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.

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.

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.

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.

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.

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.

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.