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Legends to supplementary figures and tables. Contains detailed legends for supplementary figures S1-5 and supplementary tables 1 and 2

Supplementary Figure S4. Representative images of healthy lungs and lung tumors from EGFR/rhob mice stained with CD34 antibody, and corresponding quantification

Supplemental Methods. This file contains additional methodology (Tumor/lung ratio quantification, Tumor size measurement and grading, CD34 staining and quantification, lung tissue processing for protein extraction for Western Blotting and PAS diastase staining)

Supplementary Table S2. Kras and EGFR mutations and EGFR expression according to RhoB expression in tumors

Purpose: A crucial event in lung adenocarcinoma progression is the switch from an aerogenous spread toward an infiltrating tumor. Loss of RhoB expression has been suggested to be critical for lung cancer invasion. Here, we tested RhoB expression as a prognostic biomarker in non–small cell lung cancer (NSCLC) with a special focus on lepidic pattern.Experimental Design: We analyzed RhoB expression using both IHC and RT-qPCR in two series of operated patients (n = 100 and 48, respectively) and in a series of advanced lepidic adenocarcinoma (n = 31) from different hospitals. Next, we examined the role of RhoB in lung cancer progression in transgenic mice that express inducible EGFRL858R crossed with Rhob null mice.Results: We identified that loss of RhoB expression was strongly associated with worse survival (P = 0.0001) and progression-free survival (P < 0.001) in the first series. We then confirmed these results after multivariate analyses of the second series. In the series of adenocarcinoma with lepidic features issued from a clinical trial (IFCT-0401), we showed that loss of RhoB expression was associated with higher aggressiveness of stage IV. Finally, we showed that EGFRL858R/Rhob+/+ mice developed mainly diffuse lung tumors with a lepidic pattern, whereas EGFRL858R/Rhob+/− and EGFRL858R/Rhob−/− developed a greater number of tumors, and aggressive adenocarcinomas with invasive properties.Conclusions: We showed that RhoB is not only a strong prognostic factor in NSCLC but it is also critical for the acquisition of an aggressive phenotype of adenocarcinoma. Clin Cancer Res; 20(24); 6541–50. ©2014 AACR.

HBEC-3 cells were transfected with non-silencing shRNA (Control), shRASSF1A-777 and shRASSF1A-779. A) Changes in RASSF1A and Yap expression assessed by immunofluorescence (Red=RASSF1A, Green=Yap, Blue=DAPI) experiments. Scale bar represents 50 µm. B) The migration speed (µm/h) of transfected cells was determined by wound assay on collagen IV. Mitomycin C (1 µg/ml) was added to medium 12h before wounding. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with Control. C) Representative images of colony formation of transfected cells in soft agar. Scale bar represents 20 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

HBEC-3 2D-migration induced by RASS1A-knockdown showed features evoking both collective migration and multicellular streaming

Plasmids used in this study.

The cells were transiently transfected with siNeg, siRASSF1A or siRASSF1AC. The experiences were performed 48 hours after transfection A. The trans-endothelial invasion capacity of transfected cells was assessed using a cell culture insert (BioCoat Matrigel Invasion Chamber) on which a monolayer of endothelial cells has been grown previously. The total number of cells that invaded to the lower side of the filter after 48h was counted under the microscope (10X). The relative invasion was normalized to that of the cells transfected with siNeg. B. The migration speed (µm/h) of transfected HBEC-3 RasV12, H1975 and BEAS-2B cells was determined by wound healing assay on Fibronectin (HBEC-3) or collagen IV (H1975 and BEAS-2B). Mitomycin C (1 µg/ml) was added to medium 12h before wounding. C. The invasion capacity of transfected HBEC-3RasV12, H1975, BEAS-2B, BEAS-2B RasV12 cells was assessed using BioCoat Matrigel Invasion Chambers. The total number of cells that invaded to the lower side of the filter after 48h was counted under the microscope (10X). The relative invasion was normalized to that of the cells transfected with siNeg. D. The capacity of growth without adherence in soft agar of HBEC-3 RasV12 and BEAS-2B cells transfected by an inactive siRNA or SiRASSF1A was evaluated by quantification of colony formation. The number of colonies was scored after 7 days. One representative experiment is shown in a right panel. Scale bar represents 80 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Three independent air-liquid cultures, each requiring 21 days of growth before fixation and observation, were analyzed. For each culture, 2 slides through the porous membrane and the cells have been performed, those slides being processed at the center of the membrane to avoid artifacts linked to a peripheral location. For siNeg condition (only one stratum of cells) 50 to 70 cells were observed, whereas for siRASSF1A conditions, 50 to 250 cells (several strata of cells) were analyzed. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

The HBEC-3 cells were transiently transfected with siNeg, si RASSF1A or siRASSF1AC. The experiences were performed 48 hours after transfection. A. Expression of focal adhesion complex proteins was examined by RT-PCR analysis. B & C. Adhesion strength of the HBEC-3 cells on fibronectin (B) and laminin (C). For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

HBEC-3 cells were transiently transfected with siNeg or siRASSF1A in association with either pcDNA3-RASSF1A or pcDNA3-RASSF1C. The experiences were performed 48 hours after transfection. A. Representative images for RASSF1A expression changes by immunofluorescence and confocal microscopy. RASSF1A primary antibody (Red) followed by Alexa 555 conjugated secondary antibody along with DAPI for DNA (Blue) was used as a control along with DAPI for DNA (Blue). Scale bar represents 50 µm. B. Representative blots for quantification of Twist and Vimentin proteins levels by western blotting in HBEC-3 cells. Actin was used as an internal control. (n=2) C. The migration speed (µm/h) of transfected HBEC-3 cells was determined by wound healing assay on collagen I & IV. Mitomycin C (1 µg/ml) was added to medium 12h before scratch in order to limit cell proliferation. D. Representative images for nuclear localization of Yap in HBEC-3 cell by immunofluorescence and confocal microscopy. The cells stained by Yap primary antibody (Green) followed by Alexa488 conjugated secondary antibody, along with DAPI for DNA (Blue). Scale bar represents 50 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

HBEC-3 cells were transiently transfected with siNeg or siRASSF1A-1 in combination or not with siRhoB A) The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with siNeg. B) Quantification of nuclear localization of Yap by immunofluorescence and confocal microscopy. The cells stained by Yap primary antibody (Green) followed by Alexa488 conjugated secondary antibody along with DAPI for DNA (Blue). Scale bar represents 50 µm. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Representative images are shown for YAP localization changes, by immunofluorescence and confocal microscopy. HBEC-3 cells were transfected by inactive siNeg (upper lane), siRASS1A, siYAP or siRASSF1A plus siYAP (lower lane) and monitored 12, 24, 36 and 48h after transfection for YAP. The cells were stained by Yap primary antibody (Green) followed by Alexa488 conjugated secondary antibody, along with DAPI for DNA (Blue).

legends of all supplemental figures

A) GST pull-down assay in HBEC-3 cells. Quantification of active GTP-bound RhoA levels as determined by western blotting. One representative experiment is shown in a top panel of histogram. B & C) HBEC-3 cells were transiently transfected with non-silencing siRNA (siNeg), siRASSF1A-1 in combination or not with B) three plasmids coding for wt-RhoA, RhoA N19 (dominant negative form) & RhoAV14 (constitutively active form) C) Cells were assayed for their invasive capacities with Narciclasine treatment. The invasion capacity was assessed using a BioCoat Matrigel Invasion Chamber. The relative invasion was normalized to that of the cells transfected with siNeg. D) Nuclear intensity of YAP was assessed by immunofluorescence and confocal microscopy (right panel), upon depletion of RASSF1A, transfection of RhoAwt expressing plasmid, co-transfection of siRASSF1A and RhoAwt plasmid, transfection of dominant negative RhoAN19 plasmid alone, or upon RASSF1A depletion, transfection of constitutively active RhoAV14 plasmid alone or upon RASSF1A depletion. For all of the histograms, error bars indicate the standard error of the mean (SEM) of at least three independent experiments. *p

Inactivation of the tumor suppressor gene RASSF1A by promoter hypermethylation represents a key event underlying the initiation and progression of lung cancer. RASSF1A inactivation is also associated with poor prognosis and may promote metastatic spread. In this study, we investigated how RASSF1A inactivation conferred invasive phenotypes to human bronchial cells. RNAi-mediated silencing of RASSF1A induced epithelial-to-mesenchymal transition (EMT), fomenting a motile and invasive cellular phenotype in vitro and increased metastatic prowess in vivo. Mechanistic investigations revealed that RASSF1A blocked tumor growth by stimulating cofilin/PP2A–mediated dephosphorylation of the guanine nucleotide exchange factor GEF-H1, thereby stimulating its ability to activate the antimetastatic small GTPase RhoB. Furthermore, RASSF1A reduced nuclear accumulation of the Hippo pathway transcriptional cofactor Yes-associated protein (YAP), which was reinforced by RhoB activation. Collectively, our results indicated that RASSF1 acts to restrict EMT and invasion by indirectly controlling YAP nuclear shuttling and activation through a RhoB-regulated cytoskeletal remodeling process, with potential implications to delay the progression of RASSF1-hypermethylated lung tumors. Cancer Res; 76(6); 1627–40. ©2016 AACR.

RASSF1 eight coding exon regions are represented along with the eight different transcripts arising from alternative splicing with the use of two different promoters. Only siRASSF1A and shRASSF1A-777, by annealing to 1alpha exon are able to deplete the mRNA species transcribed from the first promoter proceeding 1alpha(RASSF1A, D, E , F, G, H), whereas siRASSF1AC and shRASSF1A-779, by annealing to exon 4 or 4-5 respectively, are able to deplete all species, since they all contain exon 4. The 737 bp CpG island methylated in human cancers spans the first promoter for RASSF1A, D, E, F, G, H.

Drug-tolerant “dormant” cells (DTC) have emerged as one of the major non-genetic mechanisms driving resistance to targeted therapy in lung cancer, although the sequence of events leading to entry and exit from dormancy remain poorly described. Here, we performed real-time monitoring of the cell cycle dynamics during the adaptive response to Epidermal Growth Factor Receptor tyrosine kinase inhibitors (EGFR-TKi) in a panel of EGFR-mutated lung cancer cell lines. We identified a rare population of S/G2 cycling cells (referred to as early escapers) that emerged in the first hours of treatment amongst stably arrested and progressively dying G1 cells. We determined that early escapers evolved from a non-proliferative differentiated alveolar type 1 (AT1) phenotype which was invariably associated with cytoskeletal remodeling through Rho/ROCK pathway activation. Using a panel of Rho-pathway inhibitors, we found that the farnesyltransferase inhibitor tipifarnib induced a complete clearance of EGFR-TKi-induced DTC thus fully preventing relapse in vitro. Using a xenograft model and a PDX model of EGFRL858R/T790M lung cancer, co-treatment with tipifarnib prevented relapse to osimertinib for up to 6 months with no evidence of toxicity. Among the farnesylated proteins regulated during osimertinib treatment, concomitant inhibition of RHOE and LaminB1 was sufficient to recapitulated FTIs’ effect. Osimertinib and tipifarnib co-treatment completely suppressed the emergence of AT1 phenotype, prevented mitosis of S/G2-treated cells and increased the apoptotic response through activation of ATF4-CHOP-dependent Integrated Stress Response (ISR) pathway. Our data strongly support the use of tipifarnib in combination with osimertinib in patients to effectively and durably prevent relapse.

Purpose: Drug-tolerance has emerged as one of the major non-genetic mechanism that drives resistance to targeted therapies in lung cancers. Identification of drug-tolerant cells (DTC) vulnerabilities thus represents an attractive strategy to prevent relapse in patients. We observed that EGFR-TKI-derived DTC displayed an increased level of DNA Double Strand Breaks (DSB), raising the hypothesis that genomic instability in these cells could favor the onset of resistance mechanisms. We tested AsiDNA, a small molecule that interferes with the DNA repair machinery, to prevent the emergence of resistances Experimental design: AsiDNA is a double stranded DNA molecule (decoy oligonucleotide) that mimics DNA DSB to interfere with DNA repair, by over-activating a false signaling of DNA damage through DNA-PK and PARP enzymes. We used EGFR-mutated (PC9 and HCC827) or ALK-translocated (H3122) lung tumor cells as well as a PC9 xenograft model, to assess whether a long-term combination of AsiDNA and TKI (erlotinib or osimertinib for EGFR-mutated and alectinib for ALK-translocated cells) could prevent the emergence of resistances arising from DTC. We also performed RNAseq to identify molecular mechanisms underlying AsiDNA effect on TKI-derived DTC. Results:Concomitant treatment of TKI with AsiDNA strongly prevented the emergence of TKI-resistant clones in the three cell lines tested, whereas AsiDNA alone did not display cytotoxic activity. Interestingly, AsiDNA didn’t increase the apoptotic activity of TKI but rather prevented the re-entry of drug-tolerant “dormant” cells into a proliferative state, ultimately resulting in DTC death. RNAseq data revealed a strong downregulation of gene signatures associated with cell cycle, DNA repair but also several pathways known to promote DTC survival such as Epithelial-to-Mesenchymal Transition (EMT) or Interferon pathway in AsiDNA+osimertinib vs osimertinib alone. In vivo, AsiDNA strongly interfered with the emergence of resistances in a PC9 xenograft model treated with erlotinib, resulting in a delayed relapse for the majority of tumors and a complete tumor regression for 20% of them, with no evidence of toxicity for mice. Conclusion: Inhibition of the DNA repair machinery using AsiDNA efficiently prevented the emergence of resistances to tyrosine kinase inhibitors and point to the therapeutic opportunity of combining AsiDNA and TKI to overcome resistance in clinical situation. These findings should have immediate implications to design drug combination strategies to prevent TKI-induced resistance in lung cancers. Citation Format: Sarah Figarol, Sandra Pagano, Wael Jdey, Hugo Guillaume, Gilles Favre, Olivier Calvayrac. DNA repair-interfering molecule AsiDNA® overcomes resistance to tyrosine kinase inhibitors in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 656.

International audience; Lung cancer is the leading cause of cancer-related deaths among men and women worldwide. Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective therapies for advanced non-small-cell lung cancer (NSCLC) patients harbouring EGFR-activating mutations, but are not curative due to the inevitable emergence of resistances. Recent in vitro studies suggest that resistance to EGFR-TKI may arise from a small population of drug-tolerant persister cells (DTP) through non-genetic reprogramming, by entering a reversible slow-to-non-proliferative state, before developing genetically derived resistances. Deciphering the molecular mechanisms governing the dynamics of the drug-tolerant state is therefore a priority to provide sustainable therapeutic solutions for patients. An increasing number of molecular mechanisms underlying DTP survival are being described, such as chromatin and epigenetic remodelling, the reactivation of anti-apoptotic/survival pathways, metabolic reprogramming, and interactions with their micro-environment. Here, we review and discuss the existing proposed mechanisms involved in the DTP state. We describe their biological features, molecular mechanisms of tolerance, and the therapeutic strategies that are tested to target the DTP.

EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFR(T790M) and EGFR(C797S). It is generally assumed that these secondary mutations render EGFR completely unresponsive to the inhibitors, but contrary to this, we uncovered here that gefitinib and osimertinib increased STAT3 phosphorylation (p-STAT3) in EGFR(T790M) and EGFR(C797S) tumoral cells. Interestingly, we also found that concomitant Notch inhibition with gefitinib or osimertinib treatment induced a p-STAT3–dependent strong reduction in the levels of the transcriptional repressor HES1. Importantly, we showed that tyrosine kinase inhibitor–resistant tumors, with EGFR(T790M) and EGFR(C797S) mutations, were highly responsive to the combined treatment of Notch inhibitors with gefitinib or osimertinib, respectively. Finally, in patients with EGFR mutations treated with tyrosine kinase inhibitors, HES1 protein levels increased during relapse and correlated with shorter progression-free survival. Therefore, our results offer a proof of concept for an alternative treatment to chemotherapy in lung adenocarcinoma osimertinib-treated patients after disease progression.

Although lung cancer patients harboring EGFR mutations benefit from treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKI), most of them rapidly relapse. RHOB GTPase is a critical player in both lung carcinogenesis and the EGFR signaling pathway; therefore, we hypothesized that it could play a role in the response to EGFR-TKI. In a series of samples from EGFR-mutated patients, we found that low RHOB expression correlated with a good response to EGFR-TKI treatment while a poor response correlated with high RHOB expression (15.3 versus 5.6 months of progression-free survival). Moreover, a better response to EGFR-TKI was associated with low RHOB levels in a panel of lung tumor cell lines and in a lung-specific tetracycline-inducible EGFR L858R transgenic mouse model. High RHOB expression was also found to prevent erlotinib-induced AKT inhibition in vitro and in vivo. Furthermore, a combination of the new-generation AKT inhibitor G594 with erlotinib induced tumor cell death in vitro and tumor regression in vivo in RHOB-positive cells. Our results support a role for RHOB/ AKT signaling in the resistance to EGFR-TKI and propose RHOB as a potential predictor of patient response to EGFR-TKI treatment.

Introduction Les pneumopathies interstitielles diffuses (PID) representent un groupe heterogene de maladies dont certaines sont caracterisees par la presence d’une fibrose evolutive et mutilante. Elles sont connues pour representer un facteur de risque independant de cancer primitif bronchique dont le mecanisme physiopathologique est mal connu et la prise en charge therapeutique complexe. Notre objectif est d’apporter la description d’une cohorte de patients presentant un cancer broncho-pulmonaire compliquant une PID fibrosante et d’en caracteriser le profil moleculaire. Methodes L’etude Fibro-K est une etude de cohorte, monocentrique, realisee au CHU de Toulouse, incluant des patients presentant un cancer broncho-pulmonaire associe a une PID fibrosante preexistante ou synchrone. Les donnees cliniques ont ete analysees de facon retrospective. Une analyse immunohistochimique (PD-L1, CD8) et moleculaire par sequencage a « haut debit » (FoundationOne®CDx, Foundation Medicine Inc) a ete realisee sur le materiel tumoral archive. La base de donnees Reactome a permis l’analyse des voies de signalisation moleculaires impactees. Resultats Quarante-neuf patients ont ete analyses. L’histologie la plus representee etait l’adenocarcinome (65.3 %), suivi du carcinome epidermoide (30.6 %). Il existait une majorite de fibroses pulmonaires idiopathiques (30.6 %) et de PID associees aux connectivites (22.4 %), notamment a la sclerodermie. L’aspect scanographique de pneumopathie interstitielle commune dominait. Une proportion importante de stades tumoraux precoces etait observee et la reponse aux traitements etait comparable aux donnees de population generale mais avec une survie inferieure (34.5 mois pour les stades localises et 11.2 mois pour les stades metastatiques). La principale cause de mortalite etait une complication evolutive du cancer. L’etude immunohistochimique realisee chez 28 patients retrouvait une expression de PD-L1 faible (0 %) a intermediaire (1–49 %). La charge mutationnelle etait basse dans 69.2 % des cas et le statut microsatellitaire stable dans 100 % des cas sur 14 prelevements tumoraux eligibles a une analyse moleculaire. Le genotypage de ces echantillons montrait une atteinte frequente du gene TP53 et l’implication de voies de signalisation communes avec les processus fibrosants telles que TGFβ et PI3 K/AKT. Conclusion Ces observations suggerent un continuum dans le developpement de la fibrose vers le cancer et peuvent etre la base de strategies therapeutiques specifiques.

Lung cancer is the leading cause of cancer mortality worldwide, with adenocarcinomas of the non‐small cell lung carcinoma (NSCLC) subtype accounting for the majority of cases. Therefore, an urgent need exists for a more detailed dissection of the molecular events driving NSCLC development and the identification of clinically relevant biomarkers. Even though originally identified as a tumour suppressor, recent studies associate the cytoplasmically (mis)localised CDK inhibitor p27Kip1 (p27) with unfavourable responses to chemotherapy and poor outcomes in NSCLC, supporting the hypothesis that the protein can execute oncogenic activities. In a recent issue of The Journal of Pathology, Calvayrac and coworkers uncover a novel molecular mechanism that can explain this oncogenic role of p27. They demonstrate that cytoplasmic p27 binds and inhibits the small GTPase RhoB and thereby relieves a selection pressure for RhoB loss that is frequently observed in NSCLC. This is supported not only by studies with genetically modified mice, but also through identification of a cohort of human lung cancer patients with cytoplasmic p27 and continued RhoB expression, where this signature correlates with decreased survival. This not only establishes a potentially useful biomarker, but also provides yet another facet of the complex roles p27 undertakes in tumourigenesis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

International audience

Purpose: A crucial event in lung adenocarcinoma progression is the switch from an aerogenous spread toward an infiltrating tumor. Loss of RhoB expression has been suggested to be critical for lung cancer invasion. Here, we tested RhoB expression as a prognostic biomarker in non–small cell lung cancer (NSCLC) with a special focus on lepidic pattern. Experimental Design: We analyzed RhoB expression using both IHC and RT-qPCR in two series of operated patients (n = 100 and 48, respectively) and in a series of advanced lepidic adenocarcinoma (n = 31) from different hospitals. Next, we examined the role of RhoB in lung cancer progression in transgenic mice that express inducible EGFRL858R crossed with Rhob null mice. Results: We identified that loss of RhoB expression was strongly associated with worse survival (P = 0.0001) and progression-free survival (P < 0.001) in the first series. We then confirmed these results after multivariate analyses of the second series. In the series of adenocarcinoma with lepidic features issued from a clinical trial (IFCT-0401), we showed that loss of RhoB expression was associated with higher aggressiveness of stage IV. Finally, we showed that EGFRL858R/Rhob+/+ mice developed mainly diffuse lung tumors with a lepidic pattern, whereas EGFRL858R/Rhob+/− and EGFRL858R/Rhob−/− developed a greater number of tumors, and aggressive adenocarcinomas with invasive properties. Conclusions: We showed that RhoB is not only a strong prognostic factor in NSCLC but it is also critical for the acquisition of an aggressive phenotype of adenocarcinoma. Clin Cancer Res; 20(24); 6541–50. ©2014 AACR.

Resumen Introduccion Estudios previos sugieren que la perdida de funcion de NOR-1 modula la activacion de las celulas musculares lisas vasculares (CMLV). En este estudio utilizamos un raton que sobreexpresa NOR-1 en CMLV para analizar su efecto en la activacion celular y en la hiperplasia de la intima inducida por estres hemodinamico. Metodos Para generar el modelo animal el ADNc de NOR-1 humano se situo bajo el control del promotor de SM22α. La expresion de NOR-1 se analizo mediante PCR a tiempo real, Western-blot, inmunohistoquimica e inmunocitoquimica, y su funcionalidad se determino mediante ensayos de actividad luciferasa. Como indice de proliferacion celular se determino la incorporacion de timidina tritiada. La carotida izquierda se sometio a ligadura y en secciones de la misma se realizaron analisis morfometricos e inmunohistoquimicos. Resultados El transgenico desarrollado exhibia niveles significativos de NOR-1 humano en la aorta y las arterias carotidas. En las CMLV de los animales transgenicos se detecto un aumento de la actividad transcripcional de la ciclina D2, una mayor actividad proliferativa y niveles incrementados de Myh10. En estos animales la ligadura de la carotida indujo mayor formacion de neointima y de estenosis que en los animales control, en consonancia con el marcaje de Myh10 e histona H3 fosforilada. Conclusiones Estos resultados refuerzan el papel de NOR-1 en la proliferacion de las CMLV y en el remodelado vascular, y permiten proponer este modelo como una herramienta util para estudiar la implicacion de este receptor en la funcion vascular y en enfermedades como la arteriosclerosis y la reestenosis.

International audience; We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22a minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [ 3 H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

The identification of oncogenic driver alterations that underlie sensitivity to small inhibitors has led to growing interest in identifying additional targetable oncogenes in nonsmall cell lung cancer. Although the therapeutic impact of the discovery of these alterations has now been widely demonstrated, the epidemiological data associated with each of these biomarkers remain insufficiently studied. In this review, we discuss the techniques used to discover each of these candidate oncogenes, their prevalence in nonsmall cell lung cancer, and briefly outline the epidemiological features of the major oncogenes and ways in which their identification can determine therapeutic strategies.

International audience; Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, which is mainly due to its high risk of metastatic dissemination. One critical point of this process is the ability of cancer cells to detach from the primary tumor and migrate through the extracellular matrix; however, the underlying molecular mechanisms are not yet fully understood. In the present study, we identified the small GTPase RhoB as a key regulator of bronchial cell morphology in a three-dimensional (3D) matrix. RhoB loss, which is frequently observed during lung cancer progression, induced an epithelial-mesenchymal transition (EMT) characterized by an increased proportion of invasive elongated cells in 3D. The process was mediated by Slug induction and E-cadherin repression. In addition, downregulation of RhoB induced Akt1 activation, which in turn activated Rac1 through the guanine-exchange factor Trio to control cell shape rearrangement. Further, we provide evidence that RhoB interacted with and positively regulates phosphatase PP2A through the recruitment of its regulatory subunit B55, which was found to be crucial for Akt dephosphorylation. B55 inhibition completely suppressed RhoB-mediated PP2A regulation. Finally, we show that PP2A inactivation, by targeting either its catalytic or its regulatory B55 subunit, completely reversed RhoB-dependent morphological changes and also fully prevented the ability of RhoB to decrease the invasiveness of bronchial cells. Altogether, these results highlight a novel signaling axis and describe new molecular mechanisms that could explain the tumor suppressor role of RhoB in lung cancer. Therefore, we propose that RhoB could be responsible for early metastatic prevention by inhibiting the EMT-derived invasiveness of lung cells through the control of PP2A activity.

Objective— Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). Methods and Results— In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P Conclusion— This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.

Resumen Introduccion La lisil oxidasa (LOX), enzima implicada en la maduracion de la matriz extracelular, juega un papel clave en el mantenimiento de la homeostasis del endotelio. Sin embargo, se desconoce el mecanismo a traves del cual la LOX regula la funcion de la celula endotelial. Nuestro objetivo ha sido caracterizar los procesos celulares controlados por la LOX en celulas endoteliales. Metodos La LOX se sobreexpreso en celulas endoteliales humanas de vena de cordon umbilical (HUVEC) utilizando un sistema lentiviral. Las consecuencias de la sobreexpresion de LOX sobre el patron de expresion se determinaron mediante microarrays. La expresion de LOX y de la α 2 -macroglobulina (A2 M) se evaluo mediante PCR a tiempo real y/o western-blot. Resultados El sistema de expresion lentiviral incremento muy significativamente la expresion de LOX en HUVEC (mas de 20 veces). En concordancia, el analisis por western-blot evidencio un marcado aumento del nivel de proteina LOX en el extracto celular, tanto de la forma madura y cataliticamente activa como de la proenzima. Asimismo, se indujo la secrecion de LOX al medio de cultivo. El analisis del perfil de expresion mediante microarrays demostro que la sobreexpresion de LOX en HUVEC modula genes implicados en senalizacion, comunicacion y adhesion celular. Uno de los genes mas fuertemente inhibidos por la sobreexpresion de LOX es la A2 M, un inhibidor de proteasas de amplio espectro que puede desempenar multiples actividades biologicas. Conclusiones La sobreexpresion de LOX en celulas endoteliales altera el perfil de expresion genica y reduce significativamente la expresion de la A2 M, lo que puede tener importantes repercusiones sobre la funcion del endotelio.

Objective— Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. Methods and Results— Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)–dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti–PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1–deficient mice. Interestingly, circulating MMP-10 levels ( P Conclusion— Thrombin induces MMP-10 through a PAR-1–dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

SummaryMatrix metalloproteinases (MMPs) are associated with tissue remodelling and repair. In non-vascular tissues, NR4A receptors have been involved in the regulation of MMPs by transcriptional repression mechanisms. Here, we analyse alternative mechanisms involving NR4A receptors in the modulation of MMP activity in vascular smooth muscle cells (VSMC). Lentiviral overexpression of NR4A receptors (NOR-1, Nurr1 and Nur77) in human VSMC strongly decreased MMP-2 and MMP-9 activities (analysed by zymography and DQ-gelatin assays) and protein levels. NR4A receptors also down-regulated MMP-2 mRNA levels. Real-time PCR analysis evidenced that alpha-2-macroglobulin (A2M), but not other MMP inhibitors (TIMP-1 and TIMP-2) were up-regulated in NR4A-transduced cells. Interestingly, A2M was expressed in human vascular tissues including the smooth muscle media layer. While NR4A receptors increased A2M expression and secretion in VSMC, NR4A knockdown significantly reduced basal A2M expression in these cells. The direct transcriptional regulation of the human A2M promoter by NR4A receptors was characterised in luciferase reporter assays, electrophoretic mobility shift assays and by chromatin immunoprecipitation, identifying a NGFI-B response element (NBRE-71/-64) essential for the NR4A-mediated induction. The blockade of A2M partially prevented the reduction of MMPs activity observed in NR4A-transduced cells. Although mouse A2M promoter was unresponsive to NR4A receptors, vascular MMP expression was attenuated in transgenic mice over-expressing human NOR-1 in VSMC challenged with lipopolysaccharide. Our results show that the panproteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M.

Previous studies have shown that the loss of NOR-1 function modulates the activation of vascular smooth muscle cells (VSMC). In this study we use a mouse that over-expresses human NOR-1 in VSMC to analyze the effect of a gain of NOR-1 function on the activation of VSMC and in the hyperplasia of the intima induced by hemodynamic stress.To generate the transgenic animal the human NOR-1 cDNA was placed under the control of the SM22α promoter. The expression of NOR-1 was analyzed by real time PCR, Western blot, immunohistochemistry and immunocitochemistry, and NOR-1 functionality was evaluated by luciferase activity assays. The incorporation of tritiated thymidine was determined as a cell proliferation index. The left carotid artery was ligated, and cross-sections were subjected to morphometric and immunostaining analysis.The transgenic mouse exhibited significant levels of human NOR-1 in aorta and carotid arteries. In aortic VSMC from transgenic mice an increase in the transcriptional activity of ciclin D2 was detected, as well as higher proliferative rates and increased levels of the marker Myh10. In these animals, carotid artery ligation induced a greater neointimal formation and a higher stenotic grade than in wild-type animals, in accordance with the labelling detected for Myh10 and phosphorylated Histone H3.These results reinforce the role of NOR-1 in VSMC proliferation and in vascular remodelling, and allow us to propose this model as a useful tool to study the involvement of NOR-1 in vascular function and in vascular diseases such as atherosclerosis and restenosis.

Objective— Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo. Methods and Results— Human endothelial cells were stimulated with thrombin (0.1–10 U/mL), CD40L (0.25–1 μg/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P P N -terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L. Conclusion— Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.

SummaryThrombin is a multifunctional serine protease generated at the site of vascular injury that transforms fibrinogen into fibrin, activates blood platelets and elicits multiple effects on a variety of cell types including endothelial cells, vascular smooth muscle cells (VSMC), monocytes,T lymphocytes and fibroblasts. Cellular effects of thrombin are mediated by protease-activated receptors (PARs), members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage. Thrombin signalling in platelets contributes to haemostasis and thrombosis. In normal arteries PARs are mainly expressed in endothelial cells, while their expression in VSMC is limited. Endothelial PARs participate in the regulation of vascular tone, vascular permeability and endothelial secretory activity while in VSMC they mediate contraction, migration, proliferation, hypertrophy and production of extra-cellular matrix. PARs contribute to the pro-inflammatory phenotype observed in endothelial dysfunction and their up-regulation inVSMC seems to be a key element in the pathogenesis of atherosclerosis and restenosis. In the last years a myriad of studies have emphasized the critical role of PAR signalling in thrombin mediated effects in haemostasis, inflammation, cancer and embryonic development. Lately, PARs have become a therapeutic target to inhibit platelet aggregation and thrombosis. Early data from a clinical trial (TRA-PCI) to evaluate safety and efficacy of a potent new oral thrombin receptor antagonist (TRA) have promisingly indicated that overallTRA treatment reduces adverse event rates without an increase in bleeding risk. In this paper we review cellular responses triggered by thrombin and their implication in vascular pathophysiology.

Atherosclerosis is the common pathophysiological substrate of ischemic vascular diseases and their thrombotic complications. The unbalance between matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) has been hypothesized to be involved in the growth, destabilization, and eventual rupture of atherosclerotic lesions. Different MMPs have been assigned relevant roles in the pathology of vascular diseases and MMP-10 (stromelysin-2) has been involved in vascular development and atherogenesis. This article examines the pathophysiological role of MMPs, particularly MMP-10, in the onset and progression of vascular diseases and their regulation by pro-inflammatory stimuli. MMP-10 over-expression has been shown to compromise vascular integrity and it has been associated with aortic aneurysms. MMP-10 is induced by C-reactive protein in endothelial cells, and it is over-expressed in atherosclerotic lesions. Additionally, higher MMP-10 serum levels are associated with inflammatory markers, increased carotid intima-media thickness and the presence of atherosclerotic plaques. We have cloned the promoter region of the MMP-10 gene and studied the effect of inflammatory stimuli on MMP-10 transcriptional regulation, providing evidences further supporting the involvement of MMP-10 in the pathophysiology of atherothrombosis.

Neuron-derived orphan receptor-1 (NOR-1) is a nuclear receptor recently involved in the onset and development of atherosclerosis. NOR-1 is induced in a cell-specific manner by extracellular stimuli. NOR-1 is over-expressed in human atherosclerotic plaques and in porcine arteries subjected to angioplasty, is induced by growth factors in vascular cells and it has been involved in cell migration and proliferation. This article examines the mechanisms that regulate NOR-1 in vascular cells and the effects of NOR-1 knockdown on cell growth induced by mitogens, in particular thrombin. Mitogenic stimuli up-regulates NOR-1 in endothelial cells (ECs) through multiple pathways, including increase of cytosolic calcium, activation of protein kinase C, mitogen-activated protein kinase (MAPK) (ERK1/2 and p38 MAPK) and downstream activation of cAMP response element binding protein (CREB). Inhibition of protease-activated receptor-1 (PAR-1) abolished thrombin-induced NOR-1 up-regulation and DNA synthesis. NOR-1 knockdown reduces DNA synthesis and EC re-growth in an in vitro model of wound repair. NOR-1 could be regarded as a new target to prevent endothelial effects triggered by thrombin and other mitogens.

Recent work has highlighted the role of NR4A receptors in atherosclerosis and inflammation. In vascular smooth muscle cell (VSMC) proliferation, however, NOR-1 (neuron-derived orphan receptor-1) exerts antagonistic effects to Nur77 and Nurr1. The aim of this study was to analyse the effect of NOR-1 in VSMC inflammatory response. We assessed the consequence of a gain-of-function of this receptor on the response of VSMC to inflammatory stimuli. In human VSMC, lentiviral over-expression of NOR-1 reduced lipopolysaccharide (LPS)-induced up-regulation of cytokines (IL-1β, IL-6 and IL-8) and chemokines (MCP-1 and CCL20). Similar effects were obtained in cells stimulated with TNFα or oxLDL. Conversely, siRNA-mediated NOR-1 inhibition significantly increased the expression of pro-inflammatory mediators. Interestingly, in the aortas from transgenic mice that over-express human NOR-1 in VSMC (TgNOR-1), the up-regulation of cytokine/chemokine by LPS was lower compared to wild-type littermates. Similar results were obtained in VSMC from transgenic animals. NOR-1 reduced the transcriptional activity of NFκB sensitive promoters (in transient transfections), and the binding of NFκB to its responsive element (in electrophoretic mobility shift assays). Furthermore, NOR-1 prevented the activation of NFκB pathway by decreasing IκBα phosphorylation/degradation and inhibiting the phosphorylation and subsequent translocation of p65 to the nucleus (assessed by Western blot and immunocytochemistry). These effects were associated with an attenuated phosphorylation of ERK1/2, p38 MAPK and Jun N-terminal kinase, pathways involved in the activation of NFκB. In mouse challenged with LPS, the activation of the NFκB signalling was also attenuated in the aorta from TgNOR-1. Our data support a role for NOR-1 as a negative modulator of the acute response elicited by pro-inflammatory stimuli in the vasculature.