Differential methylation landscape of pancreatic ductal adenocarcinoma and its precancerous lesions

Background: Pancreatic cancer is one of the most lethal diseases with an incidence almost equal to the mortality. In addition to having genetic causes, cancer can also be considered an epigenetic disease. DNA methylation is the premier epigenetic modification and patterns of aberrant DNA methylation are recognized to be a common hallmark of human tumor. In the multistage carcinogenesis of pancreas starting from precancerous lesions to pancreatic ductal adenocarcinoma(PDAC), the epigenetic changes play a significant role. Data sources: Relevant studies for this review were derived via an extensive literature search in Pub Med via using various keywords such as pancreatic ductal adenocarcinoma, precancerous lesions, methylation profile, epigenetic biomarkers that are relevant directly or closely associated with the concerned area of our interest. The literature search was intensively done considering a time frame of 20 years(1998–2018). Result: In this review we have highlighted the hypermethylation and hypomethylation of the precancerous PDAC lesions(pancreatic intra-epithelial neoplasia, intraductal papillary mucinous neoplasm, mucinous cystic neoplasm and chronic pancreatitis) and PDAC along with the potential biomarkers. We have also achieved the early epigenetic driver that leads to progression from precancerous lesions to PDAC. A bunch of epigenetic driver genes leads to progression of precancerous lesions to PDAC( pp ENK, APC, p14/5/16/17, h MLH1 and MGMT) are also documented. We summarized the importance of these observations in therapeutics and diagnosis of PDAC hence identifying the potential use of epigenetic biomarkers in epigenetic targeted therapy. Epigenetic inactivation occurs by hypermethylation of Cp G islands in the promoter regions of tumor suppressor genes. We listed all hyper-and hypomethylation of Cp G islands of several genes in PDAC including its precancerous lesions. Conclusions: The concept of the review would help to understand their biological effects, and to determine whether they may be successfully combined with other epigenetic drugs. However, we need to continue our research to develop more specific DNA-demethylating agents, which are the targets for hypermethylated Cp G methylation sites.     

Alterations of epigenetics and microRNA in hepatocellular carcinoma

Studies have shown that alterations of epigenetics and microRNA (miRNA) play critical roles in the initiation and progression of hepatocellular carcinoma (HCC). Epigenetic silencing of tumor suppressor genes in HCC is generally mediated by DNA hypermethylation of CpG island promoters and histone modifications such as histone deacetylation, methylation of histone H3 lysine 9 (H3K9) and tri‐methylation of H3K27. Chromatin‐modifying drugs such as DNA methylation inhibitors and histone deacetylase inhibitors have shown clinical promise for cancer therapy. miRNA are small non‐coding RNA that regulate expression of various target genes. Specific miRNA are aberrantly expressed and play roles as tumor suppressors or oncogenes during hepatocarcinogenesis. We and other groups have demonstrated that important tumor suppressor miRNA are silenced by epigenetic alterations, resulting in activation of target oncogenes in human malignancies including HCC. Restoring the expression of tumor suppressor miRNA by inhibitors of DNA methylation and histone deacetylase may be a promising therapeutic strategy for HCC.     

Interplay among epigenetic alterations and crosstalk between genetic and epigenetic alterations in esophageal squamous cell carcinoma

Epigenetic alterations that do not involve a change in the DNA sequence have been increasingly recognized to be important key events in the regulation of the gene expression and carcinogenesis. Major epigenetic mechanisms include the methylation of cytosine in DNA, changes in the histone and chromatin structure due to covalent posttranslational modification of histone proteins and the RNA-mediated regulation of the gene expression. Esophageal squamous cell carcinoma (ESCC) continues to be associated with a very poor prognosis, indicating that obtaining a clear understanding of the pathogenesis of ESCC is desired for improving clinical outcomes. In this review, we discuss the recent progress in research on epigenetic alterations in ESCC, with respect to the following points: (1) DNA methylation, including global hypomethylation and DNA hypermethylation at CpG islands in the promoters of tumor suppressor genes, (2) histone acetylation/deacetylation and histone methylation with the alteration of histone-modifying enzymes and (3) alterations in the expression of microRNA and the recently emerging long non-coding RNA. We then discuss the interplay among these epigenetic events and the crosstalk between epigenetic and genetic changes in ESCC. It is therefore important to understand the molecular mechanisms underlying the development and progression of ESCC to improve the treatment outcome of this devastating disease, although this information is quite complicated and confusing.     

Hemizygous deletion and hypermethylation of RUNX3 gene in hepatocellular carcinoma

AIM：To analyze the genetic and epigenetic alterations of RUNX3 gene, a potential putative tumor suppressor gene,in hepatocellular carcinoma (HCC).METHODS: PCR-based loss of heterozygosity (LOH) detection, analysis of mutation with PCR-single strand conformational polymorphism (SSCP) and sequencing, and methylation study with methylation specific PCR (MSP) were performed on RUNX3 gene in a series of 62 HCCs along with their matched normal tissues.RESULTS:Mutation of RUNX3 gene was not found, but one single nucleotide polymorphism with T to A transversion at the second nucleotide of the 18th condon was found.Nine of 26 informative cases (34.6%) showed allelic loss on the polymorphic site and 30 cases (48.4%) revealed hypermethylation of RUNX3 gene in promoter CpG islands.Furthermore,of the 9 cases with LOH, 8 (88.9%) also had hypermethylation.CONCLUSION:Our findings indicate that inactivation of RUNX3 gene through allelic loss and promoter hypermethylation might be one of the major mechanisms in hepatocellualr carcinogenesis.     

一例Prader-Willi综合征的临床及表观遗传学研究

目的分析Prader—Willi综合征（PWS）的临床及表观遗传学特征。方法应用重硫酸盐测序法对一例临床高度疑似PWS的患者及其父母的15q11—q13印迹中心SNRPN基因alpha外显子的10个CpG位点的甲基化情况进行分析。结果患者SNRPN基因位点的10个CpG位点完全甲基化，而其父母则是部分甲基化，结论SNRPN基因位点的DNA甲基化异常可能是该PWS患者的分子遗传缺陷。     

Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.     

DNA Methylation as a Therapeutic Target in Hematologic Disorders: Recent Results in Older Patients with Myelodysplasia and Acute Myeloid Leukemia

DNA methylation provides a major epigenetic code (besides histone modification) of the lineage- and development-specific genes (such as regulators of differentiation in the hematopoietic lineages) that control expression of normal cells. However, DNA methylation is also involved in malignancies because aberrant methylating gene activity occurs during leukemic transformation. Thus, genes such as tumor suppressor genes, growth-regulatory genes, and adhesion molecules are often silenced in various hematopoietic malignancies by epigenetic inactivation via DNA hypermethylation. This inactivation is frequently seen not only in transformed cell lines but also in primary leukemia cells. Because this defect is amenable to reversion by pharmacologic means, agents that inhibit DNA methylation have been developed to specifically target this hypermethylation defect in leukemia and preleukemia cases. The most clinically advanced agents, the azanucleosides 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine), were discovered more than 25 years ago, when their methylation-inhibitory activities, even at low concentrations, became apparent. Although both of these agents, like cytarabine, had been clinically used until then at high doses, the redevelopment of these agents for low-dose schedules has revealed very interesting clinical activities for treating myelodysplasia (MDS) and acute myeloid leukemia (AML). Because these diseases occur mostly in patients over 60 years of age, low-dose schedules with these compounds provide a very promising approach in such patient groups by virtue of their low nonhematologic toxicity profiles. In the present review, we describe the development of treatments that target DNA hypermethylation in MDS and AML, and clinical results are presented. In addition, pharmacologic DNA demethylation may be viewed as a platform for biological modification of malignant cells to become sensitized (or resensitized) to secondary signals, such as differentiating signals (retinoids, vitamin D3) and hormonal signals (eg, estrogen receptor in breast cancer cells, androgen receptor in prostate cancer cells). Finally, an in vitro synergism between the reactivating potency of demethylating agents and inhibitors of histone deacetylation has been tested in several pilot studies of AML and MDS treatment. Finally, gene reactivation by either group of compounds results in therapeutically meaningful reactivation of fetal hemoglobin in patients with severe hemoglobinopathies, extending the therapeutic range of derepressive epigenetic agents to nonmalignant hematopoietic disorders.     

Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomas

Aim: The aim of the present study was to examine DNA methylation and histone modification changes in hepatocellular carcinomas (HCC). Methods: DNA methylation in the P16, RASSF1a, progesterone receptor (PGR) and estrogen receptor alpha (ERalpha) promoters was determined by quantitative bisulfite-pyrosequencing technique in HCC patients. Histone H3-lysine (K) 4, H3-K9 and H3-K27 modifications in all these four genes were examined by chromatin immunoprecipitation (ChIP) assay in HCC cell lines. Expression of two DNA methyltransferases (DNMT1 and DNMT3b) and three histone methyltransferases (SUV39H1, G9a and EZH2) in HCC patients was measured by real-time polymerase chain reaction. Results: Aberrant DNA methylation was detected in all the HCC. Patients with DNA methylation in the RASSF1a, PGR andERalpha promoters in cancers also had substantial DNA methylation in their non-cancerous liver tissues, whereas DNA methylation in the P16 promoter was cancer specific. Epigenetic states in HCC cell lines showed that silencing of P16 and RASSF1a depended on DNA methylation and histone H3-K9 methylation. However, silencing of the PGR and ERalpha genes was more closely related to H3-K27 methylation rather than DNA methylation. Consistent with the alteration of histone status, higher expression of G9a and EZH2 was found in HCC than in non-cancerous liver tissues (P < 0.01). Conclusion: These data suggest that multiple epigenetic silencing mechanisms are inappropriately active in HCC cells.     

The potential role of epigenetic therapy in multiple myeloma

This review describes the role that epigenetic changes play in the pathogenesis of cancer, concentrating on the plasma cell malignancy multiple myeloma, and highlights recent findings regarding the efficacy of epigenetic therapeutic agents in laboratory studies and clinical trials. DNA methylation is altered in a wide range of cancers with hypermethylation of CpG islands associated with silencing of tumour suppressor genes. Genes found to be silenced by methylation in myeloma samples include VHL, TP53, CDKN2A, and TGFBR2. Myeloma is linked to the overexpression of a histone methylatransferase (MMSET) and inactivating mutations of a histone demethylase (UTX), suggesting that the regulation of histone methylation is a potential therapeutic target. Abnormal expression of histone deacetylases (HDACs) has been widely described in solid tumours and haematological malignancies. In myeloma, histone deacetylase inhibitors show promising results both in laboratory‐based cell culture studies and in clinical trials, where they demonstrate particularly good therapeutic outcome when administered in combination with other standard chemotherapeutic agents. The study of epigenetics shows great promise for understanding the alterations in gene expression that underlie malignancies and provides exciting novel drugable targets.     

Epigenetics and chronic lymphocytic leukemia

The DNA methylation level in patients with chronic lymphocytic leukemia is generally lower than healthy individuals. Although DNA methylation is globally decreased, regional hypermethylation of gene promoters leads to gene silencing. Many of these genes have tumor suppressor phenotypes. Unlike mutations or deletions, hypermethylation is potentially reversible after inhibition with DNA methylation modulators. Myelodysplastic syndrome has been a model disease in which treatment of patients results in demethylation of specific genes. The story in patients with chronic lymphocytic leukemia is slowly unraveling as epigenetic modifications likely also play an important role. Ongoing clinical trials correlating clinical response to gene expression after treatment with DNA methylation inhibitors will ultimately allow us to better risk stratify and predict the subgroup of patients who will benefit from treatment with this class of drugs.     

DNA methylation in hepatocellular carcinoma

As for many other tumors, development of hepatocellular carcinoma （HCC） must be understood as a multistep process with accumulation of genetic and epigenetic alterations in regulatory genes, leading to activation of oncogenes and inactivation or loss of tumor suppressor genes （TSG）. In the last decades, in addition to genetic alterations, epigenetic inactivation of （tumor suppressor） genes by promoter hypermethylation has been recognized as an important and alternative mechanism in tumorigenesis. In HCC, aberrant methylation of promoter sequences occurs not only in advanced tumors, it has been also observed in premalignant conditions just as chronic viral hepatitis B or C and cirrhotic liver. This review discusses the epigenetic alterations in hepatocellular carcinoma focusing DNA methylation.