Tissue Factor Prothrombotic Activity Is Regulated by Integrin-arf6 Trafficking.

OBJECTIVE: Coagulation initiation by tissue factor (TF) is regulated by cellular inhibitors, cell surface availability of procoagulant phosphatidylserine, and thiol-disulfide exchange. How these mechanisms contribute to keeping TF in a noncoagulant state and to generating prothrombotic TF remain incompletely understood. APPROACH AND RESULTS: Here, we study the activation of TF in primary macrophages by a combination of pharmacological, genetic, and biochemical approaches. We demonstrate that primed macrophages effectively control TF cell surface activity by receptor internalization. After cell injury, ATP signals through the purinergic receptor P2rx7 induce release of TF+ microvesicles. TF cell surface availability for release onto microvesicles is regulated by the GTPase arf6 associated with integrin α4ß1. Furthermore, microvesicles proteome analysis identifies activation of Gαi2 as a participating factor in the release of microvesicles with prothrombotic activity in flowing blood. ATP not only prevents TF and phosphatidylserine internalization but also induces TF conversion to a conformation with high affinity for its ligand, coagulation factor VII. Although inhibition of dynamin-dependent internalization also exposes outer membrane procoagulant phosphatidylserine, the resulting TF+ microvesicles distinctly lack protein disulfide isomerase and high affinity TF and fail to produce fibrin strands typical for microvesicles generated by thrombo-inflammatory P2rx7 activation. CONCLUSIONS: These data show that procoagulant phospholipid exposure is not sufficient and that TF affinity maturation is required to generate prothrombotic microvesicles from a variety of cell types. These findings are significant for understanding TF-initiated thrombosis and should be considered in designing functional microvesicles-based diagnostic approaches.

Tissue factor and PAR1 promote microbiota-induced intestinal vascular remodelling.

The gut microbiota is a complex ecosystem that has coevolved with host physiology. Colonization of germ-free (GF) mice with a microbiota promotes increased vessel density in the small intestine, but little is known about the mechanisms involved. Tissue factor (TF) is the membrane receptor that initiates the extrinsic coagulation pathway, and it promotes developmental and tumour angiogenesis. Here we show that the gut microbiota promotes TF glycosylation associated with localization of TF on the cell surface, the activation of coagulation proteases, and phosphorylation of the TF cytoplasmic domain in the small intestine. Anti-TF treatment of colonized GF mice decreased microbiota-induced vascular remodelling and expression of the proangiogenic factor angiopoietin-1 (Ang-1) in the small intestine. Mice with a genetic deletion of the TF cytoplasmic domain or with hypomorphic TF (F3) alleles had a decreased intestinal vessel density. Coagulation proteases downstream of TF activate protease-activated receptor (PAR) signalling implicated in angiogenesis. Vessel density and phosphorylation of the cytoplasmic domain of TF were decreased in small intestine from PAR1-deficient (F2r(-/-)) but not PAR2-deficient (F2rl1(-/-)) mice, and inhibition of thrombin showed that thrombin-PAR1 signalling was upstream of TF phosphorylation. Thus, the microbiota-induced extravascular TF-PAR1 signalling loop is a novel pathway that may be modulated to influence vascular remodelling in the small intestine.

Single cell tracking assay reveals an opposite effect of selective small non-peptidic α5ß1 or αvß3/ß5 integrin antagonists in U87MG glioma cells.

BACKGROUND: Integrins are extracellular matrix receptors involved in several pathologies. Despite homologies between the RGD-binding α5ß1 and αvß3 integrins, selective small antagonists for each heterodimer have been proposed. Herein, we evaluated the effects of such small antagonists in a cellular context, the U87MG cell line, which express both integrins. The aim of the study was to determine if fibronectin-binding integrin antagonists are able to impact on cell adhesion and migration in relationships with their defined affinity and selectivity for α5ß1 and αvß3/ß5 purified integrins. METHODS: Small antagonists were either selective for α5ß1 integrin, for αvß3/ß5 integrin or non-selective. U87MG cell adhesion was evaluated on fibronectin or vitronectin. Migration assays included wound healing recovery and single cell tracking experiments. U87MG cells stably manipulated for the expression of α5 integrin subunit were used to explore the impact of α5ß1 integrin in the biological assays. RESULTS: U87MG cell adhesion on fibronectin or vitronectin was respectively dependent on α5ß1 or αvß3/ß5 integrin. Wound healing migration was dependent on both integrins. However U87MG single cell migration was highly dependent on α5ß1 integrin and was inhibited selectively by α5ß1 integrin antagonists but increased by αvß3/ß5 integrin antagonists. CONCLUSIONS: We provide a rationale for testing new integrin ligands in a cell-based assay to characterize more directly their potential inhibitory effects on integrin cellular functions. GENERAL SIGNIFICANCE: Our data highlight a single cell tracking assay as a powerful cell-based test which may help to characterize true functional integrin antagonists that block α5ß1 integrin-dependent cell migration.

Dendritic cell PAR1-S1P3 signalling couples coagulation and inflammation.

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor 1 (PAR1) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR1 signalling. The sphingosine 1-phosphate (S1P) axis is a downstream component of PAR1 signalling, and by combining chemical and genetic probes for S1P receptor 3 (S1P3) we show a critical role for dendritic cell PAR1-S1P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR1-S1P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-1beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

Cooperation of tissue factor cytoplasmic domain and PAR2 signaling in breast cancer development.

Constitutive expression of tissue factor (TF) by cancer cells triggers local activation of the coagulation cascade and promotes breast cancer progression through cell signaling involving protease activated receptor (PAR)2. In human breast cancer, TF and PAR2 are up-regulated and TF cytoplasmic domain phosphorylation is correlated with relapse. Here we show that cancer cell PAR2 signaling promotes angiogenesis independent of PAR2 phosphorylation at the recognized ß-arrestin recruitment site. Similar to PAR2(-/-) mice, TF cytoplasmic domain-deleted (TF(ΔCT)) mice have delayed spontaneous breast cancer development in the polyoma middle T model. Simultaneous deletion of PAR2 in TF(ΔCT) mice did not further delay tumor appearance, consistent with overlapping roles of TF and PAR2 in promoting the angiogenic switch in early stages of breast cancer. In advanced carcinomas, tumor-associated macrophages were reduced in TF(ΔCT) and TF(ΔCT)/PAR2(-/-) mice, and increased tumor vessel diameters of TF(ΔCT) mice were partially reversed by PAR2-deficiency, indicating that the TF cytoplasmic domain has additional roles that are interdependent with PAR2 signaling in regulating host angiogenic responses. These experiments demonstrate a crosstalk of tumor cell TF cytoplasmic domain and PAR2 signaling and provide a possible mechanism for the close correlation between TF phosphorylation and cancer recurrence of TF and PAR2-positive clinical breast cancer.

Evidence for tissue factor phosphorylation and its correlation with protease-activated receptor expression and the prognosis of primary breast cancer.

Tissue factor (TF)-mediated protease-activated receptor (PAR)-2 signaling is associated with a promigratory, invasive and proangiogenic phenotype in experimental models of breast cancer and has been mechanistically coupled to phosphorylation of the TF cytoplasmic domain (pTF). However, the clinical relevance of these findings is unknown. Here, we provide the first in vivo evidence of TF phosphorylation in experimental as well as clinical breast cancer tumors. pTF was demonstrated in MDA-MB-231 xenografts and in tumors from the MMTV-PyMT transgene model of spontaneous murine breast adenocarcinoma. Tumors from PAR-2-deficient transgenic mice were negative for pTF, thus linking pTF to PAR-2 signaling. The clinical correlation between TF, pTF, PAR-1, PAR-2 and vascular endothelial growth factor (VEGF)-A was determined by immunohistochemistry on tumors from a cohort of 172 consecutive primary breast cancer patients, with a median follow-up time of 50 months. In 160 evaluable patient tumors, pTF was associated with TF (p = 0.01) and cancer cell expression of PAR-1 (p = 0.001), PAR-2 (p = 0.014) and VEGF-A (p = 0.003) using chi(2) test. PAR-2 and VEGF-A were coexpressed (p = 0.013) and associated with a more aggressive phenotype. Interestingly, all patients experiencing recurrences had tumors expressing pTF and PAR-2, and pTF alone as well as coexpression of pTF and PAR-2 were significantly correlated with shorter recurrence-free survival (log rank test, p = 0.04 and p = 0.02, respectively). This study provides the first evidence to link PAR-2 expression and TF phosphorylation to clinical data in human breast cancer. In conjunction with experimental tumor models, these data support an important role of TF-PAR-2 signaling in breast cancer recurrence.

Tissue factor and PAR2 signaling in the tumor microenvironment.

Diverse oncogenic transformations result in the constitutive expression of tissue factor (TF) in cancer cells. The local and systemic activation of the coagulation cascade has long been a recognized hallmark for aggressive cancer, but genetic mouse models and new experimental therapeutics have only recently demonstrated crucial roles for TF initiated cell signaling in the pathogenesis of cancer. On tumor cells, the TF-VIIa binary complex mediates activation of protease activated receptor (PAR) 2 and thereby shapes the tumor microenvironment by inducing an array of proangiogenic and immune modulating cytokines, chemokines, and growth factors. PAR2 also uniquely triggers tumor cell migration by G protein-independent pathways through beta-arrestin scaffolding. Metastatic tumor cells use additional signaling networks of the coagulation cascade by activating PAR1 through thrombin or the ternary TF-VIIa-Xa signaling complex in the vascular and potentially lymphatic system. Selective antagonists of TF-VIIa-PAR2 signaling may be used as antiangiogenic therapy without increasing the risk of bleeding, whereas coagulation and associated signaling pathways on platelets and other host cells may be targeted for therapeutic benefit in advanced cancer and metastatic disease.

Endothelial ephrinB2 is controlled by microenvironmental determinants and associates context-dependently with CD31.

OBJECTIVE: The EphB ligand ephrinB2 has been identified as a critical determinant of arterial endothelial differentiation and as a positive regulator of invading endothelial cells during angiogenesis. This study was aimed at identifying determinants of endothelial cell ephrinB2 expression. METHODS AND RESULTS: Arteriovenous asymmetrical endothelial cell ephrinB2 expression in vivo is lost on transfer into culture with aortic endothelial cells becoming partially ephrinB2-negative and saphenous vein endothelial cells becoming partially ephrinB2-positive. Contact with smooth muscle cells and angiogenic stimulation by vascular endothelial growth factor lead to an increased endothelial cell ephrinB2 expression. Quiescent, smooth muscle-contacting endothelial cells express ephrinB2 uniformly on their luminal surface. In contrast, monolayer endothelial cells translocate ephrinB2 to interendothelial cell junctions, which is strongly enhanced by EphB4-Fc-mediated receptor body activation. Junctional ephrinB2 colocalizes and coimmunoprecipitates with CD31. CONCLUSIONS: This study identifies distinct regulatory mechanisms of endothelial ephrinB2 expression and cellular distribution in quiescent and activated endothelial cells. The data demonstrate that endothelial cell ephrinB2 expression is controlled by microenvironmental determinants rather than being an intrinsic endothelial cell differentiation marker.

Inhibition of tumor growth and angiogenesis by soluble EphB4.

EphB receptors and their ephrinB ligands play a key role in the formation of a regular vascular system. Recent studies have also shown the involvement of Eph/ephrin interactions in malignant tumor progression and angiogenesis. We have generated soluble monomeric EphB4 (sEphB4)-expressing A375 melanoma cells to study the effect of dominant negatively acting sEphB4 on tumor growth and angiogenesis. Soluble EphB4-expressing A375 tumors grown subcutaneously in nude mice show dramatically reduced tumor growth compared to control tumors. The proliferative capacity of sEphB4-expressing cells in monolayer culture is not altered. Yet, sEphB4-expressing A375 cells cannot establish proper cell-cell contacts in three-dimensional spheroids. However, sEphB4 transfectants have reduced proliferation and apoptosis rates when grown in three-dimensional culture in vitro or in subcutaneous tumors in vivo. Analysis of the vascular phenotype of the tumors revealed a reduction of intratumoral microvessel density in sEphB4-expressing tumors. Corresponding to these mouse experiments, a matched pair analysis of EphB4 and ephrinB2 expression in human colon carcinomas revealed significantly upregulated levels of EphB4 expression compared to adjacent normal tissue. Taken together, the data identify dual effects of sEphB4 on the tumor and the vascular compartment that collectively inhibit tumor growth.

Role of the protein C receptor in cancer progression.

The hemostatic system plays pleiotropic roles in cancer progression by shaping the tumor microenvironment and metastatic niches through thrombin-dependent fibrin deposition and platelet activation. Expanding experimental evidence implicates coagulation protease receptors expressed by tumor cells as additional players that directly influence tumor biology. Pro-angiogenic G protein-coupled signaling of TF through protease activated receptor 2 and regulation of tumor cell and vascular integrins through ligation by alternative spliced TF are established pathways driving tumor progression. Our recent work shows that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is also crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. In aggressive triple-negative breast cancer cells, EPCR expression is a characteristic of cancer stem cell-like populations that have tumor initiating properties in vivo. Blocking antibodies to EPCR attenuate in vivo tumor growth and proliferation specifically of EPCR(+) cells on defined integrin matrices in vitro. We also showed that tumor-associated macrophages are a source for upstream coagulation proteases that can activate TF- and EPCR-dependent cellular responses, suggesting that tumor cells utilize the tumor microenvironment for tumor promoting coagulation protease signaling.

Quantitative measurement of delivery and gene silencing activities of siRNA polyplexes containing pyridylthiourea-grafted polyethylenimines.

The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H(+)/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of "simple"/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom-up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity.

Integrin α5ß1, the Fibronectin Receptor, as a Pertinent Therapeutic Target in Solid Tumors.

Integrins are transmembrane heterodimeric proteins sensing the cell microenvironment and modulating numerous signalling pathways. Changes in integrin expression between normal and tumoral cells support involvement of specific integrins in tumor progression and aggressiveness. This review highlights the current knowledge about α5ß1 integrin, also called the fibronectin receptor, in solid tumors. We summarize data showing that α5ß1 integrin is a pertinent therapeutic target expressed by tumoral neovessels and tumoral cells. Although mainly evaluated in preclinical models, α5ß1 integrin merits interest in particular in colon, breast, ovarian, lung and brain tumors where its overexpression is associated with a poor prognosis for patients. Specific α5ß1 integrin antagonists will be listed that may represent new potential therapeutic agents to fight defined subpopulations of particularly aggressive tumors.

Activation of p53 pathway by Nutlin-3a inhibits the expression of the therapeutic target α5 integrin in colon cancer cells.

Integrins emerge nowadays as crucial actors of tumor aggressiveness and resistance to therapies. Integrin α5ß1, the fibronectin receptor, determines malignant properties of colon carcinoma which is one of the most important causes of cancer-related deaths in the world. Here we show that inhibition of α5 integrin subunit expression by siRNA or α5ß1 integrin function by specific antagonist affects the survival of HCT116 colon cancer cells. We also evidence that pharmacological reactivation of the tumor suppressor p53 by Nutlin-3a inhibits specifically the expression of the α5 integrin subunit both at the transcriptional and protein level. Inversely repression of α5 integrin modulates p53 activity. A clear relationship between p53 activation by Nutlin-3a, α5 repression and cell survival is shown. No such effects are obtained in cells lacking p53 or when another non-genotoxic activator of p53, RITA, is used. Our results emphasize the crucial role of α5ß1 integrin in colon tumors. Data also suggest that interfering with the integrin α5ß1 through the reactivation of p53 by Nutlin-3a may be of valuable interest as a new therapeutic option for colon tumors expressing high level of the integrin and a wild type p53.

Endothelial protein C receptor function in murine and human breast cancer development.

Several markers identify cancer stem cell-like populations, but little is known about the functional roles of stem cell surface receptors in tumor progression. Here, we show that the endothelial protein C receptor (EPCR), a stem cell marker in hematopoietic, neuronal and epithelial cells, is crucial for breast cancer growth in the orthotopic microenvironment of the mammary gland. Mice with a hypomorphic allele of EPCR show reduced tumor growth in the PyMT-model of spontaneous breast cancer development and deletion of EPCR in established PyMT tumor cells significantly attenuates transplanted tumor take and growth. We find expansion of EPCR(+) cancer stem cell-like populations in aggressive, mammary fat pad-enhanced human triple negative breast cancer cells. In this model, EPCR-expressing cells have markedly increased mammosphere- and tumor-cell initiating activity compared to another stable progenitor-like subpopulation present at comparable frequency. We show that receptor blocking antibodies to EPCR specifically attenuate in vivo tumor growth initiated by either EPCR(+) cells or the heterogenous mixture of EPCR(+) and EPCR(-) cells. Furthermore, we have identified tumor associated macrophages as a major source for recognized ligands of EPCR, suggesting a novel mechanism by which cancer stem cell-like populations are regulated by innate immune cells in the tumor microenvironment.

Tissue factor proangiogenic signaling in cancer progression.

Cancer progression from a dormant, non-vascularized benign tumor to metastatic disease is a multiple steps process that critically depends on contributions from the hemostatic system. Tissue factor (TF), protease activated receptors (PARs), factor VIIa, and the endothelial protein C receptor (EPCR) are expressed by tumor cells as well as the host compartment. These components of the hemostatic system regulate tumor growth, angiogenesis and metastasis. Here we review the evidence that TF-dependent signaling is the major driver of primary tumor growth, whereas TF-initiated coagulation and interactions of procoagulant tumor cells with the host compartments initiate multiple pathways that support and regulate the efficiency of metastatic tumor dissemination.

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

PURPOSE: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions. METHODS: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4. RESULTS: Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia. CONCLUSION: The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.

Tissue factor in cancer progression and angiogenesis.

Constitutive expression of tissue factor (TF) by cancer cells triggers local and systemic activation of the coagulation cascade and is a major cause of cancer-associated thrombosis. Primary breast cancer biopsies show a marked upregulation of TF and protease activated receptor (PAR) 2, as well as increased TF cytoplasmic domain phosphorylation that is correlated with cancer relapse. TF signaling involving PAR2 and integrins has multiple effects on angiogenesis and tumor progression. The non-coagulant, alternatively spliced form of TF retains an integrin-binding site and, upon deposition into the tumor stroma, stimulates angiogenesis by ligating endothelial integrins alpha(v)beta(3) and alpha(6)beta(1). On tumor cells, full-length TF is constitutively associated with laminin-binding beta(1) integrins that support TF-VIIa-PAR2 signaling leading to upregulation of pro-angiogenic and immune modulatory cytokines and growth factors. Deficiency of PAR2, but not of the thrombin receptor PAR1, delays spontaneous breast cancer development and the angiogenic switch in mice. In addition, human xenograft breast cancer growth and angiogenesis is suppressed by selective antibody inhibition of TF-VIIa-PAR2 signaling, but not by blocking TF initiated coagulation. Thus, interruption of TF signaling represents a potential anti-angiogenic strategy that does not carry an increased risk of bleeding associated with prolonged inhibition of the TF coagulation pathway.

EphB4 promotes site-specific metastatic tumor cell dissemination by interacting with endothelial cell-expressed ephrinB2.

The tyrosine kinase receptor EphB4 interacts with its ephrinB2 ligand to act as a bidirectional signaling system that mediates adhesion, migration, and guidance by controlling attractive and repulsive activities. Recent findings have shown that hematopoietic cells expressing EphB4 exert adhesive functions towards endothelial cells expressing ephrinB2. We therefore hypothesized that EphB4/ephrinB2 interactions may be involved in the preferential adhesion of EphB4-expressing tumor cells to ephrinB2-expressing endothelial cells. Screening of a panel of human tumor cell lines identified EphB4 expression in nearly all analyzed tumor cell lines. Human A375 melanoma cells engineered to express either full-length EphB4 or truncated EphB4 variants which lack the cytoplasmic catalytic domain (ΔC-EphB4) adhered preferentially to ephrinB2-expressing endothelial cells. Force spectroscopy by atomic force microscopy confirmed, on the single cell level, the rapid and direct adhesive interaction between EphB4 and ephrinB2. Tumor cell trafficking experiments in vivo using sensitive luciferase detection techniques revealed significantly more EphB4-expressing A375 cells but not ΔC-EphB4-expressing or mock-transduced control cells in the lungs, the liver, and the kidneys. Correspondingly, ephrinB2 expression was detected in the microvessels of these organs. The specificity of the EphB4-mediated tumor homing phenotype was validated by blocking the EphB4/ephrinB2 interaction with soluble EphB4-Fc. Taken together, these experiments identify adhesive EphB4/ephrinB2 interactions between tumor cells and endothelial cells as a mechanism for the site-specific metastatic dissemination of tumor cells. AACR.

Tissue factor and protease-activated receptor signaling in cancer.

The activation of the coagulation cascade in the tumor microenvironment is a key feature of advanced malignancies. On tumor cells, tissue factor (TF) plays a central role to initiate cross-talk through the release of procoagulant microparticles or through direct, protease-activated receptor (PAR)-mediated cell signaling that leads to the production of soluble cytokines and angiogenic growth factors. In addition, the hemostatic system in the host compartment sustains crucial circuits that promote metastasis and support tumor growth and angiogenesis. Experimental tumor and genetic models have defined specific pathways that are supported by tumor cell and host TF and have identified potential therapeutic modalities to specifically interrupt TF signaling in tumor biology without impairment of hemostatic functions.

Protease-activated receptor (PAR) 2, but not PAR1, signaling promotes the development of mammary adenocarcinoma in polyoma middle T mice.

The G protein-coupled protease-activated receptors (PAR) are key signaling components for proteases in vascular biology and tumor progression. To address the contributions of PAR1 and PAR2 to breast cancer development, we established cohorts of mouse mammary tumor virus-polyoma middle T (PyMT) PAR1(-/-) and PAR2(-/-) mice, considering that the PyMT model recapitulates aspects of human disease. Appearance of palpable tumors, tumor expansion, and metastasis was indistinguishable between wild-type and PAR1(-/-) mice. PAR1(-/-) breast cancer cells were no longer responsive to thrombin in vitro, excluding compensatory up-regulation of alternative thrombin receptors and indicating that thrombin-PAR1 signaling is dispensable in breast tumor microenvironments. In contrast, palpable tumors and multifocal disease developed slower in PAR2(-/-) mice, and as a consequence of delayed tumor onset, metastasis was reduced. Analysis of early tumors showed persistence of adenomas with delayed appearance of vascularized adenocarcinomas in PAR2(-/-) mice. Furthermore, CXCL1 production by early PAR2(-/-) tumors was reduced. These results are consistent with previous xenograft data that implicated breast cancer PAR2 signaling in the induction of proangiogenic growth factors and chemokines. This study establishes that protease signaling contributes to mammary tumor development and that PAR2, rather than the thrombin receptor PAR1, plays a crucial role in the angiogenic switch.