Both hypomethylation and hypermethylation in a 0.2-kb region of a DNA repeat in cancer

NBL2 is a tandem 1.4-kb DNA repeat, whose hypomethylation in hepatocellular carcinomas was shown previously to be an independent predictor of disease progression. Here, we examined methylation of all cytosine residues in a 0.2-kb subregion of NBL2 in ovarian carcinomas, Wilms' tumors, and diverse control tissues by hairpin-bisulfite PCR. This new genomic sequencing method detects 5-methylcytosine on covalently linked complementary strands of a DNA fragment. All DNA clones from normal somatic tissues displayed symmetrical methylation at seven CpG positions and no methylation or only hemimethylation at two others. Unexpectedly, 56% of cancer DNA clones had decreased methylation at some normally methylated CpG sites as well as increased methylation at one or both of the normally unmethylated sites. All 146 DNA clones from 10 cancers could be distinguished from all 91 somatic control clones by assessing methylation changes at three of these CpG sites. The special involvement of DNA methyltransferase 3B in NBL2 methylation was indicated by analysis of cells from immunodeficiency, centromeric region instability, and facial anomalies syndrome patients who have mutations in the gene encoding DNA methyltransferase 3B. Blot hybridization of 33 cancer DNAs digested with CpG methylation-sensitive enzymes confirmed that NBL2 arrays are unusually susceptible to cancer-linked hypermethylation and hypomethylation, consistent with our novel genomic sequencing findings. The combined Southern blot and genomic sequencing data indicate that some of the cancer-linked alterations in CpG methylation are occurring with considerable sequence specificity. NBL2 is an attractive candidate for an epigenetic cancer marker and for elucidating the nature of epigenetic changes in cancer.     

Potential applications of DNA methylation testing technology in female tumors and screening methods

DNA methylation is a common epigenetic modification, and the current commonly used methods for DNA methylation detection include methylation-specific PCR, methylation-sensitive restriction endonuclease-PCR, and methylation-specific sequencing. DNA methylation plays an important role in genomic and epigenomic studies, and combining DNA methylation with other epigenetic modifications, such as histone modifications, may lead to better DNA methylation. DNA methylation also plays an important role in the development of disease, and analyzing changes in individual DNA methylation patterns can provide individualized diagnostic and therapeutic solutions. Liquid biopsy techniques are also increasingly well established in clinical practice and may provide new methods for early cancer screening. It is important to find new screening methods that are easy to perform, minimally invasive, patient-friendly, and affordable. DNA methylation mechanisms are thought to have an important role in cancer and have potential applications in the diagnosis and treatment of female tumors. This review discussed early detection targets and screening methods for common female tumors such as breast, ovarian, and cervical cancers and discussed advances in the study of DNA methylation in these tumors. Although existing screening, diagnostic, and treatment modalities exist, the high morbidity and mortality rates of these tumors remain challenging.     

Stat3 is a candidate epigenetic biomarker of perinatal Bisphenol A exposure associated with murine hepatic tumors with implications for human health

Bisphenol A (BPA) is an endocrine disrupting chemical (EDC) that has been implicated as a potential carcinogen and epigenotoxicant. We have previously reported dose-dependent incidence of hepatic tumors in 10-month-old isogenic mice perinatally exposed to BPA. Here, we evaluated DNA methylation at 3 candidate genes (Esr1, Il-6st, and Stat3) in liver tissue of BPA-exposed mice euthanized at 2 time points: post-natal day 22 (PND22; n = 147) or 10-months of age (n = 78, including n = 18 with hepatic tumors). Additionally, DNA methylation profiles were analyzed at human homologs of murine candidate genes in human fetal liver samples (n = 50) with known liver tissue BPA levels. Candidate genes were chosen based on reported expression changes in both rodent and human hepatocellular carcinoma (HCC). Regions for bisulfite sequencing were chosen by mining whole genome next generation sequencing methylation datasets of both mice and human liver samples with known perinatal BPA exposures. One of 3 candidate genes, Stat3, displayed dose-dependent DNA methylation changes in both 10-month mice with liver tumors as compared to those without liver tumors and 3-week sibling mice from the same exposure study, implicating Stat3 as a potential epigenetic biomarker of both early life BPA exposure and adult disease in mice. DNA methylation profiles within STAT3 varied with liver tissue BPA level in human fetal liver samples as well, suggesting STAT3 may be a translationally relevant candidate biomarker. These data implicate Stat3 as a potential early life biomarker of adult murine liver tumor risk following early BPA exposure with early evidence of relevance to human health.     

Current progress in epigenetic research for hepatocarcinomagenesis

Hepatocellular carcinoma is the main type of primary liver cancer, and also one of the most malignant tumors. At present, the pathogenesis mechanisms of liver cancer are not entirely clear. It has been shown that inactivation of tumor suppressor genes and activation of oncogenes play a significant role in carcinogenesis, caused by the genetic and epigenetic aberrance. In the past, people generally thought that genetic mutation is a key event of tumor pathogenesis, and somatic mutation of tumor suppressor genes is in particular closely associated with oncogenesis. With deeper understanding of tumors in recent years, increasing evidence has shown that epigenetic silencing of those genes, as a result of aberrant hypermethylation of CpG islands in promoters and histone modification, is essential to carcinogenesis and metastasis. The term epigenetics refers to heritable changes in gene expression caused by regulation mechanisms, other than changes in the underlying DNA sequence. Specific epigenetic processes include DNA methylation, genome imprinting, chromotin remodeling, histone modification and microRNA regulations. This paper reviews recent epigenetics research progress in the hepatocellular carcinoma study, and tries to depict the relationships between hepatocellular carcinomagenesis and DNA methylation as well as microRNA regulation. Supported by National Basic Research Program of China (Grant No. 2006CD910402) and Science and Technology Commission of Shanghai Municipality (Grant No. 05DZ22201 and 08JC1416400).     

Dose-dependence, sex- and tissue-specificity, and persistence of radiation-induced genomic DNA methylation changes

Radiation is a well-known genotoxic agent and human carcinogen that gives rise to a variety of long-term effects. Its detrimental influence on cellular function is actively studied nowadays. One of the most analyzed, yet least understood long-term effects of ionizing radiation is transgenerational genomic instability. The inheritance of genomic instability suggests the possible involvement of epigenetic mechanisms, such as changes of the methylation of cytosine residues located within CpG dinucleotides. In the current study we evaluated the dose-dependence of the radiation-induced global genome DNA methylation changes. We also analyzed the effects of acute and chronic high dose (5Gy) exposure on DNA methylation in liver, spleen, and lung tissues of male and female mice and evaluated the possible persistence of the radiation-induced DNA methylation changes. Here we report that radiation-induced DNA methylation changes were sex- and tissue-specific, dose-dependent, and persistent. In parallel we have studied the levels of DNA damage in the exposed tissues. Based on the correlation between the levels of DNA methylation and DNA damage we propose that radiation-induced global genome DNA hypomethylation is DNA repair-related.     

The first mitochondrial 5-methylcytosine map in a non-model teleost (Oreochromis niloticus) reveals extensive strand-specific and non-CpG methylation

DNA methylation is one of the main epigenetic mechanisms that regulate gene expression in a manner that depends on the genomic context and varies considerably across taxa. This DNA modification was first found in nuclear genomes of eukaryote several decades ago and it has also been described in mitochondrial DNA. It has recently been shown that mitochondrial DNA is extensively methylated in mammals and other vertebrates. Our current knowledge of mitochondrial DNA methylation in fish is very limited, especially in non-model teleosts. In this study, using whole-genome bisulfite sequencing, we determined methylation patterns within non-CpG (CH) and CpG (CG) contexts in the mitochondrial genome of Nile tilapia, a non-model teleost of high economic importance. Our results demonstrate the presence of mitochondrial DNA methylation in this species predominantly within a non-CpG context, similarly to mammals. We found a strand-specific distribution of methylation, in which highly methylated cytosines were located on the minus strand. The D-loop region had the highest mean methylation level among all mitochondrial loci. Our data provide new insights into the potential role of epigenetic mechanisms in regulating metabolic flexibility of mitochondria in fish, with implications in various biological processes, such as growth and development.     

Global DNA Hypomethylation in Liver Cancer Cases and Controls: A Phase I Preclinical Biomarker Development Study

Background: Global genomic DNA hypomethylation is a feature of genomic DNA derived from solid and hematologic tumors in animal models and human carcinogenesis. Global genomic DNA hypomethylation may be the earliest epigenetic change from a normal to a pre-malignant cell. Objectives: To test if global hypomethylation is a good marker for early detection of cancer we used a novel quantification method of 2’-deoxynucleosides to evaluate DNA methylation in liver cancer cases and controls. Methods: Frozen tissue from liver cancer patients and controls were obtained from the Cooperative Human Tissue Network. DNA was extracted using standard methods. Genomic DNA samples were boiled and treated with nuclease P1 and alkaline phosphatase. Global genomic DNA methylation patterns were obtained using HPLC for fraction separation and mass spectrometry for quantification. A two-sample t test was performed using Welch’s approximation for samples with unequal variances. A Wilcoxon rank sum test was also performed. Results: A global genomic DNA methylation index measuring methylated cytidine relative to global cytidine in the genome was significantly lower (p-value = 0.001) for all cases, mean = 2.43 (95% CI, 2.08, 2.78), when compared to controls, mean = 3.55 (95% CI, 3.16, 3.93). Discussion: A correlation between global genomic DNA methylation patterns and type of liver tissue was observed. These results add to the accumulating body of evidence suggesting that global DNA hypomethylation may be a useful biomarker to distinguish between liver cancer cases and controls.     

Initiation of aberrant DNA methylation patterns and heterogeneity in precancerous lesions of human hepatocellular cancer

While intratumor heterogeneity contributes to disease progression, metastasis, and resistance to chemotherapy, it also provides a route to understanding the evolution and drivers of disease. Defects in epigenetic landscapes are intimately linked to pathogenesis of a variety of human diseases, with epigenetic deregulation promoting tumorigenesis. Understanding epigenetic heterogeneity is crucial in hepatocellular carcinoma (HCC), where epigenetic alterations are frequent, early, and pathogenic events. We determined genome-wide DNA methylation and copy number variation leveraging the Infinium 450K in a series of regenerative nodules from within single patient livers. Bioinformatics strategies were used to ascertain within-patient heterogeneity, link epigenetic changes to clinical features, and determine their relevance to disease pathogenesis. Our data demonstrate that DNA methylation and copy number alterations evolve during the pre-neoplastic phase of HCC and independently segregate regenerative nodules into distinct clusters. Regenerative nodules with a high frequency of epigenetic changes have significantly lower copy number variation, suggesting that individual nodules have differential enrichment of epigenetic and genetic components, with both contributing to disease progression. Regenerative nodules were scored based on ‘epigenetic progression’ with higher scores associated with increased proliferation measured by Ki67 staining. Early events observed in epigenetically ‘aggressive’ nodules are enriched for genes involved in liver cancer. Our study demonstrates that marked epigenetic and genetic heterogeneity exists in early pre-neoplastic liver tissue within individual patients, emphasizing the potential contributions of each mechanism to driving liver disease progression, and it unveils strategies for identifying epigenetic drivers of hepatocellular carcinoma.     

Methylome sequencing for fibrolamellar hepatocellular carcinoma depicts distinctive features

With the goal of studying epigenetic alterations in fibrolamellar hepatocellular carcinoma (FLC) and establish an associated DNA methylation signature, we analyzed LINE-1 methylation in a cohort of FLC and performed next-generation sequencing of DNA methylation in a training set of pure-FLCs and non-cirrhotic hepatocellular carcinomas (nc-HCC). DNA methylation was correlated with gene expression. Furthermore, we established and validated an epigenetic signature differentiating pure-FLC from other HCCs. LINE-1 methylation correlated with shorter recurrence-free survival and overall survival in resected pure-FLC patients. Unsupervised clustering using CG sites located in islands distinguished pure-FLC from nc-HCC. Major DNA methylation changes occurred outside promoters, mainly in gene bodies and intergenic regions located in the vicinity of liver developmental genes (i.e., SMARCA4 and RXRA). Partially methylated domains were more prone to DNA methylation changes. Furthermore, we identified several putative tumor suppressor genes (e.g., DLEU7) and oncogenes (e.g., DUSP4). While ∼70% of identified gene promoters gaining methylation were marked by bivalent histone marks (H3K4me3/H3K27me3) in embryonic stem cells, ∼70% of those losing methylation were marked by H3K4me3. Finally, we established a pure FLC DNA methylation signature and validated it in an independent dataset. Our analysis reveals a distinct epigenetic signature of pure FLC as compared to nc-HCC, with DNA methylation changes occurring in the vicinity of liver developmental genes. These data suggest new options for targeting FLC based on cancer epigenome aberrations.     

DNA Methylation Differences Associated with Tumor Tissues Identified by Genome Scanning Analysis

Most investigations on the role of DNA methylation in cancer have focused on epigenetic changes associated with known tumor suppressor genes. This may have led to an underestimation of the number of CpG islands altered by DNA methylation, since it is possible that a subset of unknown genes relevant to cancer development may preferentially be affected by epigenetic rather than genetic means and would not be identified as familial deletions, mutations, or loss of heterozygosity. We used a recently developed screening procedure (methylation-sensitive arbitrarily primed-polymerase chain reaction to scan genomic DNA for CpG islands methylated in white blood cells (WBCs) and in tumor tissues. DNA methylation pattern analysis showed little interindividual differences in the WBCs and normal epithelium (adjacent to colon, bladder, and prostate cancer cells), but with some tissue-specific differences. Cancer cells showed marked methylation changes that varied considerably between different tumors, suggesting variable penetrance of the methylation phenotype in patients. Direct sequencing of 8 of 45 bands altered in these cancers showed that several of them were CpG islands, and 2 of these sequences were identified in GenBank. Surprisingly, three of the bands studied corresponded to transcribed regions of genes. Thus, hypermethylation of CpG islands in cancer cells is not confined to the promoters of growth regulatory genes but is also found in actively transcribed regions.     

Genetic and epigenetic alterations in hepatitis B virus-associated hepatocellular carcinoma

Hepatitis B virus(HBV) is a major cause of hepatocellular carcinoma(HCC). Its chronic infection can lead to chronic liver inflammation and the accumulation of genetic alterations to result in the oncogenic transformation of hepatocytes. HBV can also sensitize hepatocytes to oncogenic transformation by causing genetic and epigenetic changes of the host chromosomes. HBV DNA can insert into host chromosomes and recent large-scale whole-genome sequencing studies revealed recurrent HBV DNA integrations sites that may play important roles in the initiation of hepatocellular carcinogenesis. HBV can also cause epigenetic changes by altering the methylation status of cellular DNA, the post-translational modification of histones, and the expression of micro RNAs. These changes can also lead to the eventual hepatocellular transformation. These recent findings on the genetic and epigenetic alterations of the host chromosomes induced by HBV opened a new avenue for the development of novel diagnosis and treatments for HBV-induced HCC.     

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.     

Promoter hypermethylation mediated downregulation of FBP1 in human hepatocellular carcinoma and colon cancer

FBP1, fructose-1,6-bisphosphatase-1, a gluconeogenesis regulatory enzyme, catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. The mechanism that it functions to antagonize glycolysis and was epigenetically inactivated through NF-kappaB pathway in gastric cancer has been reported. However, its role in the liver carcinogenesis still remains unknown. Here, we investigated the expression and DNA methylation of FBP1 in primary HCC and colon tumor. FBP1 was lowly expressed in 80% (8/10) human hepatocellular carcinoma, 66.7% (6/9) liver cancer cell lines and 100% (6/6) colon cancer cell lines, but was higher in paired adjacent non-tumor tissues and immortalized normal cell lines, which was well correlated with its promoter methylation status. Methylation was further detected in primary HCCs, gastric and colon tumor tissues, but none or occasionally in paired adjacent non-tumor tissues. Detailed methylation analysis of 29 CpG sites at a 327-bp promoter region by bisulfite genomic sequencing confirmed its methylation. FBP1 silencing could be reversed by chemical demethylation treatment with 5-aza-2'-deoxycytidine (Aza), indicating direct epigenetic silencing. Restoring FBP1 expression in low expressed cells significantly inhibited cell growth and colony formation ability through the induction of G2-M phase cell cycle arrest. Moreover, the observed effects coincided with an increase in reactive oxygen species (ROS) generation. In summary, epigenetic inactivation of FBP1 is also common in human liver and colon cancer. FBP1 appears to be a functional tumor suppressor involved in the liver and colon carcinogenesis.     

肝癌基因组变异与分子分型

肿瘤是机体在各种致癌因素刺激下,基因组发生变异导致细胞失去正常生长调控而异常增殖的一种恶性疾病.肿瘤具有维持细胞增殖信号、逃避生长抑制、抗细胞凋亡、无限复制、诱导血管生成、激活侵袭和转移、能量代谢的重编程和免疫逃避等特点.原发性肝癌是一种高致死性的癌症类型,在中国发病率高,约占全世界发病人数的一半.肝细胞癌是原发性肝癌中的主要组织学亚型,与乙型和丙型肝炎病毒感染、酒精刺激、肥胖以及饮食污染等有关.遗传学和表观遗传突变事件的研究有助于理解肝癌的发病机制并对患者进行分子分型,而分子分型则可以指导临床个体化治疗和预后判断.     

Bisphenol A Exposure Disrupts Genomic Imprinting in the Mouse

Exposure to endocrine disruptors is associated with developmental defects. One compound of concern, to which humans are widely exposed, is bisphenol A (BPA). In model organisms, BPA exposure is linked to metabolic disorders, infertility, cancer, and behavior anomalies. Recently, BPA exposure has been linked to DNA methylation changes, indicating that epigenetic mechanisms may be relevant. We investigated effects of exposure on genomic imprinting in the mouse as imprinted genes are regulated by differential DNA methylation and aberrant imprinting disrupts fetal, placental, and postnatal development. Through allele-specific and quantitative real-time PCR analysis, we demonstrated that maternal BPA exposure during late stages of oocyte development and early stages of embryonic development significantly disrupted imprinted gene expression in embryonic day (E) 9.5 and 12.5 embryos and placentas. The affected genes included Snrpn, Ube3a, Igf2, Kcnq1ot1, Cdkn1c, and Ascl2; mutations and aberrant regulation of these genes are associated with imprinting disorders in humans. Furthermore, the majority of affected genes were expressed abnormally in the placenta. DNA methylation studies showed that BPA exposure significantly altered the methylation levels of differentially methylated regions (DMRs) including the Snrpn imprinting control region (ICR) and Igf2 DMR1. Moreover, exposure significantly reduced genome-wide methylation levels in the placenta, but not the embryo. Histological and immunohistochemical examinations revealed that these epigenetic defects were associated with abnormal placental development. In contrast to this early exposure paradigm, exposure outside of the epigenetic reprogramming window did not cause significant imprinting perturbations. Our data suggest that early exposure to common environmental compounds has the potential to disrupt fetal and postnatal health through epigenetic changes in the embryo and abnormal development of the placenta.     

Effects of cadmium on DNA-(Cytosine-5) methyltransferase activity and DNA methylation status during cadmium-induced cellular transformation

Cadmium is a human carcinogen that likely acts via epigenetic mechanisms. Since DNA methylation alterations represent an important epigenetic event linked to cancer, the effect of cadmium on DNA methyltransferase (MeTase) activity was examined using in vitro (TRL1215 rat liver cells) and ex vivo (M.SssI DNA MeTase) systems. Cadmium effectively inhibited DNA MeTases in a manner that was noncompetitive with respect to substrate (DNA), indicating an interaction with the DNA binding domain rather than the active site. Based on these results, the effects of prolonged cadmium exposure on DNA MeTase and genomic DNA methylation in TRL1215 cells were studied. After 1 week of exposure to 0-2.5 microM cadmium, DNA MeTase activity was reduced (up to 40%) in a concentration-dependent fashion, while genomic DNA methylation showed slight but significant reductions at the two highest concentrations. After 10 weeks of exposure, the cells exhibited indications of transformation, including hyperproliferation, increased invasiveness, and decreased serum dependence. Unexpectedly, these cadmium-transformed cells exhibited significant increases in DNA methylation and DNA MeTase activity. These results indicate that, while cadmium is an effective inhibitor of DNA MeTase and initially induces DNA hypomethylation, prolonged exposure results in DNA hypermethylation and enhanced DNA MeTase activity.     

Differential DNA methylation alterations in radiation-sensitive and -resistant cells

An understanding of cellular processes that determine the response to ionizing radiation (IR) exposure is essential to improve radiotherapy and to assess risks to human health after accidental radiation exposure. Exposure to IR induces a multitude of biological effects. Recent studies have indicated the involvement of epigenetic events in regulating the responses of irradiated cells. DNA methylation, where the cytosine bases in CpG dimers are converted to 5-methyl cytosine, is an epigenetic event that has been shown to regulate a variety of biological processes. We investigated the DNA methylation changes in irradiated TK6 and WTK1 human cells that differ in sensitivity to IR. The global DNA methylation alterations as measured by an enzyme-linked immunosorbent assay-based assay showed hypomethylation in both type of cells. Using an arbitrarily primed polymerase chain reaction (AP-PCR) approach, we observed time-dependent dynamic changes in the regional genomic DNA methylation patterns in both cell lines. The AP-PCR DNA methylation profiles were different between TK6 and WTK1 cells, indicating the involvement of differential genomic DNA responses to radiation treatment. The analysis of the components of the DNA methylation machinery showed the modulation of maintenance and de novo methyltransferases in irradiated cells. DNMT1 mRNA levels were increased in TK6 cells after irradiation but were repressed in WTK1 cells. DNMT3A and DNMT3B were induced in both cells after radiation treatment. TET1, involved in the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), was induced in both cells. This study demonstrates that irradiated cells acquire epigenetic changes in the DNA methylation patterns, and the associated cellular machinery are involved in the response to radiation exposure. This study also shows that DNA methylation patterns change at different genomic regions and are dependent on time after irradiation and the genetic background of the cell.     

Age-related DNA methylation in normal breast tissue and its relationship with invasive breast tumor methylation

Age is a key risk factor for breast cancer and epigenetic alterations may contribute to age-related increases in breast cancer risk, though the relation of age-related methylation in normal breast tissues with altered methylation in breast tumors is unclear. We investigated the relation of age with DNA methylation in normal breast tissues genome-wide using two data sets from the Gene Expression Omnibus (GEO) database (GSE32393 and GSE31979). We validated our observations in an independent set of normal breast tissues, examined age-related methylation in normal breast for enrichment of genomic features, and compared age-related methylation in normal tissue with methylation alterations in breast tumors. Between the two array-based methylation data sets, there were 204 CpG loci with significant (P < 0.05) and consistent age-related methylation, 97% of which were increases in methylation. Our validation sets confirmed the direction of age-related DNA methylation changes in all measured regions. Among the 204 age-related CpG loci, we observed a significant enrichment for CpG islands (P = 8.7E-6) and polycomb group protein target genes (P = 0.03). In addition, 24 of the 204 CpGs with age-related methylation in normal breast were significantly differentially methylated between normal and breast tumor tissues. We identified consistent age-related methylation changes in normal breast tissue that are further altered in breast tumors and may represent early events contributing to breast carcinogenesis. This work identifies age-related methylation in normal breast tissue and begins to deconstruct the contribution of aging to epigenetic alterations present in breast tumors.