Lung cancer is the most common diagnosed cancer, and a leading cause of cancer-associated deaths worldwide. It is imperative to understand the molecular mechanisms underlying early lung cancer development, to identify better diagnostic and treatment options. We have conducted a multi-platform genomic screen of sixty paired early stage tumor and non-tumor adjacent tissues excised from a cohort of lung cancer patients recruited in our longstanding NCI-MD Case-Control Study. Tissues were characterized through miRNA and mRNA expression, and methylation profiling. Our aim is to provide a framework that can be interrogated to illuminate biological differences associated with racial disparities, to find diagnostic/prognostic biomarkers of lung cancer, and to define miRNA transcriptional networks operational in lung cancer. MicroRNAs (miRNAs) are 20-22 nucleotide-long non-coding RNAs that regulate mRNA transcription and translation through sequence-specific base pairing. MiRNAs are transcribed from large pri-miRNAs, as independent transcripts or within introns of protein-coding genes. It is unclear whether they possess independent promoter regions. However, several miRNAs are adjacent to CpG islands (regions rich in CG dinucleotides). CpG island methylation is a well-described mechanism by which genes with tumor-suppressive function are silenced in cancer. It has been reported that some tumor-suppressive miRNAs are epigenetically controlled through CpG island methylation. Such regulation could have important implications for progression of early stage lung cancer. Here, tumor and adjacent non-tumor tissue DNA were analyzed using the Illumina Infinium HumanMethylation27 BeadChip assays, featuring genome-wide coverage of CpG sites. This platform covers 110 miRNA promoters, which are annotated by being close to gene-associated CpGs. We found seven miRNA loci hypermethylated in tumors. Of these, 4 miRNAs are located within homeobox gene clusters, miR-196b (HOXA9), miR-615 (HOXC5), miR-10a (HOXB4), and miR-10b (HOXD4). Two other miRNAs are located adjacent to genes with significant changes in methylation, miR-638 (DNM2) and miR-639 (GPSN2). Lastly, methylation of miR-34b/c (BTG4) was consistent with prior studies. A comparative analysis of methylation at these loci showed a high concordance between our study and methylation profiling in lung adenocarcinoma conducted by TCGA (The Cancer Genome Atlas), with r2: 0.8. Therefore, methylation at these sites is a generalized finding. Additionally, we confirmed the methylation of miR-615 in A549 lung cancer cell line and its demethylation and reactivation by exposure to 5-aza/TSA. Downstream targets of miR-615 are being explored using available transcriptomic data to gain insight into the functional role of miRNAs in lung cancer progression. We are also expanding the analysis to other lung cancer cohorts to evaluate the association of miRNA methylation with patient survival.

Citation Format: Saikartik A. Kumar, Ana I. Robles, Hirokazu Okayama, Curtis C. Harris. Epigenetic regulation of microRNAs in early stage lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3087. doi:10.1158/1538-7445.AM2013-3087