The Epstein-Barr virus oncogene product, latent membrane protein 1, induces the downregulation of E-cadherin gene expression via activation of DNA methyltransferases

Abstract

The latent membrane protein (LMP1) of Epstein-Barr virus (EBV) is expressed in EBV-associated nasopharyngeal carcinoma, which is notoriously metastatic. Although it is established that LMP1 represses E-cadherin expression and enhances the invasive ability of carcinoma cells, the mechanism underlying this repression remains to be elucidated. In this study, we demonstrate that LMP1 induces the expression and activity of the DNA methyltransferases 1, 3a, and 3b, using real-time reverse transcription-PCR and enzyme activity assay. This results in hypermethylation of the E-cadherin promoter and down-regulation of E-cadherin gene expression, as revealed by methylation-specific PCR, real-time reverse transcription-PCR and Western blotting data. The DNA methyltransferase inhibitor, 5'-Aza-2'dC, restores E-cadherin promoter activity and protein expression in LMP1-expressing cells, which in turn blocks cell migration ability, as demonstrated by the Transwell cell migration assay. Our findings suggest that LMP1 down-regulates E-cadherin gene expression and induces cell migration activity by using cellular DNA methylation machinery.

Figure 1

Repression of E-cadherin gene expression by LMP1. (A) Western blotting of E-cadherin and its associated complex in cells stably expressing LMP1. Cell extracts (40 μg) of LMP1-expressing cells (MDCKLMP, NPC076LMP), neo-control cell clones (MDCK-neo, NPC076-neo) and parental NPC076 were separated on a SDS/8% PAGE gel, followed by electroblotting onto nitrocellulose membrane. The membrane was incubated with individual antibodies for LMP1, E-cadherin, and γ-catenin, as described in Materials and Methods. Tubulin detected by α-tubulin antibody was used to normalize the amount of total protein in each preparation. (B) Immunostaining studies. Expression of E-cadherin and γ-catenin of the E-cadherin-associated complex in LMP1-expressing MDCK cells was examined by immunfluorescence staining with antibodies specific for these proteins, as described in A. Anti-mouse-FITC antibody was used as the secondary antibody. DAPI staining of MDCK-neo cells revealed the nuclei of the cells. (C) Quantitative analysis of E-cadherin transcripts. Poly(A)⁺ RNA purified from parental NPC076, NPC076-neo, and NPC076-LMP1 cells was subjected to real time RT-PCR for measuring E-cadherin transcripts, as described in Materials and Methods. Western blotting data on LMP1 and tubulin for individual cell clones are additionally shown. The data represent an average of three independent experiments. The vertical bar indicates SD.

Figure 2

Identification of the LMP1-responsive region of the E-cadherin promoter. (A) Schematic representation of human E-cadherin promoter-containing reporter construct and its deletion mutants. The +1 position represents the transcriptional initiation site and restriction enzyme sites used to generate deletion mutants are indicated. (B and C) LMP1 down-regulation of E-cadherin promoter activity in MDCK and MCF-7 cells. The pE-cad constructs were individually cotransfected with pCMV-LMP1 or pCMV2-FLAG. Luciferase activity was normalized by the β-galactosidase activity of pSV2-gal per transfection. Western blotting analysis of the LMP1 protein is shown at the bottom of the figure. The data are an average of at least six independent experiments, and SD is indicated by the vertical bar.

Figure 3

Identification of the LMP1 domain critical for E-cadherin promoter regulation. (A) Schematic representation of LMP1 and its mutant constructs. The Src homology 3 (SH3)-like domain, CTAR1 and CTAR2 are indicated. (B) Regulation of pEcad−164/+49 by LMP1 and its mutants. Individual LMP1-expressing constructs were cotransfected with the pEcad−164/+49, and relative luciferase activity was determined and normalized as described in Materials and Methods. The percentage of promoter activity in each transfection was determined with respect to the vector control value, taken as 100%. The LMP1 protein in each transfection was determined by Western blotting analysis using α-flag antibody, M5 (Sigma).

Figure 4

LMP1-mediated repression of E-cadherin and cell migration ability are restored by 5′-Aza-2′dC. (A) Restoration of E-cadherin promoter activity by 5′-Aza-2′dC. NPC cells were cotransfected individually with pEcad−164/+49 with pCMV-LMP1 or pCMV2-FLAG in the presence or absence of 5′-Aza-2′dC. Luciferase activity was normalized by the β-gal activity of pSV2-gal per transfection. The data are an average of five independent experiments, and SD is indicated by the vertical bar. (B) De-repression of E-cadherin by 5′-Aza-2′dC. NPC076-LMP1 cells were treated with 5 μM 5′-Aza-2′dC for 5 days. Cells were harvested, and E-cadherin expression was analyzed by Western blotting using α-E-cadherin antibody. LMP1 expression was detected with an LMP1-specific monoclonal antibody, S12. Tubulin (identified by the α-tubulin antibody) was used as the internal control. (C and D) Inhibition of LMP1-mediated cell migration by 5′-Aza-2′dC. MDCK-LMP1 and MDCK-neo cells were treated with 5 μM 5′-Aza-2′dC for 5 days or left untreated before subjection to the Transwell migration assay. Cells migrating to the other side of the membrane were stained with Giemsa and counted under the microscope. Data shown in D represent an average of 10 wells. The experiment was repeated three times, with reproducible results. The vertical bar in the figure indicates SD.

Figure 5

LMP1 activates DNMT. (A) Western blot analysis of DNA methyltransferase 1 (DNMT1) in NPC076-LMP1 and NPC076-neo cells. Western blot analysis was performed as described in Fig. 1A. The protein level of DNMT1, PCNA, and LMP1 were detected by α-DNMT1 antibody 60B1220, α-PCNA antibody PC10, and α-LMP1 antibody S12, individually. Tubulin detected by α-tubulin antibody was used to normalize the amount of total protein in each preparation. (B) DNA methyltransferase activity assay in LMP1-expressing stable cell clones. DNA methyltransferase activity was measured in NPC076-LMP1 and MDCK-LMP1 or neo-control cells. (C) Kinetics of DNA methyltransferase activity in rAdLMP1-infected MCF-7 cells. Cells were infected with either rAdLMP1 or rAdLacZ at a multiplicity of infection of 100. Methyltransferase activity was determined as described in Materials and Methods. The data are an average of at least three independent experiments. The vertical bar represents standard deviation. Increase in C was determined by dividing the activity of cells infected with rAdLMP1 with that of cells infected with rAdLacZ. LMP1 and tubulin expression, shown at the bottom, were analyzed by Western blotting. (D) Methylation-specific PCR of E-cadherin promoter. MDA-MB-468 and MCF-7 cells were infected with rAdLMP1 or control virus, rAdLacZ, at multiplicity of infection of 100. Methylation-PCR analysis was carried out as described in Materials and Methods. U, unmethylated; M, methylated. The 116-bp PCR product represents the methylated state of island 1 within the E-cadherin promoter sequence, whereas the 97-bp product represents the unmethylated state. Uninfected cells were used as the unmethylated controls.