Therapeutic strategies targeting uPAR potentiate anti-PD-1 efficacy in diffuse-type gastric cancer.

The diffuse-type gastric cancer (DGC) is a subtype of gastric cancer (GC) associated with low HER2 positivity rate and insensitivity to chemotherapy and immune checkpoint inhibitors. Here, we identify urokinase-type plasminogen activator receptor (uPAR) as a potential therapeutic target for DGC. We have developed a novel anti-uPAR monoclonal antibody, which targets the domains II and III of uPAR and blocks the binding of urokinase-type plasminogen activator to uPAR. We show that the combination of anti-uPAR and anti-Programmed cell death protein 1 (PD-1) remarkably inhibits tumor growth and prolongs survival via multiple mechanisms, using cell line-derived xenograft and patient-derived xenograft mouse models. Furthermore, uPAR chimeric antigen receptor-expressing T cells based on the novel anti-uPAR effectively kill DGC patient-derived organoids and exhibit impressive survival benefit in the established mouse models, especially when combined with PD-1 blockade therapy. Our study provides a new possibility of DGC treatment by targeting uPAR in a unique manner.

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma.

With a 5-year recurrence rate of 30-78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. METHODS: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. RESULTS: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. CONCLUSIONS: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.

Development of inhibitors for uPAR: blocking the interaction of uPAR with its partners.

Urokinase-type plasminogen activator receptor (uPAR) mediates a multitude of biological activities, has key roles in several clinical indications, including malignancies and inflammation, and, thus, has attracted intensive research over the past few decades. The pleiotropic functions of uPAR can be attributed to its interaction with an array of partners. Many inhibitors have been developed to intervene with the interaction of uPAR with these partners. Here, we review the development of these classes of uPAR inhibitor and their inhibitory mechanisms to promote the translation of these inhibitors to clinical applications.

Small-Molecule Inhibition of the uPAR ⋠uPA Interaction by Conformational Selection.

The urokinase receptor (uPAR) is a cell surface receptor that binds to the serine protease urokinase-type plasminogen activator (uPA) with high affinity. This interaction is beneficial for extravascular fibrin clearance, but it has also been associated with a broad range of pathological conditions including cancer, atherosclerosis, and kidney disease. Here, starting with a small molecule that we previously discovered by virtual screening and cheminformatics analysis, we design and synthesize several derivatives that were tested for binding and inhibition of the uPAR ⋅ uPA interaction. To confirm the binding site and establish a binding mode of the compounds, we carried out biophysical studies using uPAR mutants, among them uPARH47C-N259C , a mutant previously developed to mimic the structure of uPA-bound uPAR. Remarkably, a substantial increase in potency is observed for inhibition of uPARH47C-N259C binding to uPA compared to wild-type uPAR, consistent with our use of the structure of uPAR in its uPA-bound state to design small-molecule uPAR ⋅ uPA antagonists. Combined with the biophysical studies, molecular docking followed by extensive explicit-solvent molecular dynamics simulations and MM-GBSA free energy calculations yielded the most favorable binding pose of the compound. Collectively, these results suggest that potent inhibition of uPAR binding to uPA with small molecules will likely only be achieved by developing small molecules that exhibit high-affinity to solution apo structures of uPAR, rather than uPA-bound structures of the receptor.

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.

Bispecific Targeting of EGFR and Urokinase Receptor (uPAR) Using Ligand-Targeted Toxins in Solid Tumors.

Ligand-targeted toxins (LTTs) are bioengineered molecules which are composed of a targeting component linked to a toxin that induces cell death once the LTT binds its target. Bispecific targeting allows for the simultaneous targeting of two receptors. In this review, we mostly focus on the epidermal growth factor receptor (EGFR) as a target. We discuss the development and testing of a bispecific LTT targeting EGFR and urokinase-type plasminogen activator receptor (uPAR) as two attractive targets implicated in tumor growth and in the regulation of the tumor microvasculature in solid tumors. In vitro and mouse xenograft studies have shown that EGFR-targeted bispecific angiotoxin (eBAT) is effective against human solid tumors. Canine studies have shown that eBAT is both safe and effective against canine hemangiosarcoma, which is physiologically similar to human angiosarcoma. Finding the appropriate dosing strategy and sequencing of eBAT administration, in combination with other therapeutics, are among important factors for future directions. Together, the data indicate that eBAT targets cancer stem cells, it may have a role in inhibiting human tumor vasculature, and its bispecific conformation may have a role in reducing toxicity in comparative oncologic trials in dogs.

A uPAR targeted nanoplatform with an NIR laser-responsive drug release property for tri-modal imaging and synergistic photothermal-chemotherapy of triple-negative breast cancer.

In the present work, an iridium (Ir) complex loaded theranostic nanoplatform was designed for high-efficiency triple-negative breast cancer (TNBC) therapy. For this purpose, the Ir complex was firstly loaded on a photothermal agent of gold nanostars (GNS) by simply mixing followed by functionalization using a urokinase-type plasminogen activator receptor (uPAR) targeted polyetherimide-AE105 peptide conjugate (P-AE105) with the formation of GNS@Ir@P-AE105. It was demonstrated that the resultant GNS@Ir@P-AE105 was a multifunctional nanoplatform with advantages of (1) NIR laser controlled release of the Ir complex; (2) precise delivery of the Ir complex to TNBC cells; (3) excellent photothermal (PT)/photoacoustic (PA)/X-ray computed tomography (CT) tri-modal imaging ability; and (4) a synergistic photothermal-chemotherapeutic effect. An in-depth investigation of the mechanism revealed that binding forces of the Ir complex-GNS and P-AE105-GNS were significantly diminished upon NIR laser irradiation, which conferred an NIR laser-responsive Ir complex release property to the nanoplatform. Moreover, the nanoplatform exerted high efficiency anti-TNBC effects via a ROS-induced p53 apoptotic pathway. Specifically, combinational photothermal-chemotherapeutic treatments stimulated intracellular ROS generation, which significantly up-regulated apoptotic-relative p53 gene expression either by causing severe DNA damage or inducing an arrest effect on the sub-G1 phase of the cell cycle. Taken together, our work provides a novel theranostic nanoplatform for efficient and simultaneous diagnosis and therapy of TNBC.

The uPAR System as a Potential Therapeutic Target in the Diseased Eye.

Dysregulation of vascular networks is characteristic of eye diseases associated with retinal cell degeneration and visual loss. Visual impairment is also the consequence of photoreceptor degeneration in inherited eye diseases with a major inflammatory component, but without angiogenic profile. Among the pathways with high impact on vascular/degenerative diseases of the eye, a central role is played by a system formed by the ligand urokinase-type plasminogen activator (uPA) and its receptor uPAR. The uPAR system, although extensively investigated in tumors, still remains a key issue in vascular diseases of the eye and even less studied in inherited retinal pathologies such as retinitis pigmantosa (RP). Its spectrum of action has been extended far beyond a classical pro-angiogenic function and has emerged as a central actor in inflammation. Preclinical studies in more prevalent eye diseases characterized by neovascular formation, as in retinopathy of prematurity, wet macular degeneration and rubeosis iridis or vasopermeability excess as in diabetic retinopathy, suggest a critical role of increased uPAR signaling indicating the potentiality of its modulation to counteract neovessel formation and microvascular dysfunction. The additional observation that the uPAR system plays a major role in RP by limiting the inflammatory cascade triggered by rod degeneration rises further questions about its role in the diseased eye.

Therapeutic Targeting of the Notch Pathway in Glioblastoma Multiforme.

BACKGROUND: Glioblastoma (GBM) is the most common and deadly form of brain tumor. After standard treatment of resection, radiotherapy, and chemotherapy, the 5-year survival is <5%. In recent years, research has uncovered several potential targets within the Notch signaling pathway, which may lead to improved patient outcomes. METHODS: A literature search was performed for articles containing the terms "Glioblastoma" and "Receptors, Notch" between 2003 and July 2015. Of the 62 articles retrieved, 46 met our criteria and were included in our review. Nine articles were identified from other sources and were subsequently included, leaving 55 articles reviewed. RESULTS: Of the 55 articles reviewed, 47 used established human GBM cell lines. Seventeen articles used human GBM surgical samples. Forty-five of 48 articles that assessed Notch activity showed increased expression in GBM cell lines. Targeting the Notch pathway was carried out through Notch knockdown and overexpression and targeting δ-like ligand, Jagged, γ-secretase, ADAM10, ADAM17, and Mastermindlike protein 1. Arsenic trioxide, microRNAs, and several other compounds were shown to have an effect on the Notch pathway in GBM. Notch activity in GBM was also shown to be associated with hypoxia and certain cancer-related molecular pathways such as PI3K/AKT/mTOR and ERK/MAPK. Most articles concluded that Notch activity amplifies malignant characteristics in GBM and targeting this pathway can bring about amelioration of these effects. CONCLUSIONS: Recent literature suggests targeting the Notch pathway has great potential for future therapies for GBM.

Resveratrol Targets Urokinase-Type Plasminogen Activator Receptor Expression to Overcome Cetuximab-Resistance in Oral Squamous Cell Carcinoma.

Drug resistance to anti-cancer agents is a major concern regarding the successful treatment of malignant tumors. Recent studies have suggested that acquired resistance to anti-epidermal growth factor receptor (EGFR) therapies such as cetuximab are in part caused by genetic alterations in patients with oral squamous cell carcinoma (OSCC). However, the molecular mechanisms employed by other complementary pathways that govern resistance remain unclear. In the current study, we performed gene expression profiling combined with extensive molecular validation to explore alternative mechanisms driving cetuximab-resistance in OSCC cells. Among the genes identified, we discovered that a urokinase-type plasminogen activator receptor (uPAR)/integrin ß1/Src/FAK signal circuit converges to regulate ERK1/2 phosphorylation and this pathway drives cetuximab-resistance in the absence of EGFR overexpression or acquired EGFR activating mutations. Notably, the polyphenolic phytoalexin resveratrol, inhibited uPAR expression and consequently the signaling molecules ERK1/2 downstream of EGFR thus revealing additive effects on promoting OSCC cetuximab-sensitivity in vitro and in vivo. The current findings indicate that uPAR expression plays a critical role in acquired cetuximab resistance of OSCC and that combination therapy with resveratrol may provide an attractive means for treating these patients.

Targeting LC3 and Beclin-1 autophagy genes suppresses proliferation, survival, migration and invasion by inhibition of Cyclin-D1 and uPAR/Integrin ß1/ Src signaling in triple negative breast cancer cells.

Autophagy is a catabolic process for degrading dysfunctional proteins and organelles, and closely associated with cancer cell survival under therapeutic, metabolic stress, hypoxia, starvation and lack of growth factors, contributing to resistance to therapies. However, the role of autophagy in breast cancer cells is not well understood. In the present study, we investigated the role of autophagy in highly aggressive and metastatic triple negative breast cancer (TNBC) and non-metastatic breast cancer cells and demonstrated that the knockdown of autophagy-related genes (LC3 and Beclin-1) inhibited autophagy and significantly suppressed cell proliferation, colony formation, migration/invasion and induced apoptosis in MDA-MB-231 and BT-549 TNBC cells. Knockdown of LC3 and Beclin-1 led to inhibition of multiple proto-oncogenic signaling pathways, including cyclin D1, uPAR/integrin-ß1/Src, and PARP1. In conclusion, our study suggests that LC3 and Beclin-1 are required for cell proliferation, survival, migration and invasion, and may contribute to tumor growth and progression of highly aggressive and metastatic TNBC cells and therapeutic targeting of autophagy genes may be a potential therapeutic strategy for TNBC in breast cancer.

EGF-induced urokinase plasminogen activator receptor promotes epithelial to mesenchymal transition in human gastric cancer cells.

Epidermal growth factor (EGF) signaling has been shown to induce epithelial to mesenchymal transition (EMT) in many types of cancer cells. However, the molecular mechanism of EGF-induced EMT in gastric cancer remains largely unknown. In the present study, we found that human gastric cancer cell lines SGC-7901 and BGC-823 underwent EMT phenotypic changes upon exposure to EGF. The induction of EMT was consistent with aggressive characteristics such as increased cell migration, invasion and clonogenic growth. Additionally, EGF stimulation also led to the upregulation of urokinase plasminogen activator receptor (uPAR) both at mRNA and protein levels. Knockdown of uPAR by siRNA significantly attenuated EMT induction by EGF in SGC-7901 and BGC-823 cells. Furthermore, EGF increased ERK1/2 activity and blocking ERK1/2 signaling with its inhibitor, U0126, markedly inhibited EGF-induced uPAR expression and consequently EMT. Collectively, the present study demonstrated that EGF induced aggressiveness of gastric cancer cells by activating EMT, which involved the activation of the ERK1/2 pathway and, subsequently, uPAR expression.

Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells.

The urokinase receptor (uPAR) is a glycosylphosphatidylinositol (GPI)-anchored protein that promotes tissue remodeling, tumor cell adhesion, migration and invasion. uPAR mediates degradation of the extracellular matrix through protease recruitment and enhances cell adhesion, migration and signaling through vitronectin binding and interactions with integrins. Full-length uPAR is released from the cell surface, but the mechanism and significance of uPAR shedding remain obscure. Here we identify transmembrane glycerophosphodiesterase GDE3 as a GPI-specific phospholipase C that cleaves and releases uPAR with consequent loss of function, whereas its homologue GDE2 fails to attack uPAR. GDE3 overexpression depletes uPAR from distinct basolateral membrane domains in breast cancer cells, resulting in a less transformed phenotype, it slows tumor growth in a xenograft model and correlates with prolonged survival in patients. Our results establish GDE3 as a negative regulator of the uPAR signaling network and, furthermore, highlight GPI-anchor hydrolysis as a cell-intrinsic mechanism to alter cell behavior.

Urokinase-type plasminogen activator receptor (uPAR) expression enhances invasion and metastasis in RAS mutated tumors.

The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor that focuses urokinase (uPA) proteolytic activity on the cell surface. Its expression is increased in many human cancers, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and correlates with a poor prognosis and early invasion and metastasis. uPAR is able to control, through a cross-talk with tyrosine kinase receptors, the shift between tumor dormancy and proliferation, that usually precedes metastasis formation. Therefore, we investigated the role of uPAR expression in RAS mutated NSCLC and CRC cells. In this study we provided evidence, for the first time, that RAS mutational condition is functionally correlated to uPAR overexpression in NSCLC and CRC cancer cell lines and patient-derived tissue samples. Moreover, oncogenic features related to uPAR overexpression in RAS mutated NSCLC and CRC, such as adhesion, migration and metastatic process may be targeted, in vitro and in vivo, by new anti-uPAR small molecules, specific inhibitors of uPAR-vitronectin interaction. Therefore, anti-uPAR drugs could represent an effective pharmacological strategy for NSCLC and CRC patients carrying RAS mutations.

Retro-inverso Urokinase Receptor Antagonists for the Treatment of Metastatic Sarcomas.

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.

Safe and Effective Sarcoma Therapy through Bispecific Targeting of EGFR and uPAR.

Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo "ontarget" companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I-II study of 23 dogs with spontaneous, stage I-II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 µg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956-65. ©2017 AACR.

Growth inhibition of MDA-MB-231 cell line by peptides designed based on uPA.

Interaction between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) plays an important role in the progression of numerous cancer types including breast cancer by promoting tumor initiating, proliferation, invasion and metastasis. Hence, disruption of this interaction inhibits their downstream cascades and subsequently tumor growth. For this, we created two series of 8 and 10 amino acids linear peptides, derived from uPA binding region to target uPAR and studied the inhibition of proliferation in MDA-MB-231 cell line. Results revealed that all of the 10-mer peptides inhibited breast cancer cell proliferation significantly with maximum 40% inhibition of 103 peptides. Meanwhile, none of the 8-mer peptides showed significant toxicity. Current results indicate that the linear 10-mer peptides which mimic a small part of a sequence of a binding domain of uPA to uPAR could be exploited to design a novel class of anti-cancer agents.

Dual functionality of phosphonic-acid-appended phthalocyanines: inhibitors of urokinase plasminogen activator and anticancer photodynamic agents.

Phthalocyanines (Pcs) bearing phosphonic acid groups at the periphery exhibit a potential photodynamic effect to induce phototoxicity on human bladder cancer epithelial cells (UM-UC-3). In vitro photophysical and biological studies show high intrinsic ability to inhibit the activity of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9).

Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.