Circulating Biomarkers for Monitoring Chemotherapy-Induced Cardiotoxicity in Children.

Most commonly diagnosed cancer pathologies in the pediatric population comprise leukemias and cancers of the nervous system. The percentage of cancer survivors increased from approximatively 50% to 80% thanks to improvements in medical treatments and the introduction of new chemotherapies. However, as a consequence, heart disease has become the main cause of death in the children due to the cardiotoxicity induced by chemotherapy treatments. The use of different cardiovascular biomarkers, complementing data obtained from electrocardiogram, echocardiography cardiac imaging, and evaluation of clinical symptoms, is considered a routine in clinical diagnosis, prognosis, risk stratification, and differential diagnosis. Cardiac troponin and natriuretic peptides are the best-validated biomarkers broadly accepted in clinical practice for the diagnosis of acute coronary syndrome and heart failure, although many other biomarkers are used and several potential markers are currently under study and possibly will play a more prominent role in the future. Several studies have shown how the measurement of cardiac troponin (cTn) can be used for the early detection of heart damage in oncological patients treated with potentially cardiotoxic chemotherapeutic drugs. The advent of high sensitive methods (hs-cTnI or hs-cTnT) further improved the effectiveness of risk stratification and monitoring during treatment cycles.

Posttranscriptional Regulation of the Plasminogen Activation System by Non-Coding RNA in Cancer.

Various species of non-coding RNAs (ncRNAs) may act as functional molecules regulating diverse biological processes. In cancer cell biology, ncRNAs include RNAs that regulate the expression of oncogenes and tumor suppressor genes through various mechanisms. The urokinase (uPA)-mediated plasminogen activation system (PAS) includes uPA, its inhibitors PAI-1 and PAI-2 and its specific cellular receptor uPAR; their increased expression represents a negative prognostic factor in several cancers. Here, we will briefly describe the main uPA-mediated PAS components and ncRNA species; then, we will review more recent evidence of the roles that ncRNAs may play in regulating the expression and functions of uPA-mediated PAS components in cancer.

Identification of uPAR Variants Acting as ceRNAs in Leukaemia Cells.

The 3'untranslated region (3'UTR) of the urokinase (uPA) receptor (uPAR) mRNA can act as a competitive endogenous RNA (ceRNA) in acute myeloid leukaemia (AML) cells, promoting the expression of pro-tumoral targets, including uPAR. Here, we identified three variants of uPAR mRNA containing the 3'UTR, in KG1 and U937 leukaemia cells expressing low and high uPAR levels, respectively. Identified variants lack exon 5 (uPAR Δ5) or exon 6 (uPAR Δ6) or part of exon 6, exon 7 and part of 3'UTR (uPAR Δ6/7). uPAR Δ5 and uPAR Δ6 transcript levels were higher in U937 cells compared to KG1 cells. Both uPAR variants were expressed also in AML blasts, at higher levels as compared to CD34 hematopoietic cells from healthy donors. The presence of the 3'UTR conferred high instability to the uPAR Δ5 variant transcript, preventing its translation in protein. Overexpression of the uPAR Δ5-3'UTR variant regulated the expression of some pro-tumoral factors previously reported to be regulated by the 3'UTR of uPAR and increased KG1 cell adhesion, migration and proliferation. These results demonstrate the expression of uPAR mRNA variants containing the 3'UTR in AML cells and the ceRNA activity and the biological effects of the uPAR Δ5-3'UTR variant.

CRISPR/Cas9 uPAR Gene Knockout Results in Tumor Growth Inhibition, EGFR Downregulation and Induction of Stemness Markers in Melanoma and Colon Carcinoma Cell Lines.

uPAR is a globular protein, tethered to the cell membrane by a GPI-anchor involved in several cancer-related properties and its overexpression commonly correlates with poor prognosis and metastasis. We investigated the consequences of uPAR irreversible loss in human melanoma and colon cancer cell lines, knocking out its expression by CRISPR/Cas9. We analyzed through flow cytometry, western blotting and qPCR, the modulation of the most known cancer stem cells-associated genes and the EGFR while we observed the proliferation rate exploiting 2D and 3D cellular models. We also generated uPAR "rescue" expression cell lines as well as we promoted the expression of only its 3'UTR to demonstrate the involvement of uPAR mRNA in tumor progression. Knocking out PLAUR, uPAR-encoding gene, we observed an inhibited growth ratio unexpectedly coupled with a significant percentage of cells acquiring a stem-like phenotype. In vivo experiments demonstrated that uPAR loss completely abrogates tumorigenesis despite the gained stem-like profile. Nonetheless, we proved that the reintroduction of the 3'UTR of PLAUR gene was sufficient to restore the wild-type status validating the hypothesis that such a region may act as a "molecular sponge". In particular miR146a, by binding PLAUR 3' UTR region might be responsible for uPAR-dependent inhibition of EGFR expression.

The Urokinase Receptor: A Multifunctional Receptor in Cancer Cell Biology. Therapeutic Implications.

Proteolysis is a key event in several biological processes; proteolysis must be tightly controlled because its improper activation leads to dramatic consequences. Deregulation of proteolytic activity characterizes many pathological conditions, including cancer. The plasminogen activation (PA) system plays a key role in cancer; it includes the serine-protease urokinase-type plasminogen activator (uPA). uPA binds to a specific cellular receptor (uPAR), which concentrates proteolytic activity at the cell surface, thus supporting cell migration. However, a large body of evidence clearly showed uPAR involvement in the biology of cancer cell independently of the proteolytic activity of its ligand. In this review we will first describe this multifunctional molecule and then we will discuss how uPAR can sustain most of cancer hallmarks, which represent the biological capabilities acquired during the multistep cancer development. Finally, we will illustrate the main data available in the literature on uPAR as a cancer biomarker and a molecular target in anti-cancer therapy.

New Pieces in the Puzzle of uPAR Role in Cell Migration Mechanisms.

The urokinase (uPA) receptor (uPAR) plays a key role in cell migration. We previously showed that uPAR-negative HEK-293 cells efficiently migrate toward serum but, after uPAR ectopic expression, migrate only in a uPAR-dependent manner. In fact, migration of uPAR-transfected HEK-293 (uPAR-293) cells is impaired by anti-uPAR antibodies, without recovery of the uPAR-independent migration mechanisms formerly active. Prostate carcinoma PC3 cells, which express high endogenous uPAR levels, migrated only through a uPAR-dependent mechanism; in fact, the silencing of uPAR expression inhibited their migration. We hypothesize a crucial role of the uPAR glycosyl-phosphatidyl-inositol (GPI) tail, which promotes uPAR partitioning to lipid rafts, in uPAR-controlled cell migration. Here, we show that removal of the uPAR GPI-tail, or lipid rafts disruption by methyl-beta-cyclodextrin impairs migration of PC3 cells, incapable of uPAR-independent migration, whereas it restores uPAR-independent migration in uPAR-293 cells. We then show that, in PC3 cells, both uPAR signaling partners, ß1 integrins and receptors for formylated peptides (FPRs), partly associate with lipid rafts. Inhibition of their interaction with uPAR impairs this association and impairs cell migration. Interestingly, blocking uPAR association with FPRs also impairs ß1 integrin partitioning to lipid rafts, whereas blocking its association with ß1 integrins has no effect on FPRs partitioning. On these bases, we propose that uPAR controls cell migration by connecting ß1 integrins and FPRs and, through its GPI tail, by driving them into lipid rafts, thus promoting pro-migratory signals. uPAR-mediated partitioning of integrins to lipid rafts is strictly dependent on uPAR association with FPRs.

TLR4 Response to LPS Is Reinforced by Urokinase Receptor.

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Though uPAR role in inflammatory processes is documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. Downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NFκB, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR-/- mice demonstrated better survival, strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Mechanistically, GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

A novel oncogenic role for urokinase receptor in leukemia cells: molecular sponge for oncosuppressor microRNAs.

Urokinase receptor (uPAR) expression is up-regulated and represents a negative prognostic marker in most cancers. We previously reported that uPAR and CXCR4 can be regulated by common microRNAs in leukemia cells. Transcripts containing response elements for shared microRNAs in their 3'UTR may regulate their availability. We investigated uPAR 3'UTR capability to recruit microRNAs, thus regulating the expression of their targets. uPAR 3'UTR transfection in KG1 leukemia cells up-regulates the expression of endogenous uPAR. Transfection of uPAR 3'UTR, inserted downstream a reporter gene, increases uPAR expression and simultaneously down-regulates the reporter gene expression. Transfection of uPAR 3'UTR also increases CXCR4 expression; accordingly, uPAR silencing induces down-regulation of CXCR4 expression, through a mechanism involving Dicer, the endoribonuclease required for microRNA maturation. Transfection of uPAR 3'UTR also increases the expression of pro-tumoral factors and modulates cell adhesion and migration, consistently with the capability of uPAR3'UTR-recruited microRNAs to target several and different transcripts and, thus, functions. Finally, we found 3'UTR-containing variants of uPAR transcript in U937 leukemia cells, which show higher levels of uPAR expression as compared to KG1 cells, in which these variants are not detected. These results suggest that uPAR mRNA may recruit oncosuppressor microRNAs, allowing the expression of their targets.

N-Formyl Peptide Receptors Induce Radical Oxygen Production in Fibroblasts Derived From Systemic Sclerosis by Interacting With a Cleaved Form of Urokinase Receptor.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis, alteration in the microvasculature and immunologic abnormalities. It has been hypothesized that an abnormal redox state could regulate the persistent fibrotic phenotype in SSc patients. N-Formyl peptide receptors (FPRs) are chemotactic receptors overexpressed in fibroblasts derived from SSc patients. In this study, we demonstrated that stimulation of FPRs promotes the generation of reactive oxygen species (ROS) in skin fibroblasts. In fibroblast cells, ROS production was due to FPRs interaction with the urokinase receptor (uPAR) and to ß1 integrin engagement. FPRs cross-talk with uPAR and integrins led to Rac1 and ERKs activation. FPRs stimulation increased gp91phox and p67phox expression as well as the direct interaction between GTP-Rac1 and p67phox, thus promoting assembly and activation of the NADPH oxidase complex. FPRs functions occur through interaction with a specific domain of uPAR (residues 88SRSRY92) that can be exposed on the cell membrane by protease-mediated receptor cleavage. Immunohistochemistry analysis with a specific anti-SRSRY antibody showed increased expression of uPAR in a cleaved form, which exposes the SRSRY sequence at its N-terminus (DIIDIII-uPAR88-92) in skin biopsies from SSc patients. As expected by the increased expression of both FPRs and DII-DIII-uPAR88-92, fibroblasts derived from SSc patients showed a significantly increase in ROS generation both at a basal level than after FPRs stimulation, as compared to fibroblasts from normal subjects. C37, a small molecule blocking the interaction between FPRs and uPAR, and selumetinib, a clinically approved MAPKK/ERK inhibitor, significantly inhibited FPRs-mediated ROS production in fibroblasts derived from SSc patients. Thus, FPRs, through the interaction with the uPA/uPAR system, can induce ROS generation in fibroblasts by activating the NADPH oxidase, playing a role in the alteration of the redox state observed in SSc.

Recent Advances in the Function of the 67 kDa Laminin Receptor and its Targeting for Personalized Therapy in Cancer.

The 67 kDa high affinity laminin receptor (67LR) is a non-integrin cell surface receptor for laminin, the major component of basement membranes. Interactions between 67LR and laminin play a major role in mediating cell adhesion, migration, proliferation and survival. 67LR derives from homo- or hetero-dimerization of a 37 kDa cytosolic precursor (37LRP), most probably by fatty acid acylation. Interestingly, 37LRP, also called p40 or OFA/iLR (oncofetal antigen/immature laminin receptor), is a multifunctional protein with a dual activity in the cytoplasm and in the nucleus. In the cytoplasm, 37LRP it is associated with the 40S subunit of ribosome, playing a critical role in protein translation and ribosome biogenesis while in the nucleus it is tightly associated with nuclear structures, and bound to components of the cytoskeleton, such as tubulin and actin. 67LR is mainly localized in the cell membrane, concentrated in lipid rafts. Acting as a receptor for laminin is not the only function of 67LR; indeed, it also acts as a receptor for viruses, bacteria and prions. 67LR expression is increased in neoplastic cells and correlates with an enhanced invasive and metastatic potential. The primary function of 67LR in cancer is to promote tumor cell adhesion to basement membranes, the first step in the invasion-metastasis cascade. Thus, 67LR is overexpressed in neoplastic cells as compared to their normal counterparts and its overexpression is considered a molecular marker of metastatic aggressiveness in cancer of many tissues, including breast, lung, ovary, prostate, stomach, thyroid and also in leukemia and lymphoma. Thus, inhibiting 67LR binding to laminin could be a feasible approach to block cancer progression. Here, we review the current understanding of the structure and function of this molecule, highlighting its role in cancer invasion and metastasis and reviewing the various therapeutic options targeting this receptor that could have a promising future application.

Urokinase type plasminogen activator receptor (uPAR) as a new therapeutic target in cancer.

The urokinase (uPA)-type plasminogen activator receptor (uPAR) is a GPI-anchored receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. uPAR also regulates cell adhesion, migration and proliferation, protects from apoptosis and contributes to epithelial mesenchymal transition (EMT), independently of uPA enzymatic activity. Indeed, uPAR interacts with beta1, beta2 and beta3 integrins, thus regulating their activities. uPAR cross-talks with receptor tyrosine kinases through integrins and regulates cancer cell dormancy, proliferation and angiogenesis. Moreover, uPAR mediates uPA-dependent cell migration and chemotaxis induced by fMet-Leu-Phe (fMLF), through its association with fMLF-receptors (fMLF-Rs). Further, uPAR is an adhesion receptor because it binds vitronectin (VN), a component of provisional extracellular matrix. High uPAR expression predicts for more aggressive disease in several cancer types for its ability to increase invasion and metastasis. In fact, uPAR has been hypothesized to be the link between tumor cell dormancy and proliferation that usually precedes the onset of metastasis. Thus, inhibiting uPAR could be a feasible approach to affect tumor growth and metastasis. Here, we review the more recent advances in the development of uPAR-targeted anti-cancer therapeutic agents suitable for further optimization or ready for the evaluation in early clinical trials.

Involvement of urokinase receptor in the cross-talk between human hematopoietic stem cells and bone marrow microenvironment.

Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and can be induced to mobilize into the circulation for transplantation. Homing and lodgement into BM of transplanted HSCs are the first critical steps in their engraftment and involve multiple interactions between HSCs and the BM microenvironment.uPAR is a three domain receptor (DIDIIDIII) which binds urokinase, vitronectin, integrins. uPAR can be cleaved and shed from the cell surface generating full-length and cleaved soluble forms (suPAR and DIIDIII-suPAR). DIIDIII-suPAR can bind fMLF receptors through the SRSRY sequence (residues 88-92).We previously reported the involvement of soluble uPAR in HSC mobilization. We now investigate its possible role in HSC homing and engraftment.We show similar levels of circulating full-length suPAR in healthy donors and in acute myeloid leukemia (AML) patients before and after the pre-transplant conditioning regimen. By contrast, levels of circulating DIIDIII-suPAR in AML patients are higher as compared to controls and significantly decrease after the conditioning.We found that suPAR and uPAR84-95, a uPAR-derived peptide which mimics active DIIDIII-suPAR, induce a significant increase in Long Term Culture (LTC)-Initiating Cells (ICs) and in the release of clonogenic progenitors from LTCs of CD34+ HSCs. Further, suPAR increases adhesion and survival of CD34+ KG1 AML cells, whereas uPAR84-95 increases their proliferation.Thus, circulating DIIDIII-suPAR, strongly increased in HSC mobilization, is indeed down-regulated by pre-transplant conditioning, probably to favour HSC homing. BM full-length suPAR and DIIDIII-suPAR may be involved in HSC lodgement within the BM by contributing to a suitable microenvironment.

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells.

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3'untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo.

Discovery of new small molecules inhibiting 67 kDa laminin receptor interaction with laminin and cancer cell invasion.

The 67 kDa laminin receptor (67LR) is a non-integrin receptor for laminin (LM) that derives from a 37 kDa precursor (37LRP). 67LR expression is increased in neoplastic cells and correlates with an enhanced invasive and metastatic potential. We used structure-based virtual screening (SB-VS) to search for 67LR inhibitory small molecules, by focusing on a 37LRP sequence, the peptide G, able to specifically bind LM. Forty-six compounds were identified and tested on HEK-293 cells transfected with 37LRP/67LR (LR-293 cells). One compound, NSC47924, selectively inhibited LR-293 cell adhesion to LM with IC50 and Ki values of 19.35 and 2.45 µmol/L. NSC47924 engaged residues W176 and L173 of peptide G, critical for specific LM binding. Indeed, NSC47924 inhibited in vitro binding of recombinant 37LRP to both LM and its YIGSR fragment. NSC47924 also impaired LR-293 cell migration to LM and cell invasion. A subsequent hierarchical similarity search with NSC47924 led to the identification of additional four compounds inhibiting LR-293 cell binding to LM: NSC47923, NSC48478, NSC48861, and NSC48869, with IC50 values of 1.99, 1.76, 3.4, and 4.0 µmol/L, respectively, and able to block in vitro cancer cell invasion. These compounds are promising scaffolds for future drug design and discovery efforts in cancer progression.

Upregulation of the N-formyl Peptide receptors in scleroderma fibroblasts fosters the switch to myofibroblasts.

Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis. N-Formyl peptide (fMLF) receptors (FPRs) are chemotactic receptors involved in inflammation. Three FPRs have been identified: FPR1, FPR2, and FPR3. We have examined, by RT-PCR, Western blot and immunohistochemistry, FPRs expression in skin fibroblasts from 10 normal subjects and 10 SSc patients, showing increased expression in SSc fibroblasts. Several functions of FPRs occur through the interaction with a region of the urokinase-type plasminogen activator receptor (uPAR88-92), able to interact with FPRs and to mediate urokinase (uPA) or fMLF-dependent cell migration. Soluble uPAR84-95 peptide can act as a direct ligand of FPRs. Furthermore, uPA or its aminoterminal fragment (ATF) can promote the exposure of the uPAR88-92 region. The WKYMVm peptide is a FPRs pan-agonist. We investigated the functional effects of these agonists on normal and SSc fibroblasts. ATF, uPAR84-95, and WKYMVm regulated adhesion, migration, and proliferation of normal fibroblasts. Despite FPR overexpression, the response of SSc fibroblasts to the same agonists was greatly reduced, except for the proliferative response to ATF. SSc fibroblasts showed increased α-smooth muscle actin expression and improved capability to induce wound closure. Indeed, they overexpressed a cleaved uPAR form, exposing the uPAR88-92 region, and vitronectin, both involved in fibrosis and in the fibroblast-to-myofibroblast transition. FPR stimulation promoted α-smooth muscle actin expression in normal fibroblasts as well as motility, matrix deposition, αvß5 integrin expression, and radical oxygen species generation in normal and SSc fibroblasts. This study provides evidence that FPRs may play a role in fibrosis and in the fibroblast-to-myofibroblast transition.

Urokinase receptor promotes ovarian cancer cell dissemination through its 84-95 sequence.

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 urokinase receptor takes control of cell migration by recruiting integrins and FPR1 on the cell surface.

The receptor (uPAR) of the urokinase-type plasminogen activator (uPA) is crucial in cell migration since it concentrates uPA proteolytic activity at the cell surface, binds vitronectin and associates to integrins. uPAR cross-talk with receptors for the formylated peptide fMLF (fMLF-Rs) has been reported; however, cell-surface uPAR association to fMLF-Rs on the cell membrane has never been explored in detail. We now show that uPAR co-localizes at the cell-surface and co-immunoprecipitates with the high-affinity fMLF-R, FPR1, in uPAR-transfected HEK-293 (uPAR-293) cells. uPAR/ß1 integrin and FPR1/ß1 integrin co-localization was also observed. Serum or the WKYMVm peptide (W Pep), a FPR1 ligand, strongly increased all observed co-localizations in uPAR-293 cells, including FPR1/ß1 integrin co-localization. By contrast, a low FPR1/ß1 integrin co-localization was observed in uPAR-negative vector-transfected HEK-293 (V-293) cells, that was not increased by serum or W Pep stimulations. The role of uPAR interactions in cell migration was then explored. Both uPAR-293 and V-293 control cells efficiently migrated toward serum or purified EGF. However, cell treatments impairing uPAR interactions with fMLF-Rs or integrins, or inhibiting specific cell-signaling mediators abrogated uPAR-293 cell migration, without exerting any effect on V-293 control cells. Accordingly, uPAR depletion by a uPAR-targeting siRNA or uPAR blocking with an anti-uPAR polyclonal antibody in cells constitutively expressing high uPAR levels totally impaired their migration toward serum. Altogether, these results suggest that both uPAR-positive and uPAR-negative cells are able to migrate toward serum; however, uPAR expression renders cell migration totally and irreversibly uPAR-dependent, since it is completely inhibited by uPAR blocking. We propose that uPAR takes control of cell migration by recruiting fMLF-Rs and ß1 integrins, thus promoting their co-localization at the cell-surface and driving pro-migratory signaling pathways.

Role of uPA/uPAR in the modulation of angiogenesis.

Blood vessels connect all districts of the body and allow blood oxygen and nutrients to reach every cell in the organism. Dysregulation of blood vessel formation or functionality is the origin of a large number of diseases. During new vessel formation, endothelial cells degrade their basement membrane, migrate into the interstitial matrix and proliferate. Migrating endothelial cells need to be polarized, to focus at their leading edge the proteolytic machinery, which is essential for extracellular matrix degradation; thus, proteases and their receptors play a crucial role in angiogenesis. The urokinase-mediated plasminogen activation system is a complex system of serine proteases strongly involved in angiogenesis. The plasminogen activation system includes plasminogen/plasmin, activators, inhibitors and cell receptors. In the last decades, a large body of evidence has clearly indicated that the role of this system is not limited to extracellular matrix proteolysis but can contribute to all phases of the angiogenic process.

Discovery of new small molecules targeting the vitronectin-binding site of the urokinase receptor that block cancer cell invasion.

Besides focusing urokinase (uPA) proteolytic activity on the cell membrane, the uPA receptor (uPAR) is able to bind vitronectin, via a direct binding site. Furthermore, uPAR interacts with other cell surface receptors, such as integrins, receptor tyrosine kinases, and chemotaxis receptors, triggering cell-signaling pathways that promote tumor progression. The ability of uPAR to coordinate binding and degradation of extracellular matrix (ECM) and cell signaling makes it an attractive therapeutic target in cancer. We used structure-based virtual screening (SB-VS) to search for small molecules targeting the uPAR-binding site for vitronectin. Forty-one compounds were identified and tested on uPAR-negative HEK-293 epithelial cells transfected with uPAR (uPAR-293 cells), using the parental cell line transfected with the empty vector (V-293 cells) as a control. Compounds 6 and 37 selectively inhibited uPAR-293 cell adhesion to vitronectin and the resulting changes in cell morphology and signal transduction, without exerting any effect on V-293 cells. Compounds 6 and 37 inhibited uPAR-293 cell binding to vitronectin with IC50 values of 3.6 and 1.2 µmol/L, respectively. Compounds 6 and 37 targeted S88 and R91, key residues for uPAR binding to vitronectin but also for uPAR interaction with the fMLF family of chemotaxis receptors (fMLF-Rs). As a consequence, compounds 6 and 37 impaired uPAR-293 cell migration toward fetal calf serum (FCS), uPA, and fMLF, likely by inhibiting the interaction between uPAR and FPR1, the high affinity fMLF-R. Both compounds blocked in vitro ECM invasion of several cancer cell types, thus representing new promising leads for pharmaceuticals in cancer.

uPAR regulates pericellular proteolysis through a mechanism involving integrins and fMLF-receptors.

The expression of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) can be regulated by several hormones, cytokines, and tumour promoters. uPAR is a glycosyl-phosphatidyl inositol (GPI)-linked cell-surface protein; however, it is capable to transduce signals inside the cell by interacting with other cell-surface proteins, such as integrins and G-protein coupled (GPC) receptors. We previously reported that uPAR cell-surface expression can be positively regulated by its ligand, uPA, independently of its proteolytic activity. We now demonstrate that uPAR overexpression induces or increases uPA secretion both in uPAR-negative and in uPAR-expressing cells. Accordingly, uPAR depletion impairs uPA expression in cells which constitutively express both uPA and its receptor. uPAR exerts its regulatory effect through the activation of the ERK mitogen-activated protein kinases (MAPKs), whereas the p-38 MAPK is not involved. Overexpression of truncated forms of uPAR, lacking the N-terminal domain (DI) and not able to interact with membrane co-receptors, failed to increase uPA expression. Inhibition of uPAR-integrin interaction by the specific P-25 peptide, as well as Gi-protein inhibition by cholera pertussin toxin or depletion of the GPC receptors for fMLF (fMLF-Rs) also impaired uPAR capability to regulate uPA expression. These findings demonstrate that uPAR, whose expression is regulated by uPA, can, in turn, regulate uPA expression through a mechanism involving its functional interaction with integrins and fMLF-Rs.