Epigenetic Deregulation Of Gene Expression Research Articles

Decitabine potentiates efficacy of doxorubicin in a preclinical trastuzumab-resistant HER2-positive breast cancer models

Acquired drug resistance and metastasis in breast cancer (BC) are coupled with epigenetic deregulation of gene expression. Epigenetic drugs, aiming to reverse these aberrant transcriptional patterns and sensitize cancer cells to other therapies, provide a new treatment strategy for drug-resistant tumors. Here we investigated the ability of DNA methyltransferase (DNMT) inhibitor decitabine (DAC) to increase the sensitivity of BC cells to anthracycline antibiotic doxorubicin (DOX). Three cell lines representing different molecular BC subtypes, JIMT-1, MDA-MB-231 and T-47D, were used to evaluate the synergy of sequential DAC + DOX treatment in vitro. The cytotoxicity, genotoxicity, apoptosis, and migration capacity were tested in 2D and 3D cultures. Moreover, genome-wide DNA methylation and transcriptomic analyses were employed to understand the differences underlying DAC responsiveness. The ability of DAC to sensitize trastuzumab-resistant HER2-positive JIMT-1 cells to DOX was examined in vivo in an orthotopic xenograft mouse model. DAC and DOX synergistic effect was identified in all tested cell lines, with JIMT-1 cells being most sensitive to DAC. Based on the whole-genome data, we assume that the aggressive behavior of JIMT-1 cells can be related to the enrichment of epithelial-to-mesenchymal transition and stemness-associated pathways in this cell line. The four-week DAC + DOX sequential administration significantly reduced the tumor growth, DNMT1 expression, and global DNA methylation in xenograft tissues. The efficacy of combination therapy was comparable to effect of pegylated liposomal DOX, used exclusively for the treatment of metastatic BC. This work demonstrates the potential of epigenetic drugs to modulate cancer cells' sensitivity to other forms of anticancer therapy.

IDH3α regulates one-carbon metabolism in glioblastoma.

Mutation or transcriptional up-regulation of isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) promotes cancer progression through metabolic reprogramming and epigenetic deregulation of gene expression. Here, we demonstrate that IDH3α, a subunit of the IDH3 heterotetramer, is elevated in glioblastoma (GBM) patient samples compared to normal brain tissue and promotes GBM progression in orthotopic glioma mouse models. IDH3α loss of function reduces tricarboxylic acid (TCA) cycle turnover and inhibits oxidative phosphorylation. In addition to its impact on mitochondrial energy metabolism, IDH3α binds to cytosolic serine hydroxymethyltransferase (cSHMT). This interaction enhances nucleotide availability during DNA replication, while the absence of IDH3α promotes methionine cycle activity, S-adenosyl methionine generation, and DNA methylation. Thus, the regulation of one-carbon metabolism via an IDH3α-cSHMT signaling axis represents a novel mechanism of metabolic adaptation in GBM.

Epigenetics and Adult Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is the most common leukemia diagnosed in adults and curative treatments remain currently a great challenge. AML occur in about 60% of patients over 60 years with unsatisfactory survival results, which have not improved significantly over the last two decades, in spite of improved supportive care. Global genomic instability is the hallmark of the disease, leading to chromosomal/molecular alterations, proliferative advantage and clonal heterogeneity. Epigenetic deregulation of gene expression is considered as a consequence and/or the initiating cause of this instability and DNA methylation is one of the most commonly occurring epigenetic events in this setting. The plasticity of the epigenome, compared to the conserved status of the genome, designed it as a therapeutic target especially in AML. Among novel therapeutic agents, the availability of compounds targeting epigenetic modifiers offers the opportunity to have new therapeutic insights with improvement of outcome, especially in older patients, while reducing toxicity. We reviewed herein some of the epigenetic mechanisms involved in normal hematopoiesis, which may be deregulated in AML, and therapeutic potentials of cell re-programming. Keywords: Acute myeloid leukemia, azacitidine, decitabine, epigenetics, hypomethylating agents, targeted therapies.

Silencing of dominant genes in heterozygous genotypes of interspecific hybrids Fagopyrum esculentum Moench. × C2026 F. homotropicum Ohnishi

Fagopyrum homotropicum Ohnishi is a very polymorphic self-pollinating species with homostylous flowers, which morphologically different lineages are differ also in ability to hybridize with F. esculentum Moench. (closely related outcrosser with heterostyly). A lineage C2026 F. homotropicum diverged from F. esculentum with forming noticeable pre-zygotic and post-zygotic barriers: the most successful interspecific crossing F esculentum x C2026 resulted wrinkled hybrid seeds germinated in Petri dishes. These interspecific hybrids and backcrosses F. esculentum x F₁, being heterozygous at loci DET/det, SHT/sht and homostyly gene of F. homotropicum, in our experiments often formed phenotype like a recessive homozygote for at least one of these genes, i.e. dominant alleles were silenced. Apparently, these effects can be caused by disorders of epigenetic regulation associated with the divergence of hybridized species. Such disorders, especially those that occur at the stage of seed development, represent one of the main experimentally confirmed mechanisms of pre-zygotic isolation between species. Apparently, F. esculentum and the lineage C2026 of F. homotropicum represent an example of intermediate stage of post-zygotic isolation development process which based on epigenetic deregulation of gene expression in the hybrids. Sometimes it may be revealed not only at the stage of seed development, but also at later stages of ontogenesis.

Abstract 71: Epigenetic control of signature gene expression in the inducible differentiation of acute promyelocytic leukemia cells

Abstract [Background] Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), all-trans retinoic acid (ATRA) or dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still not well determined. [Methods and Results] We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, ATRA/DMSO and D3 and found that around 3000 genes are significantly altered commonly under those treatments in the process. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of common down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression. [Conclusion] We identified the signature genes controlling the inducible differentiation of promyelocytic leukemia cells and found the epigenetic mechanism regulating the gene expression in the process. Citation Format: Chunhua Song, Xiaokang Pan, Zhanjun Li, Yali Ding, Sunil Mudhusami, Chandrika Gowda, Zheng Ge, Sadie Steffens, Bi-hua Tan, Sinisa Dovat. Epigenetic control of signature gene expression in the inducible differentiation of acute promyelocytic leukemia cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 71. doi:10.1158/1538-7445.AM2015-71

Role of histone deacetylases in pancreas: Implications for pathogenesis and therapy.

In the last years, our knowledge of the pathogenesis in acute and chronic pancreatitis (AP/CP) as well as in pancreatic cancerogenesis has significantly diversified. Nevertheless, the medicinal therapeutic options are still limited and therapeutic success and patient outcome are poor. Epigenetic deregulation of gene expression is known to contribute to development and progression of AP and CP as well as of pancreatic cancer. Therefore, the selective inhibition of aberrantly active epigenetic regulators can be an effective option for future therapies. Histone deacetylases (HDACs) are enzymes that remove an acetyl group from histone tails, thereby causing chromatin compaction and repression of transcription. In this review we present an overview of the currently available literature addressing the role of HDACs in the pancreas and in pancreatic diseases. In pancreatic cancerogenesis, HDACs play a role in the important process of epithelial-mesenchymal-transition, ubiquitin-proteasome pathway and, hypoxia-inducible-factor-1-angiogenesis. Finally, we focus on HDACs as potential therapeutic targets by summarizing currently available histone deacetylase inhibitors.

Chromatin State Shaping the Gene Expression Profiling in the Inducible Differentiation of Promyelocytic Leukemia Cells

Background Epigenetic changes in DNA and chromatin are regarded as emerging major players for hematopoietic stem cells development and lineage differentiation. Epigenetic deregulation of gene expression leads to leukemia and reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. The promyelocytic leukemia HL-60 cell can differentiate into microphage, granulocyte and monocyte with stimulation of phorbol myristic acid (PMA), dimethylsulfoxide (DMSO), and Vitamin D3, respectively, by affecting the gene expression on cell cycle and cell differentiation. However, how epigenetic regulation on gene expression in the inducible differentiation is still undetermined.Methods and Results We did the microarray analysis to identify the genome-wide gene expression profile of HL-60 cells in various time points(0h, 6h, 1d,2d,4d and 6d) under the treatment of PMA, DMSO and D3 and found that around 3000 genes are significantly altered commonly in the cells upon the 3 treatments. The percentage of down-regulated genes in the all commonly altered genes is significantly higher than that of up-regulated genes, and the significantly altered genes showed significantly physical clustering on chromosome loci, indicating the epigenetic regulation involved in the regulation of the gene expression. We did observed the expression changes of epigenetic enzymes in the process and further did the ChIP-on-ChIP analysis by using the customer array tiling the selected changed genes and 5 genomic loci hybridizing with ChIP’d DNA of histone markers (H3K4me3, H3K9me3 and H3K27me3). We found that the obvious decrease of H3K4me3 binding and increase of H3K9me3 and H3K27me3 are observed in the promoter of the commonly down-regulated genes. These data indicated histone H3K4, H3K9 and H3K27 marker play important roles in shaping the chromatin state and regulating gene expression.Conclusion We identified the signature genes controlling the inducible differentiation of promyelocytic leukemiacells and found the epigenetic mechanism regulating the gene expression in the process. DisclosuresNo relevant conflicts of interest to declare.

Abstract IA20: Epigenetic deregulation of gene expression

Abstract Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss of function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCPG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using alpha-ketoglutarate (aKG) as a substrate. 2HG can disrupt the activity of enzymes that use aKG as a cofactor, including TET2 and the Jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibit TET2 hydroxymethylation and Jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards reduced histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is abundance in hematopoietic stem cells, and displays specific distribution patterns, and yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B-cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Citation Format: Ari Melnick. Epigenetic deregulation of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA20.

Epigenetic effects of nano-sized materials

The term epigenetics includes several phenomena such as DNA methylation, histone tail modifications, and microRNA mediated mechanisms, which are able to mold the chromatin structure and/or gene expression levels, without altering the primary DNA sequence. Environmental agents can exert epigenetic properties and there is increasing evidence of epigenetic deregulation of gene expression in several human diseases, including cancer, cardiovascular diseases, autism spectrum disorders, autoimmune diseases, and neurodegeneration, among others. Given the widespread use and dispersion in the environment of nano-sized materials, this article summarizes the studies performed so far to evaluate their potential epigenetic properties. Those studies highlight the ability of certain nano-sized compounds to induce an impaired expression of genes involved in DNA methylation reactions leading to global DNA methylation changes, as well as changes of gene specific methylation of tumor suppressor genes, inflammatory genes, and DNA repair genes, all potentially involved in cancer development. Moreover, some nano-sized compounds are able to induce changes in the acetylation and methylation of histone tails, as well as microRNA deregulated expression. We also provided a detailed description of currently available methodologies to evaluate epigenetic modifications. Standard protocols are currently available to evaluate cytotoxic and genotoxic effects of nano-sized materials. By contrast, there are at present no available standard protocols to evaluate the epigenetic potential of any given compound. The currently available methodologies offer different, but often complementary information to characterize potential epigenetic changes induced by exposure to nano-sized compounds. Given the widespread use and dispersion in the environment of nano-sized materials, at present and foreseeable in the near future, and in light of the indication of potential epigenetic properties here reviewed, more attention should be paid to unravel the consequences of such effects in future studies.

The histone methyltransferase MMSET/WHSC1 activates TWIST1 to promote an epithelial–mesenchymal transition and invasive properties of prostate cancer

Epigenetic deregulation of gene expression plays a role in the initiation and progression of prostate cancer (PCa). The histone methyltransferase MMSET/WHSC1 (Multiple Myeloma Set Domain) is overexpressed in a number of metastatic tumors, but its mechanism of action has not been defined. In this work, we found that PCa cell lines expressed significantly higher levels of MMSET compared to immortalized, non-transformed prostate cells. Knockdown experiments showed that, in metastatic PCa cell lines, dimethylation of lysine 36 and trimethylation of lysine 27 on histone H3 (H3K36me2 and H3K27me3, respectively) depended on MMSET expression, while depletion of MMSET in benign prostatic cells did not affect chromatin modifications. Knockdown of MMSET in DU145 and PC-3 tumor cells decreased cell proliferation, colony formation in soft agar, and strikingly diminished cell migration and invasion. Conversely, overexpression of MMSET in immortalized, non-transformed RWPE-1 cells promoted cell migration and invasion, accompanied by an epithelial to mesenchymal transition (EMT). Among a panel of EMT-promoting genes analyzed, TWIST1 expression was strongly activated in response to MMSET. Chromatin immunoprecipitation analysis demonstrated that MMSET binds to the TWIST1 locus, leading to an increase in H3K36me2, suggesting a direct role of MMSET in the regulation of this gene. Depletion of TWIST1 in MMSET-overexpressing RWPE-1 cells blocked cell invasion and EMT, indicating that TWIST1 was a critical target of MMSET, responsible for the acquisition of an invasive phenotype. Collectively, these data suggest that MMSET plays a role in PCa pathogenesis and progression through epigenetic regulation of metastasis-related genes.

Cytogenetic and Molecular Abnormalities in Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematological disorders characterized by ineffective hematopoiesis which causes peripheral cytopenias and a risk of progression to acute myeloid leukemia. Although various forms of chromosomal abnormalities have been detected in approximately 50-60% of patients with de novo MDS and in up to 80% of patients with therapy-related MDS, their molecular significance for pathogenesis and disease progression is not yet fully understood. Recent technical advances in molecular biology have disclosed more accurately details of pathological chromosomal and molecular aberrations in MDS. Such details could not be identified with conventional cytogenetical techniques, including G-banding. In particular, with recent technical advances in comparative genome hybridization or single nucleotide polymorphism array technology, several candidate genes for the pathogenesis of MDS have been identified, which are located in minimally deleted or uniparental disomy segments. Moreover, epigenetic deregulation of gene expression is also likely to be involved in the pathogenesis of MDS. Accordingly, in addition to classical oncogenic abnormalities, such as p53 abnormalities, or NRAS mutation, various molecular abnormalities, such as TET2, RPS14, or c-CBL, have been identified and/or proposed as the novel candidates for molecular basis of the development and progression of MDS. A better understanding of the causative molecular events underlying MDS pathogenesis is essential for the development and establishment of a more effective treatment resulting in a complete cure for MDS. We here review current knowledge regarding the molecular significance of chromosomal and genetic aberrations in MDS and the proposed molecular mechanisms of action of new agents for MDS, such as lenalidomide or azacitidine.

Abstract B48: Xanthohumol from hops (Humulus lupulus L.) reactivates epigentically silenced genes by potential DNA methyltransferase inhibition

Abstract Epigenetic silencing of tumor suppressor genes by DNA hypermethylation is now recognized as a hallmark of cancer. Thus, reversal of DNA hypermethylation by demethylating agents and DNA methyltranserase (Dnmt) inhibitors has been extensively investigated. Recently, several studies have shown that natural dietary compounds have the potential to reactivate epigenetically silenced genes by various mechanisms, which might have an impact on chemoprevention strategies. Xanthohumol (XN), a prenylated chalcone from hops, exerts a broad spectrum of chemopreventive actions and inhibits mammary carcinogenesis in vivo. In this study, we report for the first time that epigenetic deregulation of gene expression during carcinogenesis is a novel target of XN action. We demonstrated that XN reactivates a series of candidate genes silenced by DNA promoter hypermethylation in human cancer cell lines. By treating human breast and prostate cancer cell lines MCF-7 and LNCaP with subtoxic XN-concentrations for 48 and 72h, respectively, we observed significant dose-dependent re-expression of glutathione-S-transferase Π (GSTPi), Cyclin D2 and RassF1A mRNA, measured by quantitative RT-PCR. The demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) was used as positive control. To address if the XN-mediated mechanism of gene re-expression involved inhibition of Dnmt activity, we tested XN in an in vitro system using the bacterial methlytransferase M.SssI as an enzyme source. XN at concentrations ranging from 3.13–50 µM dose-dependently inhibited M.SssI activity with a halfmaximal inhibitory concentration of 35.2 µM (p<0.05). Computational modelling of XN docking to Dnmt1 indicates steric interaction with the active site. In addition, in MCF-7 and LNCaP cells treated with XN, expression of Dnmt1, 3a and 3b was inhibited by XN-treatment in a dose-dependent manner, both at the transcriptional (p<0.05) and protein level. Next, demethylating potential of XN was investigated. DNA of XN-treated MCF-7 and LNCaP cells was subjected to quantitative methylation analyis using the Sequenom Massarray platform. Whereas treatment for 72h with 1µM of 5-aza-dC significantly reduced DNA hypermethylation of promoter CpG islands of GSTPi, Cyclin D2 and RassF1A by 10–20% (p<0.05), XN-treatment with 5–10µM did not show general DNA demethylation. However, individual CpG sites in the analyzed regions were strongly demethylated upon XN-treatment. Ongoing studies investigate whether demethylation of these single CpG sites is crucial for active transcription, and whether this can be correlated to induced gene-expression. In conclusion, we demonstrated that XN reactivates the expression of hypermethlyated genes by inhibition of Dnmt activity, expression and demethylation of selected CpG sites, which represents a novel chemopreventive mechanism by XN. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B48.

The rationale and use of hypomethylation agents in adult acute myeloid leukemia

Background: Epigenetic deregulation of gene expression is a newly recognized mechanism that leads to hematologic malignancies such as leukemia and myelodysplastic syndromes. DNA methylation is one of the most commonly occurring epigenetic events. Objective/methods: The rationale and use of hypomethylation agents in adult acute myeloid leukemia are discussed. Data in this review came from the published literature. Results/conclusion: In leukemias, alterations in DNA methylation are characterized by the hypermethylation of several genes. Hypermethylation represses transcription of the promoter regions of tumor suppressor genes leading to gene silencing. This change is reversible making it an important therapeutic target. Drugs such as methyltranferase inhibitors including 5-azacytidine and 5-aza-2′-deoxycytidine, and histone deacetylase inhibitors are being used in the treatment of these hematological malignancies.

Epigenetic plasticity of chromatin in embryonic and hematopoietic stem/progenitor cells: therapeutic potential of cell reprogramming

During embryonic development and adult life, the plasticity and reversibility of modifications that affect the chromatin structure is important in the expression of genes involved in cell fate decisions and the maintenance of cell-differentiated state. Epigenetic changes in DNA and chromatin, which must occur to allow the accessibility of transcriptional factors at specific DNA-binding sites, are regarded as emerging major players for embryonic and hematopoietic stem cell (HSC) development and lineage differentiation. Epigenetic deregulation of gene expression, whether it be in conjunction with chromosomal alterations and gene mutations or not, is a newly recognized mechanism that leads to several diseases, including leukemia. The reversibility of epigenetic modifications makes DNA and chromatin changes attractive targets for therapeutic intervention. Here we review some of the epigenetic mechanisms that regulate gene expression in pluripotent embryonic and multipotent HSCs but may be deregulated in leukemia, and the clinical approaches designed to target the chromatin structure in leukemic cells.