Induction of alpha2-antiplasmin inhibits E-cadherin processing mediated by the plasminogen activator/plasmin system, leading to suppression of progression of oral squamous cell carcinoma via upregulation of cell-cell adhesion

The plasminogen activator/plasmin system is one of the main protease systems involved in tumor cell invasion and metastasis. Our previous study has shown that plasmin degrades E-cadherin and promotes cell dissemination by downregulation of E-cadherin-mediated cell-cell adhesion in oral squamous cell carcinoma (SCC) cells. To examine the effect of downregulation of the plasminogen activator/plasmin system by alpha2-antiplasmin (alpha2-AP) on cell-cell adhesion mediated by E-cadherin in oral SCC cells, the oral SCC cell line SCCKN was stably transfected with alpha2-AP cDNA. Induction of alpha2-AP expression led to the inhibition of the proteolysis of E-cadherin by plasminogen activator/plasmin in SCC cells, resulting in the enhancement of the cell aggregation and the suppression of the cell motility. Moreover, alpha2-AP also reduced the ability of SCC cells to invade type I collagen gel, and suppressed tumorigenicity in vivo. These results suggested that downregulation of the plasminogen activator/ plasmin system by alpha2-AP might be a potent therapeutic approach to prevent the progression of oral SCC.     

Plasminogen promotes sarcoma growth and suppresses the accumulation of tumor-infiltrating macrophages

The specific functions of plasminogen, stromal plasminogen activator, stromal plasminogen activator receptor, and stromal plasminogen activator inhibitor in the progression of the murine soft tissue sarcoma, T241 were investigated. Negation of plasminogen to the tumor blunted the orthotopic growth of the sarcoma in syngeneic mice. The reduced tumor growth was associated with a dramatic increase in tumor-infiltrating F4/80-positive macrophages and a diminution of vessel density, but not with obvious differences in fibrin and collagen deposition, or invasiveness of the tumor. Ablation of plasminogen activation by the tumor stroma only modestly impaired the prolonged growth of the sarcoma, suggesting that tumor cell-produced plasminogen activator is sufficient to mediate productive plasminogen activation. Plasminogen facilitated sarcoma progression, angiogenesis, and suppression of macrophage infiltration in the absence of either stromal urokinase plasminogen activator receptor or stromal plasminogen activator inhibitor. These data demonstrate that tumor cell-produced plasminogen activator and host plasminogen cooperate to facilitate soft tissue sarcoma growth and suppress the accumulation of tumor-infiltrating macrophages.     

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.     

Revisiting the biological roles of PAI2 (SERPINB2) in cancer

Tumour expression of the urokinase plasminogen activator correlates with invasive capacity. Consequently, inhibition of this serine protease by physiological inhibitors should decrease invasion and metastasis. However, of the two main urokinase inhibitors, high tumour levels of the type 1 inhibitor actually promote tumour progression, whereas high levels of the type 2 inhibitor decrease tumour growth and metastasis. We propose that the basis of this apparently paradoxical action of two similar serine protease inhibitors lies in key structural differences controlling interactions with components of the extracellular matrix and endocytosis-signalling co-receptors.     

Transforming growth factor beta 1 and sodium butyrate differentially modulate urokinase plasminogen activator and plasminogen activator inhibitor-1 in human breast normal and cancer cells.

The effects of transforming growth factor beta 1 (TGF-beta1) and sodium butyrate on cell proliferation and the urokinase plasminogen activator (uPA) system were examined in normal human breast epithelial cells (HBECs) and in a breast cancer cell line, MDA-MB-231. In HBECs, TGF-beta1 inhibited cell proliferation and uPA activity, while it augmented plasminogen activator inhibitor-1 (PAI-1) antigen level. Sodium butyrate inhibited both cell proliferation and uPA activity but did not affect the level of PAI-1. In MDA-MB-231, TGF-beta1 had no effect on cell proliferation but increased uPA activity and PAI-1 antigen level; sodium butyrate inhibited both cell proliferation and uPA activity but had no effect on PAI-1 level. Moreover, in the presence of plasminogen, cell detachment could be modulated by the level of cell-associated uPA. Our results indicate (1) that the effects of TGF-beta1 on cell growth can be dissociated from its effects on the uPA/PAI system; (2) that, while TGF-beta1 is a potent inhibitor of cell proliferation and uPA activity in normal cells, it may promote invasion and metastasis of tumour cells by increasing uPA activity and PAI-1 levels; and (3) that sodium butyrate offers a potential approach to preventing tumour development by inhibiting both cell proliferation and invasion.     

Melanoma cell migration on vitronectin: Regulation by components of the plasminogen activation system

Tumor cell migration and invasion require complex interactions between tumor cells and the surrounding extracellular matrix. These interactions are modified by cell adhesion receptors, as well as by proteolytic enzymes and their receptors. Here, we study the influence of the protease urokinasetype plasminogen activator (uPA) and its receptor (uPAR) on melanoma cell adhesion to, and migration on, the extracellular matrix protein vitronectin (VN). Cell adhesion to VN, but not to type I collagen, is significantly enhanced in the presence of either uPA or its amino-terminal fragment (ATF). Soluble uPAR can inhibit this effect, indicating that uPA/uPAR on melanoma cells can function as a VN receptor. In the absence of bivalent cations, uPA/uPAR can promote cell attachment on VN, but not cell spreading, suggesting that the glycosylphosphatidylinositol (GPI)-anchored uPAR alone is unable to organize the cytoskeleton. Chemotactic melanoma cell migration on a uniform VN matrix is inhibited by uPA and ATF, implying that cell motility decreases when uPA/uPAR acts as a VN receptor. In contrast, plasminogen activator inhibitor I (PAI-I) can stimulate melanoma cell migration on VN, presumably by inhibiting uPA/uPAR-mediated cell adhesion to VN and thereby releasing the inhibition of cell migration induced by uPA. Together, our data implicate components of the plasminogen activation system in the direct regulation of cell adhesion and migration, thereby modulating the behavior of malignant tumor cells. Int. J. Cancer, 71:116–122, 1997. © 1997 Wiley-Liss, Inc.     

Effect of reoxygenation on the hypoxia-induced up-regulation of serine protease inhibitor PAI-1 in head and neck cancer cells

In squamous cell carcinoma of the head and neck (SCCHN), hypoxia is considered a crucial physiological modulator for malignant progression, wherebythe plasminogen activation system is involved in overlapping functions such as moulding of the extracellular matrix, cell proliferation and signal transduction. Little is known about the effects of reoxygenation on the plasminogen activation system in SCCHN cells. Three human SCCHN cell lines (BHY, CAL27, FaDu) and a non-transformed human fibroblast cell line (VH7) were exposed to hypoxic (<0.5% O(2)) conditions for up to 72 h and subsequently reoxygenated at normoxic conditions for 24 h. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) protein concentration and former protein activity were determined by ELISA and complex ELISA, respectively. Reoxygenation induced significant changes in cell-associated and secreted PAI-1 protein compared to the normoxic control. Significant increase in cell-associated and secreted uPA protein after reoxygenation was only observed for some of the cell lines. Determination of uPA-PAI-1 complex formation revealed the release of active protein into the cell supernatant. The beneficial role of reoxygenation during radiation therapy is widely accepted. However, reoxygenation does not seem to counteract the effects induced by hypoxia on the plasminogen activation system. Fatally irradiated reoxygenat- ed tumour cells might still produce sufficient amounts of 'harmful' protein and thus initiate a path for invasion and metastasis for surviving tumour cells.     

Molecular competition between plasminogen activator inhibitors type -1 and -2 for urokinase: Implications for cellular proteolysis and adhesion in cancer

Up-regulation of the urokinase plasminogen activation (uPA) system leads to increased cancer invasion and metastasis. Plasminogen activator inhibitors type-1 (PAI-1/SERPINE1) and type-2 (PAI-2/SERPINB2) have similar uPA inhibitory properties yet PAI-1 promotes cell invasion by modulating cell adhesion and migration. High tumour PAI-2 levels are associated with improved prognoses. Herein we show that PAI-2 is capable of inhibiting uPA in the presence of PAI-1, particularly on adherent cells in the presence of vitronectin. This suggests that elevated levels of PAI-2 in the tumour microenvironment could outcompete PAI-1 for uPA inhibition through its inhibitory serpin function. However, PAI-1 modulated cell adhesion in a largely uPA-independent manner consequently PAI-2 could not counteract the effects of PAI-1 on adhesion/migration. Thus studies aimed at further characterising the interplay between PAI-1 and PAI-2 on uPA-dependent pro-invasive processes are warranted.     

Endostatin-induced modulation of plasminogen activation with concomitant loss of focal adhesions and actin stress fibers in cultured human endothelial cells

Endostatin, a M(r) 20,000 fragment of collagen XVIII, is able to inhibit angiogenesis and induce apoptosis in endothelial cells in vivo. We analyzed the effectsof recombinant endostatin on human microvascular endothelial cells, focusing on pericellular plasminogen activation and its targeting by the focal adhesion-associated cytoskeletal structures. Analysis of the proteolytic plasminogen activator system revealed that endostatin modulates the distribution of soluble and cell surface-associated urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor, type 1 (PAI-1). Casein zymographic and immunoprecipitation analyses indicated that endostatin exerts its effects by decreasing the levels of soluble uPA and PAI-1 and their complexes in a dose-dependent manner. Immunofluorescence analysis of cell surface-associated uPA indicated that endostatin treatment caused the redistribution of receptor-bound uPA from focal contacts, resulting in diffuse cell surface staining. In accordance with this observation, immunofluorescence staining of the urokinase receptor revealed that endostatin treatment removed uPAR from focal adhesions. Accordingly, endostatin caused a rapid disassembly of focal adhesions as observed by immunofluorescence analysis of the focal adhesion proteins vinculin and paxillin. A prominent change in the cytoskeletal architecture was observed as the actin stress fiber network was dissociated in response to endostatin treatment. The effect of focal adhesion disassembly was reversible, persisting from 1 h up to 6 h. Our results suggest that the antiangiogenic activity of endostatin involves the modulation of focal adhesions and actin stress fibers and the down-regulation of the urokinase plasminogen activator system.     

Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10(-8)M EGF stimulated AR expression in HMECs to 140-300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5-10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system.     

Secretion of proteinase inhibitors by tumorigenic and nontumorigenic guinea pig and Syrian hamster fibroblasts: evidence for autocrine regulation of local proteolysis

A cytokine that inhibits fibrinolysis has been detected in the serum-free culture medium of guinea pig and hamster fibroblasts. This proteinase inhibitor was also present in Triton X-100 extracts of guinea pig cells. It was stable at pH 3.0 for 2 hr and was produced by cells rather than assimilated from serum in the culture medium as evidenced by: (a) an apparent molecular size (less than 45 kilodaltons) less than that of the principal serum-derived proteinase inhibitors; (b) its continued secretion after several passages of the cells in serum-free medium; and (c) the lack of inhibitory activity in the medium of mitomycin C-treated cells. The cytokine inhibited the proteinase activity of human urokinase, soluble TPA-stimulated guinea pig plasminogen activator, and the cell-associated plasminogen activator of tumorigenic guinea pig cells. Soluble plasminogen activator appeared to be inhibited to a greater degree than the cell-associated enzyme. The fluorogenic substrate (7-(N-carbobenzoxyglycylglycylargininamido)-4-methylcoumarin was used in a direct assay of proteinase activity and demonstrated that the cytokine inhibited both plasminogen activator and plasmin, the two proteinases of the fibrinolytic cascade. Tumorigenic guinea pig and hamster fibroblasts as well as nontransformed guinea pig fibroblasts were found to produce the inhibitory cytokine, and the amount of inhibitor secreted was independent of the tumorigenic potential of the cells. Production of the inhibitor by normal cells may be related to contact inhibition of growth, and this cytokine may contribute to the fine regulation of local proteolysis within tissues.     

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.     

Expression pattern of the urokinase-plasminogen activator system in rat DS-sarcoma: role of oxygenation status and tumour size

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT-PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia.     

Role of the urokinase receptor in facilitating extracellular matrix invasion by cultured colon cancer.

There is increasing evidence that urokinase secreted by tumor cells can be bound to a cell surface receptor retaining its full potential to activate plasminogen and subsequently cleave basement membrane constituents. This study was undertaken to discriminate between soluble and cell surface bound urokinase as a potential mediator of in vitro invasion by cultured colon cancer. Extracellular matrix invasion by a colon cancer cell line GEO, characterized as being a poor secretor of urokinase and having few receptors (less than 10(4) receptors/cell) was not augmented when these cells were made to secrete up to 8 times as much urokinase, in response to an exogenous urokinase gene driven by the Rous sarcoma virus long terminal repeat promoter. The majority of the plasminogen activator (greater than 95%) appeared in the culture medium, this reflecting the low numbers of binding sites displayed by GEO cells. In contrast, the cell line HCT 116 equipped with 10 times as many binding sites, (greater than 10(5)/cell), the majority of which are occupied with endogenous ligand, was an efficient invader of the extracellular matrix. Inhibition of urokinase binding to the cell surface receptors using an antibody to the A chain of the plasminogen activator reduced invasion by 65%. The cell line RKO is equipped with 3 x 10(5) receptors/cell, 15% of which are tagged with endogenous urokinase. Pretreatment of these cells with a concentration range of urokinase known to result in the majority of these binding sites being charged with the plasminogen activator led to a dose dependent increase in extracellular matrix invasion. Together, these data suggest that for cultured colon cancer, at least, invasion is a function of the amount of cell surface receptor bound urokinase.     

Plasminogen activator system and its clinical significance in patients with a malignant disease

Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, a serine protease participating in the activation of matrixmetaloproteinases, latent elastases, growth factors and cytokines involved in the degradation of extracellular matrix elements. Together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI-1, PAI-2, PAI-3 and protease nexin), it forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. Plasminogen activator inhibitor 1 inhibits uPA-dependent invasiveness of some cancer cell lines. The vitronectin-PAI-1 complex inhibits migration of smooth muscle cells by binding alpha(v)beta3 integrin to vitronectin. PAI-1 or its deficiency interferes with signalling pathways such as PI3K/Akt and JAK/STAT and it is included in the processes of maintaining the integrity of the endothelial cells and thereby regulation of cell death. PAI-1 affects apoptosis by reducing cell adhesion and functioning of intracellular signalling pathways. The individual components of PAS undoubtedly play an important role in angiogenesis and metastasising of malignant tumours. In the near future, results of published studies with various types of cancer could be reflected in diagnostic and therapeutic algorithms and, at the same time, could serve as the goal for targeted therapies.