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The article presents a case study of a 61-year-old man who was taken to the hospital because of melena and progressive anemia requiring transfusion. His colonoscopy and upper gastrointestinal endoscopy were all normal. He was performed with laparoscopy because both anterograde and retrograde single-balloon enteroscopy failed to reach the tumor. The article discusses pyogenic granuloma, a disease of the gastrointestinal tract which arises on the oral mucosa and extremities.

Background: Aberrant DNA methylation is prevalent in colorectal serrated lesions. We previously reported that the CpG island of SMOC1 is frequently methylated in traditional serrated adenomas (TSAs) and colorectal cancers (CRCs) but is rarely methylated in sessile serrated lesions (SSLs). In the present study, we aimed to further characterize the expression of SMOC1 in early colorectal lesions., Methods: SMOC1 expression was analyzed immunohistochemically in a series of colorectal tumors (n = 199) and adjacent normal colonic tissues (n = 112)., Results: SMOC1 was abundantly expressed in normal colon and SSLs while it was significantly downregulated in TSAs, advanced adenomas and cancers. Mean immunohistochemistry scores were as follows: normal colon, 24.2; hyperplastic polyp (HP), 18.9; SSL, 23.8; SSL with dysplasia (SSLD)/SSL with early invasive cancer (EIC), 15.8; TSA, 5.4; TSA with high grade dysplasia (HGD)/EIC, 4.7; non-advanced adenoma, 21.4; advanced adenoma, 11.9; EIC, 10.9. Higher levels SMOC1 expression correlated positively with proximal colon locations and flat tumoral morphology, reflecting its abundant expression in SSLs. Among TSAs that contained both flat and protruding components, levels of SMOC1 expression were significantly lower in the protruding components., Conclusion: Our results suggest that reduced expression of SMOC1 is associated with progression of TSAs and conventional adenomas and that SMOC1 expression may be a biomarker for diagnosis of serrated lesions and risk prediction in colorectal tumors., (© 2024. The Author(s).)

We previously showed that upregulation of adipocyte enhancer-binding protein 1 ( AEBP1 ) in vascular endothelial cells promotes tumor angiogenesis. In the present study, we aimed to clarify the role of stromal AEBP1 /ACLP expression in oral squamous cell carcinoma (OSCC). Immunohistochemical analysis showed that ACLP is abundantly expressed in cancer-associated fibroblasts (CAFs) in primary OSCC tissues and that upregulated expression of ACLP is associated with disease progression. Analysis using CAFs obtained from surgically resected OSCCs showed that the expression of AEBP1 /ACLP in CAFs is upregulated by co-culture with OSCC cells or treatment with TGF-β1, suggesting cancer-cell-derived TGF-β1 induces AEBP1 /ACLP in CAFs. Collagen gel contraction assays showed that ACLP contributes to the activation of CAFs. In addition, CAF-derived ACLP promotes migration, invasion, and in vivo tumor formation by OSCC cells. Notably, tumor stromal ACLP expression correlated positively with collagen expression and correlated inversely with CD8+ T cell infiltration into primary OSCC tumors. Boyden chamber assays suggested that ACLP in CAFs may attenuate CD8+ T cell migration. Our results suggest that stromal ACLP contributes to the development of OSCCs, and that ACLP is a potential therapeutic target., Competing Interests: All authors declare no conflict of interest.

Long noncoding RNAs (lncRNAs) play pivotal roles in tumor development. To identify dysregulated lncRNAs in gastric cancer (GC), we analyzed genome-wide trimethylation of histone H3 lysine 4 (H3K4me3) to screen for transcriptionally active lncRNA genes in the non-tumorous gastric mucosa of patients with GC and healthy individuals. We found that H3K4me3 at TM4SF1-AS1 was specifically upregulated in GC patients and that the expression of TM4SF1-AS1 was significantly elevated in primary and cultured GC cells. TM4SF1-AS1 contributes to GC cell growth in vitro and in vivo, and its oncogenic function is mediated, at least in part, through interactions with purine-rich element-binding protein α (Pur-α) and Y-box binding protein 1 (YB-1). TM4SF1-AS1 also activates interferon signaling in GC cells, which is dependent on Pur-α and RIG-I. Chromatin isolation by RNA purification (ChIRP)-mass spectrometry demonstrated that TM4SF1-AS1 was associated with several stress granule (SG)-related proteins, including G3BP2, RACK1, and DDX3. Notably, TM4SF1-AS1 promoted SG formation and inhibited apoptosis in GC cells by sequestering RACK1, an activator of the stress-responsive MAPK pathway, within SGs. TM4SF1-AS1-induced SG formation and apoptosis inhibition are dependent on Pur-α and YB-1. These findings suggested that TM4SF1-AS1 contributes to tumorigenesis by enhancing SG-mediated stress adaptation., (© 2023. The Author(s).)

Background: The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs., Methods: Publicly available single-cell RNA-sequencing (RNA-seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α-smooth muscle antigen (α-SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co-cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse-transcription PCR., Results: Analysis of the RNA-seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12-positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α-SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co-cultured with OSCC cells., Conclusion: Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background and Aim: The tumor microenvironment plays an essential role in the development and progression of colorectal cancer (CRC). We recently reported that crosstalk between CRC cells and tumor-associated macrophages (TAMs) via serum amyloid A1 (SAA1) promotes invasion by T1 CRCs. In the present study, we aimed to clarify the role of neutrophils in early CRCs., Methods: Immunohistochemical analysis of CD66b, chemokine CXC motif ligand 8 (CXCL8 or interleukin-8, IL-8) and matrix metalloproteinase-9 (MMP-9) was performed using primary T1 CRCs (n = 49). The HL-60 human promyelocytic leukemia cell line and THP-1 human monocytic leukemia cell line were used to obtain neutrophil-like and macrophage-like cells, respectively. Boyden chamber assays were used to analyze cell migration and invasion, and quantitative RT-PCR was used to analyze gene expression., Results: Immunohistochemical analysis revealed accumulation of neutrophils at the SAA1-positive invasive front of T1 CRCs. Experiments using HL-60 cells suggested that treatment with SAA1 induced neutrophil migration and expression of CXCL8 and MMP-9 in neutrophils and that neutrophils promote CRC cell migration and invasion. Immunohistochemistry confirmed accumulation of CXCL8- or MMP-9-positive neutrophils at the SAA1-positive invasive front of T1 CRCs. Moreover, co-culture experiments using CRC, THP-1 and HL-60 cells suggested that CRC cells activated by macrophages upregulate CXCL8 and MMP-9 in neutrophils., Conclusions: Our results suggest that interplay between macrophages and CRC cells leads to recruitment of neutrophils to the invasive front of T1 CRCs and that SAA1 secreted by CRC cells activate neutrophils to promote invasion., (© 2022 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Background: Although MicroRNAs (miRNAs) play important roles in various biological processes, the biological functions of miRNAs are achieved through mRNAs. The aim of this study is to identify dysregulated miRNA/mRNA expression patterns in colorectal tumors., Methods: We examined 42 colorectal tumors [15 adenomas, 8 intramucosal cancers (IMCs), and 19 invasive colorectal cancers (CRCs)] with the microsatellite stable (MSS) phenotype (first cohort). The first cohort was used for genome-wide miRNA and mRNA expression arrays, whereas the second cohort (37 colorectal neoplasias) was used for validation analyses. Finally, we used 15 cases of "adenoma in/with carcinoma" to identify network patterns of miRNAs/mRNAs that were directly associated with neoplastic progression. In addition, simple regression analysis for array-based and RT-PCR analyses was performed to select candidate miRNA-mRNA pairs. Transfection of miRNA mimics was also performed to confirm whether target mRNA expression is affected by specific miRNAs., Results: Specific paired miRNA/mRNA networks, including hsa-miR-34a-5p/SLC12A2, hsa-miR-15b-5p/SLC12A2, hsa-miR-195-5p/SLC12A2, hsa-miRNA-502-3p/OLFM4, hsa-miRNA-6807-5p/ZG16, and hsa-miRNA 3064-5p/SH3BGRL3, were identified in samples of adenoma, IMC, and CRC with the MSS phenotype. In adenomatous lesions obtained from the same tumor with a carcinomatous lesion, we identified pairs of miRNA-130a-3p/HSPA8 and miRNA-22-3p/RP53 that were linked to multiple pathways. On the other hand, 2 pairs of miRNA/mRNA (miRNA-660-5p and miRNA-664a-5p/APP) were found in isolated carcinomatous glands. Ectopic expression of miRNA 3064-5p suppressed SH3BGRL3 expression., Conclusions: We found that networks based on specific pairs of miRNAs/mRNAs contribute to progression from adenomatous and carcinomatous lesions. Our results provide insights into the molecular tumorigenesis of colorectal tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Sugai, Osakabe, Niinuma, Sugimoto, Eizuka, Tanaka, Yanagawa, Otsuka, Sasaki, Matsumoto and Suzuki.)

mRNA expression varies in human cancers. Such altered mRNA expression is negatively regulated by the expression of microRNAs (miRNAs), which play an important role in human tumorigenesis. According to this theory, inverse mRNA/miRNA expression may be a direct driver of cancer development, and certain genetic events may occur prior to the development of any discernible histological abnormalities. We examined the inverse expression between mRNAs and their corresponding miRNAs in colorectal cancer (CRC) and adjacent normal mucosa and performed pathway analysis to identify mRNA/miRNA networks. The cancer samples were divided into first (20 cases) and second (24 cases) cohorts, and 48 samples were obtained from two sections of the normal mucosa adjacent to the tumors from the second cohort. We investigated mRNAs with commonly altered expression in CRC and adjacent normal mucosa using isolated cancer glands and normal crypts from the first cohort, compared with that of distal normal crypts, using an array-based method. As a result, significant inverse correlations between CEACAM1 and miRNA-7114-5p and between AK1 and miRNA-6780-5p were found in CRC and adjacent normal mucosa. We validated these correlations in the second cohort using RT-PCR. To confirm these findings, transfection and immunohistochemical assays were also performed, which verified the inverse correlation between CEACAM1 and miRNA-7114-5p. Our findings suggest that the inverse correlations between the CEACAM1/miRNA-7114-5p and possibly AK1/miRNA-6780-5p pairs play an important role in early CRC development, and may help identify potential molecular targets for early detection of CRC., (© 2022 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland & John Wiley & Sons, Ltd.)

MicroRNA (miRNA) expression is dysregulated in human tumors, thereby contributing to tumorigenesis through altered expression of mRNA. Thus, identification of the relationships between miRNAs and mRNAs is important for evaluating the molecular mechanisms of tumors. In addition, elucidation of the molecular features of serrated lesions is essential in colorectal tumorigenesis. Here, we examined the relationships of miRNA and mRNA expressed in serrated lesions, including 26 sessile serrated lesions (SSLs), 12 traditional serrated adenomas (TSAs), and 11 colorectal cancers (CRCs) with a microsatellite instability (MSI) phenotype using crypt isolation. We divided the samples into the first and second cohorts for validation. Array-based expression analyses were used to evaluate miRNAs and mRNAs with opposite expression patterns in isolated tumor glands. In addition, we validated the relationships of miRNA/mRNA pairs in the second cohort using real-time polymerase chain reaction. We found that the expression of miRNA-5787 was correlated with reciprocal expression of two mRNAs, that is, SRRM2 and POLR2J3, in SSL samples. In TSA samples, two pairs of miRNAs/mRNAs showing opposite expression patterns, that is, miRNA-182-5p/ETF1 and miRNA-200b-3p/MYB, were identified. Ultimately, three pairs of miRNAs/mRNAs with opposite expression patterns, including miRNA-222-3p/SLC26A3, miRNA-6753-3p/FABP1, and miRNA-222-3p/OLFM4, were retained in CRC with an MSI phenotype. Finally, we performed transfection with an miR-222-3p mimic to confirm the expression of SLC26A3 and OLFM4; the results showed that ectopic expression of miR-222-3p moderately suppressed OLFM4 and downregulated SLC26A3 to some extent. Overall, our results provided basic insights into the evaluation of colorectal tumorigenesis of serrated lesions and CRC with an MSI phenotype., (© 2021 Wiley Periodicals LLC.)

Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells., (© 2021. The Author(s).)

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA-sequencing (RNA-seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP-1 cells suggested that interleukin 1β (IL-1β) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL-1β. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1-like/M2-like macrophages at SAA1-positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase-9 in macrophages. Our data suggest that tumor-associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background/aim: Epigenetic alterations play an important role in the pathogenesis of gastrointestinal stromal tumors (GISTs). To obtain further insight into the GIST epigenome, we analyzed genome-wide histone modification and DNA methylation in GIST cells., Materials and Methods: To reverse epigenetic silencing, GIST-T1 cells were treated with a DNA methyltransferase inhibitor and a histone deacetylase inhibitor, and subsequently H3K4me3 levels, the DNA methylome, and the transcriptome were analyzed., Results: Treatment with epigenetic inhibitors not only up-regulated genes with DNA methylation, but also genes related to interferon signaling. ChIP-seq analysis revealed that drug treatment up-regulated H3K4me3 levels in retrotransposons, including endogenous retroviruses (ERV). Finally, utilizing the omics data, we found that hypermethylation of MEG3 is a frequent event and an indicator of poorer prognosis in GIST patients., Conclusion: Epigenetic inhibitors may activate interferon signaling via viral mimicry in GIST cells. Moreover, epigenome data could be a useful resource to identify novel GIST-related genes., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Epigenetic mechanisms such as histone modification play key roles in the pathogenesis of multiple myeloma (MM). We previously showed that EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, and G9, a H3K9 methyltransferase, are potential therapeutic targets in MM. Moreover, recent studies suggest EZH2 and G9a cooperate to regulate gene expression. We therefore evaluated the antitumor effect of dual EZH2 and G9a inhibition in MM. A combination of an EZH2 inhibitor and a G9a inhibitor strongly suppressed MM cell proliferation in vitro by inducing cell cycle arrest and apoptosis. Dual EZH2/G9a inhibition also suppressed xenograft formation by MM cells in vivo. In datasets from the Gene Expression Omnibus, higher EZH2 and EHMT2 (encoding G9a) expression was significantly associated with poorer prognoses in MM patients. Microarray analysis revealed that EZH2/G9a inhibition significantly upregulated interferon (IFN)-stimulated genes and suppressed IRF4-MYC axis genes in MM cells. Notably, dual EZH2/G9a inhibition reduced H3K27/H3K9 methylation levels in MM cells and increased expression of endogenous retrovirus (ERV) genes, which suggests that activation of ERV genes may induce the IFN response. These results suggest that dual targeting of EZH2 and G9a may be an effective therapeutic strategy for MM.

Rationale: Aggressive variant of splenic marginal zone lymphoma (AV-SMZL) is a very rare disease that is often associated with TP53 mutations and has a poor prognosis. On the other hand, recent advances in genome sequencing techniques enable us to understand the molecular characteristics of rare cancers such as AV-SMZL. Here we present a case of AV-SMZL analyzed using a genetic test., Patient Concerns: A 66-year-old woman was admitted with splenomegaly and lymphocytosis. Computed tomography revealed marked splenomegaly without lymphadenopathy in any other areas. The serum soluble interleukin-2 receptor (sIL-2R) level was significantly elevated. Peripheral and bone marrow blood tests showed an increase in abnormal lymphocytes., Diagnosis: A splenectomy revealed an SMZL pattern with increased numbers of large cells and mitotic cells and a high Ki-67 positivity rate, which led to a diagnosis of AV-SMZL. Although TP53 mutation was not detected, mutations in NOTCH2, NCOA4, PTEN, EPHA3, and KMT2D were identified. Among these, the mutations in NCOA4, PTEN, and EPHA3 were novel pathogenic mutations in SMZL, which suggests they may be related to the aggressiveness and persistence of the disease., Interventions: The patient was administered a rituximab-containing regimen and rituximab-maintenance therapy., Outcomes: The patient continues to exhibit a complete response., Lessons: This is a case of AV-SMZL in which a cancer panel test successfully detected genetic alterations that are potentially associated with its pathogenesis. These findings suggest that genetic analysis is useful for making diagnoses as well as for determining treatment strategies in AV-SMZL.

Tumor angiogenesis is an important therapeutic target in colorectal cancer (CRC). We aimed to identify novel genes associated with angiogenesis in CRC. Using RNA sequencing analysis in normal and tumor endothelial cells (TECs) isolated from primary CRC tissues, we detected frequent upregulation of adipocyte enhancer-binding protein 1 (AEBP1) in TECs. Immunohistochemical analysis revealed that AEBP1 is upregulated in TECs and stromal cells in CRC tissues. Quantitative RT-PCR analysis showed that there is little or no AEBP1 expression in CRC cell lines, but that AEBP1 is well expressed in vascular endothelial cells. Levels of AEBP1 expression in Human umbilical vein endothelial cells (HUVECs) were upregulated by tumor conditioned medium derived from CRC cells or by direct coculture with CRC cells. Knockdown of AEBP1 suppressed proliferation, migration, and in vitro tube formation by HUVECs. In xenograft experiments, AEBP1 knockdown suppressed tumorigenesis and microvessel formation. Depletion of AEBP1 in HUVECs downregulated a series of genes associated with angiogenesis or endothelial function, including aquaporin 1 (AQP1) and periostin (POSTN), suggesting that AEBP1 might promote angiogenesis through regulation of those genes. These results suggest that upregulation of AEBP1 contributes to tumor angiogenesis in CRC, which makes AEBP1 a potentially useful therapeutic target., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Ubiquitin-like protein containing PHD and RING finger domains 1 (UHRF1) is a major regulator of epigenetic mechanisms and is overexpressed in various human malignancies. In this study, we examined the involvement of UHRF1 in aberrant DNA methylation and gene silencing in colorectal cancer (CRC)., Results: CRC cell lines were transiently transfected with siRNAs targeting UHRF1, after which DNA methylation was analyzed using dot blots, bisulfite pyrosequencing, and Infinium HumanMethylation450 BeadChip assays. Gene expression was analyzed using RT-PCR and gene expression microarrays. Depletion of UHRF1 rapidly induced genome-wide DNA demethylation in CRC cells. Infinium BeadChip assays and bisulfite pyrosequencing revealed significant demethylation across entire genomic regions, including CpG islands, gene bodies, intergenic regions, and repetitive elements. Despite the substantial demethylation, however, UHRF1 depletion only minimally reversed CpG island hypermethylation-associated gene silencing. By contrast, the combination of UHRF1 depletion and histone deacetylase (HDAC) inhibition reactivated the silenced genes and strongly suppressed CRC cell proliferation. The combination of UHRF1 depletion and HDAC inhibition also induced marked changes in the gene expression profiles such that cell cycle-related genes were strikingly downregulated., Conclusions: Our results suggest that (i) maintenance of DNA methylation in CRC cells is highly dependent on UHRF1; (ii) UHRF1 depletion rapidly induces DNA demethylation, though it is insufficient to fully reactivate the silenced genes; and (iii) dual targeting of UHRF1 and HDAC may be an effective new therapeutic strategy.

Epigenetic alterations play an important role in the pathogenesis in multiple myeloma, but their biological and clinical relevance is not fully understood. Here, we show that DOT1L, which catalyzes methylation of histone H3 lysine 79, is required for myeloma cell survival. DOT1L expression levels were higher in monoclonal gammopathy of undetermined significance and smoldering multiple myeloma than in normal plasma cells. Treatment with a DOT1L inhibitor induced cell cycle arrest and apoptosis in myeloma cells, and strongly suppressed cell proliferation in vitro The anti-myeloma effect of DOT1L inhibition was confirmed in a mouse xenograft model. Chromatin immunoprecipitation-sequencing and microarray analysis revealed that DOT1L inhibition downregulated histone H3 lysine 79 dimethylation and expression of IRF4-MYC signaling genes in myeloma cells. In addition, DOT1L inhibition upregulated genes associated with immune responses and interferon signaling. Myeloma cells with histone modifier mutations or lower IRF4/MYC expression were less sensitive to DOT1L inhibition, but with prolonged treatment, anti-proliferative effects were achieved in these cells. Our data suggest that DOT1L plays an essential role in the development of multiple myeloma and that DOT1L inhibition may provide new therapies for myeloma treatment., (Copyright© 2019 Ferrata Storti Foundation.)

Background: Recent studies revealed that colorectal tumors are composed of genetically diverse subclones. We aimed to clarify whether the surface microstructures of colorectal tumors are associated with genetic intratumoral heterogeneity (ITH)., Methods: The surface microstructures (pit patterns) of colorectal tumors were observed using magnifying endoscopy, and biopsy specimens were obtained from respective areas when tumors exhibited multiple pit patterns. A total of 711 specimens from 477 colorectal tumors were analyzed for BRAF, KRAS and TP53 mutations using pyrosequencing and direct sequencing. A panel of cancer-related genes was analyzed through targeted sequencing in 7 tumors., Results: Colorectal tumors with multiple pit patterns exhibited more advanced pit patterns and higher frequencies of KRAS and/or TP53 mutations than tumors with a single pit pattern. In tumors with multiple pit patterns, mutations were observed as public (common to all areas) or private (specific to certain areas), and private KRAS and/or TP53 mutations were often variable and unrelated to the pit pattern grade. Notably, invasive CRCs frequently exhibited public TP53 mutations, even in adenomatous areas, which is indicative of their early malignant potential. Targeted sequencing revealed additional public and private mutations in tumors with multiple pit patterns, indicating their single clonal origin., Conclusions: Our results suggest intratumoral pit pattern variation does not simply reflect the process of colorectal tumor evolution, but instead represents genetically diverse subclones, and this diversity may be associated with malignant potential.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC). Nucleos(t)ide analogue (NA) therapy effectively reduces the incidence of HCC, but it does not completely prevent the disease. Here, we show that dysregulation of microRNAs (miRNAs) is involved in post-NA HCC development. We divided chronic hepatitis B (CHB) patients who received NA therapy into two groups: 1) those who did not develop HCC during the follow-up period after NA therapy (no-HCC group) and 2) those who did (HCC group). miRNA expression profiles were significantly altered in CHB tissues as compared to normal liver, and the HCC group showed greater alteration than the no-HCC group. NA treatment restored the miRNA expression profiles to near-normal in the no-HCC group, but it was less effective in the HCC group. A number of miRNAs implicated in HCC, including miR-101, miR-140, miR-152, miR-199a-3p, and let-7g, were downregulated in CHB. Moreover, we identified CDK7 and TACC2 as novel target genes of miR-199a-3p. Our results suggest that altered miRNA expression in CHB contributes to HCC development, and that improvement of miRNA expression after NA treatment is associated with reduced HCC risk., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recent studies have shown that long noncoding RNAs (lncRNAs) have pivotal roles in human malignancies, although their significance in oral squamous cell carcinoma (OSCC) is not fully understood. In the present study, we identified lncRNAs functionally associated with OSCC. By analyzing RNA-seq datasets obtained from primary head and neck squamous cell carcinoma (HNSCC), we identified 15 lncRNAs aberrantly expressed in cancer tissues. We then validated their expression in 18 OSCC cell lines using qRT-PCR and identified 6 lncRNAs frequently overexpressed in OSCC. Among those, we found that knocking down DLEU1 (deleted in lymphocytic leukemia 1) strongly suppressed OSCC cell proliferation. DLEU1 knockdown also suppressed migration, invasion, and xenograft formation by OSCC cells, which is suggestive of its oncogenic functionality. Microarray analysis revealed that DLEU1 knockdown significantly affects expression of a number of cancer-related genes in OSCC cells, including HAS3, CD44, and TP63, suggesting that DLEU1 regulates HA-CD44 signaling. Expression of DLEU1 was elevated in 71% of primary OSCC tissues, and high DLEU1 expression was associated with shorter overall survival of HNSCC patients. These data suggest that elevated DLEU1 expression contributes to OSCC development, and that DLEU1 may be a useful therapeutic target in OSCC.

Background: Colorectal serrated lesions (SLs) are important premalignant lesions whose clinical and biological features are not fully understood., Aims: We aimed to establish accurate colonoscopic diagnosis and treatment of SLs through evaluation of associations among the morphological, pathological, and molecular characteristics of SLs., Methods: A total of 388 premalignant and 18 malignant colorectal lesions were studied. Using magnifying colonoscopy, microsurface structures were assessed based on Kudo's pit pattern classification system, and the Type II pit pattern was subcategorized into classical Type II, Type II-Open (Type II-O) and Type II-Long (Type II-L). BRAF/KRAS mutations and DNA methylation of CpG island methylator phenotype (CIMP) markers (MINT1, - 2, - 12, - 31, p16, and MLH1) were analyzed through pyrosequencing., Results: Type II-O was tightly associated with sessile serrated adenoma/polyps (SSA/Ps) with BRAF mutation and CIMP-high. Most lesions with simple Type II or Type II-L were hyperplastic polyps, while mixtures of Type II or Type II-L plus more advanced pit patterns (III/IV) were characteristic of traditional serrated adenomas (TSAs). Type II-positive TSAs frequently exhibited BRAF mutation and CIMP-low, while Type II-L-positive TSAs were tightly associated with KRAS mutation and CIMP-low. Analysis of lesions containing both premalignant and cancerous components suggested Type II-L-positive TSAs may develop into KRAS-mutated/CIMP-low/microsatellite stable cancers, while Type II-O-positive SSA/Ps develop into BRAF-mutated/CIMP-high/microsatellite unstable cancers., Conclusions: These results suggest that Type II subtypes reflect distinct molecular subclasses in the serrated neoplasia pathway and that they could be useful hallmarks for identifying SLs at high risk of developing into CRC.

AMP deaminase (AMPD) plays a crucial role in adenine nucleotide metabolism. Recently we found that upregulated AMPD activity is associated with ATP depletion and contractile dysfunction under the condition of pressure overloading in the heart of a rat model of type 2 diabetes mellitus (T2DM), OLETF. Here we examined the mechanism of AMPD upregulation by T2DM. The protein level of 90-kDa full-length AMPD3 was increased in whole myocardial lysates by 55% in OLETF compared to those in LETO, a non-diabetic control. In contrast, the mRNA levels of AMPD3 in the myocardium were similar in OLETF and LETO. AMPD3 was comparably ubiquitinated in OLETF and LETO, and its degradation ex vivo was more sensitive to MG-132, a proteasome inhibitor, in OLETF than in LETO. MicroRNA array analysis revealed downregulation (>50%) of 57 microRNAs in OLETF compared to those in LETO, among which miR-301b was predicted to interact with the 3'UTR of AMPD3 mRNA. AMPD3 protein level was significantly increased by a miR-301b inhibitor and was decreased by a miR-301b mimetic in H9c2 cells. A luciferase reporter assay confirmed binding of miR-301b to the 3'UTR of AMPD3 mRNA. Transfection of neonatal rat cardiomyocytes with a miR-301b inhibitor increased 90-kDa AMPD3 and reduced ATP level. The results indicate that translational regulation by miR-301b mediates upregulated expression of cardiac AMPD3 protein in OLETF, which potentially reduces the adenine nucleotide pool at the time of increased work load. The miR-301b-AMPD3 axis may be a novel therapeutic target for intervening enegy metabolism in diabetic hearts., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

In this study, we identified microRNAs (miRNAs) involved in cisplatin (CDDP) resistance in bladder cancer (BCa). After establishing CDDP-resistant BCa cell lines (T24RC and EJ138RC), TaqMan arrays revealed that members of the miR-200 family (miR-200b, miR-200a and miR-429) were downregulated in T24RC as compared to parental T24 cells. miR-200b was associated with CDDP sensitivity in BCa cells, and its downregulation was associated with CpG island hypermethylation. Pharmacological demethylation using 5-aza-2'-deoxycytidine restored miR-200b expression, and the combination of 5-aza-2'-deoxycytidine + CDDP strongly inhibited T24RC cell proliferation. Microarray analysis revealed that miR-200b + CDDP induced genes involved in CDDP sensitivity or cytotoxicity, including IGFBP3, ICAM1 and TNFSF10, in the resistant cells. Expression and DNA methylation of miR-200b were inversely associated in primary BCa, and low expression/high methylation was associated with poor overall survival. These results suggest downregulation of miR-200b is associated with CDDP resistance in BCa. Epigenetic silencing of miR-200b may be a marker of CDDP resistance and a useful therapeutic target for overcoming CDDP resistance in BCa., Competing Interests: CONFLICTS OF INTEREST All authors declare no conflicts of interest.

Objective: To clarify the clinical utility of urinary DNA methylation for detection of intravesical recurrence of non-muscle invasive BCa (NMIBC), we performed a 2-center prospective study., Patients and Methods: A series of 207 self-voided urine samples were prospectively collected from 132 patients with NMIBC who had undergone transurethral resection of BCa. Methylation of miRNA genes (miR-9-3, miR-124-2, miR-124-3, and miR-137) was analyzed using bisulfite pyrosequencing. The primary end point was detection of intravesical recurrence; the secondary end point was prediction of late recurrence. The number of methylated genes (M-score) or quantitative level of methylation were compared with outcomes., Results: Twenty-six urine specimens were collected on the same day intravesical recurrence was detected, and 14 were collected from patients whose recurrences were found during the subsequent follow-up period (0-632 days, mean, 342.2 days). For detection of current recurrence, M-scores achieved 61.5% sensitivity and 74.0% specificity, and the area under the ROC curve was 0.71. Regarding prediction of late recurrence, patients with a high M-score (≥3) showed worse recurrence-free survival (P <.01). Multivariate analysis revealed that high M-scores were independently associated with current (P = .028) and late recurrence (P = .026). Elevated levels of urinary DNA methylation were also strongly associated with recurrence and radical cystectomy., Conclusion: Our data suggest that urinary methylation of miRNA genes may be a useful marker for detecting and predicting BCa recurrence., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Most gastrointestinal stromal tumors (GISTs) harbor activating mutations in the receptor tyrosine kinase gene KIT or platelet-derived growth factor receptor alpha ( PDGFRA ), and the resultant activation of downstream signals plays a pivotal role in the development of GISTs. The sites of the tyrosine kinase gene mutations are associated with the biological behavior of GISTs, including risk category, clinical outcome and drug response. Mutations in RAS signaling pathway genes, including KRAS and BRAF, have also been reported in KIT/PDGFRA wild-type GISTs, though they are rare. Neurofibromin 1 ( NF1 ) is a tumor suppressor gene mutated in neurofibromatosis type 1. Patients with NF1 mutations are at high risk of developing GISTs. Recent findings suggest that altered expression or mutation of members of succinate dehydrogenase (SDH) heterotetramer are causally associated with GIST development through induction of aberrant DNA methylation. At present, GISTs with no alterations in KIT, PDGFRA , RAS signaling genes or SDH family genes are referred to as true wild-type GISTs. KIT and PDGFRA mutations are thought as the earliest events in GIST development, and subsequent accumulation of chromosomal aberrations and other molecular alterations are required for malignant progression. In addition, recent studies have shown that epigenetic alterations and noncoding RNAs also play key roles in the pathogenesis of GISTs., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare.

Colorectal sessile serrated adenoma/polyps (SSA/Ps) are well-known precursors of colorectal cancer (CRC) characterized by BRAF mutation and microsatellite instability. By contrast, the molecular characteristics of traditional serrated adenoma (TSAs) are not fully understood. We analyzed genome-wide DNA methylation in TSAs having both protruding and flat components. We identified 11 genes, including SMOC1 , methylation of which progressively increased during the development of TSAs. SMOC1 was prevalently methylated in TSAs, but was rarely methylated in SSA/Ps ( p < 0.001). RT-PCR and immunohistochemistry revealed that SMOC1 was expressed in normal colon and SSA/Ps, but its expression was decreased in TSAs. Ectopic expression of SMOC1 suppressed proliferation, colony formation and in vivo tumor formation by CRC cells. Analysis of colorectal lesions ( n = 847) revealed that SMOC1 is frequently methylated in TSAs, high-grade adenomas and CRCs. Among these, SMOC1 methylation was strongly associated with KRAS mutation and CpG island methylator phenotype (CIMP)-low. These results demonstrate that epigenetic silencing of SMOC1 is associated with TSA development but is rarely observed in SSA/Ps. SMOC1 expression could thus be a diagnostic marker of serrated lesions, and SMOC1 methylation could play a role in neoplastic pathways in TSAs and conventional adenomas., Competing Interests: CONFLICTS OF INTEREST All authors declare no conflict of interest.

Although dysregulation of microRNAs (miRNAs/miRs) is a common feature of human malignancies, its involvement in gastrointestinal stromal tumors (GISTs) is not fully understood. The present study aimed to identify the miRNAs that perform a role in GIST metastasis. miRNA expression profiles from a series of 32 primary GISTs were analyzed using microarrays, and miR-186 was observed to be downregulated in tumors exhibiting metastatic recurrence. Reverse transcription-quantitative polymerase chain reaction analysis of an independent cohort of 100 primary GISTs revealed that low miR-186 expression is associated with metastatic recurrence and a poor prognosis. Inhibition of miR-186 in GIST-T1 cells promoted cell migration. Gene expression microarray analysis demonstrated that miR-186 inhibition upregulated a set of genes implicated in cancer metastasis, including insulin-like growth factor-binding protein 3, AKT serine/threonine kinase 2, hepatocyte growth factor receptor, CXC chemokine receptor 4 and epidermal growth factor-containing fibulin-like extracellular matrix protein 1. These results suggest that the downregulation of miR-186 is involved in the metastatic recurrence of GISTs, and that miR-186 levels could potentially be a predictive biomarker for clinical outcome.

Diacylglycerol kinases (DGKs) are important regulators of cell signaling and have been implicated in human malignancies. Whether epigenetic alterations are involved in the dysregulation of DGKs in cancer is unknown, however. We therefore analyzed methylation of the promoter CpG islands of DGK genes in colorectal cancer (CRC) cell lines. We found that DGKG, which encodes DGKγ, was hypermethylated in all CRC cell lines tested (n = 9), but was not methylated in normal colonic tissue. Correspondingly, DGKG expression was suppressed in CRC cell lines but not in normal colonic tissue, and was restored in CRC cells by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC). DGKG methylation was frequently observed in primary CRCs (73/141, 51.8%) and was positively associated with KRAS and BRAF mutations and with the CpG island methylator phenotype (CIMP). DGKG methylation was also frequently detected in colorectal adenomas (89 of 177, 50.3%), which suggests it is an early event during colorectal tumorigenesis. Ectopic expression of wild-type DGKγ did not suppress CRC cell proliferation, but did suppress cell migration and invasion. Notably, both constitutively active and kinase-dead DGKγ mutants exerted inhibitory effects on CRC cell proliferation, migration and invasion, and the wild-type and mutant forms of DGKγ all suppressed Rac1 activity in CRC cells. These data suggest DGKG may play a tumor suppressor role in CRC., (© 2017 Wiley Periodicals, Inc.)

Aberrant DNA methylation is commonly observed in colorectal cancer (CRC), but the underlying mechanism is not fully understood. 5-hydroxymethylcytosine levels and TET1 expression are both reduced in CRC, while epigenetic silencing of TET1 is reportedly associated with the CpG island methylator phenotype. In the present study, we aimed to clarify the relationship between loss of TET1 and aberrant DNA methylation in CRC. Stable TET1 knockdown clones were established using Colo320DM cells, which express high levels of TET1, and HCT116 cells, which express TET1 at a level similar to that in normal colonic tissue. Infinium HumanMethylation450 BeadChip assays revealed increased levels of 5-methylcytosine at more than 10,000 CpG sites in TET1-depleted Colo320DM cells. Changes in DNA methylation were observed at various positions within the genome, including promoters, gene bodies and intergenic regions, and the altered methylation affected expression of a subset of genes. By contrast, TET1 knockdown did not significantly affect DNA methylation in HCT116 cells. However, TET1 depletion was associated with attenuated effects of 5-aza-2'-deoxycytidine on gene expression profiles in both cell lines. These results suggest that loss of TET1 may induce aberrant DNA methylation and may attenuate the effect of 5-aza-2'-deoxycytidine in CRC cells., Competing Interests: The authors have declared that no competing interests exist.

To clarify the molecular and clinicopathological characteristics of colorectal serrated lesions, we assessed the DNA methylation of cancer-associated genes in a cohort of BRAF-mutant precancerous lesions from 94 individuals. We then compared those results with the lesions' clinicopathological features, especially colorectal subsites. The lesions included hyperplastic polyps (n = 16), traditional serrated adenomas (TSAs) (n = 15), TSAs with sessile serrated adenomas (SSAs) (n = 6), SSAs (n = 49) and SSAs with dysplasia (n = 16). The prevalence of lesions exhibiting the CpG island methylator phenotype (CIMP) was lower in the sigmoid colon and rectum than in other bowel subsites, including the cecum, ascending, transverse and descending colon. In addition, several cancer-associated genes showed higher methylation levels within lesions in the proximal to sigmoid colon than in the sigmoid colon and rectum. These results indicate that the methylation status of lesions with BRAF mutation is strongly associated with their location, histological findings and neoplastic pathways. By contrast, no difference in aberrant DNA methylation was observed in normal-appearing background colonic mucosa along the bowel subsites, which may indicate the absence of an epigenetic field defect., Competing Interests: All authors declare no conflicts of interest.

Long noncoding RNAs (lncRNAs) have emerged as key components in multiple cellular processes, although their physiological and pathological functions are not fully understood. To identify cancer-related lncRNAs, we screened for those that are epigenetically silenced in colorectal cancer (CRC). Through a genome-wide analysis of histone modifications in CRC cells, we found that the transcription start sites (TSSs) of 1,027 lncRNA genes acquired trimethylation of histone H3 lysine 4 (H3K4me3) after DNA demethylation. Integrative analysis of chromatin signatures and the DNA methylome revealed that the promoter CpG islands (CGIs) of 66 lncRNA genes contained cancer-specific methylation. By validating the expression and methylation of lncRNA genes in CRC cells, we ultimately identified 20 lncRNAs, including ZNF582-AS1, as targets of epigenetic silencing in CRC. ZNF582-AS1 is frequently methylated in CRC cell lines (87.5%), primary CRCs (77.2%), colorectal adenomas (44.7%) and advanced adenomas (87.8%), suggesting that this methylation is an early event during colorectal tumorigenesis. Methylation of ZNF582-AS1 is associated with poor survival of CRC patients, and ectopic expression of ZNF582-AS1 suppressed colony formation by CRC cells. Our findings offer insight into the association between epigenetic alterations and lncRNA dysregulation in cancer and suggest that ZNF582-AS1 may be a novel tumor-suppressive lncRNA.

Epigenetic alterations, including aberrant DNA methylation and histone modification, play key roles in the dysregulation of tumor-related genes, thereby affecting numerous cellular processes, including cell proliferation, cell adhesion, apoptosis, and metastasis. In recent years, studies have demonstrated that short and long noncoding RNAs (ncRNAs) are key players in the initiation and progression of cancer, and epigenetic mechanisms are deeply involved in their dysregulation. Indeed, the growing list of microRNA (miRNA) genes aberrantly methylated in cancer suggests that a large number of miRNAs act as tumor suppressors or oncogenes. In addition, emerging evidence suggests that dysregulation of long ncRNAs (lncRNAs) plays critical roles in tumorigenesis. And because ncRNAs are involved in regulating gene expression through interaction with epigenetic modifiers, their dysregulation appears causally related to epigenetic alterations in cancer. Dissection of the interrelationships between ncRNAs and epigenetic alterations has the potential to reveal novel approaches to the diagnosis and treatment of cancer.

Our aim was to identify DNA methylation changes associated with the growth pattern and invasiveness of colorectal cancers (CRCs). Comparison of the methylation statuses of large (≥ 20 mm in diameter along the colonic surface) noninvasive tumors (NTs) and small (<20 mm in diameter along the colonic surface) invasive tumors (ITs) using CpG island microarray analysis showed neurotensin receptor 1 (NTSR1) to be hypermethylated in large NTs. Quantitative bisulfite pyrosequencing revealed that NTSR1 is frequently methylated in colorectal tumors, with large NTs exhibiting the highest methylation levels. The higher NTSR1 methylation levels were associated with better prognoses. By contrast, NTSR1 copy number gains were most frequent among small ITs. Methylation of NTSR1 was associated with the gene's silencing in CRC cell lines, whereas ectopic expression of NTSR1 promoted proliferation and invasion by CRC cells. Analysis of primary tumors composed of adenomatous and malignant portions revealed that NTSR1 is frequently methylated in the adenomatous portion, while methylation levels are generally lower in the cancerous portions. These results suggest that NTSR1 methylation is associated with lateral and noninvasive growth of colorectal tumors, while low levels of methylation may contribute to the malignant potential through activation of NTSR1. Our data also indicate that NTSR1 methylation may be a prognostic biomarker in CRC.

Background: The common disease-common variant hypothesis is insufficient to explain the complexities of Crohn's disease (CD) genetics; therefore, rare variants are expected to be important in the disease. We explored rare variants associated with susceptibility to CD in Japanese individuals by personal genomic analysis., Methods: Two-step analyses were performed. The first step was a trio analysis with whole-exome sequence (WES) analysis and the second was a follow-up case-control association study. The WES analysis pipeline comprised Burrows-Wheeler Aligner, Picard, Genome Analysis Toolkit, and SAMTOOLS. Single nucleotide variants (SNVs)/indels were annotated and filtered by using programs implemented in ANNOVAR in combination with identity-by-descent (IBD), subsequently were subjected to the linkage based, and de novo based strategies. Finally, we conducted an association study that included 176 unrelated subjects with CD and 358 healthy control subjects., Results: In family members, 234,067-297,523 SNVs/indels were detected and they were educed to 106-146 by annotation based filtering. Fifty-four CD variants common to both individuals of the affected sib pair were identified. The linkage based strategy detected five candidate variants whereas the de novo based strategy identified no variants. Consequently, five candidates were analyzed in the case-control association study. CD showed a significant association with one variant in exon 4 of IL23R, G149R [rs76418789, P = 3.9E-5, odds ratio (OR) 0.21, 95% confidence interval (CI) 0.09-0.47 for the dominant model (AA + AG versus GG), and P = 7.3E-5, OR 0.21, 95% CI 0.10-0.48 for AG versus GG, and P = 7.2E-5, OR 0.23, 95% CI 0.10-0.50 for the allele model]., Conclusions: The present study, using personal genomics analysis of a small CD pedigree, is the first to show that the low-frequency non-synonymous variant of IL23R, rs76418789, protects against CD development in Japanese subjects.

Background: Dysregulation of microRNA (miRNA) has been implicated in gastrointestinal stromal tumors (GISTs) but the mechanism is not fully understood. In this study, we aimed to explore the involvement of epigenetic alteration of miRNA genes in GISTs., Methods: GIST-T1 cells were treated with 5-aza-2'-deoxycytidine (5-aza-dC) and 4-phenylbutyric acid (PBA), after which miRNA expression profiles were analyzed using TaqMan miRNA arrays. DNA methylation was then analyzed using bisulfite pyrosequencing. The functions of miRNAs were examined using MTT assays, wound-healing assays, Boyden chamber assays and Matrigel invasion assays. Gene expression microarrays were analyzed to assess effect of ectopic miRNA expression in GIST-T1 cells., Results: Of the 754 miRNAs analyzed, 61 were significantly upregulated in GIST-T1 cells treated with 5-aza-dC plus PBA. Among those, 21 miRNA genes were associated with an upstream CpG island (CGI), and the CGIs of miR-34a and miR-335 were frequently methylated in GIST-T1 cells and primary GIST specimens. Transfection of miR-34a or miR-335 mimic molecules into GIST-T1 cells suppressed cell proliferation, and miR-34a also inhibited migration and invasion by GIST-T1 cells. Moreover, miR-34a downregulated a number of predicted target genes, including PDGFRA. RNA interference-mediated knockdown of PDGFRA in GIST-T1 cells suppressed cell proliferation, suggesting the tumor suppressive effect of miR-34a is mediated, at least in part, through targeting PDGFRA., Conclusions: Our results suggest that miR-34a and miR-335 are candidate tumor suppressive miRNAs in GISTs, and that they are frequent targets of epigenetic silencing in GISTs.

Cancers exhibiting the CpG island methylator phenotype (CIMP) are found among a wide variety of human malignancies and represent a subclass of tumors showing concurrent hypermethylation of multiple CpG islands. These CIMP-positive tumors often exhibit characteristic molecular and clinicopathological features, suggesting CIMP represents a distinct carcinogenic pathway. However, marker genes to define CIMP have been largely inconsistent among studies, which has caused results to vary. Nonetheless, recent advances in genome-wide methylation analysis have enabled the existence of CIMP to be confirmed, and large-scale cancer genome analyses have begun to unravel the previously unknown molecular basis of CIMP tumors. CIMP is strongly associated with clinical outcome, suggesting it may be a predictive biomarker., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Aberrant DNA methylation could potentially serve as a biomarker for colorectal neoplasms. In this study, we assessed the feasibility of using DNA methylation detected in bowel lavage fluid (BLF) for colorectal cancer screening. A total of 508 BLF specimens were collected from patients with colorectal cancer (n = 56), advanced adenoma (n = 53), minor polyp (n = 209), and healthy individuals (n = 190) undergoing colonoscopy. Methylation of 15 genes (miR-1-1, miR-9-1, miR-9-3, miR-34b/c, miR-124-1, miR-124-2, miR-124-3, miR-137, SFRP1, SFRP2, APC, DKK2, WIF1, LOC386758, and ZNF582) was then analyzed in MethyLight assays, after which receiver operating characteristic (ROC) curves were analyzed to assess the diagnostic performance of BLF methylation. Through analyzing BLF specimens in a training set (n = 345), we selected the three genes showing the greatest sensitivity for colorectal cancer detection (miR-124-3, 71.8%; LOC386758, 79.5%; and SFRP1, 74.4%). A scoring system based on the methylation of those three genes (M-score) achieved 82% sensitivity and 79% specificity, and the area under the ROC curve (AUC) was 0.834. The strong performance of this system was then validated in an independent test set (n = 153; AUC = 0.808). No significant correlation was found between M-score and the clinicopathologic features of the colorectal cancers. Our results demonstrate that DNA methylation in BLF specimens may be a useful biomarker for the detection of colorectal cancer., (©2014 American Association for Cancer Research.)

Background: Metachronous gastric cancer (GC) can develop after endoscopic resection of GC and cannot be predicted based on clinical signature. Aberrant DNA methylation in noncancerous gastric mucosa is strongly implicated in gastric carcinogenesis and could be a useful biomarker of GC risk. We evaluated the clinical utility of DNA methylation as a biomarker of metachronous GC risk., Method: We carried out scheduled follow-up endoscopy in 129 patients after curative endoscopic resection of GC. Biopsy specimens were collected from noncancerous mucosa in the gastric antrum and body, after which quantitative methylation analysis of miR-34b/c, SFRP1, SFRP2, SFRP5, DKK2 and DKK3 was carried out using bisulfite pyrosequencing. The utility of the methylation for predicting the risk of metachronous GC development was assessed using Kaplan-Meier and Cox proportional hazards model analyses., Results: During the follow-up period, 17 patients (13%) developed metachronous GCs. The cumulative incidence of metachronous GC was significantly higher among patients with elevated miR-34b/c, SFRP2 and DKK2 methylation in their gastric body. MiR-34b/c showed the strongest association with the risk of metachronous GC, and the cumulative incidence of metachronous GC was much higher in the high-miR-34b/c-methylation group than the low-methylation group. Multivariate analysis adjusted for age, sex, H. pylori status and pathological findings showed miR-34b/c methylation in gastric body to be an independent predictor of metachronous GC risk., Conclusion: Our results suggest that methylation of miR-34b/c in the mucosa of the noncancerous gastric body may be a useful biomarker for predicting the risk of metachronous GC.

Large intergenic noncoding RNAs (lincRNA) have been less studied than miRNAs in cancer, although both offer considerable theranostic potential. In this study, we identified frequent upregulation of miR-196a and lincRNA HOTAIR in high-risk gastrointestinal stromal tumors (GIST). Overexpression of miR-196a was associated with high-risk grade, metastasis and poor survival among GIST specimens. miR-196a genes are located within the HOX gene clusters and microarray expression analysis revealed that the HOXC and HOTAIR gene were also coordinately upregulated in GISTs which overexpress miR-196a. In like manner, overexpression of HOTAIR was also strongly associated with high-risk grade and metastasis among GIST specimens. RNA interference-mediated knockdown of HOTAIR altered the expression of reported HOTAIR target genes and suppressed GIST cell invasiveness. These findings reveal concurrent overexpression of HOX genes with noncoding RNAs in human cancer in this setting, revealing miR-196a and HOTAIR as potentially useful biomarkers and therapeutic targets in malignant GISTs.

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that the silencing of some miRNAs is associated with CpG island hypermethylation. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by treatment with 5-aza-2'-deoxycytidine and 4-phenylbutyrate. We found that miR-34b and miR-34c are epigenetically silenced in GC and that their downregulation is associated with hypermethylation of the neighboring CpG island. Methylation of the miR-34b/c CpG island was frequently observed in GC cell lines (13/13, 100%) but not in normal gastric mucosa from Helicobacter pylori-negative healthy individuals. Transfection of a precursor of miR-34b and miR-34c into GC cells induced growth suppression and dramatically changed the gene expression profile. Methylation of miR-34b/c was found in a majority of primary GC specimens (83/118, 70%). Notably, analysis of non-cancerous gastric mucosae from GC patients (n = 109) and healthy individuals (n = 85) revealed that methylation levels are higher in gastric mucosae from patients with multiple GC than in mucosae from patients with single GC (27.3 versus 20.8%; P < 0.001) or mucosae from H. pylori-positive healthy individuals (27.3 versus 20.7%; P < 0.001). These results suggest that miR-34b and miR-34c are novel tumor suppressors frequently silenced by DNA methylation in GC, that methylation of miR-34b/c is involved in an epigenetic field defect and that the methylation might be a predictive marker of GC risk.

Purpose: Gastrointestinal stromal tumors (GIST) are the most important mesenchymal tumors of the gastrointestinal tract. The vast majority of GISTs exhibit activating mutations of KIT or PDGFRA, but epigenetic alteration of GISTs is largely unknown. In this study, we aimed to clarify the involvement of DNA methylation in GIST malignancy., Experimental Design: A total of 106 GIST specimens were studied. Levels of LINE-1 methylation were analyzed using bisulfite pyrosequencing. In addition, methylation of three other repetitive sequences (Alu Yb8, Satellite-α, and NBL2) was similarly analyzed, and CpG island hypermethylation was analyzed using MethyLight. Array-based comparative genomic hybridization (array CGH) was carried out in 25 GIST specimens., Results: LINE-1 hypomethylation was significantly correlated with risk, and high-risk GISTs exhibited significantly lower levels of LINE-1 methylation than low-risk (61.3% versus 53.2%; P = 0.001) or intermediate-risk GISTs (60.8% versus 53.2%; P = 0.002). Hypomethylation of Satellite-α and NBL2 was also observed in high-risk GISTs. By contrast, promoter hypermethylation was relatively infrequent (CDH1, 11.2%; MLH1, 9.8%; SFRP1, 1.2%; SFRP2, 11.0%; CHFR, 9.8%; APC, 6.1%; CDKN2A, 0%; RASSF1A, 0%; RASSF2, 0%) and did not correlate with LINE-1 methylation or risk. Array CGH analysis revealed a significant correlation between LINE-1 hypomethylation and chromosomal aberrations., Conclusions: Our data suggest that LINE-1 hypomethylation correlates significantly with the aggressiveness of GISTs and that LINE-1 methylation could be a useful marker for risk assessment. Hypomethylation may increase the malignant potential of GISTs by inducing accumulation of chromosomal aberrations., (©2010 AACR.)