ANK1 Methylation regulates expression of MicroRNA-486-5p and discriminates lung tumors by histology and smoking status

Abstract

The intragenic tumor-suppressor microRNA miR-486-5p is often down-regulated in non-small cell lung cancer (NSCLC) but the mechanism is unclear. This study investigated epigenetic co-regulation of miR-486-5p and its host gene ANK1. MiR-486-5p expression in lung tumors and cell lines was significantly reduced compared to normal lung (p < 0.001) and is strongly correlated with ANK1 expression. In vitro, siRNA-mediated ANK1 knockdown in NSCLC cells also reduced miR-486-5p while the DNA methylation inhibitor 5-aza-2'-deoxycytidine induced expression of both. ANK1 promoter CpG island was unmethylated in normal lung but methylated in 45% (118/262) lung tumors and 55% (17/31) NSCLC cell lines. After adjustment for tumor histology and smoking, methylation was significantly more prevalent in adenocarcinoma (101/200, 51%) compared to squamous cell carcinoma (17/62, 27%), p < 0.001; HR = 3.513 (CI: 1.818-6.788); and in smokers (73/128, 57%) than never-smokers (28/72, 39%), p = 0.014; HR = 2.086 (CI: 1.157-3.759). These results were independently validated using quantitative methylation data for 809 NSCLC cases from The Cancer Genome Atlas project. Together, our data indicate that aberrant ANK1 methylation is highly prevalent in lung cancer, discriminate tumors by histology and patients' smoking history, and contributes to miR-486-5p repression.

Keywords: ANK1; Epigenetics; Intronic microRNA; NSCLC; miR-486-5p.

Figure 1. Expression of miR-486-5p and its host gene ANK1 in lung cancer

Next generation sequencing reveals miR-486-5p expression is (A) reduced in lung cancer and (B) could be induced by the DNA methylation inhibitor 5-aza-2′-deoxycytidine (DAC). C) Schematic representation depicting genomic structure of ANK1, its various transcript variants, CpG islands, and miR-486 located in the last intron. Diamond headed lines indicate exons targeted by the transcript specific TaqMan assays. Double and single arrow heads indicate the binding sites for the different siRNAs used in the study and the location of miR486, respectively. D) Expression of the three ANK1 transcript variants in normal and malignant lung tissue/cells.

Figure 2. Comparison of miR-486-5p and ANK1 expression in lung cancer

A) Expression patterns of ANK1 and miR-486-5p in normal lung and NSCLC cell lines. B–E) NSCLC cell lines were transiently transfected with control or miR-486-5p mimic (B and C) and control or ANK1-specific siRNA, siANK1#4, (D and E) and the level of miR-486-5p (B and E) and ANK1 (C and D) mRNA (top) and protein (bottom) expression were determined.

Figure 3. Aberrant methylation of ANK1 promoter CpG islands in lung cancer

Methylation of (A) ANK1B and (B) ANK1E promoter CpG islands in normal (top panels) and lung cancer (middle and bottom panels) was evaluated using the semi-quantitative Combined Bisulfite Modification and Restriction Analysis (CoBRA). Partial or complete digestion of PCR products into smaller fragments following the addition (+) of the BstU1 restriction enzyme indicates partial or complete methylation. Partial demethylation of the two promoter CpG islands (bottom panels) following treatment with the methylation inhibitor DAC compared to control shown by the re-appearance of some undigested bands, indicates reversibility of these epigenetic changes.

Figure 4. Quantitative validation of ANK1 methylation in lung cancer

The methylation level (mean ± SD) of (A) ANK1B and (B) ANK1E promoter CpG islands was quantitatively determined using whole genome methylation data from the HumanMethylation450 beadchip (HM450K). The location of each probe with respect to the promoter CpG island region and first exon of the specific transcript variant is depicted below the x-axis labels. C) HM450K data for large lung tumor and normal samples from the publicly available TCGA database validated the tumor-specific (not present in normal lung) methylation of ANK1B promoter in lung cancer and revealed its strong association with lung adenocarcinoma (AC) than squamous cell carcinoma SCC.

Figure 5. ANK1 knockdown in lung cancer impacts cancer related pathways

A) ANK1 expression in two NSCLC cell lines 48 h after transfection with control (siCon) or ANK1-specific (siANK1#4) siRNA. B) The genome-wide impact of these knockdowns was evaluated using whole transcriptome array and the top pathways affected by significantly altered genes were identified using pathway analysis.