Twist and epithelial-mesenchymal transition are induced by the EBV oncoprotein latent membrane protein 1 and are associated with metastatic nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC), an EBV-associated malignancy, is highly metastatic compared with other head and neck tumors, perhaps because of its viral link. Here, we show that the principal EBV oncoprotein, latent membrane protein 1 (LMP1), induces epithelial-mesenchymal transition (EMT) via Twist, a master transcriptional regulator in embryogenesis and newly implicated in metastasis, which, in turn, are likely to contribute to the highly metastatic character of NPC. LMP1 could induce EMT and its associated cell motility and invasiveness in a cell culture model, whereas expression of Twist small interfering RNA reversed LMP1-induced EMT. In diverse EBV-infected cell lines, expression of Twist correlates with expression of LMP1. Dominant-negative LMP1 could suppress Twist expression in EBV-positive cells, whereas LMP1 could induce Twist in EBV-negative nasopharyngeal cells. LMP1 signals through the nuclear factor-kappaB pathway, and an IkappaB superrepressor inhibited induction of Twist by LMP1. Finally, in human NPC tissues, expression of Twist and LMP1 is directly correlated and expression of Twist is associated with metastasis clinically. These results suggest that induction of Twist by a human viral oncoprotein LMP1 directly contributes to the metastatic nature of NPC.

MUC1 induced by Epstein-Barr virus latent membrane protein 1 causes dissociation of the cell-matrix interaction and cellular invasiveness via STAT signaling.

Disruption of cellular adhesion is an essential pathobiologic step leading to tumor dissemination. Mucin 1 (MUC1) is a mucinous glycoprotein expressed at the surfaces of epithelial cells in many tissues and their carcinomas. MUC1 plays crucial roles in tumor invasion and metastasis, especially in opposing cell adhesion. We have shown that virus infection, specifically by the human tumor virus Epstein-Barr virus (EBV) induces a spectrum of cellular invasiveness and metastasis factors. Here we show that expression of MUC1 is increased in diverse latently EBV-infected cell lines that express latent membrane protein 1 (LMP1), the main viral oncoprotein, and that the level of MUC1 was suppressed by expression of a dominant-negative mutant of LMP1. Expression of LMP1 in EBV-negative nasopharyngeal cell lines induces expression of MUC1 through activation of the MUC1 promoter via binding of STAT1 and STAT3. Finally, LMP1 reduces cell adhesion ability, which is restored by inhibition of MUC1 expression with MUC1 small interfering RNA (siRNA). In addition, LMP1 increases cell invasiveness, which is suppressed by MUC1 siRNA. Thus, LMP1 induces MUC1, a factor important in an early step of detachment and release of tumor cells, which along with induction of other invasiveness and angiogenic factors may combine to act in a complex sequential process that culminates in metastasis of EBV-infected tumor cells.

Epstein-Barr virus latent membrane protein 1 induces the matrix metalloproteinase-1 promoter via an Ets binding site formed by a single nucleotide polymorphism: enhanced susceptibility to nasopharyngeal carcinoma.

The Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) has a significant role in several malignancies, including nasopharyngeal carcinoma (NPC). LMP1 is the principal oncoprotein, and we have shown that it also induces a set of factors that mediates invasion, angiogenesis and metastasis. Matrix metalloproteinase-1 (MMP1) is also involved in several malignancies. A single guanine insertion polymorphism (2G) in the MMP1 promoter creates an Ets binding site that causes high levels of transcription and correlates with risk for some malignancies. Here, we evaluate the impact of this 2G insertion type on NPC. We genotyped 44 Japanese and 39 Taiwanese NPC patients, as well as 58 Japanese and 23 Taiwanese healthy controls. The proportion of 2G homozygotes was higher in the NPC groups than in controls (Japanese: p = 0.02, odds ratio (OR) = 2.49; Taiwanese: p = 0.02, OR = 3.66). An analysis of overall survival rates in the patients with NPC, and the 1G/1G genotype disclosed a favorable prognosis (5-year survival rate = 100%, p = 0.04). Multivariate analysis showed that 1G/1G has independent prognostic significance. We also examined whether LMP1 enhances MMP1 expression in epithelial cells in culture. LMP1-transfected cells with 2G/2G genotype expressed MMP1, which was abolished by activator protein-1 (AP1) dominant-negative (DN) and Ets-DN. LMP1 also induced active MMP3, which can cleave latent MMP1, and AP1-DN and Ets-DN suppressed the MMP3 expression. These results suggest that LMP1-induced MMP1 and MMP3 are closely linked and show that LMP1 activates MMP1 via an Ets binding site formed by 2G, which is a candidate marker for both risk and prognosis of NPC.

Expression of interleukin-8 receptor A predicts poor outcome in patients with nasopharyngeal carcinoma.

OBJECTIVES/HYPOTHESIS: The authors recently demonstrated that interleukin-8 (IL-8) is induced by Epstein-Barr virus encoding latent membrane protein 1 and that IL-8 is overexpressed in tumor cells and correlates significantly with angiogenesis in nasopharyngeal carcinoma. The present objective was to investigate the expressions and the roles of IL-8 receptors in nasopharyngeal carcinoma. STUDY DESIGN: Retrospective patient file review and immunohistochemical study of tissues of patients with nasopharyngeal carcinoma. METHODS: The authors examined the expressions of two high-affinity IL-8 receptors, IL-8 receptor A (IL8RA) and IL-8 receptor B (IL8RB), in 30 patients with nasopharyngeal carcinoma by immunohistochemical analysis. RESULTS: As expected, both IL-8 receptors were expressed on microvessels in tumor nests and surrounding stroma. Interestingly, they were also abundantly expressed on tumor cells. The expressions of IL8RA and IL8RB had no associations with gender, metastasis, or clinical stage. However, the expression of IL8RA in tumors significantly correlated with a shorter overall survival rate (P = .0045). Although the estimated 5-year overall survival rate for IL8RA-negative patients was 68.2%, that in IL8RA-positive patients was only 33.3%. CONCLUSION: The study results suggest that the expression of IL8RA in tumor cells becomes an important indicator of poor prognosis in nasopharyngeal carcinoma.

Diagnostic value of serum EBV-DNA quantification and antibody to viral capsid antigen in nasopharyngeal carcinoma patients.

We compared the amount of serum Epstein-Barr virus DNA (EBV-DNA) detected in patients with nasopharyngeal carcinoma (NPC) in a high-incidence area, represented by Taiwan, and a low-incidence area, represented by Japan, using real-time quantitative PCR. The median serum EBV-DNA value in 41 Japanese NPC cases was 5450 copies/ml, and that in in 23 Taiwanese cases was 2125 copies/ml. The median serum EBV-DNA value in all 64 NPC cases was significantly higher than in control groups. Using receiver-operating-characteristic (ROC) curves, the sensitivity and specificity of EBV-DNA quantification were determined (cut-off point, 6.87 copies/ml; sensitivity, 0.855; specificity, 0.885) and compared with those of EBV-viral-capsid-antigen (VCA) titers; the results showed that EBV-DNA was a more sensitive and specific parameter than EBV-VCA titer. Then, we analyzed 19 NPC patients in whom recurrence developed (11 Japanese and 8 Taiwanese), and 26 NPC patients in continuous remission. Although there was no significant difference in EBV-DNA values between Japanese and Taiwanese patients, the value was significantly higher in the 19 patients with recurrence than in those in remission. ROC analysis again revealed a higher diagnostic value of EBV-DNA than EBV-VCA. These results suggest EBV-DNA is a more reliable tumor marker than EBV-VCA in both high-incidence and low-incidence areas of NPC.