Demethylating Agent Research Articles

Prognostic significance of DNMT3A alterations in Middle Eastern papillary thyroid carcinoma

BackgroundThyroid cancer is the second most common cancer affecting Saudi women after breast cancer, with papillary thyroid carcinoma (PTC) accounting for 80–90% of thyroid cancers. DNA methyltransferases affect DNA methylation, and it is thought that they play an important role in the malignant transformation of various cancers. MethodsWe sought to evaluate the frequency of DNA methyltransferase 3A (DNMT3A) alterations in a large cohort of >1000 PTC cases using exome sequencing, capture sequencing, immunohistochemistry and methylation-specific polymerase chain reaction. We also performed in vitro analysis to investigate the role of DNMT3A methylation in PTC cell lines. ResultsDNMT3A pathogenic mutations were noted in 1.2% (12/1013) of PTC cases. Reduced/loss of DNMT3A expression was seen in 59.8% (579/968) of PTC cases and was significantly associated with the DNMT3A mutation (p = 0.0120). DNMT3A alterations (mutation and/or loss of expression) were associated with aggressive clinical parameters and a poor outcome. The promoter region of the DNMT3A gene was methylated in 57.1% of PTC cases tested and was significantly associated with reduced DNMT3A protein expression (p = 0.0253). Treatment of the methylated PTC cell line with 5-aza-2′-deoxycytidine resulted in demethylation of the DNMT3A gene, leading to restoration of its expression. Demethylation significantly potentiated the TRAIL-mediated apoptosis in PTC cells. Interestingly, silencing of DNMT3A using siRNA suppressed TRAIL-mediated apoptosis. ConclusionThese findings suggest that DNMT3A alterations play an important role in PTC pathogenesis and demethylation agents can be used to restore the function of DNMT3A in a subset of patients with PTC.

Abstract 583: DNA demethylating agents enhance susceptibility of lung cancer cells to γδ T cell-based immunotherapy via MHC-independent mechanisms

Abstract Immune-based therapy is revolutionizing lung cancer management in the clinic. In addition to rejuvenating T cells through immune checkpoint blockade, enhancing the immunogenicity of cancer cells and their interactions with immune cells may also be key to boosting the clinical efficacy of immunotherapy. Previous transcriptomic studies have shown that DNA demethylating agents may alter antigen processing/presentation machinery, upregulate cancer-testis antigens, reactivate endogenous retroviruses to facilitate efficacy of immunotherapy. Nevertheless, how DNA demethylating agents modulate surface immune synapse proteins and affect interactions between cancer and immune cells remain unclear. We utilized stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative membrane proteomics to identify surface proteins altered in decitabine-treated human lung cancer cells. The results showed that decitabine upregulated multiple immune synapse proteins. Gene enrichment analyses discovered that these surface proteins are predominantly involved in γδT cell-mediated cytotoxicity. γδT cells are a distinct subgroup of T cells that bridge between the innate and the adaptive immune systems and do not require the major histocompatibility complexes for antigen recognition. Co-culture of decitabine-pretreated human lung cancer cells with allogenic γδT cells in vitro confirmed sensitization effects of decitabine for the killing of γδT cells. A similar effect was also observed in immunocompromised mice bearing xenograft tumors in vivo. Moreover, the comparison of decitabine-induced surface proteomes between sensitizable and unsensitizable lung cancer cells revealed that intercellular adhesion molecule 1 (ICAM1), was among the top differentially expressed proteins. Imaging analyses confirmed that ICAM1 participated in the formation of immune synapses between cancer cells and γδT cells. Increased expression of ICAM1 was also observed in xenograft tumors from mice receiving combination therapy of decitabine and γδT cells. Overall, our data characterized decitabine-induced surface proteomes and uncovered an immunomodulatory effect of decitabine for enhancing MHC-independent killing by γδT cells. These findings have great clinical implications and provide a novel molecular basis for coupling DNA demethylation agents with γδT cell-based immunotherapy in the treatment of lung cancer patients. Citation Format: Rueyhung Weng, Chien-Ting Lin, Tai-Chung Huang, Hsuan-Hsuan Lu, Yi-Chieh Wu, Xuan-Hui Lin, Rong-Shan Lin, Chong-Jen Yu, Hsing-Chen Tsai. DNA demethylating agents enhance susceptibility of lung cancer cells to γδ T cell-based immunotherapy via MHC-independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 583.

Abstract 947: Defining UHRF1 domains that support maintenance of human colon cancer DNA methylation and tumorigenicity

Abstract Reversing the DNA methylation abnormalities by targeting the maintenance DNA methylation machinery represents a sought-after therapy paradigm in both liquid and solid tumors. UHRF1, a multi-domain protein with both chromatin reader and writer functions, is essential for targeting DNA methyltransferase 1 (DNMT1) to replicating DNA to maintain DNA methylation in both normal and cancer cells. Dysregulation of the DNMT1-UHRF1 axis is being increasingly linked to malignant transformation and progression across cancer types. Our recent study shows that UHRF1 depletion, alone or in combination with low doses of a DNMT inhibitor, effectively induces DNA demethylation and tumor suppressor genes (TSGs) reactivation. These findings collectively suggest UHRF1 represents a promising target for developing next-generation DNA demethylation agents for cancer therapy. Understanding the collective contribution of UHRF1’s domains for maintenance methylation is essential for the major translational goal of blocking maintenance of abnormal DNA methylation in established cancers and throughout cancer initiation and progression. While the UHRF1 domains responsible for de novo or re-methylation have been identified, the domain requirements specifically for maintaining normal and cancer-specific DNA methylation are poorly characterized. Herein, by developing a carefully timed genetic complementation assay, we systematically interrogate the roles for each major domain of UHRF1 for maintaining genome-wide DNA methylation in human colorectal cancer (CRC) cells. Distinct from the domain required for establishing methylation, we find that UHRF1 histone-binding and hemimethylated DNA reader domains, but not E3 ligase activity, are essential for maintaining cancer-specific DNA methylation in both HCT116 and RKO cells. Disrupting either one of the essential domains phenocopies UHRF1 depletion for global DNA demethylation and reactivation of epigenetically silenced TSGs. Supporting the essential roles of abnormal DNA methylation in sustaining the key oncogenic properties of CRC cells, we further show genetic perturbation of either histone- or hemimethylated DNA-binding activities of UHRF1 dramatically impairs CRC proliferation, invasion, and metastasis in vitro and in vivo. Moreover, for eight UHRF1 chromatin-reader domain regulated TSGs, we reveal a strong negative correlation between high UHRF1 expression, promoter hypermethylation, and their low expression with disease progression and overall survival in CRC patients in both TCGA and two independent CRC cohorts (n=363 and 390, respectively). Taken together, our data identify important differences from domains dominant for de novo DNA methylation, and provide important implications for the oncogenic functions of UHRF1 in CRC, and for credentialing UHRF1 as a desirable target for cancer drug development and means to personalize this. Citation Format: Xiangqian Kong, Jie Chen, Wenbing Xie, Stephen M. Brown, Yi Cai, Kaichun Wu, Daiming Fan, Yongzhan Nie, Yong Tao, Ray-Whay Chiu Yen, Hariharan Easwaran, Michael J. Topper, Scott B. Rothbart, Limin Xia, Stephen B. Baylin. Defining UHRF1 domains that support maintenance of human colon cancer DNA methylation and tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 947.

Synergistic Efficacy of the Demethylation Agent Decitabine in Combination With the Protease Inhibitor Bortezomib for Treating Multiple Myeloma Through the Wnt/β-Catenin Pathway.

Multiple myeloma (MM) is a hematopoietic malignancy characterized by the clonal proliferation of antibody-secreting plasma cells. Bortezomib (BZM), the first FDA-approved proteasome inhibitor, has significant antimyeloma activity and prolongs the median survival of MM patients. However, MM remains incurable predominantly due to acquired drug resistance and disease relapse. β-Catenin, a key effector protein in the canonical Wnt signaling pathway, has been implicated in regulating myeloma cell sensitivity to BZM. Decitabine (DAC) is an epigenetic modulating agent that induces tumor suppressor gene reexpression based on its gene-specific DNA hypomethylation. DAC has been implicated in modulating Wnt/β-catenin signaling by promoting the demethylation of the Wnt/β-catenin antagonists sFRP and DKK. In this study, we report the effects of single reagent DAC therapy and DAC combined with BZM on β-catenin accumulation, myeloma cell survival, apoptosis, and treatment sensitivity. Our study proved that DAC demethylated and induced the reexpression of the Wnt antagonists sFRP3 and DKK1. DAC also reduced GSK3β (Ser9) phosphorylation and decreased β-catenin accumulation in the nucleus, which were induced by BZM. Thus, the transcription of cyclin D1, c-Myc, and LEF/TCF was reduced, which synergistically inhibited cell proliferation, enhanced BZM-induced apoptosis, and promoted BZM-induced cell cycle arrest in myeloma cells. In summary, these results indicated that DAC could synergistically enhance myeloma cell sensitivity to BZM at least partly by regulating Wnt/β-catenin signaling. Our results can be used to optimize therapeutic regimens for MM.

Effect of Different DNA Demethylating Agents on In vitro Cultures of Peach Rootstock GF 677

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and, in some cases, provides a valuable source of new phenotypes suitable for crop improvement. Numerous studies have confirmed that these changes can be explained by alterations of DNA methylation. Interestingly, a group of chemical compounds termed ‘demethylating agents’ (DMT agents) enable artificial changes to be made in the DNA methylation state. Thus, these agents are theoretically able to induce new phenotypes or more favourable properties. The objective of the present study was to verify suitable conditions for the application of different DMT agents within in vitro protocols for micropropagation using the stone fruit rootstock GF 677 as an example. The impact of these agents on the properties of plant regenerants was evaluated, and their DNA methylation state was controlled by using an AFLP protocol based on a restriction endonuclease that differed in its sensitivity to methylated cytosines. Moreover, the effect of newly synthesised derivates was compared with that of conventional compounds with a well-documented DNA-demethylating impact. Based on the results, the suitable concentration for treatment by a DMT agent was established as approximately 50 µM. Promising results were generated using a combination of DMT agents with different mechanisms of action, such as azacytidine and dihydroxypropyladenine; under these conditions, probable synergy between methyltransferase interception by the cytosine analogue and interruption of methyl group donation by dihydroxypropyladenine significantly changed the DNA methylation state of treated plants. Regarding newly synthesised compounds, the 5,6-dihydro-5-azacytosine nucleoside showed the most promising results, which can likely be explained by its higher stability in the media used for in vitro cultivation.
 
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 In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.
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Gene-specific DNA demethylation changes associates with ethylene induced germination of soybean [Glycine max (L.) Merrill

The present work aims to evaluate epigenetic changes during ethylene (ET) induced germination in soybean [Glycine max (L.) Merrill]. Our results demonstrated that ethephon (2-chloroethylphosphonic acid, an ET-releasing compound) primed seeds exhibited: (1) DNA hypo-methylation of representative candidate genes (viz. ERF1b and AS-α1) and (2) functional restoration of DNA methylation dependent phenotype and expression of transcriptional silenced reporter transgene. Based on these observations, we proposed the novel and generic role of ET as a DNA de-methylating agent during the germination, which may lead to early developmental phenotypes in plants.

Threshold Inhibition of Methyltransferase G9a/Glp Exacerbates Neuropathic Hypersensitivity through Mediating GRIN2B Methylation

Background In this study, we investigated whether G9a related DNA methylation and histone modification is involved in the transcription alteration of NR2B following peripheral nerve injury and subsequently contributes to pain facilitation of G9a inhibition. Methods After approval by the institutional ethical committee on pain research in conscious animals, C57BL/6 mice were used to induce neuropathic pain with spared nerve injury (SNI). G9a/Glp expression, GRIN2B gene 5’-regulatory region CpG sites methylation profile, as well as NR2B expression in the spinal dorsal horn following SNI was detected with immunofluorescence, bisulfite sequencing, and western blot, respectively. For mechanism study, threshold doses of G9a/Glp inhibitors BIX01294/UNC0638 or direct DNA demethylation agent 5-Aza was intrathecal injected through the pre-buried catheter daily in bolus for 3 days, G9a/Glp and its enzymatic substrate H3K9me2/H3K9me3 expression, GRIN2B gene methylation alteration, as well as NR2B expression were observed. Nociceptive behavior was depicted in response to von Frey filaments following SNI with or without intrathecal BIX/UNC and 5-Aza treatments. Results Ipsilateral mechanical withdrawal threshold rather than thermal withdrawal latency prominently decreased and peaked at day 7 to 14 post SNI. Besides, nerve injury consistently increased G9a/Glp, H3K9me2 expression, GRIN2B gene 5’-regulatory region CpG sites methylation, as well as NR2B expression in the spinal dorsal horn at day 7 post SNI. Furthermore, either G9a/Glp inhibition by BIX/UNC or H3K9me2 blockade by 5-Aza independently reversed GRIN2B gene high methylation, followed with disinhibition of NR2B transcription inhibition. Consistent with molecular changes, either BIX/UNC or 5-Aza further worsens nerve injury-induced allodynia. Conclusion G9a/Glp contributes to the pathogenesis of neuropathic pain via methylating GRIN2B gene affecting NR2B transcription. G9a/Glp at an elevated setpoint may prevent the over-sensitization following peripheral nerve injury.

Effects of parental light environment on growth and morphological responses of clonal offspring.

Environments experienced by parent ramets of clonal plants can potentially influence fitness of clonal offspring ramets. Such clonal parental effects may result from heritable epigenetic changes, such as DNA methylation, which can be removed by application of DNA de-methylation agents such as 5-azacytidine. To test whether parental shading effects occur via clonal generation and whether DNA methylation plays a role in such effects, parent plants of the clonal herb Alternanthera philoxeroides were first subjected to two levels of light intensity (high versus low) crossed with two levels of DNA de-methylation (no or with de-methylation by application of 5-azacytidine), and then clonal offspring taken from each of these four types of parent plant were subjected to the same two light levels. Parental shading effects transmitted via clonal generation decreased growth and modified morphology of clonal offspring. Offspring responses were also influenced by DNA methylation level of parent plants. For clonal offspring growing under low light, parental shading effects on growth and morphology were always negative, irrespective of the parental de-methylation treatment. For clonal offspring growing under high light, parental shading effects on offspring growth and morphology were negative when the parents were not treated with 5-azacytidine, but neutral when they were treated with 5-azacytidine. Overall, parental shading effects on clonal offspring performance of A.philoxeroides were found, and DNA methylation is likely to be involved in such effects. However, parental shading effects contributed little to the tolerance of clonal offspring to shading.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.

The methylation of Notch1 promoter mediates the osteogenesis differentiation in human aortic valve interstitial cells through Wnt/β-catenin signaling.

Aortic valve interstitial cells (AVICs) have the potential to undergo calcification, which has been regarded as a critical issue during the pathology of calcific aortic valve disease (CAVD). In the past decade, epigenetics, in particular, DNA methylation dysregulation, has been reported to play a vital role in the occurrence and development of CAVD. In the present study, the expression of Notch1, which can inhibit the osteogenesis differentiation of valve interstitial cells, was downregulated whereas the expression of methyltransferases was upregulated in CAVD tissues, suggesting the potential role of DNA methylation in Notch1 expression and CAVD progression. As revealed by DNA extraction and bisulfite sequencing polymerase chain reaction (PCR), the methylation level in Notch1 promoter was much higher in CAVD tissues and human AVICs on Day 14 of osteogenesis differentiation induction. The silence of Notch1 intercellular domain (NICD) promoted while the treatment of demethylation agent, 5-Aza-dC, inhibited the osteogenesis differentiation. Moreover, NICD overexpression significantly suppressed the transcriptional activity of β-catenin on TCF4, and the expression of osteogenesis differentiation factors, indicating the involvement of Wnt/β-catenin signaling in Notch1 modulating the osteogenesis differentiation in human AVICs (hAVICs). Taken together, Notch1 promoter methylation leads to a decreased Notch1 expression and subsequent decreased release of NICD in the nucleus of hAVICs, therefore promoting the activation of Wnt/β-catenin signaling and the expression of osteogenesis differentiation factors, finally promoting the osteogenesis differentiation in hAVICs. DNA methylation might act as an important bridge to link epigenetic variation and CAVD progression.

Hypermethylation of mitochondrial transcription factor A induced by cigarette smoke is associated with chronic obstructive pulmonary disease

Purpose of the study: Cigarette smoking is a leading environmental contributor to chronic obstructive pulmonary disease (COPD), but its epigenetic regulation of mtTFA gene remains elusive. This study aims to explore the relationship of DNA methylation of mtTFA and cigarette smoking in COPD. Materials and Methods: We analyzed DNA methylation on mtTFA promoters in clinical samples from COPD patients and subjects with normal pulmonary function. Expression of mtTFA mRNA in the clinical samples and mtTFA mRNA and protein in human umbilical vein endothelial cells(HUVECs) treated with cigarette smoke extract (CSE) was evaluated. mtTFA mRNA and protein levels were measured to determine effects of demethylation agents on CSE-treated HUVECs. Results: The DNA methylation level of the mtTFA promoter was significantly increased in COPD group. Expression of mtTFA mRNA was downregulated in the lungs as a consequence of hypermethylation of mtTFA promoter. Expression of mtTFA mRNA and protein was downregulated in CSE-treated HUVECs as a consequence of hypermethylation of the mtTFA promoter. mtTFA expression in CSE-treated HUVECs was restored by the methylation inhibitor, 5-aza-2’-deoxycytidine(AZA). Conclusions: Cigarette smoke-induced hypermethylation of the mtTFA promoter is related to the initiation and progression of COPD. Our finding may provide a new strategy for the intervention of COPD by developing demethylation agents targeting mtTFA hypermethylation.

Epigenome-wide association study identifies Behçet's disease-associated methylation loci in Han Chinese.

The aetiology of Behçet's disease (BD), known as a systemic vasculitis, is not completely understood. Increasing evidence suggests that aberrant DNA methylation may contribute to the pathogenesis of BD. The aim of this epigenome-wide association study was to identify BD-associated methylation loci in Han Chinese. Genome-wide DNA methylation profiles were compared between 60 BD patients and 60 healthy controls using the Infinium Human Methylation 450 K Beadchip. BD-associated methylation loci were validated in 100 BD patients and 100 healthy controls by pyrosequencing. Gene expression and cytokine production was quantified by real-time PCR and ELISA. A total of 4332 differentially methylated CpG sites were associated with BD. Five differentially methylated CpG sites (cg03546163, cg25114611, cg20228731, cg23261343 and cg14290576) revealed a significant hypomethylation status across four different genes (FKBP5, FLJ43663, RUNX2 and NFIL3) and were validated by pyrosequencing. Validation results showed that the most significant locus was located in the 5'UTR of FKBP5 (cg03546163, P = 3.81E-13). Four CpG sites with an aberrant methylation status, including cg03546163, cg25114611, cg23261343 and cg14290576, may serve as a diagnostic marker for BD (area under the receiver operating curve curve = 83.95%, 95% CI 78.20, 89.70%). A significantly inverse correlation was found between the degree of methylation at cg03546163 as well as cg25114611 and FKBP5 mRNA expression. Treatment with a demethylation agent, 5-Aza-2'-deoxycytidine resulted in an increase of FKBP5 mRNA expression and a stimulated IL-1β production. Our findings suggest that aberrant DNA methylation, independently of previously known genetic variants, plays a vital role in the pathogenesis of BD. Chinese Clinical Trial Registry, chictr.org.cn, ChiCTR-CCC-12002184.

Decitabine enhances tumor recognition by T cells through upregulating the MAGE-A3 expression in esophageal carcinoma

Cancer testis (CT) antigens are expressed in various types of tumors and represent the potential targets for T cell-based immunotherapy. Analysis of CT gene expression and DNA methylation have indicated that certain CT genes are epigenetically regulated and studies have confirmed that certain CT antigens are regulated by DNA methylation. In this study, we explored the epigenetic regulation of MAGE-A3 and improved the clinical outcome of MAGE-A3-specific T cell therapy in esophageal squamous cell carcinoma (ESCC).We used molecular profiling datasets in The Cancer Genome Atlas to analyze CT gene expression in ESCC and its regulation by DNA methylation. We performed quantitative reverse transcription PCR (qRT-PCR), immunohistochemistry and bisulfite sequencing in ESCC cell lines and ESCC tissues. Functional assays, such as flow cytometry, cytotoxicity assays and ELISA, were performed to determine the demethylation agent, decitabine (5-aza-2′-deoxycytidine, DAC)-treated cancer cell improved antigen specific T cells response. ESCC tumor cell-xenograft mouse model and enzyme-linked immunospot (ELISPOT) assays were used to determine the function of DAC treatment in enhancing anti-MAGE-3 T cell responses in ESCC. Furthermore, we performed qRT-PCR and flow cytometry in the peripheral blood mononuclear cells (PBMC) of myelodysplastic syndromes (MDS) patients.MAGE-A3, one of the CT antigens, expressed at various levels in ESCC and was interfered by DNA methylation. We observed an efficient increase in MAGE-A3 expression in tumor cells and tissues after the treatment of decitabine and the expression of MAGE-A3 was affected by DNA methylation. Functional assays showed enhanced secretion of IFN-γ and cytolysis of MAGE-A3 antigen-specific T cells by DAC-treated target cells. In the tumor cell-xenograft mouse model and ELISPOT assays, DAC increased the expression of MAGE-A3 and T cell mediated tumor clearance in ESCC as well. Notably, the proportions of MAGE-A3-responsive T cells were elevated in DAC-treated patients with MDS, indicating DAC dismissed the epigenetic inhibition of MAGE-A3.DAC would probably improve the clinical outcome of MAGE-A3-specific T cell therapy by augmenting the expression of target gene.

Helicobacter pylori-induced DNA Methylation as an Epigenetic Modulator of Gastric Cancer: Recent Outcomes and Future Direction.

Gastric cancer is ranked fifth in cancer list and has the third highest mortality rate. Helicobacter pylori is a class I carcinogen and a predominant etiological factor of gastric cancer. H. pylori infection may induce carcinogenesis via epigenetic alterations in the promoter region of various genes. H. pylori is known to induce hypermethylation-silencing of several tumor suppressor genes in H. pylori-infected cancerous and H. pylori-infected non-cancerous gastric mucosae. This article presents a review of the published literature mainly from the last year 15 years. The topic focuses on H. pylori-induced DNA methylation linked to gastric cancer development. The authors have used MeSH terms “Helicobacter pylori” with “epigenetic,” “DNA methylation,” in combination with “gastric inflammation”, gastritis” and “gastric cancer” to search SCOPUS, PubMed, Ovid, and Web of Science databases. The success of epigenetic drugs such as de-methylating agents in the treatment of certain cancers has led towards new prospects that similar approaches could also be applied against gastric cancer. However, it is very important to understand the role of all the genes that have already been linked to H. pylori-induced DNA methylation in order to in order to evaluate the potential benefits of epigenetic drugs.

Non-inflammatory emphysema induced by NO2 chronic exposure and intervention with demethylation 5-Azacytidine

AimsA rat model of emphysema was established that mimics the features of the human emphysema subtype and explores the effects of demethylation on lung function and blood tests. Materials and methodsRats were randomly assigned to NO2, NO2 + 5-Azacytidine, and normal air groups based on a emphysema rat model induced by chronic NO2 exposure. This study estimates the characteristics of emphysema by conducting an analysis for IL-6 and TNF-α levels in bronchoalveolar lavage fluids (BALF) and plasma. Furthermore, CD68 macrophage immunofluorescent staining and inflammatory cell counts in BALF were compared between rats exposed to NO2 and normal air. Key findings5-Azacytidine treatment led to restored ∆weight at 14 and 75 days of intervention and NO2 + 5-Azacytidine significantly reversed the effect of NO2 exposure on ∆weight. Intervention with 5-Azacytidine alleviated the decline of pulmonary function with a significant increase in FEV100/FVC% at 75 days in NO2 + 5-Azacytidine rats compared to NO2 rats. 5-Azacytidine reduced the counts of white blood cells (WBCs), granulocytes, lymphocytes, and monocytes at 14 days, but increased WBC, granulocyte, and monocyte counts at 45 days. Red blood cell counts, hemoglobin, and hematocrit concentrations were significantly reduced in NO2 + 5-Azacytidine rats. SignificanceThis non-inflammatory rat emphysema model (induced by chronic NO2 exposure with global DNA hypomethylation and demethylation therapy with 5-Azacytidine) effectively improved emphysema by alleviating the decline of lung function and hypoxia, and slightly reinforced immune function. These results indicate the therapeutic potential of demethylation agents for the prevention and treatment of emphysema induced by the air pollutant NO2.

Recombinant Methioninase as a DNA Demethylation Agent.

This chapter reviews the effect of methionine (MET) restriction, via treatment with recombinant methioninase (rMETase), on DNA methylation of cancer cells. CCRF-CEM human cancer cells were treated with rMETase under subcytotoxic conditions. The rMETase-treated cells contained significantly lower levels of genomic methylated DNA than did untreated control cells. DNA methylation was measured by incorporation of the methyl group of [3H]methyl-S-adenosylmethionine into DNA and by methylation-sensitive arbitrarily-primed PCR. DNA hypomethylation effected by rMETase was of similar extent to that effected by treatment of the cells with the DNA methyltransferase inhibitor 5-azacytidine.

Association of BAX hypermethylation with coronary heart disease is specific to individuals aged over 70.

As a member of B-cell lymphoma-2 (BCL-2) gene family, BCL-2 associated X (BAX) is important for cell apoptosis. In this work, we investigated the association of BAX promoter DNA methylation with coronary heart disease (CHD) in Han Chinese. A SYBR green-based quantitative methylation specific PCR (qMSP) was used to test BAX methylation levels in 959 CHD cases and 514 controls. Although BAX methylation was not associated with CHD in the total samples, further breakdown analysis by age showed that BAX hypermethylation was significantly associated with CHD for individuals aged over 70 (median percentage of methylation ratio [PMR], 10.70% in cases versus (vs) 2.25% in controls, P =.046). Moreover, BAX methylation was associated with smoking and lipoprotein A (Lp(a)) for individuals aged over 70 (CHD: smoking P = .012, Lp(a) P = .001; non-CHD: smoking P = .051, Lp(a) P = .004). Further analysis of Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data showed BAX expression was upregulated by 5-aza-2'-deoxycytidine demethylation agent (fold = 1.66, P = .038) and inversely correlated with BAX methylation (r = -0.428, P = 8E-05). Our study supported that BAX hypermethylation might contribute to CHD risk via downregulation of BAX expression for individuals aged over 70.

Establishment of Stable and Secretable TATκ-GFP Recombinant Protein: A Preliminary Report of Promoter Methylation in 293T Cell Line

Induced pluripotent stem cells (iPSC) is a novel technology useful for therapeutic and research applications. To date, iPSCs is produced through genetic modification that can promote mutation; making it harmful for therapeutic use. Therefore, application of non-genetic modification through direct delivery of recombinant proteins aided by protein transduction domain (PTD) enable a safer production of iPSC. This study is aimed to establish a stable production of secretable recombinant protein via recombination of green fluorescence protein (GFP) and a novel PTD peptide, namely TATκ-GFP. 293Tcell line was transfected with 20 μg/ml of TATκ-GFP plasmid and the stably transfected 293T cells were then cultured for 54 days to determine the stability of expression and secretion of TATκ-GFP recombinant protein in prolonged culture. Methylation at the CMV promoter of the TATκ-GFP plasmid was investigated following treatment of transfected cells with 3 μM/mL of demethylation agent, namely 5-Azacytidine for 72 h in three cycles. Flow cytometry analysis demonstrated a transfection efficiency of 9.33% and successful secretion of TATκ-GFP proteins into the culture medium as analysed by Western blot at 72 h post-transfection. However, the transfected cells exhibited a decreasing level of GFP expression and secretion following prolonged culture with notable stability that only sustained for two weeks. 5-Azacytidine-treated cells showed a slight increase of GFP expression compared to non-treated control, suggesting possible promoter methylation which could cause instability of TATκ-GFP expression. Conclusively, promoter methylation should be considered for future establishment of iPSCs as it could inhibit stable expression and secretion of recombinant proteins.

Epigenetically regulated miR-1247 functions as a novel tumour suppressor via MYCBP2 in methylator colon cancers

BackgroundColorectal cancer (CRC) is a heterogeneous disease with distinct clinical subsets based on underlying genetic and epigenetic changes. DNA hypermethylation yields a unique CRC subset with a distinct phenotype and clinical behaviour, but this oncogenic pathway is not fully characterised. This study identifies and characterises miR-1247 as a novel tumour suppressor microRNA in methylated human colon cancers.MethodTumour samples from patients with hypermethylated and non-methylated colon cancer and cell lines were evaluated for miR-1247 expression and function. A murine subcutaneous xenograft model was used for in vivo functional studies.ResultsmiR-1247 was methylated and underexpressed in methylator colon cancers. Overexpression of miR-1247 significantly inhibited cell proliferation, decreased tumour cell motility, induced apoptosis, and mitigated tumour formation capacity both in vivo and in vitro. Pharmacologic demethylation increased miR-1247 expression and produced similar anti-tumour activities. Mechanistic investigations revealed that MYCBP2, a member of the c-myc oncogene family, is a direct functional target of miR-1247. Furthermore, in CRC patients, MYCBP2 protein levels are associated with miR-1247 levels and survival.ConclusionsmiR-1247 acts as a tumour suppressor by inhibiting MYCBP2 in methylator colon cancer. The MYCBP2/c-myc axis may underlie the anti-tumour activities of miR-1247 and is a potential therapeutic target via demethylation agents.

Abstract PR12: Epigenetic reprograming promotes an immunogenic ovarian tumor microenvironment and synergizes with adoptive transfer of engineered T cells expressing NY-ESO-1 specific T cell receptors

Abstract The ability of the immune system to recognize and reject tumors is dependent on several factors that include the expression of immunogenic target antigens on tumor cells, generation of high frequencies of tumor antigen-specific T cells with potent effector function, and capacity to overcome several mechanisms by which tumors escape immune attack. The immunosuppressive environment in the tumor bearing host and at the tumor site restricts persistence and function of antigen-specific T cells, and represent a major obstacle to a durable adoptive engineered TCR (T-cell receptor) cell transfer immunotherapy. DNA demethylation agents have been shown to change immune gene signatures of tumor cells and activate the expression of suppressed tumor antigens by relieving promotor hyper-methylation. In the current study, we aim to determine the immunomodulatory activity of demethylation agents on both ovarian cancer cells and immune cell components in tumor microenvironment, and identify strategies to enhance adoptive T-cell transfer (ACT) immunotherapy in ovarian cancer models. Using a preclinical xenograft model, NY-ESO-1 negative ovarian cancer, OVCAR3, we investigated therapeutic effects of three HLA-A*02 restricted clones of NY-ESO-1 specific CD8 TCR gene-engineered T cells in combination with the demethylation agents decitabine and SGI-110. Demethylation agent treatment followed by ACT elicited synergistic antitumor responses, ranging from inhibition to regression of tumor growth, with curative effect in some animals. In the NY-ESO-1 negative OVCAR3 model, demethylation agents not only induced expression of NY-ESO-1 tumor antigen and MHC I and II, rendering the tumor visible for recognition by CD8 T cells, but also dramatically promoted persistence and accumulation of adoptively transferred T cells at tumor site, as well as reduction of suppressive myeloid cells in the tumor. To further optimize the therapeutic effect of NY-ESO-1 specific CD8 T cells, we explored the activity of TCR gene-engineered NY-ESO-1 specific CD4 T cells that directly recognize cancer cells in a MHC II-restricted manner, in combination with decitabine. We also demonstrated that decitabine rendered the tumor visible for recognition by NY-ESO-1 specific CD4 T cells leading to significant tumor inhibition, and improved persistency of CD4 T cells at peripheral and tumor sites. To maximize curative effect and durability of the therapy, we are currently investigating the immunomodulatory role of decitabine in combination with both CD8 and CD4 T cells specific to NY-ESO-1 in ovarian cancer. Our findings provide insights into novel modes of action of demethylation agents, and indicate that, with optimized therapeutic strategies, demethylation agents can dramatically augment efficacy of adoptive T-cell transfer immunotherapy and counter the immunosuppressive mechanisms in ovarian cancer models. This abstract is also being presented as Poster B34. Citation Format: Li Shen, Bob McGray, Anthony Miliotto, Ariel Francois, Cheryl Eppolito, Junko Matsuzaki, Takemasa Tsuji, Richard Koya, Adekunle Odunsi. Epigenetic reprograming promotes an immunogenic ovarian tumor microenvironment and synergizes with adoptive transfer of engineered T cells expressing NY-ESO-1 specific T cell receptors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR12.