Supplementary Figures 4 - 6 from DNA Methyltransferase Inhibition Reverses Epigenetically Embedded Phenotypes in Lung Cancer Preferentially Affecting Polycomb Target Genes

Hascher, Antje; Haase, Ann-Kristin; Hebestreit, Katja; Rohde, Christian; Klein, Hans-Ulrich; Rius, Maria; Jungen, Dominik; Witten, Anika; Stoll, Monika; Schulze, Isabell; Ogawa, Seishi; Wiewrodt, Rainer; Tickenbrock, Lara; Berdel, Wolfgang E.; Dugas, Martin; Thoennissen, Nils H.; Müller-Tidow, Carsten

doi:10.1158/1078-0432.22449636.v1

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Supplementary Figures 4 - 6 from DNA Methyltransferase Inhibition Reverses Epigenetically Embedded Phenotypes in Lung Cancer Preferentially Affecting Polycomb Target Genes

journal contribution

posted on 2023-03-31, 17:32 authored by Antje Hascher, Ann-Kristin Haase, Katja Hebestreit, Christian Rohde, Hans-Ulrich Klein, Maria Rius, Dominik Jungen, Anika Witten, Monika Stoll, Isabell Schulze, Seishi Ogawa, Rainer Wiewrodt, Lara Tickenbrock, Wolfgang E. Berdel, Martin Dugas, Nils H. Thoennissen, Carsten Müller-Tidow

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.

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ARTICLE ABSTRACT

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.