The Urokinase Receptor (uPAR) as a “Trojan Horse” in Targeted Cancer Therapy: Challenges and Opportunities

One of the largest challenges to the implementation of precision oncology is identifying and validating selective tumor-driving targets to enhance the therapeutic efficacy while limiting off-target toxicity. In this context, the urokinase-type plasminogen activator receptor (uPAR) has progressively emerged as a promising therapeutic target in the management of aggressive malignancies. By focalizing the plasminogen activation cascade and subsequent extracellular proteolysis on the cell surface of migrating cells, uPAR endows malignant cells with a high proteolytic and migratory potential to dissolve the restraining extracellular matrix (ECM) barriers and metastasize to distant sites. uPAR is also assumed to choreograph multiple other neoplastic stages via a complex molecular interplay with distinct cancer-associated signaling pathways. Accordingly, high uPAR expression is observed in virtually all human cancers and is frequently associated with poor patient prognosis and survival. The promising therapeutic potential unveiled by the pleiotropic nature of this receptor has prompted the development of distinct targeted intervention strategies. The present review will focus on recently emerged cytotoxic approaches emphasizing the novel technologies and related limits hindering their application in the clinical setting. Finally, future research directions and emerging opportunities in the field of uPAR targeting are also discussed.

Urokinase type plasminogen activator receptor (uPAR) and human epidermal growth factor receptor 2 (HER2) expression in metastasis of breast cancer

BACKGROUND: The plasminogen urokinase activation system consists of urokinase plasminogen activator (uPA), its receptor uPAR, and plasminogen activator inhibitor type 1 (PAI-1), which are considered to have a relationship with cancer aggressiveness. Several studies have found correlations between HER2 mRNA and uPAR in disseminated tumor cells (DTCs) in breast cancer patients. They are associated with a more aggressive primary tumor phenotype and recurrence/metastasis. OBJECTIVE: This study aims to determine the relationship between the expression of urokinase-type plasminogen activator receptor (uPAR) and human epidermal growth factor receptor type 2 (HER2) with the incidence of distant metastases in breast cancer. METHODS: This study was an observational study using a cross-sectional method and was conducted at Wahidin Sudirohusodo Hospital and the network. Immunohistochemical methods carry out examination of uPAR and HER2 expression from tissues of breast cancer patients. The relationship of uPAR, HER2 expression, and metastasis was tested with the Mann Whitney test. RESULTS: The study results found that the proportion of patients with metastasis was significantly higher in high uPAR expression compared to low uPAR (77.8% compared to 36.8%). The negative HER2 expression was significantly higher in the low uPAR expression than the high uPAR (78.9% compared to 33.3%). In comparison, the positive HER2 expression was significantly higher in the high uPAR expression than the low uPAR (66.7% compared to 21.1%). In positive HER2 expression, the mean percentage of uPAR expression was significantly higher in metastases than those without metastasis (72.7% compared to 42.1%). CONCLUSIONS: uPAR expression is associated with metastasis in HER2 positive breast cancer.

Ulinastatin Inhibits Nasopharyngeal Carcinoma Metastasis by Suppressing uPA/uPAR Signaling

Background: Distant metastasis is the main reason for treatment failure in patients with nasopharyngeal carcinoma (NPC). In this study, we evaluated the effect of ulinastatin (UTI) on NPC metastasis and investigated its underlying mechanism.Methods: Highly-metastatic NPC cell lines S18 and 5-8F were treated with UTI and the effect on cellular growth, migration, and invasion of NPC cells was determined using MTS and transwell assays. The luciferase-expressing S18 cells (S18-1C3) were injected into the left hind footpad of nude mice to establish a spontaneous footpad to popliteal lymph node (LN) metastasis model. The luciferase mRNA was measured by qPCR to calculate the metastatic inhibition rate. The UTI-related uPA, uPAR and the main molecular members of the JAT/STAT3 pathway were detected by qPCR and immunoblotting.Results: UTI inhibited the migration and invasion of S18 and 5-8F cells with no effect on cellular growth in vitro, and suppressed metastasis of S18 cells in vivo. uPAR expression was reduced from 24 to 48 hours after UTI treatment. The anti-metastasis effect of UTI partially relies on the suppression of uPA and uPAR.Conclusion: UTI partially inhibits NPC metastasis via downregulating the expression of uPA and uPAR.

CEA, EpCAM, αvβ6 and uPAR Expression in Rectal Cancer Patients with a Pathological Complete Response after Neoadjuvant Therapy

Rectal cancer patients with a complete response after neoadjuvant therapy can be monitored with a watch-and-wait strategy. However, regrowth rates indicate that identification of patients with a pathological complete response (pCR) remains challenging. Targeted near-infrared fluorescence endoscopy is a potential tool to improve response evaluation. Promising tumor targets include carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), integrin αvβ6, and urokinase-type plasminogen activator receptor (uPAR). To investigate the applicability of these targets, we analyzed protein expression by immunohistochemistry and quantified these by a total immunostaining score (TIS) in tissue of rectal cancer patients with a pCR. CEA, EpCAM, αvβ6, and uPAR expression in the diagnostic biopsy was high (TIS > 6) in, respectively, 100%, 100%, 33%, and 46% of cases. CEA and EpCAM expressions were significantly higher in the diagnostic biopsy compared with the corresponding tumor bed (p < 0.01). CEA, EpCAM, αvβ6, and uPAR expressions were low (TIS < 6) in the tumor bed in, respectively, 93%, 95%, 85%, and 62.5% of cases. Immunohistochemical evaluation shows that CEA and EpCAM could be suitable targets for response evaluation after neoadjuvant treatment, since expression of these targets in the primary tumor bed is low compared with the diagnostic biopsy and adjacent pre-existent rectal mucosa in more than 90% of patients with a pCR.

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.

Suppression of Urokinase-Type Plasminogen Activator Receptor by Docosahexaenoic Acid Mediated by Heme Oxygenase-1 in 12-O-Tetradecanoylphorbol-13-Acetate-Induced Human Endothelial Cells

Urokinase-type plasminogen activator receptor (uPAR) plays a crucial role in inflammation and tumor metastasis. Docosahexaenoic acid (DHA), a representative omega-3 polyunsaturated fatty acid, has been shown to exhibit anti-inflammatory and anti-tumor properties. However, the mechanism by which DHA negatively regulates uPAR expression is not yet understood. The aim of this study was to investigate the effect of DHA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced uPAR expression and potential role of heme oxygenase-1 (HO-1) in DHA-induced inhibition of uPAR in human endothelial ECV304 cells. Results showed that TPA induced uPAR expression in a time dependent manner, while DHA inhibited uPAR expression in a concentration-dependent manner. Moreover, treatment with DHA induced HO-1 expression in a time- and concentration-dependent manner. In addition, DHA-induced inhibition of uPAR expression and cell invasion in TPA-stimulated cells was reversed by si-HO-1 RNA. Induction of HO-1 by ferric protoporphyrin IX (FePP) inhibited TPA-induced uPAR expression, and this effect was abolished by treatment with the HO-1 inhibitor tin protoporphyrin IX (SnPP). Additionally, carbon monoxide, an HO-1 product, attenuated TPA-induced uPAR expression and cell invasion. Collectively, these data suggest a novel role of DHA-induced HO-1 in reducing uPAR expression and cell invasion in human endothelial ECV304 cells.

uPAR-expressing melanoma exosomes promote angiogenesis by VE-Cadherin, EGFR and uPAR overexpression and rise of ERK1,2 signaling in endothelial cells

Exosomes (Exos) have been reported to promote pre-metastatic niche formation, proliferation, angiogenesis and metastasis. We have investigated the role of uPAR in melanoma cell lines-derived Exos and their pro-angiogenic effects on human microvascular endothelial cells (HMVECs) and endothelial colony-forming cells (ECFCs). Melanoma Exos were isolated from conditioned media of A375 and M6 cells by differential centrifugation and filtration. Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle tracking analysis were performed to analyze dimension and concentration of Exos. The CRISPR–Cas 9 technology was exploited to obtain a robust uPAR knockout. uPAR is expressed in melanoma Exos that are internalized by HMVECs and ECFCs, enhancing VE-Cadherin, EGFR and uPAR expression in endothelial cells that undergo a complete angiogenic program, including proliferation, migration and tube formation. uPAR loss reduced the pro-angiogenic effects of melanoma Exos in vitro and in vivo by inhibition of VE-Cadherin, EGFR and uPAR expression and of ERK1,2 signaling in endothelial cells. A similar effect was obtained with a peptide that inhibits uPAR–EGFR interaction and with the EGFR inhibitor Gefitinib, which also inhibited melanoma Exos-dependent EGFR phosphorylation. This study suggests that uPAR is required for the pro-angiogenic activity of melanoma Exos. We propose the identification of uPAR-expressing Exos as a potentially useful biomarker for assessing pro-angiogenic propensity and eventually monitoring the response to treatment in metastatic melanoma patients.

Helicobacter pylori Colonization Drives Urokinase Receptor (uPAR) Expression in Murine Gastric Epithelium During Early Pathogenesis

(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.

Evaluation of the Diagnostic Potential of uPAR as a Biomarker in Renal Biopsies of Patients with FSGS

Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are primary glomerulopathies leading to proteinuria, known as podocytopathies, which share syndromic and morphological similarities. Morphological similarity occurs in cases of FSGS in which the sclerotic lesion was not sampled in renal biopsy, due to the focal nature of the disease. Differentiating these entities is very important, especially in cases of suspected FSGS but with sclerotic lesion not sampled, as they are diseases that apparently have different pathogenic mechanisms and prognosis. The difference in uPAR expression in situ among these two entities may be related to a distinct molecular mechanism involved in pathogenesis. Thus, finding biomarkers involved in the pathogenesis and that can also help in differential diagnosis is very relevant. The aim of this work was to evaluate the potential of urokinase-type plasminogen activator receptor (uPAR) as a biomarker in renal biopsies of patients with podocytopathies (n=38). Immunohistochemistry showed that FSGS (n=22) had increased uPAR expression in podocytes compared with both the MCD group (n=16; p=0.0368) and control group (n=21; p=0.0076). ROC curve (p=0.008) showed that this biomarker has 80.95% of specificity in biopsies of patients with FSGS. Therefore, uPAR presented a high specificity in cases of podocytopathies associated with sclerosis and it can be considered a potential biomarker for FSGS.