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Glioblastoma (GBM) is the most aggressive type of brain tumor, characterized by poor outcome and limited therapeutic options. During tumor progression, GBM may undergo the process of vasculogenic mimicry (VM), consisting of the formation of vascular-like structures which further promote tumor aggressiveness and malignancy. The resulting resistance to anti-angiogenetic therapies urges the identification of new compounds targeting VM. Extracts of natural plants may represent potential therapeutic tools. Among these, components of Ruta graveolens water extract (RGWE) display a wide range of biological activities. To test the effect of RGWE on human GBM and rat glioma cell line VM, tube formation on a gelled matrix was monitored. Quantitative assessment of VM formation shows the clear-cut inhibitory activity of RGWE. Unlike rutin, one of the most abundant extract components, the whole RGWE strongly reduced the migration and invasion of GBM tumor cells. Moreover, RGWE induced cell death of GBM patient-derived cancer stem cells and impaired VM at sub-lethal doses. Overall, our data reveal a marked RGWE-dependent inhibition of GBM cell survival, migration, invasion, and VM formation. Thus, the clear-cut ability of RGWE to counteract GBM malignancy deserves attention, holding the promise to bring natural products to clinical use, thus uncovering new therapeutic opportunities.

Multiple oncogenic alterations contribute to breast cancer development. Metabolic reprogramming, deeply contributing to tumor microenvironment (TME) education, is now widely recognized as a hallmark of cancer. The reverse Warburg effect induces cancer-associated fibroblasts (CAFs) to produce and secrete L-lactate, enhancing malignant characteristics such as neoangiogenesis, metastatic dissemination, and treatment resistance. Monocarboxylate transporter (MCT) 4 is involved in lactate efflux from CAFs into stromal and epithelial cells. Here, we first assess the expression of miR-425-5p and its target MCT4 in breast cancer CAFs and normal fibroblasts. We analyzed the metabolic changes induced by miR-425-5p in CAFs and its role in the education of breast cancer epithelial cells. We show that miR-425-5p-induced MCT4 knockdown decreased lactate extrusion from CAFs and its availability in the TME. miR-425-5p overexpression induced profound metabolic transformation in CAFs, ultimately influencing breast cancer metabolism. Furthermore, miR-425-5p impaired the capacity of CAFs to sustain vessel formation and breast cancer cell migration, viability, and proliferation. These findings emphasize the key role of miR-425-5p in breast cancer metabolism and aggressiveness, and its possible importance for breast cancer therapy and monitoring., (© 2024. The Author(s).)

Among the deadliest human cancers is glioblastoma (GBM) for which new treatment approaches are urgently needed. Here, the effects of the cyclic decapeptide, uPAcyclin, are investigated using the U87-MG, U251-MG, and U138-MG human GBM and C6 rat cell models. All GBM cells express the αV-integrin subunit, the target of uPAcyclin, and bind specifically to nanomolar concentrations of the decapeptide. Although peptide exposure affects neither viability nor cell proliferation rate, nanomolar concentrations of uPAcyclin markedly inhibit the directional migration and matrix invasion of all GBM cells, in a concentration- and αV-dependent manner. Moreover, wound healing rate closure of U87-MG and C6 rat glioma cells is reduced by 50% and time-lapse videomicroscopy studies show that the formation of vascular-like structures by U87-MG in three-dimensional matrix cultures is markedly inhibited by uPAcyclin. A strong reduction in the branching point numbers of the U87-MG, C6, and U251-MG cell lines undergoing vasculogenic mimicry, in the presence of nanomolar peptide concentrations, was observed. Lysates from matrix-recovered uPAcyclin-exposed cells exhibit a reduced expression of VE-cadherin, a prominent factor in the acquisition of vascular-like structures. In conclusion, these results indicate that uPAcyclin is a promising candidate to counteract the formation of new vessels in novel targeted anti-GBM therapies.

Tumor growth and metastasis strongly rely on cell-cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

Urokinase-type plasminogen activator receptor (uPAR or CD87) is a glycosyl-phosphatidyl-inositol anchored (GPI) membrane protein. The uPAR primary ligand is the serine protease urokinase (uPA), converting plasminogen into plasmin, a broad spectrum protease, active on most extracellular matrix components. Besides uPA, the uPAR binds specifically also to the matrix protein vitronectin and, therefore, is regarded also as an adhesion receptor. Complex formation of the uPAR with diverse transmembrane proteins, including integrins, formyl peptide receptors, G protein-coupled receptors and epidermal growth factor receptor results in intracellular signalling. Thus, the uPAR is a multifunctional receptor coordinating surface-associated pericellular proteolysis and signal transduction, thereby affecting physiological and pathological mechanisms. The uPAR-initiated signalling leads to remarkable cellular effects, that include increased cell migration, adhesion, survival, proliferation and invasion. Although this is beyond the scope of this review, the uPA/uPAR system is of great interest to cancer research, as it is associated to aggressive cancers and poor patient survival. Increasing evidence links the uPA/uPAR axis to epithelial to mesenchymal transition, a highly dynamic process, by which epithelial cells can convert into a mesenchymal phenotype. Furthermore, many reports indicate that the uPAR is involved in the maintenance of the stem-like phenotype and in the differentiation process of different cell types. Moreover, the levels of anchor-less, soluble form of uPAR, respond to a variety of inflammatory stimuli, including tumorigenesis and viral infections. Finally, the role of uPAR in virus infection has received increasing attention, in view of the Covid-19 pandemics and new information is becoming available. In this review, we provide a mechanistic perspective, via the detailed examination of consolidated and recent studies on the cellular responses to the multiple uPAR activities., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Alfano, Franco and Stoppelli.)

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. TNBC progression is sustained by recruitment of a strong tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAFs) able to endorse tumor hallmarks. Increasing evidences demonstrate that exosomes mediate the crosstalk between cancer cells and the TME. We examined TNBC-derived exosomes and their microRNA (miRNA) cargo in activation of normal fibroblasts (NFs) toward CAFs. We demonstrated that TNBC cell-derived exosomes increased NF collagen contraction and migration alongside CAF molecular markers. Exosome-activated fibroblasts promoted the invasion potential of normal breast epithelial cells, as assessed by an organotypic co-culture assay that resembled the in vivo context. We also investigated TNBC cell-derived exosome cargo in activating NFs to CAFs by performing small RNA sequencing. We found that the synergistic action of miR-185-5p, miR-652-5p, and miR-1246 boosted fibroblast migration and contraction, promoting specific CAF subspecialization toward a pro-migratory functional state. These data highlight the role of breast cancer cells in re-education of the TME and their contribution to tumor evolution., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Notch1 plays a key role in epithelial-mesenchymal transition (EMT) and in the maintenance of cancer stem cells. In the present study we tested whether high levels of activated Notch1 in oncogene-driven NSCLC can induce a reversible shift of driver dependence from EGFR to Notch1, and thus causing resistance to EGFR inhibitors. Adherent cells (parental) and tumor spheres (TS) from NSCLC H1975 cells and patient-derived CD133-positive cells were tested for EGFR and Notch1 signaling cascade. The Notch1-dependent modulation of EGFR, NCID, Hes1, p53, and Sp1 were then analyzed in parental cells by binding assays with a Notch1 agonist, DLL4. TS were more resistant than parental cells to EGFR inhibitors. A strong upregulation of Notch1 and a concomitant downregulation of EGFR were observed in TS compared to parental cells. Parental cell exposure to DLL4 showed a dose-dependent decrease of EGFR and a simultaneous increase of NCID, Hes1, p53, and Sp1, along with the dislocation of Sp1 from the EGFR promoter. Furthermore, an enhanced interaction between p53 and Sp1 was observed in TS. In NSCLC cells, high levels of active Notch1 can promote a reversible shift of driver dependence from EGFR to Notch1, leading to resistance to EGFR inhibitors.

Among peritumoral cells, cancer-associated fibroblasts (CAFs) are major facilitators of tumor progression. This study describes the effects of two urokinase-derived, novel decapeptides, denoted as Pep 1 and its cyclic derivative Pep 2. In a mouse model of tumor dissemination, using HT1080 fibrosarcoma cells, Pep 2 reduced the number and size of lung metastases. Specific binding of fluoresceinated Pep 2 to HT1080 and telomerase immortalised fibroblasts (TIF) cell surfaces was enhanced by αv overexpression or abolished by excess vitronectin, anti-αv antibodies or silencing of ITGAV αv gene, identifying αv-integrin as the Pep 2 molecular target. In 3D-organotypic assays, peptide-exposed TIFs and primary CAFs from breast carcinoma patients both exhibited a markedly reduced pro-invasive ability of either HT1080 fibrosarcoma or MDA-MB-231 mammary carcinoma cells, respectively. Furthermore, TIFs, either exposed to Pep 2, or silenced for αv integrin, were impaired in their ability to chemoattract cancer cells and to contract collagen matrices, exhibiting reduced α-smooth muscle actin (α-SMA) levels. Finally, peptide exposure of αv-expressing primary CAFs led to the downregulation of α-SMA protein and to a dramatic reduction of their pro-invasive capability. In conclusion, the ability of the novel decapeptides to interfere with tumor cell invasion directly and through the down-modulation of CAF phenotype suggests their use as lead compounds for co-targeting anti-cancer strategies.

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH 2 , denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.

Fibroblasts are among the most abundant stromal cells in the tumor microenvironment (TME), progressively differentiating into activated, motile, myofibroblast-like, protumorigenic cells referred to as Cancer-Associated Fibroblasts (CAFs). To investigate the mechanisms by which epithelial cells direct this transition, the early stages of tumorigenesis were exemplified by indirect cocultures of WI-38 or human primary breast cancer fibroblasts with human mammary epithelial cells expressing an inducible c-Myc oncogene (MCF10A-MycER). After c-Myc activation, the conditioned medium (CM) of MCF10A-MycER cells significantly enhanced fibroblast activation and mobilization. As this was accompanied by decreased insulin-like growth factor binding protein-6 (IGFBP-6) and increased insulin-like growth factor-1 and IGF-II (IGF-I, IGF-II) in the CM, IGFs were investigated as key chemotactic factors. Silencing IGFBP-6 or IGF-I or IGF-II expression in epithelial cells or blocking Insulin-like growth factor 1 receptor (IGF-1R) activity on fibroblasts significantly altered fibroblast mobilization. Exposure of WI-38 fibroblasts to CM from induced MCF10A-MycER cells or to IGF-II upregulated FAK phosphorylation on Tyr 397 , as well as the expression of α-smooth muscle actin (α-SMA), features associated with CAF phenotype and increased cell migratory/invasive behavior. In three-dimensional (3D)-organotypic assays, WI-38 or human primary fibroblasts, preactivated with either CM from MCF10A-MycER cells or IGFs, resulted in a permissive TME that enabled nontransformed MCF10A matrix invasion. This effect was abolished by inhibiting IGF-1R activity. Thus, breast epithelial cell oncogenic activation and stromal fibroblast transition to CAFs are linked through the IGFs/IGF-1R axis, which directly promotes TME remodeling and increases tumor invasion., (© 2019 UICC.)

Background: The biological behavior of epithelial ovarian cancer (EOC) is unique since EOC cells metastasize early to the peritoneum. Thereby, new anti-target agents designed to block trans-coelomic dissemination of EOC cells may be useful as anti-metastatic drugs. The Urokinase Plasminogen Activator Receptor (uPAR) is overexpressed in EOC tissues, and its truncated forms released in sera and/or ascitic fluid are associated with poor prognosis and unfavorable clinical outcome. We documented that uPAR triggers intra-abdominal dissemination of EOC cells through the interaction of its 84-95 sequence with the Formyl Peptide Receptor type 1 (FPR1), even as short linear peptide Ser-Arg-Ser-Arg-Tyr (SRSRY). While the pro-metastatic role of uPAR is well documented, little information regarding the expression and role of FPR1 in EOC is currently available., Methods: Expression levels of uPAR and FPR1 in EOC cells and tissues were assessed by immunofluorescence, Western blot, or immunohystochemistry. Cell adhesion to extra-cellular matrix proteins and mesothelium as well as mesothelium invasion kinetics by EOC cells were monitored using the xCELLigence technology or assessed by measuring cell-associated fluorescence. Cell internalization of FPR1 was identified on multiple z-series by confocal microscopy. Data from in vitro assays were analysed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons. Tissue microarray data were analyzed with the Pearson's Chi-square (χ 2 ) test., Results: Co-expression of uPAR and FPR1 by SKOV-3 and primary EOC cells confers a marked adhesion to vitronectin. The extent of cell adhesion decreases to basal level by pre-exposure to anti-uPAR84-95 Abs, or to the RI-3 peptide, blocking the uPAR84-95/FPR1 interaction. Furthermore, EOC cells exposed to RI-3 or desensitized with an excess of SRSRY, fail to adhere also to mesothelial cell monolayers, losing the ability to cross them. Finally, primary and metastatic EOC tissues express a high level of FPR1., Conclusions: Our findings identify for the first time FPR1 as a potential biomarker of aggressive EOC and suggests that inhibitors of the uPAR84-95/FPR1 crosstalk may be useful for the treatment of metastatic EOC.

Glioblastoma (GBM), a high-grade glioma (WHO grade IV), is the most aggressive form of brain cancer. Available treatment options for GBM involve a combination of surgery, radiation and chemotherapy but result in a poor survival outcome. GBM is a high-vascularized tumor and antiangiogenic drugs are widely used in GBM therapy as adjuvants to control abnormal vasculature. Vasculogenic mimicry occurs in GBM as an alternative vascularization mechanism, providing a means whereby GBM can escape anti-angiogenic therapies. Here, using an in vitro tube formation assay on Matrigel ® , we evaluated the ability of different histone deacetylase inhibitors (HDACis) to interfere with vasculogenic mimicry. We found that vorinostat (SAHA) and MC1568 inhibit tube formation by rat glioma C6 cells. Moreover, at sublethal doses for GBM cells, SAHA, trichostatin A (TSA), entinostat (MS275), and MC1568 significantly decrease tube formation by U87MG and by patient-derived human GBM cancer stem cells (CSCs). The reduced migration and invasion of HDACis-treated U87 cells, at least in part, may account for the inhibition of tube formation. In conclusion, our results indicate that HDACis are promising candidates for blocking vascular mimicry in GBM.

Background: Accumulating evidence demonstrates that the Urokinase Receptor (uPAR) regulates tumor cell migration through its assembly in composite regulatory units with transmembrane receptors, and uPAR 88-92 is the minimal sequence required to induce cell motility through the Formyl Peptide Receptor type 1 (FPR1). Both uPAR and FPR1 are involved in melanoma tumor progression, suggesting that they may be targeted for therapeutic purposes. In this study, the role of the uPAR-FPR1 cross-talk to sustain melanoma cell ability to invade extracellular matrix and cross endothelial barriers is investigated. Also, the possibility that inhibition of the uPAR mediated FPR1-dependent signaling may prevent matrix invasion and transendothelial migration of melanoma cells was investigated., Methods: Expression levels of uPAR and FPR1 were assessed by immunocytochemistry, Western Blot and qRT-PCR. Cell migration was investigated by Boyden chamber and wound-healing assays. Migration and invasion kinetics, trans-endothelial migration and proliferation of melanoma cells were monitored in real time using the xCELLigence technology. The agonist-triggered FPR1 internalization was visualized by confocal microscope. Cell adhesion to endothelium was determined by fluorometer measurement of cell-associated fluorescence or identified on multiple z-series by laser confocal microscopy. The 3D-organotypic models were set up by seeding melanoma cells onto collagen I matrices embedded dermal fibroblasts. Data were analyzed by one-way ANOVA and post-hoc Dunnett t-test for multiple comparisons., Results: We found that the co-expression of uPAR and FPR1 confers to A375 and M14 melanoma cells a clear-cut capability to move towards chemotactic gradients, to cross extracellular matrix and endothelial monolayers. FPR1 activity is required, as cell migration and invasion were abrogated by receptor desensitization. Finally, melanoma cell ability to move toward chemotactic gradients, invade matrigel or fibroblast-embedded collagen matrices and cross endothelial monolayers are prevented by anti-uPAR 84-95 antibodies or by the RI-3 peptide which we have previously shown to inhibit the uPAR 84-95 /FPR1 interaction., Conclusions: Collectively, our findings identify uPAR and FPR1 as relevant effectors of melanoma cell invasiveness and suggest that inhibitors of the uPAR 84-95 /FPR1 cross-talk may be useful for the treatment of metastatic melanoma.

Purpose: One of the hallmarks of cancer cells is the excessive conversion of glucose to lactate under normoxic conditions, also known as the Warburg effect. Here, we tested whether the targeted inhibition of EGFR may revert this effect and reactivate mitochondrial oxidative phosphorylation in non-small cell lung cancer (NSCLC)., Experimental Design: Sensitive (HCC827) and resistant (H1975 and H1993) NSCLC cells were treated with a panel of EGFR or MET inhibitors, and then tested for changes of EGFR signaling, glycolytic cascade, and mitochondrial function. Silencing of key glycolytic enzymes was then performed with targeted siRNAs. Furthermore, tumor-bearing nude mice treated with EGFR inhibitors were evaluated with (18)F-FDG PET/CT and tumors were analyzed for glycolytic and mitochondrial proteins., Results: Effective inhibition of EGFR signaling in NSCLC cells induced a dramatic reduction of hexokinase II (HKII) and phospho-pyruvate kinase M2 (p-PKM2, Tyr105) levels as well as an upregulation of mitochondrial complexes subunits (OXPHOS). Accordingly, a decreased lactate secretion and increased intracellular ATP levels were also observed in response to EGFR inhibitors. Downregulation of HKII and PKM2 by targeted siRNA transfection did not cause upregulation of OXPHOS but enhanced the effects of EGFR TKIs. Conversely, selective inhibition of AKT and ERK1/2 caused OXPHOS upregulation and glycolysis inhibition, respectively. Similar findings were obtained in tumors from animals treated with appropriate EGFR inhibitors., Conclusions: Our findings indicate that EGFR inhibitors may reactivate oxidative phosphorylation of cancer cells and provide a mechanistic clue for the rational combination of agents targeting EGFR-dependent proliferation and glucose metabolism in cancer therapy., (©2015 American Association for Cancer Research.)

The clinical relevance of the urokinase receptor (uPAR) as a prognostic marker in ovarian cancer is well documented. We had shown that the uPAR sequence corresponding to 84-95 residues, linking D1 and D2 domains (uPAR84-95), drives cell migration and angiogenesis in a protease-independent manner. This study was aimed at defining the contribution of uPAR84-95 sequence to invasion of ovarian cancer cells. Now, we provide evidence that the ability of uPAR-expressing ovarian cancer cells to cross extra-cellular matrix and mesothelial monolayers is prevented by specific inhibitors of the uPAR84-95 sequence. To specifically investigate uPAR84-95 function, uPAR-negative CHO-K1 cells were stably transfected with cDNAs coding for uPAR D2 and D3 regions exposing (uPARD2D3) or lacking (uPAR∆D2D3) the 84-95 sequence. CHO-K1/D2D3 cells were able to cross matrigel, mesothelial and endothelial monolayers more efficiently than CHO-K1/∆D2D3 cells, which behave as CHO-K1 control cells. When orthotopically implanted in nude mice, tumor nodules generated by CHO-K1/D2D3 cells spreading to peritoneal cavity were more numerous as compared to CHO-K1/∆D2D3 cells. Ovarian tumor size and intra-tumoral microvessel density were significantly reduced in the absence of uPAR84-95. Our results indicate that cell associated uPAR promotes growth and abdominal dissemination of ovarian cancer cells mainly through its uPAR84-95 sequence.

The receptor for the urokinase-type plasminogen activator (uPAR) is a widely recognized master regulator of cell migration, and uPAR₈₈₋₉₂ is the minimal sequence required to induce cell motility. We previously showed that soluble forms of uPAR elicit angiogenic responses through their uPAR₈₈₋₉₂ chemotactic sequence and that the synthetic peptide SRSRY exerts similar effects. By a drug design approach, based on the conformational analysis of the uPAR₈₈₋₉₂ sequence, we developed peptides (pERERY, RERY, and RERF) that potently inhibit signaling triggered by uPAR₈₈₋₉₂. In this study, we present evidence that these peptides are endowed also with a clear-cut antiangiogenic activity, although to different extents. The most active, RERF, prevents tube formation by human endothelial cells exposed to SRSRY. RERF also inhibits VEGF-triggered endothelial cell migration and cord-like formation in a dose-dependent manner, starting in the femtomolar range. RERF prevents F-actin polymerization, recruitment of αvβ3 integrin at focal adhesions, and αvβ3/VEGFR2 complex formation in endothelial cells exposed to VEGF. At molecular level, the inhibitory effect of RERF on VEGF signaling is shown by the decreased amount of phospho-FAK and phospho-Akt in VEGF-treated cells. In vivo, RERF prevents VEGF-dependent capillary sprouts originating from the host vessels that invaded angioreactors implanted in mice and neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Consistently, RERF reduced the growth and vascularization rate of tumors formed by HT1080 cells injected subcutaneously in the flanks of nude mice, indicating that RERF is a promising therapeutic agent for the control of diseases fuelled by excessive angiogenesis such as cancer., (©2013 AACR.)

Functional analysis of isolated protein domains may uncover cryptic activities otherwise missed. The serine protease urokinase (uPA) has a clear-cut motogen activity that is catalytically independent and resides in its amino-terminal growth factor domain (GFD, residues 1-49) and connecting peptide region (CP, residues 132-158). To functionally dissect the CP region, we analysed the biological activity of two synthetic peptides corresponding to the N-terminal [uPA-(135-143), residues 135-143] and C-terminal [uPA-(144-158), residues 144-158] CP subregions. Most of the chemotactic activity of connecting peptide-derived peptide (CPp, [uPA-(135-158)]) for embryonic kidney HEK293/uPAR-25 cells is retained by uPA-(144-158) at nanomolar concentrations. In contrast, uPA-(135-143) inhibits basal, CPp -, vitronectin- and fibronectin-induced cell migration. Radioreceptor binding assays on intact HEK293 cells revealed that uPA-(135-143) and uPA-(144-158) are both able to compete with [(125)I]-CPp, albeit with different binding affinities. The consequences of phospho-mimicking, S138E substitution, were studied using [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. Unlike CPp, [138E]uPA-(135-158) and [138E]uPA-(135-143) exhibit remarkable inhibitory properties. Finally, analysis of the conformational preferences of the peptides allowed to identify secondary structure elements exclusively characterising the stimulatory CPp and uPA-(144-158) versus the inhibitory uPA-(135-143), [138E]uPA-(135-158) and [138E]uPA-(135-143) peptides. In conclusion, these data shed light on the cryptic activities of uPA connecting peptide, revealing the occurrence of two adjacent regions, both competing for binding to cell surface but conveying opposite signalling on cell migration., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Urokinase-type plasminogen activator (uPA) and its receptor, uPAR, play important roles in promoting cancer cell adhesion, migration and invasion. Rho GTPases are key coordinators of these processes; the Rho GTPase Rac1 has previously been implicated in uPA- and/or uPAR-induced migratory or morphological cell responses. We used RNAi to deplete 12 different Rho GTPases to screen for effects on uPA-stimulated migration, and found that depletion of RhoB significantly reduces uPA-induced migration and invasion of prostate carcinoma cells. RhoB depletion did not affect the expression or surface levels of uPAR but reduced the uPAR-induced increase in levels of several integrins and inhibited uPAR signalling to the actin regulator cofilin, the cell-adhesion signal-transduction adaptor molecule paxillin and the serine/threonine kinase Akt. uPAR rapidly activated RhoB and increased RhoB expression. RhoB depletion also reduced cell adhesion to and spreading on vitronectin, which is a uPAR ligand. This correlated with decreased association between integrins and uPAR and reduced integrin β1 activity. Our results indicate that RhoB is a key regulator of uPAR signalling in cell adhesion, migration and invasion.

The receptor for urokinase-type plasminogen activator (uPAR) plays an important role in controlling cell migration. uPAR binds urokinase and vitronectin extracellular ligands, and signals in complex with transmembrane receptors such as Formyl-peptide Receptors (FPR)s and integrins. Previous work from this laboratory has shown that synthetic peptides, corresponding to the uPAR(88-92) chemotactic sequence, when carrying the S90P or S90E substitutions, up- or down-regulate cell migration, respectively. To gain mechanistic insights into these opposite cell responses, the functional consequences of S90P and S90E mutations in full-length uPAR were evaluated. First, (HEK)-293 embryonic kidney cells expressing uPAR(S90P) exhibit enhanced FPR activation, increased random and directional cell migration, long-lasting Akt phosphorylation, and increased adhesion to vitronectin, as well as uPAR/vitronectin receptor association. In contrast, the S90E substitution prevents agonist-triggered FPR activation and internalization, decreases binding and adhesion to vitronectin, and inhibits uPAR/vitronectin receptor association. Also, 293/uPAR(S90P) cells appear quite elongated and their cytoskeleton well organized, whereas 293/uPAR(S90E) cells assume a large flattened morphology, with random orientation of actin filaments. Interestingly, when HT1080 cells co-express wild type uPAR with uPAR S90E, the latter behaves as a dominant-negative, impairing uPAR-mediated signaling and reducing cell wound repair as well as lung metastasis in nude mice. In contrast, signaling, wound repair and in vivo lung metastasis of HT1080 cells bearing wild type uPAR are enhanced when they co-express uPAR(S90P). In conclusion, our findings indicate that Ser(90) is a critical residue for uPAR signaling and that the S90P and S90E exert opposite effects on uPAR activities. These findings may be accommodated in a molecular model, in which uPAR(S90E) and uPAR(S90P) are forced into inactive and active forms, respectively, suggesting important implications for the development of novel drugs targeting uPAR function.

Proteins secreted by cancer cells are a major component of tumor microenvironment. However, little is known on the impact of single oncogenic lesions on the expression of secreted proteins at early stages of tumor development. Because c-Myc overexpression is among the most frequent alterations in cancer, here we investigated the effect of sustained c-Myc expression on the secretome of a nontransformed human epithelial cell line (hT-RPE). By using a quantitative proteomic approach, we have identified 125 proteins in conditioned media of hT-RPE/MycER cells upon c-Myc induction. Analysis of the 49 proteins significantly down-regulated by c-Myc revealed a marked enrichment of factors associated with growth inhibition and cellular senescence. Accordingly, media conditioned by hT-RPE cells expressing c-Myc show an increased ability to sustain hT-RPE cellular proliferation/viability. We also find a marked down-regulation of several structural and regulatory components of the extracellular matrix (ECM), which correlates with an increased chemotactic potency of the conditioned media toward fibroblasts, a major cellular component of tumor stroma. In accordance with these data, the expression of the majority of the genes encoding proteins down-regulated in hT-RPE was significantly reduced also in colorectal adenomatous polyps, early tumors in which c-Myc is invariably overexpressed. These findings help to elucidate the significance of c-Myc overexpression at early stages of tumor development and uncover a remarkable autocrine/paracrine component in the ability of c-Myc to stimulate proliferation, sustain tumor maintenance, and modulate cell migration.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Urokinase (uPA) was originally identified in human urine for its ability to catalyse the transformation of plasminogen into its active form, plasmin which degrades fibrin and extracellular matrix components. Two major, functionally independent regions have been identified in the uPA molecule: a non-catalytic N-terminal region (residues 1-135) and a large catalytic region (residues 159-411) spaced by the "connecting peptide" (residues 136-158). Binding of uPA to its specific surface receptor (uPAR) amplifies cell surface plasminogen activation, thus enhancing pericellular proteolysis. The uPAR, linked to the lipid bilayer via a glycosylphosphatidylinositol anchor, mediates signaling through the assembly of a multiprotein complex with transmembrane receptors, like the integrins, EGFR, GPCRs. Receptor engagement with uPA results in a variety of cell responses, including increased proliferation, survival, migration and invasion. These responses may be enhanced by the concomitant binding of the uPA "connecting peptide" region to αvβ5 integrin, thus favoring uPAR-integrin association. Receptors engaged with uPA exhibit a high affinity for vitronectin, stimulating cell adhesion. The uPA/uPAR system is regarded as one of the key systems driving tumour invasion and metastases. Different strategies to prevent the activity of the protease, as well as the interactions of uPAR with integrins and GPCRs have been designed. Many preclinical studies are ongoing and, at least, two uPA-related compounds have reached Phase II clinical trials. The aim of this review is to provide a comprehensive picture of the functionally relevant interactions, together with a description of the promising compounds and strategies to control uPA activity and signaling in human pathologies.

Increased expression of Bcl-xL in cancer has been shown to confer resistance to a broad range of apoptotic stimuli and to modulate a number of other aspects of cellular physiology, including energy metabolism, cell cycle, autophagy, mitochondrial fission/fusion and cellular adhesion. However, only few of these activities have a mechanistic explanation. Here we used Tandem Affinity purification to identify novel Bcl-xL interacting proteins that could explain the pleiotropic effects of Bcl-xL overexpression. Among the several proteins co-purifying with Bcl-xL, we focused on Praf2, a protein with a predicted role in trafficking. The interaction of Praf2 with Bcl-xL was found to be dependent on the transmembrane domain of Bcl-xL. We found that Bcl-2 also interacts with Praf2 and that Bcl-xL and Bcl-2 can interact also with Arl6IP5, an homologue of Praf2. Interestingly, overexpression of Praf2 results in the translocation of Bax to mitochondria and the induction of apoptotic cell death. Praf2 dependent cell death is prevented by the co-transfection of Bcl-xL but not by its transmembrane domain deleted mutant. Accordingly, knock-down of Praf2 increases clonogenicity of U2OS cells following etoposide treatment by reducing cell death. In conclusion a screen for Bcl-xL-interacting membrane proteins let us identify a novel proapoptotic protein whose activity is strongly counteracted exclusively by membrane targeted Bcl-xL.

Background: The urokinase plasminogen activator receptor (u-PAR) focuses the proteolytic activity of the urokinase plasminogen activator (u-PA) on the endothelial cell surface, thus promoting angiogenesis in a protease-dependent manner. The u-PAR may exist in a glycophosphatidylinositol-anchored and in a soluble form (soluble u-PAR [Su-PAR]), both including the chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² internal sequence., Objective: To investigate whether Su-PAR may trigger endothelial cell signaling leading to new vessel formation through its chemotactic Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² sequence., Methods and Results: In this study, the formation of vascular-like structures by human umbilical vein endothelial cells was assessed by using a matrigel basement membrane preparation. First, we found that Su-PAR protein promotes the formation of cord-like structures, and that this ability is retained by the isolated Ser(88) -Arg-Ser-Arg-Tyr⁹² chemotactic sequence, the maximal effect being reached at 10 nmol L⁻¹ SRSRY peptide (SRSRY). This effect is mediated by the α(v) β₃ vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formyl-peptide receptor in endothelial cells. Furthermore, exposure of human saphenous vein rings to Su-PAR or SRSRY leads to a remarkable degree of sprouting. Finally, we show that Su-PAR and SRSRY promote a marked response in angioreactors implanted into the dorsal flank of nude mice, retaining 91% and 66%, respectively, of the angiogenic response generated by a mixture of vascular endothelial growth factor and fibroblast growth factor type 2., Conclusions: Our results show a new protease-independent activity of Su-PAR that stimulates in vivo angiogenesis through its Ser⁸⁸ -Arg-Ser-Arg-Tyr⁹² chemotactic sequence., (© 2010 International Society on Thrombosis and Haemostasis.)

It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser(88)-Arg-Ser-Arg-Tyr(92) is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH(2) (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe-dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an alphav integrin-dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/alphav association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein-expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy.

We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention., (Copyright (c) 2008 Wiley-Liss, Inc.)

Urokinase (uPA) is a serine protease which converts plasminogen to plasmin, a broad-spectrum protease active on extracellular matrix (ECM) components. Like many components of the blood coagulation, fibrinolytic and complement cascades, uPA has a modular structure, including three conserved domains: a growth factor-like domain (GFD, residues 1 - 49), a kringle domain (residues 50 - 131), linked by an interdomain linker or "connecting peptide" (CP, residues 132 - 158) to the serine protease domain (residues 159 - 411). Although direct molecular interactions with urokinase receptor and integrins have been extensively described, the function of single uPA domains is not completely understood. Because of the causal involvment of uPA in cancer invasion and metastasis, the blockade of uPA interactions and activity with specific inhibitors is of interest for novel strategies in cancer therapy. New inhibitors derived from the interdomain linker or "connecting peptide" are coming into focus. This review summarizes the recent findings on the uPA structure-function relationship and provides further information on existing inhibitors of uPA multiple functions.

Urokinase receptor (uPAR) plays a key role in physiological and pathological processes sustained by an altered cell migration. We have developed peptides carrying amino acid substitutions along the Ser(88)-Arg-Ser-Arg-Tyr(92) (SRSRY) uPAR chemotactic sequence. The peptide pyro glutamic acid (pGlu)-Arg-Glu-Arg-Tyr-NH2 (pERERY-NH(2)) shares the same binding site with SRSRY and competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the G-protein-coupled N-formyl-peptide receptor (FPR). pERERY-NH(2) is a dose-dependent inhibitor of both SRSRY- and fMLF-directed cell migration, and prevents agonist-induced FPR internalization and fMLF-dependent ERK1/2 phosphorylation. pERERY-NH(2) is a new and potent uPAR inhibitor which may suggest the generation of new pharmacological treatments for pathological conditions involving increased cell migration.

The serine protease urokinase (uPA) binds to the urokinase receptor (uPAR) through its growth-factor domain (GFD, residues 1-49), affecting cell migration, adhesion and growth. Here, we show that uPA can promote cytoskeletal rearrangements and directional cell migration in a GFD-independent manner, through a new and specific interaction between an internal uPA domain coined ;connecting peptide' (residues 132-158) and cell-surface integrin alpha v beta 5. Remarkably, a peptide corresponding to this region (CPp, residues 135-158) retains the ability to bind to alpha v beta 5, eliciting cytoskeletal rearrangements and directing cell migration at a concentration as low as 1-10 pM. These effects are lost in cells not expressing uPAR, indicating that the uPAR is required for CPp-dependent signaling. Furthermore, the CPp-alpha v beta 5-integrin interaction enhances F-actin-enriched protrusions and cell migration induced by the well-established interaction between the uPAR-binding peptide (GFDp, residues 12-32) of uPA and uPAR. These results provide new insight into the function of uPA, which--through individual domains--can engage two different surface receptors (uPAR and alpha v beta 5 integrin), thus initiating and potentiating intracellular signaling and migration.

The acquired capabilities of resistance to apoptotic cell death and tissue invasion are considered to be obligate steps in tumor progression. The binding of the serine protease urokinase (uPA) to its receptor (uPAR) plays a central role in the molecular events coordinating tumor cell adhesion, migration, and invasion. Here we investigate whether uPAR signaling may also prevent apoptosis following loss of anchorage (anoikis) or DNA damage. If nontransformed human retinal pigment epithelial cells are pre-exposed to uPA or to its noncatalytic amino-terminal region (residues 1-135), they exhibit a markedly reduced susceptibility to anoikis as well as to UV-induced apoptosis. This anti-apoptotic effect is retained by a uPA-derived synthetic peptide corresponding to the receptor binding domain and is inhibited by anti-uPAR polyclonal antibodies. Furthermore, the stable reduction of uPA or uPAR expression by RNA interference leads to an increased susceptibility to UV-, cisplatin-, and detachment-induced apoptosis. In particular, the level of uPAR expression positively correlates with cell resistance to anoikis. The protective ability of uPA is prevented by UO126, LY294002, by an MAPK targeting small interference RNA, and by a dominant negative Akt variant. Accordingly, incubation of retinal pigment epithelial cells with uPA elicits a time-dependent enhancement of MAPK and phosphatidylinositol 3-kinase activities as well as the transcriptional activation of Bcl-xL anti-apoptotic factor. Vice versa, the silencing of Bcl-xL expression prevents uPA protection from anoikis. In conclusion, the data show that ligand engagement of uPAR promotes cell survival by activating Bcl-xL transcription through the MEK/ERK- and phosphatidylinositol 3-kinase/Akt-dependent pathways.

Sp1 regulates the activation of many genes involved in tumor growth, apoptosis, and angiogenesis. We have previously shown the involvement of Sp1 in the up-regulation of urokinase receptor (uPAR) expression, a key molecule in tumor invasion and metastasis. Here, we investigated whether a marked down-regulation of Sp1 activity may inhibit uPAR expression and migration ability of MDA-MB-231 breast cancer cells. To this end, we tested the decoy ability of a novel peptide nucleic acid (PNA)-DNA chimera which carries a central DNA strand, containing Sp1-binding sequence, covalently linked to two PNA fragments at both ends (PNA-DNA-PNA, PDP). The chimera was synthesized, annealed with complementary DNA (PDP-DNA), and then tested for its ability to bind Sp1 both in vitro and in living MDA-MB-231 breast cancer cells in the presence of urokinase (uPA). This PDP-DNA decoy molecule efficiently competes for the binding to endogenous Sp1 in nuclear extracts, and upon transfection with liposomal vectors, causes a marked decrease of available Sp1 in both untreated and uPA-treated MDA-MB-231 cells. Accordingly, both uPA-dependent enhancement of uPAR expression and cell migration were strongly reduced in transfected cells. Interestingly, a detectable inhibitory effect is also observed in breast cancer cells exposed to PDP-DNA in the absence of transfection reagents. Finally, the inhibitory effect of PDP-DNA appeared to be stronger than that observed with oligonucleotides carrying Sp1 consensus sequence. Our findings show that this novel PNA-DNA chimera, containing Sp1 consensus sequence, effectively inhibits Sp1 activity, uPAR expression, and motility of breast cancer cells indicating its potential therapeutic use to prevent tumor dissemination.

The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis). The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.

We have previously shown that urokinase receptor physically and functionally interacts with alpha(v)beta5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and alpha(v)beta5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and alpha(v)beta5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate alpha(v)beta5 expression. Here, we report the receptor-dependent ability of catalytically inactive urokinase to upregulate the alpha(v) and beta5 chains in MDA-MB-231 and MCF-7 breast carcinoma cell lines in a time- and concentration-dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled alpha(v)beta5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the "phosphorylation-like" urokinase receptor antagonist His-uPA(138E/303E), the DNA-binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti-alpha(v)beta5 antibodies. Finally, urokinase enables benign breast MCF-10A cells to cross Matrigel in a alpha(v)beta5- and urokinase receptor-dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression., (Copyright 2002 Wiley-Liss, Inc.)

We have previously reported that phosphorylation of human urokinase on Ser138/303 abolishes its catalytic-independent motogen and proadhesive abilities, whereas receptor binding is not affected. Here we show that substitution of the two relevant serines with glutamic acid residues impairs the ability of urokinase to mobilize a variety of human and mouse cell lines as well as human primary T lymphocytes. Accordingly, urokinase receptor-dependent signaling, leading to cytoskeletal rearrangements and paxillin re-distribution, does not occur in MCF-7 breast carcinoma cells exposed to 'phosphorylation-like' urokinase. Unlike the wild-type form, di-substituted urokinase is unable to induce the physical association of urokinase receptor with alphavbeta5 vitronectin receptor, which is required for MCF-7 urokinase-dependent cell migration. Finally, the di-substituted variant fails to activate p55fgr, a member of the Src tyrosine kinase family, which mediates cell migration and adhesion of U937 myelomonocytic cells. In conclusion, the finding that specific amino acid substitutions strongly interfere with the ability of urokinase to stimulate cell migration, and the associated intracellular events uncover a novel way to regulate urokinase receptor-dependent signaling.

In this study we show that SV-IV, a major immunomodulatory, anti-inflammatory, and sperm immunoprotective protein secreted from the rat seminal vesicle epithelium, acts in vitro as a substrate of protein kinase C (PKC) competing efficiently with H1 histone, a very well known PKC substrate. Electrospray mass spectrometry (ES-MS) analysis demonstrated that approximately 10% of the native SV-IV molecules were phosphorylated by PKC and that such a modification involved only a single serine residue (Ser58) out of the 22 occurring in the protein. Interestingly, this modification produced a substantial enhancement (approximately 50%) of the native SV-IV's ability to stimulate the activity of both horseradish peroxidase (POD) and selenium-dependent glutathione peroxidase (GPX), an enzyme that is known to protect the mammalian spermatozoa from oxidative stress and loss of motility in the female genital tract following ejaculation. In contrast, the phosphorylation of SV-IV on Ser58 did not produce any effect on the anti-inflammatory properties of SV-IV, as measured by its ability to inhibit the phospholipase A2.

The regulatory mechanisms underlying the overexpression of urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in highly invasive breast carcinomas remain poorly understood. In this study, we have simultaneously determined the level of uPAR and the activity of the transcription factor Sp1 in 14 breast carcinomas and 5 benign lesions. We found that uPAR levels and Sp1-binding activity are coordinately elevated in malignant tumors (r, 0.94; P < 0.001). On the contrary, undetectable or only barely detectable levels of uPAR and Sp1 activity were found in benign breast lesions. Finally, the engagement of uPAR by catalytically inactive uPA in the MDA-MB-231 breast carcinoma cell line results in a rapid up-regulation of Sp1-binding activity followed by an increase of uPAR protein. These results, taken together, suggest the existence of a uPA-dependent positive regulatory loop that may progressively enhance malignant breast cell invasiveness.

Perturbation of adhesive interactions at cell-substratum and cell-cell contact sites is a critical event in the multistep process of cancer invasion. Recent studies indicate that the urokinase receptor (uPAR) is associated in large molecular complexes with other molecules, such as integrins. To test the possibility that uPAR may physically and functionally interact with vitronectin (Vn) receptors, we determined the expression level of uPAR, alpha(v)beta3, and alpha(v)beta5 Vn receptors in 10 human breast carcinomas. Here, we show the ability of uPAR to physically associate with alpha(v)beta5 in the breast carcinomas examined. The functional effects of this interaction were studied using HT1080 human fibrosarcoma and MCF-7 human breast carcinoma cell lines, both exhibiting a urokinase-dependent physical association between uPAR and alpha(v)beta5. Both cell lines respond to urokinase or to its noncatalytic amino-terminal fragment by exhibiting remarkable cytoskeletal rearrangements that are mediated by alpha(v)beta5 and require protein kinase C activity. On the contrary, binding of Vn to alpha(v)beta5 results in the protein kinase C-independent formation of F-actin containing microspike-type structures. Furthermore, alpha(v)beta5 is required for urokinase-directed, receptor-dependent MCF-7 and HT1080 cell migration. These data show that uPAR association with alpha(v)beta5 leads to a functional interaction of these receptors and suggest that uPAR directs cytoskeletal rearrangements and cell migration by altering alpha(v)beta5 signaling specificity.

Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.

We recently reported that in vivo phosphorylation of urokinase-type plasminogen activator on Ser138/303 prevents its catalytic-independent ability to promote myelomonocytic cell adherence and motility. We now show that Ca2+ activated, phospholipid-dependent protein kinase C from rat brain phosphorylates in vitro a peptide corresponding to prourokinase residues 133-143 (DGKKPSSPPEE) and the full-length molecule on Ser138/139. The in vivo involvement of the protein kinase C isoenzyme family is supported by the finding that inhibition of kinase C activity prevents prourokinase phosphorylation on Ser138/303 in A431 human carcinoma cells. Conversely, a short treatment of A431 cells with phorbol myristate acetate increases the extent of phosphorylated prourokinase and, concomitantly, affects its function; under these conditions, the capability of prourokinase to up-regulate U937 monocyte-like cell adherence is severely impaired, although receptor binding is unaltered. By the aid of a "phosphorylation-like" variant (Ser138 to Glu) we show that modification of Ser138 is sufficient to confer to prourokinase the antagonistic properties observed following in vivo stimulation of protein kinase C activity. These observations provide the first evidence that protein kinase C directs the formation of a receptor competitive antagonist by regulating the in vivo phosphorylation state of prourokinase.

Functional assembly of the plasminogen-dependent proteolytic system on the cell surface requires multiple interactions involving urokinase (uPA), urokinase receptor (uPAR), plasminogen activator inhibitors, and other molecules that mediate cell migration and adhesion. We analyzed the in vitro interaction of uPAR-containing particulate cell fractions with the amino-terminal fragment (ATF) of human urokinase and the matrix-like form of vitronectin. Binding and cross-linking of 125I-labeled ATF to crude membrane extracts from LB6-19 mouse cells overexpressing human uPARs in the presence of 25 nM urea-denatured vitronectin led to the formation of Mr 137,000, 92, 000, and 82,000 covalent complexes. Immunoprecipitation of the preformed cross-linked 125I-labeled complexes with anti-vitronectin, anti-uPA, or anti-uPAR antibodies revealed that the Mr 82,000 and 92, 000 species do contain ATF and vitronectin and identified the Mr 137, 000 species as a ternary complex formed by ATF, uPAR, and vitronectin. A similar electrophoretic pattern was displayed by acid-pretreated membranes extracted from MCF-7 breast carcinoma or HT1080 fibrosarcoma cell lines, as well as a ductal breast carcinoma specimen; the latter exhibited complex formation at concentrations of vitronectin lower than 10 nM. Finally, uPAR-vitronectin interaction was further documented by the decreased reactivity of an anti-uPAR polyclonal antibody to acid-pretreated sections of 10 breast carcinomas that had been preincubated with vitronectin. Our findings highlight the ability of uPAR to interact simultaneously with vitronectin and uPA in breast cancer, supporting a dynamic coupling of the molecular mechanisms underlying plasminogen-dependent matrix degradation and cell adhesion.

Serine phosphorylation of human pro-urokinase (pro-uPA) by A431 human carcinoma cells results in a catalytically active molecule with reduced sensitivity to plasminogen activator inhibitor type 1. We mapped the phosphorylated seryl residues by analyzing the in vivo phosphorylation state of engineered pro-uPA variants carrying a COOH-terminal poly-histidine tag. Stably transfected A431 cells do not incorporate radioactive phosphate into tagged pro-uPA in which the serines 138 and 303 have been replaced with glutamic residues, although endogenous nontagged pro-uPA is 32P-labeled on A and B chains. Moreover, the catalytic-independent ability of the mono- and di-substituted "phosphorylation-like" variants to bind to the GPI-anchored urokinase receptor (uPAR) and promote adherence of differentiating U937, HL-60, and THP-1 myelomonocytic cells was examined. We found that glutamic residues as well as the naturally occurring phosphoserines at positions 138 and 303 abolish proadhesive ability, although they do not interfere with receptor binding. In addition, pro-uPA carrying Glu138/303 lacks the capability to induce a chemotactic response of THP-1 cells. The exclusive presence of Glu138 reduces pro-uPA proadhesive and chemotactic ability by 70-80%, indicating that a phosphoserine residue at the same position plays a major inhibitory role of myeloid cell response to pro-urokinase. The di-substitution does not affect pro-uPA ability to interact with vitronectin or to enhance binding of urea-denatured vitronectin to uPAR. However, unlike wild-type tagged pro-uPA, the di-substituted variant does not induce receptor polarization in pre-adherent U937 cells. Taken together, the data support the possibility that pro-uPA phosphorylation on Ser138/303 can modulate uPAR transducing ability.

Current evidence regarding the regulation of urokinase-dependent extracellular proteolysis indicates that plasminogen activation is a surface-associated process. We have compared the histological localization of urokinase plasminogen activator (uPA) and urokinase plasminogen activator receptor (uPAR) in breast cancer sections using a panel of monoclonal antibodies. First, the ability of six different anti-uPA monoclonal antibodies to recognize pro-uPA, uPA, and in vitro-formed complexes of uPA with either soluble uPAR or with plasminogen activator inhibitor type 1 was compared. Then the reactivity of the anti-uPAR antibodies was tested, and the occurrence of an uPA receptor of about M(r) 55,000 in samples from breast carcinoma was assessed by immunoprecipitating the uPA receptor from an in vitro 125I-labeled tumor extract. Immunocytochemical data from adjacent sections of 10 tumor specimens showed that antibodies recognizing free and bound uPA mostly stain the cytoplasm and the membrane of epithelial tumor cells in confined areas of the tumor and some fibroblast-like stromal cells. Acid pretreatment of tumor sections, which removes receptor-bound uPA, causes a strong reduction of the immunocytochemical reactivity of epithelial tumor cells, whereas staining of fibroblast-like cells is not considerably affected. Consistent with these results, epithelial tumor cells were mostly unreactive to anti-uPAR antibodies unless pretreated with acidic buffer, whereas fibroblast-like stromal cells showed a faint but acid-resistant staining with all anti-uPARs. In conclusion, these results show that occupied uPA receptors are definitely present on the membrane of epithelial tumor cells and suggest the occurrence of uPA-uPAR-dependent proteolytic activity on the surface of breast cancer cells.

Since many of the factors involved in tumor growth and progression act through a receptor-mediated mechanism, we applied in vitro receptor imaging techniques to study the intratumoral distribution and concentration of receptor-molecules having experimental biological relevance in such processes. Here we summarize the results of a study concerning the role of urokinase receptor (uPAR) in the acquisition of an invasive phenotype by tumor cells. Independently of the system studied, we demonstrated that in vitro receptor imaging techniques can be used to define the biological characteristics of human solid tumors and can contribute to clarify the complex network of ligand/receptor interactions leading to tumor spread.

We measured the tissue concentration of human urokinase receptor (uPAR) in 22 breast carcinomas and 9 benign breast lesions using in vitro quantitative autoradiography. Tissue sections were incubated with increasing concentrations of 125I-pro-urokinase in the presence or absence of unlabeled competitor. Breast carcinomas were found to contain 5 times more uPAR than benign breast lesions with respect to their protein content [523 +/- 72 versus 108 +/- 20 (SE) fmol/mg (P < 0.001)]. Simultaneous quantitation of urokinase (uPA) by immunoenzymatic assay on tissue extracts from the same specimens showed that breast carcinomas also contain 19 times more uPA than benign tumors (611 +/- 134 versus 32 +/- 8 fmol/mg) (P < 0.01). The reliability of quantitative autoradiography measurements was confirmed by uPAR cross-linking assay on membrane fraction from either U937 histiocytic lymphoma cells or breast carcinomas and immunoperoxidase staining with an anti-uPAR antibody on tumor sections. Also, immunoperoxidase staining with an anti-uPA monoclonal antibody showed that uPA is indeed localized on the plasma membrane of epithelial tumor cells in confined areas of breast carcinomas whereas cells from benign breast lesions were devoid of uPA under the same experimental conditions. In conclusion, our findings support the hypothesis that uPAR plays a central role in the acquisition of an invasive phenotye and favor its potential use as a prognostic factor in patients with breast carcinoma.

Urokinase synthesized by human A431 epidermoid carcinoma cells is phosphorylated on serine (Mastronicola, M. R., Stoppelli, M. P., Migliaccio, A., Auricchio, F., and Blasi, F. (1990) FEBS Lett. 266, 109-114). To test the possibility that phosphorylation may have specific effects on urokinase function, the phosphorylated and nonphosphorylated forms of urokinase were separated by Fe(3+)-Sepharose chromatography. Both forms exhibit indistinguishable Km and kcat for plasminogen activation. On the other hand, their sensitivity toward the specific plasminogen activator inhibitor type 1 is different as assessed by measuring both the stability of the covalent complex and the residual enzymatic activity. Phosphorylated urokinase was 50% inhibited at a concentration of plasminogen activator inhibitor type 1 4-fold higher than nonphosphorylated urokinase (0.7 versus 0.15 nM). Furthermore about 10% of phosphorylated urokinase was resistant to plasminogen activator inhibitor type 1 at a concentration as high as 20 nM. Thus, phosphorylation affects urokinase sensitivity to plasminogen activator inhibitor type 1, therefore resulting in a net, although indirect, increase of urokinase activity. These results suggest the existence of a novel cellular regulatory mechanism of extracellular proteolysis.

Unregulated or increased expression of epidermal growth factor receptor (EGF-R) is a common event in neoplastic transformation; modulation of such a receptor by physiological agents could be, therefore, of clinical interest. We have studied the binding ability, the availability at cell surface, and the synthesis of EGF-R in the A431 and KB human epidermoid cancer cell lines after treatment with recombinant alpha-interferon (IFN-alpha). After 48 h of treatment, IFN-alpha induces, in both cell lines, growth inhibition and enhances class I major histocompatibility HLA complex expression, which is a common marker of IFN action. [125I]EGF total binding assessed after 48 h of treatment with IFN-alpha shows a dose-dependent upregulation of EGF-R binding capacity. Saturation plots of the binding data show that IFN-alpha treatment does not dramatically alter the affinity of the EGF-R and indicate that IFN-alpha only increases the number of low affinity receptors. We show that this effect is due to a specific increase in the synthesis of the receptor protein, as assessed by immunoprecipitation of [35S]methionine-labeled cell extracts. Electron microscopy analysis has confirmed an increase of EGF-R proteins at cell surface without major changes in the morphology of the cells. Taken together, these results indicate that IFN-alpha consistently induces both the binding capacity and the synthesis of EGF-R in human epidermoid cancer cells and suggest the use of such a mechanism for new anticancer therapies.