Abstract 5384: Aberrant methylation of ANK1, the host gene for miR-486, distinguishes lung tumors by histology and smoking status
Authors: Tessema Mathewos , Yingling Christin M. , Picchi Maria A. , Wu Guodong , Ryba Tyron , Lin Young , Belinsky Steven A. 
Published: July 2017

Abstract
MicroRNAs (miRs) regulate many fundamental biological processes primarily through inhibiting the transcription and/or translation of their target genes. MiR-486 is an intragenic miR located within ANK1, a gene that encodes for the adapter protein ankyrin-1. Although the tumor suppressor role of miR-486-5p and its down-regulation in non-small cell lung cancer (NSCLC) is well established, the mechanism(s) leading to its repression is unclear. In this study, we investigated epigenetic regulation of ANK1 and its impact on miR-486-5p expression in lung cancer. DNA methylation and expression levels of ANK1 and miR-486-5p in lung tumor-normal pairs, NSCLC cell lines, as well as various normal blood and lung epithelial cell controls were evaluated using qualitative and quantitative assays. The genome-wide impact of epigenetic repression of ANK1 was evaluated using siRNA. The results show that miR-486-5p expression in lung tumors and NSCLC cell lines was significantly reduced compared to distant normal lung tissue pairs (p = 0.0006) and strongly correlated with ANK1 expression. In NSCLC cell lines with strongly reduced expression of miR-486-5p and ANK1, treatment with the DNA methylation inhibitor 5-aza-2-deoxycytidine significantly increased the expression of both the miR and its host gene. Human ANK1 is a large (~230kb) gene with three promoter regions regulating tissue specific expression of three (ANK1B, ANK1E, and ANK1A) transcript variants. ANK1B (the longest of the three variants) is the primary transcript expressed in lung tissue, while ANK1E and ANK1A are weakly and rarely expressed in lung epithelial cells. ANK1B promoter contains a large CpG island that is methylated in 45% (118/262) of lung tumors but not in normal lung or blood samples. The prevalence for ANK1B methylation was significantly higher (p &lt; 0.001) in lung adenocarcinoma (101/200, 51%) compared to lung squamous cell carcinomas (17/62, 27%) after adjustment for smoking. Comparison of methylation by patients’ smoking history also revealed that ANK1B methylation in lung adenocarcinoma was significantly more prevalent in smokers (57%, 73/128) than in never smokers (39%, 28/72), p = 0.014; HR = 2.086 (CI: 1.157 - 3.759). These findings were confirmed by quantitative methylation assays and independently validated using publicly available methylation data for large (n = 809) NSCLC cases from the Cancer Genome Atlas database. Finally, siRNA mediated knockdown of ANK1 in lung cancer cell lines followed by transcriptome-wide expression and pathway analysis revealed that ANK1 repression primarily alters cancer development and progression pathways. Taken together, our data indicates that aberrant methylation of ANK1B promoter is highly prevalent in lung cancer, discriminates lung tumors by histology and smoking history, and represses the expression of ANK1 along with the tumor-suppressor miR-486-5p it hosts within its last intron.
Citation Format: Mathewos Tessema, Christin M. Yingling, Maria A. Picchi, Guodong Wu, Tyron Ryba, Young Lin, Steven A. Belinsky. Aberrant methylation of ANK1, the host gene for miR-486, distinguishes lung tumors by histology and smoking status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5384. doi:10.1158/1538-7445.AM2017-5384