ENHANCED MALIGNANT PROGRESSION OF NASOPHARYNGEAL CARCINOMA CELLS MEDIATED BY THE EXPRESSION OF EPSTEIN–BARR NUCLEAR ANTIGEN 1 IN VIVO

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein–Barr virus (EBV) and mostly classified as poorly differentiated squamous cell carcinoma or undifferentiated carcinoma with early metastasis and a rapidly progressive clinical course. The EBV-encoded latent proteins, Epstein–Barr nuclear antigen 1 (EBNA 1) and latent membrane proteins (LMPs), may be expressed in NPC, but their biological effects are poorly understood. EBNA 1 may predispose B lymphocytes to lymphomagenesis in transgenic mice, but its biological effects in NPC are still unknown. This study investigated the biological effects of EBNA 1 by expressing it in an EBV-negative NPC cell line (HONE-1), which was then inoculated into both nude and severe combined immunodeficiency mice. The EBNA 1 caused HONE-1 cells to grow in a less differentiated pattern and to progress more rapidly, as well as increasing their tumourigenicity and metastatic capability. These data suggest that EBNA 1 may play a critical role in the progressive evolution of NPC.     

CLINICOPATHOLOGICAL,IMMUNOLOGICAL AND GENETIC STUDIES OF CD30+ ANAPLASTIC LARGE CELL LYMPHOMA OF B-CELL TYPE; ASSOCIATION WITH EPSTEIN–BARR VIRUS IN A JAPANESE POPULATION

The clinicopathological features, the immunophenotype, and the presence of Epstein–Barr virus (EBV)-associated genomes and gene products were examined in 17 cases of CD30⁺ anaplastic large cell lymphoma (ALCL) of B-cell type. Microscopically, the 17 cases were divided into ten cases of the monomorphic type and seven cases of the pleomorphic type. EBV was detected in 6 of 17 cases (38 per cent) by RNA in situ hybridization (ISH) with EBV-encoded RNA (EBER1). EBER1⁺ cases consisted of two cases (20 per cent) of the monomorphic type and four cases (57 per cent) of the pleomorphic type. The five EBER1⁺ cases showed clonality of the EBV genome by Southern blotting, consistent with the presence of EBV in a monoclonal proliferation. The EBV-encoded latent membrane protein 1 (LMP1) was found in all six EBER1⁺ cases and EBV-encoded nuclear antigen 2 (EBNA2) was present in two cases by immunohistochemistry. No expression of LMP1 or EBNA2 was observed in the EBER1⁻ cases. The EBER1⁺ cases had a tendency for a more favourable prognosis than the EBER1⁻ cases. It is concluded that EBV has an association with CD30⁺ ALCL of B-cell type in the Japanese population studied, and especially with the large pleomorphic type. EBV infection may play a pathoaetiological role and may influence clinical behaviour.     

Incidence of latent infection of Epstein–Barr virus in lung cancers—an analysis of EBER1 expression in lung cancers by in situ hybridization

To evaluate the presence of Epstein–Barr virus (EBV) in lung cancers of Japanese patients, 81 lung cancers were examined using a highly sensitive in situ hybridization (ISH) method, employing an antisense oligonucleotide probe for EBV-encoded small nuclear RNA-1 (EBER). EBER1 expression was demonstrated in one poorly differentiated squamous cell carcinoma associated with marked lymphoid stroma (PDSCC-LS), two well differentiated adenocarcinomas, and two moderately differentiated squamous cell carcinomas, but was not detectable in other lung cancers, including small cell carcinomas. Unlike lymphoepithelioma-like undifferentiated carcinoma (LELC) of the lung, the PDSCC-LS consisted of poorly differentiated cells with distinct cell borders and nuclei with a coarse chromatin pattern and some prominent nucleoli. Most of the cancer cells expressed intense EBER1 signals. Although small to moderate numbers of cells positive for EBER1 were present in two adenocarcinomas and two squamous cell carcinomas, EBER1 signals varied in intensity and number in these four cases. Although polymerase chain reaction (PCR) and Southern blot hybridization with a ³²P-labelled probe internal to the primers were conducted to detect the EBV genome in 24 lung cancers, including five EBER1-positive cases, the genome was found to be positive in the five cases with EBER1-positive staining, including the PDSCC-LS, two adenocarcinomas and two squamous cell carcinomas, but not in the other cases. This study indicates that the morphological features of EBV-associated lung cancers are not restricted to the typical LELC type.     

PHENOTYPIC MODULATION OF HODGKIN AND REED–STERNBERG CELLS BY EPSTEIN–BARR VIRUS

Expression of the Epstein–Barr virus (EBV) gene product LMP1 is found in tumour cells in varying proportions of Hodgkin's disease (HD) cases. It is not clear which cellular genes are influenced by EBV in HD. A total of 387 HD cases were tested for differences among LMP1-positive and -negative cases with respect to age, sex, histotype and immunophenotypic parameters (CD2, CD3, CD4, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD43, CD45RA, CD45R0, CD70, HLA-DR, T-cell receptor β-chain, and p53 expression). Comparison of patient age and sex as well as distribution of histotype and tumour cell immunophenotype with published data suggests that the cases in this study are representative of the spectrum of HD in developed countries. LMP1 expression was found in 131/387 HD cases (36·4 per cent) with non-homogeneous distribution among HD histotypes, the mixed cellularity type (HDmc) being most frequently EBV-associated (71/129 cases, 55 percent). No relationship was found to age and sex. Significant phenotypic differences were restricted to the HDmc histotype, where the tumour cells expressed the activation marker CD30 in a larger proportion, and CD20 in a smaller proportion, when harbouring EBV. These results suggest that EBV may influence the tumour cell phenotype in HD.     

Epstein–Barr virus in Reed–Sternberg G-like cells in non Hodgkin's lymphomas

In the course of our study on Hodgkin's disease (HD), ten cases of non-Hodgkin's lymphomas (NHL) containing Hodgkin and Reed-Sternberg-like (MRS) cells were encountered. Many of these cases had initially been diagnosed as HD, but on careful review of the histology, with the aid of immunophenotyping studies, they were reclassified as NHL. The presence of Epstein–Barr virus (EBV) in these HRS-like cells was investigated using a combination of EBER in situ hybridization (ISH) and immunostaining for the detection of EBV-encoded latent membrane protein (LMP). HRS-like cells in four cases (two lymphoplasmacytoid lymphomas, one Richter's transformation of lymphoplasmacytoid lymphoma, and one immunoblastic lymphoma of T-cell type) were found to be EBV-positive. In two of these cases, a second biopsy taken up to 10 years later also contained EBV in the HRS-like cells. In three of the four cases, HRS-like cells expressed the activation antigen CD30, but the expression of B- or T-cell antigens was variable. All cases of T-cell-rich B-cell lymphomas were negative for EBV. In conclusion, EBV may play a role in the development of HRS-like cells i some cases of NHL. The relationship of HRS-like cells to HRS cells of HD is discussed.     

Frequent expansion of Epstein–Barr virus (EBV) infected cells in germinal centres of tonsils from an area with a high incidence of EBV-associated lymphoma

Burkitt's lymphoma (BL) and Hodgkin's disease (HD) occurring in developing regions are frequently associated with Epstein–Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV-infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV-encoded small nuclear RNA (EBER) and EBV-encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER-positive lymphoid cells were found in the GCs of 8/20 EBV-positive tonsils from Brazil (9–89 cells/GC; mean: 14·5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1–9 cells/GC; mean: 0·5 cell/GC per case) in this compartment (p < 0·007). In addition, the EBV-infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV-encoded latent membrane protein (LMP)-1, findings not present in German samples. These data show that latently EBV-infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP-1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD. Copyright © 1999 John Wiley & Sons, Ltd.     

New variations of Epstein–Barr virus‐encoded small RNA genes in nasopharyngeal carcinomas,gastric carcinomas,and healthy donors in northern China

It has been generally believed that the Epstein–Barr virus (EBV)‐encoded small RNA 1 and 2 (EBER1 and EBER2) genes are conserved as two families that correlated with type 1 (B95‐8) and type 2 (AG876 or P3HR‐1) EBV strains. EBER polymorphism and its association with EBV‐associated disease have not received much attention. To explore the variations of EBER genes in different malignancies and healthy donors, the sequences of EBER genes were analyzed in 154 EBV‐positive samples, including 47 nasopharyngeal carcinoma (NPC), 50 EBV‐associated gastric carcinoma (EBVaGC) biopsies and 57 throat washing (TW) samples from healthy donors in northern China, where NPC is non‐endemic. Three main distinct variants of EBER genes, designated as EB‐6m, EB‐8m, and EB‐10m, were identified. EB‐6m had a previously identified EBER sequence identical to P3HR‐1 and was found in 33/47 (70.2%) NPC, 48/50 (96.0%) EBVaGC, and 54/57 (94.7%) TW isolates. EB‐8m and EB‐10m were new EBER variants with more mutations in EBER2 genes. They were found in 13/47 (27.7%) NPC cases, whereas in only 1/50 (2.0%) EBVaGC cases and not found in TW cases. The distributions were significantly different (P < 0.05). Other five isolates (one NPC, one EBVaGC and three TW cases) showed different sequences and could not be assigned to any of the three groups. Type 1 EBV strains showed heterogeneous in terms of EBER variants. These results suggest that EBER locus can be useful to identify different EBV isolates, and it would be interesting to evaluate the association of EBER polymorphisms with EBV‐associated tumors. J. Med. Virol. 82: 829–836, 2010. © 2010 Wiley‐Liss, Inc.     

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty-six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well-differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV-positive OSCCs. However, no signal of the EBV-encoded small RNA (EBER)-1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow-up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV-positive patients, which thus suggested a good prognosis for EBV-positive OSCC patients. Copyright © 1999 John Wiley & Sons, Ltd.     

Epstein–Barr virus (EBV) provides survival factors to EBV+ diffuse large B‐cell lymphoma (DLBCL) lines and modulates cytokine induced specific chemotaxis in EBV+ DLBCL

Diffuse large B‐cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non‐Hodgkin lymphomas. Epstein–Barr virus (EBV) ‐positive DLBCL of the elderly is a newly recognized subtype that accounts for 8–10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL‐derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin‐4 and interleukin‐21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4‐ or CCR7‐mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant‐negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV‐positive lines. These results show that EBV plays an important role in EBV‐positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV‐carrying DLBCL cells and might have therapeutic implications .     

IgH AND TcR-γ GENE REARRANGEMENTS IDENTIFIED IN HODGKIN'S DISEASE BY PCR DEMONSTRATE LACK OF CORRELATION BETWEEN GENOTYPE,PHENOTYPE, AND EPSTEIN–BARR VIRUS STATUS

Analysis of IgH and TcR-γ genes using consensus primers identifying junctional regions of rearranged genes by polymerase chain reaction (PCR) was performed on tissues involved by Hodgkin's disease (HD) in 90 cases and was correlated with the immunophenotype of Hodgkin and Reed–Sternberg (HRS) cells and the presence of Epstein–Barr virus (EBV) within these cells. Clonal IgH gene rearrangements were found in 1/5 cases of lymphocyte predominance (LP) subtype and none was positive for EBV. In 85 cases of classic HD, no IgH or TcR-γ gene rearrangements were found in 51 (60 per cent) cases. A similar percentage, but not the same cases, were of null (non-B, non-T) phenotype. Of 30 cases where a B phenotype was assigned to HRS cells, nine had IgH gene rearrangements, three had TcR-γ gene rearrangements, and two had both genes rearranged. None of the five cases assigned to T phenotype of HRS cells showed rearrangement of TcR-γ genes, but two cases showed rearranged IgH genes. Among 41 cases of null phenotype, ten had IgH gene rearrangements, five had TcR-γ gene rearrangements, and three cases had both genes rearranged. Whereas EBV was detectable in HRS cells in 17/43 classic HD cases of assigned B phenotype, EBV was also detectable in 2/5 cases of assigned T phenotype and in 21 cases with the null phenotype. Furthermore, there was no correlation of EBV with the presence or lack of IgH or TCR-γ gene rearrangements. Of the remainder, half (30 per cent) expressed antigens associated with lymphocytes without an appropriate genotype. The results confirm lymphocyte-lineage committed cells at the origin of HRS cells in 40 per cent of cases. Any hypothesis of a non-lymphocytic origin of HRS cells will require the inducibility of CD30 on candidate precursors and the methodology for probing genetic events in such cells to be addressed. © 1997 by John Wiley & Sons, Ltd.     

Serum sCD23 Level in Patients with AIDS-Related Non-Hodgkin''s Lymphoma Is Associated with Absence of Epstein–Barr Virus in Tumor Tissue

The cytokine soluble CD23 (sCD23) acts as a B cell growth factor and is associated with Epstein–Barr virus (EBV) infection. To elucidate the role sCD23 might play in the pathogenesis of AIDS-related non-Hodgkin's lymphoma (AIDS NHL), 101 AIDS NHL patients from the Multicenter AIDS Cohort Study were studied. Serum sCD23 within 18 months prior to lymphoma diagnosis was measured for all patients and EBV in tumor tissue was ascertained for 49 patients. Tumor morphology and primary site were verified from pathology reports and tumor specimens. Bivariate tests and multivariate regression were employed to determine whether serum sCD23 correlated with tumor EBV, morphology, and primary site. Higher levels of serum sCD23 were associated with the absence of tumor EBV and with small noncleaved cell morphology. Thus, the serum sCD23 level does not appear to be mediated by EBV in these patients, but could be related to a pathogenetic mechanism of small noncleaved cell lymphoma.     

EPSTEIN–BARR VIRUS (EBV) INFECTION IN INFECTIOUS MONONUCLEOSIS: VIRUS LATENCY,REPLICATION AND PHENOTYPE OF EBV-INFECTED CELLS

Primary Epstein–Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infectious mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2⁻/LMP1⁻) and III (EBNA2⁺/LMP1⁺), cells displaying a latency II pattern (EBNA2⁻/LMP1⁺) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients. © 1997 John Wiley & Sons, Ltd.     

Analysis of single EBER-positive and negative tumour cells in EBV-harbouring B-cell non-Hodgkin lymphomas.

In situ hybridization for Epstein-Barr virus (EBV) encoded small nuclear RNAs (EBERs) is the method of choice for the detection of EBV infection at the single cell level. With the application of this technique it was shown that non-Hodgkin's lymphomas of B-cell type may be associated with an EBV infection of tumour cells. Interestingly, in many EBV-positive cases, only a proportion of the tumour cell population has been found to be EBER-positive. To clarify whether EBV is absent or whether EBER gene expression is downregulated in EBER-negative tumour cells, single EBER-positive and negative tumour cells were isolated from paraffin sections from four B-cell non-Hodgkin's lymphoma cases with partial EBER expression in the neoplastic cells. These single cells were then screened for the presence of EBV DNA by a nested single cell PCR. EBV DNA was undetectable in all but one of the 86 EBER-negative cells, whereas in the EBER-positive cells EBV-specific DNA amplificates could be generated following single cell PCR. This finding prompts the conclusion that the inability to detect EBERs in a proportion of tumour cells is not due to a down-regulation of gene expression but to a real absence of EBV from these cells. This partial absence of EBV is thought to be caused by a loss of the EBV episomes during cell division, rather than by infection of only a proportion of the tumour cells.     

Epstein–Barr virus BZLF1 gene promoter variants in pediatric patients with acute infectious mononucleosis: Its comparison with pediatric lymphomas

Epstein–Barr virus genotypes can be distinguished by polymorphic variations in the genes encoding EBNA2, 3A, 3B, and 3C. The immediate early gene BZLF1 plays a key role in modulating the switch from latency to lytic replication and therefore enabling viral propagation. The aim of this study was to investigate and compare BZLF1 promoter sequence (Zp) variation in pediatric infectious mononucleosis (IM) and in pediatric EBV positive lymphoma biopsies. Zp was sequenced from peripheral blood mononuclear cells (PBMC) and throat swabs from 10 patients with IM at the time of diagnosis (D0) and during convalescence; and from 13 lymphoma biopsies. Zp ? P and Zp ? V3 variants were found in eight and one IM patients, as well as in five and six tumor biopsies, respectively. A correlation between viral genotype and Zp variant was found significant for Zp ? V3 and EBV2 (P = 0.0002). One IM patient harbored two concomitant Zp variants. Regardless of anatomical compartment or stage of disease all IM patients displayed the same Zp variant along the course of the study. No new infections or adaptative selection of different variants was evidenced. A new Zp variant (Zp ? V3 + 49) was described in two Hodgkin lymphomas, but not in IM. This is the first study to describe Zp variants compartmentalization in children with acute EBV infection and convalescence in a developing country; and comparing them with Zp variants in pediatric lymphomas from the same geographic area. J. Med. Virol. 81:1912–1917, 2009. © 2009 Wiley‐Liss, Inc.     

Non-Hodgkin's lymphoma,coeliac disease,and Epstein–Barr virus: A study of 13 cases of enteropathy-associated T- and B-cell lymphoma

A group of 166 patients with coeliac disease was followed for a period of up to 25 years. During this time, 17 patients developed intestinal tumours that were diagnosed as lymphoma, of which 15 cases were available for review. Eleven of the lymphomas were of T-cell type (enteropathy-associated T-cell lymphoma, EATL) and two were of B-cell type. Two cases were reclassified as undifferentiated carcinoma. The interval between the diagnosis of enteropathy and the onset of lymphoma varied from less than 2 months in four patients to more than 5 years in seven. Seven of the T-cell and both B-cell lymphomas were investigated for the presence of Epstein–Barr virus (EBV) by in situ hybridization (ISH) using probes against Epstein–Barr virus-encoded RNAs (EBERs) and by immuno-histochemistry with EBV-specific monoclonal antibodies. All EATL cases were negative, suggesting that EBV is not an important factor in these cases. In one of the B-cell cases, EBV was detectable by ISH and immunohistochemistry in most tumour cells in the mesenteric lymph nodes, but not in any of the tumour cells in the primary ileal tumour, indicating that in this case EBV infection was a late event in the neoplastic process. These results show that lymphoma may develop any time after the onset of coeliac disease and that in our cases of EATL, EBV was not an important factor. In some cases of EBV-related neoplasia, virus infection may be a late event.     

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein–Barr virus (EBV)

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro re-stimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV-specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL-2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual-colour flow cytometry revealed two subpopulations, one CD4⁺ CD8⁺ population and the other CD4⁻ CD8⁺. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4⁺ CD8⁺ fraction was also shown to be MHC class II-restricted; the MHC restriction of the CD8⁺ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine-induced immune protection.     

A20 RNA expression is associated with undifferentiated nasopharyngeal carcinoma and poorly differentiated head and neck squamous cell carcinoma

A20 is an anti-apoptotic gene that can be induced in human epithelial cell lines in response to expression of the Epstein–Barr virus (EBV) gene product latent membrane protein 1 (LMP1). EBV is a ubiquitous, persistent human herpesvirus that is consistently associated with undifferentiated nasopharyngeal carcinoma (NPC), in which antigen expression includes LMP1. Consistent with a potential role in the development of NPC, LMP1 has profound effects on epithelial cell growth. A20 may be a key downstream effector of LMP1 in NPC, as LMP1-induced A20 blocks p53-mediated apoptosis in H1299 epithelial cells and most NPCs have wild-type p53. Moreover, the potential role of A20 in the development of epithelial malignancies may extend to tumours not associated with EBV. The purpose of this study was to develop an in situ hybridization assay to assess expression of A20 RNA in undifferentiated NPC and in non-EBV-associated poorly differentiated head and neck squamous cell carcinomas (SCCs) and well-differentiated SCCs of the skin. A20 RNA expression was also examined in normal samples of oral mucosa and skin. Expression of A20 was demonstrated in 76 per cent of undifferentiated NPCs and in 80 per cent of poorly differentiated head and neck SCCs, suggesting a role for A20 in the pathogenesis of these epithelial malignancies. By contrast, A20 RNA was not detected in well-differentiated SCCs of the skin, or in any normal samples of squamous epithelial tissue. The pathway leading to A20 expression in non-EBV-associated poorly differentiated head and neck SCCs is clearly LMP1-independent. LMP1 expression was demonstrated in 29 per cent of NPC biopsies, suggesting an LMP1-independent pathway to A20 induction in undifferentiated NPC. Copyright © 1999 John Wiley & Sons, Ltd.     

Activation of sterol regulatory element‐binding protein 1 (SREBP1)‐mediated lipogenesis by the Epstein–Barr virus‐encoded latent membrane protein 1 (LMP1) promotes cell proliferation and progression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein–Barr virus (EBV) infection. The EBV‐encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element‐binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV‐infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1‐mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1‐mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo. Our findings demonstrate that LMP1 activation of SREBP1‐mediated lipogenesis promotes tumor cell growth and is involved in EBV‐driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.