Association of Dietary Antioxidant Vitamin Intake and Gastric Cancer Risk According to Smoking Status and Histological Subtypes of Gastric Cancer: A Case-Control Study in Korea.

Smoking is a risk factor for gastric cancer (GC) and causes oxidative stress. Antioxidant vitamins may protect against oxidative stress. This study aimed to determine the association between dietary antioxidant vitamin intake and GC risk according to smoking status and the histological subtype. This case-control study included 286 pairs of patients with GC and controls aged 20-79 years enrolled at two hospitals from 2002 to 2006, matched by age (± 2 years), sex, hospital, and participation period (± 1 years). Dietary information was collected using a quantitative food frequency questionnaire (FFQ). When stratified by smoking status, increased intake of vitamin C (OR = 0.38; 95% CI = 0.17-0.84 for highest vs. lowest; P for trend = 0.033) and folate (OR = 0.28; 95% CI = 0.12-0.64 for highest vs. lowest; P for trend = 0.003) decreased GC risk in nonsmokers. Vitamin C (P for interaction = 0.043) and folate (P for interaction =0.015) levels were significantly associated with smoking status. Similar results were observed in nonsmokers with diffuse and mixed types of GC, but not in those with intestinal type of GC. Therefore, we found an inverse association between higher intake of dietary vitamin C and folate with the risk of GC among nonsmokers. These protective associations were strong in nonsmokers with diffuse and mixed types of GC.

The Associations of Dietary Intake of High Sodium and Low Zinc with Gastric Cancer Mortality: A Prospective Cohort Study in Korea.

Sodium and zinc display opposite effects on immune cells, such as regulatory T cells (Tregs) and T helper 17 cells (Th17), resulting in an altered immune response. Immune cells have a pivotal role in regulating tumor progression, which may affect gastric cancer (GC) mortality. Thus, this cohort study investigated the associations between the combination of sodium and zinc intake and GC mortality and whether these associations differ by histological type by following up deaths of GC cases in Korea. A total of 490 patients with GC were enrolled between 2002 and 2006. Survival or death was prospectively followed up until December 31, 2016. Finally, 300 patients with the two main histological types of GC were included; 99 GC deaths occurred during a median follow-up period of 7.1 years. Patients with high sodium and low zinc intake had a significantly higher GC mortality than those with low sodium and high zinc intake (hazard ratio [HR], 2.07; 95% confidence interval [CI], 1.09-3.93). However, no significant association was found between the histological types of GC. In conclusion, we found that high sodium and low zinc intake may worsen the survival rate of patients with GC.

Sex-dependent associations between MAP3K1 gene polymorphisms and soy products with the gastric cancer risk in Korea: a case-control study.

BACKGROUND/OBJECTIVES: The hormone-dependent effect of MAP3K1 gene polymorphisms may explain sex-specific differences in gastric cancer (GC) risk. Phytoestrogens have been shown to interact with this genetic factor. Here, we investigated the association between MAP3K1 gene polymorphisms and GC risk by sex and whether these associations differ depending on soy products intake. METHODS: Participants aged 20-79 years were recruited from two hospitals between December 2002 and September 2006. In all, 440 cases and 485 controls were recruited, among, 246 pairs of cases and controls, matched by sex, age (± 5 years), study admission period (± 1 years), and hospital, were included for the analysis. RESULTS: In dominant model, men with the A allele of rs252902 showed significantly increased GC risk (odd ratio; OR=2.19, 95% confidence interval; CI=1.31-3.64) compared to GG homozygotes. When stratified by intake of soy products, men with the A allele of rs252902 and low intake of soy products showed significantly higher GC risk (OR=3.29, 95% CI=1.55-6.78) than that in GG homozygotes. CONCLUSIONS: Men with the risk allele of MAP3K1 had a significantly increased GC risk compared to GG homozygotes; this trend was more pronounced in those with low intake of soy products.

Methylation of the CDX2 promoter in Helicobacter pylori-infected gastric mucosa increases with age and its rapid demethylation in gastric tumors is associated with upregulated gene expression.

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.

ONECUT2 upregulation is associated with CpG hypomethylation at promoter-proximal DNA in gastric cancer and triggers ACSL5.

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.

LAD1 expression is associated with the metastatic potential of colorectal cancer cells.

BACKGROUND: Anchoring filament protein ladinin-1 (LAD1) was related to the aggressive progression of breast, lung, laryngeal and thyroid cancers. However, the association of LAD1 with colorectal cancer remained unknown. Here, to determine the relationship of LAD1 with colorectal cancer progression, we explored the effect of LAD1 loss on the malignant features of colorectal cancer cells. METHODS: We constructed LAD1-depleted cell lines and examined the effect of LAD1 deficiency on the phenotypic and molecular features of colorectal cancer cells in vitro. The function of LAD1 in metastasis in vivo was examined by establishing a spleen-to-liver metastasis mouse model. LAD1 protein expression in colorectal cancer patient specimens was assessed by immunohistochemistry of tumor microarrays. RESULTS: We found that LAD1 was abundant in most colorectal cancer cells. In addition, high expression of LAD1 significantly correlated with poor patient outcome. LAD1 depletion inhibited the migration and invasion of two different colorectal cancer cell lines, SW620 and Caco-2, without affecting their proliferation. In addition, LAD1 loss led to defects in liver metastasis of SW620 cells in the mouse model. Immunohistochemistry of colorectal cancer tissues revealed LAD1 enrichment in metastatic tissues compared to that in primary tumor and normal tissues. CONCLUSION: These results suggest that LAD1 expression is associated with the metastatic progression of colorectal cancer by promoting the migration and invasion of cancer cells.

Identification of a molecular signature of prognostic subtypes in diffuse-type gastric cancer.

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.

Subepithelial Spread of Early Gastric Signet Ring Cell Carcinoma: How Far They Can Reach?

INTRODUCTION: Although signet ring cell carcinoma (SRC) is a poorly differentiated cancer subtype, recent studies suggest that endoscopic resection can be applied in small, mucosal early gastric SRC. However, other studies report frequent positive lines at the lateral resection margin after endoscopic treatment. Subepithelial spread beneath normal mucosa can exist in SRC, and such lesions may be the cause of positive margins after endoscopic resection. Thus, we conducted a retrospective study in order to evaluate the significance of subepithelial spread in early gastric SRC. METHOD: Medical records of early gastric SRC patients who underwent surgery or endoscopic resection from January 2011 to December 2016 at a single tertiary hospital (Daejeon, South Korea) were reviewed to examine subepithelial spread and clinical datum. Two expert pathologists reviewed all pathologic specimens, and only patients showing a pure SRC component were included. RESULTS: Eighty-six patients were initially enrolled, and subepithelial spread existed in 62 patients (72.1%). The mean distance of subepithelial spread was 1,132.1 µm, and the maximal distance was 6,000 µm. Only discoloration was significantly associated with the presence of a subepithelial spread (p < 0.05, χ2 test, and logistic regression test). Distance of subepithelial spread did not correlate with total lesion size. CONCLUSION: Subepithelial spread of early gastric SRC occurs frequently and can reach up to 6 mm. Lesion discoloration may be associated with the presence of subepithelial spread. Our results suggest that careful decision of the margin is needed when performing endoscopic resection of early gastric SRC.

Sporadic Early-Onset Diffuse Gastric Cancers Have High Frequency of Somatic CDH1 Alterations, but Low Frequency of Somatic RHOA Mutations Compared With Late-Onset Cancers.

BACKGROUND & AIMS: Early-onset gastric cancer, which develops in patients younger than most gastric cancers, is usually detected at advanced stages, has diffuse histologic features, and occurs more frequently in women. We investigated somatic genomic alterations associated with the unique characteristics of sporadic diffuse gastric cancers (DGCs) from younger patients. METHODS: We conducted whole exome and RNA sequence analyses of 80 resected DGC samples from patients 45 years old or younger in Korea. Patients with pathogenic germline mutations in CDH1, TP53, and ATM were excluded from the onset of this analysis, given our focus on somatic alterations. We used MutSig2CV to evaluate the significance of mutated genes. We recruited 29 additional early-onset Korean DGC samples and performed SNP6.0 array and targeted sequencing analyses of these 109 early-onset DGC samples (54.1% female, median age, 38 years). We compared the SNP6.0 array and targeted sequencing data of the 109 early-onset DGC samples with those from diffuse-type stomach tumor samples collected from 115 patients in Korea who were 46 years or older (late onset) at the time of diagnosis (controls; 29.6% female, median age, 67 years). We compared patient survival times among tumors from different subgroups and with different somatic mutations. We performed gene silencing of RHOA or CDH1 in DGC cells with small interfering RNAs for cell-based assays. RESULTS: We identified somatic mutations in the following genes in a significant number of early-onset DGCs: the cadherin 1 gene (CDH1), TP53, ARID1A, KRAS, PIK3CA, ERBB3, TGFBR1, FBXW7, RHOA, and MAP2K1. None of 109 early-onset DGC cases had pathogenic germline CDH1 mutations. A higher proportion of early-onset DGCs had mutations in CDH1 (42.2%) or TGFBR1 (7.3%) compared with control DGCs (17.4% and 0.9%, respectively) (P < .001 and P = .014 for CDH1 and TGFBR1, respectively). In contrast, a smaller proportion of early-onset DGCs contained mutations in RHOA (9.2%) than control DGCs (19.1%) (P = .033). Late-onset DGCs in The Cancer Genome Atlas also contained less frequent mutations in CDH1 and TGFBR1 and more frequent RHOA mutations, compared with early-onset DGCs. Early-onset DGCs from women contained significantly more mutations in CDH1 or TGFBR1 than early-onset DGCs from men. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times in patients with early-onset DGCs (hazard ratio, 3.4; 95% confidence interval, 1.5-7.7). RHOA activity was reduced by an R5W substitution-the RHOA mutation most frequently detected in early-onset DGCs. Silencing of CDH1, but not RHOA, increased migratory activity of DGC cells. CONCLUSIONS: In an integrative genomic analysis, we found higher proportions of early-onset DGCs to contain somatic mutations in CDH1 or TGFBR1 compared with late-onset DGCs. However, a smaller proportion of early-onset DGCs contained somatic mutations in RHOA than late-onset DGCs. CDH1 alterations, but not RHOA mutations, were associated with shorter survival times of patients, which might account for the aggressive clinical course of early-onset gastric cancer. Female predominance in early-onset gastric cancer may be related to relatively high rates of somatic CDH1 and TGFBR1 mutations in this population.

Gene polymorphisms in the ornithine decarboxylase-polyamine pathway modify gastric cancer risk by interaction with isoflavone concentrations.

BACKGROUND: The study aimed to examine the association between genes encoding molecules in the ornithine decarboxylase (ODC)-polyamine pathway (ODC1, AMD1, NQO1, NOS2A, and OAZ2) and gastric cancer risk and whether the gene-phytoestrogen interaction modifies gastric cancer risk. METHODS: Among 76 gastric cancer cases and their 1:4 matched controls within the Korean Multi-center Cancer Cohort, a total of 30 SNPs in five genes involved in the ODC pathway were primarily analyzed. The second-stage genotyping in 388 matched case-control sets was conducted to reevaluate the significant SNPs interacting with phytoestrogens during the primary analysis. The summary odds ratios (ORs) [95 % confidence intervals (CIs)] for gastric cancer were estimated. Interaction effects between the SNPs and plasma concentrations of phytoestrogens (genistein, daidzein, equol, and enterolactone) were evaluated. RESULTS: In the pooled analysis, NQO1 rs1800566 showed significant genetic effects on gastric cancer without heterogeneity [OR 0.83 (95 % CI 0.70-0.995)] and a greater decreased risk at high genistein/daidzein levels [OR 0.36 (95 % CI 0.15-0.90) and OR 0.26 (95 % CI 0.10-0.64), respectively; p interaction < 0.05]. Risk alleles of AMD1 rs1279599, AMD1 rs7768897, and OAZ2 rs7403751 had a significant gene-phytoestrogen (genistein and daidzein) interaction effect to modify the development of gastric cancer. They had an increased gastric cancer risk at low isoflavone levels, but a decreased risk at high isoflavone levels (p interaction < 0.01). CONCLUSIONS: Our findings suggest that common variants in the genes involved in the ODC pathway may contribute to the risk of gastric cancer possibly by modulating ODC polyamine biosynthesis or by interaction between isoflavones and NQO1, OAZ2, and AMD1.

Integrative genomics analysis reveals the multilevel dysregulation and oncogenic characteristics of TEAD4 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.

Differences in gastric mucosal microbiota profiling in patients with chronic gastritis, intestinal metaplasia, and gastric cancer using pyrosequencing methods.

BACKGROUND: Helicobacter pylori (H. pylori) infection plays an important role in the early stage of cancer development. However, various bacteria that promote the synthesis of reactive oxygen and nitrogen species may be involved in the later stages. We aimed to determine the microbial composition of gastric mucosa from the patients with chronic gastritis, intestinal metaplasia, and gastric cancer using 454 GS FLX Titanium. METHODS: Gastric mucosal biopsy samples were collected from 31 patients during endoscopy. After the extraction of genomic DNA, variable region V5 of the 16S rRNA gene was amplified. PCR products were sequenced using 454 high-throughput sequencer. The composition, diversity, and richness of microbial communities were compared between three groups. RESULTS: The composition of H. pylori-containing Epsilonproteobacteria class appeared to be the most prevalent, but the relative increase in the Bacilli class in the gastric cancer group was noticed, resulting in a significant difference compared with the chronic gastritis group. By analyzing the Helicobacter-dominant group at a family level, the relative abundance of Helicobacteraceae family was significantly lower in the gastric cancer group compared with chronic gastritis and intestinal metaplasia groups, while the relative abundance of Streptococcaceae family significantly increased. In a UPGMA clustering of Helicobacter-dominant group based on UniFrac distance, the chronic gastritis group and gastric cancer group were clearly separated, while the intestinal metaplasia group was distributed in between the two groups. The evenness and diversity of gastric microbiota in the gastric cancer group was increased compared with other groups. CONCLUSIONS: In Helicobacter predominant patients, the microbial compositions of gastric mucosa from gastric cancer patients are significantly different to chronic gastritis and intestinal metaplasia patients. These alterations of gastric microbial composition may play an important, as-yet-undetermined role in gastric carcinogenesis of Helicobacter predominant patients.

Soluble c-Met protein as a susceptible biomarker for gastric cancer risk: A nested case-control study within the Korean Multicenter Cancer Cohort.

This study was conducted to evaluate the relevance of the soluble form of c-Met protein, a truncated form of the c-Met membrane receptor involved in the CagA pathway, as a potential biomarker for gastric cancer. Among 290 gastric cancer case-control sets selected from the Korean Multicenter Cancer Cohort, the plasma concentrations of soluble c-Met protein were measured with enzyme-linked immunosorbent assays. Using analysis of variance and covariance models with age, sex, smoking, Helicobacter pylori infection, and CagA seropositivity, the mean concentrations of soluble c-Met protein between cases and controls were compared. To evaluate the association between gastric cancer and a c-Met protein level, odds ratios and 95% confidence intervals were estimated using conditional logistic regression models. Interactions between CagA-related genes and the soluble c-Met protein concentration were also investigated. The overall median plasma concentration of soluble c-Met among cases was significantly lower than those of controls (1.390 vs. 1.610 ng/mL, p < 0.0001). Closer to the onset of gastric cancer, the soluble c-Met protein level decreased linearly in a time-dependent manner (p for trend = 0.0002). The combined effects between the CagA-related genes and the soluble c-Met protein concentration significantly intensified risks for gastric cancer. Restricted analyses including cases that had been diagnosed within 1 year after entering the cohort had a fair degree of ability (area under the receiver operating characteristic curve of 0.73-0.77) to discriminate gastric cancer cases from normal controls. Our findings demonstrate the potential of the soluble form of c-Met protein as a novel biomarker for gastric cancer. The beneficial effects of a high soluble c-Met concentration in human plasma are strongly supported.

Aberrant upregulation of ASCL2 by promoter demethylation promotes the growth and resistance to 5-fluorouracil of gastric cancer cells.

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.

Association between soy products, fruits, vegetables, and dairy products and gastric cancer risk in Helicobacter pylori-infected subjects: a case-control study in Korea.

BACKGROUND/OBJECTIVES: Consumption of certain protective foods may help inhibit Helicobacter pylori (H. pylori) associated gastric pathologies. However, studies conducted to assess the efficacy of protective foods in H. pylori-infected subjects are either limited or inconsistent. This study evaluated the association of individual or a combination of protective foods on the incidence of gastric cancer (GC) in H. pylori-positive subjects through a case-control study. MATERIALS/METHODS: Subjects aged 20-79 years were selected from 2 hospitals between December 2002 and September 2006. In total, 134 patients and 212 controls tested positive for H. pylori infection. Among these, we included 82 pairs of cases and controls matched by sex, age (± 5 years), enrollment period (± 1 years), and hospital. RESULTS: A higher intake of soy products was associated with a significantly lower risk of GC than a lower intake of soy products (odds ratio [OR] = 0.37, 95% confidence interval [CI] = 0.14-0.96). Additionally, a higher fruit intake resulted in a significantly lower risk of GC than a lower fruit intake (OR = 0.35, 95% CI = 0.13-0.94). A combination of food groups was evaluated, and a lower risk of GC was observed with a high intake of both soy products and fruits (OR = 0.20, 95% CI = 0.06-0.67), high intake of soy and dairy products (OR = 0.28, 95% CI = 0.10-0.78) and high intake of fruits and dairy products (OR = 0.28, 95% CI = 0.09-0.83). CONCLUSIONS: A high intake of soy products or fruits was associated with a lower risk of GC. A combination of soy products or fruits with dairy products was associated with a lower risk of GC. A balanced intake of soy products, fruits, and dairy products may help reduce GC risk.

Epigenetic Activation of Tensin 4 Promotes Gastric Cancer Progression.

Gastric cancer (GC) is a complex disease influenced by multiple genetic and epigenetic factors. Chronic inflammation caused by Helicobacter pylori infection and dietary risk factors can result in the accumulation of aberrant DNA methylation in gastric mucosa, which promotes GC development. Tensin 4 (TNS4), a member of the Tensin family of proteins, is localized to focal adhesion sites, which connect the extracellular matrix and cytoskeletal network. We identified upregulation of TNS4 in GC using quantitative reverse transcription PCR with 174 paired samples of GC tumors and adjacent normal tissues. Transcriptional activation of TNS4 occurred even during the early stage of tumor development. TNS4 depletion in GC cell lines that expressed high to moderate levels of TNS4, i.e., SNU-601, KATO III, and MKN74, reduced cell proliferation and migration, whereas ectopic expression of TNS4 in those lines that expressed lower levels of TNS4, i.e., SNU-638, MKN1, and MKN45 increased colony formation and cell migration. The promoter region of TNS4 was hypomethylated in GC cell lines that showed upregulation of TNS4. We also found a significant negative correlation between TNS4 expression and CpG methylation in 250 GC tumors based on The Cancer Genome Atlas (TCGA) data. This study elucidates the epigenetic mechanism of TNS4 activation and functional roles of TNS4 in GC development and progression and suggests a possible approach for future GC treatments.

Clinical Manifestations and Genotype of Primary Ciliary Dyskinesia Diagnosed in Korea: Multicenter Study.

PURPOSE: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder that leads to secondary ciliary dysfunction. PCD is a rare disease, and data on it are limited in Korea. This study systematically evaluated the clinical symptoms, diagnostic characteristics, and treatment modalities of pediatric PCD in Korea. METHODS: This Korean nationwide, multicenter study, conducted between January 2000 and August 2022, reviewed the medical records of pediatric patients diagnosed with PCD. Prospective studies have been added to determine whether additional genetic testing is warranted in some patients. RESULTS: Overall, 41 patients were diagnosed with PCD in 15 medical institutions. The mean age at diagnosis was 11.8 ± 5.4 years (range: 0.5 months-18.9 years). Most patients (40/41) were born full term, 15 (36.6%) had neonatal respiratory symptoms, and 12 (29.3%) had a history of admission to the neonatal intensive care unit. The most common complaint (58.5%) was chronic nasal symptoms. Thirty-three patients were diagnosed with transmission electron microscopy (TEM) and 12 patients by genetic studies. TEM mostly identified outer dynein arm defects (alone or combined with inner dynein arm defects, n = 17). The genes with the highest mutation rates were DNAH5 (3 cases) and DNAAF1 (3 cases). Rare genotypes (RPGR, HYDIN, NME5) were found as well. Chest computed tomography revealed bronchiectasis in 33 out of 41 patients. Among them, 15 patients had a PrImary CiliAry DyskinesiA Rule score of over 5 points. CONCLUSIONS: To our knowledge, this is the first multicenter study to report the clinical characteristics, diagnostic methods, and genotypes of PCD in Korea. These results can be used as basic data for further PCD research.

Epigenetic alteration of CCDC67 and its tumor suppressor function in gastric cancer.

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.

Regulation of glycogen synthase kinase-3 by thymosin beta-4 is associated with gastric cancer cell migration.

Thymosin beta-4 (Tß4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tß4 and it is associated with Tß4-mediated migration in gastric cancer cells. Various expression level of Tß4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tß4. Cell migration was higher in SNU668 with a higher expression level of Tß4 than that in SNU638 with a lower Tß4. Although the level of phosphorylated(p)-GSK-3α (inactive), ß-catenin, E-cadherin and E-cadherin:ß-catenin complex was relatively higher, p-GSK-3ß (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/ß inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:ß-catenin complex formation through a decrease in ß-catenin and E-cadherin. Expression level of GSK-3α/ß, ß-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tß4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tß4, respectively. E-cadherin expression in SNU638 cells was decreased by ß-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, ß-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, ß-catenin and E-cadherin could be negatively regulated by Tß4-induced ERK phosphorylation. It suggests that Tß4 could be a novel regulator to control Wnt signaling pathways.

Pyruvate kinase M2 promotes the growth of gastric cancer cells via regulation of Bcl-xL expression at transcriptional level.

PKM2 is an isoenzyme of the glycolytic enzyme pyruvate kinase that promotes aerobic glycolysis. Here, we describe an important role for PKM2 in regulating the survival of gastric cancer (GC) cells. We showed that PKM2 was overexpressed in gastric tumor tissues compared to normal tissues and its expression level was associated with poor survival of gastric cancer patients. We also showed that PKM2 affected cell survival by regulating Bcl-xL at the transcriptional level. PKM2 knockdown partially affected the stability of NF-kB subunit p65, suggesting that post-translational regulation of p65 by PKM2 is one of plausible mechanisms for the increased cell growth. Therefore, PKM2 may function as an upstream molecule that regulates p65 function and thus enhances the growth of tumor cells.