Metformin Reduces Histone H3K4me3 at the Promoter Regions of Positive Cell Cycle Regulatory Genes in Lung Cancer Cells

SIMPLE SUMMARY: To understand the effect of metformin on epigenetic regulation, we analyzed histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin showed little effect on DNA methylation or chromatin accessibility but significantly reduced H3K4me3 levels at the promoters of positive cell cycle regulatory genes. Metformin downregulated H3K4 methyltransferase MLL2 expression and knockdown of MLL2 resulted in suppression of H3K4me3 expression and lung cancer cell proliferation. We further evaluated the clinicopathological significance of MLL2 in tumor and matched normal tissues from 42 non-small cell lung cancer patients. MLL2 overexpression was significantly associated with poor recurrence-free survival in lung adenocarcinoma. Our study facilitates the understanding of the effect of metformin on the regulation of histone H3K4me3 at promoter regions of cell cycle regulatory genes in lung cancer cells, and MLL2 may be a potential therapeutic target for lung cancer therapy. ABSTRACT: This study aimed at understanding the effect of metformin on histone H3 methylation, DNA methylation, and chromatin accessibility in lung cancer cells. Metformin significantly reduced H3K4me3 level at the promoters of positive cell cycle regulatory genes such as CCNB2, CDK1, CDK6, and E2F8. Eighty-eight genes involved in cell cycle showed reduced H3K4me3 levels in response to metformin, and 27% of them showed mRNA downregulation. Metformin suppressed the expression of H3K4 methyltransferases MLL1, MLL2, and WDR82. The siRNA-mediated knockdown of MLL2 significantly downregulated global H3K4me3 level and inhibited lung cancer cell proliferation. MLL2 overexpression was found in 14 (33%) of 42 NSCLC patients, and a Cox proportional hazards analysis showed that recurrence-free survival of lung adenocarcinoma patients with MLL2 overexpression was approximately 1.32 (95% CI = 1.08–4.72; p = 0.02) times poorer than in those without it. Metformin showed little effect on DNA methylation and chromatin accessibility at the promoter regions of cell cycle regulatory genes. The present study suggests that metformin reduces H3K4me3 levels at the promoters of positive cell cycle regulatory genes through MLL2 downregulation in lung cancer cells. Additionally, MLL2 may be a potential therapeutic target for reducing the recurrence of lung adenocarcinoma.