Publications by authors named "Yves Courty"

34 Publications

Prognostic value of kallikrein-related peptidase 12 (KLK12) mRNA expression in triple-negative breast cancer patients.

Mol Med 2020 02 7;26(1):19. Epub 2020 Feb 7.

Clinical Research Unit, Department of Obstetrics and Gynecology, Technical University of Munich, Ismaninger Str. 22, 81576, Munich, Germany.

Background: The serine protease KLK12 belongs to the human fifteen-member family of kallikrein-related peptidases. Differential expression accompanied by either increased or decreased enzymatic activity has been linked to several diseases including cancer. Triple-negative breast cancer (TNBC) represents a very aggressive subgroup of breast cancer with high tumor recurrence rates and poor patient prognosis. Here, we quantified the KLK12 mRNA expression levels in tumor tissue of TNBC patients and analyzed their prognostic value.

Methods: In the present study, KLK12 mRNA expression in tumor tissue of TNBC patients (n = 116) was determined by quantitative real-time PCR assay. The association of KLK12 mRNA levels with clinical parameters, and patients' outcome was analyzed using Chi-square tests, Cox regression models and Kaplan-Meier survival analysis.

Results: Positive, but low KLK12 mRNA levels were detected in about half of the cases (54 out of 116; 47%), the other samples were negative for KLK12 mRNA expression. No significant association was observed between KLK12 mRNA levels and clinicopathological variables (age, lymph node status, tumor size, and histological grade). In univariate Cox analyses, positive KLK12 mRNA expression was significantly associated with shortened disease-free survival (DFS; hazard ratio [HR] = 2.12, 95% CI = 1.19-3.78, p = 0.010) as well as overall survival (OS; HR = 1.91, 95% CI = 1.04-3.50, p = 0.037). In multivariable Cox analysis, including all clinical parameters plus KLK12 mRNA, the latter - together with age - remained an independent unfavorable predictive marker for DFS (HR = 2.33, 95% CI = 1.28-4.24, p = 0.006) and showed a trend towards significance in case of OS (HR = 1.80, 95% CI = 0.96-3.38, p = 0.066).

Conclusions: Positive KLK12 expression is remarkably associated with shortened DFS and OS, suggesting that KLK12 plays a tumor-supporting role in TNBC.
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http://dx.doi.org/10.1186/s10020-020-0145-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7006133PMC
February 2020

Cigarette smoke induces overexpression of active human cathepsin S in lungs from current smokers with or without COPD.

Am J Physiol Lung Cell Mol Physiol 2019 11 25;317(5):L625-L638. Epub 2019 Sep 25.

Université de Tours, Tours, France.

Cigarette smoking has marked effects on lung tissue, including induction of oxidative stress, inflammatory cell recruitment, and a protease/antiprotease imbalance. These effects contribute to tissue remodeling and destruction resulting in loss of lung function in chronic obstructive pulmonary disease (COPD) patients. Cathepsin S (CatS) is a cysteine protease that is involved in the remodeling/degradation of connective tissue and basement membrane. Aberrant expression or activity of CatS has been implicated in a variety of diseases, including arthritis, cancer, cardiovascular, and lung diseases. However, little is known about the effect of cigarette smoking on both CatS expression and activity, as well as its role in smoking-related lung diseases. Here, we evaluated the expression and activity of human CatS in lung tissues from never-smokers and smokers with or without COPD. Despite the presence of an oxidizing environment, CatS expression and activity were significantly higher in current smokers (both non-COPD and COPD) compared with never-smokers, and correlated positively with smoking history. Moreover, we found that the exposure of primary human bronchial epithelial cells to cigarette smoke extract triggered the activation of P2X7 receptors, which in turns drives CatS upregulation. The present data suggest that excessive CatS expression and activity contribute, beside other proteases, to the deleterious effects of cigarette smoke on pulmonary homeostasis.
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http://dx.doi.org/10.1152/ajplung.00061.2019DOI Listing
November 2019

Tissue kallikrein regulates alveolar macrophage apoptosis early in influenza virus infection.

Am J Physiol Lung Cell Mol Physiol 2019 06 25;316(6):L1127-L1140. Epub 2019 Mar 25.

INSERM, U1100-Centre d'Etude des Pathologies Respiratoires , Tours , France.

Host cell proteases are involved in influenza pathogenesis. We examined the role of tissue kallikrein 1 (KLK1) by comparing wild-type (WT) and KLK1-deficient mice infected with influenza H3N2 virus. The levels of KLK1 in lung tissue and in bronchoalveolar lavage (BAL) fluid increased substantially during infection. KLK1 did not promote virus infectivity despite its trypsin-like activity, but it did decrease the initial virus load. We examined two cell types involved in the early control of pathogen infections, alveolar macrophages (AMs) and natural killer (NK) cells to learn more about the antiviral action of KLK1. Inactivating the gene or treating WT mice with an anti-KLK1 monoclonal antibody to remove KLK1 activity accelerated the initial virus-induced apoptotic depletion of AMs. Intranasal instillation of deficient mice with recombinant KLK1 (rKLK1) reversed the phenotype. The levels of granulocyte-macrophage colony-stimulating factor in infected BAL fluid were significantly lower in KLK1-deficient mice than in WT mice. Treating lung epithelial cells with rKLK1 increased secretion of this factor known to enhance AM resistance to pathogen-induced apoptosis. The recruitment of NK cells to the air spaces peaked 3 days after infection in WT mice but not in KLK1-deficient mice, as did increases in several NK-attracting chemokines (CCL2, CCL3, CCL5, and CXCL10) in BAL. Chronic obstructive pulmonary disease (COPD) patients are highly susceptible to viral infection, and we observed that the mRNA levels decreased with increasing COPD severity. Our findings indicate that KLK1 intervenes early in the antiviral defense modulating the severity of influenza infection. Decreased KLK1 expression in COPD patients could contribute to the worsening of influenza.
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http://dx.doi.org/10.1152/ajplung.00379.2018DOI Listing
June 2019

Comprehensive clinical profiling of the Gauting locoregional lung adenocarcinoma donors.

Cancer Med 2019 04 25;8(4):1486-1499. Epub 2019 Feb 25.

Comprehensive Pneumology Center and Institute for Lung Biology and Disease, University Hospital, Ludwig-Maximilians University of Munich (LMU) and Helmholtz Center Munich, Member of the German Center for Lung Research (DZL), Munich, Bavaria, Germany.

A comprehensive characterization of lung adenocarcinoma (LADC) clinical features is currently missing. We prospectively evaluated Caucasian patients with early-stage LADC. Patients with LADC diagnosed between 2011 and 2015 were prospectively assessed for lung resection with curative intent. Fifty clinical, pathologic, radiologic, and molecular variables were recorded. Patients were followed till death/study conclusion. The main findings were compared to a separate cohort from France. Of 1943 patients evaluated, 366 were enrolled (18.8%; 181 female; 75 never-smokers; 28% of registered Bavarian cases over the study period). Smoking and obstruction were significantly more prevalent in GLAD compared with adult Bavarians (P < 0.0001). Ever-smoker tumors were preferentially localized to the upper lobes. We observed 120 relapses and 74 deaths over 704 cumulative follow-up years. Median overall and disease-free survival were >7.5 and 3.6 years, respectively. Patients aged <45 or >65 years, resected >60 days postdiagnosis, with abnormal FVC/DL V , N2/N3 stage, or solid histology had significantly decreased survival estimates. These were fit into a weighted locoregional LADC death risk score that outperformed pTNM7 in predicting survival in the GLAD and in our second cohort. We define the clinical gestalt of locoregional LADC and provide a new clinical tool to predict survival, findings that may aid future management and research design.
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http://dx.doi.org/10.1002/cam4.2031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488114PMC
April 2019

Highlight: The 7th International Symposium on Kallikreins and Kallikrein-Related Peptidases.

Authors:
Yves Courty

Biol Chem 2018 09;399(9):921

Centre d'Etude des Pathologies Respiratoires (CEPR) INSERM U1100, Faculté de Médecine, Université de Tours, 10 bvd Tonnellé, F-37032 Tours Cedex, France.

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http://dx.doi.org/10.1515/hsz-2018-0326DOI Listing
September 2018

Kallikrein-related peptidase 5 and seasonal influenza viruses, limitations of the experimental models for activating proteases.

Biol Chem 2018 09;399(9):1053-1064

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, F-37032 Tours, France.

Every year, influenza A virus (IAV) affects and kills many people worldwide. The viral hemagglutinin (HA) is a critical actor in influenza virus infectivity which needs to be cleaved by host serine proteases to exert its activity. KLK5 has been identified as an activating protease in humans with a preference for the H3N2 IAV subtype. We investigated the origin of this preference using influenza A/Puerto Rico/8/34 (PR8, H1N1) and A/Scotland/20/74 (Scotland, H3N2) viruses. Pretreatment of noninfectious virions with human KLK5 increased infectivity of Scotland IAV in MDCK cells and triggered influenza pneumonia in mice. These effects were not observed with the PR8 IAV. Molecular modeling and in vitro enzymatic studies of peptide substrates and recombinant HAs revealed that the sequences around the cleavage site do not represent the sole determinant of the KLK5 preference for the H3N2 subtype. Using mouse Klk5 and Klk5-deficient mice, we demonstrated in vitro and in vivo that the mouse ortholog protease is not an IAV activating enzyme. This may be explained by unfavorable interactions between H3 HA and mKlk5. Our data highlight the limitations of some approaches used to identify IAV-activating proteases.
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http://dx.doi.org/10.1515/hsz-2017-0340DOI Listing
September 2018

Kallikrein-related peptidases in lung diseases.

Biol Chem 2018 09;399(9):959-971

Centre d'Etude des Pathologies Respiratoires, INSERM UMR 1100, Faculté de Médecine, 10 Boulevard Tonnellé, F-37032 Tours, France.

Human tissue kallikreins (KLKs) are 15 members of the serine protease family and are present in various healthy human tissues including airway tissues. Multiple studies have revealed their crucial role in the pathophysiology of a number of chronic, infectious and tumour lung diseases. KLK1, 3 and 14 are involved in asthma pathogenesis, and KLK1 could be also associated with the exacerbation of this inflammatory disease caused by rhinovirus. KLK5 was demonstrated as an influenza virus activating protease in humans, and KLK1 and 12 could also be involved in the activation and spread of these viruses. KLKs are associated with lung cancer, with up- or downregulation of expression depending on the KLK, cancer subtype, stage of tumour and also the microenvironment. Functional studies showed that KLK12 is a potent pro-angiogenic factor. Moreover, KLK6 promotes malignant-cell proliferation and KLK13 invasiveness. In contrast, KLK8 and KLK10 reduce proliferation and invasion of malignant cells. Considering the involvement of KLKs in various physiological and pathological processes, KLKs appear to be potential biomarkers and therapeutic targets for lung diseases.
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http://dx.doi.org/10.1515/hsz-2018-0114DOI Listing
September 2018

Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs.

J Virol 2017 08 27;91(16). Epub 2017 Jul 27.

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, Tours, France

Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype , but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans. Influenza A viruses (IAVs) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered promising targets for the development of new antivirals. However, the specific proteases that activate seasonal influenza viruses, especially H3N2 viruses, in the human respiratory tract have remain undefined despite many years of work. Here we demonstrate that the secreted, extracellular protease KLK5 (kallikrein-related peptidase 5) is efficient in promoting the infectivity of H3N2 IAV and Furthermore, we found that its secretion was selectively enhanced in the human lower respiratory tract during a seasonal outbreak dominated by an H3N2 virus. Collectively, our data support the clinical relevance of this protease in human influenza pathogenesis.
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http://dx.doi.org/10.1128/JVI.00421-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533929PMC
August 2017

Downregulation of the neonatal Fc receptor expression in non-small cell lung cancer tissue is associated with a poor prognosis.

Oncotarget 2016 08;7(34):54415-54429

Université François Rabelais, UMR 1100, Tours, France.

Lung cancer is the leading cause of cancer-related death worldwide. Although the recommended tumor, node and metastasis (TNM) classification and stage determination are important to select therapeutic options for patients with non-small cell lung carcinoma (NSCLC), additional molecular markers are required to indicate the prognosis, in particular within a specific stage, and help with the management of patients.Because neonatal Fc receptor (FcRn) has recently been involved in colon cancer immunosurveillance, we measured its expression in non-cancerous and NSCLC lung tissues and evaluated its prognostic value in overall survival for patient with NSCLC. FcRn expression was determined at both mRNA and protein levels on cancerous and adjacent non-cancerous tissues from 80 NSCLC patients. In NSCLC, FcRn was mainly found in resident and tumor infiltrating immune cells. The corresponding mRNA and protein were significantly less abundant in lung tumor than non-cancerous tissue. Moreover, analysis of our cohort and datasets from the public data bases show that FCGRT mRNA down-regulation is a robust and independent, unfavorable predictive factor of NSCLC patient survival. We conclude that FCGRT mRNA expression may be a useful additional marker for immunoscoring, reflecting tumor immune system, and help in the decision-making process for NSCLC patients.
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http://dx.doi.org/10.18632/oncotarget.10074DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5342352PMC
August 2016

Altered expression of the CCN genes in the lungs of mice in response to cigarette smoke exposure and viral and bacterial infections.

Gene 2016 Jul 11;586(1):176-83. Epub 2016 Apr 11.

INSERM U1100, Centre d'Etude des Pathologies Respiratoires, Faculté de Médecine, Tours, France; Université François Rabelais, Tours, France. Electronic address:

The CCN proteins are key signaling and regulatory molecules involved in many biological functions and contribute to malignant and non-malignant lung diseases. Despite the high morbidity and mortality of the lung respiratory infectious diseases, there is very little data related to the expression of the CCNs during infection. We investigated in mice the pulmonary mRNA expression levels of five CCNs (1 to 5) in response to influenza A virus (IAV) and bacterial agents (Nontypeable Haemophilus influenzae (NTHi), lipopolysaccharide (LPS) and lipoteichoic acid (LTA)). IAV, NTHi, LPS or LTA were instilled intranasally into mice. Mice were also exposed for 4days or 8weeks to cigarette smoke alone or prior infection to IAV in order to determine if CS modifies the CCN response to a viral infection. All challenges induced a robust inflammation. The mRNA expression of CCN1, CCN2 and CCN3 was decreased after short exposure to CS whereas prolonged exposure altered the expression of CCN1, CCN3 and CCN4. Influenza A virus infection increased CCN1, 2, 4 and 5 mRNA levels but expression of CCN3 was significantly decreased. Acute CS exposure prior infection had little effect on the expression of CCN genes but prolonged exposure abolished the IAV-dependent induction. Treatment with LPS or LTA and infection with NTHi revealed that both Gram-positive and Gram-negative bacteria rapidly modulate the expression of the CCN genes. Our findings reveal that several triggers of lung inflammation influence differently the CCN genes. CCN3 deserves special attention since its mRNA expression is decreased by all the triggers studied.
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http://dx.doi.org/10.1016/j.gene.2016.04.022DOI Listing
July 2016

Interleukin-22 receptor is overexpressed in nonsmall cell lung cancer and portends a poor prognosis.

Eur Respir J 2016 Apr 4;47(4):1277-80. Epub 2016 Feb 4.

INSERM, Centre d'Etude des Pathologies Respiratoires, U1100, Tours, France Université François Rabelais de Tours, Tours, France

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http://dx.doi.org/10.1183/13993003.01580-2015DOI Listing
April 2016

Neutrophil proteases alter the interleukin-22-receptor-dependent lung antimicrobial defence.

Eur Respir J 2015 Sep 6;46(3):771-82. Epub 2015 Aug 6.

INSERM, Centre d'Etude des Pathologies Respiratoires, U1100, Tours, France Université François Rabelais de Tours, Tours, France

Chronic obstructive pulmonary disease (COPD) is punctuated by episodes of infection-driven acute exacerbations. Despite the life-threatening nature of these exacerbations, the underlying mechanisms remain unclear, although a high number of neutrophils in the lungs of COPD patients is known to correlate with poor prognosis. Interleukin (IL)-22 is a cytokine that plays a pivotal role in lung antimicrobial defence and tissue protection. We hypothesised that neutrophils secrete proteases that may have adverse effects in COPD, by altering the IL-22 receptor (IL-22R)-dependent signalling.Using in vitro and in vivo approaches as well as reverse transcriptase quantitative PCR, flow cytometry and/or Western blotting techniques, we first showed that pathogens such as the influenza virus promote IL-22R expression in human bronchial epithelial cells, whereas Pseudomonas aeruginosa, bacterial lipopolysaccharide or cigarette smoke do not. Most importantly, neutrophil proteases cleave IL-22R and impair IL-22-dependent immune signalling and expression of antimicrobial effectors such as β-defensin-2. This proteolysis resulted in the release of a soluble fragment of IL-22R, which was detectable both in cellular and animal models as well as in sputa from COPD patients with acute exacerbations.Hence, our study reveals an unsuspected regulation by the proteolytic action of neutrophil enzymes of IL-22-dependent lung host response. This process probably enhances pathogen replication, and ultimately COPD exacerbations.
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http://dx.doi.org/10.1183/09031936.00215114DOI Listing
September 2015

Kallikrein-related peptidase 13: an independent indicator of favorable prognosis for patients with nonsmall cell lung cancer.

Tumour Biol 2015 Jul 13;36(7):4979-86. Epub 2015 Feb 13.

INSERM, UMR 1100, F-37032, Tours, France.

The KLK13 gene is dysregulated in several carcinomas, and its expression levels seem to be associated with disease prognosis. The aim of our study was to investigate the prognostic potential of KLK13 mRNA expression for patients with nonsmall cell lung cancer (NSCLC). Total RNA was isolated from cancerous and normal tissues from a cohort of 128 NSCLC patients. The KLK13 mRNA transcription levels were measured using a sensitive quantitative RT-PCR method. The results were normalized by dividing the KLK13 mRNA values with the geometric mean of mRNA expression from four reference genes: beta-actin, TATA-binding protein, hypoxanthine phosphoribosyltransferase 1, and acidic ribosomal phosphoprotein P0. The malignant tissues from the majority of patients (59.3 %) contained significantly more KLK13 mRNA transcripts than did the paired nonmalignant tissues (median difference 11.1-fold, P = 0.008). KLK13 was expressed at higher levels in females than that in males (P = 0.021). No other statistically significant association with clinicopathological data was observed. Kaplan-Meier survival analyses demonstrated that patients with KLK13-positive tumors survived significantly longer than those with KLK13-negative ones (P = 0.009). KLK13 expression was also shown to be able to stratify high-risk individuals among patients with early disease stages (P = 0.030). Multivariate Cox regression analysis showed that KLK13 expression is a favorable, independent prognostic indicator of overall survival (OS) (P = 0.024). Our results suggest that KLK13 mRNA expression constitutes a novel biomarker for the prediction of overall survival in NSCLC and that its quantitative assessment in tumor tissues can aid in treatment decision making.
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http://dx.doi.org/10.1007/s13277-015-3148-1DOI Listing
July 2015

Development of monoclonal antibodies to human kallikrein-related peptidase 6 (KLK6) and their use in an immunofluorometric assay for free KLK6.

Biol Chem 2014 Sep;395(9):1119-26

We have raised monoclonal antibodies against KLK6 and constructed a 'sandwich' type immunoassay using 8A8G3 as capture and 3H3E9 as tracer antibodies. 8A8G3 bound to one side of KLK6, whereas 3H3E9 probably bound near its catalytic site. The assay did not detect complexed forms of KLK6, indicating that it quantifies only free KLK6 (fKLK6). fKLK6 was higher in serum of patients with ovarian cancer (11.34 μg/l±1.37) than in controls (3.39 μg/l±0.42). The cerebrospinal fluid contained high concentrations of fKLK6 (257-965 μg/l). This assay could facilitate the evaluation of fKLK6 in human diseases.
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http://dx.doi.org/10.1515/hsz-2014-0133DOI Listing
September 2014

Growth and survival of lung cancer cells: regulation by kallikrein-related peptidase 6 via activation of proteinase-activated receptor 2 and the epidermal growth factor receptor.

Biol Chem 2014 Sep;395(9):1015-25

The dysregulated expression of kallikrein-related peptidase 6 (KLK6) is involved in non-small cancer (NSCLC) cell growth. However, the mechanism that sustains KLK6 signaling remains unknown. We used an isogenic non-small cell lung cancer (NSCLC) cell model system to demonstrate that KLK6 promotes the proliferation of lung tumoral cells and restrains their apoptosis in vitro via ligand-dependent EGFR transactivation. KLK6 activated the ERK and Akt pathways and triggered the nuclear translocation of β-catenin. The stimulating effects of KLK6 required its proteolytic activity and were dependent on the protease-activated receptor 2 (PAR2). These observations support the concept of a role for KLK6 in the oncogenesis of NSCLC.
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http://dx.doi.org/10.1515/hsz-2014-0124DOI Listing
September 2014

Angiogenesis stimulated by human kallikrein-related peptidase 12 acting via a platelet-derived growth factor B-dependent paracrine pathway.

FASEB J 2014 Feb 13;28(2):740-51. Epub 2013 Nov 13.

2CEPR INSERM U1100/EA 6305, Faculté de Médecine, 10 Blvd. Tonnellé, F-37032 Tours cedex, France.

KLK12, a kallikrein peptidase, is thought to take part in the control of angiogenesis. Our analysis of the secretome of endothelial cells (ECs) that had been treated with KLK12 showed that KLK12 converts the extracellular matrix- or membrane-bound precursor of platelet-derived growth factor B (PDGF-B) into a soluble form. Both PDGF-B and vascular endothelial growth factor A (VEGF-A) take part in the induction of angiogenesis by KLK12 in a coculture model of angiogenesis that mimics endothelial tubule formation. We used a cellular approach to analyze the interplay between KLK12, PDGF-B, and VEGF-A and showed that release of PDGF-B by KLK12 leads to the fibroblast-mediated secretion of VEGF-A. This then stimulates EC differentiation and the formation of capillary tube-like structures. Thus, KLK12 favors the interaction of ECs and stromal cells. The released PDGF-B acts as a paracrine factor that modulates VEGF-A secretion by stromal cells, which ultimately leads to angiogenesis. Moreover, the genes encoding KLK12 and PDGFB are both expressed in ECs and up-regulated in tumor cells kept under hypoxic conditions, which is consistent with the physiological involvement of KLK12 in PDGF-B maturation.
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http://dx.doi.org/10.1096/fj.13-237503DOI Listing
February 2014

Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer.

FEBS Open Bio 2013 27;3:291-301. Epub 2013 Jun 27.

EA 6305, Université François Rabelais de Tours, Tours F-37032, France ; Centre d'Etude des Pathologies Respiratoires, UMR 1100/EA6305, Tours F-37032, France.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC.
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http://dx.doi.org/10.1016/j.fob.2013.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3722576PMC
August 2013

Benign lower limb amyotrophy due to TARDBP mutation or post-polio syndrome?

Amyotroph Lateral Scler Frontotemporal Degener 2013 Sep 28;14(5-6):476-8. Epub 2013 Jan 28.

Centre SLA, CHU Bretonneau, 2 Boulevard Tonnellé, Tours Cedex 1, France.

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http://dx.doi.org/10.3109/21678421.2013.764567DOI Listing
September 2013

Pro-angiogenic effect of human kallikrein-related peptidase 12 (KLK12) in lung endothelial cells does not depend on kinin-mediated activation of B2 receptor.

Biol Chem 2013 Mar;394(3):385-91

Université François Rabelais, EA 6305, F-37032 Tours, France.

Kallikrein-12 (KLK12) may play an important role in angiogenesis modulating proangiogenic factor bioavailability and activating the kinin receptor B2 pathway. We studied whether KLK12 had an impact on angiogenesis and the activation of kinin receptor B2 results from the KLK12-dependent generation of kinins. KLK12 efficiently hydrolyzed high molecular weight kininogen, liberating a fragment containing the carboxy-terminal end of kinins. The kininogenase activity of KLK12 was poor, however, due to the cleavage resistance of the N-terminal side of the kinin sequence. A very low amount of kinins was accordingly released after in vitro incubation of high molecular weight kininogen with KLK12 and thus the proangiogenic activity of KLK12 in lung endothelial cells was not related to a kinin release.
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http://dx.doi.org/10.1515/hsz-2012-0291DOI Listing
March 2013

Tenascin-W is a better cancer biomarker than tenascin-C for most human solid tumors.

BMC Clin Pathol 2012 Sep 4;12:14. Epub 2012 Sep 4.

Friedrich Miescher Institute for Biomedical Research, Novartis Research Foundation, Basel, Switzerland.

Background: Tenascins are large glycoproteins found in the extracellular matrix of many embryonic and adult tissues. Tenascin-C is a well-studied biomarker known for its high overexpression in the stroma of most solid cancers. Tenascin-W, the least studied member of the family, is highly expressed in the stroma of colon and breast tumors and in gliomas, but not in the corresponding normal tissues. Other solid tumors have not been analyzed. The present study was undertaken to determine whether tenascin-W could serve as a cancer-specific extracellular matrix protein in a broad range of solid tumors.

Methods: We analyzed the expression of tenascin-W and tenascin-C by immunoblotting and by immunohistochemistry on multiple frozen tissue microarrays of carcinomas of the pancreas, kidney and lung as well as melanomas and compared them to healthy tissues.

Results: From all healthy adult organs tested, only liver and spleen showed detectable levels of tenascin-W, suggesting that tenascin-W is absent from most human adult organs under normal, non-pathological conditions. In contrast, tenascin-W was detectable in the majority of melanomas and their metastases, as well as in pancreas, kidney, and lung carcinomas. Comparing lung tumor samples and matching control tissues for each patient revealed a clear overexpression of tenascin-W in tumor tissues. Although the number of samples examined is too small to draw statistically significant conclusions, there seems to be a tendency for increased tenascin-W expression in higher grade tumors. Interestingly, in most tumor types, tenascin-W is also expressed in close proximity to blood vessels, as shown by CD31 co-staining of the samples.

Conclusions: The present study extends the tumor biomarker potential of tenascin-W to a broad range of solid tumors and shows its accessibility from the blood stream for potential therapeutic strategies.
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http://dx.doi.org/10.1186/1472-6890-12-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444373PMC
September 2012

Kallikrein-related peptidase 12 hydrolyzes matricellular proteins of the CCN family and modifies interactions of CCN1 and CCN5 with growth factors.

J Biol Chem 2011 Jul 31;286(29):25505-18. Epub 2011 May 31.

INSERM U618-Université François Rabelais, Faculté de Médecine, 2 bis bd Tonnellé, 37032 Tours, France.

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF(165) binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-β1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.
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http://dx.doi.org/10.1074/jbc.M110.213231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138267PMC
July 2011

The airways, a novel route for delivering monoclonal antibodies to treat lung tumors.

Pharm Res 2011 Sep 14;28(9):2147-56. Epub 2011 Apr 14.

INSERM U618, Université François Rabelais de Tours, Tours, France.

Purpose: Lung cancer is the leading cause of cancer-related death worldwide. The efficacy of current systemic treatments is limited, with major side effects and only modest survival improvements. Aerosols routinely used to deliver drugs into the lung for treating infectious and inflammatory lung diseases have never been used to deliver monoclonal antibodies to treat lung cancer. We have shown that cetuximab, a chimeric anticancer anti-EGFR mAb, is suitable for airway delivery as it resists the physical constraints of aerosolization, and have evaluated the aerosol delivery of cetuximab in vivo.

Methods: We developed an animal model of lung tumor sensitive to cetuximab by injecting Balb/c Nude mice intratracheally with A431 cells plus 10 mM EDTA and analyzed the distribution, pharmacokinetics and antitumor efficacy of cetuximab aerosolized into the respiratory tract.

Results: Aerosolized IgG accumulated durably in the lungs and the tumor, but passed poorly and slowly into the systemic circulation. Aerosolized cetuximab also limited the growth of the mouse tumor. Thus, administering anticancer mAbs via the airways is effective and may limit systemic side effects.

Conclusion: Delivery of aerosolized-mAbs via the airways deserves further evaluation for treating lung cancers.
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http://dx.doi.org/10.1007/s11095-011-0442-5DOI Listing
September 2011

Alternative splicing variant of kallikrein-related peptidase 8 as an independent predictor of unfavorable prognosis in lung cancer.

Clin Chem 2010 Jun 1;56(6):987-97. Epub 2010 Apr 1.

Université François-Rabelais de Tours, Tours, France.

Background: A relatively unexplored area for biomarker identification is alternative splice variants. We undertook this study to evaluate the usefulness of mRNA isoforms encoded by the KLK8 (kallikrein-related peptidase 8) gene as prognostic markers for lung cancer.

Methods: Real-time reverse-transcription PCR was used to analyze the mRNAs encoded by KLK8 (particularly 2 mRNA splice variants, KLK8-T3 and KLK8-T4) in 60 non-small-cell lung cancer (NSCLC) tumors and in paired unaffected tissues. The ratios of these mRNAs to those encoded by the KLK5, KLK6, KLK7, KLK10, KLK11, KLK13, and KLK14 genes were also determined and analyzed for correlations with various clinicopathologic variables.

Results: KLK8-T3 and KLK8-T4 were the most abundant of the 6 mRNA isoforms identified in lung tissues. The overall expression of the KLK8 gene and the amounts of the KLK8-T3 and KLK8-T4 mRNAs were significantly increased in lung tumor tissue (P < 0.0001). Univariate survival analysis revealed significant relationships of the relative concentrations of mRNA splice variants KLK8 (P = 0.043), KLK8-T3 (P = 0.037), and KLK8-T4 (P = 0.009) with overall survival (OS). Cox multivariate analysis indicated that the amount of KLK8-T4 mRNA was an independent prognostic factor for OS (relative risk = 3.90; P = 0.016) and that high KLK8-T4/KLK7, KLK8-T4/KLK10, and KLK8-T4/KLK11 mRNA ratios in NSCLC indicated increased risk of death. The increase was approximately 5-fold for the KLK8-T4/KLK7 and KLK8-T4/KLK10 ratios (P = 0.006, and P = 0.011, respectively) and 8-fold for the KLK8-T4/KLK11 ratio (P = 0.001).

Conclusions: The KLK8-T4 alternative splice variant, alone or in combination, may be a new independent marker of unfavorable prognosis in lung cancer.
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http://dx.doi.org/10.1373/clinchem.2009.138917DOI Listing
June 2010

TFPI-2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour-stromal cell interactions.

J Cell Mol Med 2011 Feb;15(2):196-208

Inserm, U618, Université François Rabelais, Tours, France.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI-2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI-2 is frequently down-regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI-2 down-regulation in the National Cancer Institute (NCI)-H460 non-small cell lung cancer cell line using specific micro interfering micro-interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI-2 down-regulation enhanced cell adhesion to collagen IV and laminin via an increase in α(1) integrin on cell surface, and increased MMP expression (mainly MMP-1 and -3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI-2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP-1, -3 and -7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00989.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3822788PMC
February 2011

High kallikrein-related peptidase 6 in non-small cell lung cancer cells: an indicator of tumour proliferation and poor prognosis.

J Cell Mol Med 2009 Sep 1;13(9B):4014-22. Epub 2009 May 1.

INSERM, Université François-Rabelais de Tours, Tours, France.

The human kallikrein-related peptidases (KLK) are serine proteases whose concentrations are often abnormal in common human malignancies and contribute to neoplastic progression through multifaceted roles. However, little attention has been paid to their synthesis and involvement in the development and dissemination of lung cancer, the leading cause of cancer mortality worldwide. We have analysed the production of KLK6 in normal lung and tumour tissues from patients with non-small cell lung cancer (NSCLC). KLK6 immunoreactivity was restricted to epithelial cells of the normal bronchi, but most of the cancer samples were moderately or highly immunoreactive, regardless of the histological subtype. In contrast, little or no KLK6 was detected in NSCLC cells. We have developed NSCLC lines expressing wild-type KLK6 in order to investigate the role of KLK6 in lung cancer biology, and analysed its impact on proliferation. Ectopic KLK6 dramatically enhanced NSCLC cell growth and KLK6-producing NSCLC cells had accelerated cell cycles, between the G1 and S phases. This was accompanied by a marked increase in cyclin E and decrease in p21. KLK6 production was also associated with enhanced synthesis of c-Myc, which is known to promote cell-cycle progression. Finally, examination of specimens from patients with NSCLC revealed that KLK6 mRNA is overexpressed in tumour tissue, and high KLK6 concentrations were associated with lower survival rates. We conclude that a high concentration of KLK6 is an indicator of tumour proliferation and an independent predictive factor in NSCLC.
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http://dx.doi.org/10.1111/j.1582-4934.2009.00763.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516548PMC
September 2009

Quantitative RT-PCR analysis and immunohistochemical localization of the kallikrein-related peptidases 13 and 14 in lung.

Biol Chem 2008 Jun;389(6):781-6

INSERM U618 Protéases et Vectorisation Pulmonaires, Faculté de Médecine, F-37000 Tours, France.

Expression of the KLK13 and KLK14 genes was examined at the mRNA and protein levels in a cohort of 57 patients with non-small-cell lung cancer (NSCLC). The mRNA levels, assessed by real-time RT-PCR, were significantly different in malignant tissues compared to adjacent non-malignant tissues (KLK13, p=0.006; KLK14, p=0.022). KLK13 and KLK14 mRNA overexpression in tumors (1/3 of the patients) was associated with a positive nodal status in multivariate analysis (p=0.018 and p=0.069, respectively). KLK13 and KLK14 were localized in the cytoplasm of epithelial cells of normal bronchus and NSCLC, as determined by immunohistochemistry. Moreover, positive staining was significantly associated with adenocarcinoma histotype (KLK13, p=0.014) and tumor size (KLK14, p=0.048). Although the results are marginally significant, patients with high KLK13 expression at the mRNA or protein level had lower overall survival.
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http://dx.doi.org/10.1515/BC.2008.089DOI Listing
June 2008

Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization.

Pharm Res 2008 Jun;25(6):1318-26

U618, Protéases et vectorisation Pulmonaires, Université François-Rabelais de Tours, Tours, France.

Purpose: Despite an increasing interest in the use of inhalation for local delivery of molecules for respiratory diseases and systemic disorders, methods to deliver therapy through airways has received little attention for lung cancer treatment. However, inhalation of anticancer drugs is an attractive alternative route to systemic administration which results in limited concentration of the medication in the lungs, and triggers whole-body toxicity. In this study, we investigated the feasibility of nebulization for therapeutic antibodies, a new class of fully-approved anticancer drugs in oncology medicine.

Materials And Methods: Cetuximab, a chimeric IgG1 targeting the epidermal growth factor receptor (EGFR), was nebulized using three types of delivery devices: a jet nebulizer PARI LC+, a mesh nebulizer AeronebPro and an ultrasonic nebulizer SYST'AM LS290. Aerosol size distribution was measured using a cascade impactor and aerosol droplets were observed under optical microscopy. The immunological and pharmacological properties of cetuximab were evaluated following nebulization using A431 cells.

Results: The aerosol particle clouds generated with the three nebulizers displayed similar aerodynamical characteristics, but the IgG formed aggregates in liquid phase following nebulization with both the jet and ultrasonic devices. Flow cytometry analyses and assays of EGFR-phosphorylation and cell growth inhibitions on A431 demonstrated that both the mesh and the jet nebulizers preserved the binding affinity to EGFR and the inhibitory activities of cetuximab.

Conclusions: Altogether, our results indicate that cetuximab resists the physical constraints of nebulization. Thus, airway delivery represents a promising alternative to systemic administration for local delivery of therapeutic antibodies in lung cancer treatment.
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http://dx.doi.org/10.1007/s11095-007-9481-3DOI Listing
June 2008

Expression of the human kallikrein genes 10 (KLK10) and 11 (KLK11) in cancerous and non-cancerous lung tissues.

Biol Chem 2006 Jun;387(6):783-8

INSERM U618'Protéases et Vectorisation Pulmonaires', F-37000 Tours, France.

Only one transcript for KLK10 was identified by RT-PCR in lung tissue, whereas KLK11 expressed at least four alternative transcripts. Quantitative analysis of KLK10 and KLK11 expression levels was assessed by real-time PCR, in a cohort of 47 patients with non-small-cell lung cancer (NSCLC). Expression levels of these genes were widely distributed in the population studied. Multivariate analysis revealed a correlation between KLK10 over-expression and the squamous cell carcinoma histotype (p=0.034). There was no correlation between gene expression and patient survival. Overall, both genes behaved similarly (p<0.001). These results suggest a co-regulation of KLK10 and KLK11 expression in lung and a lack of KLK10 suppressor role in NSCLC. Finally, our findings indicate that these genes are likely involved in normal physiology processes in bronchus.
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http://dx.doi.org/10.1515/BC.2006.098DOI Listing
June 2006

Recombinant kallikrein expression: site-specific integration for hK6 production in human cells.

Biol Chem 2006 Jun;387(6):687-95

INSERM, U618 Protéases et Vectorisation Pulmonaires, F-37032 Tours, France.

Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.
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http://dx.doi.org/10.1515/BC.2006.087DOI Listing
June 2006

A comprehensive nomenclature for serine proteases with homology to tissue kallikreins.

Biol Chem 2006 Jun;387(6):637-41

Lund University, Clinical Chemistry, Department of Laboratory Medicine, University Hospital MAS, S-205 02 Malmö, Sweden.

The human kallikrein locus on chromosome 19q13.3-13.4 contains kallikrein 1--the tissue kallikrein--and 14 related serine proteases. Recent investigations into their function and evolution have indicated that the present nomenclature for these proteins is inadequate or insufficient. Here we present a new nomenclature in which proteins without proven kininogenase activity are denoted kallikrein-related peptidase. Names are also given to the unique rodent proteins that are closely related to kallikrein 1.
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http://dx.doi.org/10.1515/BC.2006.082DOI Listing
June 2006