RESUMEN
The current targeted therapy for BRAFV600E-mutant lung cancer consists of a dual blockade of RAF/MEK kinases often combining dabrafenib/trametinib (D/T). This regimen extends survival when compared to single-agent treatments, but disease progression is unavoidable. By using whole-genome CRISPR screening and RNA sequencing, we characterize the vulnerabilities of both persister and D/T-resistant cellular models. Oxidative stress together with concomitant induction of antioxidant responses is boosted by D/T treatment. However, the nature of the oxidative damage, the choice of redox detoxification systems, and the resulting therapeutic vulnerabilities display stage-specific differences. Persister cells suffer from lipid peroxidation and are sensitive to ferroptosis upon GPX4 inhibition in vivo. Biomarkers of lipid peroxidation are detected in clinical samples following D/T treatment. Acquired alterations leading to mitogen-activated protein kinase (MAPK) reactivation enhance cystine transport to boost GPX4-independent antioxidant responses. Similarly to BRAFV600E-mutant melanoma, histone deacetylase (HDAC) inhibitors decrease D/T-resistant cell viability and extend therapeutic response in vivo.
Asunto(s)
Adenocarcinoma del Pulmón , Resistencia a Antineoplásicos , Inhibidores de Histona Desacetilasas , Neoplasias Pulmonares , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Proteínas Proto-Oncogénicas B-raf , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/patología , Adenocarcinoma del Pulmón/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Línea Celular Tumoral , Animales , Inhibidores de Histona Desacetilasas/farmacología , Ferroptosis/efectos de los fármacos , Ferroptosis/genética , Ratones , Estrés Oxidativo/efectos de los fármacos , Oximas/farmacología , Imidazoles/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Peroxidación de Lípido/efectos de los fármacos , Mutación/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Drug-tolerance has emerged as one of the major non-genetic adaptive processes driving resistance to targeted therapy (TT) in non-small cell lung cancer (NSCLC). However, the kinetics and sequence of molecular events governing this adaptive response remain poorly understood. Here, we combine real-time monitoring of the cell-cycle dynamics and single-cell RNA sequencing in a broad panel of oncogenic addiction such as EGFR-, ALK-, BRAF- and KRAS-mutant NSCLC, treated with their corresponding TT. We identify a common path of drug adaptation, which invariably involves alveolar type 1 (AT1) differentiation and Rho-associated protein kinase (ROCK)-mediated cytoskeletal remodeling. We also isolate and characterize a rare population of early escapers, which represent the earliest resistance-initiating cells that emerge in the first hours of treatment from the AT1-like population. A phenotypic drug screen identify farnesyltransferase inhibitors (FTI) such as tipifarnib as the most effective drugs in preventing relapse to TT in vitro and in vivo in several models of oncogenic addiction, which is confirmed by genetic depletion of the farnesyltransferase. These findings pave the way for the development of treatments combining TT and FTI to effectively prevent tumor relapse in oncogene-addicted NSCLC patients.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Resistencia a Antineoplásicos , Farnesiltransferasa , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Farnesiltransferasa/antagonistas & inhibidores , Farnesiltransferasa/metabolismo , Farnesiltransferasa/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Animales , Ratones , Dependencia del Oncogén/genética , Terapia Molecular Dirigida , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Ensayos Antitumor por Modelo de Xenoinjerto , Oncogenes/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , QuinolonasRESUMEN
BACKGROUND: Interstitial lung disease is a heterogeneous group of diseases, some of which are known to present an independent risk factor for lung cancer. Its pathophysiological mechanism has not been fully elucidated and therapeutic management is also complex. We aim to both describe a cohort of patients with lung cancer associated with pre-existing fibrosing interstitial lung disease and to characterize their molecular profile. METHODS: We conducted a retrospective, single centre cohort study, at Toulouse University Hospital. Immuno-histochemical (PD-L1, CD8) and molecular analysis was performed on archived tumour sample. Molecular signalling pathways involved were analysed with the Reactome Pathway Database. RESULTS: Forty-nine patients were analysed. Most common histology was adenocarcinoma (65,3%), followed by squamous cell carcinoma (30.6%). Idiopathic pulmonary fibrosis (30,6%) and interstitial lung disease associated with connective tissue disease (22,4%) were mostly diagnosed. Usual interstitial pneumonia dominated the scans patterns. A high proportion of early tumour stages was observed and overall survival was 34,5 months. In metastatic stages response rate to first line chemotherapy was 38% and overall survival was 11,2 months. Main cause of death was complex cancer progression. PD-L1 expression (n=23) was low (0%) to intermediate (1-49%). Tumour mutational burden was low in 69,2% of analysed cases (n=12) and microsatellite status was stable in all cases (n=13). Sample genotyping (n=14) showed frequent involvement of the TP53 gene and the implication of signalling pathways common to fibrotic processes such as TGFß and PI3K/AKT. CONCLUSIONS: We suggest a particular phenotype of lung cancer associated with fibrosing interstitial lung disease that could provide the basis for specific therapeutic strategies.
Asunto(s)
Fibrosis Pulmonar Idiopática , Enfermedades Pulmonares Intersticiales , Neoplasias Pulmonares , Humanos , Antígeno B7-H1/genética , Estudios Retrospectivos , Estudios de Cohortes , Fosfatidilinositol 3-Quinasas , Enfermedades Pulmonares Intersticiales/epidemiología , Enfermedades Pulmonares Intersticiales/genética , Fibrosis Pulmonar Idiopática/complicaciones , Fibrosis Pulmonar Idiopática/epidemiología , Fibrosis Pulmonar Idiopática/genética , Fibrosis , Neoplasias Pulmonares/complicaciones , Neoplasias Pulmonares/epidemiología , Neoplasias Pulmonares/genéticaRESUMEN
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.
RESUMEN
Vγ9Vδ2 T cells are known to be efficient anti-tumor effectors activated through phosphoantigens (PAg) that are naturally expressed by tumor cells or induced by amino bisphosphonates treatment. This PAg-activation which is TCR and butyrophilin BTN3A dependent can be modulated by NKG2D ligands, immune checkpoint ligands, adhesion molecules, and costimulatory molecules. This could explain the immune-resistance observed in certain clinical trials based on Vγ9Vδ2 T cells therapies. In NSCLC, encouraging responses were obtained with zoledronate administrations for 50% of patients. According to the in vivo results, we showed that the in vitro Vγ9Vδ2 T cell reactivity depends on the NSCLC cell line considered. If the PAg-pretreated KRAS mutated A549 is highly recognized and killed by Vγ9Vδ2 T cells, the EGFR mutated PC9 remains resistant to these killers despite a pre-treatment either with zoledronate or with exogenous BrHPP. The immune resistance of PC9 was shown not to be due to immune checkpoint ligands able to counterbalance NKG2D ligands or adhesion molecules such as ICAM-1 highly expressed by PC9. RHOB has been shown to be involved in the Vγ9Vδ2 TCR signaling against these NSCLC cell lines, in this study we therefore focused on its intracellular behavior. In comparison to a uniform distribution of RHOB in endosomes and at the plasma membrane in A549, the presence of large endosomal clusters of RHOB was visualized by a split-GFP system, suggesting that RHOB rerouting in the PC9 tumor cell could impair the reactivity of the immune response.
Asunto(s)
Antígenos de Neoplasias/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Células Cultivadas , Endosomas/inmunología , Endosomas/metabolismo , Humanos , Neoplasias Pulmonares/etiología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , FosforilaciónRESUMEN
EGFR-mutated lung adenocarcinoma patients treated with gefitinib and osimertinib show a therapeutic benefit limited by the appearance of secondary mutations, such as EGFRT790M and EGFRC797S. 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 EGFRT790M and EGFRC797S 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 EGFRT790M and EGFRC797S 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.
Asunto(s)
Acrilamidas/farmacología , Adenocarcinoma del Pulmón , Compuestos de Anilina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores ErbB , Gefitinib/farmacología , Neoplasias Pulmonares , Mutación Missense , Proteínas de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/metabolismo , Adenocarcinoma del Pulmón/patología , Sustitución de Aminoácidos , Animales , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Transgénicos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción HES-1/genética , Factor de Transcripción HES-1/metabolismoRESUMEN
The cell cycle inhibitor p27Kip1 is a tumor suppressor via the inhibition of CDK complexes in the nucleus. However, p27 also plays other functions in the cell and may acquire oncogenic roles when located in the cytoplasm. Activation of oncogenic pathways such as Ras or PI3K/AKT causes the relocalization of p27 in the cytoplasm, where it can promote tumorigenesis by unclear mechanisms. Here, we investigated how cytoplasmic p27 participates in the development of non-small cell lung carcinomas. We provide molecular and genetic evidence that the oncogenic role of p27 is mediated, at least in part, by binding to and inhibiting the GTPase RhoB, which normally acts as a tumor suppressor in the lung. Genetically modified mice revealed that RhoB expression is preferentially lost in tumors in which p27 is absent and maintained in tumors expressing wild-type p27 or p27CK- , a mutant that cannot inhibit CDKs. Moreover, although the absence of RhoB promoted tumorigenesis in p27-/- animals, it had no effect in p27CK- knock-in mice, suggesting that cytoplasmic p27 may act as an oncogene, at least in part, by inhibiting the activity of RhoB. Finally, in a cohort of lung cancer patients, we identified a subset of tumors harboring cytoplasmic p27 in which RhoB expression is maintained and these characteristics were strongly associated with decreased patient survival. Thus, monitoring p27 localization and RhoB levels in non-small cell lung carcinoma patients appears to be a powerful prognostic marker for these tumors. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Asunto(s)
Adenocarcinoma del Pulmón/enzimología , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Citoplasma/enzimología , Neoplasias Pulmonares/enzimología , Proteína de Unión al GTP rhoB/metabolismo , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Animales , Carcinogénesis , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Citoplasma/genética , Citoplasma/patología , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Unión Proteica , Transducción de Señal , Proteína de Unión al GTP rhoB/genéticaAsunto(s)
Resistencia a Antineoplásicos/fisiología , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteína de Unión al GTP rhoB/fisiología , Humanos , Neoplasias Pulmonares/enzimología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/enzimologíaRESUMEN
Metastatic dissemination is the cause of death in the vast majority of cancers, including lung cancers. In order to metastasize, tumor cells must undergo a well-known series of changes, however the molecular details of how they manage to overcome the barriers at each stage remain incomplete. One critical step is acquiring the ability to migrate through the extracellular matrix. Loss of expression of the RAS-related small GTPase RHOB is a common feature of lung cancer progression, and we recently reported that this induces an epithelial-to-mesenchymal transition (EMT) that is dependent on SLUG overexpression and E-Cadherin inhibition and is characterized by 3-dimensional cell shape reorganization and the increased invasiveness of bronchial cells. RHOB loss was found to induce AKT1 activation, which in turn activates RAC1 through its GEF TRIO. Further investigation of this pathway revealed that RHOB interacts with and positively regulates PP2A, one of the major cellular serine-threonine phosphatases, by recruiting its regulatory subunit B55. Here we discuss the role of this newly discovered RHOB/PP2A/AKT1/RAC1 pathway in relation to mesenchymal migration and invasion in lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Mesodermo/patología , Fenotipo , Proteína Fosfatasa 2/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Invasividad NeoplásicaRESUMEN
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.
Asunto(s)
Adenocarcinoma/genética , Biomarcadores de Tumor/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Adenocarcinoma del Pulmón , Humanos , Mutación , Oncogenes/genéticaRESUMEN
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 EGFRL858R 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.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/fisiopatología , Resistencia a Medicamentos , Receptores ErbB/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína de Unión al GTP rhoB/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones Transgénicos , Proteínas Tirosina Quinasas/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Fosfoproteínas/genética , Factores de Intercambio de Guanina Nucleótido Rho/genética , Proteínas Supresoras de Tumor/genética , Proteína de Unión al GTP rhoB/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Silenciador del Gen/fisiología , Genes Supresores de Tumor/fisiología , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Regiones Promotoras Genéticas/genética , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Matrix 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 pan-proteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , alfa-Macroglobulinas/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Proteínas de Unión al ADN/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Lipopolisacáridos/farmacología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Transgénicos , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Regiones Promotoras Genéticas , Interferencia de ARN , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transcripción Genética , Transfección , alfa-Macroglobulinas/genéticaRESUMEN
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-1ß, 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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Inflamación/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Músculo Liso Vascular/citología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , FN-kappa B/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Animales , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Mediadores de Inflamación/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Unión Proteica , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Transducción de Señal , Activación TranscripcionalRESUMEN
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 EGFR(L858R) 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 EGFR(L858R)/Rhob(+/+) mice developed mainly diffuse lung tumors with a lepidic pattern, whereas EGFR(L858R)/Rhob(+/-) and EGFR(L858R)/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.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/patología , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Mutación , Proteína de Unión al GTP rhoB/genética , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Adulto , Anciano , Animales , Biomarcadores de Tumor , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/mortalidad , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Factores de Riesgo , Carga TumoralRESUMEN
INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Remodelación Vascular/fisiología , Animales , Aorta/metabolismo , Aorta/patología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Proliferación Celular/fisiología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Hiperplasia/patología , Ratones , Ratones Transgénicos , Neointima/etiología , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Estrés Fisiológico/fisiología , Túnica Íntima/metabolismoRESUMEN
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 SM22α 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.
Asunto(s)
Arterias Carótidas/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Proteínas de Unión al ADN/biosíntesis , Neointima/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Receptores de Esteroides/biosíntesis , Receptores de Hormona Tiroidea/biosíntesis , Animales , Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/genética , Traumatismos de las Arterias Carótidas/patología , Proteínas de Unión al ADN/genética , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Hiperplasia/patología , Ratones , Ratones Transgénicos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Neointima/genética , Neointima/patología , Proteínas del Tejido Nervioso/genética , Ratas , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genéticaRESUMEN
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<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, α-CD40L, and α-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun 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. CONCLUSIONS: 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.
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Ligando de CD40/metabolismo , Micropartículas Derivadas de Células/enzimología , Células Endoteliales/enzimología , Metaloproteinasa 10 de la Matriz/metabolismo , Sepsis/enzimología , Trombina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos/farmacología , Coagulación Sanguínea , Antígenos CD40/antagonistas & inhibidores , Antígenos CD40/metabolismo , Ligando de CD40/antagonistas & inhibidores , Ligando de CD40/sangre , Estudios de Casos y Controles , Micropartículas Derivadas de Células/patología , Células Cultivadas , Modelos Animales de Enfermedad , Coagulación Intravascular Diseminada/enzimología , Coagulación Intravascular Diseminada/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Endotoxemia/enzimología , Endotoxemia/genética , Endotoxemia/patología , Femenino , Regulación Enzimológica de la Expresión Génica , Hirudinas/farmacología , Células Endoteliales de la Vena Umbilical Humana/enzimología , Humanos , Lipopolisacáridos/farmacología , Masculino , Metaloproteinasa 10 de la Matriz/sangre , Metaloproteinasa 10 de la Matriz/deficiencia , Metaloproteinasa 10 de la Matriz/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteína Quinasa 8 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Análisis Multivariante , Péptidos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/metabolismo , Receptor PAR-1/agonistas , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/metabolismo , Medición de Riesgo , Factores de Riesgo , Sepsis/mortalidad , Sepsis/patología , Transducción de Señal , EspañaRESUMEN
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<0.01). LDL induced the expression of CCL20 in VSMC in a dose- and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-κB. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-κB site (-80/-71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. CONCLUSION: This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.
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Quimiocina CCL20/metabolismo , Hipercolesterolemia/metabolismo , Lipoproteínas LDL/farmacología , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Adulto , Anciano , Aorta/metabolismo , Aorta/patología , Células Cultivadas , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Enfermedad de la Arteria Coronaria/cirugía , Relación Dosis-Respuesta a Droga , Endarterectomía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/patología , Transducción de Señal , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
Hypoxia modulates gene expression and affects multiple aspects of endothelial cell biology. Fibulin-5 (FBLN5) is an extracellular matrix protein essential for elastic fiber assembly and vasculogenesis that participates in vascular remodeling and controls endothelial cell adhesion, motility, and proliferation. In this context, we aimed to analyze FBLN5 regulation by hypoxia in endothelial cells. Hypoxia (1% O(2)) increased FBLN5 mRNA levels in endothelial cells in a time-dependent manner. Maximal induction (â¼2.5-fold) was achieved after 24 h of hypoxia. This effect paralleled an increase in both intracellular and extracellular FBLN5 protein levels. The increase in FBLN5 mRNA levels observed in hypoxic cells was blocked by inhibitors of the PI3K/Akt/mTOR pathway (LY294002 and rapamycin) and mimicked by dimethyl oxal glycine, which prevents proline hydroxylase-mediated degradation of HIF-1α. Silencing of HIF-1α completely prevented hypoxia-induced FBLN5 up-regulation. Accordingly, both hypoxia and HIF-1α overexpression increased FBLN5 transcriptional activity. Serial promoter deletion and mutagenesis studies revealed the involvement of a putative hypoxia response element (HRE) located at -78 bp. In fact, EMSA and ChIP assays demonstrated increased HIF-1 binding to this site in hypoxic cells. Interestingly, the rate of endothelial cells undergoing apoptosis in cultures exposed to hypoxia increased in FBLN5 knockdown cells, suggesting that hypoxia-induced FBLN5 expression contributes to preserve cell survival. These results provide evidence that HIF-1 signaling underlies the increase of FBLN5 expression elicited by hypoxia in endothelial cells and suggest that FBLN5 induction could be involved in the adaptive survival response of endothelial cells to hypoxia.