In vivo invasion of modified chorioallantoic membrane by tumor cells: the role of cell surface-bound urokinase

The ability of the chick embryo chorioallantoic membrane (CAM) to withstand invasion by tumor cells can be intentionally compromised by altering its morphological integrity. Using a newly developed quantitative assay of invasion we showed that intact CAMs were completely resistant to invasion by tumor cells, wounded CAMs did not pose a barrier to penetration, and CAMs that were wounded and then allowed to reseal displayed partial susceptibility to invasion. The invasion of resealed CAMs required catalytically active plasminogen activator (PA) of the urokinase type (uPA); the invasive efficiency of tumor cells was reduced by 75% when tumor uPA activity or tumor uPA production was inhibited. The invasive ability of human tumor cells, which have surface uPA receptors but which do not produce the enzyme, could be augmented by saturating their receptors with exogenous uPA. The mere stimulation of either uPA or tissue plasminogen activator production, in absence of binding to cell receptors, did not result in an enhancement of invasiveness. These findings suggest that the increased invasive potential of tumor cells is correlated with cell surface-associated proteolytic activity stemming from the interaction between uPA and its surface receptor.     

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.     

Design of novel and selective inhibitors of urokinase-type plasminogen activator with improved pharmacokinetic properties for use as antimetastatic agents

The serine protease urokinase-type plasminogen activator (uPA) interacts with a specific receptor (uPAR) on the surface of various cell types, including tumor cells, and plays a crucial role in pericellular proteolysis. High levels of uPA and uPAR often correlate with poor prognosis of cancer patients. Therefore, the specific inhibition of uPA with small molecule active-site inhibitors is one strategy to decrease the invasive and metastatic activity of tumor cells. We have developed a series of highly potent and selective uPA inhibitors with a C-terminal 4-amidinobenzylamide residue. Optimization was directed toward reducing the fast elimination from circulation that was observed with initial analogues. The x-ray structures of three inhibitor/uPA complexes have been solved and were used to improve the inhibition efficacy. One of the most potent and selective derivatives, benzylsulfonyl-D-Ser-Ser-4-amidinobenzylamide (inhibitor 26), inhibits uPA with a Ki of 20 nm. This inhibitor was used in a fibrosarcoma model in nude mice using lacZ-tagged human HT1080 cells, to prevent experimental lung metastasis formation. Compared with control (100%), an inhibitor dose of 2 x 1.5 mg/kg/day reduced the number of experimental metastases to 4.6 +/- 1%. Under these conditions inhibitor 26 also significantly prolonged survival. All mice from the control group died within 43 days after tumor cell inoculation, whereas 50% of mice from the inhibitor-treated group survived more than 117 days. This study demonstrates that the specific inhibition of uPA by these inhibitors may be a useful strategy for the treatment of cancer to prevent metastasis.     

Reversal of the hypomethylation status of urokinase (uPA) promoter blocks breast cancer growth and metastasis

Metastasis is a leading cause of mortality and morbidity in cancer. Urokinase (uPA), only expressed by the highly invasive cancer cells, has been implicated in invasion, metastases, and angiogenesis of several malignancies including breast cancer. Because uPA expression is strongly correlated with its hypomethylated state, we utilized the uPA gene in the highly invasive MDA-231 human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA promoter hypomethylation would result in its silencing and inhibition of metastasis. S-Adenosyl-l-methionine (AdoMet) has previously been shown to cause hypermethylation and inhibit demethylation. Treatment of MDA-231 cells with AdoMet, but not its unmethylated analogue S-adenosylhomocysteine, significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and metastasis in vivo. The effects of AdoMet on uPA expression were reversed by the demethylating agent 5'-azacytidine, supporting the conclusion that AdoMet effects are caused by hypermethylation. Knockdown of the methyl-binding protein 2 also causes a significant inhibition of uPA expression in vitro and tumor growth and metastasis in vivo. These treatments did not have any effects on estrogen receptor expression, suggesting that inhibition of hypomethylation will not affect genes already silenced by hypermethylation. These data are consistent with the hypothesis that hypomethylation of critical genes like uPA plays a causal role in metastasis. Inhibition of hypomethylation can thus be used as a novel therapeutic approach to silence the pro-metastatic gene uPA and block breast cancer progression into the aggressive and metastatic stages of the disease.     

Down‐regulation of uPA and uPAR by 3,3′‐diindolylmethane contributes to the inhibition of cell growth and migration of breast cancer cells

3,3′‐Diindolylmethane (DIM) is a known anti‐tumor agent against breast and other cancers; however, its exact mechanism of action remains unclear. The urokinase plasminogen activator (uPA) and its receptor (uPAR) system are involved in the degradation of basement membrane and extracellular matrix, leading to tumor cell invasion and metastasis. Since uPA‐uPAR system is highly activated in aggressive breast cancer, we hypothesized that the biological activity of B‐DIM could be mediated via inactivation of uPA‐uPAR system. We found that B‐DIM treatment as well as silencing of uPA‐uPAR led to the inhibition of cell growth and motility of MDA‐MB‐231 cells, which was in part due to inhibition of VEGF and MMP‐9. Moreover, silencing of uPA‐uPAR led to decreased sensitivity of these cells to B‐DIM indicating an important role of uPA‐uPAR in B‐DIM‐mediated inhibition of cell growth and migration. We also found similar effects of B‐DIM on MCF‐7, cells expressing low levels of uPA‐uPAR, which was due to direct down‐regulation of MMP‐9 and VEGF, independent of uPA‐uPAR system. Interestingly, over‐expression of uPA‐uPAR in MCF‐7 cells attenuated the inhibitory effects of B‐DIM. Our results, therefore, suggest that B‐DIM down‐regulates uPA‐uPAR in aggressive breast cancers but in the absence of uPA‐uPAR, B‐DIM can directly inhibit VEGF and MMP‐9 leading to the inhibition of cell growth and migration of breast cancer cells. J. Cell. Biochem. 108: 916–925, 2009. © 2009 Wiley‐Liss, Inc.     

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.     

Inhibition of receptor-bound urokinase by plasminogen-activator inhibitors

Urokinase-type plasminogen activator (uPA) binds to a specific receptor on various cell types, the bound molecule retaining its enzymatic activity against plasminogen. We have now investigated whether receptor-bound uPA also retains the ability to react with and be inhibited by plasminogen activator inhibitors (PAI-1 and PAI-2). uPA bound to its receptor on human U937 monocyte-like cells was inhibited by PAI-1 (in its active form in the presence of vitronectin fragments) with an association rate constant of 4.5 x 10(6) M-1 s-1, which was 40% lower than that obtained for uPA in solution (7.9 x 10(6) M-1 s-1). The inhibition of uPA by PAI-2 was decreased to a similar extent by receptor binding, falling from 5.3 x 10(5) to 3.3 x 10(5) M-1 s-1. Stimulation of U937 cells with phorbol 12-myristate 13-acetate was accompanied by a further reduction in receptor-bound uPA inhibition by PAI-1 and PAI-2 to 1.7 x 10(6) and 1.1 x 10(5) M-1 s-1, respectively. These constants although lower than those for uPA in solution still represent rather rapid inhibition of the enzyme, and demonstrate that uPA bound to its specific cellular receptor remains available for efficient inhibition by PAI's, which may therefore play a major role in controlling cell-surface plasminogen activation and extracellular proteolytic activity.     

Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and uPA secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN induced PI 3'-kinase dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.     

Suppression of urokinase-type plasminogen activator expression from human ovarian cancer cells by urinary trypsin inhibitor

Urinary trypsin inhibitor (UTI), a Kunitz-type protease inhibitor, efficiently inhibits tumor cell invasion and metastasis. We examined the effect of UTI on urokinase-type plasminogen activator (uPA) expression in ovarian cancer cell lines, HOC-I and HRA. By Northern blot, Western blot, ELISA, and zymographic analyses, we demonstrated that UTI inhibited the expression of uPA mRNA and protein in these cells in a time- and dose-dependent manner, independent of whether induction was triggered by phorbol ester. Monoclonal antibody 4G12, which inhibits UTI binding to the cells, produced a dose-dependent abrogation in UTI-mediated down-regulation of uPA expression. These data suggest that UTI significantly down-regulates tumor cell uPA mRNA expression and protein secretion, and that UTI binding to the cells is necessary to exert the UTI's action.     

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.     

Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis

Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS) resulting in 10–20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA) and of the uPA receptor (uPAR) exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC), and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004) inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.     

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.     

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.     

7-Allyl-8-oxoguanosine (loxoribine) inhibits the metastasis of B16 melanoma cells and has adjuvant activity in mice immunized with a B16 tumor vaccine

We have shown previously that loxoribine exhibits adjuvant activity for B cells, activates natural killer (NK) cells, and enhances the activation of lymphokine-activated killer cells by interleukin-2 (IL-2). In this study, we examined loxoribine for protective effects in a B16 melanoma lung tumor metastasis model. Significant inhibition of B16 metastasis was seen in mice given a single injection of 2 mg loxoribine as late as day 3 of tumor growth but the greatest inhibition (96%) was seen in mice given four injections of loxoribine on alternate days starting the day before tumor injection. In experiments in which both IL-2 and loxoribine were administered, both agents were active when tested alone, but the combination of IL-2 and loxoribine gave significantly greater inhibition of metastasis. Loxoribine partially inhibited the development of tumors in mice that had been depleted of NK cells by the administration of anti-asialo-GM1 or anti-NK1. 1 antibodies and in NK-deficient beige mice. In all cases, protection was seen only when smaller tumor inocula were injected. Taken together, these data suggest that both NK and non-NK cell populations or effector mechanisms with antitumor activity were activated by loxoribine. Since substituted guanosine analogs have been shown to have adjuvant activity in B cell systems, we evaluated whether loxoribine was active as an adjuvant in a tumor protection model. Mice immunized with both irradiated tumor cells and loxoribine developed a significantly lower number of lung tumors when challenged by live B16 tumor cells, whereas mice injected with either vaccine or loxoribine alone were not protected. There was a clear dose response seen with both loxoribine and the vaccine preparations. These data suggest that loxoribine may be useful in tumor therapy as an immunomodulator or as an adjuvant for use with tumor vaccines.     

Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma

High levels of heparanase are an indicator of poor prognosis in myeloma patients, and up-regulation of the enzyme enhances tumor growth, angiogenesis, and metastasis in animal models. At least part of the impact of heparanase in driving the aggressive tumor phenotype is due to its effect on increasing the expression and shedding of the heparan sulfate proteoglycan syndecan-1, a molecule known to promote myeloma progression. The present work demonstrated that elevation in heparanase expression in myeloma cells stimulates sustained ERK phosphorylation that in turn drives MMP-9 expression. In addition, urokinase-type plasminogen activator (uPA) and uPA receptor expression levels increased, and blocking the proteolytic activation of either MMP-9 or uPA inhibited the heparanase-induced increase in syndecan-1 shedding. Together these data provide a mechanism for heparanase-induced syndecan-1 shedding and, more importantly, demonstrate that heparanase activity in myeloma cells can lead to increased levels of proteases that are known to play important roles in the aggressive behavior of myeloma tumors. This in addition to its other known biological roles, indicates that heparanase acts as a master regulator of the aggressive tumor phenotype by up-regulating protease expression and activity within the tumor microenvironment.     

Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.     

Protein kinase C mediates up-regulation of urokinase and its receptor in the migrating keratinocytes of wounded cultures,but urokinase is not required for movement across a substratum in vitro

Both in cell culture and in vivo, keratinocytes that are migrating in response to a wound express enhanced levels of both urokinase-type plasminogen activator (uPA) and the uPA cell surface receptor (uPA-R). To explore the mechanism of this up-regulation, keratinocyte cultures were treated prior to wounding with a variety of metabolic and growth factor inhibitors in order to evaluate their effect on uPA and uPA-R expression. Actinomycin D and cycloheximide inhibited the up-regulation of both uPA and uPA-R, as determined by immunohistochemistry, indicating that RNA and protein syntheses are required for their induction in migrating keratinocytes. Neither removal of protein growth factors from the medium nor addition of inhibitory antibodies to a number of growth factors depressed uPA or uPA-R induction; these findings suggest that a variety of exogenous or endogenous growth factors [i.e., basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor-α (TGF-α), amphiregulin, and tumor necrosis factor-α (TNF-α)] do not have a critical role in the induction of uPA or uPA-R. In contrast, when protein kinase C (PKC) was either down-regulated with bryostatin 5 or inhibited with Ro31-8220 or staurosporine, the expression of both uPA and uPA-R was greatly decreased in migrating keratinocytes. Furthermore, pharmacologic activation of PKC enhanced uPA levels in non-wounded cultures. These data suggest that the enhanced expression of uPA and uPA-R in migrating keratinocytes is mediated by selective activation of PKC in these cells, perhaps secondary to alterations in the cytoskeleton induced by wounding. To test the requirement for uPA during keratinocyte migration in vitro, the extent of migration was quantified in the presence and absence of a variety of inhibitors in the wounded culture model. Migration was not altered by actinomycin D, cycloheximide, any of the above growth factor inhibitors, anti-uPA antibodies, a variety of inhibitors of uPA or plasmin enzymatic activity, or exogenous uPA. The independence of keratinocyte migration in vitro from uPA was further suggested by experiments which combined the phagokinetic assay of migration and the zymographic assay for pericellular uPA activity; no relationship was observed between pericellular uPA activity and the motility of individual cells. © 1996 Wiley-Liss, Inc.