Reduction in Surface Urokinase Receptor Forces Malignant Cells into a Protracted State of Dormancy

Considerable evidence links urokinase plasminogen activator (uPA) bound to its surface receptor (uPAR) with enhanced invasiveness of cancer cells. By blocking uPAR expression in human epidermoid carcinoma cells (HEp3), we have now identified an additional and novel in vivo function for this receptor by showing that receptor-deficient cells enter a state of dormancy reminiscent of that observed in human cancer metastasis. Its main characteristic is survival without signs of progressive growth. Five clones transfected with a vector expressing uPAR antisense RNA under the β-actin promoter were isolated and shown to have uPAR (at the mRNA and protein levels) reduced by 50 to 80%; four clones, transfected with vector alone and having uPAR levels similar to those of parental cells, served as controls. In confirmation of our previous results, reduced uPAR always coincided with a significantly reduced invasiveness. Each of the control clones produced rapidly growing, highly metastatic tumors within 2 wk of inoculation on chorioallantoic membranes (CAMs) of chick embryos. In contrast, each of the clones with low surface uPAR, whose proliferation rate in culture was indistinguishable from controls, remained dormant for up to 5 mo when inoculated on CAMs. Thus, the reduction in uPAR altered the phenotype of HEp3 tumor cells from tumorigenic to dormant. Although protracted, tumor dormancy was not permanent since in spite of maintaining low uPAR levels, each of the in vivo–passaged antisense clones eventually reemerged from dormancy to initiate progressive growth and to form metastases at a level of 20 to 90% of that of fully malignant control. This observation suggested that other factors, whose expression is dependent on cumulative and prolonged in vivo effects, can compensate for the lack of a full complement of surface uPAR required for the expression of malignant properties. These “reemerged,” uPAR-deficient clones were easily distinguishable from the vector-transfected controls by the fact that after only 1 wk in culture, the invasion of CAM by all five clones and tumorigenicity of four of the five clones were reduced back to the values observed before in vivo maintenance. In contrast, dissociated and in vitro–grown cells of control tumors were fully invasive and produced large, metastatic tumors when reinoculated on CAMs. Quantitation of the percent of apoptotic and S-phase cells in vivo, in the control and uPAR-deficient, dormant clones, showed that the mechanism responsible for the dormancy was a diminished proliferation.     

尿激酶受体反义RNA抑制人乳腺癌细胞的侵袭作用

将尿激酶受体 u PAR反义 RNA表达质粒 p URAS以脂质体法转染高侵袭性人乳腺癌细胞株 MDA- MB- 2 31 ,G41 8筛选抗性克隆 .Northern印迹法检测 u PAR反义 RNA的表达 ,RT- PCR法检测 u PAR的表达 ,牛奶板法测定细胞培养上清中纤溶活性 .改良 Boyden小室模型和裸小鼠乳房脂肪垫接种试验分别检测肿瘤细胞体外和体内侵袭能力 .反义克隆细胞能表达 u PAR反义RNA,其 u PAR表达水平及培养上清中纤溶活性明显降低 .反义细胞克隆体外侵袭能力比原代细胞 MDA- MB- 2 31和转染载体细胞克隆显著降低 .裸小鼠体内侵袭实验表明 ,反义细胞克隆的成瘤性、生长性和侵袭性均显著受到抑制 .u PAR至少在一部分恶性乳腺癌侵袭行为中发挥重要作用 ,反义 RNA可望成为抗肿瘤侵袭治疗的一种有效手段 .     

Downregulation of uPA, uPAR and MMP-9 using small, interfering, hairpin RNA (siRNA) inhibits glioma cell invasion, angiogenesis and tumor growth

The diffuse, extensive infiltration of malignant gliomas into the surrounding normal brain is believed to rely on modification of the proteolysis of extracellular matrix components. Our previous results clearly demonstrate that uPA, uPAR and MMP-9 concentrations increase significantly during tumor progression and that tumor growth can be inhibited with antisense stable clones of these molecules. Because antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPA, uPAR and MMP-9 in this study. We examined a cytomegalovirus (CMV) promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to inhibit uPA, uPAR and MMP-9 gene expression with a single construct. uPAR protein levels and enzymatic activity of uPA and MMP-9 were found to significantly decrease in cells transfected with a plasmid expressing hairpin siRNA for uPAR, uPA and MMP-9. pU(2)M-transfected SNB19 cells significantly decreased uPA, uPAR and MMP-9 expression compared to mock and EV/SV-transfected cells, determined by immunohistochemical analysis. Furthermore, the effect of the single constructs for these molecules was a specific inhibition of their respective protein levels, as demonstrated by immunohistochemical analysis. After transfection with a plasmid vector expressing dsRNA for uPA, uPAR and MMP-9, glioma-cell invasion was retarded compared with mock and EV/SV-treated groups, demonstrated by Matrigel-invasion assay and spheroid-invasion assay. Downregulation of uPA, uPAR and MMP-9 using RNAi inhibited angiogenesis in an in vitro (co-culture) model. Direct intratumoral injections of plasmid DNA expressing hpRNA for uPA, uPAR and MMP-9 significantly regressed pre-established intracranial tumors in nude mice. In addition, cells treated with RNAi for uPAR, uPA and MMP-9 showed reduced pERK levels compared with parental and EV/SV-treated SNB19 cells. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating gliomas.     

Involvement of the Ras/MAPK signaling pathway in the modulation of urokinase production and cellular invasiveness by transforming growth factor-beta(1) in transformed keratinocytes

Transformed PDV keratinocytes respond to TGF-beta(1) by stimulating cell motility and invasiveness concomitantly to enhancement of the urokinase-type plasminogen activator (uPA) expression/secretion. Depletion of extracellular signal-regulated kinase (ERK1, 2) proteins by treatment of PDV cells with antisense oligonucleotides reduced basal uPA production and abolished stimulation of uPA secreted levels and cell motility by TGF-beta(1). PD098059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK), decreased TGF-beta(1)-induced uPA mRNA expression, secreted activity in a dose-dependent manner, and abrogated TGF-beta(1)-stimulated cell motility and invasiveness. PDV-derived dominant-negative RasN17 cell transfectants secreted similar amounts of uPA and exhibited similar invasive abilities as the parental cells or control clones, but were unable to respond to TGF-beta(1) for stimulation of uPA-secreted levels and invasiveness. These results suggest that a Ras/MAPK transduction pathway is involved in the invasive response of transformed keratinocytes to TGF-beta(1).     

In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.     

High level synthesis of recombinant soluble urokinase receptor (CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice

Focussing of the serine protease urokinase (uPA) to the tumor cell surface via interaction with its receptor (uPAR) is an important step in tumor invasion and metastasis. The human ovarian cancer cell line OV-MZ-6#8 was stably transfected with expression plasmids either encoding cell-associated uPAR (GPI-uPAR) or a soluble form of uPAR (suPAR) lacking its glycan lipid anchor. In vitro, high level synthesis of functionally active recombinant suPAR inhibited cell proliferation and led to reduced cell-associated fibrin matrix degradation, whereas fibrinolytic activity was increased in OV-MZ-6#8 cells overexpressing GPI-uPAR. Both OV-MZ-6#8-derived clones were inoculated into the peritoneum of nude mice and tested for tumor growth and spread. High level synthesis of recombinant suPAR (without altering the physiological expression levels of GPI-uPAR and uPA in these cells) resulted in a significant reduction of tumor burden (up to 86%) in the xenogeneic mouse model. In contrast, overexpression of GPI-uPAR in tumor cells did not affect tumor growth. Our results demonstrate that high levels of suPAR in the ovarian cancer cell vicinity can act as a potent scavenger for uPA, thereby significantly reducing tumor cell growth and cancer progression in vivo.     

Proteomic profiling of proteins associated with urokinase plasminogen activator receptor in a colon cancer cell line using an antisense approach

Expression of urokinase plasminogen activator (uPA) and its receptor (uPAR) strongly correlates with a malignant tumour cell phenotype. In the multistep process of metastasis, uPA binding to uPAR influences different cellular functions. In the present study, a highly metastatic colon cancer cell line, HCT116 was transfected with an expression vector containing a 5' uPAR cDNA fragment in an antisense orientation. This construct was most effective in reducing uPAR cell surface expression as confirmed by flow cytometry analysis. Antisense transfection of HCT116 cells had no effect on proliferation but the following effects were observed: (1) a 1.3-fold decreased adhesion; (2) a two-fold decreased Erk MAP kinase activity; (3) a 2.7-fold decrease in Src kinase activity; (4) a 1.5- and two-fold decrease in uPA cell surface expression and secretion; (5) abrogation of promatrix metalloproteinase-9 secretion; and (6) a complete suppression of plasminogen-dependent matrix degradation. Using proteomic analysis, we demonstrate loss of approximately 200 proteins and quantitative differences in the expression of 141 other proteins in an antisense-clone compared to wild-type and mock-transfected control. Such changes in protein expression with the down-regulation of uPAR may be an important contributor in colon cancer progression and metastasis and may not only provide a basis to develop a proteomic data bank of uPAR-mediated signaling molecules but may also lead to the development of therapeutic approaches for the cure and better management of colon cancer.     

cDNA transfection of amino-terminal fragment of urokinase efficiently inhibits cancer cell invasion and metastasis.

Focusing of urokinase-type plasminogen activator (uPA) to the cell surface via binding to its specific receptor (uPAR, CD87) is critical for tumor invasion and metastasis. Consequently, the inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy. We examined the effects of cDNA transfection of the human uPA amino-terminal fragment (ATF) on invasion and metastasis of cancer cells. First, a highly metastatic human lung giant-cell carcinoma cell line (PG), used as the target cell for evaluation of this effect, was demonstrated to express both uPA and uPAR. Then, ATF, which contains an intact uPAR binding site but is catalytically inactive, was designed as an antagonist of uPA-uPAR interaction and was transfected into PG cells. [(3)H]-Thymidine incorporation and cell growth curves indicated that expressed ATF did not affect the proliferation of transfected cells. However, analysis by scanning electron microscopy revealed that ATF changed the host cells from the typical invasive phenotype to a noninvasive one. Correspondingly, the modified Boyden chamber test in vitro showed that ATF expression significantly decreased the invasive capacity of transfected cells. Furthermore, in the spontaneous metastasis model, it was confirmed in vivo that expressed ATF remarkably inhibited lung metastasis of implanted ATF-transfected PG cells. In summary, autocrine ATF could act as an antagonist of uPA-uPAR interaction, and ATF cDNA transfection could efficiently inhibit the invasion and metastasis of the cancer cells. Inhibition of uPA-uPAR interaction on the cell surface might be a promising anti-invasion and anti-metastasis strategy.     

Urokinase plasminogen activator/urokinase-specific surface receptor expression and matrix invasion by breast cancer cells requires constitutive p38alpha mitogen-activated protein kinase activity

Overexpression of urokinase plasminogen activator (uPA) and its receptor (uPAR) has been well documented in a wide variety of tumor cells. In breast cancer, expression of uPA/uPAR is essential for tumor cell invasion and metastasis. However, the mechanism responsible for uPA/uPAR expression in cancer cells remains unclear. In the studies reported here, we show that endogenous p38 MAPK activity correlates well with breast carcinoma cell invasiveness. Treatment of highly invasive BT549 cells with a specific p38 MAPK inhibitor SB203580 diminished both uPA/uPAR mRNA and protein expression and abrogated the ability of these cells to invade matrigel, suggesting that p38 MAPK signaling pathway is involved in the regulation of uPA/uPAR expression and breast cancer cell invasion. We also demonstrated that SB203580-induced reduction in uPA/uPAR mRNA expression resulted from the de- stabilization of uPA and uPAR mRNA. Finally, by selectively inhibiting p38alpha or p38beta MAPK isoforms, we demonstrate that p38alpha, rather than p38beta, MAPK activity is essential for uPA/uPAR expression. These studies suggest that p38alpha MAPK signaling pathway is important for the maintenance of breast cancer invasive phenotype by promoting the stabilities of uPA and uPAR mRNA.     

Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator

Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA–/–) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1) developed a novel methodology to detect pericellular proteolytic function, (2) demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3) demonstrated its usefulness in real-time studies of cell migration, and (4) showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis.     

Genetic down-regulation of phosphoinositide 3-kinase by bikunin correlates with suppression of invasion and metastasis in human ovarian cancer HRA cells

Using a cDNA microarray analysis, we previously found that exposure of a highly invasive ovarian cancer cell line HRA with bikunin, a Kunitz-type protease inhibitor, or bikunin gene overexpression markedly reduced phosphoinositide kinase (PI3K) p85 gene expression, demonstrating that PI3K may be a candidate bikunin target gene. To clarify how reduced levels of PI3K may confer repressed invasiveness, we transfected HRA cells with PI3K p85 antisense-oligodeoxynucleotide (AS-ODN) and compared the properties of the transfected cells with those of parental cells and sense (S)-ODN cells. We have also demonstrated previously that transforming growth factor-beta1 (TGF-beta1) stimulates urokinase-type plasminogen activator (uPA)-dependent invasion and metastasis of HRA cells. Here, we show that 1) TGF-beta1 induced a rapid increase of the PI3K activity that was accompanied by increased expression (5-fold) of the uPA mRNA; 2) pharmacological inhibition of PI3K or AS-PI3K ODN transfection inhibited TGF-beta1-stimulated Akt phosphorylation; 3) both PI3K pharmacological inhibitors and forced expression of AS-PI3K ODN reduced TGF-beta1-stimulated uPA mRNA and protein expression by approximately 70% compared with controls; 4) concentrations of PI3K inhibitors, sufficient to inhibit uPA up-regulation, inhibited TGF-beta1-dependent HRA cell invasion; 5) the AS-PI3K ODN cells had a decreased ability to invade the extracellular matrix layer as compared with controls; and 6) when the AS-PI3K ODN cells were injected intraperitoneally into nude mice, the mice developed smaller intraperitoneal tumors and showed longer survival. We conclude that PI3K plays an essential role in promoting uPA-mediated invasive phenotype in HRA cells. Our data identify a novel role for PI3K as a bikunin target gene on uPA up-regulation and invasion.     

Gelsolin Induces Colorectal Tumor Cell Invasion via Modulation of the Urokinase-Type Plasminogen Activator Cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.     

Urokinase induces basophil chemotaxis through a urokinase receptor epitope that is an endogenous ligand for formyl peptide receptor-like 1 and -like 2

Basophils circulate in the blood and are able to migrate into tissues at sites of inflammation. Urokinase plasminogen activator (uPA) binds a specific high affinity surface receptor (uPAR). The uPA-uPAR system is crucial for cell adhesion and migration, and tissue repair. We have investigated the presence and function of the uPA-uPAR system in human basophils. The expression of uPAR was found at both mRNA and protein levels. The receptor was expressed on the cell surface of basophils, in the intact and cleaved forms. Basophils did not express uPA at either the protein or mRNA level. uPA (10(-12)-10(-9) M) and its uPAR-binding N-terminal fragment (ATF) were potent chemoattractants for basophils, but did not induce histamine or cytokine release. Inactivation of uPA enzymatic activity by di-isopropyl fluorophosphate did not affect its chemotactic activity. A polyclonal Ab against uPAR inhibited uPA-dependent basophil chemotaxis. The uPAR-derived peptide 84-95 (uPAR84-95) induced basophil chemotaxis. Basophils expressed mRNA for the formyl peptide receptors formyl peptide receptor (FPR), FPR-like 1 (FPRL1), and FPRL2. The FPR antagonist cyclosporin H prevented chemotaxis induced by FMLP, but not that induced by uPA and uPAR84-95. Incubation of basophils with low and high concentrations of FMLP, which desensitize FPR and FPRL1, respectively, but not FPRL2, slightly reduced the chemotactic response to uPA and uPAR84-95. In contrast, desensitization with WKYMVm, which also binds FPRL2, markedly inhibited the response to both molecules. Thus, uPA is a potent chemoattractant for basophils that seems to act through exposure of the chemotactic uPAR epitope uPAR84-95, which is an endogenous ligand for FPRL2 and FPRL1.     

Expression of the urokinase receptor regulates focal adhesion assembly and cell migration in adenoid cystic carcinoma cells

Adenoid cystic carcinoma (AdCC) cell lines (ACCS and ACCT) showed higher migration responses and adhesion to the extracellular matrix (ECM), especially types I and IV collagen, than did the oral squamous cell carcinoma (SCC) lines (NA and TF). The response to collagens was largely and exclusively inhibited by anti-alpha(2) integrin antibody. Moreover, AdCC cell lines expressed higher surface levels of urokinase-type plasminogen activator receptor (uPAR) than did SCC cell lines. When AdCC cells were plated on collagen, the surface level of uPAR was increased, and numerous focal adhesions consisting of uPAR, vinculin, and paxillin were assembled; whereas collagen-stimulated SCC cell counterparts or AdCC cells plated on other types of ECM, such as fibronectin, failed to assemble such definite focal adhesions. In order to elucidate the association of uPAR with collagen-induced events, an ACCS-AS cell line transfected with a vector expressing antisense uPAR RNA was established and shown to have reduced uPAR (about 10% that of parental ACCS at both the protein and mRNA levels). ACCS-AS showed a strong reduction of collagen-stimulated migration and focal adhesion assembly of alpha(2) integrin, vinculin, and paxillin. These findings suggest that AdCC has a proclivity for migrating to types I and IV collagens due to the overexpression of uPAR, which plays a key role in focal adhesion assembly and migration.     

Characterization of low-glycosylated forms of soluble human urokinase receptor expressed in Drosophila Schneider 2 cells after deletion of glycosylation-sites

The urokinase-type plasminogen activator receptor (uPAR) is a glycolipid-anchored membrane protein that is thought to play an active role during cancer cell invasion and metastasis. We have expressed a truncated soluble form of human uPAR using its native signal peptide in stably transfected Drosophila Schneider 2 (S2) cells. This recombinant product, denoted suPAR (residues 1–283), is secreted in high quantities in serum-free medium and can be isolated in very high purity. Characterization by SDS–PAGE and mass spectrometry reveals that suPAR produced in this system carries a uniform glycosylation composed of biantennary carbohydrates. In contrast, suPAR produced in stably transfected Chinese hamster ovary (CHO) cells carries predominantly complex-type glycosylation and exhibits in addition a site-specific microheterogeneity of the individual N-linked carbohydrates. Measurement of binding kinetics for the interaction with uPA by surface plasmon resonance reveals that S2-produced suPAR exhibits binding properties similar to those of suPAR produced by CHO cells. By site-directed mutagenesis we have furthermore removed the five potential N-linked glycosylation-sites either individually or in various combinations and studied the effect thereof on secretion and ligand-binding. Only suPAR completely deprived of N-linked glycosylation exhibits an impaired level of secretion. All the other mutants showed comparable secretion levels and retained the ligand-binding properties of suPAR-wt. In conclusion, stable expression of suPAR in Drosophila S2 cells offers a convenient and attractive method for the large scale production of homogeneous preparations of several uPAR mutants, which may be required for future attempts to solve the three-dimensional structure of uPAR by X-ray crystallography.