抑癌基因 FOXO3a 協同組蛋白去乙醯酶進而透過轉錄層次調控 DNA 甲基化轉移酶3B

Abstract

Background: DNA methyltransferase 3B (DNMT3B) contributes to de novo methylation and its overexpression plays a promoting role in tumorigenesis. However, the mechanisms of DNMT3B overexpression remain unclear. Recent report showed that the expression of FOXO3a, a member of forkhead box class O (FOXO) transcription factors, negatively correlates with expression of DNA methyltransferases during the growth of oocyte. In addition, loss of FOXO3a has been shown to involve in carcinogen-induced lung adenocarcinoma. Purpose: This study aims to investigate whether FOXO3a transcriptionally represses DNMT3B expression in lung cancer cells. The inverse correlation between FOXO3a and DNMT3B expressions is examined in lung cancer patients and animal models. Results: According to the luciferase reporter assay, the repressive regulation of FOXO3a was preferentially restricted to the proximal region of DNMT3B promoter, which contains the putative binding site of FOXO3a (+166~+173; FOXO3a-E). In addition, FOXO3a and histone deacetylase HDAC3 bound corporately to the FOXO3a-E site of DNMT3B promoter by chromatin-immunoprecipitation PCR assay. However, when abundant FOXO3a was accumulated in nuclei by doxorubicin treatment, it further bound at the distal putative binding site, FOXO3a-P (-249~-242). Knockdown of FOXO3a resulted in an open chromatin structure evident by a decrease of HDAC3 binding, gain of H3K9K14 acetylation, and loss of H3K27 tri-methylation. In contrast, over-expressed FOXO3a and combined treatment with doxorubicin to induce the FOXO3a nuclear accumulation showed a repressed chromatin structure. Interaction between FOXO3a and HDAC3 proteins was confirmed by immunoprecipitation-Western analysis. Note that FOXO3a is a substrate of MDM2 E3 ligase. Co-treatment with doxorubicin and MDM2 inhibitor, nutlin-3, further enforced abundant nuclear accumulation of FOXO3a to decrease the mRNA level of DNMT3B. In addition, the combined treatment forced the FOXO3a expression and decreased the DNMT3B expression, resulting in additional inhibition of tumor growth and methylation status on tumor suppressor genes in xenograft specimens. Moreover, the inverse correlation between FOXO3a and DNMT3B (P=0.026) and between FOXO3a and MDM2 (P=0.032) and the positive correlation between MDM2 and DNMT3B (P=0.004) were confirmed in tumors from 74 lung cancer patients by immunohistochemistry. Conclusion: Our studies provide first evidence that FOXO3a transcriptionally represses DNMT3B expression by coordinating binding with HDAC3 to DNMT3B promoter in cell model. This study also shows additional inhibition of antitumor activities for co-treatment of nutlin-3 and doxorubicin in xenograft and provides a new approach for lung cancer therapy. In addition, the clinical data elucidate the inverse correlation between FOXO3a and DNMT3B as well FOXO3a and MDM2, and the positively correlation between DNMT3B and MDM2 in lung cancer patients.