Abstract
Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC through alteration of the methylome of oral cells. Here we report that cigarette smoke condensate (CSC) significantly changes the genomic 5-methyldeoxycytidine content and nuclear accumulation of DNA methyltransferase 1 (DNMT1) and DNMT3A in human untransformed oral cells. By using integrated analysis of cDNA and methylation arrays of the smoking-associated dysplastic oral cell line and OSCC tumors, respectively, we identified four epigenetic targets--UCHL1, GPX3, LXN, and LDOC1--which may be silenced by cigarette. Results of quantitative methylation-specific PCR showed that among these four genes, LDOC1 promoter was the most sensitive to CSC. LDOC1 promoter hypermethylation and gene silencing followed 3 weeks of CSC treatment. LDOC1 knockdown led to a proliferative response and acquired clonogenicity of untransformed oral cells. Immunohistochemistry showed that LDOC1 was downregulated in 53.3% (8/15) and 57.1% (20/35) of premalignant oral tissues and early stage OSCCs, respectively, whereas 76.5% (13/17) of normal oral tissues showed high LDOC1 expression. Furthermore, the microarray data showed that LDOC1 expression had decreased in the lung tissues of current smokers compared with that in those of never smokers and had significantly decreased in the lung tumors of smokers compared with that in normal lung tissues. Our data suggest that CSC-induced promoter methylation may contribute to LDOC1 downregulation, thereby conferring oncogenic features to oral cells. These findings also imply a tumor suppressor role of LDOC1 in smoking-related malignancies such as OSCC and lung cancer.
Highlights
Oral cancer, of which the vast majority of cases are oral squamous cell carcinoma (OSCC), is the major subtype of head and neck cancers
We investigated whether cigarette exposure induces profound epigenetic changes in oral cells, causing the silencing of tumor suppressor genes through promoter DNA methylation, which is involved in the development of oral cancer
To determine the effects of smoking on the global DNA methylation content of oral cells, we measured genomic 5-methyl-2-deoxycytidine (5mC) in CGHNK6 [11] and DOK [12] cells after cigarette smoke condensate (CSC) exposure by using an enzyme-linked immunoassay (EIA)-based method
Summary
Of which the vast majority of cases are oral squamous cell carcinoma (OSCC), is the major subtype of head and neck cancers. Using the mortality data from countries and regions such as Japan, Europe, Australasia, and North America, previous epidemiologic studies have indicated that the major etiological factor responsible for death from oral cancers is smoking followed by heavy alcohol consumption [5, 6]. Overall, these studies provide convincing evidence of an association between cigarette smoking and OSCC. We investigated whether cigarette exposure induces profound epigenetic changes in oral cells, causing the silencing of tumor suppressor genes through promoter DNA methylation, which is involved in the development of oral cancer
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