The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients
Ruo-Kai Lin, … , Chung-Fan Lee, Yi-Ching Wang
Ruo-Kai Lin, … , Chung-Fan Lee, Yi-Ching Wang
Published January 19, 2010
Citation Information: J Clin Invest. 2010;120(2):521-532. https://doi.org/10.1172/JCI40706.
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Research Article Oncology

The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients

Abstract

DNA methyltransferase 1 (DNMT1) catalyzes DNA methylation and is overexpressed in many human diseases, including cancer. The tobacco-specific carcinogen NNK also induces DNA methylation. However, the role of DNMT1-mediated methylation in tobacco carcinogenesis remains unclear. Here we used human and mouse lung cancer samples and cell lines to determine a mechanism whereby NNK induced DNMT1 expression and activity. We determined that in a human lung cell line, glycogen synthase kinase 3β (GSK3β) phosphorylated DNMT1 to recruit β-transducin repeat–containing protein (βTrCP), resulting in DNMT1 degradation, and that NNK activated AKT, inhibiting GSK3β function and thereby attenuating DNMT1 degradation. NNK also induced βTrCP translocation to the cytoplasm via the heterogeneous nuclear ribonucleoprotein U (hnRNP-U) shuttling protein, resulting in DNMT1 nuclear accumulation and hypermethylation of the promoters of tumor suppressor genes. Fluorescence immunohistochemistry (IHC) of lung adenomas from NNK-treated mice and tumors from lung cancer patients that were smokers were characterized by disruption of the DNMT1/βTrCP interaction and DNMT1 nuclear accumulation. Importantly, DNMT1 overexpression in lung cancer patients who smoked continuously correlated with poor prognosis. We believe that the NNK-induced DNMT1 accumulation and subsequent hypermethylation of the promoter of tumor suppressor genes may lead to tumorigenesis and poor prognosis and provide an important link between tobacco smoking and lung cancer. Furthermore, this mechanism may also be involved in other smoking-related human diseases.

Authors

Ruo-Kai Lin, Yi-Shuan Hsieh, Pinpin Lin, Han-Shui Hsu, Chih-Yi Chen, Yen-An Tang, Chung-Fan Lee, Yi-Ching Wang

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Figure 4

GSK3β and βTrCP interact with DNMT1 protein and enhance DNMT1 degradation.

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GSK3β and βTrCP interact with DNMT1 protein and enhance DNMT1 degradatio...
(A) A549 cells were transfected with pCMV-SPORT6-GSK3β, then treated with or without NNK. The GSK3β pathway promoted DNMT1 protein degradation, which was attenuated by NNK treatment. Data are mean ± SEM (n = 3). (B) Cell lysates were immunoprecipitated with anti-DNMT1 or anti–phospho-Ser antibody and then Western blotted. DNMT1 protein formed complexes with GSK3β and βTrCP proteins. Increase DNMT1 phosphorylation was mediated by exogenous GSK3β, which increased the interaction between DNMT1 and βTrCP. Normal IgG was used as a negative control. (C) βTrCP increased the ubiquitination level of DNMT1. A549 cells were transfected with pCMV-SPORT6-βTrCP for 24 hours, after which cell lysates were immunoprecipitated with anti-DNMT1 and then western blotted. (D) Site-directed mutagenesis of both Ser410 (S410A) and Ser414 (S414A) on DNMT1 protein decreased the phosphorylation level of DNMT1 protein by GSK3β and disrupted the interaction between βTrCP and DNMT1. Cells were transfected with WT or mutant His-tag DNMT1 expression vector and exogenous GSK3β or vector control. Cell lysates were immunoprecipitated with anti–His-tag antibody and then Western blotted.