Phosphatase and tensin homolog (PTEN) is a major tumor suppressor and usually silenced via the deletion, insertion and mutation. We previously discovered its inactivation via aberrant CpG island methylation. Here, we provide further evidence that EBV latent membrane protein 1(LMP1) can induce a higher intensity of DNA methylation at PTEN CpG islands, inactivating PTEN at the cellular and molecular level. Initially, increased methylation intensity of PTEN CpG islands was observed in EBV-infected nasopharyngeal carcinoma (NPC) cells, accompanied by decreased PTEN expression. In NPC tissue samples showing the methylation at PTEN promoter, LMP1 was highly expressed in higher methylation intensity group relative to lower intensity group, and DNA methyltransferase 3b (DNMT3b) expression was positively correlated with LMP1 expression. Moreover, transfection of LMP1 gene into EBV-negative NPC cells demonstrated that LMP1 up-regulated DNMT3b expression, leading to a higher intensity of PTEN CpG island methylation. Mechanistically, computational prediction and luciferase reporter assay identified a functional NF-κB binding site on DNMT3b promoter and the mutated NF-κB binding site abolished LMP1-mediated DNMT3b activation. Chromatin immunoprecipitation displayed that NF-κB p65 subunit constitutively bound to DNMT3b promoter, supporting the activation of DNMT3b by EBV LMP1 via NF-κB signaling. Furthermore, the expression level of DNMT3b was observed to be increased in the nuclei of LMP1-expressing NPC cells, and a NF-κB inhibitor, PDTC, counteracted LMP1-mediated DNMT3b overexpression. Thus, this study first reports that LMP1-mediated NF-κB can up-regulate DNMT3b transcription, thereby leading to relatively higher methylation intensity at PTEN CpG islands, and ultimately silencing major tumor suppressor PTEN.
The inactivation of phosphatase and tensin homolog (PTEN) due to its CpG island hypermethylation has been observed in some types of tumors except nasopharyngeal carcinoma (NPC). In the present study, we focused on the aberrant methylation of PTEN CpG islands in NPC. The mRNA expression of PTEN was detected by quantitative PCR in 45 NPC and 22 non-tumor nasopharyngeal epithelial (NP) tissues. The methylation status of PTEN was examined by methylation-specific polymerase chain reaction and sequencing. The mRNA expression of PTEN in three NPC cell lines treated with 5-aza-2'-deoxycytidine (5-aza-dC) was also examined. PTEN was downregulated in both NPC tissues and NPC cell lines and a relatively higher methylation level of PTEN was found in NPC specimens (82.2%) relative to NP tissues (5.3%). The PTEN mRNA expression was restored in NPC cell lines by treatment with 5-aza-dC. These results first reveal an epigenetic alteration, aberrant methylation of PTEN, in NPC, which is probably an early event and may be regarded as a novel candidate biomarker for early stage of NPC detection and prevention.
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