Epigenetic mechanisms for silencing glutathione S‐transferase m2 expression by hypermethylated specificity protein 1 binding in lung cancer

BACKGROUND: Glutathione S‐transferases M2 (GST‐M2) is a detoxifying enzyme. Low expression levels of GST‐M2 have been detected in lung cancer cells. However, little is known about the regulation of GST‐M2 in lung cancer cells. In this study, the authors investigated the epigenetic regulatory mechanisms of GST‐M2 in lung cancer cells. METHODS: The authors evaluated the promoter methylation of GST‐M2 in lung cancer cells after treatment with the DNA methyltransferase (DNMT) inhibitor 5′‐aza‐2′‐deoxycytidine (5′‐aza‐dC). Reporter activity assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility‐shift assays, and small interfering RNA (siRNA) assays were used to determine whether the methylation of specificity protein 1 (Sp1) affected binding to the GST‐M2 promoter or regulated GST‐M2 transcription. Real‐time polymerase chain reaction was used to determine GST‐M2 and DNMT‐3b messenger RNA levels in 73 nonsmall cell lung cancer (NSCLC) tissues. RESULTS: GST‐M2 expression was restored after treatment with 5′‐aza‐dC in lung cancer cells. GST‐M2 exhibited high frequency of promoter hypermethylation in lung cancer cells and NSCLC tumor tissues. CpG hypermethylation abated Sp1 binding to the GST‐M2 promoter in lung cancer. Knockdown of Sp1 in normal lung cells reduced GST‐M2 expression, and silencing of DNMT‐3b increased GST‐M2 expression in lung cancer cells. In addition, DNMT‐3b expression was significantly higher in lung tumors with low levels of GST‐M2 expression than in lung tumors with high levels of GST‐M2 expression, especially among women and among patients who had stage I disease. CONCLUSIONS: Epigenetic silencing of GST‐M2 was distinguished from Sp1‐mediated GST‐M2 transcriptional expression. The authors concluded that this represents a mechanism that leads to decreased expression of GST‐M2 in lung cancer cells. Cancer 2011. © 2011 American Cancer Society. The current findings provided evidence indicating that hypermethylation of CpG islands within the glutathione S‐transferase M2 (GST‐M2) proximal promoter is a major mechanism that mediates the silence of GST‐M2 gene expression in lung cancer cell lines.