Jan Grøndahl-Hansen

The receptor for urokinase plasminogen activator is present in plasma from healthy donors and elevated in patients with paroxysmal nocturnal haemoglobinuria

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl‐phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme‐linked immunosorbent assay (ELISA) has been applied for quantttation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma‐uPAR value ±SD was 31 ± 15 pm, median 28 (range 11‐108), and the corresponding value for six PNH patients was 116±67 pm, median 90 (range 61‐228). The elevated uPAR‐level in PNH patients was highly significant (Mann‐Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro‐uPA by uPAR in plasma may interfere with the appropriate binding of pro‐uPA to cell‐bound uPAR and therefore inhibit cell‐associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR‐mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.

Urokinase receptor in breast cancer tissue extracts. Enzyme-linked immunosorbent assay with a combination of mono- and polyclonal antibodies

SummaryUrokinase plasminogen activator (uPA) is a proteolytic enzyme involved in degradation of the extracellular matrix during cancer invasion. The levels of uPA and its inhibitor PAI-1 in tumor extracts have previously been demonstrated to be of prognostic value in breast cancer as well as other types of cancer. We have previously characterized a specific cell surface receptor for uPA (uPAR) which strongly enhances the catalytic activity of uPA and is expressed during mammary cancer invasion. In order to quantitate uPAR in breast cancer tissue, we have now developed a sensitive enzyme-linked immunosorbent assay (ELISA), with polyclonal catching antibodies and three monoclonal detecting antibodies. The detection limit of the assay is approximately 0.16 fmol of uPAR in a volume of 100 µl (1.6 pM). There is a linear relationship between signal and uPAR concentration up to at least 6.6 fmol per 100 µl (66 pM). Both free uPAR and uPAR in complex with uPA is detected. The recovery of an internal uPAR standard in breast cancer tissue extracts is above 87%. The intra-assay and inter-assay variation coefficients are 7% and 13%. In order to find a suitable buffer for extraction of various components of the uPA-system from breast cancer tissue, we tested buffers which previously have been used for optimal extraction of estrogen receptor (A), uPA (B), and uPAR (C). Buffer A and B extracted approximately 30% and 50%, respectively, of the amount of uPAR extracted with buffer C. Extracts of samples of breast cancer tissue from 94 patients all contained uPAR in amounts above the detection limit of the present assay, which appears suitable for studies of the potential prognostic value of uPAR in this disease. Significant correlations were found between uPAR, uPA and PAI-1 tumor levels.

Elevated plasma levels of urokinase plasminogen activator receptor in non-small cell lung cancer patients

The urokinase plasminogen activator (uPA) is involved in extracellular matrix degradation during cancer invasion. Binding of uPA to a specific cell surface receptor (uPAR) is a key step in this process. We have previously reported that high levels of uPAR in squamous cell lung cancer tissue extracts are associated with poor prognosis (Pedersen et al., Cancer Res 1994, 54, 4671-4675). Recently we found that uPAR is present in blood plasma from healthy donors as determined by enzyme-linked immunosorbent assay (ELISA) and chemical cross-linking. We now report that uPAR in plasma from 17 patients with non-small cell lung cancer (NSCLC) was significantly higher than in 30 healthy controls (P = 0.0004), while no significant increase was found in plasma from 14 patients with small cell lung cancer (SCLC). The increased levels of uPAR in the plasma from NSCLC patients is likely to be due to release of uPAR from the tumour tissue, and may, therefore, be related to prognosis.

Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass

Purified 54 kDa plasminogen activator inhibitor from human fibrosarcoma cells was converted to an inactive form with slightly higher electrophoretic mobility by incubation with catalytic amounts of urokinase‐type or tissue‐type plasminogen activator. Serine proteinase inhibitors and a monoclonal antibody against urokinase‐type plasminogen activator inhibited the conversion, indicating that it was caused by plasminogen activator‐catalyzed proteolysis. These findings represent the first demonstration of a well‐defined protein apart from plasminogen, constituting a substrate for plasminogen activators.

Prognostic significance of PAI-1 and uPA in cytosolic extracts obtained from node-positive breast cancer patients

Cancer cell invasion is accomplished by the concertedaction of several extracellular proteolytic enzyme systems, oneof which is the urokinase plasminogen activation system.The different components of this system, e.g. urokinaseplasminogen activator (uPA), its receptor uPAR, as wellas its main inhibitor plasminogen activator inhibitor type1 (PAI-1) have all been shown to haveprognostic value in breast cancer, i.e. high tumorlevels are associated with a poor prognosis.In orderto further substantiate the prognostic value of uPAand PAI-1, we have tested the cutpoints (medianvalues and optimized cutpoints) from our first study(Cancer Res 53: 2513–2521, 1993) in an independentgroup of breast cancer patients. Breast cancer cytosolsfrom 100 premenopausal and 150 post-menopausal node positivepatients were included. The median observation time was80 months (range 49–145). Univariate analysis showed thathigh PAI-1 levels (above the median PAI-1 value)were significantly associated with short recurrence-free survival (RR:1.65; 95% CI: 1.04–2.63; P=0.03) andshort overall survival (RR: 2.46; 95% CI: 1.52–3.96;P=0.0001) in postmenopausal patients. Postmenopausal patientswith high uPA levels (above the median uPAvalue) had a significantly shorter recurrence-free survival (RR:2.04; 95% CI: 1.17–3.56; P=0.01) andoverall survival (RR: 2.07; 95% CI: 1.16–3.70; P= 0.01) than patients with low uPA values.Nearly identical results were obtained when using theoptimized PAI-1 or uPA value.In a Cox multivariateanalysis which included other established prognostic factors, highPAI-1 was found to be an independent prognosticvariable predicting short overall survival with a relativerisk of 2.27 in postmenopausal women, and highuPA was found to be an independent prognosticvariable predicting short recurrence-free survival with a relativerisk of 1.86 in postmenopausal women. The presentstudy indicates that uPA and PAI-1 are independentand significant prognostic variables in subsets of breastcancer patients.

Low cathepsin D and low plasminogen activator type 1 inhibitor in tumor cytosols defines a group of node negative breast cancer patients with low risk of recurrence

Prognostic factors are highly needed to divide node negative breast cancer patients into groups of low versus high risk of recurrence and death. In order to invade and spread, cancer cells must degrade extracellular matrix proteins. Accordingly, tumor levels of molecules involved in this degradation might be associated with patient outcome. Previous work has demonstrated that high levels of the aspartyl protease cathepsin D in breast cancer are associated with a poor prognosis and similar findings have been reported for molecules involved in the urokinase pathway of plasminogen activation. Interactions between different protease systems have been described and data suggest that several proteolytic enzymes may be operable at the same time in a tumor. In the present study we measured cathepsin D (n=162), uPA (n=116), uPAR (n=109) and PAI-1 (n=135) in tumor cytosols obtained from a population of node negative breast cancer patients. A significant correlation was found between levels of uPA, uPAR, and PAI-1. Levels of cathepsin D were directly related to levels of uPA and uPAR. With a median observation time of 4.81 years, univariate survival analyses showed that high levels of uPA and cathepsin D significantly predicted a shorter disease free survival, while only high levels of cathepsin D were able to significantly predict a shorter overall survival. Tumor levels of uPAR and PAI-1 gave mixed results depending on the cut-off point choosen. Interestingly, multivariate analysis demonstrated that PAI-1 and cathepsin D were independent significant prognostic indicators for disease-free survival while only cathepsin D was helpful in overall survival. The five year relapse rate of patients with low PAI-1 and low cathepsin D was 13% while patients who had greater than the median value for both of these molecules had a 5 year relapse rate of 40%. These data would indicate that at least two different protease systems are active in spread of node negative breast cancer and that measurement of these molecules may aid in the decisions to be made when offering adjuvant treatment to these patients.

Plasminogen activator inhibitor‐1 as a measure of vascular remodelling in breast cancer

The generation of urokinase plasminogen activator (uPA) by tumours is an important pathway for neoplastic cell invasion and metastasis. Indeed in several tumour types, elevated levels of uPA, its receptor (uPAR) or its inhibitor plasminogen activator inhibitor‐1 (PAI‐1) is associated with a poorer prognosis. Since endothelial cells also use this proteolytic system to remodel the extracellular matrix during angiogenesis and since angiogenesis, as assessed by microvessel density, is also a predictor of patient survival, this study was designed to investigate the relationship between angiogenesis and the urokinase system in breast tumours. The aims were to assess whether the uPA, uPAR and/or PAI‐1 correlates with angiogenic activity and could therefore be a useful objective clinical measure of tumour neovascularization; and to clarify whether the poor outcome associated with high levels of the urokinase system is due to its association with angiogenesis. The study also sought to examine the relationship between the uPA system and vessel remodelling using loss of a basement membrane epitope (LH39) normally associated with established capillaries. The cytosolic levels of uPA, PAI‐1 and uPAR were therefore measured by enzyme linked immunoabsorbent assay, together with tumour vascularity, in 136 well‐characterized invasive breast carcinomas. There were significant relationships between uPA and uPAR (Spearman r=0.37, p<0.0001), uPA and PAI‐1 (Spearman r=0.19, p=0.03) and between uPAR and PAI‐1 (Spearman r=0.23 p=0.01). A significant correlation was also observed between PAI‐1 and vessel remodelling (Spearman r=0.34, p=0.04), patient age (p=0.01), nodal status (p=0.047) and tumour grade (p=0.04), but no association between tumour vascularity and PAI (p=0.96), uPA (p=0.69) or uPAR (p=0.81) was present. No significant association was seen between any of the urokinase variables and expression of the angiogenic factor thymidine phosphorylase. Furthermore, no significant associations were found between any of the studied parameters and overall survival in a univariate analysis of the cancer patients. A multivariate Cox proportional hazard model of overall survival showed that uPA (p=0.15), but not uPAR (p=0.52) or PAI‐1 (p=0.61), gave no additional prognostic information. These findings show that uPA may work via an independent pathway to angiogenesis and therefore combined blockade of uPA and angiogenesis may have additional therapeutic benefits. It also shows, as recently demonstrated in animal models, that PAI‐1 may be a key regulator of vascular remodelling in human cancer. Copyright

Enzyme-linked immunosorbent assay of urokinase-type plasminogen activator (uPA) in cytosolic extracts of human breast cancer tissue.

SummaryThe enzyme urokinase-type plasminogen activator (uPA) plays a role in cancer invasion, and high levels of uPA in detergent extracts of mammary cancer tissue have been reported to be associated with a poor prognosis. We have explored the possibility of using mammary cancer cytosol extracts routinely prepared for steroid receptor analysis for retrospective prognostic studies of uPA. A sandwich enzyme-linked immunosorbent assay (ELISA) for uPA was developed, using polyclonal catching antibodies and a mixture of three biotinylated monoclonal detecting antibodies, that were selected to recognize free uPA, inhibitor-bound uPA, and uPA bound to its cell surface receptor. The assay detects active uPA and its inactive proenzyme form, pro-uPA, equally well. The limit of detection is approximately 1 pg of pro-uPA in a volume of 100 µl, and there is a linear dose-response up to 100 pg pro-uPA. The efficiency in extracting uPA of a neutral non-detergent buffer used to prepare cytosol extracts was compared with that of 4 other buffers. There was a pronounced difference in the efficiency, the most efficient being a pH 4.2 buffer containing the non-ionic detergent Triton X-100, while the least efficient was the buffer used to prepare cytosols. Nevertheless, uPA immunoreactivity was readily measurable in the cytosols, and there was a close correlation between the amounts of uPA extracted under optimal conditions and those routinely used for steroid hormone receptor analysis. While the amount of uPA extracted under the latter conditions constituted only about 12% of that optimally extractable, we conclude that the ELISA described herein can be used to retrospectively analyze the potential prognostic value of uPA-concentrations in cytosols prepared for analysis of steroid hormone receptors.

ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

A sandwich‐type ELISA has been developed for the assessment of complexes between urokinase‐type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti‐uPA antibodies and a biotinylated mouse anti‐uPAR monoclonal antibody (MAb). The detection limit of the assay is approximately 0.5 fmol/ml. A linear dose‐response is obtained with up to 40 fmol/ml of uPA:uPAR complexes, while uPA and uPAR separately do not cause any response in the ELISA. A buffer which has been used previously for optimal extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti‐uPA or anti‐uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross‐linked uPA:uPAR complexes added to tumor extracts varies between 80% and 105%. The intra‐ and inter‐assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly indicate that de novo complex formation is a major factor to consider and that complexes analyzed in the presence of this antagonist represent original uPA:uPAR complexes present prior to tumor tissue processing. The present ELISA appears suitable for studying the potential prognostic impact of uPA:uPAR complexes in lung tumor tissue as well as other types of cancer. Int. J. Cancer 72:416–423, 1997.

Levels of plasminogen activator inhibitor type 1 and urokinase plasminogen activator receptor in non-small cell lung cancer as measured by quantitative ELISA and semiquantitative immunohistochemistry

The components of the plasminogen activation system have been reported to have prognostic impact in several cancer types, e.g. breast-, colon-, gastric- and lung cancer. Most of these studies have used quantification by enzyme-linked immunosorbent assay (ELISA) on tumour tissue extracts. However, results in non-small cell lung cancer (NSCLC) studies obtained by quantitative ELISA and semiquantitative immunohistochemistry differ. If the prognostic value of the components of the plasminogen activation system is to be exploited clinically in the future, it is important to choose an easy and valid methodology. In the present study we investigated levels of plasminogen activator inhibitor type 1 (PAI-1) and urokinase plasminogen activator receptor (uPAR), as quantitated by ELISA in tumour extracts from 64 NSCLC patients (38 squamous cell carcinomas, 26 adenocarcinomas), and compared them to staining intensity as semiquantitated by immunohistochemistry for PAI-1 and uPAR on corresponding cryostat sections. A significant association (r = 0.49, P < 0.0001) was found between the PAI-1 levels measured by ELISA and semiquantitated by immunohistochemistry. No association was found for uPAR. When correlating levels of PAI-1 and uPAR determined by ELISA and immunohistochemistry, respectively, to survival status, no significant correlation was found for any of the subgroups. At present neither of the methods examined in the present study can be recommended as superior for quantitating PAI-1 and uPAR with the aim of predicting prognosis. In conclusion, a larger comparative study is needed to clarify the relationship between ELISA and immunohistochemical results, before a methodology for clinical use can be chosen in non-small cell lung cancer.