Follistatin supplementation induces changes in CDX2 CpG methylation and improves in vitro development of bovine SCNT preimplantation embryos.

Caudal Type Homeobox 2 (CDX2) is a key regulator of trophectoderm formation and maintenance in preimplantation embryos. We previously demonstrated that supplementation of exogenous follistatin, during in vitro culture of bovine IVF embryos, upregulates CDX2 expression, possibly, via alteration of the methylation status of CDX2 gene. Here, we further investigated the effects of exogenous follistatin supplementation on developmental competence and CDX2 methylation in bovine somatic cell nuclear transfer (SCNT) embryos. SCNT embryos were cultured with or without follistatin for 72h, then transferred into follistatin free media until d7 when blastocysts were collected and subjected to CDX2 gene expression and DNA methylation analysis for CDX2 regulatory regions by bisulfite sequencing. Follistatin supplementation significantly increased both blastocyst development as well as blastocyst CDX2 mRNA expression on d7. Three different CpG rich fragments within the CDX2 regulatory elements; proximal promoter (fragment P1, -1644 to -1180; P2, -305 to +126) and intron 1 (fragment I, + 3030 to + 3710) were identified and selected for bisulfite sequencing analysis. This analysis showed that follistatin treatment induced differential methylation (DM) at specific CpG sites within the analyzed fragments. Follistatin treatment elicited hypomethylation at six CpG sites at positions -1374, -279, -163, -23, +122 and +3558 and hypermethylation at two CpG sites at positions -243 and +20 in promoter region and first intron of CDX2 gene. Motif analysis using MatInspector revealed that differentially methylated CpG sites are putative binding sites for key transcription factors (TFs) known to regulate Cdx2 expression in mouse embryos and embryonic stem cells including OCT1, AP2F, KLF and P53, or TFs that have indirect link to CDX2 regulation including HAND and NRSF. Collectively, results of the present study together with our previous findings in IVF embryos support the hypothesis that alteration of CDX2 methylation is one of the epigenetic mechanisms by which follistatin may regulates CDX2 expression in preimplantation bovine embryos.

Simultaneous silencing Aurora-A and UHRF1 inhibits colorectal cancer cell growth through regulating expression of DNMT1 and STAT1.

Aurora-A has attracted a great deal of interest as a potential therapeutic target for patients with CRC. However, the outcomes of inhibitors targeting Aurora-A are not as favorable as expected, and the basis behind the ineffectiveness remains unknown. Here, we found that signal transducer and activator of transcription 1 (STAT1) was highly expressed in colorectal cancer (CRC) xenograft mouse models that were resistant to alisertib, an Aurora-A inhibitor. Unexpectedly, we found that alisertib disrupted Aurora-A binding with ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1), leading to UHRF1 mediated ubiquitination and degradation of DNA methyltransferase 1 (DNMT1), which in turn resulted in demethylation of CpG islands of STAT1 promoter and STAT1 overexpression. Simultaneous silencing Aurora-A and UHRF1 prevented STAT1 overexpression and effectively inhibited CRC growth. Hence, concomitant targeting Aurora-A and UHRF1 can be a promising therapeutic strategy for CRC.

RAP1GAP Functions as a Tumor Suppressor Gene and Is Regulated by DNA Methylation in Differentiated Thyroid Cancer.

Inactivation of tumor suppressor genes, such as RAP1GAP, by hypermethylation of their regulatory region can give rise to thyroid tumors. The aim of this study was to investigate the expression of the RAP1GAP gene and the DNA methylation patterns of its CpG74a, CpG74b, and CpG24 in an Iranian population with differentiated thyroid cancer (DTC). In this study, 160 individuals who underwent thyroidectomy in the Tehran Erfan Hospital between 2018 and 2020 were selected. DNA methylation patterns of selected CpG islands (CpG74a, CpG74b, and CpG24) were determined using methylation-specific PCR. The mRNA expression and protein level of -RAP1GAP were also evaluated. SW1736 and B-CPAP cells were treated with 5-aza-2'-deoxycytidine (5-Aza) to demethylate these regions. The hypermethylation rates of CpG74a and CpG24 in DTC samples were significantly higher than in the control. The mRNA expression and protein level of -RAP1GAP were significantly decreased in the DTC group. In the DTC group, hypermethylation in CpG74a was correlated with decreasing RAP1GAP expression (R2: 0.34; p = 0.043). CpG74a with a specificity of 86.4% has significant prediction power to distinguish between DTC and normal thyroid tissues. Additionally, hypermethylation of CpG74a was significantly associated with higher tumor stages (stage III-IV: 77%; stage I-II: 23%; p = 0.012). Increasing expression of RAP1GAP after demethylation with 15 µM of 5-Aza was observed in both cell lines. These results indicate that DNA hypermethylation in CpG74a can be considered as an epigenetic biomarker in DTC.

Cigarette smoke-induced LKB1/AMPK pathway deficiency reduces EGFR TKI sensitivity in NSCLC.

Smoker patients with non-small cell lung cancer (NSCLC) have poorer prognosis and survival than those without smoking history. However, the mechanisms underlying the low response rate of those patients to EGFR tyrosine kinase inhibitors (TKIs) are not well understood. Here we report that exposure to cigarette smoke extract enhances glycolysis and attenuates AMP-activated protein kinase (AMPK)-dependent inhibition of mTOR; this in turn reduces the sensitivity of NSCLC cells with wild-type EGFR (EGFRWT) to EGFR TKI by repressing expression of liver kinase B1 (LKB1), a master kinase of the AMPK subfamily, via CpG island methylation. In addition, LKB1 expression is correlated positively with sensitivity to TKI in patients with NSCLC. Moreover, combined treatment of EGFR TKI with AMPK activators synergistically increases EGFR TKI sensitivity. Collectively, the current study suggests that LKB1 may serve as a marker to predict EGFR TKI sensitivity in smokers with NSCLC carrying EGFRWT and that the combination of EGFR TKI and AMPK activator may be a potentially effective therapeutic strategy against NSCLC with EGFRWT.

Epigenetic therapy induces transcription of inverted SINEs and ADAR1 dependency.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.

From tobacco smoking to cancer mutational signature: a mediation analysis strategy to explore the role of epigenetic changes.

BACKGROUND: Tobacco smoking is associated with a unique mutational signature in the human cancer genome. It is unclear whether tobacco smoking-altered DNA methylations and gene expressions affect smoking-related mutational signature. METHODS: We systematically analyzed the smoking-related DNA methylation sites reported from five previous casecontrol studies in peripheral blood cells to identify possible target genes. Using the mediation analysis approach, we evaluated whether the association of tobacco smoking with mutational signature is mediated through altered DNA methylation and expression of these target genes in lung adenocarcinoma tumor tissues. RESULTS: Based on data obtained from 21,108 blood samples, we identified 374 smoking-related DNA methylation sites, annotated to 248 target genes. Using data from DNA methylations, gene expressions and smoking-related mutational signature generated from ~ 7700 tumor tissue samples across 26 cancer types from The Cancer Genome Atlas (TCGA), we found 11 of the 248 target genes whose expressions were associated with smoking-related mutational signature at a Bonferroni-correction P < 0.001. This included four for head and neck cancer, and seven for lung adenocarcinoma. In lung adenocarcinoma, our results showed that smoking increased the expression of three genes, AHRR, GPR15, and HDGF, and decreased the expression of two genes, CAPN8, and RPS6KA1, which were consequently associated with increased smoking-related mutational signature. Additional evidence showed that the elevated expression of AHRR and GPR15 were associated with smoking-altered hypomethylations at cg14817490 and cg19859270, respectively, in lung adenocarcinoma tumor tissues. Lastly, we showed that decreased expression of RPS6KA1, were associated with poor survival of lung cancer patients. CONCLUSIONS: Our findings provide novel insight into the contributions of tobacco smoking to carcinogenesis through the underlying mechanisms of the elevated mutational signature by altered DNA methylations and gene expressions.

Thyroid Hormone Induces DNA Demethylation in Xenopus Tadpole Brain.

Thyroid hormone (T3) plays pivotal roles in vertebrate development, acting via nuclear T3 receptors (TRs) that regulate gene transcription by promoting post-translational modifications to histones. Methylation of cytosine residues in deoxyribonucleic acid (DNA) also modulates gene transcription, and our recent finding of predominant DNA demethylation in the brain of Xenopus tadpoles at metamorphosis, a T3-dependent developmental process, caused us to hypothesize that T3 induces these changes in vivo. Treatment of premetamorphic tadpoles with T3 for 24 or 48 hours increased immunoreactivity in several brain regions for the DNA demethylation intermediates 5-hydroxymethylcytosine (5-hmC) and 5-carboxylcytosine, and the methylcytosine dioxygenase ten-eleven translocation 3 (TET3). Thyroid hormone treatment induced locus-specific DNA demethylation in proximity to known T3 response elements within the DNA methyltransferase 3a and Krüppel-like factor 9 genes, analyzed by 5-hmC immunoprecipitation and methylation sensitive restriction enzyme digest. Chromatin-immunoprecipitation (ChIP) assay showed that T3 induced TET3 recruitment to these loci. Furthermore, the messenger ribonucleic acid for several genes encoding DNA demethylation enzymes were induced by T3 in a time-dependent manner in tadpole brain. A TR ChIP-sequencing experiment identified putative TR binding sites at several of these genes, and we provide multiple lines of evidence to support that tet2 contains a bona fide T3 response element. Our findings show that T3 can promote DNA demethylation in developing tadpole brain, in part by promoting TET3 recruitment to discrete genomic regions, and by inducing genes that encode DNA demethylation enzymes.

Sperm DNA methylation altered by THC and nicotine: Vulnerability of neurodevelopmental genes with bivalent chromatin.

Men consume the most nicotine and cannabis products but impacts on sperm epigenetics are poorly characterized. Evidence suggests that preconception exposure to these drugs alters offspring neurodevelopment. Epigenetics may in part facilitate heritability. We therefore compared effects of exposure to tetrahydrocannabinol (THC) and nicotine on DNA methylation in rat sperm at genes involved in neurodevelopment. Reduced representation bisulfite sequencing data from sperm of rats exposed to THC via oral gavage showed that seven neurodevelopmentally active genes were significantly differentially methylated versus controls. Pyrosequencing data revealed majority overlap in differential methylation in sperm from rats exposed to THC via injection as well as those exposed to nicotine. Neurodevelopmental genes including autism candidates are vulnerable to environmental exposures and common features may mediate this vulnerability. We discovered that autism candidate genes are significantly enriched for bivalent chromatin structure, suggesting this configuration may increase vulnerability of genes in sperm to disrupted methylation.

Enhanced cellular uptake of CpG DNA by α-helical antimicrobial peptide Kn2-7: Effects on macrophage responsiveness to CpG DNA.

DNA containing unmethylated cytosine-guanine motifs (CpG DNA) initiates innate immune responses, including the secretion of cytokines from macrophages. Some antimicrobial peptides modulate the responses to CpG DNA, although the molecular mechanisms of this process remain unclear. This study examined the effects of four α-helical antimicrobial peptides on the immune responses induced by CpG DNA. The antimicrobial peptide FIKRIARLLRKIF, known as Kn2-7, increased the CpG DNA-dependent secretion of interleukin-10 (IL-10) and tumor necrosis factor-α from mouse macrophage-like RAW264.7 cells. Kn2-7 enhanced the cellular uptake of CpG DNA; this effect was decreased by the substitution of arginine residues with alanine residues, and increased by the substitution of lysine residues with arginine residues. The degree to which these peptides enhanced the cellular uptake of CpG DNA correlated well with their ability to increase CpG DNA-dependent IL-10 secretion. In contrast, Kn2-7 synthesized with d-amino acids did not increase CpG DNA-dependent IL-10 secretion, although the ability of the D-form of Kn2-7 to enhance the cellular uptake of CpG DNA was not diminished relative to that of Kn2-7. These results indicate that enhanced cellular uptake of CpG DNA is necessary but insufficient to augment CpG DNA-dependent immune responses.

Predicting Carcinogenic Mechanisms of Non-Genotoxic Carcinogens via Combined Analysis of Global DNA Methylation and In Vitro Cell Transformation.

An in vitro cell transformation assay (CTA) is useful for the detection of non-genotoxic carcinogens (NGTXCs); however, it does not provide information on their modes of action. In this study, to pursue a mechanism-based approach in the risk assessment of NGTXCs, we aimed to develop an integrated strategy comprising an in vitro Bhas 42 CTA and global DNA methylation analysis. For this purpose, 10 NGTXCs, which were also predicted to be negative through Derek/Sarah structure-activity relationship analysis, were first tested for transforming activity in Bhas 42 cells. Methylation profiles using reduced representation bisulfite sequencing were generated for seven NGTXCs that were positive in CTAs. In general, the differentially methylated regions (DMRs) within promoter regions showed slightly more bias toward hypermethylation than the DMRs across the whole genome. We also identified 13 genes associated with overlapping DMRs within the promoter regions in four NGTXCs, of which seven were hypermethylated and six were hypomethylated. Using ingenuity pathway analysis, the genes with DMRs at the CpG sites were found to be enriched in cancer-related categories, including "cell-to-cell signaling and interaction" as well as "cell death and survival". Moreover, the networks related to "cell death and survival", which were considered to be associated with carcinogenesis, were identified in six NGTXCs. These results suggest that epigenetic changes supporting cell transformation processes occur during non-genotoxic carcinogenesis. Taken together, our combined system can become an attractive component for an integrated approach for the testing and assessment of NGTXCs.

Aloe emodin inhibits telomerase activity in breast cancer cells: transcriptional and enzymological mechanism.

BACKGROUND: Telomerase plays an essential role in cancer cell proliferation. In this study, we investigated inhibition mechanism of aloe emodin (AE) on three different types of breast cancer cell lines, MDA-MB-453, MDA-MB-231 and MCF-7. METHODS: The cells were treated with different concentrations of AE. Relative length of telomere and human telomerase reverse-transcriptase (hTERT) mRNA level was analyzed by quantitative PCR (qPCR). Protein level was assayed by Western blot. Sodium bisulfite methylation sequencing was performed to assess the methylation status of gene promoter. Enzymology kinetics was applied to reveal the interaction between AE and telomerase. Ultraviolet-visible titration and fluorescence resonance energy transfer (FRET) melting experiment were carried out to study the interaction between AE and telomeric DNA. RESULTS: Continuous AE exposure of these cells for 48 h results in shortening of telomeres and inhibition of telomerase. The transcription of hTERT was repressed by activation of E2F1 and inactivation of c-myc proteins. Significant demethylation of CpG islands in hTERT gene promoter was observed in MDA-MB-453 and MCF-7 cells. AE competed with dNTP for occupation of the enzyme active site. AE was a telomeric G-quadruplex structure stabilizer as indicated by titration test and FRET experiments. CONCLUSIONS: AE was a competitive inhibitor of telomerase and a G-quadruplex structure stabilizer. AE decreased the transcription of hTERT gene in the three breast cancer cell lines via up-regulation E2F1 and down-regulation c-myc expressions. The suppressed transcription was also related to the demethylation of the gene promoter.

Epigenome, Transcriptome, and Protection by Sulforaphane at Different Stages of UVB-Induced Skin Carcinogenesis.

Sulforaphane (SFN), a potent antioxidant and antiinflammatory agent, has been shown to protect against cancers especially at early stages. However, how SFN affects UVB-mediated epigenome/DNA methylome and transcriptome changes in skin photodamage has not been fully assessed. Herein, we investigated the transcriptomic and DNA methylomic changes during tumor initiation, promotion, and progression and its impact and reversal by SFN using next-generation sequencing (NGS) technology. The results show that SFN reduced tumor incidence and tumor number. SFN's protective effects were more dramatic in the early stages than with later stages. Bioinformatic analysis of RNA sequencing (RNA-seq) data shows differential expressed genes and identifies the top canonical pathways related to SFN treatment of UVB-induced different stages of epidermal carcinogenesis. These pathways include p53 signaling, cell cycle: G2-M DNA damage checkpoint regulation, Th1, and Th2 activation pathway, and PTEN signaling pathways. The top upstream regulators related to UVB and SFN treatment as time progressed include dextran sulfate, TP53, NFE2L2 (Nrf2), IFNB1, and IL10RA. Bioinformatic analysis of Methyl-seq data shows several differential methylation regions induced by UVB were attenuated by SFN. These include Notch1, Smad6, Gnai3, and Apc2 Integrative analysis of RNA-seq and DNA-seq/CpG methylome yields a subgroup of genes associated with ultraviolet B (UVB) and SFN treatment. The changes in gene expression were inversely correlated with promoter CpG methylation status. These genes include Pik3cd, Matk, and Adm2 In conclusion, our study provides novel insights on the impact of SFN on the transcriptomic and DNA methylomic of UVB-induced different stages of skin cancer in mice.

Triterpenoid corosolic acid modulates global CpG methylation and transcriptome of tumor promotor TPA induced mouse epidermal JB6 P+ cells.

Epigenetic regulation is one of the driving forces in the process of carcinogenesis. Corosolic acid (CA); triterpenoid abundantly found in Lagerstroemia speciosa L. is known to modulate various cellular process including cellular oxidative stress and signaling kinases in various diseases, including skin cancer. Genetic mutations in early stages of skin cancer are well-documented, the epigenetic alterations remain elusive. In the present study, we identified the transcriptomic gene expression changes with RNAseq and genome-wide DNA CpG methylation changes with DNA methylseq to profile the early stage transcriptomic and epigenomic changes using tumor promoter TPA-mediated mouse epidermal epithelial JB6 P+ cells. JB6 P+ cells were treated with TPA and Corosolic acid by 7.5uM optimized by MTS assay. Differentiated expressed genes (DEGs) and Differentially methylated genes (DMRs) were analyzed by R software. Ingenuity Pathway Analysis (IPA) was employed to understand the differential regulation of specific pathways. Novel TPA induced differentially overexpressed genes like tumor promoter Prl2c2, small prolin rich protein (Sprr2h) was reported which was downregulated by corosolic acid treatment. Several cancer related pathways were identified by Ingenuity Pathways Analysis (IPA) including p53, Erk, TGF beta signaling pathways. Moreover, differentially methylated regions (DMRs) in genes like Dusp22 (Dual specificity protein phosphatase 22), Rassf (tumor suppressor gene family, Ras association domain family) in JB6 P+ cells were uncovered which are altered by TPA and are reversed by CA treatment. Interestingly, genes like CDK1 (Cyclin-dependent kinases 1) and RASSF2 (Ras association domain family member 2) observed to be differentially methylated and expressed which was further modulated by corosolic acid treatment, validated by qPCR. Given study indicated gene expression changes to DNA CpG methylation epigenomic changes modulated various molecular pathways in TPA-induced JB6 cells and revealed that CA can potentially reverse these changes which deciphering novel molecular targets for future prevention of early stages of skin cancer studies in human.

Curcumin Activates the Nrf2 Pathway and Induces Cellular Protection Against Oxidative Injury.

Curcumin is a naturally occurring polyphenol that is isolated from the rhizome of Curcuma longa (turmeric). This medicinal compound has different biological activities, including antioxidant, antibacterial, antineoplastic, and anti-inflammatory. It also has therapeutic effects on neurodegenerative disorders, renal disorders, and diabetes mellitus. Curcumin is safe and well-tolerated at high concentrations without inducing toxicity. It seems that curcumin is capable of targeting the Nrf2 signaling pathway in protecting the cells against oxidative damage. Besides, this strategy is advantageous in cancer therapy. Accumulating data demonstrates that curcumin applies four distinct ways to stimulate the Nrf2 signaling pathway, including inhibition of Keap1, affecting the upstream mediators of Nrf2, influencing the expression of Nrf2 and target genes, and finally, improving the nuclear translocation of Nrf2. In the present review, the effects of curcumin on the Nrf2 signaling pathway to exert its therapeutic and biological activities has been discussed.

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression.

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported in 1987, as a protein crucial for the invasiveness of malignant melanoma. Recently, it has been confirmed that CD146 has been involved in progression and poor overall survival of many cancers including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify the potential involvement of epigenetic mechanism behind the regulation of CD146 expression in breast cancer cells, as it has been previously reported in prostate cancer. First, we analysed the response of breast cancer cell lines, differing in the initial CD146 mRNA and protein content, to epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at mRNA and protein level, what was accompanied by increased expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of CD146 gene expression. In conclusion, our study revealed, for the first time, that aberrant methylation maybe involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide the basis for novel therapies as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.

DNA methylome-wide alterations associated with estrogen receptor-dependent effects of bisphenols in breast cancer.

BACKGROUND: Bisphenol A (BPA), an estrogen-like endocrine disruptor used in plastics, has been associated with development and promotion of breast cancer, so plastic manufacturers shifted towards less-studied analogs, BPF and BPS. Studying the associated DNA methylome-wide mechanisms of these derivatives is timely, particularly in comparison with BPA. METHODS: We assessed proliferation, cell cycle, and migration of breast cancer cells (estrogen receptor (ER)-positive: MCF-7 and ER-negative: MDA-MB-231) treated with BPF and BPS ± estrogen receptor inhibitor (ERI) in comparison to BPA ± ERI. RNA expression and activity of DNA (de)methylation enzymes and LINE-1 methylation were quantified. DNA methylome-wide analysis was evaluated in bisphenol-exposed cells and compared to clinical breast cancer data. RESULTS: The three bisphenols caused ER-dependent increased proliferation and migration of MCF-7 but not MDA-MB-231 cells, with BPS being 10 times less potent than BPA and BPF. Although they have similar chemical structures, the three bisphenols induced differential DNA methylation alterations at several genomic clusters of or single CpG sites, with the majority of these being ER-dependent. At equipotent doses, BPA had the strongest effect on the methylome, followed by BPS then BPF. No pathways were enriched for BPF while BPA- and BPS-induced methylome alterations were enriched in focal adhesion, cGMP-PKG, and cancer pathways, which were also dysregulated in methylome-wide alterations comparing ER-positive breast cancer samples to adjacent normal tissues. CONCLUSIONS: The three bisphenols have important epigenetic effects in breast cell lines, with those of BPA and BPS overlapping with cancer-related pathways in clinical breast cancer models. Hence, further investigation of their safety is warranted.

Oral vitamin C supplementation to patients with myeloid cancer on azacitidine treatment: Normalization of plasma vitamin C induces epigenetic changes.

BACKGROUND: Patients with haematological malignancies are often vitamin C deficient, and vitamin C is essential for the TET-induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), the first step in active DNA demethylation. Here, we investigate whether oral vitamin C supplementation can correct vitamin C deficiency and affect the 5hmC/5mC ratio in patients with myeloid cancers treated with DNA methyltransferase inhibitors (DNMTis). RESULTS: We conducted a randomized, double-blinded, placebo-controlled pilot trial (NCT02877277) in Danish patients with myeloid cancers performed during 3 cycles of DNMTi-treatment (5-azacytidine, 100 mg/m2/d for 5 days in 28-day cycles) supplemented by oral dose of 500 mg vitamin C (n = 10) or placebo (n = 10) daily during the last 2 cycles. Fourteen patients (70%) were deficient in plasma vitamin C (< 23 µM) and four of the remaining six patients were taking vitamin supplements at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (< 11.4 µM; 4.997 vs 4.656% 5mC relative to deoxyguanosine, 95% CI [0.126, 0.556], P = 0.004). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (mean increase 34.85 ± 7.94 µM, P = 0.0004). We show for the first time that global 5hmC/5mC levels were significantly increased in mononuclear myeloid cells from patients receiving oral vitamin C compared to placebo (0.037% vs - 0.029%, 95% CI [- 0.129, - 0.003], P = 0.041). CONCLUSIONS: Normalization of plasma vitamin C by oral supplementation leads to an increase in the 5hmC/5mC ratio compared to placebo-treated patients and may enhance the biological effects of DNMTis. The clinical efficacy of oral vitamin C supplementation to DNMTis should be investigated in a large randomized, placebo-controlled clinical trial. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02877277 . Registered on 9 August 2016, retrospectively registered.

DNA methylation changes following DNA damage in prostate cancer cells.

Many cancer therapies operate by inducing double-strand breaks (DSBs) in cancer cells, however treatment-resistant cells rapidly initiate mechanisms to repair damage enabling survival. While the DNA repair mechanisms responsible for cancer cell survival following DNA damaging treatments are becoming better understood, less is known about the role of the epigenome in this process. Using prostate cancer cell lines with differing sensitivities to radiation treatment, we analysed the DNA methylation profiles prior to and following a single dose of radiotherapy (RT) using the Illumina Infinium HumanMethylation450 BeadChip platform. DSB formation and repair, in the absence and presence of the DNA hypomethylating agent, 5-azacytidine (5-AzaC), were also investigated using γH2A.X immunofluorescence staining. Here we demonstrate that DNA methylation is generally stable following a single dose of RT; however, a small number of CpG sites are stably altered up to 14 d following exposure. While the radioresistant and radiosensitive cells displayed distinct basal DNA methylation profiles, their susceptibility to DNA damage appeared similar demonstrating that basal DNA methylation has a limited influence on DSB induction at the regions examined. Recovery from DSB induction was also similar between these cells. Treatment with 5-AzaC did not sensitize resistant cells to DNA damage, but rather delayed recruitment of phosphorylated BRCA1 (S1423) and repair of DSBs. These results highlight that stable epigenetic changes are possible following a single dose of RT and may have significant clinical implications for cancer treatment involving recurrent or fractionated dosing regimens.

Novel predictive epigenetic signature for temozolomide in non-G-CIMP glioblastomas.

OBJECTIVE: To identify novel epigenetic signatures that could provide predictive information that is complementary to promoter methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) gene for predicting temozolomide (TMZ) response, among glioblastomas (GBMs) without glioma-CpGs island methylator phenotype (G-CIMP) METHODS: Different cohorts of primary non-G-CIMP GBMs with genome-wide DNA methylation microarray data were included for discovery and validation of a multimarker signature, combined using a RISK score model. Different statistical analyses and functional experiments were performed for clinical and biological validation. RESULTS: By employing discovery cohorts with radiotherapy (RT) and TMZ versus RT alone and a strict multistep selection strategy, we identified seven CpGs, each of which was significantly correlated with overall survival (OS) of non-G-CIMP GBMs with RT/TMZ, independent of age, MGMT promoter methylation status, and other identified CpGs. A RISK score signature of the 7 CpGs was developed and validated to distinguish non-G-CIMP GBMs with differential survival outcomes to RT/TMZ, but not to RT alone. The interaction analyses also showed differential outcomes to RT/TMZ versus RT alone within the RISK score-based subgroups. The signature could also improve the risk classification by age and MGMT promoter methylation status. Functional experiments showed that HSBP2 appeared to be epigenetically regulated by one identified CpG and was associated with TMZ resistance, but it was not associated with cell proliferation or apoptosis in GBM cell lines. The predictive value of the single CpG methylation of HSBP2 by pyrosequencing was observed in a local cohort of isocitrate dehydrogenase 1 (IDH1) R132H wild-type GBMs. CONCLUSIONS: This novel epigenetic signature might be a promising predictive (but not a general prognostic) biomarker and be helpful for refining the MGMT-based guiding approach to TMZ usage in non-G-CIMP GBMs.

Role of epigenetic regulation of Igf2 and H19 in 2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD)-induced ovarian toxicity in offspring rats.

2,3,7,8-Tetrachlorobenzo-p-dioxin (TCDD) exposure during embryonic gonadal sex determination had been demonstrated to harm the ovarian development. However, its mechanism was unclear and possibly related to epigenetic regulation. In the present study, the pregnant rats were treated with TCDD (100 ng/kg/day or 500 ng/kg/day) or only vehicle and corn oil on the day 8-14 of gestation through the gavage with a stainless-steel feeding needle. The vaginal opening time and estrous cycle of female offspring rats (F1) were monitored twice a day. The ovarian histology, follicle count, real-time PCR, Western Blotting and DNA methylation analysis for Igf2 and H19 were carried out. The results showed that maternal TCDD exposure disrupted estrous cyclicity, resulted in aberrant concentration of serum E2 and FSH, and affected the number of primordial follicles, secondary follicles and corpus luteum. However, TCDD had no effect on the number of primary follicles and atresia follicles. Furthermore, the mRAN expression of imprinted genes Igf2 and H19 was down-regulated, and the IGF2 protein was also down-regulated. TCDD exposure did not alter the mean methylation rate of Igf2 DMR2 and H19 ICR, and only some CpG sites throughout them were hypermethylated in high-dose TCDD rats. In conclusion, maternal exposure of TCDD could affect the ovary development and functions which were possibly associated with down-regulation expression of IGF2 and H19. However, it was not entirely clear whether the impairment of ovary by TCDD was related to the methylation pattern of Igf2 and H19 ICR.