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 3

NNK enhances DNMT1 protein stability through the AKT signaling pathway, which is associated with the ubiquitin-proteosome system.

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NNK enhances DNMT1 protein stability through the AKT signaling pathway, ...
(A) NNK prolonged DNMT1 protein half-life. DNMT1 protein levels were determined by Western blotting and densitometry. β-Actin was used as a loading control, and p53 served as a positive control for CHX treatment because its half-life is known to be short. White bars, cells treated with DMSO; dark gray bars, cells treated with CHX for 6 hours; light gray bars, cells treated with CHX and NNK for the indicated times. (B) Ubiquitination of DNMT1 decreased upon NNK treatment. A549 cells were treated with NNK or DMSO control for 2 hours, after which cell lysates were immunoprecipitated with anti-DNMT1 or anti-ubiquitin antibody and then Western blotted. Normal IgG was used as a negative control. (C and D) A549 cells were pretreated with or without the PI3K/AKT pathway inhibitor LY294002 (C) or AKT siRNA (D), then treated with NNK. Western blotting and densitometry showed that each reduced levels of NNK-induced DNMT1. (E) LY294002 treatment increased ubiquitination of DNMT1, leading to low levels of DNMT1 protein in A549 cells. (F) Western blotting and densitometry of A549 cells treated with NNK with or without pretreatment with LY294002 and the proteasome inhibitor MG132 showed altered DNMT1 protein levels. Data are mean ± SEM (n = 3).