients due to bleeding complications, selectively inhibition of TF signaling should be considered as an alternative approach to more safely treat tumor promoting activities of the TF pathway in the clinic.
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