Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial-mesenchymal transition in metastatic nasopharyngeal carcinoma

Abstract

Background: Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial-mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT.

Methods: We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells.

Results: In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells.

Conclusions: This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC.

Figure 1

Expression of Snail and LMP1 are associated, and Snail correlates directly with metastasis and inversely with expression of E-cadherin in NPC. (A) LMP1 and Snail are overexpressed in NPC tumour cells, but not detected in adjacent normal nasopharyngeal epithelium by immunohistochemistry. Representative results in NPC and in normal nasopharyngeal epithelium are shown. Bars, 50 μm. (B) Twist, SIP1 and Slug are expressed in nuclei of tumour cells in NPC; Twist is highly expressed in tumour-cell nests. (C) Expression scores of Snail and LMP1 correlated significantly in NPC: Pearson's correlation coefficient, r=0.415, P=0.0065. Expression levels of Snail in relation to incidence of cervical lymph-node metastasis. ^*Significance according to Mann–Whitney U test, P<0.0001. (D) Expression of Snail correlates inversely with expression of E-cadherin in NPC (r=−0.68, P<0.0001).

Figure 2

Expression of Snail protein and mRNA is induced by LMP1 in Ad-AH human nasopharyngeal epithelial cells. (A) Different amounts of LMP1 expression plasmid were transiently transfected and Snail protein levels were determined by western blotting. (B) Expression of Snail is increased in Ad-AH cells stably expressing LMP1. (C) Snail mRNA level is upregulated by LMP1. Extracts from the same cells in Figure 2B were analyzed by RT–PCR.

Figure 3

LMP1 induces EMT phenotype through Snail in a human nasopharyngeal cell line. (A) Morphological changes in NP69SV40T nasopharyngeal epithelial cells induced by transfection with LMP1 could be reversed by Snail shRNA. Snail shRNA and control shRNA were transduced by the retroviral system to generate stable clones. NP69SV40T cell clones, 2 × 10⁵ cells per dish, were plated onto 35-mm plastic dishes and observed after 24 h. Representative clones are shown. Bars, 50 μm. (B) Transduction of Twist siRNA in LMP1-transfected NP69SV40T cells did not change the cellular phenotype. (C) Scrape-wound migration assay shows that enhanced motility in LMP1-transfected cells is downregulated by silencing Snail through Snail shRNA. Confluent monolayers of NP69SV40T nasopharyngeal epithelial cell clones were scraped with a plastic pipette tip, and migration of cells was observed. Typical wounds at 0 and 10 h are shown. Bars, 200 μm. (D) Enhanced invasiveness of LMP1-transformed cells is downregulated by Snail shRNA. After 72 h in the Matrigel invasion assays, each NP69SV40T cell clone adherent on the lower surface of the filter was fixed and stained. Representative photographs are shown. Bar, 50 μm. Invasion indices were calculated from the counts of cells invading through Matrigel-coated membrane. Significance was tested by paired t-test. ^*P=0.0007 as compared with control cells (NP69SV40T+vector+control shRNA cells); ^**P=0.0041 as compared with NP69SV40T+LMP1+control shRNA cells.

Figure 4

Molecular markers confirm that LMP1 induces EMT through Snail in NP69SV40T nasopharyngeal cells. (A) Transfection of LMP1 does not change levels of Twist in NP69SV40T cells. (B) Expression of representative epithelial markers, E-cadherin and α-catenin, and mesenchymal markers, vimentin and N-cadherin, together with LMP1 and Snail in NP69SV40T cell clones is shown by western blotting. (C) Immunofluorescence staining for a representative epithelial marker, E-cadherin, and mesenchymal marker, N-cadherin, in NP69SV40T cell clones together with staining for Snail are shown. Bars, 20 μm.