Regulation of cell migration and invasion by specific modules of uPA: mechanistic insights and specific inhibitors.

Urokinase (uPA) is a 411 residues serine protease originally identified for its ability to activate plasminogen and generate plasmin, a broad-spectrum matrix- and fibrin-degrading enzyme. Later, this protease has been shown to possess also a clear-cut ability to stimulate cell migration and survival in a catalytic-independent manner. This activity turned out to be exerted through the growth factor-like domain (GFD-like, residues 1-49) of the protease binding to a GPIanchored membrane receptor (uPAR), in complex with transmembrane receptors such as integrins, the epidermal growth factor and the formyl-peptide receptors. Direct binding of uPA to integrins through its kringle (residues 50-131) and connecting peptide (residues 132-158) regions results in enhanced migration. The dual function of uPA in promoting migration while reducing the physical resistance of extracellular matrix underlies its crucial role in the invasion of malignant tumours. Consolidated evidence emerging from animal models and clinical studies shows that the overexpression of uPA is a causal determinant to tumour metastasis and is associated to a poor prognosis. Therefore, pinpointing the molecular interactions and identifying novel agents to interfere with the diverse activities of uPA is a goal of basic and applied research. In this review, we discuss the general theme of cell migration and invasion. A description of the uPA structure-function relationship and the functional effects of isolated domains is presented. Current information on molecular agonistic as well as antagonistic compounds, including the compounds which have reached clinical trials, is provided.

Preliminary study on the correlation between grading and histology of solitary pulmonary nodules and contrast enhancement and [18F]fluorodeoxyglucose standardised uptake value after evaluation by dynamic multiphase CT and PET/CT.

AIM: To evaluate whether the histology and grading of solitary pulmonary nodules (SPNs) correlated with the results of dynamic multiphase multidetector CT (MDCT) and the [(18)F]fluorodeoxyglucose standardised uptake value (SUV) in 30 patients. METHODS: Chest x-rays of 270 patients with incidentally detected SPNs were retrospectively evaluated. Thirty patients with histologically proven SPNs were enrolled. On MDCT and positron emission tomography (PET)/CT images, two experts measured the density of nodules in all perfusion phases and the SUV. Net enhancement (NE) was calculated by subtracting peak pre-contrast density from peak post-contrast density. The Pearson test was used to correlate nodule NE, SUV, grading, histology and diameter. RESULTS: Of the 30 malignant SPNs, six were classified as G1 (median NE, 31.5 Hounsfield units (HU); median SUV, 4.8 units), 15 were classified as G2 (median NE, 49 HU; median SUV, 6 units), and nine were classified as G3 (median NE, 32 HU; median SUV, 4.5 units). A highly negative correlation was found in G3 SPNs between NE and the corresponding diameters (r=-0.834; p=0.00524). NE increased with the increase in diameter (r=0.982; p=0.284). SUV increased as the SPN diameter increased (r=0.789; p=0.421). NE and SUV were higher in G2 than G1 SPNs, and lower in G2 than G3 SPNs (r=0.97; p=0.137). CONCLUSIONS: The significant correlation in dedifferentiated (G3) SPNs between NE and diameter (r=-0.834; p=0.00524) supports the theory that stroma and neoangiogenesis are fundamental in SPN growth. The highly negative correlation between NE and diameter demonstrates a net decrease in perfusion despite an increase in dimension. The multidisciplinary approach used herein may result in a more precise prognosis and consequently a better therapeutic outcome, particularly in patients with undifferentiated lung cancer.

Adenosine receptor ligands in clinical trials.

Adenosine and its receptors play different roles in normal and patho-physiological conditions. For these reasons, many laboratories have focused their attention on dissecting the molecular pathway underlying the mechanism of action of this nucleoside with the final goal being to design new drugs. Wide expression and overlapping functions have been the major problems to designing specific adenosine agonists and antagonists with few associated negative side effects. Engineered mice have helped to dissect the single adenosine receptor function in specific tissues and pathological conditions. All these efforts in the last 20 years have led to the design of more than 2000 compounds, some of them now in clinical trials for treating different pathologies. In this review, we highlight the mouse animal models that have been of use in designing new selective drugs as well as discuss the main adenosine receptor ligands in clinical trials.

Modulation of cellular migration and survival by c-Myc through the downregulation of urokinase (uPA) and uPA receptor.

It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

Structure, function and antagonists of urokinase-type plasminogen activator.

Urokinase (uPA) is a serine protease which converts plasminogen to plasmin, a broad-spectrum protease active on extracellular matrix (ECM) components. Like many components of the blood coagulation, fibrinolytic and complement cascades, uPA has a modular structure, including three conserved domains: a growth factor-like domain (GFD, residues 1 - 49), a kringle domain (residues 50 - 131), linked by an interdomain linker or "connecting peptide" (CP, residues 132 - 158) to the serine protease domain (residues 159 - 411). Although direct molecular interactions with urokinase receptor and integrins have been extensively described, the function of single uPA domains is not completely understood. Because of the causal involvment of uPA in cancer invasion and metastasis, the blockade of uPA interactions and activity with specific inhibitors is of interest for novel strategies in cancer therapy. New inhibitors derived from the interdomain linker or "connecting peptide" are coming into focus. This review summarizes the recent findings on the uPA structure-function relationship and provides further information on existing inhibitors of uPA multiple functions.

Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation.

We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.

Molecular mechanisms underlying airway smooth muscle contraction and proliferation: implications for asthma.

Airway smooth muscle (ASM) plays a key role in bronchomotor tone, as well as in structural remodeling of the bronchial wall. Therefore, ASM contraction and proliferation significantly participate in the development and progression of asthma. Many contractile agonists also behave as mitogenic stimuli, thus contributing to frame a hyperresponsive and hyperplastic ASM phenotype. In this review, the molecular mechanisms and signaling pathways involved in excitation-contraction coupling and ASM cell growth will be outlined. Indeed, the recent advances in understanding the basic aspects of ASM biology are disclosing important cellular targets, currently explored for the implementation of new, more effective anti-asthma therapies.

Effects of TGF-beta and glucocorticoids on map kinase phosphorylation, IL-6/IL-11 secretion and cell proliferation in primary cultures of human lung fibroblasts.

Transforming growth factor-beta1 (TGF-beta1) is crucially involved in the fibrotic events characterizing interstitial lung diseases (ILDs), as well as in the airway remodeling process typical of asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal and fibrotic human lung fibroblasts (HLFs), the effects of TGF-beta1 on mitogen-activated protein kinase (MAPK) phosphorylation, cell proliferation, and production of interleukins 6 (IL-6) and 11 (IL-11), in the presence or absence of a pretreatment with budesonide (BUD). MAPK phosphorylation was detected by Western blotting, cell viability and proliferation were evaluated using Trypan blue staining and [(3)H]-thymidine incorporation assay, respectively, and the release of IL-6 and IL-11 into cell culture supernatants was assessed by ELISA. TGF-beta1 (10 ng/ml) significantly stimulated MAPK phosphorylation (P < 0.01), and also enhanced cell proliferation as well as the secretion of both IL-6 and IL-11, which reached the highest increases at the 72nd h of cell exposure to this growth factor. All such effects were prevented by BUD (10(-8) M) and, with the exception of IL-6 release, also by a mixture of MAPK inhibitors. Therefore, our findings suggest that the fibrotic action exerted by TGF-beta1 in the lung is mediated at least in part by MAPK activation and by an increased synthesis of the profibrogenic cytokines IL-6 and IL-11; all these effects appear to be prevented by corticosteroids via inhibition of MAPK phosphorylation.

Analysis of HtrA1 serine protease expression in human lung cancer.

HtrA1 is a serine protease homologue to the bacterial serine-protease HtrA, also known as DegP, a heat shock-induced envelope-associated serine protease. It has been shown that over-expression of HtrA1 in human cancer cells inhibits cell growth and proliferation in vitro and in vivo, thus, suggesting a possible role as a tumor suppressor. The expression of HtrA1 was investigated in depth by means of immunohistochemistry in a large group of human lung cancer specimens and corresponding lymph node metastases. Univariate analysis showed, that the only statistically significant correlation was found between the HtrA1 expression level detected in the primary tumors and in the lymph node metastases. This result was also confirmed when the analysis was restricted only to the cases where both the primary tumor and the autologous lymph node metastasis were available. Our data suggest that HtrA1 may be involved in lung cancer progression by targeting several molecular pathways.

Evidence of angiogenesis in bronchial biopsies of smokers with and without airway obstruction.

The involvement of bronchial vasculature in the airway remodelling occurring in symptomatic smokers with normal lung function and with chronic obstructive pulmonary disease (COPD) has been poorly investigated. An immunohistochemical study was performed on bronchial biopsies taken from 8 non-smokers and 18 smokers divided, according to global health initiative on obstructive lung diseases (GOLD) classification of COPD, into two groups, GOLD 0 and GOLD 2, each of 9 subjects. The number of vessels and the percentage of vascular area in the lamina propria were evaluated by mAb anti-collagen IV. Cellular expression of VEGF and vascular expression of alphavbeta3 integrin were evaluated by the specific monoclonal antibodies. An image processing and analysis system was used to quantify the immunohistochemical data. The number of vessels, the vascular area, the cellular expression of VEGF, the number and percentage of alphavbeta3 positive vessels were significantly higher in GOLD 0 and in GOLD 2 smokers than in non-smokers. The comparison between GOLD 0 and GOLD 2 smokers did show a weak but significantly lower number of vessels in GOLD 2, while the vascular area and the percentage of alphavbeta3 positive vessels did not differ between the two groups. A higher cellular VEGF expression was detected in the GOLD 2 than in the GOLD 0 group. Angiogenesis of bronchial vessels is a component of the airway remodelling occurring in symptomatic smokers with normal lung function and with COPD, it seems independent by the development of airway obstruction and not related to its severity.

Biological targets for therapeutic interventions in COPD: clinical potential.

COPD is a widespread inflammatory respiratory disorder characterized by a progressive, poorly reversible airflow limitation. Currently available therapies are mostly based on those used to treat asthma. However, such compounds are not able to effectively reduce the gradual functional deterioration, as well as the ongoing airway and lung inflammation occurring in COPD patients. Therefore, there is an urgent need to improve the efficacy of the existing drug classes and to develop new treatments, targeting the main cellular and molecular mechanisms underlying disease pathogenesis. These therapeutic strategies will be highlighted in the present review.

Endothelin-1 induces proliferation of human lung fibroblasts and IL-11 secretion through an ET(A) receptor-dependent activation of MAP kinases.

Endothelin-1 (ET-1) is implicated in the fibrotic responses characterizing interstitial lung diseases, as well as in the airway remodeling process occurring in asthma. Within such a context, the aim of our study was to investigate, in primary cultures of normal human lung fibroblasts (NHLFs), the ET-1 receptor subtypes, and the intracellular signal transduction pathways involved in the proliferative effects of this peptide. Therefore, cells were exposed to ET-1 in the presence or absence of an overnight pre-treatment with either ET(A) or ET(B) selective receptor antagonists. After cell lysis, immunoblotting was performed using monoclonal antibodies against the phosphorylated, active forms of mitogen-activated protein kinases (MAPK). ET-1 induced a significant increase in MAPK phosphorylation pattern, and also stimulated fibroblast proliferation and IL-6/IL-11 release into cell culture supernatants. All these effects were inhibited by the selective ET(A) antagonist BQ-123, but not by the specific ET(B) antagonist BQ-788. The stimulatory influence of ET-1 on IL-11, but not on IL-6 secretion, was prevented by MAPK inhibitors. Therefore, such results suggest that in human lung fibroblasts ET-1 exerts a profibrogenic action via an ET(A) receptor-dependent, MAPK-mediated induction of IL-11 release and cell proliferation.

Role of cell-cycle regulators in lung cancer.

Lung cancer is the leading cause of cancer death worldwide. Histologically, 80% of lung cancers are classified as non-small-cell lung cancer (NSCLC), and the remaining 20% as small-cell lung cancer (SCLC). Lung carcinoma is the result of molecular changes in the cell, resulting in the deregulation of pathways controlling normal cellular growth, differentiation, and apoptosis. This review summarizes some of the most recent findings about the role of cell-cycle proteins in lung cancer pathogenesis and progression.

The urokinase plasminogen activator and its receptor: role in cell growth and apoptosis.

The urinary-type plasminogen activator, or uPA, controls matrix degradation through the conversion of plasminogen into plasmin and is regarded as the critical trigger for plasmin generation during cell migration and invasion, under physiological and pathological conditions (such as cancer metastasis). The proteolytic activity of uPA is responsible for the activation or release of several growth factors and modulates the cell survival/apoptosis ratio through the dynamic control of cell-matrix contacts. The urokinase receptor (uPAR), binding to the EGF-like domain of uPA, directs membrane-associated extracellular proteolysis and signals through transmembrane proteins, thus regulating cell migration, adhesion and cytoskeletal status. However, recent evidence highlights an intricate relationship linking the uPA/uPAR system to cell growth and apoptosis.

Mitogen-activated protein kinases and asthma.

Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes which play a key role in signal transduction mediated by cytokines, growth factors, neurotransmitters and various types of environmental stresses. In the airways, these extracellular stimuli elicit complex inflammatory and structural changes leading to the typical features of asthma including T cell activation, eosinophil and mast cell infiltration, as well as bronchial hyperresponsiveness and airway remodelling. Because MAPKs represent an important point of convergence for several different signalling pathways, they affect multiple aspects of normal airway function and also significantly contribute to asthma pathophysiology. Therefore, this review focuses on the crucial involvement of MAPKs in asthma pathogenesis, thus also discussing their emerging role as molecular targets for anti-asthma drugs.

Alpha-interferon and its effects on signal transduction pathways.

Interferon-alpha (IFNalpha) is a recombinant protein widely used in the therapy of several neoplasms such as myeloma, renal cell carcinoma, epidermoid cervical and head and neck tumors, and melanoma. IFNalpha, the first cytokine to be produced by recombinant DNA technology, has emerged as an important regulator of cancer cell growth and differentiation, affecting cellular communication and signal transduction pathways. However, the way by which tumor cell growth is directly suppressed by IFNalpha is not well known. Wide evidence exists on the possibility that cancer cells undergo apoptosis after the exposure to the cytokine. Here we will review the consolidate signal transducer and activator of transcription (STAT)-dependent mechanism of action of IFNalpha. We will discuss data obtained by us and others on the triggering of the stress-dependent kinase pathway induced by IFNalpha and its correlations with the apoptotic process. The regulation of the expression of proteins involved in apoptosis occurrence will be also described. In this regard, IFNalpha is emerging as a post-translational controller of the intracellular levels of the apoptosis-related protein tissue transglutaminase (tTG). This new way of regulation of tTG occurs through the modulation of their proteasome-dependent degradation induced by the cytokine. Until today, inconsistent data have been obtained regarding the clinical effectiveness of IFNalpha in the therapy of solid tumors. In fact, the benefit of IFNalpha treatment is limited to some neoplasms while others are completely or partially resistant. The mechanisms of tumor resistance to IFNalpha have been studied in vitro. The alteration of JAK-STAT components of the IFNalpha-induced signaling, can be indeed a mechanism of resistance to IFN. However, we have recently described a reactive mechanism of protection of tumor cells from the apoptosis induced by IFNalpha dependent on the epidermal growth factor (EGF)-mediated Ras/extracellular signal regulated kinase (Erk) signaling. The involvement of the Ras-->Erk pathway in the protection of tumor cells from the apoptosis induced by IFNalpha is further demonstrated by both Ras inactivation by RASN17 transfection and mitogen extracellular signal regulated kinase 1 (Mek-1) inhibition by exposure to PD098059. These data strongly suggest that the specific disruption of the latter could be a useful approach to potentiate the antitumour activity of IFNalpha against human tumors based on the new mechanistic insights achieved in the last years.

Effects of hydrogen peroxide on MAPK activation, IL-8 production and cell viability in primary cultures of human bronchial epithelial cells.

The airway epithelium is continuously exposed to inhaled oxidants, including airborne pollutants and cigarette smoke, which can exert harmful proinflammatory and cytotoxic effects. Therefore, the aim of our study was to investigate, in primary cultures of human bronchial epithelial cells (HBEC), the signal transduction pathways activated by increasing concentrations (0.25, 0.5, and 1 mM) of hydrogen peroxide (H(2)O(2)), as well as their effects on IL-8 production and cell viability. The reported results show that H(2)O(2) elicited, in a concentration-dependent fashion, a remarkable increase in phosphorylation-dependent activation of mitogen-activated protein kinases (MAPKs), associated with a significant induction of IL-8 synthesis and a dramatically enhanced cell death. Pre-treatment of HBEC with MAPK inhibitors was able to significantly inhibit the effects of H(2)O(2) on IL-8 secretion, and to effectively prevent cell death. Therefore, these findings suggest that MAPKs play a key role as molecular transducers of the airway epithelial injury triggered by oxidative stress, as well as potential pharmacologic targets for indirect antioxidant intervention.

Urokinase-mediated posttranscriptional regulation of urokinase-receptor expression in non small cell lung carcinoma.

The urokinase-type plasminogen activator (uPA) and its cellular receptor (uPAR) are involved in the proteolytic cascade required for tumor cell dissemination and metastasis, and are highly expressed in many human tumors. We have recently reported that uPA, independently of its enzymatic activity, is able to increase the expression of its own receptor in uPAR-transfected kidney cells at a posttranscriptional level. In fact, uPA, upon binding uPAR, modulates the activity and/or the level of a mRNA-stabilizing factor that binds the coding region of uPAR-mRNA. We now investigate the relevance of uPA-mediated posttranscriptional regulation of uPAR expression in non small cell lung carcinoma (NSCLC), in which the up-regulation of uPAR expression is a prognostic marker. We show that uPA is able to increase uPAR expression, both at protein and mRNA levels, in primary cell cultures obtained from tumor and adjacent normal lung tissues of patients affected by NSCLC, thus suggesting that the enzyme can exert its effect in lung cells. We investigated the relationship among the levels of uPA, uPAR and uPAR-mRNA binding protein(s) in NSCLC. Lung tissue analysis of 35 NSCLC patients shows an increase of both uPA and uPAR in tumor tissues, as compared to adjacent normal tissues, in 27 patients (77%); 19 of these 27 patients also show a parallel increase of the level and/or binding activity of a cellular protein capable of binding the coding region of uPAR-mRNA. Therefore, in tumor tissues, a strong correlation is observed among these 3 parameters, uPA, uPAR and the level and/or the activity of a uPAR-mRNA binding protein. We then suggest that uPA regulates uPAR expression in NSCLC at a posttranscriptional level by increasing uPAR-stability through a cellular factor that binds the coding region of uPAR-mRNA.

Loss of pRb2/p130 expression is associated with unfavorable clinical outcome in lung cancer.

Altered expression of cell cycle regulators represents a frequent event in both small cell and non-small cell lung cancer (NSCLC). Despite several studies that reported involvement of tumor suppressor genes, such as p53 and pRb, in the development and progression of lung cancer, contrasting opinions exist about the prognostic role of this protein in this neoplasm. We developed an immunohistochemical assay suitable for the detection of pRb2/p130, the last discovered member of the retinoblastoma gene family, on formalin-fixed and paraffin-embedded sections. We evaluated the immunohistochemical expression of pRb2/p130 in 135 lung cancer specimens, and performed Western blot analysis in a subset of 30 corresponding tumor lysates. A high correlation between immunohistochemical data and Western blot results (P = 0.0004) was found. We statistically analyzed the relationship between overall survival (OS) time and pRb2/p130 expression according to the different histological types in 105 patients. We did not find any correlation between pRb2/p130 expression and OS in small cell lung cancers, whereas in NSCLCs a direct relationship between pRb2 and OS was found in both adenocarcinoma (P = 0.0002) and squamous cell carcinoma (P = 0.0002) histotypes. According to univariate analysis, pRb2/p130 was a prognostic factor of which the lost or reduced expression correlated with a shorter OS (P < 0.0000). At multivariate analysis, pRb2/p130 expression was an independent predictor of OS (P = 0.0001) when considered together with histotype. This study demonstrates for the first time the potential independent prognostic value of pRb2/p130 expression on formalin-fixed, paraffin-embedded sections from lung cancer patients. pRb2/p130 immunoreactivity can be used to predict OS in patients with NSCLC and, therefore, may represent a new prognostic marker.