Current Pharmaceutical Design - Volume 20, Issue 11, 2014

In cancer cells, methylation-dependent gene silencing is at least partly mediated by the “Methyl-CpG-Binding Domain protein 2” (MBD2 protein), via the recruitment of chromatin remodeling complexes. However this repressive role was poorly investigated in normal cells. To identify the genes repressed by MBD2 in these cells, we have determined the impact of MBD2 depletion on gene expression in human embryonic MRC5 fibroblasts, using RNA inference combined with microarray analysis. The up-regulation of some randomly selected genes was confirmed and a direct association between gene repression and MBD2 binding on methylated promoters associated to these genes was subsequently established. This control of gene expression appears to depend on the CpG content of promoters as MBD2 depletion was not sufficient to induce the expression of silent genes associated with High-CpG promoters, but it was required to achieve the methyl-dependent transcriptional locking of the genes associated with promoters exhibiting intermediate CpG content. Therefore, MBD2 seems to play a selective role in gene repression depending on the CpG content of the promoter regions.

Nicotinamide phosphoribosyl transferase (Nampt) is the rate-limiting enzyme for the salvage biosynthesis of nicotinamide adenine dinucleotide (NAD). Although elevated level of Nampt expression has been observed in various cancers, the involvement of Nampt promoter regulation was not well understood. We have identified a cluster of MEF2 recognition sites upstream of the functional hypoxia response elements (HREs) within the human Nampt promoter, and demonstrated that the two MEF2 sites at -1272 and -1200 were functional to upregulate the promoter activity by luciferase reporter assays. The Nampt promoter was able to be activated cooperatively following hypoxic stimulation by CoCl2 treatment with associated MEF2C overexpression. During the investigation on MEF2C regulation of endogenous Nampt expression in HeLa cells, the most significant enhancement of Nampt expression observed was by overexpression of MEF2C in combination with sodium butyrate exposure. By chromatin immunoprecipitation with a MEF2C anti-body, we found that MEF2C indeed interacted with endogenous Nampt promoter. The requirement of HDAC inhibition for the MEF2C enhancement of Nampt transcription was verified by RNAi of HDAC. Our results were in support of reports indicating that MEF2 family transcription factors interacted with HDACs and regulated downstream gene expression at the epigenetic levels. Our study provided important evidence to demonstrate the sophisticated mechanism of endogenous Nampt promoter regulation, and therefore, will help to better understand the Nampt overexpression in cancer progression, especially in the context of MEF2C upregulation which frequently occurred in cancer development and drug resistance.

Sir2-like proteins, known as sirtuins, have been under a spotlight in the realm of aging because of their positive effect on longevity in Saccharomyces.cerevisiae. Because Sir2 has been identified as a NAD+-dependent histone deacetylase, researchers have attributed its lifespan-extending utilities to gene silencing. Similar phenomena are found in multicellular eukaryotes by seemingly different mechanisms. In mammals, seven sirtuin homologs (SIRT1-7) have been identified, with varied cellular locations and molecular functions. Sirtuins target a wide spectrum of molecules for diversified post-translational modifications, thereby exerting multiple physiological benefits. The roles of sirtuins in cancer are still ambiguous, although they have been extensively studied. In this review, we summarize the multiple physiological roles played by sirtuins and their putative mechanisms, especially in cancer.

Epigenetic regulation is essential to the well-being of developing as well as developed cells by providing tissue-specific gene expression. DNA methylation on cytosine nucleotides is one of the core elements of epigenetic machinery, and stable DNA methylation patterns are maintained by properly regulated DNA methylation and DNA demethylation. DNA methylation has been studied extensively in the past 15 years, while the DNA demethylation process has largely been unknown. In this review, we briefly survey recent advances on DNA methylation/demethylation axis with special emphasis on the aspects of DNA demethylation.

Objectives: Methylenetetrahydrofolate reductase (MTHFR) is an essential enzyme for DNA biosynthesis and the epigenetic process of DNA methylation. MTHFR gene polymorphisms have been implicated as risk factors for several types of cancers. However, reports on the association of MTHFR polymorphisms with ovarian cancers are inconclusive. The aim of this study is to summarize on the reported data and meta-analytically investigate the relationship between the MTHFR C677T and A1298C polymorphism and the risk of ovarian cancer. Methods: We searched for all published articles indexed in MEDLINE (1950-2012), EMBASE (1974-2012), and CNKI (1994-2012). Case-control or cohort studies that relating to MTHFR polymorphism and ovarian cancer women were included and data were extracted independently by two reviewers. The search yielded 21 articles, from which 7 studies met the inclusion criteria. We performed a metaanalysis involving 3493 patients with ovarian cancer and 3863 controls with Review Manager 5.1 software. Odds ratio (OR) and 95% confidence intervals (CIs) were used to evaluate the ovarian cancer risk. Results: All the available data considered together, no association between the MTHFR C677T polymorphism and ovarian cancer risk was found in any genetic variations. However, in the subgroup analysis by ethnicity of Asian and Caucasian, MTHFR 677T was associated with significantly increased ovarian cancer risk among Asian [T allele vs. C allele: OR=1.50, 95% CI: 1.25-1.81, P<0.0001; CT + TT vs. CC (dominant model): OR=1.49, 95% CI: 1.18-1.88, P=0.0009; TT vs. CT + CC (recessive model): OR=2.33, 95% CI: 1.57-3.45, P<0.00001], while, there was no significant increased risk in Caucasian. As for MTHFR A1298C polymorphism, no marked association was found in either group of Caucasian population, while no data was available to analyze in Asian population. Conclusions: The C677T polymorphism of the MTHFR gene is associated with the susceptibility of ovarian cancer in Asian population, suggesting that TT genotype may serve as a risk factor of ovarian cancer among Asian but not Caucasians. In addition, there is no association between A1298C gene polymorphism and ovarian cancer, including Caucasian and Asian women.

DNA methyltransferase 1 (DNMT1) plays a significant role in maintaining DNA methylation. Aberrant DNA methylation is a recognized feature of human cancers and folate is directly involved in DNA methylation via one-carbon metabolism. Previous reports also have suggested that folate deficiency was associated with many cancers. The aim of the present study was to evaluate the effect of folate deficiency and aberrant expression of DNA methyltransferase 1 (DNMT1) on cervical cancerization. The expression of DNMT1 protein and mRNA and levels of serum folate were detected in 238 women with a diagnosis of normal cervix (NCn = 53), cervical intraepithelial neoplasia (CIN I, n = 52; CIN II/III, n = 53), and squamous cell carcinoma of the cervix (SCC; n = 80). In addition, the expression of DNMT1 protein and mRNA was measured in cervical cancer cells (Caski and C33A) treated by different concentration of folate. Serum folate levels decreased and expression levels of DNMT1 protein and mRNA increased gradually with progressive severity of the cervix lesions (P<0.001). It was found that folate was able to reduce the viability of Caski or C33A cell (r=0.978, P=0.002; r=0.984, P<0.001) and regulated aberrant expression of DNMT1 protein (r=-0.859, P=0.01; r=-0.914, P<0.001) and mRNA (r=-0.297, P=0.159; r=0.433, P=0.034) in vitro. Our findings indicated that the low-level of serum folate and high-expression of DNMT1 protein or mRNA was significantly associated with cervical carcinogenesis. Folate deficiency and aberrant expression of DNA methyltransferase 1 had additive effect on cervical cancerization. Folate supplement and recovery of aberrant DNA methylation status may offer a new strategy for prevention and therapy of cancers.

HOXA10 plays an important role in the body structure and development. Recently, patterns of deregulated HOX expression have been identified in various cancers. Meanwhile, WT1 is closely associated with the HOXA10 gene, which is an inducible transcription factor . We hypothesized that during the process of the ovarian cancer pathogenesis, the HOXA10 promoter CpG hypomethylation coupled with decreased WT1 expression could co-regulate HOXA10 expression. After the treatment of 5-Aza-dC, ovarian cancer cell lines(SKOV-3 and HEY) significantly increased HOXA10 expression. Transfection of HOXA10 siRNA with SKOV-3 and HEY ovarian cancer cell lines significantly downregulated HOXA10 expression but upregulated WT1 expression, compared to the control siRNA transfection. In ovarian cancer samples, the HOXA10 expression is significantly high. Nevertheless, the expression of HOXA10 is low in normal ones(P<0.05). We tried to use the method of methylation-specific PCR(MSP) to find the CpG sites, which are located in the scope of the WT1’s core-binding consensus(GCGG). In ovarian cancer samples, methylation prevalence was extremely high with the low expression of HOXA10. However, in the normal ovarian samples, the methylation prevalence was lower with the high expression of HOXA10(P<0. 05). In all samples of epithelial and normal ovarian samples collected, the WT1 expression with the promoter CpG hypomethylation of HOXA10 both contribute to the regulation of HOXA10 expression. That is to say, HOXA10 promoter methylationpositive combined with the WT1-positive result in the low expression of HOXA10. On the other hand, the HOXA10 expression was the highest in methylation-negative and WT1-negative samples.

Clinical screening criteria, such as young age of endometrial cancer diagnosis and family history of signature cancers, have traditionally been used to identify women with Lynch Syndrome, which is caused by mutation of a DNA mismatch repair gene. Immunohistochemistry and microsatellite instability analysis have evolved as important screening tools to evaluate endometrial cancer patients for Lynch Syndrome. A complicating factor is that 15-20% of sporadic endometrial cancers have immunohistochemical loss of the DNA mismatch repair protein MLH1 and high levels of microsatellite instability due to methylation of MLH1. The PCR-based MLH1 methylation assay potentially resolves this issue, yet many clinical laboratories do not perform this assay. The objective of this study was to determine if clinical and pathologic features help to distinguish sporadic endometrial carcinomas with MLH1 loss secondary to MLH1 methylation from Lynch Syndrome-associated endometrial carcinomas with MLH1 loss and absence of MLH1 methylation. Of 337 endometrial carcinomas examined, 54 had immunohistochemical loss of MLH1. 40/54 had MLH1 methylation and were designated as sporadic, while 14/54 lacked MLH1 methylation and were designated as Lynch Syndrome. Diabetes and deep myometrial invasion were associated with Lynch Syndrome; no other clinical or pathological variable distinguished the 2 groups. Combining Society of Gynecologic Oncology screening criteria with these 2 features accurately captured all Lynch Syndrome cases, but with low specificity. In summary, no single clinical/pathologic feature or screening criteria tool accurately identified all Lynch Syndrome-associated endometrial carcinomas, highlighting the importance of the MLH1 methylation assay in the clinical evaluation of these patients.

A growing body of evidence supports that DNA methylation-mediated silencing of tumor suppressor genes plays a significant role in cancer development. DNA methylatransferase (DNMT) is the enzyme catalyzing the methylation modification of cytosines in a CpG dinucleotide context. In humans, this reaction is highly selective for certain gene promoters and/or genomic DNA domains. Elucidation of the intracellular targeting mechanism by DNMT has become the key task for understanding epigenetic regulation in cancers. Unfortunately, no suitable method is available to explore this important cell function. This study focuses on the development of an efficient technique for measuring the intracellular, DNMT isoform-specific, and methylated gene-specific, DNA methylation alterations. The technique, designated IMA for Intracellular DNA Methylation Assay, takes advantage of covalent arresting of active DNMT molecules by aza-deoxycytidine (ADC), a modified cytosine homologue readily incorporated into genomic DNA at the cytosine position. The DNMT-DNA complex was isolated by a modified ETOH precipitation procedure to remove cellular proteins including free DNMT. Chromatin immunoprecipitation using DNMT isoform-specific antibody was subsequently performed to collect DNMT-bound DNA fragments. PCR amplification was used to detect and quantify the isolated gene fragment. Validation of the IMA was performed by manipulating the DNMT activity, by treating with antisense oligonucleotides against DNMT1 and DNMT3B, and repeating the IMA experiment. One of the main discoveries with this technique was the observation of DNMT3B maintenance methylation activity. This new technique can be applied to examine the dynamic DNMT-specific action on diversified methylation-silenced genes, in a variety of cell culture conditions.

Hematopoietic progenitors derived from human embryonic stem cells (hESCs) present both a potential cell source for cellreplacement therapies and an in vitro model for hematopoietic stem cell (HSC) development. Current protocols for the hematopoietic differentiation of hESCs suffer from low efficiency and functional defects in the derived HSCs. Epigenetic mechanisms of HSC development should be addressed to overcome these imperfections. The focus of this review is to summarize the knowledge on the epigenetic regulation of pluripotency and lineage-specific genes with the emphasis on the hematopoietic cell lineage. The potential utilization of this knowledge to improve the generation of HSCs for clinical application is also discussed.

Prostate cancer (PCa) is a very common neoplasm, which is generally treated by chemo-, radio-, and/or hormonal-therapy. After a variable time, PCa becomes resistant to conventional treatment, leading to patient death. Prostate tumor-initiating cells (TICs) and cancer repopulating cells (CRCs) are stem-like populations, driving respectively cancer initiation and progression. Histone modifiers (HMs) control gene expression in normal and cancer cells, thereby orchestrating key physiological and pathological processes. In particular, Polycomb group genes (PcGs) are a set of HMs crucial for lineage-specific gene silencing and stem cell self renewal. PcG products are organized into two main Polycomb Repressive Complexes (PRCs). At specific loci, PRC2 catalyzes histone H3 Lys27 trimethylation, which triggers gene silencing by recruiting PRC1, histone deacetylases and DNA methyl transferases. PRC1 catalyzes addition of the repressive mark histone H2A ubiquitination. Recently, the catalytic component of PRC1 (BMI1) was shown to play critical roles in prostate CRC self-renewal and resistance to chemotherapy, resulting in poorer prognosis. Similarly, pharmacological disruption of PRC2 by a small molecule inhibitor reduced the tumorigenicity and metastatic potential of prostate CRCs. Along with PcGs, some histone lysine demethylases (KDMs) are emerging as critical regulators of TIC/CRC biology. KDMs may be inhibited by specific small molecules, some of which display antitumor activity in PCa cells at micromolar concentrations. Since epigenetic gene regulation is crucial for stem cell biology, exploring the role of HMs in prostate cancer is a promising path that may lead to novel treatments.

While the epithelial-mesenchymal transition (EMT) plays a fundamental role during development, its deregulation can adversely promote tumor metastasis. The phenotypic and cellular plasticity of EMT indicates that it is subject to epigenetic regulation. A hallmark of EMT is E-cadherin suppression. In this review, we try to embrace recent findings on the transcription factor Snail-mediated epigenetic silencing of E-cadherin. Our studies as well as those of others independently demonstrated that Snail can recruit various epigenetic machineries to the E-cadherin promoter. Based on these results, we propose a model of epigenetic regulation of EMT governed by Snail. Briefly, recruitment of the LSD1/HDAC complex by Snail facilitates histone H3K4 demethylation and H3/H4 deacetylation. Histone deacetylation may promote subsequent recruitment of PRC2 to methylate H3K27, while H3K4 demethylation favors the association of H3K9 methyltransferases G9a and Suv39H1. Finally, DNA methyltransferases (DNMTs) can be recruited to the promoter area in a G9a/Suv39H1-dependent manner. Together, these chromatin-modifying enzymes function in a Snail-mediated, highly orchestrated fashion to suppress E-cadherin. Disruption of the connection between Snail and these epigenetic machineries may represent an efficient strategy for the treatment of EMT-related diseases, including tumor metastasis.

Nutrient utilization is dramatically altered when cells receive signals to proliferate. Characteristic metabolic changes enable cells to meet the large biosynthetic demands associated with cell growth and division. Changes in rate-limiting glycolytic enzymes redirect metabolism to support growth and proliferation. Metabolic reprogramming in cancer is controlled largely by oncogenic activation of signal transduction pathways and transcription factors. Although less well understood, epigenetic mechanisms may seem to contribute to the regulation of metabolic gene expression in cancer. Reciprocally, accumulating evidence suggests that metabolic alterations may affect the epigenome. Understanding the relation between metabolism and epigenetics in cancer cells may open new avenues for anti-cancer strategies. In multi-cellular systems, molecular signals promoting cell growth and proliferation mediate the switch between catabolism and anabolism. Both normal proliferating and cancer cells must achieve high levels of macromolecular biosynthesis to provide the raw materials needed to produce new daughter cells. From a therapeutic view point, it is of great interest to determine metabolic differences that exist between normal proliferating cells and cancer cells. Cancer cells also exhibit significant alterations in the epigenome. Recent data indicate that cellular metabolism and epigenetic phenomenon are engaged in crosstalk [1, 2]. Considering current efforts to target both cancer metabolism and epigenetics, an understanding of the relationship between these two key features is of paramount importance [3, 4]. Here we discuss the role of cellular metabolism in regulation of the epigenome. Moreover, we discuss how epigenetic changes may contribute to establish cancer-specific metabolic features.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection were known to be risk factors for HCC, they were suspected to promote its development by eliciting epigenetic changes. However, the precise gene targets and underlying mechanisms have not been elucidated. Epigenetic regulation of gene expression has emerged as a fundamental aspect of cancer development and progression. The molecular mechanisms of carcinogenesis in hepatocellular carcinoma involve a complex interplay of both genetic and epigenetic factors. DNA methylation, post-translational modifications of histone proteins, chromatin remodeling, and noncoding RNAs are four major types of mechanistic layers in the field of epigenetics. HBV infection could affect methylation on p16INK4A, GSTP1, CDH1(E-cadherin), RASSF1A, p21WAF1/CIP1 genes, which may play important roles in the development of HCC. HCV infection was related to aberrant methylation on SOCS-1, Gadd45β, MGMT, STAT1 and APC. Other epigenetic alterations included histone proteins, chromatin remodeling, and noncoding RNAs were described in literature. Uncovering the epigenetic alterations of HBV/HCV-induced HCC carcinogenesis could highlight a new strategy for deciphering the mechanism of HCC tumorigenesis and development, as well as a potential diagnostic advantage.

DNA methylation is an important part of the epigenetic code governing gene expression. In human reproductive diseases, recent studies have shown the existence of deviations from the normal methylation profile at various genome loci. In this review, this type of epigenetic alterations is explored in pathological spermatogenesis, ovarian diseases, placental syndromes, such as preeclampsia and Intra- Uterine Growth Restriction, uterine diseases such as endometriosis, and putative pathophysiological effects of Assisted Reproductive Technologies. We review the notion of epigenetics, the technical methods available to analyze methylation, and the known associations between reproductive diseases and DNA methylation, focusing on human pathologies and on animal models when available. We show that imprinted genes control regions (ICRs) are a prominent and frequent target of methylation anomalies in reproductive disorders, but such alterations also affect non-imprinted genes. The mechanistic aspects of gene regulation in response to methylation anomalies are also discussed in this review when they have been investigated.

Genomic imprinting is among the most important epigenetic mechanisms whereby expression of a subset of genes is restricted to a single parental allele. Loss of imprinting (LOI) through hypo or hyper methylation is involved in various human syndromes. These LOI occur early during development and usually impair growth. Some imprinting syndromes are the consequences of genetic anomalies, such as uniparental disomies (UPD) or copy number variations (deletion or duplications) involving the imprinted domains; others are due to LOI at the imprinting control regions (ICR) regulating each domain. Imprinting disorders are phenotypically heterogeneous, although some share various common clinical features such that diagnosis may be difficult. Multilocus imprinting defects associated with several syndromes have been increasingly reported in recent years, although there are no obvious clinical differences between monolocus and multilocus LOI patients. Subsequently, some rare mutations of transacting factors have been identified in patients with multilocus imprinting defects but they do not explain the majority of the cases; this therefore implies that other factors are involved. By contrast, no mutation of a transacting factor has yet been identified in monolocus LOI. The effect of the environment on the regulation of imprinting is clearly illustrated by studies of assisted reproductive technology (ART). The regulation of imprinting is complex and involves a huge range of genetic and environmental factors; the identification of these factors will undoubtedly help to elucidate the regulation of imprinting and contribute to the understanding of imprinting disorders. This would be beneficial for diagnostics, clinical follow up and the development of treatment guidelines.

Worse reproductive health in the men born through intracytoplasmic sperm injection (ICSI) or other assisted reproductive techniques (ART) has been reported in many studies. However, owing to the interference of genetic and environmental factors, it is difficult to identify whether ICSI method would affect male reproductive health. Therefore, ART mouse models were established in this study. Besides semen quality, serum testosterone and histological analysis of testes, 6 paternally expressed imprinted genes were chosen to detect their expressions and methylation levels in testes of adult F1 and F2 mice. Although the phenotypic abnormalities weren’t found, Kcnq1ot1, Mest, Peg3, Plagl1 and Snrpn in ICSI group showed lower expressions than those in naturally conceived (NC) group. The expressions of Kcnq1ot1, Peg3 and Snrpn in in vitro fertilization (IVF) conceived mice was lower than those in controlled ovarian hyperstimulation (COH) conceived mice, but higher than those in ICSI mice. Most differences between NC and ICSI group and between IVF and ICSI group were also represented in F2 generations. During the methylation analysis, no matter there was significant difference between compared groups, the changing trends of methylation level were almost opposite to their corresponding gene expressions. These results indicated that the differential expressions of paternally expressed genes occurred in testes of ICSI mice, which may be mediated by methylation modification. Both ICSI procedure and mechanical stimulation can induce intergenerational transmission of the epigenetic changes. In vitro culture and mechanical stimulation were the main factors inducing the down regulation of paternally expressed imprinted genes in testes.

To investigate the possible mechanisms of the abnormal expression patterns of many genes in the embryos in vitro, the expression of histone acetyltransferase GCN5 and histone deacetylase 1 (HDAC1) were detected in mouse preimplantation embryos. For the in vitro group, the pronucleus embryos were obtained from superovulated mice, and cultured in vitro to get the two-cell, four-cell, eightcell, morula and blastocyst stages embryos. For the in vivo group, embryos at different stages were obtained from pregnant mice directly. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry were used to detect the mRNA and protein expression levels of GCN5 and HDAC1. Compared with the embryos in vivo, the expression levels of Gcn5 in the embryos in vitro significantly increased except those in four-cell embryos. The expression of Gcn5 in eight-cell embryos in vivo and in four-cell and eight-cell embryos in vitro was higher than those at other stages within the same group. Compared with the expressions of Hdac1 in embryo in vivo, only those at two-cell embryo in vitro showed decreased level. The expression of Hdac1 enhanced after two-cell embryo stage in vitro, but no difference showed in vivo. The protein expression of GCN5and HDAC1 in the embryo in vitro at every stage showed a lower level with the control of those in the embryos in vivo. Our studies indicated that in vitro culture could induce the expressed alteration of GCN5 and HDAC1, which might be related to the expression patterns of many other epigenetically regulated genes.

DNA methylation is an important regulatory component which influences phenotypes by modulating gene expression. Changes in DNA methylation may lead to altered phenotypes and ability of an organism to respond to stress leading to subsequent manifestation of life style diseases, cancer, etc. The human X chromosome represents a classical model for epigenetic processes governing differential regulation of homologous chromosomes. X monosomy (45, XO) leads to Turner's syndrome in human with mild to severe phenotypes. Using a novel cDNA based high throughput approach of assessing genome wide methylation; we have examined the methylation landscape in human fibroblasts in 45, XO and 46, XX individuals. We report here that as expected methylation of X linked genes is different in these two situations. It was observed that methylation of several autosomal genes is also affected in this X monosomy state. Genes involved in bone remodeling, glucose sensitivity and ovarian function appear to be altered in addition to genes involved in epigenetic regulatory processes. This opens up interesting possibility of misregulation of DNA methylation in the X monosomy state resulting in altered gene expression and altered phenotypes. This may be one of the reasons for the variance, differential severity and penetrance in case of Turner’s syndrome. We propose that a systematic analysis of the molecular genetic mechanisms governing this epigenetic regulation will open up new therapeutic interventions which will certainly help in reducing severity of the disease and help in better management of X monosomy (Turner’s syndrome).