Hiroshi Uozaki

Activation of DNA Methyltransferase 1 by EBV Latent Membrane Protein 2A Leads to Promoter Hypermethylation of PTEN Gene in Gastric Carcinoma

CpG island promoter methylation of tumor suppressor genes is one of the most characteristic abnormalities in EBV-associated gastric carcinoma (GC). Aberrant promoter methylation and expression loss of PTEN were evaluated in cancer tissues of GC by methylation-specific PCR and immunohistochemistry, respectively, showing that both abnormalities occurred concurrently in EBV-associated GC. PTEN abnormalities were reiterated in GC cell lines MKN-1 and MKN-7 infected with recombinant EBV, and DNA methyltransferase 1 (DNMT1) was commonly overexpressed in both cell lines. Stable and transient transfection systems in MKN-1 similarly showed that viral latent membrane protein 2A (LMP2A) up-regulated DNMT1, leading to an increase in methylation of the PTEN promoter. Importantly, the level of phosphorylated signal transducer and activator of transcription 3 (pSTAT3) increased in the nuclei of LMP2A-expressing GC cells, and knockdown of STAT3 counteracted LMP2A-mediated DNMT1 overexpression. Immunohistochemistry for both pSTAT3 and DNMT1 showed diffuse labeling in the nuclei of the cancer cells in GC tissues, especially in EBV-associated GC. Taken together, LMP2A induces the phosphorylation of STAT3, which activates DNMT1 transcription and causes PTEN expression loss through CpG island methylation of the PTEN promoter in EBV-associated GC. LMP2A plays an essential role in the epigenetic abnormalities in host stomach cells and in the development and maintenance of EBV-associated cancer.

CpG island methylation status in gastric carcinoma with and without infection of Epstein-Barr virus

Purpose: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma. Experimental Design: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated. Results: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number ± SD = 6.9 ± 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 ± 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313). Conclusion: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.

Classification of Epstein–Barr Virus–Positive Gastric Cancers by Definition of DNA Methylation Epigenotypes

Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illuminas Infinium BeadArray and used hierarchical clustering analysis to classify gastric cancers into 3 subgroups: EBV(-)/low methylation, EBV(-)/high methylation, and EBV(+)/high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV(+) tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV(+) and EBV(-)/high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV(-)/high-methylation genes and commonly methylated gastric cancer genes (P = 2 × 10(-15) and 2 × 10(-34), respectively), but not among EBV(+) tumor-specific methylation genes (P = 0.2), suggesting a different cause for EBV(+)-associated de novo methylation. When recombinant EBV was introduced into the EBV(-)/low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV(+) gastric cancers showed distinct methylation patterns likely attributable to EBV infection.

Epstein-Barr virus and gastric carcinoma : virus-host interactions leading to carcinoma

Epstein–Barr virus (EBV)‐associated gastric carcinoma (GC) is a distinct subgroup of GC, comprising 10% of all cases of GC. EBV‐associated carcinoma is the monoclonal growth of EBV‐infected epithelial cells, and it represents a model of virus–host interactions leading to carcinoma. EBV‐infected cells express several latent proteins (latency I program of viral latent gene expression) in EBV‐associated GC. However, latent membrane protein 2A (LMP2A) up‐regulates the cellular survivin gene through the NFkB pathway, conferring resistance to apoptotic stimuli on the neoplastic cells. EBV‐associated GC also shows characteristic abnormality, that is, global and non‐random CpG island methylation of the promoter region of many cancer‐related genes. Since the viral genes are also regulated by promoter methylation in the infected cells, the DNA methylation mechanism specific to EBV‐associated GC may be an exaggeration of the cellular mechanism, which is primarily for defense against foreign DNA. Production of several immunomodulator molecules, inducing tumor‐infiltrating lymphocyte and macrophages, serves to form the characteristic histologic pattern in EBV‐associated GC. The proposed sequence of events within the mucosa is as follows: EBV infection of certain gastric stem cells; expression of viral latent genes; abnormality of signal pathways caused by viral gene products; DNA methylation‐mediated repression of tumor suppressor genes; and monoclonal growth of EBV–infected cells through interaction with other etiologic factors. Potentially useful therapeutic approaches to EBV‐associated GC are those that utilize the virus–host interactions, such as bortezomib‐induced and viral enzyme‐targeted radiotherapy. (Cancer Sci 2008; 99: 1726–1733)

Downregulation of MicroRNA-200 in EBV-Associated Gastric Carcinoma

EBV-associated gastric carcinoma is a distinct gastric carcinoma subtype with characteristic morphologic features similar to those of cells that undergo epithelial-to-mesenchymal transition. The effect of microRNA abnormalities in carcinogenesis was investigated by measuring the expression of the epithelial-to-mesenchymal transition-related microRNAs, miR-200a and miR-200b, in 36 surgically resected gastric carcinomas using quantitative reverse transcription-PCR analysis. MiR-200 family expression was decreased in EBV-associated gastric carcinoma, as compared with that in EBV-negative carcinoma. Downregulation of the miR-200 family was found in gastric carcinoma cell lines infected with recombinant EBV (MKN74-EBV, MKN7-EBV, and NUGC3-EBV), accompanied by the loss of cell adhesion, reduction of E-cadherin expression, and upregulation of ZEB1 and ZEB2. E-cadherin expression was partially restored by transfection of EBV-infected cells with miR-200 family precursors. Reverse transcription-PCR analysis of primary precursors of miR-200 (pri-miR-200) revealed that the transcription of pri-miR-200 was decreased in EBV-infected cells. Transfection of MKN74 cells with BARF0, EBNA1, and LMP2A resulted in a decrease of pri-miR-200, whereas transfection with EBV-encoded small RNA (EBER) did not. These four latent genes contributed to the downregulation of the mature miR-200 family and the subsequent upregulation of ZEB1/ZEB2, resulting in the reduction of E-cadherin expression. These findings indicate that all the latency type I genes have a synergetic effect on the downregulation of the miR-200 family that leads to reduced E-cadherin expression, which is a crucial step in the carcinogenesis of EBV-associated gastric carcinoma.

Spectrum of Epstein-Barr virus-related diseases: a pictorial review

Epstein-Barr virus (EBV) prevails among more than 90% of the adult population worldwide. Most primary infections occur during young childhood and cause no or only nonspecific symptoms; then the virus becomes latent and resides in lymphocytes in the peripheral blood. Inactive latent EBV usually causes no serious consequences, but once it becomes active it can cause a wide spectrum of malignancies: epithelial tumors such as nasopharyngeal and gastric carcinomas; mesenchymal tumors such as follicular dendritic cell tumor/sarcoma; and lymphoid malignancies such as Burkitt lymphoma, lymphomatoid granulomatosis, pyothorax-associated lymphoma, immunodeficiency-associated lymphoproliferative disorders, extranodal natural killer (NK) cell/T-cell lymphoma, and Hodgkin’s lymphoma. The purpose of this article is to describe the spectrum of EBV-related diseases and their key imaging findings. EBV-related lymphoproliferative disorders and lymphomas are especially common in immunocompromised patients. Awareness of their clinical settings and imaging spectrum contributes to early detection and early treatment of possibly life-threatening disorders.

p73 gene promoter methylation in Epstein‐Barr virus‐associated gastric carcinoma

To clarify the significance of p73 in Epstein‐Barr virus (EBV)‐associated gastric carcinoma (GC), the immunohistochemical expression and CpG‐island methylation of p73 were evaluated in cancer tissues and adjacent nonneoplastic tissues of GC with and without EBV infection. Loss of p73 expression by immunohistochemistry was specific to EBV‐associated GC (11/13) compared to EBV‐negative GC (3/38), which was independent of abnormal p53 expression. With methylation‐specific polymerase chain reaction (MSP), the aberrant methylation of p73 exon 1 was similarly specific to EBV‐associated GC (12/13), and also rare in EBV‐negative GC (2/38). Bisulfite sequencing for p73 exon 1 and its 5′ region confirmed the MSP results, showing uniform and high‐density methylation in EBV‐associated GC. Comparative MSP analysis of p14, p16 and p73 methylation, using 20 cases each of formalin‐fixed and paraffin‐embedded tissues of early GC with and without EBV infection, confirmed 2 types of methylation: global methylation with increased rates (p14 and p16) and specific methylation of p73 in EBV‐associated GC. In nonneoplastic mucosa, p14, p16 and p73 methylation occurred in both EBV‐associated (8/33, 6/34 and 3/38, respectively) and EBV‐negative GC (6/23, 4/35, and 1/35). p73 methylation was observed in the mucosa without H. pylori infection in all 4 samples. Loss of p73 expression through aberrant methylation of the p73 promoter occurs specifically in EBV‐associated GC, together with the global methylation of p14 and p16. A specific type of gastritis, prone to a higher grade of atrophy and p73 methylation, may facilitate the development of EBV‐associated GC.

Protective role of the leukotriene B4 receptor BLT2 in murine inflammatory colitis

BLT2 is a low-affinity leukotriene B(4) (LTB(4)) receptor that is activated by 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB(4). Despite the well-defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice lacking either BLT1 or BLT2. BLT2(-/-) mice exhibited increased sensitivity to DSS as compared to wild-type and BLT1(-/-) mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)-γ, interleukin (IL)-1β, and IL-6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C-C motif chemokine 19 (CCL19), and metalloproteinases was highly up-regulated in the colons of DSS-treated BLT2(-/-) mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS-treated BLT2(-/-) mice. Madin-Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC-dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS-induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti-inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.

Promoter hypermethylation of E‐cadherin and its abnormal expression in Epstein‐Barr virus‐associated gastric carcinoma

Promoter hypermethylation of various tumor‐related genes is extremely frequent in Epstein‐Barr virus (EBV)‐associated gastric carcinoma (EBVaGC). To investigate the significance of the promoter methylation in EBVaGC, we focused on one of the important proteins in the carcinogenesis of the stomach, E‐cadherin. Methylation‐specific PCR analysis (MSP) was applied to surgically resected gastric carcinomas, together with immunohistochemistry, PCR‐based analysis of mutations and allelic loss, and site‐specific MSP of E‐cadherin gene. By MSP, nearly all of the carcinomas showed aberrant methylation of E‐cadherin promoter in EBVaGC (21/22), and the frequency of this aberration was significantly higher than that in EBV‐negative gastric carcinoma (GC; 45/81; p = 0.0003). According to immunohistochemistry of E‐cadherin, the frequency of abnormal staining pattern in EBVaGC (87%) was comparable to that in the diffuse type (80%), but higher than that in the intestinal type of EBV‐negative GC (47%). Promoter methylation was well correlated with abnormal staining pattern in EBVaGC, but not in EBV‐negative GC. Neither mutation nor allelic loss of E‐cadherin was observed in EBVaGC. Methylation status of E‐cadherin within each carcinoma was heterogeneous as far as examined. Thus, in addition to the known association involving p16, we determined that promoter methylation‐mediated silencing of E‐cadherin gene was also closely associated with the development of EBVaGC, although it becomes heterogeneous within a given tumor along its progression.

Global and non-random CpG-island methylation in gastric carcinoma associated with Epstein-Barr virus

DNA hypermethylation may play a primary role in the genesis of Epstein‐Barr virus (EBV)‐associated gastric carcinoma (GC) (EB‐VaGC). Methylation‐specific PCR targeting CpG‐islands demonstrated markedly increased methylation of specific genes, such as p14, p15 and p16 genes, in EBVaGC in vivo. A high frequency of methylation was observed in an EBVaGC strain of severe combined immunodeficiency mice, and the expression of methylated genes in the strain was apparently lower than the expression of the unmethylated genes in EBV‐negative GC strains. Although over‐expression of DNA methyltransferases (DNMTs) is known to be associated with some human cancers, real‐time PCR demonstrated that DNMTs expression was suppressed in EBVaGC. The DNA methylation of specific genes, independently of DNMTs expression, may be important in the development of EBVaGC. (Cancer Sci 2003; 94: 76–80)