Keld Danø

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.

Active outward transport of daunomycin in resistant Ehrlich ascites tumor cells.

Abstract The mechanism of a previously reported decreased accumulation of daunomycin in resistant compared to wild-type Ehrlich ascites tumor cells was investigated in vitro. Several findings indicated an active extrusion of daunomycin from resistant cells: (1) Over a certain range of daunomycin concentrations, the accumulation was considerably higher in isolated nuclei from resistant cells; than in the corresponding whole cells 2) The distribution ratio at steady state of daunomycin in resistant whole cells to that in the medium increased with concentration, in contrast to a decrease for isolated resistant nuclei. (3) Accumulation of daunomycin in resistant cells was enhanced by structural analogs ( N- acetyldaunomycin and daunorubicinol) and by metabolic inhibitors (2-deoxyglucose and iodoacetate). In wild-type cells the accumulation of daunomycin was also increased by 2-deoxyglucose, suggesting an active extrusion of daunomycin from these cells too. The initial rate of net daunomycin uptake was lower in resistant than in wild-type cells, and the decreased accumulation in resistant cells may be due either to a higher rate of active efflux, a lower rate of influx (in the presence of active efflux), or both. The nuclear binding capacity for daunomycin was about the same in the two cell types. Vincristine and vinblastine increased the accumulation of daunomycin in resistant cells. Together with previous findings of reciprocal cross-resistance between daunomycin and the vinca alkaloids, and a decreased accumulation of daunomycin in cells selected for resistance to vincristine and vinblastine, this effect suggests that these drugs are transported by the same extrusion mechanism as daunomycin.

Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis.

The surface receptor for urokinase plasminogen activator (uPAR) has been recognized in recent years as a key molecule in regulating plasminogen mediated extracellular proteolysis. Surface plasminogen activation controls the connections between cells, basement membrane and extracellular matrix, and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N‐terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human fibroblasts [Okayama and Berg (1983) Mol. Cell Biol., 3, 280‐289]. The cDNA encodes a protein of 313 amino acids, preceded by a 21 residue signal peptide. A hydrophobicity plot suggests the presence of a membrane spanning domain close to the C‐terminus. The cDNA hybridizes to a 1.4 kb mRNA from human cells, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis, direct binding studies and cross‐linking experiments show that the transfected mouse LB6 cells specifically bind human uPA, which in turn activates plasminogen. The Mr of the mature human receptor expressed in mouse cells is approximately 55,000, in accordance with the naturally occurring, highly glycosylated human uPAR. The Mr calculated on the basis of the cDNA sequence, approximately 35,000, agrees well with that of the deglycosylated receptor.

CANCER INVASION AND TISSUE REMODELING : COMMON THEMES IN PROTEOLYTIC MATRIX DEGRADATION

Analysis of extracellular matrix degradation systems has led to the insight that in cancer invasion there is often crucial interplay between cancer cells and several types of surrounding non-neoplastic stromal cells. Likewise, in normal tissue remodeling processes, the synthesis of proteolytic components is often distributed between several cell types, and there are strong similarities between neoplastic and non-neoplastic processes in the same tissue. Thus, tissue remodeling events are excellent models for studies of extracellular proteolysis in cancer. This has become even clearer by recent analyses of genetically modified mice.

Plasminogen activation and cancer

Breakdown of the extracellular matrix is crucial for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and a number of matrix metalloproteases. The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors. Thus, the generation of plasmin involves the pro-enzyme plasminogen, the urokinase type plasminogen activator uPA and its pro-enzyme pro-uPA, the uPA inhibitor PAI-1, the cell surface uPA receptor uPAR, and the plasmin inhibitor alpha(2)-antiplasmin. Furthermore, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of origin during non-neoplastic tissue remodelling processes. We propose that cancer invasion can be considered as uncontrolled tissue remodelling. Inhibition of extracellular proteases is an attractive approach to cancer therapy. Because proteases have many different functions in the normal organism, efficient inhibition will have toxic side effects. In cancer invasion, like in normal tissue remodelling processes, there appears to be a functional overlap between different extracellular proteases. This redundancy means that combinations of protease inhibitors must be used. Such combination therapy, however, is also likely to increase toxicity. Therefore for each type of cancer, a combination of protease inhibitors that is optimised with respect to both maximal therapeutic effect and minimal toxic side effects need to be identified.

Cancer invasion and tissue remodeling : cooperation of protease systems and cell types

Proteolytic degradation of the extracellular matrix plays a crucial role in both cancer invasion and non‐neoplastic tissue remodeling processes. In human cancers the components of matrix degrading protease systems (uPA, uPAR, PAI‐1 and MMPs) can be expressed by either the non‐neoplastic stromal cells, the cancer cells or both. Studies of the prognostic impact of these components in human cancer and the effect of targeted gene inactivation on cancer metastasis in mice support the assumption that proteases promote cancer progression, independent of whether they are expressed by cancer cells or stromal cells. The pattern of expression of components of protease systems is usually very similar in different cases of the same type of cancer, while it varies between different types of cancer. There are intriguing similarities between the cellular expression pattern of components of protease systems seen in cancer invasion and in certain types of non‐neoplastic tissue remodeling. We propose that cancer invasion can be viewed as tissue remodeling gone out of control. The stromal cell involvement in cancer invasion represents a new paradigm with important implications for cancer pathophysiology and cancer therapy.

Functional overlap between two classes of matrix-degrading proteases in wound healing.

Retarded wound healing was found in mice deficient in the serine protease precursor plasminogen, as well as in wild‐type mice treated with the metalloprotease inhibitor galardin, but in both cases wound closure was ultimately completed in all mice within 60 days. The expression of several matrix metalloproteases in keratinocytes migrating to cover the wound was strongly enhanced by galardin treatment. However, when plasminogen‐deficient mice were treated with galardin, healing was completely arrested and wound closure was not seen during an observation period of 100 days, demonstrating that protease activity is essential for skin wound healing. The requirement for both plasminogen deficiency and metalloprotease inhibition for complete inhibition of the healing process indicates that there is a functional overlap between the two classes of matrix‐degrading proteases, probably in the dissection of the fibrin‐rich provisional matrix by migrating keratinocytes. Each class alone is capable of maintaining sufficient keratinocyte migration to regenerate the epidermal surface, although this function would normally be performed by both classes acting in parallel. Since there are strong similarities between the proteolytic mechanisms in wound healing and cancer invasion, these results predict that complete arrest of this latter process in therapeutic settings will require the use of inhibitors of both classes of proteases.

Transforming growth factor-beta is a strong and fast acting positive regulator of the level of type-1 plasminogen activator inhibitor mRNA in WI-38 human lung fibroblasts.

We have studied the mechanism of a transforming growth factor‐beta (TGF‐beta)‐stimulated production of type‐1 plasminogen activator inhibitor (PAI‐1) in WI‐38 human lung fibroblasts. TGF‐beta causes an early increase in the PAI‐1 mRNA level which reaches a maximal 50‐fold enhancement after 8 h. Blocking of protein synthesis with cycloheximide causes an equally strong increase in the level of PAI‐1 mRNA. Quantitative studies of the effect of TGF‐beta on PAI‐1 protein levels in cell extracts and culture media by using monoclonal antibodies are consistent with the effect on PAI‐1 mRNA. The results suggest a primary effect of TGF‐beta on PAI‐1 gene transcription, and also suggest the possibility that the transcription of this gene in non‐induced cells may be suppressed by a short‐lived negatively regulating protein. Urokinase‐type (u‐PA) and tissue‐type (t‐PA) plasminogen activators are decreased in the culture media of TGF‐beta‐treated cells concomitantly with the increase in PAI‐1 accumulation. These findings show that a primary and important biological effect of TGF‐beta may be an overall decreased extracellular proteolytic activity, and give an insight into the molecular mechanisms underlying TGF‐beta action at the genetic level.

Cell-induced potentiation of the plasminogen activation system is abolished by a monoclonal antibody that recognizes the NH2-terminal domain of the urokinase receptor

We have raised four monoclonal antibodies recognizing different epitopes within the human cell‐surface receptor for urokinase‐type plasminogen activator (u‐PA). One of these antibodies completely abolishes the potentiation of plasmin generation observed upon incubation of the zymogens pro‐u‐PA and plasminogen with U937 cells. This antibody, which is also the only one to completely inhibit the binding of DFP‐inactivated [125I]‐u‐PA to U937 cells, is directed against the u‐PA binding NH2‐terminal domain of u‐PAR, a well‐defined fragment formed by limited chymotrypsin digestion of purified u‐PAR, demonstrating the functional independence of the u‐PA binding domain as well as the critical role of u‐PAR in the assembly of the cell‐surface plasminogen activation system.

Crystal structure of the human urokinase plasminogen activator receptor bound to an antagonist peptide

We report the crystal structure of a soluble form of human urokinase‐type plasminogen activator receptor (uPAR/CD87), which is expressed at the invasive areas of the tumor‐stromal microenvironment in many human cancers. The structure was solved at 2.7 Å in association with a competitive peptide inhibitor of the urokinase‐type plasminogen activator (uPA)–uPAR interaction. uPAR is composed of three consecutive three‐finger domains organized in an almost circular manner, which generates both a deep internal cavity where the peptide binds in a helical conformation, and a large external surface. This knowledge combined with the discovery of a convergent binding motif shared by the antagonist peptide and uPA allowed us to build a model of the human uPA–uPAR complex. This model reveals that the receptor‐binding module of uPA engages the uPAR central cavity, thus leaving the external receptor surface accessible for other protein interactions (vitronectin and integrins). By this unique structural assembly, uPAR can orchestrate the fine interplay with the partners that are required to guide uPA‐focalized proteolysis on the cell surface and control cell adhesion and migration.