Tetsuo Ushiku

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.

Recurrent gain-of-function mutations of RHOA in diffuse-type gastric carcinoma

Diffuse-type gastric carcinoma (DGC) is characterized by a highly malignant phenotype with prominent infiltration and stromal induction. We performed whole-exome sequencing on 30 DGC cases and found recurrent RHOA nonsynonymous mutations. With validation sequencing of an additional 57 cases, RHOA mutation was observed in 25.3% (22/87) of DGCs, with mutational hotspots affecting the Tyr42, Arg5 and Gly17 residues in RHOA protein. These positions are highly conserved among RHO family members, and Tyr42 and Arg5 are located outside the guanine nucleotide–binding pocket. Several lines of functional evidence indicated that mutant RHOA works in a gain-of-function manner. Comparison of mutational profiles for the major gastric cancer subtypes showed that RHOA mutations occur specifically in DGCs, the majority of which were histopathologically characterized by the presence of poorly differentiated adenocarcinomas together with more differentiated components in the gastric mucosa. Our findings identify a potential therapeutic target for this poor-prognosis subtype of gastric cancer with no available molecularly targeted drugs.

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.

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.

Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) has been accepted as an infective agent causing gastric carcinoma (GC). EBV-associated GC, comprising nearly 10% of all cases of GC, is the monoclonal growth of EBV-infected epithelial cells, which express only several EBV-latent genes (Latency I program). Histopathologically, there are two subtypes, lymphoepithelioma-like carcinoma and the ordinary type of GC. Other features include the lace pattern of carcinoma cells in the intramucosal stage and the dense infiltration of lymphocytes and macrophages at the invasive site of the submucosa. The primary molecular abnormality in EBV-associated GC is global and non-random CpG island methylation in the promoter region of many cancer-related genes. Experimental studies have demonstrated that viral latent membrane protein 2A (LMP2A) is responsible for the promotion of DNA methylation. LMP2A up-regulates cellular DNMT1 through the phosphorylation of STAT3, resulting in the repression of tumor suppressor genes, such as PTEN, through promoter methylation. DNA methylation in EBV-infected stomach cells may be due to overdrive of the cellular defense against foreign DNA. Further studies on the mechanisms of epigenetic abnormalities will clarify the strategies for prevention and treatment of this particular type of GC with EBV infection.

SALL4 represents fetal gut differentiation of gastric cancer, and is diagnostically useful in distinguishing hepatoid gastric carcinoma from hepatocellular carcinoma.

The novel stem cell marker SALL4 has been identified as a diagnostic marker of germ cell tumors, especially yolk sac tumors, in gonadal organs. To clarify the significance of SALL4 as an oncofetal protein, we investigated SALL4 expression by immunohistochemistry in non-neoplastic stomach and gastric carcinoma with particular emphasis on á-fetoprotein (AFP)-producing gastric carcinoma, as AFP-producing gastric carcinoma shares expression of AFP and glypican 3 (GPC3) with yolk sac tumors and hepatic neoplasms. A total of 338 gastric carcinomas, 60 hepatocellular carcinomas, and 48 cholangiocellular carcinomas were studied by immunohistochemistry on tissue microarrays. In addition, more detailed whole tissue section immunohistochemistry was performed on non-neoplastic gastric tissue from 5 adult and 8 fetal specimens, 6 hepatoblastomas, and 31 cases of AFP-producing gastric carcinomas. SALL4 expression was observed in the neofetal stomach in gestational week 9 and disappeared thereafter. It was also identified by tissue microarray study in a fraction of gastric carcinomas (51 of 338, 15%), associated with older age (P=0.0001), male sex (P=0.0033), intestinal-type histology (P=0.0001), and synchronous liver metastasis (P=0.0047). AFP and GPC3 were closely associated with SALL4 expression in gastric carcinoma (both, P<0.0001), and a full-section study indicated that SALL4 was positive in all 31 cases of AFP-producing gastric carcinoma with diffuse staining in 24 cases (78%). Diffuse SALL4 expression was observed in the histologic patterns of hepatoid (89%), glandular (57%), and clear cell (39%) AFP-producing gastric carcinoma. In addition, SALL4 expression was completely negative in hepatoblastoma (n=6) and hepatocellular carcinoma (n=60). SALL4 is an oncofetal protein similar to AFP and GPC3, but it represents fetal gut differentiation in gastric carcinoma. SALL4 is a sensitive marker for AFP-producing gastric carcinoma and is especially useful to distinguish hepatoid gastric carcinoma from hepatocellular carcinoma.

Immunohistochemical analysis of MDM2 and CDK4 distinguishes low-grade osteosarcoma from benign mimics.

Parosteal osteosarcoma and low-grade central osteosarcoma are two types of low-grade osteosarcoma that show similar clinical behaviors, histological features, and genetic background (ie, amplified sequences of 12q13–15, including MDM2 and CDK4). Low-grade osteosarcoma is often confused with benign lesions, and ancillary techniques to enhance diagnostic accuracy have been awaited. This study explores the use of MDM2 and CDK4 immunohistochemistry for the histological diagnosis of low-grade osteosarcoma. We studied 23 cases of low-grade osteosarcoma from 21 patients (parosteal osteosarcoma (n=14), low-grade central osteosarcoma (n=9)) and 40 cases of benign histological mimics (myositis ossificans (n=11), fibrous dysplasia (n=14), osteochondroma (n=6), desmoplastic fibroma (n=1), florid reactive periostitis (n=4), Noras lesion (n=3), and turret exostosis (n=1)). Low-grade osteosarcoma labeled for MDM2 in 16 cases (70%) and for CDK4 in 20 cases (87%). All low-grade osteosarcomas expressed one or both markers (100%), with 13 cases (57%) expressing both. Staining pattern was diffuse in most cases, and the majority expressed moderate or strong intensity for either antibody. MDM2/CDK4 immunostaining was shown irrespective of low-grade osteosarcoma histological subtype. In contrast, only 1 Noras lesion out of the 40 miscellaneous benign processes showed immunoreactivity for MDM2 or CDK4. The combination of these two markers thus shows 100% sensitivity and 97.5% specificity for the diagnosis of low-grade osteosarcoma. MDM2 and CDK4 immunostains therefore reliably distinguish low-grade osteosarcoma from benign histological mimics, and their combination may serve as a useful adjunct in this difficult differential diagnosis.

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.

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.