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Pathological changes in the epigenetic landscape of chromatin are hallmarks of cancer. Our previous study showed that global methylation of promoters may increase or decrease during the transition from gastric mucosa to intestinal metaplasia (IM) to gastric cancer (GC). Here, CpG hypomethylation of the serine/threonine kinase STK31 promoter in IM and GC was detected in a reduced representation bisulfite sequencing database. STK31 hypomethylation, which resulted in its upregulation in 120 cases of primary GC, was confirmed. Using public genome‑wide histone modification data, upregulation of STK31 promoter activity was detected in primary GC but not in normal mucosae, suggesting that STK31 may be repressed in gastric mucosa but activated in GC as a consequence of hypomethylation‑associated chromatin remodeling. STK31 knockdown suppressed the proliferation, colony formation and migration activities of GC cells in vitro , whereas stable overexpression of STK31 promoted the proliferation, colony formation, and migration activities of GC cells in vitro and tumorigenesis in nude mice. Patients with GC in which STK31 was upregulated exhibited significantly shorter survival times in a combined cohort. Thus, activation of STK31 by chromatin remodeling may be associated with gastric carcinogenesis and also may help predict GC prognosis.

Pathological changes in the epigenetic landscape of chromatin are hallmarks of cancer. The caudal-type homeobox gene CDX2 is not expressed in normal gastric epithelia but rather in adult intestinal epithelia, and it is overexpressed in intestinal metaplasia (IM). However, it remains unclear how CDX2 transcription is suppressed in normal gastric epithelial cells and overexpressed in IM. Here, we demonstrate that methylation of the CDX2 promoter increases with age in Helicobacter pylori-positive, noncancerous gastric tissue, whereas the promoter is demethylated in paired gastric tumors in which CDX2 is upregulated. Moreover, we also found that the CDX2 promoter is demethylated in IM as well as gastric tumor. Immunohistochemistry revealed that CDX2 is present in foci of parts of the gastric mucosae but highly expressed in IM as well as in gastric tumors, suggesting that the elevated level of CDX2 in IM and gastric tumors may be attributable to promoter demethylation. Our data suggest that CDX2 repression may be associated with promoter methylation in noncancerous H. pylori-positive mucosa but its upregulation might be attributable to increased promoter activity mediated by chromatin remodeling during gastric carcinogenesis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Many studies have focused on global hypomethylation or hypermethylation of tumor suppressor genes, but less is known about the impact of promoter hypomethylation of oncogenes. We previously showed that promoter methylation may gradually increase or decrease during the transition from gastric mucosa (GM) to intestinal metaplasia (IM) to gastric cancer (GC). In our study, we focused on regional CpG hypomethylation of the promoter-proximal DNA of the transcription factor ONECUT2 (OC2) in IM and GC cells. We validated the hypomethylation of promoter-proximal DNA of OC2 in 160 primary GCs, in which methylation level correlated negatively with OC2 mRNA level. IM and GC cells stained positively for OC2, whereas GM cells did not. Stable transfection of OC2 in GC cells promoted colony formation, cell migration, invasion and proliferation. Moreover, OC2 knockdown with a short hairpin RNA suppressed tumorigenesis in nude mice. In addition, chromatin immunoprecipitation coupled with DNA sequencing and RNA-seq analyses revealed that OC2 triggered ACSL5, which is strongly expressed in IM of the stomach but not in GM, indicating that OC2 and ACSL5 are early-stage biomarkers for GC. We also observed a high correlation between the levels of OC2 and ACSL5 mRNAs in the GENT database These results suggest that epigenetic alteration of OC2 upregulates its expression, which then activates ACSL5; thus, OC2 is induced in IM by epigenetic alteration and triggers ACSL5 expression, and thus OC2 and ACSL5 may cooperatively promote intestinal differentiation and GC progression., (© 2020 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Background: Although recent advances in high-throughput technology have provided many insights into gastric cancer (GC), few reliable biomarkers for diffuse-type GC have been identified. Here, we aim to identify a prognostic and predictive signature of diffuse-type GC heterogeneity., Methods: We analyzed RNA-seq-based transcriptome data to identify a molecular signature in 150 gastric tissue samples including 107 diffuse-type GCs. The predictive value of the signature was verified using other diffuse-type GC samples in three independent cohorts (n = 466). Log-rank and Cox regression analyses were used to estimate the association between the signature and prognosis. The signature was also characterized by somatic variant analyses and tissue microarray analysis between diffuse-type GC subtypes., Results: Transcriptomic profiling of RNA-seq data identified a signature which revealed distinct subtypes of diffuse-type GC: the intestinal-like (INT) and core diffuse-type (COD) subtypes. The signature showed high predictability and independent clinical utility in diffuse-type GC prognosis in other patient cohorts (HR 2.058, 95% CI 1.53-2.77, P = 1.76 × 10 -6 ). Integrative mutational and gene expression analyses demonstrated that the COD subtype was responsive to chemotherapy, whereas the INT subtype was responsive to immunotherapy with an immune checkpoint inhibitor (ICI). Tissue microarray analysis showed the practical utility of IGF1 and NXPE2 for predicting diffuse-type GC heterogeneity., Conclusions: We present a molecular signature that can identify diffuse-type GC patients who display different clinical behaviors as well as responses to chemotherapy or ICI treatment.

Gastric cancer is a heterogeneous cancer, making treatment responses difficult to predict. Here we show that we identify two distinct molecular subtypes, mesenchymal phenotype (MP) and epithelial phenotype (EP), by analyzing genomic and proteomic data. Molecularly, MP subtype tumors show high genomic integrity characterized by low mutation rates and microsatellite stability, whereas EP subtype tumors show low genomic integrity. Clinically, the MP subtype is associated with markedly poor survival and resistance to standard chemotherapy, whereas the EP subtype is associated with better survival rates and sensitivity to chemotherapy. Integrative analysis shows that signaling pathways driving epithelial-to-mesenchymal transition and insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1R) pathway are highly activated in MP subtype tumors. Importantly, MP subtype cancer cells are more sensitive to inhibition of IGF1/IGF1R pathway than EP subtype. Detailed characterization of these two subtypes could identify novel therapeutic targets and useful biomarkers for prognosis and therapy response.

Peritoneal carcinomatosis accompanied by malignant ascites is a major cause of death of advanced gastric cancer (GC). To comprehensively characterize the underlying genomic events involved in GC peritoneal carcinomatosis, we analyzed whole-exome sequences of normal gastric tissues, primary tumors, and malignant ascites from eight GC patients. We identified a unique mutational signature biased toward C-to-A substitutions in malignant ascites. In contrast, the patients who received treatment of adjuvant chemotherapy showed a high rate of C-to-T substitutions along with hypermutation in malignant ascites. Comparative analysis revealed several candidate mutations for GC peritoneal carcinomatosis: recurrent mutations in COL4A6, INTS2, and PTPN13; mutations in druggable genes including TEP1, PRKCD, BRAF, ERBB4, PIK3CA, HDAC9, FYN, FASN, BIRC2, FLT3, ROCK1, CD22, and PIK3C2B; and mutations in metastasis-associated genes including TNFSF12, L1CAM, DIAPH3, ROCK1, TGFBR1, MYO9B, NR4A1, and RHOA. Notably, gene ontology analysis revealed the significant enrichment of mutations in the Rho-ROCK signaling pathway-associated biological processes in malignant ascites. At least four of the eight patients acquired somatic mutations in the Rho-ROCK pathway components, suggesting the possible relevance of this pathway to GC peritoneal carcinomatosis. These results provide a genome-wide molecular understanding of GC peritoneal carcinomatosis and its clinical implications, thereby facilitating the development of effective therapeutics.

Recent evidence has shown that the level of 5-hydroxymethylcytosine (5 hmC) in chromosomal DNA is aberrantly decreased in a variety of cancers, but whether this decrease is a cause or a consequence of tumorigenesis is unclear. Here we show that, in gastric cancers, the 5 hmC decrease correlates with a decrease in ten-eleven translocation 1 (TET1) expression, which is strongly associated with metastasis and poor survival in patients with gastric cancer. In gastric cancer cells, TET1-targeted siRNA induced a decrease in 5 hmC, whereas TET1 overexpression induced an increase in 5 hmC and reduced cell proliferation, thus correlating decreased 5 hmC with gastric carcinogenesis. We also report the epigenetic signatures responsible for regulating TET1 transcription. Methyl-CpG Binding Domain Sequencing and Reduced Representation Bisulfite Sequencing identified unique CpG methylation signatures at the CpG island 3'-shore region located 1.3 kb from the transcription start site of TET1 in gastric tumor cells but not in normal mucosa. The luciferase activity of constructs with a methylated 3'-shore sequence was greatly decreased compared with that of an unmethylated sequence in transformed gastric cancer cells. In gastric cancer cells, dense CpG methylation in the 3'-shore was strongly associated with TET1 silencing and bivalent histone marks. Thus, a decrease in 5 hmC may be a cause of gastric tumorigenesis owing to a decrease in TET1 expression through DNA methylation coupled with bivalent marks in the 3'-shore of TET1.

nc886 is a 101 nucleotide long non-coding RNA that has been designated as a precursor microRNA or a vault RNA based upon it sequence. nc886 has also been suggested to be a tumor suppressor, mainly inferred by its expression pattern as well as its genomic location at human chromosome 5q31, a locus for a tumor suppressor gene(s). However, legitimate data based on nc886's correct identity for its functional cellular roles as a tumor suppressor have not been provided yet. Here we have investigated nc886 in gastric cancer where its expression is suppressed due to CpG DNA hypermethylation at its promoter region in a cohort of paired tumor/normal tissues from 88 gastric cancer patients. CpG hypermethylation of nc886 and thus its diminished expression is significantly associated with poor survival in these cancer patients. nc886 inhibits cell proliferation when ectopically expressed in gastric cancer cells. nc886's tumor suppressive role is corroborated by the induction of well-known oncogenes such as FOS, NF-κB, and MYC upon its knockdown. All these activities of nc886 are undoubtedly independent of mature microRNA or vault RNA. Our data indicate that nc886 is a putative tumor suppressor and could potentially be used as a diagnostic marker in gastric cancer.

Tumorigenesis is a consequence of failures of multistep defense mechanisms against deleterious perturbations that occur at the genomic, epigenomic, transcriptomic and proteomic levels. To uncover previously unrecognized genes that undergo multilevel perturbations in gastric cancer (GC), we integrated epigenomic and transcriptomic approaches using two recently developed tools: MENT and GENT. This integrative analysis revealed that nine Hippo pathway-related genes, including components [FAT, JUB, LATS2, TEA domain family member 4 (TEAD4) and Yes-associated protein 1 (YAP1)] and targets (CRIM1, CYR61, CTGF and ITGB2), are concurrently hypomethylated at promoter CpG sites and overexpressed in GC tissues. In particular, TEAD4, a link between Hippo pathway components and targets, was significantly hypomethylated at CpG site cg21637033 (P = 3.8 × 10(-) (20)) and overexpressed (P = 5.2 × 10(-) (10)) in 108 Korean GC tissues compared with the normal counterparts. A reduced level of methylation at the TEAD4 promoter was significantly associated with poor outcomes, including large tumor size, high-grade tumors and low survival rates. Compared with normal tissues, the TEAD4 protein was more frequently found in the nuclei of tumor cells along with YAP1 in 53 GC patients, demonstrating the posttranslational activation of this protein. Moreover, the knockdown of TEAD4 resulted in the reduced growth of GC cells both in vitro and in vivo. Finally, chromatin immunoprecipitation-sequencing and microarray analysis revealed the oncogenic properties of TEAD4 and its novel targets (ADM, ANG, ARID5B, CALD1, EDN2, FSCN1 and OSR2), which are involved in cell proliferation and migration. In conclusion, the multilevel perturbations of TEAD4 at epigenetic, transcriptional and posttranslational levels may contribute to GC development.

Achaete scute-like 2 (ASCL2), a basic helix-loop-helix transcription factor, plays an essential role in the maintenance of adult intestinal stem cells. However, the function of ASCL2 in gastric cancer (GC) is poorly understood. Therefore, we investigated the roles and regulatory transcription mechanisms of ASCL2 in GC. Gene expression and methylation data analysis showed that ASCL2 was upregulated and hypomethylated in GC tissues. Using real-time RT-PCR and pyrosequencing analysis, we confirmed that ASCL2 was overexpressed and hypomethylated in GC tissues compared to adjacent normal tissues. We then investigated the mechanisms underlying the aberrant expression of ASCL2 in GC and found that treatment with a methylation inhibitor induced ASCL2 expression in GC cell lines. MBD-sequencing assay also revealed hypermethylation of the promoter region of ASCL2 in GC cell lines, which barely expressed the ASCL2 gene. Furthermore, ASCL2 expression levels were inversely correlated with GC patient survival. Ectopic overexpression of ASCL2 showed that ASCL2 increased cell growth and promoted resistance to 5-fluorouracil in GC cells. These results suggest that ASCL2 might play an important role in gastric tumor growth and chemoresistance, and could be a useful prognostic marker for GC patients., (© 2012 Japanese Cancer Association.)

In this study, the promoter of the gene coiled-coil domain-containing 67 (CCDC67) was found to be frequently methylated in gastric cancer cell lines and in primary gastric tumors, as examined by restriction landmark genomic scanning. In addition, CCDC67 expression was down-regulated in 72.7% of gastric cancer cell lines tested. In most cases, gene down-regulation was associated with CpG hypermethylation in the CCDC67 promoter. Treatment with 5-aza-2'-deoxycytidine and/or trichostatin A restored CCDC67 expression in down-regulated cell lines. Pyrosequencing analysis of 150 paired primary gastric cancer samples revealed that promoter CpG methylation was increased in 74% of tested tumors compared with paired adjacent normal tissues, and this hypermethylation correlated significantly with down-regulation of CCDC67. CCDC67 protein was localized to the cell membrane by immunocytochemistry. Stable transfection of a CCDC67 gene in one gastric cancer cell line inhibited adhesion-dependent and -independent colony formation, and CCDC67 expression suppressed tumorigenesis in nude mice. We suggest that CCDC67 is a putative tumor suppressor gene that is silenced in gastric cancers by promoter CpG methylation and that it may play an important role in cell signaling and migration related to tumorigenesis.

MicroRNAs act as negative regulators of gene expression, and the altered expression of microRNAs by epigenetic mechanisms is strongly implicated in carcinogenesis. Here we report that the microRNA-10b gene (miR-10b) was silenced in gastric cancer cells by promoter methylation. In this study, using a methylation array and bisulfate pyrosequencing analysis, we found that miR-10b promoter CpGs were heavily methylated in gastric cancers. Clinicopathologic data showed that miR-10b methylation increased with patient age and occurred significantly more frequently in intestinal-type (28/44, 64%) than in diffuse-type (22/56, 39%) gastric cancers (P = 0.016). In addition, miR-10b methylation was also associated with an increase in expression of the oncogene that encodes microtubule-associated protein, RP/EB family, member 1 (MAPRE1; P = 0.004), which was identified as a potential miR-10b target. After 5-aza-2'-deoxycytidine treatment of gastric cancer cells, miR-10b methylation was significantly decreased, and expression of miR-10b and HOXD4, which is 1 kb downstream of miR-10b, was greatly restored. Moreover, decreased MAPRE1 expression coincided with increased miR-10b expression, suggesting that miR-10b targets MAPRE1 transcription. We also found that transfection with precursor miR-10b into gastric cancer cells dramatically decreased MAPRE1 mRNA and protein, resulting in a significant decrease in colony formation and cell growth rates. Thus, we show a tumor-suppressive role for miR-10b in gastric carcinogenesis. miR-10b methylation may be a useful molecular biomarker for assessing the risk of gastric cancer development, and modulation of miR-10b may represent a therapeutic approach for treating gastric cancer.

The LAMB3 and LAMC2 genes encode the laminin-5 β3 and γ2 chains, respectively, which are parts of laminin-5, one of the major components of the basement membrane zone. Here, we report the frequent up-regulation of LAMB3 and LAMC2 by promoter demethylation in gastric cancer. Gene expression data analysis showed that LAMB3 and LAMC2 were up-regulated in various tumor tissues. Combined analyses of DNA methylation and gene expression of both genes in gastric cancer cell lines and tissues showed that DNA hypomethylation was associated with the up-regulation of both genes. Treatment with a methylation inhibitor induced LAMB3 and LAMC2 expression in gastric cancer cell lines in which both genes were silenced. By chromatin immunoprecipitation assay, we showed the activation histone mark H3K4me3 was associated with the expression of both genes. The expression level of LAMB3 affected multiple malignant phenotypes in gastric cancer cell lines. These results suggest that epigenetic activation of LAMB3 and LAMC2 may play an important role in gastric carcinogenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)