Antibiotics suppress colon tumorigenesis through inhibition of aberrant DNA methylation in an azoxymethane and dextran sulfate sodium colitis model

Chronic inflammation is involved in the development of colon cancer by inducing mutations and aberrant DNA methylation in colon epithelial cells. Furthermore, there is growing evidence that colonic microbiota modulates the inflammation response in the host and influences colon tumorigenesis. However, the influence of colonic microbiota on aberrant DNA methylation remains unknown. Here, we show the effect of colonic microbes on DNA methylation and tumorigenicity using a mouse model of human ulcerative colitis. Mice treated with azoxymethane (AOM) and dextran sulfate sodium (DSS) showed an increase in degree of colitis, as estimated by body weight, occult blood, and stool consistency/diarrhea at 2 weeks after treatment, but treatment with antibiotics markedly reduced the severity of the colitis. Although mucosal hyperplasia and increased inflammation‐related genes were observed in the colonic epithelial cells of the AOM/DSS‐treated mice, treatment with antibiotics abrogated these changes. In addition, treatment with antibiotics significantly decreased the number of mucosal nodules from 5.9 ± 5.3 to 0.2 ± 0.6 (P < .01) and area of occupancy from 50.1 ± 57.4 to 0.5 ± 1.4 mm² (P < .01). Aberrant DNA methylation of three marker CpG islands (Cbln4, Fosb, and Msx1) was induced by AOM/DSS treatment in colonic mucosae, but this increase was suppressed by 50%‐92% (P < .05) with antibiotic treatment. Microbiome analysis showed that this change was associated with a decrease of the Clostridium leptum subgroup. These data indicate that antibiotics suppressed tumorigenesis through inhibition of aberrant DNA methylation induced by chronic inflammation.     

Epigenetic inactivation of endothelin‐2 and endothelin‐3 in colon cancer

Endothelin‐1 (ET‐1) and its receptors are overexpressed in human cancers, but much less is known about the roles of ET‐2 and ET‐3 in cancer etiology. We sought to examine human and rat colon tumors for dysregulation of ET‐2 and ET‐3 expression and determine the underlying mechanisms. Human primary colon cancers and carcinogen‐induced rat colon tumors were subjected to real‐time RT‐PCR, immunoblotting and immunohistochemistry; EDN2 and EDN3 genes were examined by methylation‐specific PCR, bisulfite sequencing and pyrosequencing; and forced expression of ET‐2 and ET‐3 was conducted in human colon cancer cells followed by real‐time cell migration and invasion assays. Rat and human colon tumors had markedly reduced expression of ET‐2 and ET‐3 mRNA and protein compared with matched controls. Mechanistic studies revealed hypermethylation of EDN2 and EDN3 genes in human primary colon cancers and in a panel of human colon cancer cell lines. Forced expression of ET‐2 and ET‐3 attenuated significantly the migration and invasion of human colon cancer cells. We conclude that epigenetic inactivation of ET‐2 and ET‐3 occurs frequently in both rat and human colon cancers. Current therapeutic strategies target overexpressed members of the ET axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the re‐expression of ET‐2 and ET‐3 as natural antagonists of ET‐1 in colon cancer.     

粪便DNA甲基化检测在结直肠癌早期诊断中的研究进展

粪便基因甲基化作为一种新的、敏感度较高、特异性中等的无创性结直肠肿瘤标志物,能根据其阳性结果对疑似病例进行进一步检测,可作为筛查诊断结直肠肿瘤的一种重要的辅助手段。目前已有较多研究报道检测粪便中基因异常甲基化对于早期结直肠癌（colorectal cancer,CRC）筛查有较高的敏感性和特异性,但尚无任何一种甲基化基因真正地应用于临床实践。本文对目前结直肠癌早期粪便DNA甲基化检测的国内外相关研究成果进行综述,总结粪便甲基化检测的潜在临床价值。     

Aberrant DNA methylation in cervical carcinogenesis

Persistent infection wit h high-risk types of human papillomavirus(HPV) is known to cause cervical cancer;however,additional genetic and epigenetic alterations are required for progression from precancerous disease to invasive cancer.DNA methylation is an early and frequent molecular alteration in cervical carcinogenesis.In this review,we summarize DNA methylation within the HPV genome and human genome and identify its clinical implications.Methylation of the HPV long control region(LCR) and L1 gene is common during cervical carcinogenesis and increases with the severity of the cervical neoplasm.The L1 gene of HPV16 and HPV18 is consistently hypermethylated in invasive cervical cancers and can potentially be used as a clinical marker of cancer progression.Moreover,promoters of tumor suppressor genes(TSGs) involved in many cellular pathways are methylated in cervical precursors and invasive cancers.Some are associated with squamous cell carcinomas,and others are associated with adenocarcinomas.Identification of methylated TSGs in Pap smear could be an adjuvant test in cervical cancer screening for triage of women with high-risk HPV,atypical squamous cells of undetermined significance,or low grade squamous intraepithelial lesion(LSIL).However,consistent panels must be validated for this approach to be translated to the clinic.Furthermore,reversion of methylated TSGs using demethylating drugs may be an alternative anticancer treatment,but demethylating drugs without toxic carcinogenic and mutagenic properties must be identified and validated.     

DNA methylation in thoracic neoplasms

Thoracic neoplasms, which include lung cancers, esophageal carcinoma, and thymic epithelial tumors, are the leading causes of tumor-related death and a major health concern worldwide. The development of neoplasms is a multistep process involving both genetic and epigenetic alterations. A growing body of research provides evidence that aberrant DNA methylation, including DNA hypermethylation in promoter regions, global DNA hypomethylation and the overexpression of DNA methyltransferases, plays an important role in tumorigenesis. In this review, we summarize published observations of methylation pattern disruptions in thoracic tumors, and discuss how these abnormalities contribute to the development of cancers. We review recent findings showing that suppressing the activity of the DNA methylating enzymes DNMTs can have potent anti-cancer effects, and discuss the possibility of developing novel therapies for thoracic tumors based on DNMT inhibition.     

DNA异常甲基化在前列腺癌发生发展及诊疗中的研究进展

前列腺癌是全球男性常发的恶性肿瘤之一。近年来研究发现前列腺癌的发生发展很大程度上是由表观遗传修饰所驱动的,其中最重要的当属DNA异常甲基化。DNA启动子的异常甲基化会造成基因的异常表达,从而调控着前列腺癌的发生发展过程。此外,目前前列腺癌的诊断仍然依靠侵入性的前列腺穿刺活检,而基于DNA异常甲基化的无创性液体活检有望成为未来前列腺癌分子诊断的发展趋势,同时也为前列腺癌提供了新的治疗靶标。本文就DNA甲基化在前列腺癌发生发展、早期诊断、预后评估以及治疗中的研究进展进行综述。     

Commonality and differences of methylation signatures in the plasma of patients with pancreatic cancer and colorectal cancer

Profiling of DNA methylation status of specific genes is a way to screen for colorectal cancer (CRC) and pancreatic cancer (PC) in blood. The commonality of methylation status of cancer‐related tumor suppressor genes between CRC and PC is largely unknown. Methylation status of 56 cancer‐related genes was compared in plasma of patients in the following cohorts: CRC, PC and healthy controls. Cross validation determined the best model by area under ROC curve (AUC) to differentiate cancer methylation profiles from controls. Optimal preferential gene methylation signatures were derived to differentiate either cancer (CRC or PC) from controls. For CRC alone, a three gene signature (CYCD2, HIC and VHL) had an AUC 0.9310, sensitivity (Sens) = 0.826, specificity (Spec) = 0.9383. For PC alone, an optimal signature consisted of five genes (VHL, MYF3, TMS, GPC3 and SRBC), AUC 0.848; Sens = 0.807, Spec = 0.666. Combined PC and CRC signature or “combined cancer signature” was derived to differentiate either CRC and PC from controls (MDR1, SRBC, VHL, MUC2, RB1, SYK and GPC3) AUC = 0.8177, Sens = 0.6316 Spec = 0.840. In a validation cohort, N = 10 CRC patients, the optimal CRC signature (CYCD2, HIC and VHL) had AUC 0.900. In all derived signatures (CRC, PC and combined cancer signature) the optimal panel used preferential VHL methylation. In conclusion, CRC and PC differ in specific genes methylated in plasma other than VHL. Preferential methylation of VHL is shared in the optimal signature for CRC alone, PC alone and combined PC and CRC. Future investigations may identify additional methylation markers informative for the presence of both CRC and PC.     

Aberrant DNA methylation of tumor-related genes in oral rinse: a noninvasive method for detection of oral squamous cell carcinoma

BACKGROUND:

The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor‐related genes with the objective of establishing a noninvasive method for the detection of OSCC.

METHODS:

Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation‐specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection.

RESULTS:

Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E‐cadherin (ECAD), transmembrane protein with epidermal growth factor‐like and 2 follistatin‐like domains 2 (TMEFF2), retinoic acid receptor beta (RARβ), and O‐6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARβ, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT.

CONCLUSIONS:

The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC. Cancer 2012. © 2012 American Cancer Society.     

甲状腺癌的DNA甲基化研究进展

DNA甲基化是一种重要的表观遗传学改变,在肿瘤的发病、诊断、预后评估及治疗中都有重要的临床意义.甲状腺癌是临床最常见的内分泌系统恶性肿瘤,目前国内外关于甲状腺癌的DNA甲基化的研究相对较少,该文总结近年来甲状腺癌领域相关的基因甲基化研究,就甲状腺癌的DNA甲基化研究进展进行综述.     

DNA甲基化在肺癌中的研究进展

表观遗传学主要是研究基因表达的变化,而这种变化能够通过减数分裂遗传,但不涉及有关基因碱基序列的改变.随着肿瘤发病机制研究在分子生物学方面的逐步深入,发现DNA甲基化、非编码RNA表达调控和组蛋白的修饰等表观遗传学(epigenetics)改变引起的调控机制异常与人体多种恶性肿瘤的发生发展过程有着密切联系.研究表明抑癌基因启动子区高度DNA甲基化状态在恶性肿瘤的发生发展和侵袭转移中发挥着重要作用.在肺癌发生的早期,出现很多DNA甲基化状态的改变.因此,甲基化检测可能对肺癌患者的早期诊断、治疗及预后评估具有重要意义.     

Aberrant DNA methylation of cancer-associated genes in gastric cancer in the European Prospective Investigation into Cancer and Nutrition (EPIC-EURGAST)

Epigenetic events have emerged as key mechanisms in the regulation of critical biological processes and in the development of a wide variety of human malignancies, including gastric cancer (GC), however precise gene targets of aberrant DNA methylation in GC remain largely unknown. Here, we have combined pyrosequencing-based quantitative analysis of DNA methylation in 98 GC cases and 64 controls nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort and in cancer tissue and non-tumorigenic adjacent tissue of an independent series of GC samples. A panel of 10 cancer-associated genes (CHRNA3, DOK1, MGMT, RASSF1A, p14ARF, CDH1, MLH1, ALDH2, GNMT and MTHFR) and LINE-1 repetitive elements were included in the analysis and their association with clinicopathological characteristics (sex, age at diagnosis, anatomical sub-site, histological sub-type) was examined. Three out of the 10 genes analyzed exhibited a marked hypermethylation, whereas two genes (ALDH2 and MTHFR) showed significant hypomethylation, in gastric tumors. Among differentially methylated genes, we identified new genes (CHRNA3 and DOK1) as targets of aberrant hypermethylation in GC, suggesting that epigenetic deregulation of these genes and their corresponding cellular pathways may promote the development and progression of GC. We also found that global demethylation of tumor cell genomes occurs in GC, consistent with the notion that abnormal hypermethylation of specific genes occurs concomitantly with genome-wide hypomethylation. Age and gender had no significant influence on methylation states, but an association was observed between LINE-1 and MLH1 methylation levels with histological sub-type and anatomical sub-site. This study identifies aberrant methylation patters in specific genes in GC thus providing information that could be exploited as novel biomarkers in clinics and molecular epidemiology of GC.     

Detection of aberrant methylation of HOXA9 and HIC1 through multiplex MethyLight assay in serum DNA for the early detection of epithelial ovarian cancer

Accumulated evidence revealed that aberrant CpG island hypermethylation plays an important role in carcinogenesis which can serve as a promising target for molecular detection in body fluids. Despite a myriad of attempts to diagnose ovarian cancer (OC) at an early stage, this clinical aim remains a major challenge. To date, no single biomarker is able to accurately detect early OC in either tissue or body fluid. Aberrant DNA methylation patterns in circulating DNA provide highly specific cancer signals. In our study, we establish a novel panel of methylation-specific genes for the development of a TaqMan based qPCR assay to quantify methylation levels. We analyzed promoter methylation of homeobox A9 (HOXA9) and hypermethylated in cancer 1 (HIC1) quantitatively in 120 tissue samples and in 70 matched serum cell-free DNA (CFDNA) of cancerous and noncancerous samples by MethyLight assay. HOXA9 and HIC1 methylation occurred in 82.3 and 80.0% of OC tissue samples in singleplex assay, thereby confirming that methylation was highly cancer-specific. When either or both gene promoter showed methylation, the sensitivity was 88.2% with a specificity of 88.6% in tissue samples. The combined sensitivity for this novel marker panel in serum CFDNA was 88.9% (area under the curve [AUC] = 0.95). In contrast, no hypermethylation was observed in serum from matched cancer-free control women. Our results confirm the elevated performance of novel epigenetic marker panel (HOXA9 and HIC1) when analyzed in tissue and matched serum samples. Our findings reveal the potential of this biomarker panel as a suitable diagnostic serum biomarker for early screening of OC.