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The majority of low‐grade isocitrate dehydrogenase‐mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten‐eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5‐hydroxymethyl cytosine at over 850,000 locations for region‐specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer‐related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation‐expression correlations. Functional analysis suggested that positively correlated genes are involved in cell‐cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif‐enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation‐based gene regulation. Our findings shed light on the significance of region‐specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer‐related gene expression and IDHmt glioma malignancy. [ABSTRACT FROM AUTHOR]

Epstein‐Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV‐associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV‐encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER‐positive and ‐negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER‐positive and ‐negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER‐negative than in EBER‐positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5‐aza‐deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome‐wide using methylation array in reference to RNA‐sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor‐suppressive role of UQCRH. Moreover, 5‐aza‐deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B‐induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC. [ABSTRACT FROM AUTHOR]

Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC. [ABSTRACT FROM AUTHOR]

Multiple hepatocellular carcinoma (HCC) is divided into two categories: intrahepatic metastasis (IM), which is a true relapse of HCC, and multicentric origin (MO), which is a second primary tumor. Clinical diagnosis of multiple HCC is usually made based on tumor location and/or time to recurrence; however, it is often difficult to distinguish the two types of multiple HCC. Using 41 matched pairs of multiple HCC specimens, we confirmed the accuracy of clinical diagnoses using exome sequence data and investigated the importance of discriminating the type of multiple HCC. Genomic analysis revealed that 18 (43.9%) patients diagnosed as having genomic IM had common mutations in a pair of HCC tumors with the main tumor of these patients being more progressive compared to those with genomic MO. The accuracy of clinical diagnosis based on lobe (Definition 1) and segment (Definition 2) were 68.3% and 78.0%, respectively. Intriguingly, recurrence ≥2 years after initial surgery for 3 patients was IM. The survival of patients with clinical IM was significantly shorter than for those with clinical MO based on both Definition 1 (P = 0.045) and Definition 2 (P = 0.043). However, mean survival was not different between the patients with genomic IM and those with MO (P = 0.364). Taken together, genomic analysis elucidated that liver cancer may spread more extensively and more slowly than previously thought. In addition, distinguishing multiple HCC as IM or MC may have provided biological information but was not of clinical importance with respect to patient prognosis. [ABSTRACT FROM AUTHOR]

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation is an important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples of oncogenic events, results from promoter CGI methylation. Interestingly, both MLH1 and BRCA1 have a divergent promoter, from which another gene on the opposite strand is also transcribed. Although studies have shown that divergent transcription is an important factor in transcriptional regulation, little is known about its implication in aberrant promoter methylation in cancer. In this study, we analyzed the methylation status of CGI in divergent promoters using a recently enriched transcriptome database. We measured the extent of CGI methylation in 119 colorectal cancer (CRC) clinical samples (65 microsatellite instability high [MSI‐H] CRC with CGI methylator phenotype, 28 MSI‐H CRC without CGI methylator phenotype and 26 microsatellite stable CRC) and 21 normal colorectal tissues using Infinium MethylationEPIC BeadChip. We found that CGI within divergent promoters are less frequently methylated than CGI within unidirectional promoters in normal cells. In the genome of CRC cells, CGI within unidirectional promoters are more vulnerable to aberrant methylation than CGI within divergent promoters. In addition, we identified three DNA sequence motifs that correlate with methylated CGI. We also showed that methylated CGI are associated with genes whose expression is low in normal cells. Thus, we here provide fundamental observations regarding the methylation of divergent promoters that are essential for the understanding of carcinogenesis and development of cancer prevention strategies. In this study, we revealed that the extent of promoter methylation was different depending on the arrangement of genes using DNA methylation data of 119 colorectal cancer specimens. Divergent promoters were hypomethylated compared to unidirectional promoters. The provide data is essential for the understanding of the carcinogenic mechanism. [ABSTRACT FROM AUTHOR]

Intestinal metaplasia induced by ectopic expression of caudal‐type homeobox (CDX)2 and/or CDX1 (CDX) is frequently observed around gastric cancer (GC). Abnormal expression of CDX is also observed in GC and suggests that inappropriate gastrointestinal differentiation plays essential roles in gastric tumorigenesis, but their roles on tumorigenesis remain unelucidated. Publicly available databases show that GC patients with higher CDX expression have significantly better clinical outcomes. We introduced CDX2 and CDX1 genes separately into GC‐originated MKN7 and TMK1 cells deficient in CDX. Marked suppression of cell growth and dramatic morphological change into spindle‐shaped flat form were observed along with induction of intestinal marker genes. G0‐G1 growth arrest was accompanied by changed expression of cell cycle‐related genes but not with apoptosis or senescence. Microarray analyses additionally showed decreased expression of gastric marker genes and increased expression of stemness‐associated genes. Hierarchical clustering of 111 GC tissues and 21 non‐cancerous gastric tissues by selected 18 signature genes based on our transcriptome analyses clearly categorized the 132 tissues into non‐cancer, "CDX signature"‐positive GC, and "CDX signature"‐negative GC. Gene set enrichment analysis indicated that "CDX signature"‐positive GC has lower malignant features. Immunohistochemistry of 89 GC specimens showed that 50.6% were CDX2‐deficient, 66.3% were CDX1‐deficient, and 44.9% were concomitant CDX2/CDX1‐deficient, suggesting that potentially targetable GC cases by induced intestinal differentiation are quite common. In conclusion, exogenous expression of CDX2/CDX1 can lead to efficient growth inhibition of CDX‐deficient GC cells. It is based on rapidly induced intestinal differentiation, which may be a future therapeutic strategy. Exogenous expression of CDX2/CDX1 can lead to G0‐G1 growth arrest in CDX‐deficient gastric cancer cells, accompanied by induction of intestinal genes and decreased expression of gastric genes. Publicly available databases and hierarchical clustering of gastric tissue showed that gastric cancer with CDX expression has significantly better clinical outcomes. A novel therapy against gastric cancer based on induced intestinal differentiation may be possible in the future. [ABSTRACT FROM AUTHOR]

Methylation is closely involved in the development of various carcinomas. However, few datasets are available for small cell lung cancer (SCLC) due to the scarcity of fresh tumor samples. The aim of the present study is to clarify relationships between clinicopathological features and results of the comprehensive genome-wide methylation profile of SCLC. We investigated the genome-wide DNA methylation status of 28 tumor and 13 normal lung tissues, and gene expression profiling of 25 SCLC tissues. Following unsupervised hierarchical clustering and non-negative matrix factorization, gene ontology analysis was performed. Clustering of SCLC led to the important identification of a CpG island methylator phenotype (CIMP) of the tumor, with a significantly poorer prognosis ( P = 0.002). Multivariate analyses revealed that postoperative chemotherapy and non-CIMP were significantly good prognostic factors. Ontology analyses suggested that the extrinsic apoptosis pathway was suppressed, including TNFRSF1A, TNFRSF10A and TRADD in CIMP tumors. Here we revealed that CIMP was an important prognostic factor for resected SCLC. Delineation of this phenotype may also be useful for the development of novel apoptosis-related chemotherapeutic agents for treatment of the aggressive tumor. [ABSTRACT FROM AUTHOR]

Dysregulated DNA methylation followed by abnormal gene expression is an epigenetic hallmark in cancer. DNA methylation is catalyzed by DNA methyltransferases, and the aberrant expression or mutations of DNA methyltransferase genes are found in human neoplasm. The enzymes for demethylating 5-methylcytosine were recently identified, and the biological significance of DNA demethylation is a current focus of scientific attention in various research fields. Ten-eleven translocation ( TET) proteins have an enzymatic activity for the conversion from 5-methylcytosine to 5-hydroxymethylcytosine (5-hm C), which is an intermediate of DNA demethylation. The loss-of-function mutations of TET2 gene were reported in myeloid malignancies, suggesting that impaired TET-mediated DNA demethylation could play a crucial role in tumorigenesis. It is still unknown, however, whether DNA demethylation is involved in biological properties in solid cancers. Here, we show the loss of 5-hm C in a broad spectrum of solid tumors: for example, a significant reduction of 5-hm C was found in 72.7% of colorectal cancers ( CRCs) and 75% of gastric cancers compared to background tissues. TET1 expression was decreased in half of CRCs, and a large part of them was followed by the loss of 5-hm C. These findings suggest that the amount of 5-hm C in tumors is often reduced via various mechanisms, including the downregulation of TET1. Consistently, in the in vitro experiments, the downregulation of TET1 was clearly induced by oncogene-dependent cellular transformation, and loss of 5-hm C was seen in the transformed cells. These results suggest the critical roles of aberrant DNA demethylation for oncogenic processes in solid tissues. ( Cancer Sci 2012; 103: 670-676) [ABSTRACT FROM AUTHOR]

Chronic infections by hepatitis B virus (HBV) and hepatitis C virus (HCV) appear to be the most significant causes of hepatocellular carcinoma (HCC). Aberrant promoter methylation is known to be deeply involved in cancer, including in HCC. In this study, we analyzed aberrant promoter methylation by methylated DNA immunoprecipitation-on-chip analysis on a genome-wide scale in six HCCs including three HBV-related and three HCV-related HCCs, six matched noncancerous liver tissues, and three normal liver tissues. Candidate genes with promoter methylation were detected more frequently in HCV-related HCC. Candidate genes methylated preferentially to HBV-related or HCV-related HCCs were detected and selected, and methylation levels of the selected genes were validated by quantitative methylation analysis using MALDI-TOF mass spectrometry using 125 liver tissue samples, including 61 HCCs (28 HBV-related HCCs and 33 HCV-related HCCs) and 59 matched noncancerous livers, and five normal livers. Among analyzed genes, preferential methylation in HBV-related HCC was validated in one gene only. However, 15 genes were found to be methylated preferentially in HCV-related HCC, which was independent from age. Hierarchical clustering of HCC using these genes stratified HCV-related HCC as a cluster of frequently methylated samples. The 15 genes included genes inhibitory to cancer-related signaling such as RAS/RAF/ERK and Wnt/β-catenin pathways. Methylation of dual specificity phosphatase 4 ( DUSP4), cytochrome P450, family 24, subfamily A, polypeptide 1 ( CYP24A1), and natriuretic peptide receptor A ( NPR1) significantly correlated with recurrence-free survival. It was indicated that genes methylated preferentially in HCV-related HCC exist, and that DNA methylation might play an important role in HCV-related HCC by silencing cancer-related pathway inhibitors, and might perhaps be useful as a prognostic marker. ( Cancer Sci 2010) [ABSTRACT FROM AUTHOR]

Laparoscopic ablation therapy is an attractive modality for localized hepatocellular carcinoma. It is an optimal treatment in patients with superficial tumors, tumors adjacent to other vital organs, or tumors difficult to access by percutaneous procedure. Laparoscopic access allows for easier detection and more accurate targeting of the tumor compared with percutaneous access and can minimize complications. In addition, this novel technique is favorable in terms of the length of hospital stay and cost-effectiveness because it is completed in a single session. Characteristics and therapeutic potential of laparoscopic ablation therapy are summarized and compared with other therapeutic modalities in this review. [ABSTRACT FROM AUTHOR]