RESUMO
The NF-E2-related factor 2 (referred to as NRF2) transcription factor binds antioxidant responsive elements within the promoters of cytoprotective genes to induce their expression. Next-generation sequencing studies in lung cancer have shown a significant number of activating mutations within the NRF2 signaling pathway. Mutations in components of the SWI/SNF chromatin-remodeling complex, a general regulator of transcription using either BRG1 or BRM as the catalytic subunit, also frequently occur in lung cancers. Importantly, low BRG1 expression levels in primary human NSCLC correlated with increased NRF2-target gene expression. Here, we show that loss of SWI/SNF complex function activated a subset of NRF2-mediated transcriptional targets. Using a series of isogenic NSCLC lines with reduced or depleted BRG1 and/or BRM expression, we observed significantly increased expression of the NRF2-target genes HMOX1 and GSTM4. In contrast, expression of the NRF2 target genes NQO1 and GCLM modestly increased following BRM reduction. Chromatin immunoprecipitation showed that BRG1 knockdown led to increased NRF2 binding at its respective ARE sites in the HMOX1 promoter but not in NQO1 and GCLM. Our data demonstrate that loss of BRG1 or BRM in lung cancer results in activation of the NRF2/KEAP1 pathway and HMOX1 expression. Therefore, we provide an additional molecular explanation for why patients harboring BRG1 or BRM mutations show poor prognoses. A better understanding of this mechanism may yield novel insights into the design of targeted treatment modalities. IMPLICATIONS: Our study identifies a novel mechanism for how mutations in the SMARCA4 gene may drive progression of human lung adenocarcinomas.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA Helicases/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Análise de Sequência de DNA/métodos , Transdução de Sinais , Fatores de Transcrição/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Regulação Neoplásica da Expressão Gênica , Glutamato-Cisteína Ligase/genética , Glutationa Transferase/genética , Heme Oxigenase-1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Mutação , NAD(P)H Desidrogenase (Quinona)/genética , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismoRESUMO
SWI/SNF chromatin remodeling complexes regulate critical cellular processes, including cell-cycle control, programmed cell death, differentiation, genomic instability, and DNA repair. Inactivation of this class of chromatin remodeling complex has been associated with a variety of malignancies, including lung, ovarian, renal, liver, and pediatric cancers. In particular, approximately 10% of primary human lung non-small cell lung cancers (NSCLC) display attenuations in the BRG1 ATPase, a core factor in SWI/SNF complexes. To evaluate the role of BRG1 attenuation in NSCLC development, we examined the effect of BRG1 silencing in primary and established human NSCLC cells. BRG1 loss altered cellular morphology and increased tumorigenic potential. Gene expression analyses showed reduced expression of genes known to be associated with progression of human NSCLC. We demonstrated that BRG1 losses in NSCLC cells were associated with variations in chromatin structure, including differences in nucleosome positioning and occupancy surrounding transcriptional start sites of disease-relevant genes. Our results offer direct evidence that BRG1 attenuation contributes to NSCLC aggressiveness by altering nucleosome positioning at a wide range of genes, including key cancer-associated genes.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Cromatina/fisiologia , DNA Helicases/fisiologia , Neoplasias Pulmonares/patologia , Proteínas Nucleares/fisiologia , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , DNA Helicases/genética , Metilação de DNA , Progressão da Doença , Feminino , Humanos , Camundongos , Proteínas Nucleares/genética , Nucleossomos , Fatores de Transcrição/genéticaRESUMO
Eukaryotic organisms package DNA into chromatin for compact storage in the cell nucleus. However, this process promotes transcriptional repression of genes. To overcome the transcriptional repression, chromatin remodeling complexes have evolved that alter the configuration of chromatin packaging of DNA into nucleosomes by histones. The SWI/SNF chromatin remodeling complex uses energy from ATP hydrolysis to reposition nucleosomes and make DNA accessible to transcription factors. Recent studies showing mutations of BRG1, one of two mutually exclusive ATPase subunits, in human tumor cell lines and primary tissue samples have implicated a role for its loss in cancer development. While most of the mutations lead to complete loss of BRG1 protein expression, others result in single amino acid substitutions. To better understand the role of these BRG1 point mutations in cancer development, we characterized SWI/SNF function in human tumor cell lines with these mutations in the absence of BRM expression, the other ATPase component. We found that the mutant BRG1 proteins still interacted with the core complex members and appeared at the promoters of target genes. However, while these mutations did not affect CD44 and CDH1 expression, known targets of the SWI/SNF complex, they did abrogate Rb-mediated cell-cycle arrest. Therefore, our results implicate that these mutations disrupt the de novo chromatin remodeling activity of the complex without affecting the status of existing nucleosome positioning.
