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.

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.

Aberrant Methylation of Somatostatin Receptor 2 Gene Is Initiated in Aged Gastric Mucosa Infected with Helicobacter pylori and Consequential Gene Silencing Is Associated with Establishment of Inflammatory Microenvironment In Vitro Study.

The loss-of-function variants are thought to be associated with inflammation in the stomach. We here aimed to evaluate the extent and role of methylation at the SSTR2 promoter in inflammation and gastric tumor formation. A whole-genome bisulfite sequencing analysis revealed that the SSTR2 promoter was significantly hypermethylated in gastric tumors, dysplasia, and intestinal metaplasia compared to non-tumor tissues from patients with gastric cancer. Using public data, we confirmed SSTR2 promoter methylation in primary gastric tumors and intestinal metaplasia, and even aged gastric mucosae infected with Helicobacter pylori, suggesting that aberrant methylation is initiated in normal gastric mucosa. The loss-of-function of SSTR2 in SNU638 cell-induced cell proliferation in vitro, while stable transfection of SSTR2 in AGS and MKN74 cells inhibited cell proliferation and tumorigenesis in vitro and in vivo. As revealed by a comparison of target genes differentially expressed in these cells with hallmark molecular signatures, inflammation-related pathways were distinctly induced in SSTR2-KO SNU638 cell. By contrast, inflammation-related pathways were inhibited in AGS and MKN74 cells ectopically expressing SSTR2. Collectively, we propose that SSTR2 silencing upon promoter methylation is initiated in aged gastric mucosae infected with H. pylori and promotes the establishment of an inflammatory microenvironment via the intrinsic pathway. These findings provide novel insights into the initiation of gastric carcinogenesis.

DNA methylome and single-cell transcriptome analyses reveal CDA as a potential druggable target for ALK inhibitor-resistant lung cancer therapy.

Acquired resistance to inhibitors of anaplastic lymphoma kinase (ALK) is a major clinical challenge for ALK fusion-positive non-small-cell lung cancer (NSCLC). In the absence of secondary ALK mutations, epigenetic reprogramming is one of the main mechanisms of drug resistance, as it leads to phenotype switching that occurs during the epithelial-to-mesenchymal transition (EMT). Although drug-induced epigenetic reprogramming is believed to alter the sensitivity of cancer cells to anticancer treatments, there is still much to learn about overcoming drug resistance. In this study, we used an in vitro model of ceritinib-resistant NSCLC and employed genome-wide DNA methylation analysis in combination with single-cell (sc) RNA-seq to identify cytidine deaminase (CDA), a pyrimidine salvage pathway enzyme, as a candidate drug target. CDA was hypomethylated and upregulated in ceritinib-resistant cells. CDA-overexpressing cells were rarely but definitively detected in the naïve cell population by scRNA-seq, and their abundance was increased in the acquired-resistance population. Knockdown of CDA had antiproliferative effects on resistant cells and reversed the EMT phenotype. Treatment with epigenome-related nucleosides such as 5-formyl-2'-deoxycytidine selectively ablated CDA-overexpressing resistant cells via accumulation of DNA damage. Collectively, our data suggest that targeting CDA metabolism using epigenome-related nucleosides represents a potential new therapeutic strategy for overcoming ALK inhibitor resistance in NSCLC.

PRMT1 Is Required for the Maintenance of Mature ß-Cell Identity.

Loss of functional ß-cell mass is an essential feature of type 2 diabetes, and maintaining mature ß-cell identity is important for preserving a functional ß-cell mass. However, it is unclear how ß-cells achieve and maintain their mature identity. Here we demonstrate a novel function of protein arginine methyltransferase 1 (PRMT1) in maintaining mature ß-cell identity. Prmt1 knockout in fetal and adult ß-cells induced diabetes, which was aggravated by high-fat diet-induced metabolic stress. Deletion of Prmt1 in adult ß-cells resulted in the immediate loss of histone H4 arginine 3 asymmetric dimethylation (H4R3me2a) and the subsequent loss of ß-cell identity. The expression levels of genes involved in mature ß-cell function and identity were robustly downregulated as soon as Prmt1 deletion was induced in adult ß-cells. Chromatin immunoprecipitation sequencing and assay for transposase-accessible chromatin sequencing analyses revealed that PRMT1-dependent H4R3me2a increases chromatin accessibility at the binding sites for CCCTC-binding factor (CTCF) and ß-cell transcription factors. In addition, PRMT1-dependent open chromatin regions may show an association with the risk of diabetes in humans. Together, our results indicate that PRMT1 plays an essential role in maintaining ß-cell identity by regulating chromatin accessibility.

A prognostic index based on an eleven gene signature to predict systemic recurrences in colorectal cancer.

Approximately half of colorectal cancer (CRC) patients experience disease recurrence and metastasis, and these individuals frequently fail to respond to treatment due to their clinical and biological diversity. Here, we aimed to identify a prognostic signature consisting of a small gene group for precisely predicting CRC heterogeneity. We performed transcriptomic profiling using RNA-seq data generated from the primary tissue samples of 130 CRC patients. A prognostic index (PI) based on recurrence-associated genes was developed and validated in two larger independent CRC patient cohorts (n = 795). The association between the PI and prognosis of CRC patients was evaluated using Kaplan-Meier plots, log-rank tests, a Cox regression analysis and a RT-PCR analysis. Transcriptomic profiling in 130 CRC patients identified two distinct subtypes associated with systemic recurrence. Pathway enrichment and RT-PCR analyses revealed an eleven gene signature incorporated into the PI system, which was a significant prognostic indicator of CRC. Multivariate and subset analyses showed that PI was an independent risk factor (HR = 1.812, 95% CI = 1.342-2.448, P < 0.001) with predictive value to identify low-risk stage II patients who responded the worst to adjuvant chemotherapy. Finally, a comparative analysis with previously reported Consensus Molecular Subgroup (CMS), high-risk patients classified by the PI revealed a distinct molecular property similar to CMS4, associated with a poor prognosis. This novel PI predictor based on an eleven gene signature likely represents a surrogate diagnostic tool for identifying high-risk CRC patients and for predicting the worst responding patients for adjuvant chemotherapy.

A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells.

Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naïve vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naïve ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naïve, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naïve mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naïve mESCs from primed mESCs. Mice generated using CDy9+ cells isolated during the conversion of mouse epiblast stem cells into naïve mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naïve mESCs.

Epigenetic silencing of miR-1271 enhances MEK1 and TEAD4 expression in gastric cancer.

Epigenetic dysregulation is a major driver of tumorigenesis. To identify tumor-suppressive microRNAs repressed by DNA methylation in gastric cancer (GC), we analyzed the genome-wide DNA methylation and microRNA expression profiles of EpCAM+/CD44+ GC cells. Among the set of microRNAs screened, miR-1271 was identified as a microRNA repressed by DNA methylation in GC. Forced miR-1271 expression substantially suppressed the growth, migration, and invasion of GC cells. To identify candidate target genes and signaling pathways regulated by miR-1271, we performed RNA sequencing. Among the genes down-regulated by miR-1271, MAP2K1 (MEK1) was significantly repressed by miR-1271, and the associated ERK/MAPK signaling pathway was also inhibited. TEAD4 was also repressed by miR-1271, and the associated YAP1 signatures within genes regulated by miR-1271 were significantly enriched. These findings uncovered MEK1 and TEAD4 as novel miR-1271 targets and suggest that the epigenetic silencing of miR-1271 is crucial for GC development.