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Purpose: The EBV oncoprotein, latent membrane protein 1 (LMP1), contributes to the metastasis of nasopharyngeal carcinoma (NPC) by inducing factors to promote tumor invasion and angiogenesis. The receptor for advanced glycation end products (RAGE) is associated with abnormal angiogenesis in diabetic microangiopathies. Moreover, some papers have suggested the association of RAGE overexpression with tumor metastasis; thus, the associations of RAGE with LMP1 and angiogenesis in NPC were examined., Experimental Design: Forty-two patients with NPC were evaluated for expressions of LMP1, RAGE, and S100 proteins and for microvessel counts by immunohistochemistry. Then, the RAGE induction by LMP1 was examined with Western blotting and luciferase reporter assay., Results: The microvessel counts were significantly higher in patients with high LMP1 expression or high RAGE expression compared with cases with low expressions (P=0.0049 and P<0.0001), respectively. Patients with advanced N classification were also significantly increased in these groups (P=0.0484 and P=0.0005). The expressions of LMP1 and RAGE proteins were clearly correlated in NPC tissues (P=0.0093). Transient transfection with LMP1 expression plasmid induced RAGE protein in Ad-AH cells. The expression of LMP1 transactivated the RAGE promoter as shown by luciferase reporter assay. Mutation of the reporter at nuclear factor-kappaB binding site (-671 to -663) abolished transactivation of the RAGE promoter by LMP1., Conclusion: These results suggest that LMP1-induced RAGE enhances lymph node metastasis through the induction of angiogenesis in NPC. Nuclear factor-kappaB binding site (-671 to -663) is essential for transactivation of the RAGE promoter by LMP1.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV) associated malignant tumor. Recently, cidofovir, an anti-viral drug which is an acyclic nucleoside analogue, has been reported to have an anti-tumor potential. Two patients with NPC, who had previously received multi-round therapy, were treated with cidofovir. Cidofovir was topically injected in and around the tumor once every 3 weeks (originally 75 mg/ml sulution, diluted to 15 mg/ml just before injection, 37.5 mg of cidofovir at a time). Tumor growth was suppressed for several months around the injection site in each patient. EBV-encoded RNAs in situ hybridization revealed the reduction of the tumor cell population; however, the EBER expression was still maintained in the NPC tumor cells. Although the anti-tumor mechanism remains unclear, these results suggest that cidofovir is actually an effective and safe agent for the treatment of NPC.

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.

Objective: Epstein-Barr virus (EBV) is a causative agent of nasopharyngeal carcinoma (NPC), and EBV gene expression is considered to be closely associated with the pathogenesis of NPC. Among EBV genes expressed in NPC, EBV-encoded non-polyadenylated RNAs, termed EBERs, are the most abundant transcripts of EBV in NPC. However, the role of EBERs still remains unclear. This study was designed to investigate the relevance of EBERs to the oncogenesis of NPC., Methods: Two types of EBERs expression vectors (EBERs-high-expression vector and EBERs-low-expression vector) were constructed and transfected into EBV-negative cells, MDCK or EBV-negative clones of NPC-KT cells. Then, malignant transformation, represented by anchor independent growth, was evaluated between the EBERs-transfected cells and EBERs-negative cells using a soft agar colony formation assay. Apoptosis was induced by serum deprivation (0.1% concentration of fetal bovine serum) and interferon-alpha (IFN-alpha) (500 U/ml) treatment. Cell viability was evaluated with a trypan blue exclusion test. The activation of cellular transcriptional factor NF-kappaB was studied with the IL-8 promoter sequence using a luciferase reporter assay., Results: EBERs-high-expression vector-transfected MDCK cells showed enhanced growth ability in soft agar compared with either EBERs-low-expression vector-transfected MDCK cells or EBERs-untransfected MDCK cells. However, they did not show the acquisition of any anti-apoptotic potential against either IFN-alpha or serum deprivation. Introduction of EBERs-low-expression vector into MDCK cells did not show anchor independent growth characteristics. Neither EBV-negative NPC-KT cells nor MDCK cells transfected with EBERs-high-expression vector showed any difference from EBERs-untransfected EBV-negative NPC-KT cells. Introduction of EBERs into MDCK cells did not transactivate the IL-8 promoter, indicating that neither NF-kappaB nor AP-1 was activated by EBERs., Conclusion: EBERs are believed to induce the initial transformation of epithelial cells, thus contributing to the oncogenesis of NPC. Expression of abundant EBERs is considered to be critical for this transforming property of EBERs.

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., ((c) 2005 Wiley-Liss, Inc.)

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.

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.

Background/objectives: Nasopharyngeal carcinoma (NPC) is a highly invasive and metastatic malignant tumor and is associated with Epstein-Barr virus (EBV) infection that exhibits type II latency. Angiogenesis is essential for tumor growth, invasion, and metastasis. Our previous studies have indicated that interleukin (IL)-8 was over-expressed in many NPC tissues and was found to be significantly correlated with angiogenesis by immunohistochemistry., Study Design: In vitro design., Methods: The influence of the EBV genome for IL-8 gene expression was studied using the EBV-genome-positive and -negative epithelial/NPC hybrid cell line NPC-KT. The EBV-positive and -negative clones were selected by polymerase chain reaction and in situ hybridization., Results: EBV-positive clones expressed abundant IL-8 mRNA compared with EBV-negative clones. This result indicated that over-expression of IL-8 depended on the presence of EBV genomes in NPC-KT cells. Two encoded genes, latent membrane protein (LMP)1 and EBV-encoded small RNAs (EBERs), expressed in NPC were transfected in EBV-negative NPC-KT cells. LMP1 transactivated the IL-8 promoter, whereas EBERs did not. Moreover, the nuclear factor (NF)-kappa B binding site in the IL-8 promoter was essential for the response to LMP1, and the activator protein (AP)-1 binding site played only a partial role., Conclusions: LMP1 induces IL-8 mainly through the activation of NF-kappa B and partly through AP-1 in NPC model cell lines, NPC-KT, and this suggests that LMP1 plays an important role in the angiogenesis of NPC.