Inactivation of 14‐3‐3 σ by promoter methylation correlates with metastasis in nasopharyngeal carcinoma

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Abstract
14‐3‐3 σ, the downstream target of p53, is a negative regulator of cell cycle G2‐M phase checkpoint in response to DNA damage. Our previous comparative proteomics study showed that 14‐3‐3 σ was downregulated or lost in nasopharyngeal carcinoma (NPC) tissue compared with non‐cancerous nasopharyngeal epithelial tissue (NNET). In this study, we further investigated for the epigenetic mechanism of 14‐3‐3 σ inactivation. Methylation‐specific PCR showed 14‐3‐3 σ promoter methylation in 100% of analyzed NPC cell lines (4/4) but not in immortalized human nasopharyngeal epithelial cell line NP69. Treatment of the four NPC cell lines with the methyltransferase inhibitor 5‐aza‐2′‐dC resulted in the demethylation and upregulation of 14‐3‐3 σ. In tissues, 14‐3‐3 σ promoter methylation occurred at a higher frequency in NPC, 63/75 (84%), compared to adjacent NNET, 7/25 (28%), and fully methylated 14‐3‐3 σ promoter was detected in NPC but not in any of adjacent NNET. RT‐PCR, Western blotting, and immunohistochemistry showed that 14‐3‐3 σ expression was downregulated or lost in NPC with methylation, and there was a negative correlation between the expression levels and methylation statuses of 14‐3‐3 σ gene. In addition, the patients with methylated 14‐3‐3 σ presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14‐3‐3 σ in NPC cell line 5‐8F with high metastatic potential was able to inhibit its in vitro invasive ability. Our data are the first to show that 14‐3‐3 σ is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis. J. Cell. Biochem. 106: 858–866, 2009.

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