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Introduction Immune checkpoint inhibitors (ICI) have significantly improved the prognosis of non-small cell lung cancer (NSCLC). However, only a limited proportion of patients can benefit from this therapy, and clinically useful predictive biomarkers remain to be elucidated. Methods Blood was collected from 189 patients with NSCLC before and six weeks after the initiation of the ICI treatment (anti-PD-1 or anti-PD-L1 antibody). Soluble PD-1 (sPD-1) and PD-L1 (sPD-L1) in plasma before and after treatment were analyzed for evaluation of their clinical significance. Results The Cox regression analysis demonstrated that higher sPD-L1 levels before treatment significantly predicted unfavorable progression-free survival (PFS; HR 15.4, 95%CI 1.10–86.7, P = 0.009) and overall survival (OS; HR 11.4, 95%CI 1.19–52.3, P = 0.007) in NSCLC patients treated with ICI monotherapy (n = 122), but not in those treated with ICI combined with chemotherapy (n = 67: P = 0.729 and P = 0.155, respectively). In addition, higher sPD-1 levels after treatment were significantly associated with better OS (HR 0.24, 95%CI 0.06–0.91, P = 0.037) in patients treated with anti-PD-1 monotherapy, whereas higher sPD-L1 levels after treatment were significantly associated with worse PFS (HR 6.09, 95%CI 1.42–21.0, P = 0.008) and OS (HR 42.6, 95%CI 6.83–226, P

Epstein‐Barr virus (EBV) is associated with approximately 10% of gastric cancers (GCs). We previously showed that EBV infection of gastric epithelial cells induces aberrant DNA methylation in promoter regions, which causes silencing of critical tumor suppressor genes. Here, we analyzed gene expressions and active histone modifications (H3K4me3, H3K4me1, and H3K27ac) genome‐widely in EBV‐positive GC cell lines and in vitro EBV‐infected GC cell lines to elucidate the transcription factors contributing to tumorigenesis through enhancer activation. Genes associated with “signaling of WNT in cancer” were significantly enriched in EBV‐positive GC, showing increased active β‐catenin staining. Genes neighboring activated enhancers were significantly upregulated, and EHF motif was significantly enriched in these active enhancers. Higher expression of EHF in clinical EBV‐positive GC compared with normal tissue and EBV‐negative GC was confirmed by RNA‐seq using The Cancer Genome Atlas cohort, and by immunostaining using our cohort. EHF knockdown markedly inhibited cell proliferation. Moreover, there was significant enrichment of critical cancer pathway–related genes (eg, FZD5) in the downstream of EHF. EBV protein LMP2A caused upregulation of EHF via phosphorylation of STAT3. STAT3 knockdown was shown to inhibit cellular growth of EBV‐positive GC cells, and the inhibition was rescued by EHF overexpression. Our data highlighted the important role of EBV infection in gastric tumorigenesis via enhancer activation., We here analyzed the global alteration of gene expressions and active histone modifications to identify activated enhancer regions after EBV infection and to predict the master transcription factors in EBV‐positive GC. We identified that EHF was a critical transcription factor at active enhancers, which promoted cell proliferation in EBV‐positive GC. In addition, we identified its downstream target gene, FZD5, which is a potential oncogene that regulates cell growth after EBV infection.

Epstein-Barr virus (EBV) is associated with several human malignancies including 8-10% of gastric cancers (GCs). Genome-wide analysis of 3D chromatin topologies across GC lines, primary tissue and normal gastric samples revealed chromatin domains specific to EBV-positive GC, exhibiting heterochromatin-to-euchromatin transitions and long-range human-viral interactions with non-integrated EBV episomes. EBV infection in vitro suffices to remodel chromatin topology and function at EBV-interacting host genomic loci, converting H3K9me3+ heterochromatin to H3K4me1+/H3K27ac+ bivalency and unleashing latent enhancers to engage and activate nearby GC-related genes (for example TGFBR2 and MZT1). Higher-order epigenotypes of EBV-positive GC thus signify a novel oncogenic paradigm whereby non-integrative viral genomes can directly alter host epigenetic landscapes ('enhancer infestation'), facilitating proto-oncogene activation and tumorigenesis.

Epstein‐Barr virus (EBV) is associated with particular forms of gastric cancer (GC). We previously showed that EBV infection into gastric epithelial cells induced aberrant DNA hypermethylation in promoter regions and silencing of tumor suppressor genes. We here undertook integrated analyses of transcriptome and epigenome alteration during EBV infection in gastric cells, to investigate activation of enhancer regions and related transcription factors (TFs) that could contribute to tumorigenesis. Formaldehyde‐assisted isolation of regulatory elements (FAIRE) sequencing (‐seq) data revealed 19 992 open chromatin regions in putative H3K4me1+ H3K4me3− enhancers in EBV‐infected MKN7 cells (MKN7_EB), with 10 260 regions showing increase of H3K27ac. Motif analysis showed candidate TFs, eg activating transcription factor 3 (ATF3), to possibly bind to these activated enhancers. ATF3 was considerably upregulated in MKN7_EB due to EBV factors including EBV‐determined nuclear antigen 1 (EBNA1), EBV‐encoded RNA 1, and latent membrane protein 2A. Expression of mutant EBNA1 decreased copy number of the EBV genome, resulting in relative downregulation of ATF3 expression. Epstein‐Barr virus was also infected into normal gastric epithelial cells, GES1, confirming upregulation of ATF3. Chromatin immunoprecipitation‐seq analysis on ATF3 binding sites and RNA‐seq analysis on ATF3 knocked‐down MKN7_EB revealed 96 genes targeted by ATF3‐activating enhancers, which are related with cancer hallmarks, eg evading growth suppressors. These 96 ATF3 target genes were significantly upregulated in MKN7_EB compared with MKN7 and significantly downregulated when ATF3 was knocked down in EBV‐positive GC cells SNU719 and NCC24. Knockdown of ATF3 in EBV‐infected MKN7, SNU719, and NCC24 cells all led to significant decrease of cellular growth through an increase of apoptotic cells. These indicate that enhancer activation though ATF3 might contribute to tumorigenesis of EBV‐positive GC., Through integrated analyses on alterations of transcriptome, histone modification, and open chromatin status during Epstein‐Barr virus (EBV) infection in gastric cells, we here identified activating transcription factor 3 (ATF3) as a critical transcriptional activator with involvement in enhancer activation. We undertook screening of putative transcription factors binding to activated enhancer regions and identified ATF3 as a transcription factor that is upregulated by EBV infection and induces aberrant enhancer activation. Effect of ATF3 expression on cellular proliferation was confirmed, suggesting a tumorigenic role of aberrant enhancer activation by ATF3 upregulation, which could help our understanding of the tumorigenic mechanisms in this particular subtype of gastric cancer.

Cinobufagin is a cardiotoxic bufanolide steroid secreted by the Asiatic toad, Bufo gargarizans. Bufanolides inhibit Na

Highlights • The entire landscape of AR-V7 target regions/genes in CRPC cells was investigated. • We identified 78 AR-V7 target genes, targeted specifically by AR-V7 e.g. SLC3A2, or commonly by AR and AR-V7 e.g. NUP210. • SLC3A2 and NUP210 were markedly upregulated in clinical CRPC tissues, leading to increased proliferation in CRPC., Primary prostate cancer (PC) progresses to castration-resistant PC (CRPC) under androgen deprivation therapy, by mechanisms e.g. expression of androgen receptor (AR) splice variant-7 (AR-V7). Here we conducted comprehensive epigenome and transcriptome analyses comparing LNCaP, primary PC cells, and LNCaP95, AR-V7-expressing CRPC cells derived from LNCaP. Of 399 AR-V7 target regions identified through ChIP-seq analysis, 377 could be commonly targeted by hormone-stimulated AR, and 22 were specifically targeted by AR-V7. Among genes neighboring to these AR-V7 target regions, 78 genes were highly expressed in LNCaP95, while AR-V7 knockdown led to significant repression of these genes and suppression of growth of LNCaP95. Of the 78 AR-V7 target genes, 74 were common AR/AR-V7 target genes and 4 were specific AR-V7 target genes; their most suppressed genes by AR-V7 knockdown were NUP210 and SLC3A2, respectively, and underwent subsequent analyses. NUP210 and SLC3A2 were significantly upregulated in clinical CRPC tissues, and their knockdown resulted in significant suppression of cellular growth of LNCaP95 through apoptosis and growth arrest. Collectively, AR-V7 contributes to CRPC proliferation by activating both common AR/AR-V7 target and specific AR-V7 target, e.g. NUP210 and SLC3A2., Graphical abstract Image, graphical abstract

Background: Gastric cancer (GC) is one of the leading causes of cancer-related deaths worldwide. GC is a pathologically and molecularly heterogeneous disease. DNA hypermethylation in promoter CpG islands causes silencing of tumor-suppressor genes and thus contributes to gastric carcinogenesis. In addition, various molecular aberrations, including aberrant chromatin structures, gene mutations, structural variants, and somatic copy number alterations, are involved in gastric carcinogenesis. Summary: Comprehensive DNA methylation analyses revealed multiple DNA methylation patterns in GCs and classified GC into distinct molecular subgroups: extremely high-methylation epigenotype uniquely observed in GC associated with Epstein-Barr virus (EBV), high-methylation epigenotype associated with microsatellite instability (MSI), and low-methylation epigenotype. In The Cancer Genome Atlas classification, EBV and MSI are extracted as independent subgroups of GC, whereas the remaining GCs are categorized into genomically stable (GS) and chromosomal instability (CIN) subgroups. EBV-positive GC, exhibiting the most extreme DNA hypermethylation in the whole human malignancies, frequently shows CDKN2A silencing, PIK3CA mutations, PD-L1/2 overexpression, and lack of TP53 mutations. MSI, exhibiting high DNA methylation, often has MLH1 silencing and abundant gene mutations. GS is generally a diffuse-type GC and frequently shows CDH1/RHOA mutations or CLDN18–ARHGAP fusion. CIN is generally an intestinal-type GC and frequently has TP53 mutations and genomic amplification of receptor tyrosine kinases. Key Messages: The frequency and targets of genetic aberrations vary depending on the epigenotype. Aberrations in the genome and epigenome are expected to synergistically interact and contribute to gastric carcinogenesis and comprehensive analyses of those in GCs may help elucidate the mechanism of carcinogenesis.

// Hiroe Namba-Fukuyo 1, 2, 3 , Sayaka Funata 1, 4 , Keisuke Matsusaka 1 , Masaki Fukuyo 1 , Bahityar Rahmutulla 1 , Yasunobu Mano 1 , Masashi Fukayama 4 , Hiroyuki Aburatani 5 , Atsushi Kaneda 1 1 Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Japan 2 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan 3 Japan Society for the Promotion of Science, Japan 4 Department of Pathology, Graduate School of Medicine, The University of Tokyo, Japan 5 Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Japan Correspondence to: Atsushi Kaneda, email: kaneda@chiba-u.jp Keywords: DNA hydroxymethylation, DNA methylation, Epstein-Barr virus, gastric cancer, TET2 Received: August 01, 2016 Accepted: October 17, 2016 Published: November 05, 2016 ABSTRACT Extensive DNA methylation is observed in gastric cancer with Epstein-Barr virus (EBV) infection, and EBV infection is the cause to induce this extensive hypermethylaton phenotype in gastric epithelial cells. However, some 5′ regions of genes do not undergo de novo methylation, despite the induction of methylation in surrounding regions, suggesting the existence of a resistance factor against DNA methylation acquisition. We conducted an RNA-seq analysis of gastric epithelial cells with and without EBV infection and found that TET family genes, especially TET2 , were repressed by EBV infection at both mRNA and protein levels. TET2 was found to be downregulated by EBV transcripts, e.g. BARF0 and LMP2A , and also by seven human miRNAs targeting TET2 , e.g., miR-93 and miR-29a, which were upregulated by EBV infection, and transfection of which into gastric cells repressed TET2 . Hydroxymethylation target genes by TET2 were detected by hydroxymethylated DNA immunoprecipitation sequencing (hMeDIP-seq) with and without TET2 overexpression, and overlapped significantly with methylation target genes in EBV-infected cells. When TET2 was knocked down by shRNA, EBV infection induced de novo methylation more severely, including even higher methylation in methylation-acquired promoters or de novo methylation acquisition in methylation-protected promoters, leading to gene repression. TET2 knockdown alone without EBV infection did not induce de novo DNA methylation. These data suggested that TET2 functions as a resistance factor against DNA methylation in gastric epithelial cells and repression of TET2 contributes to DNA methylation acquisition during EBV infection.

To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high‐methylation CRC. The methylation levels of six Group‐1 and 14 Group‐2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group‐1 and Group‐2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high‐methylation CRC. But TSA showed low‐methylation levels of Group‐1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high‐methylation CRC. High‐methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p, What's new? The serrated pathway of colorectal cancer (CRC) development is characterized by the presence of saw‐toothed colonic crypts and by a high‐methylation epigenotype. Not all serrated lesions, however, give rise to CRC. Here, only sessile serrated adenoma/polyps, a potentially malignant serrated polyp subtype, were identified as precursors of high‐methylation CRC. Moreover, MLH1 expression, silencing of which previously was linked to microsatellite instability in CRC, was found to be preserved in most serrated adenoma/polyp samples. The findings suggest that the acquisition of a hypermutation phenotype in serrated CRC likely depends on extensive MLH1 methylation and mutation of additional mismatch repair genes.

Silenced chromatin domains are restricted to specific regions. Eukaryotic chromosomes are organized into discrete domains delimited by domain boundaries. From approximately 6,000 genes in Saccharomyces cerevisiae, we previously isolated 55 boundary genes. In this study, we focus on the molecular function of one of boundary genes, YCR076C/FUB1 (function of boundary), whose function has not been clearly defined in vivo. Biochemical analysis of Fub1p revealed that it interacted with multiple subunits of the 20S proteasome core particle (20S CP). To further clarify the functional link between Fub1p and proteasome, several proteasome mutants were analyzed. Although only 20S CP subunits were isolated as Fub1p interactors, a genetic interaction was also observed for component of 19S regulatory particle (19S RP) suggesting involvement of Fub1p with the whole proteasome. We also analyzed the mechanism of boundary establishment by using proteasome composition factor-deficient strains. Deletion of pre9 and ump1, whose products have effects on the 20S CP, resulted in a decrease in boundary function. Domain analyses of Fub1p identified a minimum functional domain in the C terminus that was essential for boundary establishment and showed a limited sequence homology to the human PSMF1, which is known to inhibit proteasome activity. Finally, boundary assay showed that human PSMF1 also exhibited boundary establishment activity in yeast. Our results defined the functional correlation between Fub1p and PSMF1.

A single-cell method allows the assessment of relationships between the dynamic epigenetic behavior of yeast heterochromatin boundaries over multiple generations., Differences in gene expression between individual cells can be mediated by epigenetic regulation; thus, methods that enable detailed analyses of single cells are crucial to understanding this phenomenon. In this study, genomic silencing regions of Saccharomyces cerevisiae that are subject to epigenetic regulation, including the HMR, HML, and telomere regions, were investigated using a newly developed single cell analysis method. This method uses fluorescently labeled proteins to track changes in gene expression over multiple generations of a single cell. Epigenetic control of gene expression differed depending on the specific silencing region at which the reporter gene was inserted. Correlations between gene expression at the HMR-left and HMR-right regions, as well as the HMR-right and HML-right regions, were observed in the single-cell level; however, no such correlations involving the telomere region were observed. Deletion of the histone acetyltransferase GCN5 gene from a yeast strain carrying a fluorescent reporter gene at the HMR-left region reduced the frequency of changes in gene expression over a generation. The results presented here suggest that epigenetic control within an individual cell is reversible and can be achieved via regulation of histone acetyltransferase activity., Author Summary Although eukaryotic gene repression usually acts on individual genes, cells can also repress larger chromosomal regions via the establishment of a high order chromatin structure called heterochromatin. Once initiated, heterochromatin spreads until halted by a boundary, and in this study we focus on how this boundary is formed. Because the mechanism is epigenetic and can differ from cell to cell, we wanted to assess the dynamics of the process by tracking individual cells over multiple generations. Here we develop a novel method employing protein fluorescence to monitor gene expression at the boundaries of several yeast heterochromatic regions simultaneously. This allows us to assess whether different boundaries within a single cell fluctuate in concert or independently of each other. In addition, we use histone modification mutants to probe the specific types of epigenetic regulation responsible for fluctuations in heterochromatin boundary positioning. Using this method, we show that epigenetic gene expression within individual cells is reversible and that this process is regulated by histone acetylation state. Future work will identify connections between variation in boundary positioning and novel transcription control systems.

AIM: To investigate epigenomic and gene expression alterations during cellular senescence induced by oncogenic Raf. METHODS: Cellular senescence was induced into mouse embryonic fibroblasts (MEFs) by infecting retrovirus to express oncogenic Raf (RafV600E). RNA was collected from RafV600E cells as well as MEFs without infection and MEFs with mock infection, and a genome-wide gene expression analysis was performed using microarray. The epigenomic status for active H3K4me3 and repressive H3K27me3 histone marks was analyzed by chromatin immunoprecipitation-sequencing for RafV600E cells on day 7 and for MEFs without infection. These data for Raf-induced senescence were compared with data for Ras-induced senescence that were obtained in our previous study. Gene knockdown and overexpression were done by retrovirus infection. RESULTS: Although the expression of some genes including secreted factors was specifically altered in either Ras- or Raf-induced senescence, many genes showed similar alteration pattern in Raf- and Ras-induced senescence. A total of 841 commonly upregulated 841 genes and 573 commonly downregulated genes showed a significant enrichment of genes related to signal and secreted proteins, suggesting the importance of alterations in secreted factors. Bmp2, a secreted protein to activate Bmp2-Smad signaling, was highly upregulated with gain of H3K4me3 and loss of H3K27me3 during Raf-induced senescence, as previously detected in Ras-induced senescence, and the knockdown of Bmp2 by shRNA lead to escape from Raf-induced senescence. Bmp2-Smad inhibitor Smad6 was strongly repressed with H3K4me3 loss in Raf-induced senescence, as detected in Ras-induced senescence, and senescence was also bypassed by Smad6 induction in Raf-activated cells. Different from Ras-induced senescence, however, gain of H3K27me3 did not occur in the Smad6 promoter region during Raf-induced senescence. When comparing genome-wide alteration between Ras- and Raf-induced senescence, genes showing loss of H3K27me3 during senescence significantly overlapped; genes showing H3K4me3 gain, or those showing H3K4me3 loss, also well-overlapped between Ras- and Raf-induced senescence. However, genes with gain of H3K27me3 overlapped significantly rarely, compared with those with H3K27me3 loss, with H3K4me3 gain, or with H3K4me3 loss. CONCLUSION: Although epigenetic alterations are partly different, Bmp2 upregulation and Smad6 repression occur and contribute to Raf-induced senescence, as detected in Ras-induced senescence.