Examination of the role of the urokinase receptor in human colon cancer mediated laminin degradation

The relevance of urokinase receptors to urokinase-mediated laminin degradation was investigated in cultured colon cancer. Six colon cancer cell lines degraded laminin in a plasminogen-dependent manner. The ability of the individual cell lines to cleave the glycoprotein correlated well (r2 = 0.9242) with the amount of urokinase recovered from the cell surface by a mild acid treatment. A radioreceptor assay indicated that colon cancer cells most active in degrading the laminin, possessed the largest number of urokinase receptors. Moreover, acid treatment which depletes the receptors of endogenous plasminogen activator augmented the specific binding of radioactive urokinase to the colon cancer cells by 12-200%. A cell line (HCT 116) which displayed 1.1 x 10(5) receptors/cell the majority of which were occupied with endogenous urokinase was selected and the effects of a urokinase receptor antagonist on laminin degradation determined. The peptide antagonist reduced laminin turnover by 60-80%. Morphological observations were consistent with these findings. Plasminogen-treated HCT 116 cells retracted from the culture dish and many cells were observed in the culture medium. This effect could be largely reversed by simultaneous treatment with the peptide antagonist. A poor correlation was found between laminin degradation and soluble urokinase (r2 = 0.1074). These data strongly argue for a role of the urokinase receptor in facilitating the action of the plasminogen activator in colon cancer.     

Determination of the levels of urokinase and its receptor in human colon carcinoma cell lines

At present, there is a lack of availability of differentiation markers for colon carcinoma. This may, in part, be a consequence of the diversified function of the normal human colon. This study addresses the possibility that the expression of urokinase and its receptor is inversely related to differentiation in colon carcinoma. Six colon carcinoma cell lines including three well-differentiated (CBS, GEO, FET) and three poorly differentiated ones (HCT116, HCT116b, RKO) were screened for urokinase receptor display and secretion of the plasminogen activator. A radioreceptor assay was used to determine receptor levels. Binding of radioactive urokinase to colon cells was saturable, specific, and time dependent. Cell-bound 125I-labeled protease was unaffected by the presence of epidermal growth factor, low-molecular-weight urokinase, plasminogen, or transferrin. Time course studies revealed that maximum amounts of radioactive tracer were bound in a 30-min period with no change occurring over the course of a 90-min incubation. Scatchard analysis of ligand binding indicated that the well-and poorly differentiated cells could be separated on the basis of receptor display; the aggressive RKO, HCT116, and HCT116b expressed in excess of 10(5) sites per cell, while the more indolent CBS, GEO, and FET possessed less than 1.5 X 10(4) receptors per cell. The colon carcinoma cells were also analyzed for urokinase in the conditioned medium. Low levels of the plasminogen activator (0.8 to 1.3 ng/ml/10(6) cells/72 h) were associated with the more "mature" cells. This was in contrast to the elevated levels of the protease (3.9 to 11.4 ng/ml/10(6) cells/72 h) present in the medium derived from the more aggressive cells (HCT 116, HCT116b, RKO). Thus, secreted urokinase and/or the expression of cellular receptor for the plasminogen activator may provide useful measurements of the degree of undifferentiation of in vitro colon carcinoma.     

Insensitivity of laminin degradation directed by receptor bound urokinase to PAI-1 in cultured colon cancer

This laboratory recently reported that laminin degradation by cultured colon cancer was plasminogen dependent and reflected the presence of urokinase bound to cell surface receptors. (Schlecte, W.; Murano, G.; Boyd D. Cancer Res., 49:6064-6069; 1989). The present study was undertaken to determine the sensitivity of urokinase receptor directed proteolysis to the type I plasminogen activator inhibitor (PAI-1). Colon cancer cell types, that were highly effective in degrading laminin in vitro, elaborated into their conditioned medium an inhibitor which was indistinguishable from PAI-1 on the basis of its performance in reverse zymography, western blotting, and immunoprecipitation assays. A fraction of this PAI-1 was active, as evidenced by complex formation with the active site of radioactive urokinase. Laminin degradation by the colon cancer cells, however, did not appear to be affected by the endogenous inhibitor, since an antibody to the inhibitor, which blocked urokinase-PAI-1 interactions, had little effect on laminin turnover. Further, addition of exogenous PAI-1, activated by guanidine hydrochloride pretreatment, to the colon cancer cells did not perturb laminin degradation. Because laminin degradation by colonic cells was a function of receptor bound urokinase, presumably immobilized plasminogen activator escaped the neutralizing effect of the inhibitor. These data suggest either a shielding effect of the receptor on the plasminogen activator or a physical separation of activator and inhibitor. Either way, for cultured colon cancer at least, laminin degradation directed by urokinase receptor bound plasminogen activator appeared unaffected by the presence of this inhibitor.     

Downregulation of urokinase plasminogen activator receptor expression inhibits Erk signalling with concomitant suppression of invasiveness due to loss of uPAR-beta1 integrin complex in colon cancer cells

Cancer invasion is regulated by cell surface proteinases and adhesion molecules. Interaction between specific cell surface molecules such as urokinase plasminogen activator receptor (uPAR) and integrins is crucial for tumour invasion and metastasis. In this study, we examined whether uPAR and beta1 integrin form a functional complex to mediate signalling required for tumour invasion. We assessed the expression of uPAR/beta1 integrin complex, Erk signalling pathway, adhesion, uPA and matrix metalloproteinase (MMP) expression, migration/invasion and matrix degradation in a colon cancer cell line in which uPAR expression was modified. Antisense inhibition of the cell surface expression of uPAR by 50% in human colon carcinoma HCT116 cells (A/S) suppressed Erk-MAP kinase activity by two-fold. Urokinase plasminogen activator receptor antisense treatment of HCT116 cells was associated with a 1.3-fold inhibition of adhesion, approximately four-fold suppression of HMW-uPA secretion and inhibition of pro-MMP-9 secretion. At a functional level, uPAR antisense resulted in a four-fold decline in migration/invasion and abatement of plasmin-mediated matrix degradation. In empty vector-transfected cells (mock), uPA strongly elevated basal Erk activation. In contrast, in A/S cells, uPA induction of Erk activation was not observed. Urokinase plasminogen activator receptor associated with beta1 integrin in mock-transfected cells. Disruption of uPAR-beta1 integrin complex in mock-transfected cells with a specific peptide (P25) inhibited uPA-mediated Erk-MAP kinase pathway and inhibited migration/invasion and plasmin-dependent matrix degradation through suppression of pro-MMP-9/MMP-2 expression. This novel paradigm of uPAR-integrin signalling may afford opportunities for alternative therapeutic strategies for the treatment of cancer.     

Antitumor activity of the type 1 plasminogen activator inhibitor and cytotoxic conjugate in vitro.

Many cancer cell lines and cancers overexpress receptor bound urokinase plasminogen activator on the cell surface. The urokinase plasminogen activator bound to its receptor remains on the cell surface for a prolonged period of time. When urokinase plasminogen activator/urokinase plasminogen activator receptor complex binds plasminogen activator inhibitor (PAI-1), the inhibitor triggers a series of events leading to internalization of the entire complex. This mechanism makes a very attractive target for localization and internalization of PAI-1-based cytotoxic compounds in cancer treatment. We investigated the antitumor activity of PAI-1/A-chain cholera toxin in vitro. Fibrosarcoma-derived HT1080 cells treated with PAI-1 conjugate showed at least 4 times higher cell killing than the control KD normal fibroblast cell line.     

Increased plasminogen binding is associated with metastatic breast cancer cells: differential expression of plasminogen binding proteins.

Overexpression of urokinase-type plasminogen activator and its receptor correlates with metastatic capacity in breast cancer. In this study we show that the urokinase/urokinase receptor-overexpressing, metastatic human breast cancer cell line MDA-MB-231 (1) bound significantly more cell-surface plasminogen in a lysine-dependent manner and (2) was capable of generating large amounts of plasmin compared with the non-metastatic cell lines MCF-7 and T-47D. In addition, distinct plasminogen binding proteins were detected in the plasma membranes of the cell lines, suggesting heterogeneity of binding proteins. Plasminogen binding was analysed using a combination of dual-colour fluorescence flow cytometry and ligand histochemistry (for comparative and cellular localization of ligand binding), and fluorimetry (for Scatchard analysis). Apart from revealing the greater plasminogen binding capacity of MDA-MB-231 cells, flow cytometry and histochemistry also revealed that, in all three cell lines, non-viable or permeabilized cells bound significantly more plasminogen in a lysine-dependent manner than viable or non-permeabilized cells. Viable MDA-MB-231 cells bound plasminogen with moderate affinity and high capacity (Kd = 1.8 microM, receptor sites per cell 5.0 x 10(7). Our results indicate that differences in cell surface-specific plasminogen binding capacity between cell lines may not be detectable with binding techniques that cannot distinguish between viable and non-viable cells.     

The topology of plasminogen binding and activation on the surface of human breast cancer cells

The urokinase-dependent activation of plasminogen by breast cancer cells plays an important role in metastasis. We have previously shown that the metastatic breast cancer cell line MDA-MB-231 over-expresses urokinase and binds and efficiently activates plasminogen at the cell surface compared to non-metastatic cells. The aim of this study was to further characterise plasminogen binding and determine the topology of cell surface-bound plasminogen in terms of its potential for activation. The lysine-dependent binding of plasminogen at 4 degrees C to MDA-MB-231 cells was stable and resulted in an activation-susceptible conformation of plasminogen. Topologically, a fraction of bound plasminogen was co-localised with urokinase on the surfaces of MDA-MB-231 cells where it could be activated to plasmin. At 37 degrees C plasmin was rapidly lost from the cell surface. Apart from actin, other candidate plasminogen receptors were either not expressed or did not co-localise with plasminogen at the cell surface. Thus, based on co-localisation with urokinase, plasminogen binding is partitioned into two functional pools on the surface of MDA-MB-231 cells. In conclusion, these results shed further light on the functional organisation of the plasminogen activation cascade on the surface of a metastatic cancer cell.     

Tumour cell thrombospondin-1 regulates tumour cell adhesion and invasion through the urokinase plasminogen activator receptor

We have previously shown that platelet-produced thrombospondin-1 up-regulates the urokinase plasminogen activator and its receptor and promotes tumour cell invasion. Although tumour cells produce thrombospondin-1 in vivo, they produce only minimal amounts of thrombospondin-1 in vitro. To determine the effect of tumour cell-produced thrombospondin-1 in the regulation of the plasminogen/plasmin system and tumour cell invasion, we studied THBS-1-transfected MDA-MB-435 breast cancer cells that overexpress thrombospondin-1. The role of urokinase plasminogen receptor in thrombospondin-1-mediated adhesion and invasion was studied by antisense inhibition, enzymatic cleavage and antibody neutralization. Tumour cell adhesion to collagen and laminin was evaluated. Tumour cell invasion was studied in a modified Boyden chamber collagen invasion assay. Tumour cell thrombospondin-1 induced a 2-7 fold increase in urokinase plasminogen activator receptor and cell-associated urokinase plasminogen activator expression and a 50-65% increase in cell-associated urokinase plasminogen activator and plasmin activities. Furthermore, tumour cell thrombospondin-1 promoted tumour cell invasion and decreased tumour cell adhesion through up-regulation of urokinase plasminogen activator receptor-controlled urokinase plasminogen activator and plasmin activities. We conclude that tumour cell-produced thrombospondin-1 may play a critical role in the regulation of tumour cell adhesion and tumour cell invasion.     

UPA 系统在肿瘤患者中的检测意义及研究进展

肿瘤发生侵袭并最终转移，肿瘤细胞必须通过对基底膜及细胞外基质成分的附着、黏附和侵袭来穿透细胞和基质屏障。这个过程由细胞因子及生长因子严格调节，并需要肿瘤相关蛋白水解酶的表达。其中，一个重要的蛋白水解酶就是尿激酶系统。本文对该系统的成分，功能以及其在肿瘤中，尤其在乳腺癌，前列腺癌，胃肠道肿瘤中的作用及最新研究进展进行综述。     

Receptor for plasmin on human carcinoma cells

We have shown that cells from human tumor cell line SW 1116 have receptors for plasmin and plasminogen. These receptors are the same for the proenzyme and the enzyme, but they have a much higher affinity for plasmin (Kd = 6 X 10(-8) M) than for plasminogen (Kd = 5 X 10(-6) M). Plasminogen binding was strongly increased by preincubation of the tumor cells with urokinase and was inhibited by anti-urokinase serum. Because free plasmin is rapidly neutralized in vivo, it is likely that, under physiological conditions, plasminogen is bound by tumor cells and partially transformed into plasmin by urokinase already present at the surface of these cells. Bound plasmin retains its enzymatic activity, which demonstrates that its binding does not involve the enzyme's active site.     

Endogenous receptor-bound urokinase mediates tissue invasion of the human lung carcinoma cell lines A549 and Calu-1.

Macrophage colony-stimulating factor (CSF-1) increases the tissue invasive potential of the CSF-1 receptor-bearing lung carcinoma cell lines A549 and Calu-1 by increasing the number of endogenously bound urokinase-type plasminogen activators (u-PA)s on these cells. CSF-1, at concentrations which optimize invasion of A549 and Calu-1 cells into human amnion membranes (250 ng/ml), maximally augments the number of u-PA receptors occupied by endogenously produced urokinase. Preincubation of A549 and Calu-1 cells with the anti-u-PA monoclonal antibody MPW5UK (25 micrograms/ml) or with a 20- to 40-fold stoichiometric excess of fluid phase type 2 plasminogen activator inhibitor abrogates invasiveness, indicating that functionally active cell surface u-PA is essential for tissue invasion. In contrast, fluid phase type 1 plasminogen activator inhibitor (PAI-1, 5-15 units/ml) does not inhibit invasiveness unless preincubated with the amnion membranes. Inhibition of invasion by PAI-1 is abolished by presaturating the amnion membranes with antiitronectin monoclonal antibody (10 micrograms/ml) which prevents binding of PAI-1 to tissue-associated vitronectin. Binding of PAI-1 to tissue vitronectin is therefore a prerequisite for its inhibitory action. Thus, endogenously receptor-bound u-PA is the primary protease mediating CSF-1-induced tissue invasiveness of the lung carcinoma cell lines A549 and Calu-1.     

Inhibition of cancer cell urokinase plasminogen activator by its specific inhibitor PAI-2 and subsequent effects on extracellular matrix degradation

Isotopically labeled [( 3H]serine, [3H]proline, and [35S]sulfate) subendothelial cell basement membranes were used to determine the role of urokinase plasminogen activator (uPA) and its specific inhibitor plasminogen activator inhibitor 2 (PAI-2) in colon cancer cell extracellular matrix degradation. Recombinant PAI-2 irreversibly inhibited low and high molecular weight purified human uPA in addition to both colon cancer cell-associated and secreted uPA, particularly if pro-uPA had been preactivated. Two selected lines (COLO394 and LIM1215) preferentially degraded differently labeled matrices in a time- and plasminogen-dependent manner. This process was inhibitable by PAI-2 in the medium at levels which suggested that some degree of "shielding" of cell surface uPA from inhibitor occurred. The ability of PAI-2 to regulate the invasive phenotype of cells which express cell surface or receptor-bound uPA is discussed.     

Regulation of human squamous cell carcinoma plasma membrane associated urokinase plasminogen activator by epidermal growth factor

The amino terminal portion of urokinase type plasminogen activator (uPA) interacts with a cell surface binding protein/receptor that recognizes a region of the molecule autonomous from that of the catalytic domain of the enzyme, which mediates the conversion of plasminogen to plasmin. The expression of cell surface uPA receptors (uPA-Rs) and their association with uPA have been implicated in cellular invasion and tissue destruction. Treatment of A431 squamous carcinoma cells (SqCC) with epidermal growth factor (EGF) has previously been shown to result in an induction of the synthesis and extracellular accumulation of uPA and the plasminogen-dependent proteolysis of extracellular matrix (ECM). Regulation of cell membrane associated uPA activity by EGF and its influence on uPA-R expression in A431 cells, which possess an unusually large number of EGF-R (greater than 10(6)), and in an EGF-R expression variant (A431/A5), which contains 20-fold fewer cell surface EGF-R, were assessed. Exposure to 5-50 ng/mL of EGF for 24 hr enhanced uPA activity 2- to 3-fold in partially purified membrane preparations derived from A431 cells in a concentration dependent manner. In contrast, no changes in tissue type plasminogen activator (tPA) activity were detected under similar conditions. A431/A5 cell membrane preparations did not exhibit such an EGF mediated response. In accord with EGF enhanced uPA activity, a 2-fold increase in immunoreactive cell surface associated uPA was observed in A431 cells using indirect immunofluorescence staining. The increase in cell surface uPA produced by exposure to EGF required protein synthesis and could be blocked by cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell membrane receptors for urokinase plasminogen activator are increased in malignant ovarian tumours

The binding of ¹²⁵I-labelled urokinase plasminogen activator (uPA) to cell membranes of ovarian tumours was characterised. Binding was fast, specific to HMW-uPA, and saturated at low concentration [1.5 (range 1.2-1.6) nmol/l]. Scatchard analysis suggested a single class of binding sites with K_d 1.1 (0.9-1.3) nmol/l. These data indicate the presence of a specific cell membrane receptor for uPA in ovarian tumours, whose characteristics are similar to those reported for uPA receptors in other tissues. Endogenously occupied receptors were uncovered by exposing the membranes to acid conditions (pH 2) before assay, thereby allowing quantitation of the total amount of receptor. uPA receptors were assayed in 10 malignant and 6 benign epithelial ovarian tumours. The total number of receptors was higher in the malignant tumours. This was secondary to increases of both free and occupied receptors. We conclude that this reflects the biological function of cell surface bound plasminogen activation in tumour growth and spread.     

The plasminogen activation system in human colon cancer: messenger RNA for the inhibitor PAI-1 is located in endothelial cells in the tumor stroma.

Fourteen human colon adenocarcinomas were examined by in situ hybridization for the presence of mRNA for plasminogen activator inhibitor type 1 (PAI-1). All specimens contained PAI-1 mRNA in endothelial cells of some vessels in the stroma immediately surrounding the invasive tumor glands, in granulation tissue, and in some capillaries located under the free luminal surface of carcinomatous epithelium. In addition, a limited number of stromal cells in the cancerous areas located at the periphery of newly formed capillary networks, and presumably representing sprouting endothelial cells, contained PAI-1 mRNA. Cancer cells were devoid of detectable PAI-1 mRNA in all cases. PAI-1 mRNA was not seen in three biopsies of normal colon. Together with previous findings of urokinase-type plasminogen activator and its mRNA being located in fibroblast-like cells in the tumor stroma and mRNA for the urokinase receptor in the cancer cells at invasive foci, these results indicate a complex cooperativity among several cell types in regulation of plasminogen activation in colon cancer. A possible role of PAI-1 in protecting the extracellular matrix in the tumor tissue against degradation and a role in tumor-induced angiogenesis are discussed.     

Recombinant PAI-1 inhibits angiogenesis and reduces size of LNCaP prostate cancer xenografts in SCID mice

To understand the fundamental determinants of urokinase plasminogen activator (uPA) driven angiogenesis in cancer we studied how inhibition of uPA activity could reduce neovascularization and consequently reduce tumor size in experimental animals. Proteolytic enzymes are required to mediate tumor cell invasion to adjacent tissues and initiate the metastatic process. Many different human cancers commonly overexpress the urokinase plasminogen activator system, one of the proteolytic enzyme systems. Reduction of urokinase activity in cancer cells is evidently associated with diminished invasion and metastasis. However, it has been shown recently that inhibitors of uPA could reduce tumor size also. The mechanism of action leading to decline in tumor growth rate is not clear. Proteolysis is responsible for degradation of proteins, for invasion or metastasis, but not for the proliferate properties of the cancer cells. It is difficult to envision that diminishing the size of tumor is due to simply blocking of uPA activity of cancer cells. Instead, inhibitors of uPA may be interacting with the elements of the extracellular matrix, such the neovascular bed surrounding tumors that has been reported to contain high amounts of uPA and its receptor. Overall these data strongly suggest that inhibitors of urokinase limit cancer growth by inhibiting angiogenesis. However, it is possible also that uPA inhibitors could act on cancer cells directly or prevent angiogenesis by alternative mechanisms that are not related to uPA inhibition. Therefore, we examined if plasminogen activator inhibitor (PAI-1) could limit angiogenesis. If it does, it will provide definitive evidence of uPA/PAI-1 involvement in reduction of cancer growth. Indeed, our study demonstrates that exogenously applied 14-1b PAI-1 is a powerful inhibitor of angiogenesis in three different in vitro models and is a powerful anti-cancer agent in a SCID mice model inoculated with human LNCaP prostate cancer cells.     

213Bi-PAI2 conjugate selectively induces apoptosis in PC3 metastatic prostate cancer cell line and shows anti-cancer activity in a xenograft animal model

A novel alpha-particle emitting ((213)Bi) plasminogen activator inhibitor type 2 construct, which targets the membrane-bound urokinase plasminogen activator on prostate cancer cells, was prepared and evaluated in vitro and in a xenograft animal model. The PC3 prostate cancer cell line expresses urokinase plasminogen activator which binds to its receptor on the cell membrane; plasminogen activator inhibitor type 2 is bound to urokinase plasminogen activator/urokinase plasminogen activator receptor to form stable complexes. In vitro, the cytotoxicity of (213)Bi-plasminogen activator inhibitor type 2 against prostate cancer cells was tested using the MTS assay and apoptosis was documented using terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling (TUNEL) assay. In vivo, antiproliferative effects for tumours and prostate cancer lymph node metastasis were carried out in an athymic nude mouse model with a subcutaneous xenograft of PC3 cells. (213)Bi-plasminogen activator inhibitor type 2 was specifically cytotoxic to PC3 cells in a concentration-dependent fashion, causing the cells to undergo apoptosis. A single local or i.p. injection of (213)Bi-plasminogen activator inhibitor type 2 was able to completely regress the growth of tumours and lymph node metastases 2 days post subcutaneous inoculation, and obvious tumour regression was achieved in the therapy groups compared with control groups with (213)Bi-plasminogen activator inhibitor type 2 when the tumours measured 30-40 mm(3) and 85-100 mm(3). All control animals and one of five (20%) mice treated with 3 mCi kg(-1) (213)Bi-plasminogen activator inhibitor type 2 developed metastases in the lymph nodes while no lymphatic spread of cancer was found in the 6 mCi kg(-1) treated groups at 2 days and 2 weeks post-cell inoculation. These results demonstrate that this novel (213)Bi-plasminogen activator inhibitor type 2 conjugate selectively targets prostate cancer in vitro and in vivo, and could be considered for further development for the therapy of prostate cancer, especially for the control of micro-metastases or in minimal residual disease.