Adenovirus-mediated delivery of antisense gene to urokinase-type plasminogen activator receptor suppresses glioma invasion and tumor growth.

The urokinase-type plasminogen activator (uPA) and uPA receptor (UPAR) play important roles in the proteolytic cascade involved in the invasiveness of gliomas and other invasive tumors. High-level expression of uPAR has been correlated with high-grade glioma cell lines and tumors We report here that down-regulating uPAR levels by antisense strategy using an adenovirus construct (Ad-uPAR) inhibited glioma invasion in Matrigel and spheroid in vitro models. sc. (U87-MG) and intracranial (SNB19) injections of Ad-uPAR-infected glioma cells did not produce tumors in nude mice. However, injection of the Ad-uPAR construct into previously established so U87-MG tumors in nude mice caused regression of those tumors. Our results support the therapeutic potential of targeting the uPA-uPAR system for the treatment of gliomas and other cancers.     

RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth

In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans.     

Adenovirus-mediated expression of antisense MMP-9 in glioma cells inhibits tumor growth and invasion

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock- or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.     

Detection of cathepsin B, cathepsin L, cystatin C, urokinase plasminogen activator and urokinase plasminogen activator receptor in the sera of lung cancer patients

The present study aimed to determine the levels of cathepsin B (cath B), cathepsin L (cath L), cystatin C, urokinase plasminogen activator (u-PA) and urokinase plasminogen activator receptor (u-PAR) in the sera of patients with lung cancer compared to healthy controls using ELISA. Concomitantly, the relationship between the components and clinicopathological prognosis was analyzed. The study included 30 healthy volunteers and 105 lung cancer patients. Blood samples were collected and cath B, cath L, cystatin C, u-PA and u-PAR measurements were made using ELISA. Results showed that the levels of cath B, cath L, cystatin C, u-PA and u-PAR were significantly higher in the patient group compared to the healthy controls. The significance was marked for cath B and mild for u-PAR in correlation with lymph node metastasis. There was no significance for other parameters. Notably, patients with a combination of high cystatin C and high cath B levels had significantly lower survival probability as compared to those with cystatin C(+)/cath B(-) or with cystatin C(-)/cath B(-). Similarly, patients with a combination of high u-PA and u-PAR experienced significantly shorter survival. Furthermore, the univariate analysis revealed that cath B, u-PAR, lymph node metastases, stage and grade were related to survival. However, findings of the multivariate Cox analysis indicated that the sera levels of cath B, u-PAR and lymph node metastases may serve as independent prognostic variables in patients with lung cancer.     

Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion,angiogenesis, and tumor growth

The binding of urokinase plasminogen activator (uPA) to its receptor (uPAR) initiates a proteolytic cascade facilitating the activation of matrix metalloproteinase-9 (MMP-9), which in turn degrades the extracellular matrix. These processes have an established role in tumor invasion and metastasis. Our previous work revealed an inverse association between glioma invasion and the expression of uPAR and MMP-9. In the present study, we used the adenovirus serotype 5 vector system to generate a replication-deficient recombinant adenovirus capable of simultaneously expressing antisense uPAR and antisense MMP-9 (Ad-uPAR-MMP-9). This adenoviral construct is driven by the independent promoter elements cytomegalovirus and bovine growth hormone and SV40 polyadenylation signals to down-regulate key steps in the proteolytic cascade. Ad-uPAR-MMP-9 infection of SNB19 cells significantly decreased uPAR and MMP-9 expression as determined by immunohistochemical and Western blotting analyses. A Matrigel invasion assay revealed marked reduction in the invasiveness of the Ad-uPAR-MMP-9-infected cells compared with parental and vector controls. Tumor spheroids infected with Ad-uPAR-MMP-9 and cocultured with fetal rat brain aggregates did not invade rat brain aggregates, whereas 90-95% of the mock and empty vector-infected cells invaded the rat brain aggregates. Intracranial injection of SNB19 cells infected ex vivo with the Ad-uPAR-MMP-9 antisense bicistronic construct showed decreased invasiveness and tumorigenicity. s.c. injections of the bicistronic antisense construct into established tumors (U87 MG) caused tumor regression. These results support the therapeutic potential of targeting the individual components of the uPAR-MMP-9 by using a single adenovirus construct for the treatment of gliomas and other cancers.     

suPAR, a soluble form of urokinase plasminogen activator receptor, inhibits human prostate cancer cell growth and invasion

Urokinase-type plasminogen activator (uPA) and its specific membrane receptor (uPAR) control extracellular matrix proteolysis, cell migration, invasion and cell growth in several cancers. The uPAR released from human cancers is detected in blood as soluble uPAR (suPAR). No information is available on the mechanism(s) of action of suPAR on prostate cancer (PCa) cell growth and invasion. In order to clarify this issue, we tested the effect of a treatment with the human recombinant suPAR (comprising amino acids l-303) on the proliferation, migration and invasion of DU145 cells, a PCa cell line expressing a potent autocrine uPA-uPAR signalling system. The results indicate that suPAR significantly inhibits cell growth, promotes apoptosis and decreases both migration and Matrigel invasion of DU145 cells. The mechanism of action of suPAR seems to be linked to a decrease of ERK and FAK activation. Cleavage of suPAR by chymotripsin reverses these effects. When added to the uPA-negative LNCaP cells, suPAR was ineffective; on the contrary, when LNCaP cells were cultured on fibronectin-coated plates in order to stimulate uPA expression, suPAR significantly decreased cell proliferation. In conclusion, our data suggest that suPAR can function as a potent molecule scavenger for uPA in human PCa cells characterized by high levels of uPA/uPAR as in DU145 cells, while it is ineffective in uPA-deficient LNCaP cells. The molecular mechanism(s) through which suPAR participates in the control of PCa progression may bear relevance for the long-term goal to identify new therapeutic targets aimed at silencing tumours in vivo.     

RNAi-mediated inhibition of cathepsin B and uPAR leads to decreased cell invasion, angiogenesis and tumor growth in gliomas

RNA interference (RNAi) provides a powerful method for gene silencing in eukaryotic cells, including proliferating mammalian cells. Here, we determined whether RNAi could be utilized to inhibit the expression of proteases implicated in the extracellular matrix degradation, which is characteristic of tumor progression. We have previously shown that antisense stable clones of uPAR and cathepsin B were less invasive and did not form tumors when injected intracranially ex vivo. Since antisense-mediated gene silencing does not completely inhibit the translation of target mRNA and high molar concentrations of antisense molecules are required to achieve gene silencing, we used the RNAi approach to silence uPAR and cathepsin B in this study. We found that the expression of double-stranded RNA leads to the efficient and specific inhibition of endogenous uPAR and cathepsin B protein expression in glioma cell lines as determined by Western blotting. We also found the RNAi of uPAR and cathepsin B reduces glioma cell invasion and angiogenesis in in vitro and in vivo models. Intratumoral injections of plasmid vectors expressing hpRNA for uPAR and cathepsin B resulted in the regression of pre-established intracranial tumors. Further, RNAi for uPAR and cathepsin B inhibited cell proliferation and reduced the levels of pERK and pFAK compared to controls. Taken together, our findings indicate for the first time that RNAi operates in human glioma cells with potential application for cancer gene therapy.     

RNAi-mediated downregulation of urokinase plasminogen activator receptor and matrix metalloprotease-9 in human breast cancer cells results in decreased tumor invasion, angiogenesis and growth

The serine protease urokinase-type plasminogen activator (uPA) plays a significant role in tumor cell invasion and metastasis when bound to its specific receptor, uPAR (also known as CD87). In addition to the uPA-uPAR system, matrix metalloproteinases (MMPs) are involved in tumor cell invasion and metastasis. In this study, we achieved specific inhibition of uPAR and MMP-9 using RNAi technology. We introduced small interfering RNA to downregulate the expression of uPAR and MMP-9 (pUM) in breast cancer cell lines (MDA MB 231 and ZR 75 1). In vitro angiogenesis studies indicated a decrease in the angiogenic potential of the treated cells; in particular, a remarkable decrease was observed in the cells treated with bicistronic construct (pUM) in comparision to the controls. Additionally, bicistronic construct inhibited the formation of capillary-like structures in in vivo models of angiogenesis. Similarly, the invasive potential and migration decreased dramatically when treated with the bicistronic construct as shown by matrigel invasion and migration assays. These results suggest a synergistic effect from the simultaneous downregulation of uPAR and MMP-9. We also assessed the levels of phosphorylated forms of MAPK, ERK and AKT signaling pathway molecules and found reduction in the levels of these molecules in cells treated with the bicistronic construct as compared to the control cells. Furthermore, targeting both uPAR and MMP-9 totally regressed orthotopic breast tumors in nude mice. In conclusion, our results provide evidence that the simultaneous downregulation of uPAR and MMP-9 using RNAi technology may provide an effective tool for breast cancer therapy.     

Adenovirus-mediated antisense urokinase-type plasminogen activator receptor gene transfer reduces tumor cell invasion and metastasis in non-small cell lung cancer cell lines.

The urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the proteolytic cascade involved in the metastasis of lung and other cancers. We report that the reduction in uPAR levels produced by an antisense strategy using an adenovirus construct (Ad-uPAR) in H1299 cells, an invasive human lung cancer cell line that produces high levels of uPAR, resulted in a decrease of uPAR levels to 80-90% of those seen in cells infected with mock or adenovirus (Ad)-cytomegalovirus vector controls. In addition, increasing the multiplicity of infection from 25 to 200 caused a corresponding decrease in the level of uPAR protein within 5 days of treatment, as shown by Western blot analysis. Furthermore, the in vitro translation of total RNA levels of Ad-uPAR-infected H1299 cells in a rabbit reticulocyte lysate system caused a 50-70% decrease in uPAR immunoprecipitate in Ad-uPAR-infected cells relative to the levels in cells of mock and vector controls. The Matrigel invasion assay showed the invasion of H1299 cells and A549 cells infected with Ad-uPAR to be decreased by 70% relative to mock- and vector-infected controls. Infection of tumor cells with Ad-uPAR before implantation significantly reduced the incidence of lung metastasis by 85% as compared with the control virus-infected cells injected into nude mice through the tail vein. Our collective results show that the uPAR system is a potential target of treatment for lung cancers.     

RNAi-mediated downregulation of urokinase plasminogen activator receptor inhibits proliferation, adhesion, migration and invasion in oral cancer cells

RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Overexpression of urokinase-type plasminogen activator receptor (uPAR) has been implicated in progression and metastasis of oral cancer. In our study, RNAi was introduced to downregulate the expression of uPAR in the highly malignant oral squamous cell carcinoma (OSCC) cells. Our data demonstrated that siRNA targeting of uPAR leads to the efficient and specific inhibition of endogenous uPAR mRNA and protein expression as determined by quantitative real-time RT-PCR and Western blotting. Furthermore, simultaneous silencing of uPAR resulted in a dramatic reduction of tumor cell proliferation activity, adhesion, migration and invasion in vitro compared to the controls. These findings provide further evidence for the involvement of uPAR in a variety of cancer key cellular events as a versatile signaling orchestrator, and suggest that RNAi-directed targeting of uPAR can be used as a potent and specific therapeutic tool for the treatment of oral cancer, especially in inhibiting and/or preventing cancer cell invasion and metastasis.     

Targeting of urokinase plasminogen activator receptor in human pancreatic carcinoma cells inhibits c-Met- and insulin-like growth factor-I receptor-mediated migration and invasion and orthotopic tumor growth in mice

Pancreatic carcinomas express high levels of urokinase-type plasminogen activator (uPA) and its receptor (uPAR), both of which mediate cell migration and invasion. We investigated the hypotheses that (a) insulin-like growth factor-I (IGF-I)- and hepatocyte growth factor (HGF)-mediated migration and invasion of human pancreatic carcinoma cells require uPA and uPAR function and (b) inhibition of uPAR inhibits tumor growth, retroperitoneal invasion, and hepatic metastasis of human pancreatic carcinomas in mice. Using transwell assays, we investigated the effect of IGF-I and HGF on L3.6pl migration and invasion. We measured the induction of uPA and uPAR following treatment of cells with IGF-I and HGF using immunoprecipitation and Western blot analysis. The importance of uPA and uPAR on L3.6pl cell migration and invasion was studied by inhibiting their activities with amiloride and antibodies before cytokine treatment. In an orthotopic mouse model of human pancreatic carcinoma, we evaluated the effect of anti-uPAR monoclonal antibodies with and without gemcitabine on primary tumor growth, retroperitoneal invasion, and hepatic metastasis. IGF-I and HGF mediated cell migration and invasion in L3.6pl cells. In addition, IGF-I and HGF induced uPA and uPAR expression in L3.6pl cells. In vitro, blockade of uPA and uPAR activity inhibited IGF-I- and HGF-mediated cell migration and invasion. Treatment of mice with anti-uPAR monoclonal antibody significantly decreased pancreatic tumor growth and hepatic metastasis and completely inhibited retroperitoneal invasion. Our study shows the importance of the uPA/uPAR system in pancreatic carcinoma cell migration and invasion. These findings suggest that uPAR is a potential target for therapy in patients with pancreatic cancer.     

Expression of antisense uPAR and antisense uPA from a bicistronic adenoviral construct inhibits glioma cell invasion,tumor growth,and angiogenesis

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the invasiveness of gliomas and other infiltrative tumors. In glioma cell lines and tumors, high grade correlates with increased expression of uPAR and uPA. We report here the downregulation of uPAR and uPA by delivery of antisense sequences of uPAR and uPA in a single adenoviral vector, Ad-uPAR-uPA (Ad, adenovirus). The bicistronic construct (Ad-uPAR-uPA) infected glioblastoma cell line had significantly reduced levels of uPAR, uPA enzymatic activity and immunoreactivity for these proteins when compared to controls. The Ad-uPAR-uPA infected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Intracranial injection of SNB19 cells with the Ad-uPAR-uPA antisense bicistronic construct showed inhibited invasiveness and tumorigenicity. Subcutaneous injections of bicistronic antisense constructs into established tumors (U87 MG) caused regression of those tumors. Our results support the therapeutic potential of targeting the individual components of the uPAR-uPA system by using a single adenovirus construct for the treatment of glioma and other invasive cancers.     

Inhibition of cathepsin B and MMP-9 gene expression in glioblastoma cell line via RNA interference reduces tumor cell invasion, tumor growth and angiogenesis

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vector-expressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (i.p.) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.     

Increased expression of urokinase plasminogen activator and its cognate receptor in human seminomas

Background  

The urokinase plasminogen activating system (uPAS) is implicated in neoplastic progression and high tissue levels of uPAS components correlate with a poor prognosis in different human cancers. Despite that, relative few studies are available on the expression and function of the uPAS components in human seminomas. In the present study we characterized the expression of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and the uPA inhibitors PAI-1 and PAI-2 in normal human testis and seminomas.     

Enhanced expression of urokinase plasminogen activator and its receptor in pancreatic carcinoma.

Urokinase plasminogen activator (uPA) is a serine proteinase that has been suggested to play an important role in cancer invasion and metastasis. It binds to a specific membrane receptor denominated uPA receptor (uPAR). uPA activates plasminogen to form plasmin, which participates in tissue degradation and proteolysis. Binding of uPA to its receptor accelerates UPA''s own activation from pro-uPA, enhancing the activity of the uPA/uPAR cascade. Using immunohistochemistry and Northern blot analysis, we analysed the role of uPA and uPAR in 30 human pancreatic cancers. Immunohistochemical analysis demonstrated moderate to strong immunostaining of both factors in most pancreatic cancers. Cancer lesions with signs of invasion exhibited the strongest immunohistochemical signals for both factors. In addition, in desmoplastic areas adjacent to the cancer cells, moderate uPA and uPAR immunoreactivity was detectable. Northern blot analysis revealed a sixfold and a fourfold increase in uPA and uPAR mRNA levels in pancreatic cancer, respectively, in comparison with normal controls (P<0.01). Correlation of the Northern blot data with the clinical parameters of the patients indicated that patients with concomitant overexpression of uPA and uPAR had a shorter post-operative survival (median 9 months; mean+/-s.d. 10.2+/-3.6 months) than patients in whom only one or none of these factors were overexpressed (median 18 months; mean+/-s.d. 20.3+/-8.7 months) (P<0.002). Our data suggest that uPA and uPAR may serve as prognostic markers in human pancreatic cancer and that the marked overexpression of both factors may create an environment that enables pancreatic cancer cells to invade surrounding tissues.     

The recombinant kringle domain of urokinase plasminogen activator inhibits in vivo malignant glioma growth

In a previous report, the recombinant kringle domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, alpha-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis.     

A direct link between expression of urokinase plasminogen activator receptor, growth rate and oncogenic transformation in mouse embryonic fibroblasts

In addition to its role in invasion and metastasis of several tumors, the multifunctional urokinase receptor uPAR (urokinase plasminogen activator receptor) is directly involved in the growth of several cancer cells in vitro and in vivo. We have compared growth rate and oncogenic transformation in wild-type (wt) or uPAR-/- mouse embryonic fibroblasts (MEFs). Surprisingly, uPAR-/- MEFs grew faster than wt MEFs. This agreed with elevated levels of cell cycle mediators like extracellular signal-regulated protein kinase, p38, AP1 and Cyclin D1. Infection with a uPAR retrovirus reverted the effect, decreasing the growth rate.When MEFs were transformed with H-Ras(V12) and E1A oncogenes, the efficiency of transformation in uPAR-/- MEFs was higher than in wt. UPAR-/- MEFs grew faster at low serum, produced more colonies in agar and produced tumors in vivo in nude mice with a lower latency period. The properties of the heterozygous uPAR+/- MEFs were always intermediate. We conclude therefore that in MEFs uPAR concentration controls cell proliferation and the transforming activity of some oncogenes.