RESUMO
BACKGROUND: Squamous cell lung cancer (SCLC) arises from bronchial changes: basal cell hyperplasia (BCH), squamous metaplasia (SM), and dysplasia. However, the premalignant process preceding SCLC is not inevitable; it can stop at any of the bronchial lesions. Previously, we hypothesized that combinations of premalignant lesions observed in the small bronchi of SCLC patients can reflect the different "scenarios" of the premalignant process: BCHi-the stoppage at the stage of hyperplasia and BCHSM-the progression of hyperplasia to metaplasia. METHODS AND RESULTS: In this study, using whole-genome bisulfite sequencing we analyzed the DNA methylome of two forms of BCH: isolated BCH (BCHi) and BCH co-occurred with SM (BCHSM) in the small bronchi of SCLC patients. It was shown that BCHi harbored differentially methylated regions (DMRs) affecting genes associated with regulating phosphatase activity. In BCHSM, DMRs were found in genes involved in PI3K-Akt and AMPK signaling pathways. DMRs were also found to affect specific miRNA genes: miR-34a and miR-3648 in BCHi and miR-924 and miR-100 in BCHSM. CONCLUSIONS: Thus, this study demonstrated the significant changes in DNA methylome between the isolated BCH and BCH combined with SM. The identified epigenetic alterations may underlie different "scenarios" of the premalignant process in the bronchial epithelium.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , MicroRNAs , Lesões Pré-Cancerosas , Humanos , Hiperplasia/genética , Epigenoma , Fosfatidilinositol 3-Quinases , Lesões Pré-Cancerosas/genética , Metaplasia , MicroRNAs/genéticaRESUMO
Malignant cell transformation is accompanied with abnormal DNA methylation, such as the hypermethylation of certain gene promoters and hypomethylation of retrotransposons. In particular, the hypomethylation of the human-specific family of LINE-1 retrotransposons was observed in lung cancer tissues. It is also known that the circulating DNA (cirDNA) of blood plasma and cell-surface-bound circulating DNA (csb-cirDNA) of cancer patients accumulate tumor-specific aberrantly methylated DNA fragments, which are currently considered to be valuable cancer markers. This work compares LINE-1 retrotransposon methylation patterns in cirDNA of 16 lung cancer patients before and after treatment. CirDNA was isolated from blood plasma, and csb-cirDNA fractions were obtained by successive elution with EDTA-containing phosphate buffered saline and trypsin. Concentrations of methylated LINE-1 region 1 copies (LINE-1-met) were assayed by real-time methylation-specific PCR. LINE-1 methylation levels were normalized to the concentration of LINE-1 region 2, which was independent of the methylation status (LINE-1-Ind). The concentrations of LINE-1-met and LINE-1-Ind in csb-cirDNA of lung cancer patients exhibited correlations before treatment (r = 0.54), after chemotherapy (r = 0.72), and after surgery (r = 0.83) (P < 0.05, Spearman rank test). In the total group of patients, the level of LINE-1 methylation (determined as the LINE-1-met/LINE-1-Ind ratio) was shown to increase significantly during the follow-up after chemotherapy (P < 0.05, paired t test) and after surgery compared to the level of methylation before treatment (P < 0.05, paired t test). The revealed association between the level of LINE-1 methylation and the effect of antitumor therapy was more pronounced in squamous cell lung cancer than in adenocarcinoma (P < 0.05 and P > 0.05, respectively). These results suggest a need for the further investigation of dynamic changes in levels of LINE-1 methylation depending on the antitumor therapy.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Neoplasias Pulmonares/genética , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/mortalidade , Adenocarcinoma/cirurgia , Adenocarcinoma de Pulmão , Idoso , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Carboplatina/uso terapêutico , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/cirurgia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Paclitaxel/uso terapêutico , Análise de Sobrevida , Resultado do TratamentoRESUMO
Analysis of DNA epigenetic mutations in the blood circulating DNA is a prospective trend for creation of noninvasive methods for the diagnosis and treatment efficiency monitoring in cancer. The methylation status of target genes in circulating DNA was evaluated by methods based on preliminary bisulfite conversion of DNA. We used a different approach based on selection of hypermethylated sequences of circulating DNA by means of DNA-methyl-binding protein (methylated CpG island recovery assay, MIRA). Methylation was evaluated for RARß2 tumor suppression gene in circulating DNA in lung cancer and a trend was detected to higher methylation of this gene in the patients in comparison with healthy donors.