Peter A. Andreasen

Plasminogen Activators, Tissue Degradation, and Cancer

Publisher Summary This chapter discusses the role of plasminogen activators in various biological processes. In specific, it describes two types of plasminogen activators—namely, the urokinase-type plasminogen activator (u-PA) and the tissue-type plasminogen activator (t-PA), which are essentially different gene products. The amino acid sequences of these activators and nucleotide sequences of the corresponding cDNAs have largely been determined, and the cDNAs have been cloned using recombinant techniques. A variety of enzymatic as well as immunological assay and detection methods have also been developed that allows a precise quantification of the activators, a distinction between u-PA and t-PA, determination of whether an activator is present in its active or zymogen form, analysis of the kinetics of different steps of the cascade reaction, and immunocytochemical identification of u-PA and t-PA in tissue sections. Much of the studies on plasminogen activators and cancer has been guided by the hypothesis that proteolysis of the components of extracellular matrix, initiated by the release of plasminogen activator from the cancer cells, plays a decisive role for the degradation of normal tissue, and thereby for invasive growth and metastases.

The urokinase‐type plasminogen activator system in cancer metastasis: A review

The urokinase‐type plasminogen activator (u‐PA) system consists of the serine proteinases plasmin and u‐PA; the serpin inhibitors α2‐anti‐plasmin, PAI‐1 and PAI‐2; and the u‐PA receptor (u‐PAR). Two lines of evidence have strongly suggested an important and apparently causal role for the u‐PA system in cancer metastasis: results from experimental model systems with animal tumor metastasis and the finding that high levels of u‐PA, PAI‐1 and u‐PAR in many tumor types predict poor patient prognosis. We discuss here recent observations related to the molecular and cellular mechanisms underlying this role of the u‐PA system. Many findings suggest that the system does not support tumor metastasis by the unrestricted enzyme activity of u‐PA and plasmin. Rather, pericellular molecular and functional interactions between u‐PA, u‐PAR, PAI‐1, extracellular matrix proteins, integrins, endocytosis receptors and growth factors appear to allow temporal and spatial re‐organizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. Differential expression of components of the system by cancer and non‐cancer cells, regulated by paracrine mechanisms, appear to determine the involvement of the system in cancer cell–directed tissue remodeling. A detailed knowledge of these processes is necessary for utilization of the therapeutic potential of interfering with the action of the system in cancers. Int. J. Cancer 72:1–22, 1997.

The plasminogen activation system in tumor growth, invasion, and metastasis

Abstract. Generation of the serine proteinase plasmin from the extracellular zymogen plasminogen can be catalyzed by either of two other serine proteinases, the urokinase- and tissue-type plasminogen activators (uPA and tPA). The plasminogen activation system also includes the serpins PAI-1 and PAI-2, and the uPA receptor (uPAR). Many findings, gathered over several decades, strongly suggest an important and causal role for uPA-catalyzed plasmin generation in cancer cell invasion through the extracellular matrix. Recent evidence suggests that the uPA system is also involved in cancer cell-directed tissue remodeling. Moreover, the system also supports cell migration and invasion by plasmin-independent mechanisms, including multiple interactions between uPA, uPAR, PAI-1, extracellular matrix proteins, integrins, endocytosis receptors, and growth factors. These interactions seem to allow temporal and spatial reorganizations of the system during cell migration and a selective degradation of extracellular matrix proteins during invasion. The increased knowledge about the plasminogen activation system may allow utilization of its components as targets for anti-invasive therapy.

Plasminogen activator inhibitors: hormonally regulated serpins

Etude de la regulation hormonale des enzymes appartenant a la famille de serine protease inhibiteur (plasminogen activator et urokinase)

Receptor‐mediated endocytosis of plasminogen activators and activator/inhibitor complexes

Recent findings have elucidated the mechanism for clearance from the extracellular space of the two types of plasminogen activators, urokinase‐type plasminogen activator (u‐PA) and tissue‐type plasminogen activator (t‐PA), and their type‐1 inhibitor (PAI‐1). Activator/PAI‐1 complexes and uncomplexed t‐PA bind to the multiligand receptors α2 macroglubulin receptor/low density lipoprotein receptor‐related protein (α2MR) and epithelial glycoprotein 330 (gp330). These receptors mediate endocytosis and degradation of u‐PA/PAI‐1 complex bound to the glycosyl phosphatidyl inositol‐anchored urokinase receptor (u‐PAR) on cell surfaces, and participate, in cooperation with other receptors, in hepatic clearance of activator/PAI‐1 complexes and uncomplexed t‐PA from blood plasma. The α2MR‐ and gp330‐mediated endocytosis of a ligand (u‐PA/PAI‐1 complex) initially bound to another receptor (u‐PAR) is a novel kind of interaction between membrane receptors. Binding to α2MR and gp330 is a novel kind of molecular recognition of serine proteinases and serpins.

Human endothelial cells contain one type of plasminogen activator

At least two types of animal plasminogen activating enzymes exist, differing in amino acid sequence, molecular mass and immunological reactivity: the urokinase‐type and the tissue‐type plasminogen activators. By affinity chromatography with monoclonal antibodies, we have purified the human activators of both types to homogeneity. Using immunocytochemistry with rabbit antibodies raised against these preparations, we now demonstrate that the plasminogen activator present in endothelium of veins and other blood vessels is of the tissue‐type. No urokinase‐type plasminogen activator immunoreactivity was detected in endothelial cells in the intact organism. These findings support the assumption that mobilization of plasmin for different purposes may involve different types of plasminogen activators, and that the plasminogen activator involved in thrombolysis is of the tissue‐type.

Plasminogen activator inhibitor-1 and tumour growth, invasion, and metastasis

In recent decades, evidence has been accumulating showing the important role of urokinase-type plasminogen activator (uPA) in growth, invasion, and metastasis of malignant tumours. The evidence comes from results with animal tumour models and from the observation that a high level of uPA in human tumours is associated with a poor patient prognosis. It therefore initially came as a surprise that a high tumour level of the uPA inhibitor plasminogen activator inhibitor-1 (PAI-1) is also associated with a poor prognosis, the PAI-1 level in fact being one of the most informative biochemical prognostic markers. We review here recent investigations into the possible tumour biological role of PAI-1, performed by animal tumour models, histological examination of human tumours, and new knowledge about the molecular interactions of PAI-1 possibly underlying its tumour biological functions. The exact tumour biological functions of PAI-1 remain uncertain but PAI-1 seems to be multifunctional as PAI-1 is expressed by multiple cell types and has multiple molecular interactions. The potential utilisation of PAI-1 as a target for anti-cancer therapy depends on further mapping of these functions.

Immunohistochemical localization of urokinase‐type plasminogen activator, type‐1 plasminogen‐activator inhibitor, urokinase receptor and α2‐macroglobulin receptor in human breast carcinomas

We have investigated the localization of urokinase‐type plasminogen activator (u‐PA), type‐1 plasminogen‐activator inhibitor (PAI‐1), u‐PA receptor (u‐PAR) and α2‐macroglobulin‐receptor/low‐density‐lipoprotein‐receptor‐related protein (α2MR/LRP) in human breast tumors by immunohistochemical methods. Frozen sections of 133 primary breast carcinomas, 6 ductal carcinomas in situ and 33 lymph‐node metastases were stained with monoclonal antibodies. Formalin‐fixed sections of 15 primary tumors and 2 lymph‐node metastases were stained with polyclonal antibodies. In primary tumors, u‐PA and PAI‐1 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. α2MR/LRP and u‐PAR immunoreactivities were intense in macrophages, but apart from these cells, α2MR/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. Lymph‐node metastases showed staining for u‐PA and PAI‐1 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‐1 levels, as determined by ELISA, than extracts of metastases without this staining pattern. α2MR/LRP was present only in fibroblasts and u‐PAR only in some tumor cells. The presence of u‐PA, PAI‐1, α2MR/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.

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer

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 follow-up 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.

Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor

The latent form of type-1 plasminogen activator inhibitor (PAI-1) acquires inhibitory activity by denaturation followed by refolding. We show here that the reactions of denatured/refolded PAI-1 with plasminogen activators are affected by low concentrations of SDS, which may remain after using SDS for denaturation. Without SDS, the active fraction of denatured/refolded PAI-1 comprised around 60%. Increasing SDS concentrations led to conversions to an inert form without inhibitory activity; then to a substrate form, that is being cleaved proteolytically in the reactive centre by the activators without complex formation, and finally to a second inert form. The first two conversions were associated with changes of the reactivity with monoclonal antibodies and of the thermal stability, respectively. Our results define clearly different interconvertible forms of denatured/refolded PAI-1, distinguish these from the latent and the reactive-centre-cleaved forms, and provide conditions for reproducibly producing reactive-centre-cleaved PAI-1 and PAI-1/activator complexes.