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 dosedependent 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 uPAR⅐␣v␤5 vitronectin receptor physical association, indicating that ␣v␤5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that ␣v␤5 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 ␣v␤5.

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-ArgTyr 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 αv 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/αv 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.

Metastasis is the leading cause of death by cancer. Non-small-cell lung cancer (NSCLC) represents nearly 85% of primary malignant lung tumours. Recent researches have demonstrated that epithelial-to-mesenchymal transition (EMT) plays a key role in the early process of metastasis of cancer cells. Transforming growth factor-β1 (TGF-β1) is the major inductor of EMT. The aim of this study is to investigate TGF-β1's effect on cancer stem cells (CSCs) identified as cells positive for CD133, side population (SP) and non-cancer stem cells (non-CSCs) identified as cells negative for CD133, and SP in the A549 cell line. We demonstrate that TGF-β1 induces EMT in both CSC and non-CSC A549 sublines, upregulating the expression of mesenchymal markers such as vimentin and Slug, and downregulating levels of epithelial markers such as e-cadherin and cytokeratins. CSC and non-CSC A549 sublines undergoing EMT show a strong migration and strong levels of MMP9 except for the CD133− cell fraction. OCT4 levels are strongly upregulated in all cell fractions except CD133− cells. On the contrary, wound size reveals that TGF-β1 enhances motility in wild-type A549 as well as CD133+ and SP+ cells. For CD133− and SP− cells, TGF-β1 exposure does not change the motility. Finally, assessment of growth kinetics reveals major colony-forming efficiency in CD133+ A549 cells. In particular, SP+ and SP− A549 cells show more efficiency to form colonies than untreated corresponding cells, while for CD133− cells no change in colony number was observable after TGF-β1 exposure. We conclude that it is possible to highlight different cell subpopulations with different grades of stemness. Each population seems to be involved in different biological mechanisms such as stemness maintenance, tumorigenicity, invasion and migration.

This work is based on previous evidence showing that chemotactic sequence of the urokinase receptor (uPAR [88][89][90][91][92] ) drives angiogenesis in vitro and in vivo in a protease-independent manner, and that the peptide AcArg-Glu-Arg-Phe-NH 2 (RERF) prevents both uPAR 88-92 -and VEGF-induced angiogenesis. New N-acetylated and C-amidated peptide analogues containing a-methyl a-amino acids were designed and synthesized to optimize the biochemical properties for therapeutic applications. Among these, Ac-L-Arg-Aib-L-Arg-D-Ca(Me) Phe-NH 2 , named UPARANT, adopts in solution a turned conformation similar to that found for RERF, is stable to sterilization in 3 mg/mL sealed vials in autoclave for 20 minutes at 120 C, is stable in blood, and displays a long-time resistance to enzymatic proteolysis. UPARANT competes with N-formyl-Met-Leu-Phe (fMLF) for binding to the formyl-peptide receptor, inhibits VEGF-directed endothelial cell migration, and prevents cytoskeletal organization and avb3 activation in endothelial cells exposed to VEGF. In vitro, UPARANT inhibits VEGF-dependent tube formation of endothelial cells at a 100Â lower concentration than RERF. In vivo, UPARANT reduces to the basal level VEGF-dependent capillary sprouts originating from the host vessels that invaded Matrigel sponges implanted in mice, and completely prevents neovascularization induced by subcorneal implantation of pellets containing VEGF in rabbits. Both excellent stability and potency position UPARANT as a promising new therapeutic agent for the control of diseases fueled by excessive angiogenesis, such as cancer and inflammation.

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Methods and Results:In this study, the formation of vascularlike 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-Tyr 92 chemotactic sequence, the maximal effect being reached at 10 nmol L )1 SRSRY peptide (SRSRY). This effect is mediated by the a v b 3 vitronectin receptor, is independent of u-PA proteolytic activity, and involves the internalization of the G-protein-coupled formylpeptide 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 Ser 88-Arg-Ser-Arg-Tyr 92 chemotactic sequence.

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-LeuPhe (fMLF) for binding to the G-protein-coupled N-formylpeptide 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 fMLFdependent 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 development of metastases is a multistep process that requires the activation of physiological and biochemical processes that govern migration, invasion and entry of metastatic cells into blood vessels. The urokinase receptor (uPAR) promotes cell migration by interacting with the Formyl Peptide Receptors (FPRs). Since both uPAR and FPR1 are involved in tumor progression, the uPAR-FPR1 interaction is an attractive therapeutic target. We previously described peptide antagonists of the uPAR-FPR1 interaction that inhibited cell migration and angiogenesis. To develop enzyme-resistant analogues, we applied here the Retro-Inverso (RI) approach, whereby the topology of the side chains is maintained by inverting the sequence of the peptide and the chirality of all residues. Molecular dynamics suggests that peptide RI-3 adopts the turn structure typical of uPAR-FPR1 antagonists. Accordingly, RI-3 is a nanomolar competitor of N-formyl-Met-Leu-Phe for binding to FPR1 and inhibits migration, invasion, trans-endothelial migration of sarcoma cells and VEGF-triggered endothelial tube formation. When sarcoma cells were subcutaneously injected in nude mice, tumor size, intra-tumoral microvessel density, circulating tumor cells and pulmonary metastases were significantly reduced in animals treated daily with 6 mg/Kg RI-3 as compared to animals treated with vehicle only. Thus, RI-3 represents a promising lead for anti-metastatic drugs.