The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts.

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression.

Urokinase-type plasminogen activator (uPA) is a protease involved in the process of tissue remodelling and cell migration in vitro. To explore whether uPA is a prerequisite for human ovarian cancer spread in vivo the expression of uPA was suppressed in human ovarian cancer cells by antisense phosphorothioate oligonucleotides (PS-ODN). The suppression of uPA expression was dependent on PS-ODN concentration and only observed in the presence of liposomes. This phenomenon seemed to be due to the fact that PS-ODNs were taken up by the cancer cells only in concert with liposomes as studied by fluorescently-labeled PS-ODNs using flow cytofluorometry and laser scanning microscopy. uPA-deprived cancer cells exhibited a significantly reduced invasive capacity in vitro compared with untreated cancer cells or cells treated with control PS-ODNs (P = 0.003). The intraperitoneal spread of the cancer cells in vivo was significantly diminished when nude mice were treated with uPA antisense PS-ODNs in comparison with control mice (P = 0.009). These results suggest that uPA expression may be required for spread of human ovarian cancer and that its inhibition could provide a therapeutic approach.

Antisense inhibition of urokinase reduces spread of human ovarian cancer in mice

uPAR (CD87), the receptor for the urokinase-type plasminogen activator (uPA) facilitates tumor cell invasion and metastasis by focusing uPA proteolytic activity to the cell surface As uPAR exists in various molecular forms, it is desirable to use well defined antibodies for analyses of uPAR antigen expression in human malignant tumors by immunological methods Therefore, twelve monoclonal antibodies (MAbs) directed against uPAR were generated by using nonglycosylated, recombinant human uPAR (spanning amino acids 1 to 284), expressed in Escherichia coli, as the immunogen The reaction pattern of these MAbs with the immunogen and a series of carboxyl-terminally truncated versions of uPAR demonstrated that at least six different epitopes of uPAR are recognized All MAbs reacted under reducing conditions in immunoblot analyses with E coli-expressed uPA and also with highly glycosylated, functionally intact, recombinant human uPAR expressed in Chinese hamster ovary (CHO) cells Seven of the MAbs recognized CHO uPAR under nonreducing conditions as well By flow cytofluorometric analyses, three of these MAbs were shown to bind to native human uPAR present on the cell surface of monocytoid U937 cells with MAb IIIF10 being the best Saturation of uPAR with uPA on U937 cells completely blocked interaction of MAb IIIF10 with uPAR (mapped epitope, amino acids 52 to 60 of domain I of uPAR) In turn, preincubation of U937 cells with MAb IIIF10 efficiently reduced binding of uPA to uPAR, indicating that the epitope detected by MAb IIIF10 is located within or closely to the uPA-binding site of uPAR, and thus, this site may be a target to influence uPA/uPAR-mediated proteolysis in tumors Binding of MAbs IID7 or IIIB11 (mapped epitope, amino acids 125 to 132 of domain II of uPAR) to uPAR is not affected when uPAR is occupied by uPA As these MAbs reacted strongly with cellular uPAR antigen in formalin-fixed paraffin-embedded tumor sections, the domain-II-specific antibodies IID7 and IIIB11 may be useful for immunohistochemical studies of uPAR expression in tissue remodeling processes in tumor invasion In conclusion, we have devised well defined and epitope-mapped MAbs to uPAR that are highly specific tools for detection and targeting of uPAR in tumor tissue

Epitope-mapped monoclonal antibodies as tools for functional and morphological analyses of the human urokinase receptor in tumor tissue.

The overall survival rate of patients suffering from carcinomas has remained poor and nearly unchanged over the last decades. This is mainly due to the so-called minimal residual disease, i.e., remaining tumor cells that overcome surgery and/or radiotherapy and are the cause of locoregional and distant metastases. To metastasize, tumor cells take advantage of proteases to invade and remodel surrounding tissues. Here, we analyzed the efficiency of WX-UK1, a novel 3-amidinophenylalanine-based inhibitor of the uPA system, at inhibiting the invasive capacity of carcinoma cells. First, uPAR expression was characterized in different carcinoma cell lines, including SCCHN, breast and cervical carcinoma. Thereafter, the invasive potential of these cell lines was determined using Matrigel invasion chambers and a spheroid cocultivation model with human fibroblasts. uPAR expression levels correlated positively with invasion capacity, which could be significantly inhibited by WX-UK1. A decrease of tumor cell invasion by up to 50% was achieved in both models with the SCCHN line FaDu and the cervical carcinoma line HeLa after treatment with WX-UK1. Thus, our results demonstrate the potential of WX-UK1 in vitro as a promising adjuvant antimetastatic therapy of carcinomas. © 2004 Wiley-Liss, Inc.

https://onlinelibrary.wiley.com/doi/pdf/10.1002/ijc.20192

Inhibition of the invasion capacity of carcinoma cells by WX‐UK1, a novel synthetic inhibitor of the urokinase‐type plasminogen activator system

Extravasation and intravasation of tumor cells in solid malignant tumors is controlled by 3 steps: 1) attachment to and interaction of tumor cells with components of the basement membrane and the extracellular matrix, 2) local proteolysis, and 3) tumor cell migration. Evidence has accumulated that different types of tumor-associated proteases, their inhibitors and receptors are involved in tumor invasion and metastasis. Four different classes of proteases are known to be correlated with the malignant phenotype: 1) Matrix metalloproteases; including collagenases, gelatinases and stromelysins. 2) Cysteine proteases; including cathepsins B and L. 3) Aspartyl protease cathepsin D. 4) Serine proteases; including plasmin and tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). A strong independent prognostic value (relapse-free and/or overall survival) has especially been demonstrated for uPA and its inhibitor PAI-1 in patients with cancer of the breast, ovary, stomach, esophagus, colon, lung, and kidney thus predicting the course of the cancer disease. The strong correlation between elevated uPA and/or PAI-1 values in primary cancer tissues and the malignant phenotype of cancer cells has prompted to explore new tumor biology-oriented concepts in order to suppress uPA or uPA receptor (CD87) expression or to abrogate interaction of uPA with CD87. Various very different approaches to interfere with the expression or reactivity of uPA or CD87 at the gene or protein level were successfully tested including antisense oligonucleotides, antibodies, inhibitors and recombinant or synthetic uPA and CD87 analogues.

Urokinase-type plasminogen activator (uPA) and its receptor (CD87): a new target in tumor invasion and metastasis.

Expression of urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), their inhibitor PAI-1 and the uPA-receptor (uPAR) was characterized in six human tumor cell lines (OV-MZ-6, -10, -13, -15, -19 and OVCAR-3) established from patients with cystadenocarcinoma of the ovary. The invasive potential of the ovarian cancer cell lines determined in an in vitro invasion assay did neither correlate with the antigen level of uPA, t-PA, PAI-1 or uPAR nor with the cell surface uPA activity, however, did correlate with the cell surface-bound plasmin activity. The in vitro invasiveness of three cancer cell lines selected displaying a different pattern of uPA and uPAR expression was significantly inhibited by a recombinant soluble truncated form of the uPAR functioning as a scavenger for uPA. Our results suggest that the interference of the uPA/uPAR interaction leads to a reduced in vitro invasiveness of human ovarian cancer cells independent of the level of uPA and uPAR expression.

Expression of urokinase-type plasminogen-activator (upa) and its receptor (upar) in human ovarian-cancer cells and in-vitro invasion capacity.

The urokinase/urokinase receptor system A new target for cancer therapy?

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Expression of urokinase-type plasminogen-activator (upa) and its receptor (upar) in human ovarian-cancer cells and in-vitro invasion capacity.

The urokinase/urokinase receptor system A new target for cancer therapy?