Inactivation of 14-3-3 sigma by promoter methylation correlates with metastasis in nasopharyngeal carcinoma.
14-3-3 sigma, 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 sigma 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 sigma inactivation. Methylation-specific PCR showed 14-3-3 sigma 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 sigma. In tissues, 14-3-3 sigma 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 sigma promoter was detected in NPC but not in any of adjacent NNET. RT-PCR, Western blotting, and immunohistochemistry showed that 14-3-3 sigma 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 sigma gene. In addition, the patients with methylated 14-3-3 sigma presented a higher frequency of lymph node and distant metastasis, and an advanced clinical stage, and overexpression of 14-3-3 sigma 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 sigma is frequently inactivated by promoter methylation in NPC and this aberrant methylation correlates with lymph node and distant metastasis.
Yi
Bin
B
Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China.
Tan
Shuang-Xiang
SX
Tang
Can-E
CE
Huang
Wei-Guo
WG
Cheng
Ai-Lan
AL
Li
Cui
C
Zhang
Peng-Fei
PF
Li
Mao-Yu
MY
Li
Jian-Ling
JL
Yi
Hong
H
Peng
Fang
F
Chen
Zhu-Chu
ZC
Xiao
Zhi-Qiang
ZQ
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D019175:DNA Methylation; D004847:Epithelial Cells; D005092:Exonucleases / Q000235:genetics*; D005095:Exoribonucleases; D020868:Gene Silencing; D006801:Humans; D008207:Lymphatic Metastasis; D009303:Nasopharyngeal Neoplasms / Q000235:genetics / Q000473:pathology; D009362:Neoplasm Metastasis / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic
D048948:14-3-3 Proteins; D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human