Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells.

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3ß) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3ß might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3ß using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3ß levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3ß inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

PET in vivo generators 134Ce and 140Nd on an internalizing monoclonal antibody probe.

The in vivo-generator radionuclides 140Nd (t1/2 = 3.4 d) and 134Ce (t1/2 = 3.2 d) were used to trace a urokinase-type plasminogen activator (uPA)-targeting mouse monoclonal antibody, ATN-291, in U87 MG xenograft tumor-bearing mice. ATN-291 is known to internalize on the uPA/uPA-receptor pair, making it an appropriate targeting vector for investigating the fate of in vivo generator daughters on internalizing probes. Ante-mortem and post-mortem PET imaging at 120 h post-injection gave no indication of redistribution of the positron emitting daughter nuclides 134La and 140Pr from tumor tissue (p > 0.5). The lack of redistribution indicates that the parent radionuclides 134Ce and 140Nd could be considered as long-lived PET-diagnostic matches to therapeutic radionuclides like 177Lu, 161Tb and 225Ac when internalizing bioconjugates are employed.

Binding of high molecular weight kininogen to human endothelial cells is mediated via a site within domains 2 and 3 of the urokinase receptor.

The urokinase receptor (uPAR) binds urokinase-type plasminogen activator (u-PA) through specific interactions with uPAR domain 1, and vitronectin through interactions with a site within uPAR domains 2 and 3. These interactions promote the expression of cell surface plasminogen activator activity and cellular adhesion to vitronectin, respectively. High molecular weight kininogen (HK) also stimulates the expression of cell surface plasminogen activator activity through its ability to serve as an acquired receptor for prekallikrein, which, after its activation, may directly activate prourokinase. Here, we report that binding of the cleaved form of HK (HKa) to human umbilical vein endothelial cells (HUVEC) is mediated through zinc-dependent interactions with uPAR. These occur through a site within uPAR domains 2 and 3, since the binding of 125I-HKa to HUVEC is inhibited by vitronectin, anti-uPAR domain 2 and 3 antibodies and soluble, recombinant uPAR (suPAR), but not by antibody 7E3, which recognizes the beta chain of the endothelial cell vitronectin receptor (integrin alphavbeta3), or fibrinogen, another alphavbeta3 ligand. We also demonstrate the formation of a zinc-dependent complex between suPAR and HKa. Interactions of HKa with endothelial cell uPAR may underlie its ability to promote kallikrein-dependent cell surface plasmin generation, and also explain, in part, its antiadhesive properties.

Preclinical uPAR-targeted multimodal imaging of locoregional oral cancer.

OBJECTIVES: Establishing adequate resection margins and lymphatic mapping are crucial for the prognosis of oral cancer patients. Novel targeted imaging modalities are needed, enabling pre- and intraoperative detection of tumour cells, in combination with improved post-surgical examination by the pathologist. The urokinase-receptor (uPAR) is overexpressed in head and neck cancer, where it is associated with tumour progression and metastasis. MATERIAL AND METHODS: To determine suitability of uPAR for molecular imaging of oral cancer surgery, human head and neck tumours were sectioned and stained for uPAR to evaluate the expression pattern compared to normal mucosa. Furthermore, metastatic oral squamous carcinoma cell line OSC-19 was used for targeting uPAR in in vivo mouse models. Using anti-uPAR antibody ATN-658, equipped with a multimodal label, the in vivo specificity was investigated and the optimal dose and time-window were evaluated. RESULTS: All human oral cancer tissues expressed uPAR in epithelial and stromal cells. Hybrid ATN-658 clearly visualized tongue tumours in mice using either NIRF or SPECT imaging. Mean fluorescent TBRs over time were 4.3±0.7 with the specific tracer versus 1.7±0.1 with a control antibody. A significant difference in TBRs could be seen between 1nmol (150µg) and 0.34nmol (50µg) dose groups (n=4, p<0.05). Co-expression between BLI, GFP and the NIR fluorescent signals were seen in the tongue tumour, whereas human cytokeratin staining confirmed presence of malignant cells in the positive cervical lymph nodes. CONCLUSION: This study shows the applicability of an uPAR specific multimodal tracer in an oral cancer model, combining SPECT with intraoperative guidance.

Prevention of breast cancer growth, invasion, and metastasis by antiestrogen tamoxifen alone or in combination with urokinase inhibitor B-428.

Urokinase (urokinase plasminogen activator, uPA) and its cell surface receptor (uPA receptor, uPAR) play an important role in a variety of physiological and pathological processes requiring cell migration and tissue remodeling. Using our syngeneic model of uPAR overexpression by the rat breast cancer cell line Mat B-III, we have examined the ability of the nonsteroidal antiestrogen, tamoxifen (TAM), and of a selective synthetic inhibitor of uPA, 4-iodo benzo[b]thiophene-2-carboxamidine (B-428), to inhibit expression of uPA and uPAR as well as cell growth, invasion, and metastasis of wild-type Mat B-III cells and of cells overexpressing uPAR (Mat B-III-uPAR). Both TAM and B-428 inhibited uPAR gene transcription, mRNA expression, protein production and also decreased the proliferative and invasive capacity of Mat B-III and Mat B-III-uPAR. The effects of TAM and B-428 were more pronounced when these agents were tested in combination. Both control and experimental cells (1 x 10(6) cells) were inoculated orthotopically into the mammary fat pad of syngeneic female Fisher rats, and animals were infused i.p. with either TAM and B-428 alone or in combination for 2 weeks. Control animals receiving vehicle alone developed large tumors and macroscopic metastases to lungs, liver, and lymph nodes. In contrast to this, experimental animals receiving TAM and B-428 showed a significant decrease in primary tumor volume and metastases. Combination therapy had especially marked effects in blocking progression of the primary tumor in experimental animals inoculated with highly aggressive Mat B-III-uPAR cells. These results underscore the utility of anti-proteolytic agents (B-428) in addition to standard hormone therapy (TAM) in advanced breast cancer patients where the uPA/uPAR system plays a key role in tumor progression.

Stimulation of urokinase-type plasminogen activator receptor expression by PMA requires JNK1-dependent and -independent signaling modules.

The urokinase-type plasminogen activator receptor (u-PAR) has been implicated in tumor progression, and previous studies have shown that the expression of this gene is strongly up-regulated by PMA. Although the signaling mechanism by which PMA modulates u-PAR expression is not known, the effect of this phorbol ester on the expression of other genes has been ascribed to activation of the c-Raf-1-ERK signaling pathway. However, in the current study we examined an alternate possibility that the inductive effect of PMA on u-PAR expression also required a JNK1-dependent signaling cascade usually associated with stress-inducing stimuli. PMA treatment of the u-PAR-deficient OVCAR-3 ovarian cancer cells, which contain low JNK activities, resulted in a rapid (5 min) increase in JNK activity. Maximal JNK activity (12-fold induction) occurred after 30 min; this preceding the earliest detected rise in u-PAR protein (2 h). Dose-response studies with PMA also indicated that the increased JNK activity was tightly correlated with elevated u-PAR protein levels. The stimulation of u-PAR promoter activity by PMA required an intact upstream AP-1 motif (-184) and in PMA-treated cells this motif was bound with c-Jun as indicated from mobility shift assays. PMA up-regulated the c-Jun trans acting activity as indicated by the higher activity of a GAL4-regulated luciferase reporter in phorbol-ester-treated cells co-transfected with an expression vector encoding the c-Jun transactivation domain fused to the GAL4 DNA-binding domain. The ability of PMA to stimulate u-PAR promoter activity was effectively titrated out by the co-expression of either a kinase-defective JNK1 or a dominant negative MEKK1 the latter being an upstream activator of JNK1. Conversely, u-PAR promoter activity was stimulated by the co-expression of a constitutively active MEKK1 and this induction was antagonized by the inclusion of the kinase-defective JNK1 plasmid. We also determined the biological significance of the JNK1-dependent signaling cascade in regulating u-PAR promoter activity by c-Ha-ras since this oncogene is activated and/or overexpressed in a variety of tumors including ovarian cancer. Transfection of an activated c-Ha-ras into OVCAR-3 cells stimulated u-PAR promoter activity over 20-fold and this could be countered by the individual expression of dominant negative expression constructs to Rac-1, MEKK1 or JNK1. Taken together, these data suggest that the PMA- or c-Ha-Ras-dependent stimulation of u-PAR gene expression requires a JNK1-dependent signaling module and that, at least for PMA, the concurrent stimulation of a JNK1-independent signaling module is also required. Thus, caution should be exercised in invoking linear signaling modules to account for the regulation of inducible gene expression.

Effect of nifedipine and theophylline in asthma.

The effect of nifedipine, 10 mg po q.i.d. for 2 weeks, was studied in a randomized, double-blind, crossover trial in nine patients with asthma receiving theophylline. Nifedipine did not significantly affect the mean (+/- SD) morning peak expiratory flow rate (PEFR; 336 +/- 130 L/min for drug vs. 349 +/- 92 L/min for placebo), evening PEFR (393 +/- 69 L/min for drug vs. 367 +/- 66 L/min for placebo), symptom score (27.4% +/- 22.9% for drug vs. 33.8% +/- 26.4% for placebo), or the number of albuterol inhalations per day (5.8 +/- 3.5 for drug vs. 6.2 +/- 4.1 for placebo). Furthermore, there was no change in PEFR 30, 60, or 120 minutes after nifedipine dosing. Nifedipine did not significantly affect the steady-state serum theophylline trough levels (9.1 +/- 2.2 mg/ml for drug vs. 10.2 +/- 1.9 micrograms/ml for placebo) or the theophylline pharmacokinetic parameters, such as the elimination t1/2, peak serum concentration, time to peak, and AUC(0-24). We conclude that nifedipine has little, if any, effect on the clinical status, PEFR, or theophylline serum levels in patients with asthma who receive theophylline.

Expression of urokinase and its receptor in invasive and non-invasive prostate cancer cell lines.

We previously reported that extracellular matrix invasion by the prostate cancer cell lines, PC-3 and DU-145 was contingent on endogenous urokinase being bound to a specific cell surface receptor. The present study was undertaken to characterize the expression of both urokinase and its receptor in the non-invasive LNCaP and the invasive PC-3 and DU-145 prostate cells. Northern blotting indicated that the invasive PC-3 cells, which secreted 10 times more urokinase (680 ng/ml per 10(6) cells per 48 h) than DU-145 cells (63 ng/ml per 10(6) cells per 48 h), had the most abundant transcript for the plasminogen activator. This, at least, partly reflected a 3 fold amplification of the urokinase gene in the PC-3 cells. In contrast, urokinase-specific transcript could not be detected in the non-invasive LNCaP cells previously characterized as being negative for urokinase protein. Southern blotting indicated that this was not a consequence of deletion of the urokinase gene. Crosslinking of radiolabelled aminoterminal fragment of urokinase to the cell surface indicated the presence of a 51 kDa receptor in extracts of the invasive PC-3 and DU-145 cells but not in extracts of the non-invasive LNCaP cells. The amount of binding protein correlated well with binding capacities calculated by Scatchard analysis. In contrast, the steady state level of urokinase receptor transcript was a poor predictor of receptor display. PC-3 cells, which were equipped with 25,000 receptors per cell had 2.5 fold more steady state transcript than DU-145 cells which displayed 93,000 binding sites per cell.

uPA and uPAR contribute to NK cell invasion through the extracellular matrix.

BACKGROUND: The urokinase plasminogen activator (uPA) system has been implicated in cellular invasiveness of tumor cells and immune cells. Herein we provide evidence for the production by natural killer (NK) cells of both uPA and its receptor (uPAR). MATERIALS AND METHODS: Western blot analysis, RTPCR, casein/plasminogen zymography, and fluorescence microscopy were employed to detect uPA and uPAR on NK cells. NK cell invasiveness was examined using Matrigel invasion assays. RESULTS: NK cell uPA appeared at its characteristic molecular weights, is enzymatically active in casein/plasminogen zymography, and is recognized by monoclonal antibodies. uPAR was detected by RTPCR and fluorescence microscopy. Matrigel invasion assays demonstrated an active role of uPA in NK cell invasion. CONCLUSION: The uPA system contributes to extracellular matrix (ECM) degradation by NK cells, which may be essential for NK cell accumulation into metastases, and may be prerequisite for their killing of tumor cells following NK cell adoptive transfer.

Urokinase inhibits HL-60 cell proliferation in vitro.

The binding of urokinase-type plasminogen activator (u-PA) to its receptor (u-PA-R) is required for morphological and functional maturation during monocyte differentiation of the promyelocytic leukaemia line HL-60. This paper reports that monocyte differentiation of HL-60 cells induced by 1,25 dihydroxyvitamin D2 (vitamin D2) results in a marked increase in expression of u-PA and u-PA-R. This increase in u-PA expression is of greater magnitude than is observed after culture with interferon-gamma (IFN gamma), another potent inducer of monocytic differentiation. Dimethyl sulphoxide (DMSO), an agent that induces granulocytic differentiation, also increased expression of u-PA. However, culture with the granulocyte-inducing all-trans retinoic acid (RA) did not induce an increase in surface expression of u-PA or u-PA-R. The vitamin D2-induced increase in cell-surface u-PA was not coincident with an increase in steady-state levels of u-PA mRNA, suggesting that intracellular stores of this protein, translational or post-translational mechanisms of regulation, or some other regulatory mechanism may be responsible for the increase in u-PA during differentiation. To ascertain an association between the increased expression of cell-surface u-PA and reduced proliferation that accompanies differentiation, the effect of u-PA on cellular proliferation of HL-60 cells was measured. Both pro-u-PA (whole molecule) and fragments of u-PA that retained receptor-binding capability caused a marked inhibition of HL-60 proliferation in the absence of vitamin D2.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of the plasminogen activation system in angiogenesis and metastasis.

The urokinase plasminogen activator system (uPA) has been demonstrated to be required for the movement of cells through a matrix. These observations have been extended to the migration of endothelial cells during the process of angiogenesis, and recent data suggest that the uPA system is central to this process. uPA is a serine protease that is capable of initiating an extracellular cascade of proteolysis that involves the activation of plasminogen and matrix metalloproteases. These proteolytic cascades remodel extracellular matrix (ECM) and basement membrane (BM), allowing for the movement of cells across and through these barriers. In addition, these proteolytic cascades process and release various growth and differentiation factors that are sequestered on the cell surface or within the ECM, which contribute to the evolution of a migratory or invasive cell phenotype. uPA is also able to modulate signaling and cell adhesion through its specific cell surface receptor, uPAR. Recent data suggest that the nonproteolytic activities of the uPA system are coupled to adhesion, migration and signaling through various integrins. This article reviews the evidence for the role of this system in tumor angiogenesis and metastasis, which suggests that the uPA system initiates multiple cascades that contribute to these processes.

Metastatic chondrosarcoma of the gingiva mimicking a reactive exophytic lesion: a case report.

Metastases of chondrosarcoma to the oral mucosa are very rare. The present case describes such a metastasis to the maxillary gingiva which mimics a benign reactive exophytic lesion. The literature concerning this matter is reviewed. The differential diagnosis and pathogenesis of such lesions are discussed.

Pulmonary alveolar proteinosis in Israel: ethnic clustering.

BACKGROUND: Pulmonary alveolar proteinosis is a rare disease in which a surfactant-like phospholipid-rich protein accumulates in the lungs. The disease is amenable to effective therapy by total lung lavage. OBJECTIVES: To investigate the prevalence, ethnic distribution and course of PAP in Israel. METHODS: A countrywide survey was conducted during which pulmonologists were questioned about patients with PAP. The patients were examined and their charts, radiological images, pathological slides and physiological data were reviewed. RESULTS: The survey yielded 15 patients (8 females) during the period 1976-98 (14 in the last decade), giving a prevalence of 3.7 x 10(6) and an incidence of 0.36 x 10(6)/year. Mean age of the patients was 33 +/- 13 years (range 0.5-46 years). Seven patients were North African (two were siblings), four were from Iraq and two were Arabs; there was only one Ashkenazi Jew (a child). Symptoms at the onset were dyspnea and chest pain. Spontaneous remission occurred in at least 3 patients, and 10 patients required 1-4 bronchoalveolar lavage treatments. The subjective and physiological response was favorable, but there was less consistent radiological improvement. CONCLUSION: The prevalence of PAP in Israel is approximately 3.7 x 10(6). Most cases occurred in Jews who had immigrated from North Africa or Iraq, and two were siblings. The prevalence among the Arab population appears to be similar. This clustering suggests the existence of a genetic predisposition. The course of the disease appears to be similar to that reported elsewhere.

Sarcoidosis with oral involvement.

A case of sarcoidosis with bilateral hilar lymphadenopathy and multiple submucosal papular oral mucosa lesions is presented. The clinical signs and symptoms described in this case are not uncommon, and their distinguishing features may expedite accurate diagnosis. Early incisional biopsy of the oral mucosa appears useful for histopathologic proof of the diagnosis.

Identification of biomarkers for the antiangiogenic and antitumour activity of the superoxide dismutase 1 (SOD1) inhibitor tetrathiomolybdate (ATN-224).

Tetrathiomolybdate (choline salt; ATN-224), a specific, high-affinity copper binder, is currently being evaluated in several phase II cancer trials. ATN-224 inhibits CuZn superoxide dismutase 1 (SOD1) leading to antiangiogenic and antitumour effects. The pharmacodynamics of tetrathiomolybdate has been followed by tracking ceruloplasmin (Cp), a biomarker for systemic copper. However, at least in mice, the inhibition of angiogenesis occurs before a measurable decrease in systemic copper is observed. Thus, the identification and characterisation of other biomarkers to follow the activity of ATN-224 in the clinic is of great interest. Here, we present the preclinical evaluation of two potential biomarkers for the activity of ATN-224: (i) SOD activity measurements in blood cells in mice and (ii) levels of endothelial progenitor cells (EPCs) in bonnet macaques treated with ATN-224. The superoxide dismutase activity in blood cells in mice is rapidly inhibited by ATN-224 treatment at doses at which angiogenesis is maximally inhibited. Furthermore, ATN-224 dosing in bonnet macaques causes a profound and reversible decrease in EPCs without significant toxicity. Thus, both SOD activity measurements and levels of EPCs may be useful biomarkers of the antiangiogenic activity of ATN-224 to be used in its clinical development.

Phase 1 trial of the antiangiogenic peptide ATN-161 (Ac-PHSCN-NH(2)), a beta integrin antagonist, in patients with solid tumours.

To evaluate the toxicity, pharmacological and biological properties of ATN-161, a five -amino-acid peptide derived from the synergy region of fibronectin, adult patients with advanced solid tumours were enrolled in eight sequential dose cohorts (0.1-16 mg kg(-1)), receiving ATN-161 administered as a 10-min infusion thrice weekly. Pharmacokinetic sampling of blood and urine over 7 h was performed on Day 1. Twenty-six patients received from 1 to 14 4-week cycles of treatment. The total number of cycles administered to all patients was 86, without dose-limiting toxicities. At dose levels above 0.5 mg kg(-1), mean total clearance and volume of distribution showed dose-independent pharmacokinetics (PKs). At 8.0 and 16.0 mg kg(-1), clearance of ATN-161 was reduced, suggesting saturable PKs. Dose escalation was halted at 16 mg kg(-1) when drug exposure (area under the curve) exceeded that associated with efficacy in animal models. There were no objective responses. Six patients received more than four cycles of treatment (>112 days). Three patients received 10 or more cycles (> or =280 days). ATN-161 was well tolerated at all dose levels. Approximately, 1/3 of the patients in the study manifested prolonged stable disease. These findings suggest that ATN-161 should be investigated further as an antiangiogenic and antimetastatic cancer agent alone or with chemotherapy.

The urokinase plasminogen activator receptor (uPAR) as a target for the diagnosis and therapy of cancer.

The identification and characterization of validated molecular targets for cancer drug and diagnostic development is rapidly changing the way that promising new anti-cancer compounds are developed and evaluated. A significant body of in vitro and in vivo data has established the urokinase plasminogen activator (uPA) system as a promising target for cancer drug development. The uPA system has been demonstrated to have pleiotropic activities in the development of tumors, and in tumor progression and angiogenesis. There are multiple ways to target this system, the most straightforward being the development of small molecule active site inhibitors of the serine protease, uPA. However, compounds of this type have not entered into clinical trials, and issues related to selectivity and specificity of this class of inhibitors have yet to be satisfactorily resolved. Recent evidence suggests that in addition to uPA, its specific cell surface receptor (uPAR) may also be a suitable target for the design and development of cancer therapeutic and diagnostic agents. uPAR is central to several pathways implicated in tumor progression and angiogenesis. The binding of the uPA zymogen (scuPA) to uPAR appears to be a pre-requisite for efficient cell-surface activation of scuPA to the active two-chain form (tcuPA) by plasmin, and simple ligand occupancy of uPAR by scuPA initiates various signaling pathways leading to alterations in cell motility and adhesion. One therapeutic rationale that is currently being investigated is the simple displacement of scuPA or tcuPA from suPAR, which may effectively inhibit both the proteolytic and signal-transducing cascades. In addition, other approaches to the modulation of the activity of this system that may also be useful include blocking the interaction of uPAR with integrins and extracellular matrix proteins as well as strategies to down-regulate the expression of uPA and uPAR in target cells. This review will summarize these approaches, and also describe the targeting of uPAR for diagnosis and imaging.

The urokinase plasminogen activator system in cancer: implications for tumor angiogenesis and metastasis.

Substantial evidence exists which implicates the urokinase plasminogen activator system [urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1)] in the neo-vascularization, invasion and metastasis of many solid tumors. Clinical studies have demonstrated an association between high levels of expression of the components of this system in tumors and poor patient prognosis and outcome. Components of the uPA/uPAR system are differentially expressed or activated on motile cells including invading tumor cells and leukocytes, and migrating endothelial cells. In contrast, there is little or no expression on most normal, quiescent cells. Studies performed in vitro have demonstrated the regulation of the expression of uPA and uPAR by growth and differentiation factors as well as by oncogenes. In this review, we summarize recent findings on the role of the components of the uPA/uPAR system in angiogenesis, invasiveness and tumor metastasis. The activities of this system in endothelial and leukocyte cell biology and the relevance of these activities to angiogenesis and tumor metastasis will be considered. Recent experimental evidence obtained using inhibitors of uPA and uPAR has validated this system as a therapeutic target for the development of anti-angiogenic and anti-metastatic therapeutic agents. These studies, as well as additional therapeutic and diagnostic implications for uPAR targeting, will be discussed.