RESUMEN
Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation of specific loci is largely unexplored. In genome-wide DNA methylation analyses, we identified the KCa 3.1 channel gene (KCNN4) promoter to be hypomethylated in an aggressive non-small-cell lung carcinoma (NSCLC) cell line and in patient samples. Accordingly, KCa 3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for poor prognosis in lung adenocarcinoma. Increased KCa 3.1 expression was associated with aggressive features of NSCLC cells. Proliferation and migration of pro-metastatic NSCLC cells depended on KCa 3.1 activity. Mechanistically, elevated KCa 3.1 expression hyperpolarized the membrane potential, thereby augmenting the driving force for Ca(2+) influx. KCa 3.1 blockade strongly reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography-computed tomography. Thus, loss of DNA methylation of the KCNN4 promoter and increased KCa 3.1 channel expression and function are mechanistically linked to poor survival of NSCLC patients.
Asunto(s)
Epigénesis Genética/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Metilación de ADN/genética , Epigenómica/métodos , Femenino , Xenoinjertos , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Pronóstico , Regiones Promotoras Genéticas/genéticaRESUMEN
With the use of ChIP on microarray assays in primary leukemia samples, we report that acute myeloid leukemia (AML) blasts exhibit significant alterations in histone H3 acetylation (H3Ac) levels at > 1000 genomic loci compared with CD34(+) progenitor cells. Importantly, core promoter regions tended to have lower H3Ac levels in AML compared with progenitor cells, which suggested that a large number of genes are epigenetically silenced in AML. Intriguingly, we identified peroxiredoxin 2 (PRDX2) as a novel potential tumor suppressor gene in AML. H3Ac was decreased at the PRDX2 gene promoter in AML, which correlated with low mRNA and protein expression. We also observed DNA hypermethylation at the PRDX2 promoter in AML. Low protein expression of the antioxidant PRDX2 gene was clinically associated with poor prognosis in patients with AML. Functionally, PRDX2 acted as inhibitor of myeloid cell growth by reducing levels of reactive oxygen species (ROS) generated in response to cytokines. Forced PRDX2 expression inhibited c-Myc-induced leukemogenesis in vivo on BM transplantation in mice. Taken together, epigenome-wide analyses of H3Ac in AML led to the identification of PRDX2 as an epigenetically silenced growth suppressor, suggesting a possible role of ROS in the malignant phenotype in AML.
Asunto(s)
Metilación de ADN , Histonas/metabolismo , Peroxirredoxinas/genética , Proteínas Supresoras de Tumor/genética , Acetilación , Enfermedad Aguda , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular , Células Cultivadas , Epigénesis Genética , Femenino , Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Células HL-60 , Humanos , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Peroxirredoxinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Células U937 , Adulto JovenRESUMEN
The nucleoside analog 5-azacytidine is an archetypical drug for epigenetic cancer therapy, and its clinical effectiveness has been demonstrated in the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). However, therapy resistance in patients with MDS/AML remains a challenging issue. Membrane proteins that are involved in drug uptake are potential mediators of drug resistance. The responsible proteins for the transport of 5-azacytidine into MDS/AML cells are unknown. We have now systematically analyzed the expression and activity of various nucleoside transporters. We identified the human equilibrative nucleoside transporter 1 (hENT1) as the most abundant nucleoside transporter in leukemia cell lines and in AML patient samples. Transport assays using [¹4C]5-azacytidine demonstrated Naâº-independent uptake of the drug into the cells, which was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBTI), a hENT1 inhibitor. The cellular toxicity of 5-azacytidine and its DNA demethylating activity were strongly reduced after hENT1 inhibition. In contrast, the cellular activity of the 5-azacytidine derivative 5-azacytidine-5'-elaidate (CP-4200), a nucleoside transporter-independent drug, persisted after hENT1 inhibition. A strong dependence of 5-azacytidine-induced DNA demethylation on hENT1 activity was also confirmed by array-based DNA methylation profiling, which uncovered hundreds of loci that became demethylated only when hENT1-mediated transport was active. Our data establish hENT1 as a key transporter for the cellular uptake of 5-azacytidine in leukemia cells and raise the possibility that hENT1 expression might be a useful biomarker to predict the efficiency of 5-azacytidine treatments. Furthermore, our data suggest that CP-4200 may represent a valuable compound for the modulation of transporter-related 5-azacytidine resistances.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Azacitidina/análogos & derivados , Metilasas de Modificación del ADN/antagonistas & inhibidores , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Leucemia/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacocinética , Azacitidina/farmacología , Transporte Biológico , Línea Celular Tumoral , Metilación de ADN , Perros , Resistencia a Antineoplásicos , Tranportador Equilibrativo 1 de Nucleósido/antagonistas & inhibidores , Humanos , Leucemia Mieloide Aguda/metabolismo , Células de Riñón Canino Madin DarbyRESUMEN
Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.
Asunto(s)
Histonas/metabolismo , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/metabolismo , Lisina/metabolismo , Adolescente , Antígenos CD34/inmunología , Niño , Preescolar , Supervivencia sin Enfermedad , Femenino , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/inmunología , Histonas/genética , Humanos , Lactante , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Masculino , Metilación , Pronóstico , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
PURPOSE: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non-small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. EXPERIMENTAL DESIGN: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. RESULTS: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). CONCLUSIONS: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Factores Quimiotácticos/fisiología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia , Proteínas S100/fisiología , Animales , Factores Quimiotácticos/genética , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , ARN Mensajero/análisis , Proteínas S100/genética , TransfecciónRESUMEN
Decreased expression levels of EPHB6, a member of the receptor tyrosine kinases (RTKs), are associated with an increased risk of metastasis development in early stage non-small cell lung cancer (NSCLC). However, the signaling properties of the kinase-defective EPHB6 receptor are not well-understood. Here, we show that expression of EPHB6 in A549 lung adenocarcinoma cells led to phosphorylation of the MAP kinase ERK. Conversely, siRNA based knockdown of EPHB6 reversed ERK phosphorylation. Intriguingly, EPHB6-induced phosphorylation of ERK was uncoupled by activation of the Elk-1 transcriptional factor. These analyses suggest that kinase defective EPHB6 can lead to MAPK activation.
Asunto(s)
Adenocarcinoma/enzimología , Neoplasias Pulmonares/enzimología , Sistema de Señalización de MAP Quinasas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Adenocarcinoma/genética , Línea Celular Tumoral , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/genética , Fosforilación , Interferencia de ARN , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de la Familia Eph , Factores de Tiempo , Transfección , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Tau is one of the two main proteins involved in the pathology of Alzheimer's disease via formation of beta-sheet rich intracellular aggregates named paired helical filaments (PHFs). Given that tau is a natively unfolded protein with no folded core (even upon binding to physiological partners such as microtubules), its structural analysis by high-resolution techniques has been difficult. In this study, employing solution small-angle X-ray scattering from the full length isoforms and from a variety of deletion and point mutants the conformation of tau in solution is structurally characterized. A recently developed ensemble optimization method was employed to generate pools of random models and to select ensembles of coexisting conformations, which fitted simultaneously the scattering data from the full length protein and deletion mutants. The analysis of the structural properties of these selected ensembles allowed us to extract information about residual structure in different domains of the native protein. The short deletion mutants containing the repeat domain (considered the core constituent of the PHFs) are significantly more extended than random coils, suggesting an extended conformation of the repeat domain. The longer tau constructs are comparable in size with the random coils, pointing to long-range contacts between the N- and C-termini compensating for the extension of the repeat domain. Moreover, most of the aggregation-promoting mutants did not show major differences in structure from their wild-type counterparts, indicating that their increased pathological effect is triggered only after an aggregation core has been formed.
Asunto(s)
Proteínas tau/química , Enfermedad de Alzheimer/patología , Humanos , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Isoformas de Proteínas/química , Soluciones , Difracción de Rayos X , Proteínas tau/genéticaRESUMEN
The histopathological diagnosis of Alzheimer's disease relies on two kinds of proteinaceous aggregates: the extracellular plaques built from filaments of the Abeta-peptide and the intracellular tangles consisting of tau polymerized into Paired Helical Filaments (PHFs). The order of aggregation events is still under debate, but it is well accepted that tau-related changes have an important impact on the viability of neurons. In neurons, early morphological changes are seen in axons which begin to loose and retract synapses. This process is accompanied by an increase of aggregated tau protein. Thus the prevention of tau aggregation seems to be a valuable target for therapy of Alzheimer's disease. Here we present a screening procedure by which we identified inhibitors of tau polymerization. In the primary screen we used a thioflavin-S based assay which detects PHF formation in solution. These initial hits were further analyzed for their capacity to depolymerize preformed PHFs. These results were confirmed by several secondary assays (tryptophan fluorescence, pelleting, filter trapping and electron microscopy). By this approach it is possible to identify small molecule compounds which prevent or reverse the aggregation of tau and thereby might improve the viability of neurons in a therapeutic approach.
Asunto(s)
Proteínas tau/antagonistas & inhibidores , Proteínas tau/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Evaluación Preclínica de Medicamentos/métodos , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Fosforilación/efectos de los fármacosRESUMEN
External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.
Asunto(s)
Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Leucemia/genética , Receptor PAR-1/genética , Enfermedad Aguda , Animales , Hematopoyesis/genética , Humanos , Inmunohistoquímica , Leucemia/metabolismo , Leucemia/patología , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor PAR-1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: Cancer cell phenotypes are partially determined by epigenetic specifications, such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. EXPERIMENTAL DESIGN: An in vivo selection approach was used to generate highly aggressive non-small cell lung cancer (NSCLC) cell lines (A549 and HTB56) followed by genome-wide DNA methylation analysis. Furthermore, the therapeutic effects of the epigenetic agent azacytidine on DNA methylation patterns and the in vivo phenotypes were explored. RESULTS: Widespread changes of DNA methylation were observed during development of highly aggressive cell lines. Up to 2.5% of the CpG-rich region was differentially methylated as identified by reduced representation bisulfite sequencing compared with the less aggressive parental cell lines. DNA methyltransferase inhibition by azacytidine reversed the prometastatic phenotype; this was highly associated with the preferential loss of DNA methylation at sites that were hypermethylated during the in vivo selection. Of note, polycomb (PRC2) binding sites were particularly affected by DNA methylation changes after azacytidine exposure that persisted over time. CONCLUSIONS: We could show that metastatic capability of NSCLC is closely associated with DNA methylome alterations. Because inhibition of DNA methyltransferase reversed metastasis-prone phenotype, epigenetic modulation seems to be a potential therapeutic approach to prevent metastasis formation.
Asunto(s)
Adenocarcinoma/genética , ADN-Citosina Metilasas/antagonistas & inhibidores , Epigénesis Genética , Neoplasias Pulmonares/genética , Proteínas del Grupo Polycomb/fisiología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/enzimología , Adenocarcinoma/secundario , Animales , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Sitios de Unión , Línea Celular Tumoral , Metilación de ADN , ADN-Citosina Metilasas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , FenotipoRESUMEN
Alterations of Eph receptor tyrosine kinases are frequent events in human cancers. Genetic variations of EPHB6 have been described but the functional outcome of these alterations is unknown. The current study was conducted to screen for the occurrence and to identify functional consequences of EPHB6 mutations in non-small cell lung cancer. Here, we sequenced the entire coding region of EPHB6 in 80 non-small cell lung cancer patients and 3 tumor cell lines. Three potentially relevant mutations were identified in primary patient samples of NSCLC patients (3.8%). Two point mutations led to instable proteins. An in frame deletion mutation (del915-917) showed enhanced migration and accelerated wound healing in vitro. Furthermore, the del915-917 mutation increased the metastatic capability of NSCLC cells in an in vivo mouse model. Our results suggest that EPHB6 mutations promote metastasis in a subset of patients with non-small cell lung cancer.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/genética , Metástasis de la Neoplasia/genética , Mutación Puntual , Receptor EphB6/genética , Secuencia de Bases , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Cartilla de ADN , Humanos , Neoplasias Pulmonares/patología , Fenotipo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
INTRODUCTION: The functions of large noncoding RNAs (ncRNAs) have remained elusive in many cases. Metastasis-Associated-in-Lung-Adenocarcinoma-Transcript-1 (MALAT-1) is an ncRNA that is highly expressed in several tumor types. METHODS: Overexpression and RNA interference (RNAi) approaches were used for the analysis of the biological functions of MALAT-1 RNA. Tumor growth was studied in nude mice. For prognostic analysis, MALAT-1 RNA was detected on paraffin-embedded non-small cell lung cancer (NSCLC) tissue probes (n = 352) using in situ hybridization. RESULTS: MALAT-1 was highly expressed in several human NSCLC cell lines. MALAT-1 expression was regulated by an endogenous negative feedback loop. In A549 NSCLCs, RNAi-mediated suppression of MALAT-1 RNA suppressed migration and clonogenic growth. Forced expression of MALAT-1 in NIH 3T3 cells significantly increased migration. Upon injection into nude mice, NSCLC xenografts with decreased MALAT-1 expression were impaired in tumor formation and growth. In situ hybridization on paraffin-embedded lung cancer tissue probes revealed that high MALAT-1 RNA expression in squamous cell carcinoma of the lung was associated with a poor prognosis. On genetic level, MALAT-1 displays the strongest association with genes involved in cancer like cellular growth, movement, proliferation, signaling, and immune regulation. CONCLUSIONS: These data indicate that MALAT-1 expression levels are associated with patient survival and identify tumor-promoting functions of MALAT-1.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Células Escamosas/genética , Movimiento Celular , Neoplasias Pulmonares/genética , ARN no Traducido/análisis , ARN no Traducido/genética , Anciano , Análisis de Varianza , Animales , Carcinoma de Pulmón de Células no Pequeñas/química , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Células Escamosas/química , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Regulación hacia Abajo , Femenino , Fibroblastos , Expresión Génica , Humanos , Hibridación in Situ , Neoplasias Pulmonares/química , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Desnudos , Persona de Mediana Edad , Células 3T3 NIH , Metástasis de la Neoplasia , Pronóstico , Modelos de Riesgos Proporcionales , Interferencia de ARN , ARN Largo no Codificante , ARN no Traducido/fisiologíaRESUMEN
Epigenetic changes play a crucial role in leukemogenesis. HDACs are frequently recruited to target gene promoters by balanced translocation derived oncogenic fusion proteins. As important epigenetic effector mechanisms, histone deacetylases (HDAC) have emerged as potential therapeutic targets. However, the patterns of HDAC1 localization and the role of HDACs in leukemia pathogenesis remain to be elucidated. Using ChIP-Chip analyses we analyzed HDAC1 deposition patterns at more than 10,000 gene promoters in a large cohort of leukemia patients and CD34+ controls. HDAC1 binding was significantly increased in AML blasts compared to CD34+ progenitor cells at 130 gene promoters whereas decreased binding was observed at 66 gene promoters. Distinct HDAC1 binding patterns occurred in AML subtypes with balanced translocations t(15;17), t(8;21) and inv(16). In addition, a more generalized signature was established, that revealed an AML specific pattern of HDAC1 distribution. Many of the HDAC1-binding altered promoters regulate genes involved in hematopoiesis, transcriptional regulation and signal transduction. HDAC1 binding patterns were associated with patients' event free survival. This is the first study to determine HDAC1 modification patterns in a large number of AML and ALL specimens. Our findings suggest that dyslocalization of HDAC1 is a common feature in AML. Importantly, HDAC1 modifications possess prognostic power for patient survival. Our findings suggest that altered HDAC1 localization is an explanation for the observed benefit of HDAC inhibitors in AML therapy.
Asunto(s)
Hematopoyesis/genética , Histona Desacetilasa 1/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/mortalidad , Regiones Promotoras Genéticas , Adolescente , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Niño , Preescolar , Cromatina/metabolismo , Femenino , Humanos , Lactante , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/genética , Masculino , Persona de Mediana Edad , Pronóstico , Regiones Promotoras Genéticas/fisiología , Unión Proteica/fisiología , Análisis de Supervivencia , Adulto JovenRESUMEN
PURPOSE: Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. EXPERIMENTAL DESIGN: The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. RESULTS: EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. CONCLUSIONS: Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Metilación de ADN , Silenciador del Gen , Neoplasias Pulmonares/genética , Regiones Promotoras Genéticas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/secundario , Línea Celular Tumoral , Islas de CpG , Epigénesis Genética , Femenino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Pronóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de la Familia Eph , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Tau, a neuronal microtubule-associated protein that aggregates in Alzheimer disease is a natively unfolded protein. In solution, Tau adopts a "paperclip" conformation, whereby the N- and C-terminal domains approach each other and the repeat domain ( Jeganathan, S., von Bergen, M., Brutlach, H., Steinhoff, H. J., and Mandelkow, E. (2006) Biochemistry 45, 2283-2293 ). In AD, Tau is in a hyperphosphorylated state. The consequences for microtubule binding or aggregation are a matter of debate. We therefore tested whether phosphorylation alters the conformation of Tau. To avoid the ambiguities of heterogeneous phosphorylation we cloned "pseudo-phosphorylation" mutants of Tau where combinations of Ser or Thr residues were converted into Glu. These mutations were combined with FRET pairs inserted in different locations to allow distance measurements. The results show that the paperclip conformation becomes tighter or looser, depending on the pseudo-phosphorylation state. In particular, pseudo-phosphorylation at the epitope of the diagnostic antibody AT8* (S199E + S202E + T205E) moves the N-terminal domain away from the C-terminal domain. Pseudo-phosphorylation at the PHF1 epitope (S396E + S404E) moves the C-terminal domain away from the repeat domain. In both cases the paperclip conformation is opened up. By contrast, the combination of AT8* and PHF1 sites leads to compaction of the paperclip, such that the N-terminus approaches the repeat domain. The compaction becomes even stronger by combining pseudo-phosphorylated AT8*, AT100, and PHF1 epitopes. This is accompanied by a strong increase in the reaction with conformation-dependent antibody MC1, suggesting the generation of a pathological conformation characteristic for Tau in AD. Furthermore, the compact paperclip conformation enhances the aggregation to paired helical filaments but has little influence on microtubule interactions. The data provide a framework for the global folding of Tau dependent on proline-directed phosphorylation in the domains flanking the repeats and the consequences for pathological properties of Tau.
Asunto(s)
Enfermedad de Alzheimer/patología , Anticuerpos/inmunología , Epítopos/metabolismo , Prolina/metabolismo , Pliegue de Proteína , Proteínas tau/química , Proteínas tau/metabolismo , Dicroismo Circular , Epítopos/inmunología , Guanidina/farmacología , Microscopía Electrónica de Transmisión , Mutación/genética , Fosforilación , Prolina/genética , Conformación Proteica , Desnaturalización Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Proteínas tau/ultraestructuraRESUMEN
Development of distant metastasis is the major reason for cancer-related deaths worldwide. Adjuvant therapy approaches after local therapies are most effective when specific targets are inhibited. Recently, we identified S100P overexpression as a strong predictor for metastasis development in early-stage non-small cell lung cancer (NSCLC) patients. Here, we show that S100P overexpression increased angiogenesis in and metastasis formation from s.c. xenotransplants of NSCLC cells. Plasmid-derived short hairpin RNAs (shRNA) were developed as specific adjuvant therapy. I.v. injected shRNA against S100P significantly decreased S100P protein expression in xenograft tumors and inhibited tumor angiogenesis in vivo. Metastasis formation 8 weeks after primary tumor resection was significantly reduced. Lung metastases developed in 31% of mice treated with S100P-targeting shRNAs compared with 64% in control shRNA-treated mice (P < 0.05). These findings suggest that RNA interference-based therapy approaches can be highly effective in the adjuvant setting.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , ARN Interferente Pequeño/genética , Adenocarcinoma/genética , Adenocarcinoma/secundario , Adenocarcinoma/terapia , Animales , Proteínas de Unión al Calcio/genética , Carcinoma de Pulmón de Células no Pequeñas/irrigación sanguínea , Carcinoma de Pulmón de Células no Pequeñas/secundario , Línea Celular Tumoral , Terapia Genética/métodos , Humanos , Neoplasias Pulmonares/irrigación sanguínea , Neoplasias Pulmonares/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Neovascularización Patológica/terapia , Interferencia de ARN , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The abnormal aggregation of tau protein into paired helical filaments (PHFs) is one of the hallmarks of Alzheimer's disease. Aggregation takes place in the cytoplasm and could therefore be cytotoxic for neurons. To find inhibitors of PHF aggregation we screened a library of 200,000 compounds. The hits found in the PHF inhibition assay were also tested for their ability to dissolve preformed PHFs. The results were obtained using a thioflavin S fluorescence assay for the detection and quantification of tau aggregation in solution, a tryptophan fluorescence assay using tryptophan-containing mutants of tau, and confirmed by a pelleting assay and electron microscopy of the products. Here we demonstrate the feasibility of the approach with several compounds from the family of anthraquinones, including emodin, daunorubicin, adriamycin, and others. They were able to inhibit PHF formation with IC50 values of 1-5 microm and to disassemble preformed PHFs at DC50 values of 2-4 microm. The compounds had a similar activity for PHFs made from different tau isoforms and constructs. The compounds did not interfere with the stabilization of microtubules by tau. Tau-inducible neuroblastoma cells showed the formation of tau aggregates and concomitant cytotoxicity, which could be prevented by inhibitors. Thus, small molecule inhibitors could provide a basis for the development of tools for the treatment of tau pathology in AD and other tauopathies.