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Purpose: Cancer cell phenotypes are partially determined by epigenetic specifications, such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations.Experimental Design: An in vivo selection approach was used to generate highly aggressive non–small cell lung cancer (NSCLC) cell lines (A549 and HTB56) followed by genome-wide DNA methylation analysis. Furthermore, the therapeutic effects of the epigenetic agent azacytidine on DNA methylation patterns and the in vivo phenotypes were explored.Results: Widespread changes of DNA methylation were observed during development of highly aggressive cell lines. Up to 2.5% of the CpG-rich region was differentially methylated as identified by reduced representation bisulfite sequencing compared with the less aggressive parental cell lines. DNA methyltransferase inhibition by azacytidine reversed the prometastatic phenotype; this was highly associated with the preferential loss of DNA methylation at sites that were hypermethylated during the in vivo selection. Of note, polycomb (PRC2) binding sites were particularly affected by DNA methylation changes after azacytidine exposure that persisted over time.Conclusions: We could show that metastatic capability of NSCLC is closely associated with DNA methylome alterations. Because inhibition of DNA methyltransferase reversed metastasis-prone phenotype, epigenetic modulation seems to be a potential therapeutic approach to prevent metastasis formation. Clin Cancer Res; 20(4); 814–26. ©2013 AACR.

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XLSX file - 655K, Supplementary Table S4 - annotated DNA methylation in confirmed promoter DMRs (RRBS) in HTB56.

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PDF file - 1572K, Figure S4 4A+B): Column charts depict hypo- and hypermethylated regions detected by RRBS in high metastatic A549 (A) and HTB56 (B) cells after 6 days of 5-Azacytidine-treatment and after 7 days of 5-Azacytidine-release. 4C+D) Smoothed scatter plot of 5-Azacytidine-treated (250nM) versus untreated high metastatic HTB56 cells. Shown are methylation levels for CpG-sites analyzed by RRBS. C: X-axis: Methylation levels in HTB56 high metastatic cells (d0=untreated) Y-axis: Methylation levels in HTB56 high metastatic cells (d6_250= after six days of 5-Azacytidinetreatment, 5-Azacytidine-concentration of 250 nM) D: X-axis: Methylation levels in HTB56 high metastatic cells (d0=untreated) Y-axis: Methylation levels in HTB56 high metastatic cells (d13_250= after thirteen days of 5- Azacytidine-treatment, 5-Azacytidine-concentration of 250 nM) Figure S5 Unsupervised hierarchical clustering shows that 5-Azacytidine exposed cells cluster more closely together than individual cell lines. Figure S6 Chromosomal distribution of clusters and DMRs. The curves show the density distribution of hypomethylated DMRs and CpG clusters in relation to their chromosomal position: 0 indicates chromosomal end and 1 the centromeric region. Hypomethylated DMRs (green), hypermethylated DMRs (red) and CpG clusters (black) are shown. DNA demethylation upon 5-Azacytidine occurs primarily on chromosome ends.

PDF file - 247K, Supplementary Figure 3 Unsupervised hierarchical clustering shows that independent samples at each step of selection cluster more closely together than individual cell lines A549 (A) and HTB56 (B). A high correlation between biological replicates from normal and highly aggressive cell lines from both cell types were observed. The clustering is based on a distance matrix, which has been derived from pearson product-moment correlation coefficients (r) by calculating 1-r.

PDF file - 219K, Supplementary Figure 2 Chart depicts viability of high and low metastatic cells before 5-Azacytidine-treatment and after 7 days of 5-Azacytidine-release.

PDF file - 233K, Supplementary Figure 1 Genomic changes in low and high metastatic A549 cells were analyzed by 500K SNP arrays.

PDF file - 96K, Supplementary Table 1: Mutations detected in parental and highly metastatic NSCLC cell lines A549 (A) and HTB56 (B) by SNP analysis Supplementary Table 2: Mutations detected in parental and highly metastatic NSCLC cell lines A549 and HTB56 by Exome sequencing Supplementary Table 3: Conversion rate uniquely mapped reads

Epigenomic changes are an important feature of malignant tumors. How tumor aggressiveness is affected by DNA methylation of specific loci is largely unexplored. In genome-wide DNA methylation analyses, we identified the KCa 3.1 channel gene (KCNN4) promoter to be hypomethylated in an aggressive non-small-cell lung carcinoma (NSCLC) cell line and in patient samples. Accordingly, KCa 3.1 expression was increased in more aggressive NSCLC cells. Both findings were strong predictors for poor prognosis in lung adenocarcinoma. Increased KCa 3.1 expression was associated with aggressive features of NSCLC cells. Proliferation and migration of pro-metastatic NSCLC cells depended on KCa 3.1 activity. Mechanistically, elevated KCa 3.1 expression hyperpolarized the membrane potential, thereby augmenting the driving force for Ca(2+) influx. KCa 3.1 blockade strongly reduced the growth of xenografted NSCLC cells in mice as measured by positron emission tomography-computed tomography. Thus, loss of DNA methylation of the KCNN4 promoter and increased KCa 3.1 channel expression and function are mechanistically linked to poor survival of NSCLC patients.