Demethylating Agent Research Articles

The effect of zebularine on the heat-activated retrotransposon ONSEN in Arabidopsis thaliana and Vigna angularis.

The Ty1/copia-like retrotransposon ONSEN is conserved among Brassica species, as well as in beans, including adzuki bean (Vigna angularis (Willd.) Ohwi & Ohashi), which is one of the economically important crops in Japan. ONSEN has acquired a heat-responsive element that is recognized by plant heat stress defense factors, resulting in its transcription and the production of full-length extrachromosomal DNA under conditions with elevated temperatures. DNA methylation plays an important role in regulating the activation of this transposon in plants. Therefore, chemical inhibition of DNA methyltransferases has been utilized to study the effect of DNA methylation on transposon activation. To understand the effect of DNA methylation on ONSEN activation, Arabidopsis thaliana and adzuki bean seedlings were treated with zebularine, which is known to be an effective chemical demethylation agent. The results showed that ONSEN transcription levels were upregulated in zebularine-treated plants. Extrachromosomal DNA of ONSEN also accumulated in the treated plants.

Activation-Induced Cytidine Deaminase Expression Facilitates the Malignant Phenotype and Epithelial-to-Mesenchymal Transition in Clear Cell Renal Cell Carcinoma.

Although advances have been made in the development of antiangiogenesis targeted therapy and surgery, metastatic clear cell renal cell carcinoma (ccRCC) is still incurable. Activation-induced cytidine deaminase (AID) is mainly expressed in a variety of germ and somatic cells, and induces somatic hypermutation and class-switch recombination, playing a vital role in antibody diversification. We confirmed that AID was expressed at a higher level in ccRCC tissues than in the corresponding nontumor renal tissues. We explored the impact of AID on ccRCC proliferation, invasion, and migration. In 769-p and 786-0 cells, expression of an AID-specific short hairpin RNA significantly reduced AID expression, which markedly inhibited tumor cell invasion, proliferation, and migration. Previous studies showed that AID is associated with Wnt ligand secretion mediator (WLS/GPR177), cyclin-dependent kinase 4 (CDK4), and stromal cell-derived factor-1 (SDF-1/CXCL12) regulation, which was further confirmed in human ccRCC tissues. Therefore, we studied the relationship between AID and these three molecules, and the impact of AID on epithelial-to-mesenchymal transition in ccRCC. WLS/GPR177, SDF-1/CXCL12, and CDK4 were sensitive to 5-azacytidine (a DNA demethylation agent), which reverted the inhibition of carcinogenesis caused by AID repression. In summary, AID is an oncogene that might induce tumorigenesis through DNA demethylation. Targeting AID may represent a novel therapeutic approach to treat metastatic ccRCC.

Loss of PTEN sensitizes head and neck squamous cell carcinoma to 5-AZA-2'-deoxycytidine.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer associated with poor survival. Phosphatase and tensin homolog (PTEN) is a tumor suppressor gene involved in the maintenance of stem cells. DNA methylation is a known epigenetic modification involved in tumor progression. In this study, we investigated the effect of the DNA demethylation agent 5-AZA-2'-deoxycytidine (5-AZA) over HNSCC and its population of cancer stem cells (CSCs) presenting dysfunctional PTEN. The effects of 5-AZA on HNSCC were evaluated by using WSU-HN13 cells. CSC was assessed by sphere-forming assays, along with the endogenous levels of aldehyde dehydrogenase. The clonogenic potential of tumors was evaluated, along with the protein expression of mTOR signaling and the identification of nuclear factor-κB (NF-κB) and epithelial-mesenchymal transition (EMT)-associated genes, using real-time polymerase chain reaction (PCR). We observed that loss of PTEN enhances tumor biologic behavior, including colony- and tumor sphere-forming abilities. We also found that 5-AZA has an inhibitory effect over the CSCs and molecular markers associated with the NF-κB and EMT pathways. Our findings suggest that the stratification of treatment of HNSCC based on PTEN status may identify a subset of patients who can benefit from the coadministration of 5-AZA.

Zebularine-induced myeloma cell death is accompanied by decreased c-Myc expression.

Epigenetic therapies have proven to be clinically effective in several hematological malignancies. Here, we aimed to evaluate the effect of a second-generation DNA demethylation agent, zebularine, on multiple myeloma (MM). Western blot, ELISA, qRT-PCR, proliferation assays and cell transfection were used to investigate the mechanism of action of zebularine in MM. We found that zebularine induced apoptosis and DNA demethylation in most of the MM cell lines tested. Its cytotoxic effect was associated with a time-dependent decrease in the level of c-Myc protein. Moreover, zebularine induced H2AX phosphorylation, a surrogate marker of DNA damage, in five out of eight MM cell lines tested. Our study revealed novel effects of zebularine on MM that may have potential implications for DNA methylation-based therapies.

Murine Mesenchymal Stromal Cells Retain Biased Differentiation Plasticity Towards Their Tissue of Origin.

Mesenchymal stromal/stem cells (MSCs) reside in many human tissues and comprise a heterogeneous population of cells with self-renewal and multi-lineage differentiation potential, making them useful in regenerative medicine. It remains inconclusive whether MSCs isolated from different tissue sources exhibit variations in biological features. In this study, we derived MSCs from adipose tissue (AT-MSC) and compact bone (CB-MSC). We found that early passage of MSCs was readily expandable ex vivo, whereas the prolonged culture of MSCs showed alteration of cell morphology to fibroblastoid and reduced proliferation. CB-MSCs and AT-MSCs at passage 3 were CD29+, CD44+, CD105+, CD106+, and Sca-1+; however, passage 7 MSCs showed a reduction of MSC markers, indicating loss of stem cell population after prolonged culturing. Strikingly, CB-MSC was found more efficient at undergoing osteogenic differentiation, while AT-MSC was more efficient to differentiate into adipocytes. The biased differentiation pattern of MSCs from adipogenic or osteogenic tissue source was accompanied by preferential expression of the corresponding lineage marker genes. Interestingly, CB-MSCs treated with DNA demethylation agent 5-azacytidine showed enhanced osteogenic and adipogenic differentiation, whereas the treated AT-MSCs are less competent to differentiate. Our results suggest that the epigenetic state of MSCs is associated with the biased differentiation plasticity towards its tissue of origin, proposing a mechanism related to the retention of epigenetic memory. These findings facilitate the selection of optimal tissue sources of MSCs and the ex vivo expansion period for therapeutic applications.

Experimental DNA Demethylation Associates with Changes in Growth and Gene Expression of Oak Tree Seedlings

Epigenetic modifications such as DNA methylation, where methyl groups are added to cytosine base pairs, have the potential to impact phenotypic variation and gene expression, and could influence plant response to changing environments. One way to test this impact is through the application of chemical demethylation agents, such as 5-Azacytidine, which inhibit DNA methylation and lead to a partial reduction in DNA methylation across the genome. In this study, we treated 5-month-old seedlings of the tree, Quercus lobata, with foliar application of 5-Azacytidine to test whether a reduction in genome-wide methylation would cause differential gene expression and change phenotypic development. First, we demonstrate that demethylation treatment led to 3–6% absolute reductions and 6.7–43.2% relative reductions in genome-wide methylation across CG, CHG, and CHH sequence contexts, with CHH showing the strongest relative reduction. Seedlings treated with 5-Azacytidine showed a substantial reduction in new growth, which was less than half that of control seedlings. We tested whether this result could be due to impact of the treatment on the soil microbiome and found minimal differences in the soil microbiome between two groups, although with limited sample size. We found no significant differences in leaf fluctuating asymmetry (i.e., deviations from bilateral symmetry), which has been found in other studies. Nonetheless, treated seedlings showed differential expression of a total of 23 genes. Overall, this study provides initial evidence that DNA methylation is involved in gene expression and phenotypic variation in seedlings and suggests that removal of DNA methylation affects plant development.

Epigenetically silenced LINC02381 functions as a tumor suppressor by regulating PI3K-Akt signaling pathway

Recent advances have revealed that lncRNAs play important roles in tumorigenesis. However, only a small number of functional lncRNAs have been well characterized, particularly in colorectal cancer. Therefore, more extensive studies are needed to identify and characterize these lncRNAs to better understand cancer progression. In the present study, using available RNA-seq data, we found that LINC02381 (NR_026656.1) differentially expresses in CRC tissues compared to normal pairs. Consistently, RT-qPCR results showed that LINC02381 was down regulated in CRC tissues and also in different cancerous cell lines. CRC cells treatment with de-methylating and chemotherapy agents indicated that DNA methylation of LINC02381 may be responsible for the transcriptional silencing of LINC02381 in colorectal cancer cells. Then, the functional changes of the cells in response to LINC02381 alteration were assessed and the data indicated that LINC02381 up-regulation suppressed cell viability and proliferation while increasing the apoptosis in CRC-originated cell lines. Mechanistically, LINC02381 overexpression was increased PTEN protein levels but decreased phospho-Akt levels. Finally, we proposed a hypothesized model for PI3K signaling regulation by LINC02381. Altogether, the result of this study suggests that LINC02381 might have suppressive effects on human colorectal cancer tumorigenesis partly by regulating PI3K signaling pathway.

19q13 KRAB zinc-finger protein ZNF471 activates MAPK10/JNK3 signaling but is frequently silenced by promoter CpG methylation in esophageal cancer.

Zinc-finger proteins (ZFPs) are the largest transcription factor family in mammals, involved in the regulation of multiple physiologic processes including cell differentiation, proliferation, apoptosis and neoplastic transformation. Approximately one-third of ZFPs are Krüppel-associated box domain (KRAB)-ZFPs.Methods: ZNF471 expression and methylation were detected by reverse-transcription PCR and methylation-specific PCR. The impact and mechanism of ectopic ZNF471 expression in esophageal squamous cell carcinoma (ESCC) cells was evaluated in vitro and in vivo.Results: We identified a 19q13 KRAB-ZFP, ZNF471, as a methylated target in ESCC. We further found that ZNF471 is significantly downregulated in ESCC tissues compared with adjacent non-cancer tissues, due to its aberrant promoter CpG methylation, and further confirmed by methylation analysis and treatment with demethylation agent. Restoration of ZNF471 expression in silenced ESCC cells significantly altered cell morphology, induced apoptosis and G0/G1 arrest, and inhibited tumor cell colony formation, viability, migration and invasion. Importantly, ZNF471 was found to activate the expression of MAPK10/JNK3 and PCDH family genes, and further enhance MAPK10 signaling and downstream gene expression through binding to the MAPK10/JNK3 promoter.Conclusion: Our results demonstrate that ZNF471 is an important tumor suppressor and loss of ZNF471 functions hampers MAPK10/JNK3 signaling during esophageal carcinogenesis.

Conjugation of TLR7 Agonist Combined with Demethylation Treatment Improves Whole-Cell Tumor Vaccine Potency in Acute Myeloid Leukemia.

Background: Acute myeloid leukemia (AML) is a malignant hematological disease with high refractory rate. Immune escape of AML cells is one of the underlying mechanisms mediating the relapse of the cancers. Various immunotherapies based on the 'patients' immune response to tumor cells have been developed to targeting the immune escape of AML cells, which lead to the minimal residual disease (MRD) after treatment. But the efficacy of those treatments or the combination of treatments remains unsatisfactory.Methods: A Toll-like receptor (TLR)-7 agonist SZU-106 was chemically synthesized. SZU-106 was conjugated to Decitabine (DAC), a demethylation agent, treated AML cells to construct tumor vaccine. The potency of the newly constructed AML cell vaccine, SZU-106-DAC-AML was tested in vitro and in vivo for the expression of tumor antigens and the activation level of immune responses.Results: Compared to the well characterized TLR7 agonist R848, SZU-106 has a comparable potency to activate TLR7 signaling pathway. SZU-106-DAC-AML, constructed by conjugating SZU-106 to DAC treated tumor cells, exhibited increased expression of tumor antigens, and enhanced the activation of DC cells and T cells in vitro and in vivo. The result of xenograft tumor model showed that SZU-106-DAC-AML tumor vaccine greatly inhibited tumor growth and prolonged animal survival.Conclusions: Conjugation of TLR7 agonist combined with up-regulation of tumor antigen expression improved the effectiveness of whole-cell tumor vaccine in AML.

Reactivation of Atp4a concomitant with intragenic DNA demethylation for cancer inhibition in a gastric cancer model

Accumulating evidence suggests that aberrant DNA methylation and gene silencing of tumor suppressors are pervasive in gastric malignancies, supporting reactivation of tumor suppressors through DNA demethylation as a potential therapeutic opportunity. Atp4a is an important tumor suppressor gene, encoding H+, K+-ATPase, and mediating gastric acid secretion in the stomach. Using transgenic gastric cancer model K19-Wnt1/C2mE (Gan) mice, by combining the transcriptome and MeDIP (methylated DNA immunoprecipitation) sequencing, together with qRT-PCR, we showed that Atp4a was expressed at low levels in tumor tissues and multiple GC cells, while both 5-aza-CdR and 18β-glycyrrhetinic acid (GRA) pharmacological treatment triggered Atp4a activation with downregulation of DNMT1. In addition, CpG island (CGI) search showed that the CpG rich region is absent in the promoter region but present in exons 9–14 of Atp4a. Methylation specific PCR (MSP) indicated that Atp4a was fully or partly methylated in multiple GC cells. Further MassArray suggested that the demethylation in the CpG site 75, 183, 196, 262–268 might be responsible for the reactivation of Atp4a. Our research identified that GRA, a bioactive component found in abundance in Radix Glycyrrhiza, reactivated Atp4a expression and inhibited gastric tumorigenesis as a potential demethylation agent.

DNA demethylation agent 5azadC downregulates HPV16 E6 expression in cervical cancer cell lines independently of TBX2 expression.

HPV16 is the most carcinogenic human papillomavirus and causes >50% of cervical cancers, the majority of anal cancers and 30% of oropharyngeal squamous cell carcinomas. HPV carcinogenesis relies on the continuous expression of the two main viral oncoproteins E6 and E7 that target >150 cellular proteins. Among them, epigenetic modifiers, including DNA Methyl Transferases (DNMT), are dysregulated, promoting an aberrant methylation pattern in HPV-positive cancer cells. It has been previously reported that the treatment of HPV-positive cervical cancer cells with DNMT inhibitor 5-aza-2'-deoxycytidine (5azadC) caused the downregulation of E6 expression due to mRNA destabilization that was mediated by miR-375. Recently, the T-box transcription factor 2 (TBX2) has been demonstrated to repress HPV LCR activity. In the current study, the role of TBX2 in E6 repression was investigated in HPV16 cervical cancer cell lines following 5azadC treatment. A decrease of E6 expression was accompanied by p53 and p21 restoration. While TBX2 mRNA was upregulated in 5azadC-treated SiHa and Ca Ski cells, TBX2 protein was not detectable. Furthermore, the overexpression of TBX2 protein in cervical cancer cells did not allow the repression of E6 expression. The TBX2 transcription factor is therefore unlikely to be associated with the repression of E6 following 5azadC treatment of SiHa and Ca Ski cells.

Transposable element expression in tumors is associated with immune infiltration and increased antigenicity

Profound global loss of DNA methylation is a hallmark of many cancers. One potential consequence of this is the reactivation of transposable elements (TEs) which could stimulate the immune system via cell-intrinsic antiviral responses. Here, we develop REdiscoverTE, a computational method for quantifying genome-wide TE expression in RNA sequencing data. Using The Cancer Genome Atlas database, we observe increased expression of over 400 TE subfamilies, of which 262 appear to result from a proximal loss of DNA methylation. The most recurrent TEs are among the evolutionarily youngest in the genome, predominantly expressed from intergenic loci, and associated with antiviral or DNA damage responses. Treatment of glioblastoma cells with a demethylation agent results in both increased TE expression and de novo presentation of TE-derived peptides on MHC class I molecules. Therapeutic reactivation of tumor-specific TEs may synergize with immunotherapy by inducing inflammation and the display of potentially immunogenic neoantigens.

Synergistic Effect of DHODH Inhibitor PTC299 with DNA Demethylating Agent Decitabine in Myelodysplastic Syndromes

Background:Myelodysplastic Syndrome (MDS) is a clonal bone marrow disorder characterized by ineffective and clonal hematopoiesis accompanied by morphological dysplasia and variable cytopenia. There are few treatment options for MDS, and allogenic hematopoietic stem cell transplantation is the only curative option. Dihydroorotate dehydrogenase (DHODH) catalyzes a rate-limiting step in de novo pyrimidine synthesis, the conversion of DHO to orotate. DHODH inhibition has been described recently as a new approach for treating acute myeloid leukemia (AML) by inducing differentiation of diverse AML subclasses. PTC299 represents a novel potent DHODH inhibitor and recently clinical development of PTC299 as a potential treatment option for acute leukemia was initiated. Here, we explored the efficacy of DHODH inhibitor PTC299 for MDS. Methods:Anti-MDS efficacy of PTC299 was studied using human MDS cell lines and primary MDS cells in vitro. PTC299 was synthesized at PTC Therapeutics Inc. Mechanistic studies were conducted via flow cytometric analysis and RNA-sequencing (RNA-seq). Gene expression levels were analyzed by quantitative PCR (qPCR). Results:PTC299 inhibited proliferation of AML cell lines and induced their differentiation. As previously reported in other DHODH inhibitors, upregulation of CD11b was observed after PTC299 treatment in both HL-60 and THP-1 cells. In addition, PTC299 inhibited the proliferation of MDS cell lines, MDSL and SKM-1 cells, with EC50s of 12.6 nM in MDSL cells and 19.7 nM in SKM-1 cells. The inhibitory effect was reversed by the exogenous addition of 100 µM uridine, which bypasses the requirement for de novo pyrimidine synthesis by feeding into the salvage pathway, thereby negating the need for DHODH. Because the basal expression levels of CD11b are high in MDS cells, we examined the expression levels of CD38. Both cell lines showed dose-dependent upregulation of CD38 after PTC299 treatment. To investigate the possible synergism between PTC299 and decitabine, we treated MDSL and SKM-1 cells with increasing concentrations of PTC299 and decitabine as single agents or in combination. After 3 days of culture, cells were analyzed by MTS assays. PTC299 and decitabine exerted a enhanced cytotoxic effect on MDSL and SKM-1 cells. Similar results were obtained with primary MDS samples. In Annexin/PI assays, the percentage of apoptotic cells was increased by combination of PTC299 with decitabine in both cell lines. Mechanistically, treatment with PTC299 induced an intra-S-phase arrestfollowed by entry intoapoptotic cell death. It has also been reported that the expression of p53 is increased in response to the intra-S-phase arrest. To understand the genome-wide effects and target genes of PTC299 and the combination with decitabine, we performed RNA-seq of MDSL and SKM-1 cells treated with PTC299, decitabine, or the combination of both agents versus DMSO-treated cells. Gene set enrichment analysis (GSEA) using our RNA-seq data confirmed that MYC target gene sets were negatively enriched in both PTC299-, decitabine- and combination- treated cells. KEGG pathway enrichment analysis revealed activation of genes associated with apoptosis in both cell lines. To better elucidate a synergistic effect of PTC299 and decitabine, we performed qPCR of CDKN1A, which is a major target of p53 activity. The mRNA expression levels of CDKN1A were upregulated after treatment with PTC299, which was further enhanced by the combination with decitabine. Conclusions:Our result indicate that the DHODH inhibitor PTC299 suppresses the growth of MDS cells in vitro and acts in at least an additive and possibly synergistic manner with decitabine in this process. This combination therapy could be a new therapeutic option for the treatment of MDS. Disclosures Lennox: PTC Therapeutics: Employment. Weetall:PTC Therapeutics: Employment. Sheedy:PTC Therapeutics: Employment.

Epigenetic Coactivation of MAGEA6 and CT-GABRA3 Defines Orientation of a Segmental Duplication in the Human X Chromosome

The human genome harbors many duplicated segments, which sometimes show very high sequence identity. This may complicate assignment during genome assembly. One such example is in Xq28, where the arrangement of 2 recently duplicated segments varies between genome assembly versions. The duplicated segments comprise highly similar genes, including MAGEA3 and MAGEA6, which display specific expression in testicular germline cells, and also become aberrantly activated in a variety of tumors. Recently, a new gene was identified, CT-GABRA3, the transcription of which initiates inside the segmental duplication but extends far outside. According to the latest genome annotation, CT- GABRA3 starts near MAGEA3, with which it shares a bidirectional promoter. In an earlier annotation, however, the duplicated segment was positioned in the opposite orientation, and CT-GABRA3 was instead coupled with MAGEA6. To resolve this discrepancy, and based on the contention that genes connected by a bidirectional promoter are almost always co-expressed, we decided to compare the expression profiles of CT-GABRA3, MAGEA3, and MAGEA6. We found that in tumor tissues and cell lines of different origins, the expression of CT-GABRA3 was better correlated with that of MAGEA6. Moreover, in a cellular model of experimental induction with a DNA demethylation agent, activation CT-GABRA3 was associated with that of MAGEA6, but not with that of MAGEA3. Together these results support a connection between CT-GABRA3 and MAGEA6 and illustrate how promoter-sharing genes can be exploited to resolve genome assembly uncertainties.

DNA demethylation agents as a therapeutic approach in endocrine-resistant breast cancer

Searchable abstracts of presentations at key conferences in oncology ISSN 2631-4657 (online)

A male-specific association between AGTR1 hypermethylation and coronary heart disease.

The AGTR1 gene encodes angiotensin II receptor type 1, which is involved in cardiovascular diseases such as coronary heart disease (CHD). In the current study, we analyzed AGTR1 promoter methylation level in a Han Chinese population by SYBR green-based quantitative methylation-specific PCR (qMSP). We collected blood samples from 761 CHD patients and 398 non-CHD controls at the Ningbo First Hospital. A data mining analysis was also performed to explore the association between AGTR1 methylation and AGTR1 gene expression, using datasets from the cBioPortal for Cancer Genomics and the Gene Expression Omnibus (GEO) database. Our results showed a significantly higher percentage of methylated reference (PMR) of AGTR1 in male CHD patients compared with male non-CHD controls (median PMR: 2.12% vs. 0.59%, p = 0.037). The data mining analysis showed that AGTR1 expression was significantly increased in human hepatoma HepG2 cells treated with the demethylation agent 5-aza-2’-deoxycytidine (fold = 3.12, p = 0.009). Further data mining analysis using the cholangiocarcinoma (TCGA, PanCancer Atlas) data indicated an inverse association between AGTR1 methylation and AGTR1 expression (r = -0.595, p = 1.29E-04). Overall, our results suggest that AGTR1 methylation is involved in the regulation of AGTR1 gene expression and that AGTR1 hypermethylation is associated with CHD in males. These findings may provide new clues about the pathogenesis of CHD.

Curcumin Inhibits Joint Contracture through PTEN Demethylation and Targeting PI3K/Akt/mTOR Pathway in Myofibroblasts from Human Joint Capsule.

Joint contracture is increasingly regarded as a clinical problem that leads to irreversible dysfunction of the joint. It is a pathophysiological process following joint injury, which is marked by the activation of myofibroblasts. There is currently no effective treatment for the prevention of joint contracture. Curcumin is a polyphenol pigment extracted from turmeric, which possesses anti-inflammatory, antioxidative, and antitumor properties. In the present study, we demonstrated that curcumin exerts a protective effect against joint contracture via the inhibition of myofibroblast proliferation and migration in a time- and concentration-dependent manner. Moreover, we indicated that phosphatase and tension homolog (PTEN) was downregulated in myofibroblasts in vitro and in the contracture capsule tissues of patients in vivo. Additionally, western blot analysis revealed a negative correlation between the expression levels of PTEN and the fibrosis marker protein alpha smooth muscle cell actin. Methylation-specific PCR results suggested that curcumin was able to demethylate PTEN in a similar manner to the demethylation agent 5-azacytidine, increasing PTEN expression and further inhibiting phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling. In conclusion, our data illustrate part of the mechanism of curcumin inhibition in joint contracture. These results support the hypothesis that curcumin may potentially be used as a novel candidate for the treatment of joint contracture.

No evidence for local adaptation and an epigenetic underpinning in native and non-native ruderal plant species in Germany.

Many invasive species have rapidly adapted to different environments in their new ranges. This is surprising, as colonization is usually associated with reduced genetic variation. Heritable phenotypic variation with an epigenetic basis may explain this paradox.Here, we assessed the contribution of DNA methylation to local adaptation in native and naturalized non‐native ruderal plant species in Germany. We reciprocally transplanted offspring from natural populations of seven native and five non‐native plant species between the Konstanz region in the south and the Potsdam region in the north of Germany. Before the transplant, half of the seeds were treated with the demethylation agent zebularine. We recorded survival, flowering probability, and biomass production as fitness estimates.Contrary to our expectations, we found little evidence for local adaptation, both among the native and among the non‐native plant species. Zebularine treatment had mostly negative effects on overall plant performance, regardless of whether plants were local or not, and regardless of whether they were native or non‐native. Synthesis. We conclude that local adaptation, at least at the scale of our study, plays no major role in the success of non‐native and native ruderal plants. Consequently, we found no evidence yet for an epigenetic basis of local adaptation.

Epigenetic modifiers 5-aza-2'-deoxycytidine and valproic acid differentially change viability, DNA damage and gene expression in metastatic and non-metastatic colon cancer cell lines.

DNA methylation and histone modifications are major components of cellular epigenetic pattern determining gene expression. Cancer cells have their own epigenetic array, which can be different in cells of primary and metastatic tumors. In the present work we investigated effects of 1 mM valproic acid (VPA), a histone deacetylase inhibitor and 0.2 μM 5-aza-2'-deoxycytidine (5-aza-dC), a DNA demethylation agent, singly or in combination in two colorectal cancer cell lines Caco-2 (non-metastatic) and LoVo (metastatic). Cell viability, DNA damage and the mRNA expression of the CDC25C (cell division cycle 25C), CDKN1A (cyclin dependent kinase inhibitor 1A) and CHEK1 (checkpoint kinase 1), SQSTM1 (sequestosome 1) and ULK1 (unc-51 like autophagy activating kinase 1), RELA (RELA proto-oncogene, NF-κB subunit) and TP53BP1 (tumor protein p53 binding protein 1) genes important in cell cycle regulation, autophagy and cancer progression were investigated. Both drugs induced a moderate decrease in cell viability and significant DNA damage in both cell lines. LoVo cells were more sensitive to VPA and combined treatment than Caco-2. LoVo cells also showed higher expression of genes that are often associated with more aggressive tumors than Caco-2 cells and treatment with the modifiers increased this difference. In conclusion, 5-aza-dC and VPA can induce different effects in metastatic and non-metastatic cancer cell lines and this may be important in determination of epigenetic profile responsible for metastatic properties of cancer cells.

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.