Cleaved high-molecular-weight kininogen and its domain 5 inhibit migration and invasion of human prostate cancer cells through the epidermal growth factor receptor pathway.

Upregulation and activation of epidermal growth factor receptor and/or urokinase-type plasminogen activator receptor in a variety of cancers have been shown to be associated with poor prognosis. High-molecular-weight kininogen can be hydrolysed by plasma kallikrein to bradykinin and cleaved high-molecular-weight kininogen (HKa). HKa and its domain 5 (D5) both have been shown to have potent anti-angiogenic activity. We now show that HKa blocks human prostate cancer cell (DU145) migration by 76.0+/-2.4% at 300 nM and invasion by 78.0+/-12.9% at 11.1 nM. D5 inhibits tumor migration and invasion in a concentration-dependent manner. Stimulation by basic fibroblast growth factor (bFGF) or vascular endothelial growth factor results in clustering of urokinase-type plasminogen activator receptor (uPAR) and epidermal growth factor receptor (EGFR) on the surface of DU145 cells. The co-localization of uPAR and EGFR is prevented by HKa. Immunoprecipitation suggests that uPAR, EGFR and alpha5beta1 integrin formed a ternary complex. Immunoblotting shows that HKa significantly decreases the bFGF-transactivated phosphorylation of EGFR at Tyr 1173 between 30 min and 4 h. The phosphorylation of extracellular signal-regulated kinase (ERK) and AKT, which are downstream effectors of EGFR, is also inhibited by HKa. These novel data indicate that HKa and D5 inhibit migration and invasion of human prostate cancer cells through an EGFR/uPAR pathway, suggesting the therapeutic potential of HKa and D5 to decrease metastasis of human prostate cancer.

Formation of tissue factor-factor VIIa-factor Xa complex promotes cellular signaling and migration of human breast cancer cells.

Tissue factor (TF) is a transmembrane glycoprotein that initiates blood coagulation when complexed with factor (F)VIIa. Recently, TF has been shown to promote cellular signaling, tumor growth, angiogenesis, and metastasis. In the present study, we examined the pathway by which TF-FVIIa complex induces cellular signaling in human breast cancer cells using the Adr-MCF-7 cell line. This cell line has high endogenous TF expression as measured by flow cytometry and expression of protease-activated receptors 1 and 2 (PAR1 and PAR2) as determined by reverse transcriptase-polymerase chain reaction analysis. Both PAR1 and PAR2 are functionally active as determined by induction of p44/42 mitogen-activated protein kinase (MAPK) phosphorylation using specific agonist peptides. We found that MAPK phosphorylation in this cell line was strongly induced by the combination of FVIIa and factor (F)X, but not by FVIIa alone at a concentration of FVIIa that approaches physiological levels. Induction of MAPK phosphorylation involved the formation of TF-FVIIa-FXa complex and occurred by a pathway that did not require thrombin formation, indicating a critical role for FXa generation. In addition, induction of MAPK phosphorylation was found to be independent of PAR1 activation. We then examined whether TF-FVIIa complex formation could promote tumor cell migration using a modified Boyden chamber chemotaxis assay. The combination of FVIIa and FX, but not FVIIa alone, strongly induced migration of tumor cells by a pathway that probably involves PAR2, but not PAR1 activation. MAPK phosphorylation was found to be required for the induction of cell migration by the combination of FVIIa and FX. These data suggest that TF-FVIIa-mediated signaling in human breast cancer cells occurs most efficiently by formation of the TF-FVIIa-FXa complex. One of the physiological consequences of this signaling pathway is enhanced cell migration that is probably mediated by PAR2, but not PAR1 activation.

Role of protease-activated receptor 1 in tumor metastasis promoted by tissue factor.

Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. We previously reported that expression of high levels of TF in a human melanoma cell line promotes metastasis. Both the cytoplasmic domain of TF and its extracellular domain complexed with factor VIIa are required for the metastatic effect. To further explore the mechanism of TF-mediated metastasis, we investigated the possibility that a protease-activated receptor (PAR) might play a role. For this purpose, we first determined the expression levels of the known PARs (PAR1-4) in a human melanoma cell line, SIT1, that has low endogenous levels of TF and low metastatic potential. We found negligible levels of all of the known PARs and transfection of this cell line with human TF cDNA did not alter expression of the known PARs. To study the possible role of PAR1 in TF-mediated metastasis, we prepared a panel of transfected cell lines with varying levels of TF and PAR1. Our studies show that TF promotes metastasis by a pathway that does not involve high expression of known PARs by tumor cells. In addition, while overexpression of PAR1 is insufficient to induce metastasis in cells with low TF expression, it enhances the metastatic potential of cells with high TF expression, indicating a possible synergy between TF and PAR1 in promoting metastasis.

Role of tissue factor in metastasis: functions of the cytoplasmic and extracellular domains of the molecule.

Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. It was reported in an earlier study that expression of high levels of TF in a human melanoma cell line promotes metastasis, and that the cytoplasmic domain of TF is required for this metastatic effect. To analyze the functions of the cytoplasmic and extracellular domains of TF in metastasis, two TF mutants were constructed; in one mutant alanine was substituted for each of the three serine residues in the cytoplasmic domain, preventing phosphorylation; in the other mutant alanine was substituted for four key residues in the extracellular domain, preventing binding of factor VIIa and consequently eliminating the initiation of blood coagulation by the TF-VIIa complex. Melanoma lines expressing high levels of either mutant form of TF were weakly metastatic in SCID mice, indicating that phosphorylation of the cytoplasmic domain and formation of a complex with VIIa by the extracellular domain are required for the full metastatic effect of TF. It was also found that increasing TF expression in human melanoma cells does not increase expression of vascular endothelial growth factor or promote growth and vascularization of tumors derived from the melanoma cells, suggesting that TF acts by a mechanism other than angiogenesis to promote metastasis.

Ancylostoma caninum anticoagulant peptide blocks metastasis in vivo and inhibits factor Xa binding to melanoma cells in vitro.

We evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP's anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme's active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP's antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

Tissue factor promotes melanoma metastasis by a pathway independent of blood coagulation.

Several studies have established a link between blood coagulation and cancer, and more specifically between tissue factor (TF), a transmembrane protein involved in initiating blood coagulation, and tumor metastasis. In the study reported here, a murine model of human melanoma metastasis was used for two experiments. (i) The first experiment was designed to test the effect of varying the level of TF expression in human melanoma cells on their metastatic potential. Two matched sets of cloned human melanoma lines, one expressing a high level and the other a low level of the normal human TF molecule, were generated by retroviral-mediated transfections of a nonmetastatic parental line. The metastatic potential of the two sets of transfected lines was compared by injecting the tumor cells into the tail vein of severe combined immunodeficiency (SCID) mice and later examining the lungs and other tissues for tumor development. Metastatic tumors were detected in 86% of the mice injected with the high-TF lines and in 5% of the mice injected with the low-TF lines, indicating that a high TF level promotes metastasis of human melanoma in the SCID mouse model. This TF effect on metastasis occurs with i.v.-injected melanoma cells but does not occur with primary tumors formed from s.c.-injected melanoma cells, suggesting that TF acts at a late stage of metastasis, after tumor cells have escaped from the primary site and entered the blood. (ii) The second experiment was designed to analyze the mechanism by which TF promotes melanoma metastasis. The procedure involved testing the effect on metastasis of mutations in either the extracellular or cytoplasmic domains of the transmembrane TF molecule. The extracellular mutations introduced two amino acid substitutions that inhibited initiation by TF of the blood-coagulation cascade; the cytoplasmic mutation deleted most of the cytoplasmic domain without impairing the coagulation function of TF. Several human melanoma lines expressing high levels of either of the two mutant TF molecules were generated by retroviral-mediated transfection of the corresponding TF cDNA into the nonmetastatic parental melanoma line, and the metastatic potential of each transfected line was tested in the SCID mouse model. Metastases occurred in most mice injected with the melanoma lines expressing the extracellular TF mutant but were not detected in most mice injected with the melanoma lines expressing the cytoplasmic TF mutant. Results with the extracellular TF mutant indicate that the metastatic effect of TF in the SCID mouse model does not involve products of the coagulation cascade. Results with the cytoplasmic TF mutant indicate that the cytoplasmic domain of TF is important for the metastatic effect, suggesting that the TF could transduce a melanoma cell signal that promotes metastasis.

Role of myosin phosphorylation in contractility of a platelet aggregate.

The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin-activated, irreversibly aggregated platelets known as the platelet strip. Steady-state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets.