Prognostic relevance of tumour cell-associated uPAR expression in invasive ductal breast carcinoma.

AIMS: The urokinase-type plasminogen activator receptor (uPAR) is a key molecule for pericellular proteolysis in tumour cell invasion and metastasis. The aim was to evaluate the prognostic impact of uPAR in invasive breast cancer dependent on which cell types within the tumour express uPAR. METHODS AND RESULTS: uPAR expression was analysed by immunohistochemistry in 270 tumour tissue specimens of invasive ductal breast carcinomas using tissue microarrays. For evaluation of uPAR immunoexpression we used the epitope-mapped, uPAR domain II-specific monoclonal antibody IID7. High uPAR score values in both tumour cells (uPAR-Tc) and stromal cells were significantly related to high tumour grade (G3), and inversely correlated with oestrogen receptor status. On multivariate analysis, high uPAR-Tc values contributed independent prognostic information for disease-free survival (hazard ratio 1.93, P = 0.007) when adjusted for prognostically relevant clinicopathological parameters, whereas uPAR expression in stromal cells was not related to prognosis. In addition, elevated uPAR-Tc values were found to be prognostic indicators in clinically relevant subgroups of patients with invasive breast cancer. CONCLUSIONS: In invasive breast cancer uPAR expression in invasive carcinoma cells, but not in stromal cells, has a significant impact on patients' prognosis, and contributes to a more aggressive tumour phenotype.

Tissue factor and tumor: clinical and laboratory aspects.

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.

Soluble tissue actor interferes with angiostatin-mediated inhibition of endothelial cell proliferation by lysine-specific interaction with plasminogen kringle domains.

Experimental and clinical data suggest that tissue factor (TF), the major initiator of blood coagulation cascade, as well as proteases and components of the fibrinolytic system are involved in tumor growth at least in some solid tumors via effects on angiogenesis. Whereas the pro- and anti-angiogenic effects of the plasminogen/plasmin system and plasminogen kringle domains, respectively, are well characterized, the pathways responsible for the pro-angiogenic properties of TF remain poorly understood. To learn more about the biological significance of the recently described binding of plasminogen to the extracellular domain of TF, we examined the effects of soluble TF (sTF) on angiostatin-inhibited proliferation of endothelial cells. In solid phase binding assays, we found that sTF binds specifically to plasminogen, to the plasminogen kringle domains K1-3, K1-5, K4, as well as to mini-plasminogen. Inhibition of binding of plasminogen and its kringle domains to sTF by the lysine analog 6-aminohexanoic acid (AHA) suggests that lysine-binding sites are involved in plasminogen interaction with TF. Moreover, in the presence of sTF, the inhibitory effect of K1-5 on bFGF-mediated HUVEC proliferation was dose-dependently and saturably abolished. This suggests that TF can interfere with the antagonistic effect of K1-5 on endothelial cell proliferation. In contrast, sTF by itself had no effect on the endothelial cell proliferation. Whereas the interference of TF with K1-5-mediated effect was prevented by AHA, this lysine analog did not abolish the proliferation inhibition of K1-5. In conclusion, the binding of sTF to the plasminogen fragment K1-5 seems to antagonize the anti-angiogenic effects of this plasminogen fragment.

Identification of a novel urokinase receptor splice variant and its prognostic relevance in breast cancer.

The cellular receptor for urokinase-type plasminogen activator, uPAR, plays a central role in both cell surface-associated proteolysis and cellular adhesion. In the present study, we systematically searched for splice variants of uPAR mRNA in human cells and tumor tissues by qualitative RT-PCR using specific primers for uPAR exons 1 and 6. Beside the wild-type (wt) uPAR mRNA and the previously described splice variant lacking exon 5 (uPAR-del5), a novel splice variant lacking both exons 4 and 5 (uPAR-del4/5) was found predominantly in various cancer cell lines. To elucidate whether alternatively spliced uPAR mRNA may be translated and post-translationally processed, we generated stably transfected Chinese hamster ovary cells, which harbor expression plasmids of wt uPAR and various uPAR variants including uPAR-del5 and uPAR-del4/5. By ELISA, flow cytofluorometry, and Western blot analysis, we confirmed synthesis and secretion of wt uPAR and the uPAR variants by the use of domain-specific monoclonal antibodies against uPAR. For quantification of uPAR mRNA variants, we established two highly sensitive real-time RT-PCR assays based on LightCycler technology. Study of their expression in a representative set of breast cancer tissues indicated that the novel mRNA variant uPAR-del4/5 was expressed very frequently and independently of uPAR mRNA variants covering exon 4 (uPAR-wt and uPAR-del5). Higher uPAR-del4/5 expression was significantly associated with shorter disease-free survival (p = 0.0004) of breast cancer patients. These results suggest that uPAR-del4/5 mRNA may serve as a novel prognostic marker in breast cancer.

Tissue specific expression and serum levels of human tissue factor in patients with urological cancer.

Human tissue factor (TF) is involved in tumor angiogenesis and metastasis. However, little is known about the distribution of TF in urological cancer. In this study we investigated the TF expression in tumor tissue and autologous non-malignant tissue as well as in serum of patients with renal cell carcinoma (RCC), bladder cancer, and prostate cancer (PCa). To study the distribution of TF in tumor tissue and in the surrounding non-malignant tissue, we measured TF protein by ELISA in tissue specimens obtained intraoperatively from 18 RCC, seven bladder cancer and six PCa patients. Differences in TF expression were found between tumor tissue and nonmalignant tissue for the three tumor types at the protein level (in the order RCC < bladder cancer < PCa). In all but one of the 18 RCC patients (94 %) higher TF protein level was observed in non-malignant tissue as compared to the tumor tissue. In addition, the relative TF mRNA expression analyzed by a quantitative RT-PCR assay in the same RCC tissue sample pairs was higher in 78% of non-malignant tissues in comparison to the tumor tissue specimens. Moreover, using enzyme linked immunosorbent assay the TF protein content was measured in serum samples of 66 patients with bladder cancer, 75 RCC patients and 157 PCa patients, and was compared with the TF serum level of 92 healthy volunteers. Whereas no differences were detected between normal volunteers and patients with PCa or RCC, patients with bladder cancer showed a significantly increased level of serum TF (P=0.0076). However, no causal association between TF levels in serum and TF content in tissue extracts for all three tumor types of urological tumors was found. Our results suggest that TF in non-malignant renal tissues was expressed at a higher level compared to the supposed de novo TF expression in RCC tissue specimens. This indicates a tumor-associated induction of TF expression in the TF-negative RCC progenitor cells. The increased serum TF levels are alike the reported higher urinary TF levels found in bladder cancer patients. The potential clinical relevance of this finding should be further elucidated.

Gene expression analysis of HUVEC in response to TF-binding.

INTRODUCTION: Tissue factor (TF), the cofactor for factor VII/VIIa (FVII/FVIIa) and initiator of the extrinsic pathway, is transiently expressed on intravascular cells under control of cytokines and growth factors. In addition, endothelial cells express a binding site for external TF. In the present study, we investigated gene expression of endothelial cells derived from human umbilical veins (HUVEC) in response to TF-binding to identify differentially expressed genes. MATERIALS AND METHODS: HUVEC were treated with recombinant relipidated TF (Innovin) versus nontreated cells, as well as TF/FVIIa versus FVIIa alone. TF binding was measured by ELISA. Gene expression profiles were examined using HG-U133 plus 2.0 arrays (Affymetrix). RESULTS: Gene expression analysis of HUVEC showed 148 up-regulated and 29 down-regulated genes 4h after TF binding. Notably, the genes, which were significantly up- and down-regulated, either by TF alone or by the complex of TF/FVIIa, exhibited a complete overlap, indicating that activation of endothelial cells after binding of external added TF does not depend on FVIIa as has been demonstrated for TF-expressing cells. TF-mediated regulation of gene expression of several genes, involved in regulation of apoptosis, cell adhesion, cell motility, and angiogenesis, was confirmed by qPCR. Furthermore, in case of SELE, TGFB2, TNFAIP3, TNFSF4, TNFSF18, TAGLN, CXCL1, PCF11 antibodies directed to TF clearly inhibited TF-mediated regulation of gene expression. CONCLUSIONS: The results demonstrate that interaction of TF with HUVEC via a binding site, independent from FVIIa, may result in regulation of a variety of genes involved in arteriosclerosis, cancer, and cardiovascular diseases.

Eosinophils are a major intravascular location for tissue factor storage and exposure.

Blood cell progenitors were scanned for the presence of the coagulation starter protein tissue factor (TF) by immunoelectron microscopy. Thereby, substantial TF expression was observed in the precursor cells of eosinophils. TF levels were lower in basophil precursors and barely detectable in neutrophil progenitors. In peripheral blood immediately processed to avoid activation of the TF gene, mature eosinophils were found to considerably express TF, unique among the granulocyte and monocyte fractions. TF was preferentially located in the specific granules in resting eosinophils. Platelet-activating factor (PAF), and more pronounced, granulocyte-macrophage colony-stimulating factor (GM-CSF) plus PAF, caused translocation of preformed TF to the eosinophil cell membrane. GM-CSF/PAF also increased the TF transcript levels. The activated eosinophils exhibited procoagulant activity that was abrogated by TF inhibition. Targeting the extracellular domain of TF with specific antibodies markedly suppressed the initial phase of the eosinophil passage across the IL-4-activated endothelium. Eosinophil rolling and firm adhesion remained unaffected. This suggests that TF specifically facilitates the early transendothelial migration of the eosinophils. In summary, eosinophils maintain a high TF expression during maturation, providing a main source of preformed TF in blood, which might be relevant for the thrombogenesis promoted by hypereosinophilic conditions.