Very low density lipoprotein receptor (VLDL-R) was found to be expressed in bovine mammary gland and the human breast carcinoma cell line MCF-7 as an M(r) 105,000 variant, and in Chinese hamster ovary (CHO) cells transfected with human VLDL-R cDNA as an M(r) 130,000 variant. The receptor was purified by ligand affinity chromatography with immobilized M(r) 40,000 receptor-associated protein (RAP). The purified receptor was found to bind urokinase-type plasminogen activator-type-1 plasminogen activator inhibitor complex (u-PA.PAI-1), while there was no or very weak binding of active site blocked u-PA (DFP-u-PA), PAI-1 or u-PA-type-2 plasminogen activator inhibitor complex. The binding of u-PA.PAI-1 was blocked by RAP. The transfected CHO cells had an efficient, RAP-sensitive endocytosis of u-PA.PAI-1, severalfold higher than non-transfected parental CHO cells. u-PA.PAI-1 endocytosis was partially inhibited by DFP-u-PA, which blocks binding of the complex to the u-PA receptor. RAP and DFP-u-PA sensitive u-PA.PAI-1 endocytosis was also observed in MCF-7 cells, which were without detectable levels of other RAP-binding endocytosis receptors. These results show that VLDL-R represents a novel endocytosis mechanism for u-PA receptor-bound u-PA.PAI-1.

Summary The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median followup of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay.The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg-1 protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.Keywords: urokinase; plasminogen; PAI-1; breast neoplasm mortality; prognosis Cancer cells undergo the following steps during metastasis: detachment from the primary tumour; migration; invasion of the blood and lymphatic vessels; adhesion to and penetration of the endothelium, allowing colonization at distant sites (Liotta et al, 1991). Tumour progression and metastasis also involve various processes that may be called cancer cell-directed tissue remodelling. Examples are angiogenesis (Folkman, 1995) and desmoplasia (Dvorak et al, 1995).Extracellular proteolysis has been implicated in cancer metastasis for many years, with the basic idea that release of proteolytic enzymes from a tumour leads to breakdown of basement membranes and extracellular matrix (ECM), thus allowing cancer cell invasion into the surrounding normal tissue. This is true for plasminogen activators and metalloproteinases (Dan0 et al, 1985;Liotta et al, 1991;Mignatti and Rifkin, 1993;Andreasen et al, 1997). There are two types of plasminogen activator, the urokinase-type uPA and the tissue-type tPA. Both are capable of catalysing the formation of the broad spectrum proteinase plasmin from the inactive zymogen plasminogen. There seems to be general agreement that uPA is most important for generation of plasmin in events involving degradation of ECM, while the primary role of tPA is to generate plasmin for thrombolysis. In relation to cancer metastasis, therefore, uPA is of main interest. There are two main types of inhibitors of plasminogen activator, PAI-1 and PAI-2. The urokinase receptor (uPAR) serves to localize plasminogen activation to cell surfaces (Dan0 et al, 1985;Andreasen et al, 1990Andreasen et al, , 1997Mignatti and Rifkin, 1993 Recent studies have shown that the levels of uPA and PAI-I in malignant tumours vary considerably and are related to the prognosis of the patient (Andreasen et al, 1997). Whether uPA and PAI-I are related to local recurrence, metastatic spread or both is not clear. None of the research groups has to our knowledge looked specifically into the levels of...

We have investigated the localization of urokinase-type plasminogen activator (u-PA), type-I plasminogen-activator inhibitor (PAL I), U-PA receptor (u-PAR) and q-macroglobulinreceptor/low-density-lipoprotein-receptor-related protein (a2MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of I33 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph-node metastases were stained with monoclonal antibodies. Formalin-fixed sections of I 5 primary tumors and 2 lymph-node metastases were stained with polyclonal antibodies. In primary tumors, u-PA and PAI-I immunoreactivities were intense in macrophages and mast cells, and moderate in benign and malignant epithelial cells as well as in myofibroblasts and endothelial cells. A sub-group of poorly differentiated tumors showed particularly strong staining of stromal fibroblasts. U-PA immunoreactivity was also present in lymphocytes. a,MR/LRP and U-PAR immunoreactivities were intense in macrophages, but apart from these cells, a,MR/LRP was found only in fibroblasts, and U-PAR only in tumor cells located peripherally in tumor-cell clusters and glands and some myofibroblasts in the adjacent stroma. Lymphnode metastases showed staining for u-PA and PAI-I both of cancer cells and of stromal fibroblasts, also staining for U-PA of lymphocytes. Similarly to some of the poorly differentiated primary tumors, approximately half of the metastases showed very strong staining of stromal fibroblasts, and extracts of these metastases had higher U-PA and PAI-I levels, as determined by ELISA, than extracts of metastases without this staining pattern. qMR/LRP was present only in fibroblasts and U-PAR only in some tumor cells. The presence of u-PA, PAI-I, alMR/LRP and U-PAR was controlled biochemically by immunoblotting analyses, ligand-blotting analyses, and direct and reverse zymography. The spatial distribution and the variation in concentration of the various components of the plasminogen-activation system point to a complex, multifunctional role for the 4 proteins in and/or during the development and spread of breast cancer.o 1996 Wiley-Liss, Inc.

We have investigated the localization of urokinase-type plasminogen activator (u-PA), type-I plasminogen-activator inhibitor (PAL I), U-PA receptor (u-PAR) and q-macroglobulinreceptor/low-density-lipoprotein-receptor-related protein (a2MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of I33 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph-node metastases were stained with monoclonal antibodies. Formalin-fixed sections of I 5 primary tumors and 2 lymph-node metastases were stained with polyclonal antibodies. In primary tumors, u-PA and PAI-I immunoreactivities were intense in macrophages and mast cells, and moderate in benign and malignant epithelial cells as well as in myofibroblasts and endothelial cells. A sub-group of poorly differentiated tumors showed particularly strong staining of stromal fibroblasts. U-PA immunoreactivity was also present in lymphocytes. a,MR/LRP and U-PAR immunoreactivities were intense in macrophages, but apart from these cells, a,MR/LRP was found only in fibroblasts, and U-PAR only in tumor cells located peripherally in tumor-cell clusters and glands and some myofibroblasts in the adjacent stroma. Lymphnode metastases showed staining for u-PA and PAI-I both of cancer cells and of stromal fibroblasts, also staining for U-PA of lymphocytes. Similarly to some of the poorly differentiated primary tumors, approximately half of the metastases showed very strong staining of stromal fibroblasts, and extracts of these metastases had higher U-PA and PAI-I levels, as determined by ELISA, than extracts of metastases without this staining pattern. qMR/LRP was present only in fibroblasts and U-PAR only in some tumor cells. The presence of u-PA, PAI-I, alMR/LRP and U-PAR was controlled biochemically by immunoblotting analyses, ligand-blotting analyses, and direct and reverse zymography. The spatial distribution and the variation in concentration of the various components of the plasminogen-activation system point to a complex, multifunctional role for the 4 proteins in and/or during the development and spread of breast cancer.o 1996 Wiley-Liss, Inc.

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