Abstract A47: Epigenetic regulation of microRNAs in early stage lung adenocarcinoma
Authors: Robles Ana I. , Okayama Hirokazu , Kumar Saikartik A. , Franklin Zaneta , Bowman Elise D. , Edelman Daniel , Stevenson Holly , Petersen David , Mathé Ewy , Kanai Yae , Kohno Takashi , Tsuchiya Naoto , Meltzer Paul , Yokota Jun , Harris Curtis C. 
Published: August 2014

Abstract
Lung cancer is the most commonly diagnosed cancer and a leading cause of cancer-associated deaths worldwide. It is imperative to understand the molecular mechanisms underlying early lung cancer development so that better diagnostic and treatment options can be identified. We conducted a multi-platform genomic screen of stage I/II lung adenocarcinoma and non-tumor adjacent tissue excised from patients recruited into our ongoing NCI-MD Case-Control Study. MicroRNAs (miRNAs) are 20-22 nucleotide 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. Although it is unclear whether they possess independent promoter regions, several miRNAs are adjacent to CpG islands. CpG island methylation is a well-described mechanism by which genes and miRNAs with tumor-suppressive function are silenced in cancer. Such regulation could have important implications for progression of early stage lung cancer. Methods: Forty paired tumor and adjacent non-tumor tissues were characterized through methylation profiling using Illumina Infinium HumanMethylation27 BeadChip assays, miRNA expression profiling using Nanostring nCounter miRNA Expression Assay and mRNA expression profiling using Illumina HumanRef-8 v3 Expression Beadchip arrays. Consistency of methylation profiling was compared across three other cohorts of lung adenocarcinoma: TCGA (The Cancer Genome Atlas) lung adenocarcinoma (LUAD) consisting of 23 matched-tissue pairs downloaded from the TCGA data portal; GSE32867, comprising 60 matched-tissue pairs published by Selamat et al. (Genome Res. 2012 Jul;22(7)) and downloaded from GEO; and a lung adenocarcinoma cohort from Japan consisting of 76 matched-tissue pairs. Additionally, in order to functionally validate miRNA-associated methylation we exposed A549 lung cancer cell line to a chromatin demethylation protocol of 5-aza/TSA. Results: We found seven miRNA loci hypermethylated in tumors. Four of these are located within homeobox gene clusters, miR-196b (HOXA9), miR-615 (HOXC5), miR-10a (HOXB4), and miR-10b (HOXD4). Two others are located adjacent to genes with significant changes in methylation, miR-638 (DNM2) and miR-639 (GPSN2). Increased methylation of miR-34b/c (BTG4) in lung tumors was consistent with prior studies. A comparative analysis of methylation at these loci showed a high concordance between our study and methylation profiling in three other cohorts of lung adenocarcinoma. Through microRNA expression profiling, we determined that miR-520e is the top downregulated miRNA in tumors. Interestingly, miR-520e belongs to a large microRNA cluster in 19q13.42 (miR-515 family) flanked by a CpG island. Functional validation of putative tumor-suppressor microRNAs is ongoing. We have confirmed that miR-615 and miR-520e are methylated and become demethylated and reactivated by exposure to a combination of 5-aza and TSA in-vitro. Conclusions: CpG sites associated with miRNAs are methylated in early stage lung adenocarcinoma across independent cohorts of lung cancer. Therefore, tumor suppressive miRNAs maybe epigenetically controlled through CpG island methylation in early stage lung adenocarcinoma. Analysis of their association with patient outcome is forthcoming.
Citation Format: Ana I. Robles, Hirokazu Okayama, Saikartik A. Kumar, Zaneta Franklin, Elise D. Bowman, Daniel Edelman, Holly Stevenson, David Petersen, Ewy Mathé, Yae Kanai, Takashi Kohno, Naoto Tsuchiya, Paul Meltzer, Jun Yokota, Curtis C. Harris. Epigenetic regulation of microRNAs in early stage lung adenocarcinoma. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A47.