Suppression of inhibitors

Details Suppression of inhibitors Suppression of inhibitors

1996-05-15

2001-05-01

Ungar, Susan (Department: 1642)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

42, 42, 42, 42, 42, 42, 42, 42, 42, 42, C514S002600

Reexamination Certificate

active

06224865

ABSTRACT:

BRIEF DESCRIPTION OF THE INVENTION
In order to invade and spread, cancer cells must degrade extracellular matrix proteins. This degradation is catalyzed by the concerted action of several enzymes including proteases and non-proteolytic matrix-degrading enzymes e.g. metalloproteases such as interstitial collagenases, type IV collagenases and stromelysins, aspartic proteases such as cathepsin D, other degradative enzymes such as heperanase, and serine proteases such as plasmin (1, 73-76). Plasmin is formed from its precursor, plasminogen, by two activators, tissue-type plasminogen activator (tPA) which is involved in thrombolysis, and urokinase-type plasminogen activator (uPA) which plays a central role in tissue remodelling, including cancer invasion. The activation of plasminogen is regulated by two specific plasminogen activator inhibitors (PAI-1 and PAI-2). Both uPA and PAI-1 are present in various types of cancer tissue, and it was recently found that high levels of uPA and PAI-1 in breast cancer tissue each is associated with poor prognosis.
Immunohistochemical and in situ hybridization studies have revealed that in breast and colon cancer tissue, PAI-1 is expressed predominantly in stromal cells.
According to the invention it is contemplated that the high PAI-1 content found in malignant tumours from patients with poor prognosis as described in detail later is involved in promotion of tumour growth, invasion and/or metastasis, most likely by protecting the stromal element of the tumour from degradation due to plasminogen activation formed in the microenvironment during invasion. This invention relates to the suppression of inhibitors of proteases and/or of other matrix-degrading enzymes, in particular to the suppression of plasminogen activator inhibitors such as PAI-1 in cancer tissue resulting in inhibition of growth and/or invasion and/or metastasis of tumour e.g. by allowing autodegradation of the tumour tissue, impairment of cancer cell migration and/or impairment of tumour angiogenesis.
GENERAL BACKGROUND
Urokinase-Type Plasminogen Activator
The biochemistry of uPA has been reviewed previously (1). It is a serine protease, which is synthesized as an approximately 50 kD glycosylated single polypeptide chain pro-enzyme, pro-uPA, that is virtually catalytically inactive. The human uPA gene is located on chromosome 10 and is transcribed into a 2.5 kb long mRNA. Pro-uPA is converted into active uPA, consisting of two polypeptide chains (A and B) held together by a disulphide bond, the A-chain arising from the amino-terminal part of pro-uPA, and the B-chain arising from the carboxy-terminal part. The A-chain consists of two structural domains, a growth factor domain with homology to EGF, and a kringle domain (2). The B-chain is homologous to the catalytic part of other serine proteases, such as trypsin, chymotrypsin, and plasmin. Two-chain uPA can, by the metalloproteinase matrilysin (or PUMP-1) (3), be converted into a 33 kD catalytic active form of uPA consisting of the B-chain and the carboxy-terminal part of the A-chain (low molecular weight uPA), and a 17 kD non-catalytic fragment consisting of the N-terminal part of the A-chain.
uPA cleaves a single peptide bond in plasminogen, converting it into plasmin, that degrades a broad spectrum of proteins, including fibronectin, fibrin, and laminin (for a review see ref.(1)). In addition, plasmin activates latent forms of some metalloproteases (4) and affects various growth factor systems, e.g. by activating latent TGF-&bgr; (5,6) and dissociating IGF-I from its binding protein (7) and bFGF from the surface of some cell types (8).
Many cytokines and hormones control the expression of uPA in a cell specific way (see reference (9)).
uPA is produced by many cultured cell types of neoplastic origin. It has been found that explants of tumour tissue released more uPA than the corresponding normal tissue. uPA has been identified in extracts from human lung, colon, endometrial, breast, prostate and renal carcinomas, human melanomas, murine mammary tumours, the murine Lewis lung tumour, and in ascites from human peritoneal carcinomatosis. An immunohistochemical study of invasively growing and metastasing Lewis lung carcinomas in mice consistently showed the presence of uPA, but also a pronounced heterogenecity in the content of uPA in different parts of the individual tumours. A high uPA content was found in areas with invasive growth and degradation of surrounding normal tissue, while other areas were devoid of detectable uPA. The uPA was located in the cytoplasm of the tumour cells and extracelluarly surrounding the tumour cells.
Degradation of the surrounding normal tissue is a central feature of invasiveness of malignant tumours. The constant finding of uPA in malignant tumours and the findings indicating that uPA plays a role in tissue degradation in normal physiological events have led to the assumption that uPA plays a similar role in cancer development. The hypothesis of uPA playing a role in tissue destruction involves the assumption that plasmin, together with other proteolytic enzymes, degrades the extracellular matrix. It is noteworthy in this context that most components of the extracellular matrix can be degraded by plasmin. These include laminin, fibronectin, proteoglycans, and possibly some types of collagen, but not all. In addition, plasmin can activate latent collagenases which in turn can degrade the other types of collagen (4).
Many research groups have proposed that invasive tumour cells secrete matrix-degrading proteinases and that one of the crucial cascades is the plasminogen activation system. Regulation of the proteolysis can take place at many levels including tumour cell-host cell interactions and protease inhibitors produced by the host or by the tumour cells themselves. Expression of matrix-degrading enzymes is not tumour cell specific. The actively invading tumour cells may merely respond to different regulatory signals compared to their non-invasive counterparts (10).
The assumption that the plasminogen activation system, through a breakdown of extracellular matrix proteins, plays a role in invasiveness and destruction of normal tissue during growth of malignant tumours is supported by a variety of findings. These include a close correlation between transformation of cells with oncogenic viruses and synthesis of uPA, the finding that uPA is involved in tissue destruction in many non-malignant conditions, and the immunohistochemical localization of uPA in invading areas of tumours (see (1) for review).
Further support for this hypothesis has come from studies with anti-catalytic antibodies to uPA in model systems for invasion and metastasis. Such antibodies were found to decrease metastasis to the lung from a human uPA producing tumour, HEp-3, transplanted onto the chorioallantoic membrane of chicken embryos (11,12), penetration of amniotic membranes by B16 melanoma cells (13), basement membrane invasion by several human and murine cell lines of neoplastic origin (14), and formation of lung metastasis after intravenous injection of B16 melanoma cells in mice (15). In some of these studies (13,14), a plasmin-catalyzed activation of procollagenases appeared to be a crucial part of the effect of plasminogen activation.
Urokinase-Thype Plasminogen Activator Receptor
A specific cell surface receptor for uPA (uPAR) was first detected by a saturable binding of uPA to monocytes and monocyte-like cells (16) and has since been found on many types of cultured cancer cells (17). Human uPAR is a single polypeptide-chain, highly glycosylated protein with a molecular weight of 55-60 kD (18). It is translated from a 1.4 kb mRNA (19), encoded by a single gene located on chromosome 19.
It consists of three homologous domains. The amino-terminal domain (domain 1) contains the ligand binding region (20), which binds to the EGF-like domain in the uPA molecule (21). uPAR is carboxy-terminally anchored to the cell surface by a glycosyl-phosphatidylinositol moiety (22). A possible function of this lipid anchor is to facilit

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