Dataset Information

Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

ABSTRACT: BACKGROUND:The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. KEAP1 silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer. METHODS:We performed a detailed methylation density map of 13 CpGs located into the KEAP1 promoter region by analyzing a set of 25 cell lines from different histologies of lung cancer. The methylation status was assessed using quantitative methylation specific PCR (QMSP) and pyrosequencing, and the performance of the two assays was compared. RESULTS:Hypermethylation at the promoter region of the KEAP1 was detected in one third of cell lines and its effect on the modulation KEAP1 mRNA levels was also confirmed by in vitro 5-Azacytidine treatment on lung carcinoid, small lung cancer and adenocarcinoma cell lines. QMSP and pyrosequencing showed a high rate of concordant results, even if pyrosequencing revealed two different promoter CpGs sub-islands (P1a and P1b) with a different methylation density pattern. CONCLUSIONS:Our results confirm the effect of methylation on KEAP1 transcription control across multiple histologies of lung cancer and suggest pyrosequencing as the best approach to investigate the pattern of CpGs methylation in the promoter region of KEAP1. The validation of this approach on lung cancer patient cohorts is mandatory to clarify the prognostic value of the epigenetic deregulation of KEAP1 in lung tumors.

SUBMITTER: Fabrizio FP

PROVIDER: S-EPMC6600322 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Methylation Density Pattern of <i>KEAP1</i> Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

Fabrizio Federico Pio FP Sparaneo Angelo A Centra Flavia F Trombetta Domenico D Storlazzi Clelia Tiziana CT Graziano Paolo P Maiello Evaristo E Fazio Vito Michele VM Muscarella Lucia Anna LA

International journal of molecular sciences 20190531 11

<h4>Background</h4>The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. <i>KEAP1</i> silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer.<h4>Methods</h4>We performed a detailed methylation density map of 13 CpGs located into the <i>KEAP1</i> ...[more]

PMID: 31159323

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Project description:BackgroundLung cancers are the most common type of human malignancy and are intractable. Lung cancers are generally classified into four histopathological subtypes: adenocarcinoma (AD), squamous cell carcinoma (SQ), large cell carcinoma (LC), and small cell carcinoma (SC). Molecular biological characterization of these subtypes has been performed mainly using DNA microarrays. In this study, we compared the gene expression profiles of these four subtypes using twelve human lung cancer cell lines and the more reliable quantitative real-time PCR (qPCR).ResultsWe selected 100 genes from public DNA microarray data and examined them by DNA microarray analysis in eight test cell lines (A549, ABC-1, EBC-1, LK-2, LU65, LU99, STC 1, RERF-LC-MA) and a normal control lung cell line (MRC-9). From this, we extracted 19 candidate genes. We quantified the expression of the 19 genes and a housekeeping gene, GAPDH, with qPCR, using the same eight cell lines plus four additional validation lung cancer cell lines (RERF-LC-MS, LC-1/sq, 86-2, and MS-1-L). Finally, we characterized the four subtypes of lung cancer cell lines using principal component analysis (PCA) of gene expression profiling for 12 of the 19 genes (AMY2A, CDH1, FOXG1, IGSF3, ISL1, MALL, PLAU, RAB25, S100P, SLCO4A1, STMN1, and TGM2). The combined PCA and gene pathway analyses suggested that these genes were related to cell adhesion, growth, and invasion. S100P in AD cells and CDH1 in AD and SQ cells were identified as candidate markers of these lung cancer subtypes based on their upregulation and the results of PCA analysis. Immunohistochemistry for S100P and RAB25 was closely correlated to gene expression.ConclusionsThese results show that the four subtypes, represented by 12 lung cancer cell lines, were well characterized using qPCR and PCA for the 12 genes examined. Certain genes, in particular S100P and CDH1, may be especially important for distinguishing the different subtypes. Our results confirm that qPCR and PCA analysis provide a useful tool for characterizing cancer cell subtypes, and we discuss the possible clinical applications of this approach.

Dataset Information

Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

Publications

Methylation Density Pattern of <i>KEAP1</i> Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing.

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