DNA hypermethylation as a chemotherapy target

Epigenetics refers to partially reversible, somatically inheritable, but DNA sequence-independent traits that modulate gene expression, chromatin structure, and cell functions such as cell cycle and apoptosis. DNA methylation is an example of a crucial epigenetic event; aberrant DNA methylation patterns are frequently found in human malignancies. DNA hypermethylation and the associated expression silencing of tumor suppressor genes represent a hallmark of neoplastic cells. The cancer methylome is highly disrupted, making DNA methylation an excellent target for anti-cancer therapies. Several small synthetic and natural molecules, are able to reverse the DNA hypermethylation through inhibition of DNA methyltransferase (DNMT). DNMT is the enzyme catalyzing the transfer of methyl groups to cytosines in genomic DNA. These reagents are studied intensively in cell cultures, animal models, and clinical trials for potential anti-cancer activities. It was found that accompanying DNA demethylation is a dramatic reactivation of the silenced genes and inhibition of cancer cell proliferation, promotion of cell apoptosis, or sensitization of cells to other chemotherapeutic reagents. During the last few decades, an increasing number of DNMT inhibitors (DNMTi) targeting DNA methylation have been developed to increase efficacy with reduced toxicity. This review provides an update on new findings on cancer epigenetic mechanisms, the development of new DNMTi, and their application in the clinical setting. Current challenges, potential solutions, and future directions concerning the development of DNMTi are also discussed in this review.     

Association between hypermethylation of DNA repetitive elements in white blood cell DNA and pancreatic cancer

Pancreatic cancer is a leading cause of cancer-related deaths worldwide. Methylation of DNA may influence risk or be a marker of early disease. The aim of this study was to measure the association between methylation of three DNA repetitive elements in white blood cell (WBC) DNA and pancreatic cancer.DNA from WBCs of pancreatic cancer cases (n = 559) and healthy unrelated controls (n = 603) were tested for methylation of the LINE-1, Alu and Sat2 DNA repetitive elements using MethyLight quantitative PCR assays. Odds ratios (ORs) and 95% confidence intervals (95%CI) between both continuous measures of percent of methylated sample compared to a reference (PMR) or quintiles of PMR and pancreatic cancer, adjusted for age, sex, smoking, BMI, alcohol and higher education, were estimated.The PMR for each of the three markers was higher in cases than in controls, although only LINE-1 was significantly associated with pancreatic cancer (OR per log unit = 1.37, 95%CI = 1.16–1.63). The marker methylation score for all three markers combined was significantly associated with pancreatic cancer (p-trend = 0.0006). There were no associations between measures of PMR and either presence of metastases, or timing of blood collection in relation to diagnosis, surgery, chemotherapy or death (all p > 0.1).We observed an association between methylation of LINE-1 in WBC DNA and risk of pancreatic cancer. Further studies are needed to confirm this association.     

Association between hypermethylation of DNA repetitive elements in white blood cell DNA and early-onset colorectal cancer

Changes in the methylation levels of DNA from white blood cells (WBCs) are putatively associated with an elevated risk for several cancers. The aim of this study was to investigate the association between colorectal cancer (CRC) and the methylation status of three DNA repetitive elements in DNA from peripheral blood. WBC DNA from 539 CRC cases diagnosed before 60 years of age and 242 sex and age frequency-matched healthy controls from the Australasian Colorectal Cancer Family Registry were assessed for methylation across DNA repetitive elements Alu, LINE-1 and Sat2 using MethyLight. The percentage of methylated reference (PMR) of cases and controls was calculated for each marker. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using multivariable logistic regression adjusted for potential confounders. CRC cases demonstrated a significantly higher median PMR for LINE-1 (p < 0.001), Sat2 (p < 0.001) and Alu repeats (p = 0.02) when compared with controls. For each of the DNA repetitive elements, individuals with PMR values in the highest quartile were significantly more likely to have CRC compared with those in the lowest quartile (LINE-1 OR = 2.34, 95%CI = 1.48–3.70; p < 0.001, Alu OR = 1.83, 95%CI = 1.17–2.86; p = 0.01, Sat2 OR = 1.72, 95%CI = 1.10–2.71; p = 0.02). When comparing the OR for the PMR of each marker across subgroups of CRC, only the Alu marker showed a significant difference in the 5-fluoruracil treated and nodal involvement subgroups (both p = 0.002). This association between increasing methylation levels of three DNA repetitive elements in WBC DNA and early-onset CRC is novel and may represent a potential epigenetic biomarker for early CRC detection.     

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders.In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed.In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.     

DNA hypermethylation in prostate cancer is a consequence of aberrant epithelial differentiation and hyperproliferation

Prostate cancer (CaP) is mostly composed of luminal-like differentiated cells, but contains a small subpopulation of basal cells (including stem-like cells), which can proliferate and differentiate into luminal-like cells. In cancers, CpG island hypermethylation has been associated with gene downregulation, but the causal relationship between the two phenomena is still debated. Here we clarify the origin and function of CpG island hypermethylation in CaP, in the context of a cancer cell hierarchy and epithelial differentiation, by analysis of separated basal and luminal cells from cancers. For a set of genes (including GSTP1) that are hypermethylated in CaP, gene downregulation is the result of cell differentiation and is not cancer specific. Hypermethylation is however seen in more differentiated cancer cells and is promoted by hyperproliferation. These genes are maintained as actively expressed and methylation-free in undifferentiated CaP cells, and their hypermethylation is not essential for either tumour development or expansion. We present evidence for the causes and the dynamics of CpG island hypermethylation in CaP, showing that, for a specific set of genes, promoter methylation is downstream of gene downregulation and is not a driver of gene repression, while gene repression is a result of tissue-specific differentiation.     

癌症DNA甲基化调控位点的识别

DNA甲基化是一种重要的表观遗传学修饰,在基因的转录调控方面具有重要的作用。异常的DNA甲基化可以导致癌症等复杂疾病发生,癌基因相关的DNA甲基化调控位点的识别对于解析癌症的发生发展机制及识别新的癌症标记具有重要意义。本研究通过整合The Cancer Genome Atlas(TCGA)的泛癌症基因组的高通量甲基化谱和基因表达谱,识别癌基因相关的DNA甲基化调控位点。对于每种癌症分批次计算Cp G位点甲基化与相关基因表达之间的相关性,并筛选调控下游基因的Cp G位点(包括强调控位点、弱调控位点和不调控位点),结果表明仅有一半的Cp G位点对下游基因具有调控作用;对癌症间共享的调控位点的分析发现不同癌症间共享的调控位点不尽相同,表明癌症特异的甲基化调控位点的存在。进一步地,对差异甲基化和差异表达基因的功能富集分析揭示了受甲基化调控的基因确实参与了癌症发生发展相关的功能。本研究的结果是对当前甲基化调控位点集的重要补充,也是识别癌症新型分子标记特征的重要资源。     

Identification of differentially methylated sites within unmethylated DNA domains in normal and cancer cells

Altered DNA methylation has been linked to neoplastic cell transformation and is a hallmark of cancer progression. Therefore, the screening for differentially methylated sequences as tumor biomarkers has a significant implication in the clinical setting. To determine the cancer-linked alterations in DNA methylation pattern, we have applied an endonuclease, McrBC, to the existing methylation-sensitive arbitrarily primed polymerase chain reaction (msAP-PCR) method and developed McrBC-msAP-PCR. This modified approach allows detection of differentially methylated sites within unmethylated DNA domains enriched by regulatory sequences and CpG islands. In this method, we used digestion of DNA with the McrBC methylation-sensitive endonuclease to selectively exclude the methylated fraction of DNA, which comprises interspersed and tandem-repeated sequences and exons other than first exons, from analysis. The subsequent digestion of unmethylated DNA fragments with SmaI and HpaII methylation-sensitive restriction endonucleases followed by AP-PCR amplification resulted in the detection of unknown unique sequences associated with cancer-linked methylation changes in genomic DNA. Hypermethylation and hypomethylation are visualized by the increase or decrease in the band intensity of DNA fingerprints. By using this technique, we were able to differentiate clearly, identify, and characterize a number of novel unique DNA sequences with differentially methylated sites in normal and breast cancer cell lines and in normal and rat tumor liver tissues.     

癌症相关的DNA甲基化连锁区域

DNA甲基化是重要的表观遗传标记之一,在转录调控中起直接作用。DNA甲基化的异常与癌症的发生发展密切相关。高通量测序使得在单碱基分辨率下检测全基因组的DNA甲基化水平成为可能。本文基于临近CpGs位点甲基化水平的相关性挖掘DNA甲基化连锁区域。结果发现DNA甲基化连锁区域的甲基化水平和模式在癌症中存在异常,而且显著富集到分化/发育相关的生物学功能。DNA甲基化连锁区域的挖掘有助于对具有生物学功能的表观遗传标记的进一步理解,有助于对癌症诊断的表观遗传标记的挖掘。     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

GHSR DNA hypermethylation is a new epigenetic biomarker for gastric adenocarcinoma and beyond

Aberrations of DNA methylation are early events in the development of tumors. In this study, we investigated the DNA methylation status of growth hormone secretagogue receptor (GHSR), a promising pan-cancer biomarker, in gastric cancer (GC). Initially, data sets from DNA methylation and gene expression studies available at Gene Expression Omnibus (GEO) were analyzed. Confirmation was done on primary tumor specimens and adjacent normal stomach tissue samples. Both analyses showed significant hypermethylation of GHSR. For further validation, The Cancer Genome Atlas data on stomach cancer was used. A receiver operating characteristic curve analysis yielded an area under the curve value of 0.85, corroborating its usefulness as a diagnostic marker. A genome-wide comethylation analysis revealed several correlated genes. CREB1 was found to act as an upstream regulator of this gene network. Furthermore, GHSR methylation was found to be a biomarker in several other tumor entities, namely cancers of the bladder, endometrium, esophagus, head and neck, liver, thyroid, kidney, and ovary. Our findings along with previous reports on other types of cancer suggest a high potential of GHSR gene methylation as a pan-cancer biomarker, which could be considered for liquid biopsy applications.     

鼠肝细胞癌变中DNA甲基化作用的研究

本文用~3H标记的S-腺苷酰甲硫氨酸(~3H-SAM)为甲基供体,以同位素掺入法测定了正常小鼠肝细胞、H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力。并用HPLC法测定了上述细胞的DNA甲基化水平。发现:H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力和DNA甲基化水平明显低于正常肝细胞。当以抗肝癌药物去甲斑蝥素和斑蝥酸钠处理荷癌小鼠6天后,可使H_(223)腹水癌细胞及荷癌肝细胞的DNA甲基化酶活力回升,但并未检出DNA甲基化水平的回升。     

Liquid biopsies: DNA methylation analyses in circulating cell-free DNA

Analysis of patient's materials like cells or nucleic acids obtained in a minimally invasive or noninvasive manner through the sampling of blood or other body fluids serves as liquid biopsies, which has huge potential for numerous diagnostic applications. Circulating cell-free DNA (cfDNA) is explored as a prognostic or predictive marker of liquid biopsies with the improvements in genomic and molecular methods. DNA methylation is an important epigenetic marker known to affect gene expression. cfDNA methylation detection is a very promising approach as abnormal distribution of DNA methylation is one of the hallmarks of many cancers and methylation changes occur early during carcinogenesis. This review summarizes the various investigational applications of cfDNA methylation and its oxidized derivatives as biomarkers for cancer diagnosis, prenatal diagnosis and organ transplantation monitoring. The review also provides a brief overview of the technologies for cfDNA methylation analysis based on next generation sequencing.     

The role of arginine methylation in the DNA damage response

Post-translational modifications are well-known modulators of DNA damage signaling and epigenetic gene expression. Protein arginine methylation is a covalent modification that results in the addition of methyl groups to the nitrogen atoms of the arginine side chains and is catalyzed by a family of protein arginine methyltransferases (PRMTs). In the past, arginine methylation was mainly observed on abundant proteins such as RNA-binding proteins and histones, but recent advances have revealed a plethora of arginine methylated proteins implicated in a variety of cellular processes including RNA metabolism, epigenetic regulation and DNA repair pathways. Herein, we discuss these recent advances, focusing on the role of PRMTs in DNA damage signaling and its importance for maintaining genomic stability.     

MTSS1 hypermethylation is associated with prostate cancer progression

This study was conducted to evaluate the influence of DNA methylation of metastasis suppressor 1 (MTSS1) on prostate cancer (PCa) progression. Forty-nine paired PCa tissue samples and normal tissue samples from The Cancer Genome Atlas were analyzed. Methylome analysis, CpG island arrays and Hierarchical clustering were used to analyze methylation profiles of PCa tissues. MTSS1 methylation level was detected by methylation-specific PCR. Relative messenger RNA and the expression level of MTSS1 protein were identified by quantitative real-time PCR (qRT-PCR) and western blot analysis. The migration, invasion, proliferation, and cell cycle were detected separately by wound-healing assay, transwell chamber assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry. The roles of MTSS1 in PCa progression were demonstrated in vivo by tumor formation assays in nude mice. MTSS1 expression was decreased in PCa tissues in comparison with paired adjacent normal prostate tissues. Compared to the methylation of MTSS1 in normal prostate tissues based on the MethHC website, the MTSS1 in PCa tissues was hypermethylated. The expression of MTSS1 detected by qRT-PCR and western blot analysis was found to be downregulated in PCa cells and tissues. The reduced expression of MTSS1 by small interfering RNA-MTSS1 was recovered by 5-aza-2′-deoxycytidine treatment. Besides, MTSS1 demethylation inhibited migration, invasion, and proliferation of PCa cells, and induced cell cycle to be arrested at G0/G1 phase. Furthermore, it was shown by tumor xenograft assay that MTSS1 inhibited the growth of tumor in vivo. Hypermethylated MTSS1 promoted PCa cells migration, invasion, and proliferation, and suppressed cell cycle arrest at the G0/G1 phase.     

A suite of DNA methylation markers that can detect most common human cancers

Cancer-specific DNA methylation from the tumor derived fraction of cell free DNA found in blood samples could be used for minimally invasive detection and monitoring of cancer. The knowledge of marker regions with cancer-specific DNA methylation is necessary to the success of such a process. We analyzed the largest cancer DNA methylation dataset available—TCGA Illumina HumanMethylation450 data with over 8,500 tumors—in order to find cancer-specific DNA methylation markers for most common human cancers. First, we identified differentially methylated regions for individual cancer types and those were further filtered against data from normal tissues to obtain marker regions with cancer-specific methylation, resulting in a total of 1,250 hypermethylated and 584 hypomethylated marker CpGs. From hypermethylated markers, optimal sets of six markers for each TCGA cancer type were chosen that could identify most tumors with high specificity and sensitivity [area under the curve (AUC): 0.969-1.000] and a universal 12 marker set that can detect tumors of all 33 TCGA cancer types (AUC >0.84). In addition to hundreds of new DNA methylation markers, our approach also identified markers that are in current clinical use, SEPT9 and GSTP1, indicating the validity of our approach and a significant potential utility for the newly discovered markers. The hypermethylated markers are linked to polycomb associated loci and a significant fraction of the discovered markers is within noncoding RNA genes; one of the best markers is MIR129-2. Future clinical testing of herein discovered markers will confirm new markers that will improve minimally invasive diagnosis and monitoring for multiple cancers.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.