The Synergistic Effect of Chemical Carcinogens Enhances Epstein-Barr Virus Reactivation and Tumor Progression of Nasopharyngeal Carcinoma Cells

PLoS One. 2012;7(9):e44810

Chih-Yeu Fang, Sheng-Yen Huang, Chung-Chun Wu, Whey-Yu Hsu, Sheng-Ping Chou, Ching-Hwa Tsai, Yao Chang, Kenzo Takada, Jen-Yang Chen

National Institute of Cancer Research, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan


Seroepidemiological studies imply a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). N-nitroso compounds, phorbols, and butyrates are chemicals found in food and herb samples collected from NPC high-risk areas. These chemicals have been reported to be risk factors contributing to the development of NPC, however, the underlying mechanism is not fully understood. We have demonstrated previously that low dose N-methyl-N’-nitro-N-nitrosoguanidine (MNNG, 0.1 ug/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (SB) in enhancing EBV reactivation and genome instability in NPC cells harboring EBV. Considering that residents in NPC high-risk areas may contact regularly with these chemical carcinogens, it is vital to elucidate the relation between chemicals and EBV and their contributions to the carcinogenesis of NPC. In this study, we constructed a cell culture model to show that genome instability, alterations of cancer hallmark gene expression, and tumorigenicity were increased after recurrent EBV reactivation in NPC cells following combined treatment of TPA/SB and MNNG. NPC cells latently infected with EBV, NA, and the corresponding EBV-negative cell, NPC-TW01, were periodically treated with MNNG, TPA/SB, or TPA/SB combined with MNNG. With chemically-induced recurrent reactivation of EBV, the degree of genome instability was significantly enhanced in NA cells treated with a combination of TPA/SB and MNNG than those treated individually. The Matrigel invasiveness, as well as the tumorigenicity in mouse, was also enhanced in NA cells after recurrent EBV reactivation. Expression profile analysis by microarray indicates that many carcinogenesis-related genes were altered after recurrent EBV reactivation, and several aberrations observed in cell lines correspond to alterations in NPC lesions. These results indicate that cooperation between chemical carcinogens can enhance the reactivation of EBV and, over recurrent reactivations, lead to alteration of cancer hallmark gene expression with resultant enhancement of tumorigenesis in NPC.


Supplementary Information

NPC patients at an early stage of this disease usually achieve a relatively higher cure rate, however, those at a late stage frequently relapse or develop metastasis after remission and this leads to the major cause of mortality. Elevation of antibodies against EBV has been considered to be a marker of EBV reactivation [Clin Microbiol Rev, 1988. 1: p. 300-12]. Antibody titers against EBV have been shown to increase with the stage of NPC [Int J Cancer, 1977. 20: p. 663-72; Jpn J Exp Med, 1973. 43: p. 121-33], decrease after therapy with remission [Int J Cancer, 1977. 20: p. 663-72], and increase again prior to relapse and metastasis [Int J Cancer, 1977. 20: p. 663-72; Int J Cancer, 1988. 42: p. 176-81]. These observations suggest that EBV reactivation may contribute to relapse and metastasis of NPC after remission.

We have demonstrated previously that recurrent reactivation of EBV in NPC cells enhanced genome instability and tumorigenicity and many carcinogenesis related genes are altered in copy numbers, including amplifications and deletions. In this study, through gene expression profiling, we provide further evidence that many cancer hallmark genes are altered in their expression. In addition, these alterations can be validated in patient’s tissue.

Based on the results of our study, we propose a model to elucidate the underlying mechanism of how EBV reactivations may contribute to the relapse and metastasis of NPC after remission (see Figure). Patients with primary NPC usually are treated with radiochemotherapy, sometimes combined with surgery. Although the outcome usually is remission, still there might be residual NPC cells remaining. These residual cells, if continuing stimulate by lytic-inducing chemicals, will accumulate genome instability and result in cancer hallmark gene alterations via recurrent EBV reactivation. The consequence could be tumor relapses with enhanced malignancy or metastasis, eventually leading to an untreatable situation. Based on this hypothesis, we proposed that the genomic alterations resulting from the recurrent EBV reactivation may serve to increase the mutability of the host genome, thereby accelerating the acquisition of alterations of cancer hallmark genes and promoting the carcinogenesis of NPC.

Jen-Yang Chen-1



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