RESUMO
BACKGROUND: The UHRF1 gene possesses an essential role in DNA methylation maintenance, but its contribution to tumor suppressor gene hypermethylation in primary human cancers currently remains unclear. METHODS: mRNA expression levels of UHRF1, DNMT1, DNMT3A, DNMT3B, and E2F1 were evaluated in 105 primary nonsmall cell lung carcinomas by quantitative polymerase chain reaction. The methylation status of CDKN2A and RASSF1 promoters was examined by pyrosequencing. UHRF1 was knocked down by short hairpin RNA in A549 lung adenocarcinoma cells. RESULTS: All 4 genes were overexpressed in a coordinated manner in the lung tumor tissues, and their expression correlated with that of E2F1. Higher UHRF1 expression in tumor tissues correlated with the hypermethylation of CDKN2A (P = .005) and RASSF1 promoters (P = .034), and the relationship with a combined epigenotype was even stronger (P = 2.3 × 10(-4) ). When UHRF1 was knocked down in A549 lung adenocarcinoma cells, lower methylation levels of RASSF1, CYGB, and CDH13 promoters were observed. Also, UHRF1 knockdown clones demonstrated reduced proliferation and decreased cell migration properties. CONCLUSIONS: Our data demonstrate that UHRF1 is a key epigenetic switch, which controls cell cycle in nonsmall cell lung carcinoma through its ability to sustain the transcriptional silencing of tumor suppressor genes by maintaining their promoters in a hypermethylated status. Thus, UHRF1 should be considered, along with DNMTs, among the potential targets for cancer treatment and/or therapeutic stratification.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Inativação Gênica , Neoplasias Pulmonares/genética , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Metiltransferase 3A , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica/fisiologia , Genes Supressores de Tumor/fisiologia , Humanos , Neoplasias Pulmonares/patologia , Masculino , Transfecção , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , DNA Metiltransferase 3BRESUMO
p73 possesses an extrinsic P1 promoter and an intrinsic P2 promoter controlling the expression of the pro-apoptotic TAp73 isoforms and the anti-apoptotic ΔΝp73 isoforms respectively. In this study, we investigated the DNA methylation status of both promoters as a means of epigenetic transcriptional control of their corresponding isoforms in 102 primary non-small cell lung carcinomas (NSCLCs). We demonstrated that while P1 hypermethylation-associated reduction of TAp73 mRNA levels is relatively infrequent, the P2 hypomethylation-associated over-expression of ΔΝp73 mRNA is a frequent event, particularly among squamous cell carcinomas. P2 hypomethylation strongly correlated with LINE-1 element hypomethylation, indicating that ΔΝp73 over-expression may be a passive consequence of global DNA hypomethylation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Proteínas Nucleares/genética , Ativação Transcricional , Proteínas Supressoras de Tumor/genética , Adenocarcinoma/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Proteína Tumoral p73RESUMO
The p73 gene possesses an extrinsic P1 promoter and an intrinsic P2 promoter, resulting in TAp73 and DeltaNup73 isoforms, respectively. The ultimate effect of p73 in oncogenesis is thought to depend on the apoptotic TA to antiapoptotic DeltaN isoforms' ratio. This study was aimed at identifying novel transcription factors that affect TA isoform synthesis. With the use of bioinformatics tools, in vitro binding assays, and chromatin immunoprecipitation analysis, a region extending -233 to -204 bp upstream of the transcription start site of the human p73 P1 promoter, containing conserved Sp1-binding sites, was characterized. Treatment of cells with Sp1 RNAi and Sp1 inhibitor functionally suppress TAp73 expression, indicating positive regulation of P1 by the Sp1 protein. Notably Sp1 inhibition or knockdown also reduces DeltaNup73 protein levels. Therefore, Sp1 directly regulates TAp73 transcription and affects DeltaNup73 levels in lung cancer. TAp73gamma was shown to be the only TA isoform overexpressed in several lung cancer cell lines and in 26 non-small cell lung cancers, consistent with Sp1 overexpression, thereby questioning the apoptotic role of this specific p73 isoform in lung cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/metabolismo , Fator de Transcrição Sp1/metabolismo , Proteínas Supressoras de Tumor/genética , Sequência de Bases/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Biologia Computacional , Sequência Conservada/genética , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Plicamicina/análogos & derivados , Plicamicina/farmacologia , Ligação Proteica/genética , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Proteína Tumoral p73 , Proteínas Supressoras de Tumor/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
LINE-1 and Alu elements are non-LTR retrotransposons, constituting together over 30% of the human genome and they are frequently hypomethylated in human tumors. A relationship between global hypomethylation and genomic instability has been shown, however, there is little evidence to suggest active role for hypomethylation-mediated reactivation of retroelements in human cancer. In our study, we examined by Pyrosequencing the methylation levels of LINE-1 and Alu sequences in 48 primary nonsmall cell carcinomas and their paired adjacent tissues. We demonstrate a significant reduction of the methylation levels of both elements (p = 7.7 x 10(-14) and 9.6 x 10(-7), respectively). The methylation indices of the 2 elements correlated (p = 0.006), suggesting a possible common mechanism for their methylation maintenance. Genomic instability was measured utilizing 11 fluorescent microsatellite markers located on lung cancer hot-spot regions such as 3p, 5q 9p, 13q and 17p. Hypomethylation of both transposable elements was associated with increased genomic instability (LINE, p = 7.1 x 10(-5); Alu, p = 0.008). The reduction of the methylation index of LINE-1 and Alu following treatment of 3 lung cell lines with 5-aza-2'-deoxycitidine, consistently resulted in increased expression of both elements. Our study demonstrates the strong link between hypomethylation of transposable elements with genomic instability in non-small cell lung cancer and provides early evidence for a potential active role of these elements in lung neoplasia. As demethylating agents are now entering lung cancer trials, it is imperative to gain a greater insight into the potential reactivation of silent retrotransposons in order to advance for the clinical utilization of epigenetics in cancer therapy.