RESUMEN
The maintenance cytosine DNA methyltransferase DNMT1 and de novo methyltransferase DNMT3b cooperate to establish aberrant DNA methylation and chromatin complexes to repress gene transcription during cancer development. The expression of DNMT3b was constitutively increased 5-20-fold in hTERT/CDK4-immortalized human bronchial epithelial cells (HBECs) before treatment with low doses of tobacco carcinogens. Overexpression of DNMT3b increased and accelerated carcinogen-induced transformation. Genome-wide profiling of transformed HBECs identified 143 DNMT3b-target genes, many of which were transcriptionally regulated by the polycomb repressive complex 2 (PRC2) complex and silenced through aberrant methylation in non-small-cell lung cancer cell lines. Two genes studied in detail, MAL and OLIG2, were silenced during transformation, initially through enrichment for H3K27me3 and H3K9me2, commonly methylated in lung cancer, and exert tumor suppressor effects in vivo through modulating cancer-related pathways. Re-expression of MAL and OLIG2 to physiological levels dramatically reduced the growth of lung tumor xenografts. Our results identify a key role for DNMT3b in the earliest stages of initiation and provide a comprehensive catalog of genes targeted for silencing by this methyltransferase in non-small-cell lung cancer.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis Genética/genética , Neoplasias Pulmonares/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Carcinógenos/toxicidad , Cromatina/genética , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Glicoproteínas de Membrana/biosíntesis , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Factor de Transcripción 2 de los Oligodendrocitos , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Transporte de Proteínas , Receptores de Interleucina-1/biosíntesis , Telomerasa/metabolismo , Nicotiana/toxicidad , Ensayos Antitumor por Modelo de Xenoinjerto , ADN Metiltransferasa 3BRESUMEN
The heparan sulfate 6-O-endosulfatase (SULF2) promotes growth and metastasis of solid tumors. We recently identified that cytosine methylation of the SULF2 promoter is associated with better survival of resected lung adenocarcinoma patients, and now also demonstrates a marginal improvement in survival of advanced non-small cell lung cancer (NSCLC) patients receiving standard chemotherapy (hazard ratio=0.63, P=0.07). Subsequent studies focused on investigating the effect of methylation on SULF2 expression and its genome-wide impact. The genes and pathways modulated by epigenetic inactivation of SULF2 and the effects on sensitivity to chemotherapy were characterized in vitro and in vivo. Silencing SULF2 through small interfering RNA or methylation primarily increased expression of interferon-inducible genes including ISG15, a marker for increased sensitivity to topoisomerase-1 inhibitors such as camptothecin (CPT). NSCLC cell lines with methylated SULF2 (SULF2M) express 60-fold higher ISG15 compared with SULF2 unmethylated (SULF2U) NSCLC cell lines and normal human bronchial epithelial cells. In vitro, SULF2M and high ISG15 (ISG15H)-expressing NSCLC cell lines were 134-fold more sensitive to CPT than SULF2U and low ISG15 (ISG15L)-expressing cell lines. Topotecan, a soluble analog of CPT and FDA-approved anticancer drug, dramatically arrested the growth of SULF2M-ISG15H, but not SULF2U-ISG15L lung tumors in nude mice (P<0.002). Similarly, high ISG15 expression that is comparable to the topotecan (TPT)-sensitive NSCLC cell lines was found in tumors from 25% of NSCLC patients compared with normal lung, indicating a potential to identify and target the most sensitive NSCLC subpopulation for personalized TPT therapy.
Asunto(s)
Citocinas/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Regiones Promotoras Genéticas , Sulfotransferasas/genética , Sulfotransferasas/metabolismo , Ubiquitinas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/farmacología , Camptotecina/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Línea Celular Tumoral , Cisplatino/farmacología , Citocinas/genética , Metilación de ADN , ADN-Topoisomerasas de Tipo I/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidad , Ratones , Ratones Desnudos , Pronóstico , Interferencia de ARN , ARN Interferente Pequeño , Sulfatasas , Inhibidores de Topoisomerasa I/farmacología , Topotecan/farmacología , Ubiquitinas/genéticaRESUMEN
Chemokines are important regulators of directional cell migration and tumor metastasis. A genome-wide transcriptome array designed to uncover novel genes silenced by methylation in lung cancer identified the CXC-subfamily of chemokines. Expression of 11 of the 16 known human CXC-chemokines was increased in lung adenocarcinoma cell lines after treatment with 5-aza-2'-deoxycytidine (DAC). Tumor-specific methylation leading to silencing of CXCL5, 12 and 14 was found in over 75% of primary lung adenocarcinomas and DAC treatment restored the expression of each of the silenced gene. Forced expression of CXCL14 in H23 cells, where this gene is silenced by methylation, increased cell death in vitro and dramatically reduced the in vivo growth of lung tumor xenografts through necrosis of up to 90% of the tumor mass. CXCL14 re-expression had a profound effect on the genome altering the transcription of over 1000 genes, including increased expression of 30 cell-cycle inhibitor and pro-apoptosis genes. In addition, CXCL14 methylation in sputum from asymptomatic early-stage lung cancer cases was associated with a 2.9-fold elevated risk for this disease compared with controls, substantiating its potential as a biomarker for early detection of lung cancer. Together, these findings identify CXCL14 as an important tumor suppressor gene epigenetically silenced during lung carcinogenesis.
Asunto(s)
Adenocarcinoma/patología , Quimiocinas CXC/genética , Epigénesis Genética , Silenciador del Gen , Neoplasias Pulmonares/patología , Adenocarcinoma/genética , Metilación de ADN , Humanos , Neoplasias Pulmonares/genética , Necrosis , Transcripción GenéticaRESUMEN
Inactivation of the p16INK4a (p16) tumor suppressor gene by promoter hypermethylation and mutation within exon 3 of beta-catenin represent two of the more common gene alterations in human hepatocellular carcinoma (HCC). One exposure implicated in the development of liver cancer is hepatitis B or C viral infection, which causes chronic destruction and regeneration of liver parenchyma. Treatment of rats with high doses of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) also causes liver toxicity and a high incidence of tumors. The purpose of the current investigation was to define the prevalence of genetic alterations in p16 and beta-catenin in NNK-induced rat liver cancer to determine if the molecular mechanisms seen in human tumors are the same in this animal model. DNA isolated from 15 adenomas and 14 carcinomas was examined for methylation of p16 by methylation-specific PCR. p16 methylation was detected in five of 15 adenomas and eight of 14 carcinomas (45% of all tumors). Methylation of p16 was extensive within the 5'-untranslated region and exon 1alpha, areas shown to correlate with loss of gene transcription. Liver tumors were also screened for mutations within exon 3 of beta-catenin. Single strand conformation polymorphism and DNA sequencing revealed five mutations in four of 29 tumors (14%). Mutations were present in three adenomas and one carcinoma and were located within codons 33, 36 or 37. All mutations resulted in amino acid substitutions; three of these mutations occurred at potential serine phosphorylation sites. Our results link two important regulatory pathways altered in human HCC to cancer induced in the rat NNK model. The fact that common genetic alterations are observed between rodent and human HCC suggests that the rat NNK model could be useful for identifying additional genetic alterations critical to the initiation of HCC.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Proteínas del Citoesqueleto/genética , Neoplasias Hepáticas Experimentales/genética , Mutación , Nitrosaminas/toxicidad , Transactivadores , Animales , Secuencia de Bases , Metilación de ADN , Humanos , Neoplasias Hepáticas Experimentales/inducido químicamente , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Ratas , Ratas Endogámicas F344 , beta CateninaRESUMEN
Structural variation of the endothelin A-selective antagonist (S)-3-methoxy-2-(4,6-dimethoxypyrimidin-2-yloxy)-3, 3-diphenylpropionic acid (LU 135252) led to analogues which retain ET(A) affinity but exhibit substantial ET(B) affinity as well. The most active derivative obtained is (S)-3-[2-(3, 4-dimethoxyphenyl)ethoxy]-2-(4,6-dimethylpyrimidin-2-yloxy)- 3, 3-diphenylpropionic acid (LU 302872), which can be prepared in enantiomerically pure form in eight steps via an acid-catalyzed transetherification. It has a K(i) = 2.15 nM for binding to the ET(A) receptor and a K(i) = 4.75 nM for binding to the ET(B) receptor, is orally available, and antagonizes the big ET-induced blood pressure increase in rats and the big ET-induced bronchospasm in guinea pigs each time at a dose of 10 mg/kg.
Asunto(s)
Antagonistas de los Receptores de Endotelina , Propionatos/síntesis química , Pirimidinas/síntesis química , Administración Oral , Animales , Presión Sanguínea/efectos de los fármacos , Broncoconstricción/efectos de los fármacos , Células CHO , Cricetinae , Cobayas , Masculino , Propionatos/administración & dosificación , Propionatos/química , Propionatos/farmacología , Pirimidinas/administración & dosificación , Pirimidinas/química , Pirimidinas/farmacología , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A , Receptor de Endotelina B , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A novel class of endothelin-A receptor ligands was discovered by high-throughput screening. Lead structure optimization led to highly potent antagonists which can be synthesized in a short sequence. The compounds are endothelin-A-selective, are orally available, and show a long duration of action.
Asunto(s)
Antagonistas de los Receptores de Endotelina , Endotelinas/toxicidad , Pirimidinas/síntesis química , Administración Oral , Animales , Compuestos de Dansilo/farmacología , Muerte Súbita , Diseño de Fármacos , Endotelinas/antagonistas & inhibidores , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Pirimidinas/farmacología , Ratas , Receptor de Endotelina A , Receptores de Endotelina/metabolismo , Relación Estructura-Actividad , Sulfonamidas/farmacologíaRESUMEN
The new endothelin (ET) receptor antagonist LU 127043 shows higher ETA affinity than BQ 123, Ro 46-2005, and BMS 182874, with a Ki of 6 nmol/L vs. 19, 28, and 57 nmol/L. ETA/ETB selectivity of LU 127043 of about 160 is comparable to that of BQ 123 (200) and is much greater than that of Ro 46-2005 (0.93) and SB 209670 (0.74). In rabbit aortic segments, LU 127043 shows ET antagonistic potency similar to that of BQ 123 and BMS 182874 (pA2 7.34 vs. 7.36 and 7.09), whereas SB 209670 is more potent (9.80). In rats, LU 127043 completely prevents the ET-1-induced sudden death due to coronary constriction, as indicated by a pronounced T-wave increase. With i.v. pretreatment, LU 127043 is as effective as SB 209670, whereas it is three times more active using 4 h oral pretreatment. Even 8 h after oral administration, LU 127043, in contrast to SB 209670, provides dose-dependent protection. Hence, LU 127043 is an example of a selective ETA antagonist with high oral availability and long duration of action. Because the in vivo efficacy of other high affinity ET antagonists is relatively low, further optimization for therapeutic use should concentrate on pharmacokinetic properties.