TET2 Expression Research Articles

α-Lipoic Acid Increases Collagen Synthesis and Deposition in Nondiabetic and Diabetic Rat Kidneys.

α-Lipoic acid (ALA) is widely used as a nutritional supplement and therapeutic agent in diabetes management. Well-established antioxidant and hypoglycemic effects of ALA were considered to be particularly important in combating diabetic complications including renal injury. The present study evaluated the potential of ALA to affect profibrotic events in kidney that could alter its structure and functioning. ALA was administered intraperitoneally (10 mg/kg) to nondiabetic and streptozotocin-induced diabetic male Wistar rats for 4 and 8 weeks. The effects of ALA were assessed starting from structural/morphological alterations through changes that characterize profibrotic processes, to regulation of collagen gene expression in kidney. Here, we demonstrated that ALA improved systemic glucose and urea level, reduced formation of renal advanced glycation end products (AGEs), and maintained renal structural integrity in diabetic rats. However, profibrotic events provoked in diabetes were not alleviated by ALA since collagen synthesis/deposition and expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) remained elevated in ALA-treated diabetic rats, especially after 8 weeks of diabetes onset. Moreover, 8 weeks treatment of nondiabetic rats with ALA led to the development of profibrotic features reflected in increased collagen synthesis/deposition. Besides the TGF-β1 downstream signaling, the additional mechanism underlying the upregulation of collagen IV in nondiabetic rats treated with ALA involves decreased DNA methylation of its promoter that could arise from increased Tet1 expression. These findings emphasize the therapeutic caution in the use of ALA, especially in patients with renal diabetic complication.

Regulation of Oncogenic Targets by the Tumor-Suppressive miR-139 Duplex (miR-139-5p and miR-139-3p) in Renal Cell Carcinoma.

We previously found that both the guide and passenger strands of the miR-139 duplex (miR-139-5p and miR-139-3p, respectively) were downregulated in cancer tissues. Analysis of TCGA datasets revealed that low expression of miR-139-5p (p < 0.0001) and miR-139-3p (p < 0.0001) was closely associated with 5-year survival rates of patients with renal cell carcinoma (RCC). Ectopic expression assays showed that miR-139-5p and miR-139-3p acted as tumor-suppressive miRNAs in RCC cells. Here, 19 and 22 genes were identified as putative targets of miR-139-5p and miR-139-3p in RCC cells, respectively. Among these genes, high expression of PLXDC1, TET3, PXN, ARHGEF19, ELK1, DCBLD1, IKBKB, and CSF1 significantly predicted shorter survival in RCC patients according to TCGA analyses (p < 0.05). Importantly, the expression levels of four of these genes, PXN, ARHGEF19, ELK1, and IKBKB, were independent prognostic factors for patient survival (p < 0.05). We focused on PXN (paxillin) and investigated its potential oncogenic role in RCC cells. PXN knockdown significantly inhibited cancer cell migration and invasion, possibly by regulating epithelial–mesenchymal transition. Involvement of the miR-139-3p passenger strand in RCC molecular pathogenesis is a new concept. Analyses of tumor-suppressive-miRNA-mediated molecular networks provide important insights into the molecular pathogenesis of RCC.

Targeted expression profiling reveals distinct stages of early canine fibroblast reprogramming are regulated by 2-oxoglutarate hydroxylases

BackgroundEctopic expression of a defined set of transcription factors allows the reprogramming of mammalian somatic cells to pluripotency. Despite continuous progress in primate and rodent reprogramming, limited attention has been paid to cell reprogramming in domestic and companion species. Previous studies attempting to reprogram canine cells have mostly assessed a small number of presumptive canine induced pluripotent stem cell (iPSC) lines for generic pluripotency attributes. However, why canine cell reprogramming remains extremely inefficient is poorly understood.MethodsTo better characterize the initial steps of pluripotency induction in canine somatic cells, we optimized an experimental system where canine fetal fibroblasts (cFFs) are transduced with the Yamanaka reprogramming factors by Sendai virus vectors. We use quantitative PCR arrays to measure the expression of 80 target genes at various stages of canine cell reprogramming. We ask how cFF reprogramming is influenced by small molecules affecting the epigenomic modification 5-hydroxymethylcytosine, specifically L-ascorbic acid and retinoic acid (AA/RA).ResultsWe found that the expression and catalytic output of a class of 2-oxoglutarate-dependent (2-OG) hydroxylases, known as ten-eleven translocation (TET) enzymes, can be modulated in canine cells treated with AA/RA. We further show that AA/RA treatment induces TET1 expression and facilitates early canine reprogramming, evidenced by upregulation of epithelial and pluripotency markers. Using a chemical inhibitor of 2-OG hydroxylases, we demonstrate that 2-OG hydroxylase activity regulates the expression of a subset of genes involved in mesenchymal-to-epithelial transition (MET) and pluripotency in early canine reprogramming. We identify a set of transcription factors depleted in maturing reprogramming intermediates compared to pluripotent canine embryonic stem cells.ConclusionsOur findings highlight 2-OG hydroxylases have evolutionarily conserved and divergent functions regulating the early reprogramming of canine somatic cells and show reprogramming conditions can be rationally optimized for the generation of maturing canine iPSC.

Role of Ten eleven translocation-2 (Tet2) in modulating neuronal morphology and cognition in a mouse model of Alzheimer's disease.

Abnormal expression of Ten eleven translocation-2 (Tet2) contributes to the pathogenesis of Alzheimer's disease (AD). However, to date, the role of Tet2 in modulating neuronal morphology upon amyloid-β (Aβ)-induced neurotoxicity has not been shown in a mouse model of AD. Here, we have developed a model of injured mouse hippocampal neurons induced by Aβ42 oligomers in vitro. We also investigated the role of Tet2 in injured neurons using recombinant plasmids-induced Tet2 inhibition or over-expression. We found that the reduced expression of Tet2 exacerbated neuronal damage, whereas the increased expression of Tet2 was sufficient to protect neurons against Aβ42 toxicity. Our results indicate that the brains of aged APPswe/PSEN1 double-transgenic (2×Tg-AD) mice exhibit an increase in Aβ plaque accumulation and a decrease in Tet2 expression. As a result, we have also explored the underlying mechanisms of Tet2 in cognition and amyloid load in 2×Tg-AD mice via adeno-associated virus-mediated Tet2 knockdown or over-expression. Recombinant adeno-associated virus was microinjected into bilateral dentate gyrus regions of the hippocampus of the mice. Knocking down Tet2 in young 2×Tg-AD mice resulted in the same extent of cognitive dysfunction as aged 2×Tg-AD mice. Importantly, in middle-aged 2×Tg-AD mice, knocking down Tet2 accelerated the accumulation of Aβ plaques, whereas over-expressing Tet2 alleviated amyloid burden and memory loss. Furthermore, our hippocampal RNA-seq data, from young 2×Tg-AD mice, were enriched with aberrantly expressed lncRNAs and miRNAs that are modulated by Tet2. Tet2-modulated lncRNAs (Malat1, Meg3, Sox2ot, Gm15477, Snhg1) and miRNAs (miR-764, miR-211, and miR-34a) may play a role in neuron formation. Overall, these results indicate that Tet2 may be a potential therapeutic target for repairing neuronal damage and cognitive impairment in AD.

New insight in molecular mechanisms regulating SIRT6 expression in diabetes: Hyperglycaemia effects on SIRT6 DNA methylation.

Conflicting data are reported on the relationship between hyperglycaemia, diabetesand SIRT6 expression. To elucidate hyperglycaemia-induced molecular mechanisms regulating SIRT6 expression, the effect of hyperglycaemia on DNA methylation and SIRT6 expressionhas been evaluated in human aortic endothelial cells exposed to high glucose. DNA methylation of SIRT6 and any potential clinical implication was also evaluated in type 2 diabeticpatients and compared with healthy controls. Endothelial cells exposed to high glucose showed lower methylation levels in SIRT6 promoter and increased SIRT6 and TET2 expression. The high glucose-induced epigenetic changes persisted after 48 h of glucose normalization. Diabetic patients showed lower levels of SIRT6 DNA methylation compared withnondiabetic patients. SIRT6 DNA methylation levels inversely correlated with plasma glucose. Our results firstly demonstrate the involvement of epigenetic mechanisms in regulating SIRT6 expression. Further experiments are necessary to clarify metabolic memory mechanisms driving to diabetic complications and how SIRT6 is potentially involved.

The KRAS/Lin28B axis maintains stemness of pancreatic cancer cells via the let-7i/TET3 pathway.

Increasing evidence demonstrates that Lin28B plays critical roles in numerous biological processes including cell proliferation and stemness maintenance. However, the molecular mechanisms underlying Lin28B nuclear translocation remain poorly understood. Here, we found for the first time that KRAS promoted Lin28B nuclear translocation through PKCβ, which directly bound to and phosphorylated Lin28B at S243. Firstly, we observed that Lin28B was upregulated in pancreatic cancer, contributing to cellular migration and proliferation. Furthermore, nuclear Lin28B upregulated TET3 messenger RNA and protein levels by blocking the production of mature let-7i. Subsequently, increased TET3 expression could also promote the expression of Lin28B, thereby forming a Lin28B/let-7i/TET3 feedback loop. Our results suggest that the KRAS/Lin28B axis drives the let-7i/TET3 pathway to maintain the stemness of pancreatic cancer cells. These findings illuminate the distinct mechanism of Lin28B nuclear translocation and its important roles in KRAS-driven pancreatic cancer, and have important implications for development of novel therapeutic strategies for this cancer.

Fisetin: An anticancer perspective.

Despite the provision of safe and cost-effective chemopreventive cancer approaches, still there are requirements to enhance their efficiency. The use of dietary agents as phytochemicals plays an imperative role against different human cancer cell lines. Among these novel dietary agents, fisetin (3,3',4',7-tetrahydroxyflavone) is present in different fruits and vegetables such as apple, persimmon, grape, strawberry, cucumber, and onion. Being a potent anticancer agent, fisetin has been used to inhibit stages in the cancer cells (proliferation, invasion), prevent cell cycle progression, inhibit cell growth, induce apoptosis, cause polymerase (PARP) cleavage, and modulate the expressions of Bcl-2 family proteins in different cancer cell lines (HT-29, U266, MDA-MB-231, BT549, and PC-3M-luc-6), respectively. Further, fisetin also suppresses the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways, reduces the NF-κB activation, and down-regulates the level of the oncoprotein securin. Fisetin also inhibited cell division and proliferation and invasion as well as lowered the TET1 expression levels. The current review article highlights and discusses the anticancer role of fisetin in cell cultures and animal and human studies. Conclusively, fisetin as a polyphenol with pleiotropic pharmacological properties showed promising anticancer activity in a wide range of cancers. Fisetin suppresses the cancer cell stages, prevents progression in cell cycle and cell growth, and induces apoptosis.

Effects of Ten-Eleven Translocation-2 (Tet2) on myogenic differentiation of chicken myoblasts.

Skeletal muscle development is an orchestrated progress that is primarily regulated by temporospatial expression of myogenic regulatory factors (MRFs). Recent studies demonstrated that DNA demethylation also exerted a critical role in myogenesis. However, the function of Tet2 in the regulation of chicken myogenesis still remains unknown. In the present study, the role of Tet2 in regulating myogenic differentiation was determined by using a model of primary myoblasts from chickens. The expression of Tet2 was significantly elevated during myoblast differentiation. Meanwhile, the level of 5hmC in genomic DNA was increased, but H3K9me2 and H3K27me3 were markedly reduced following differentiation. Knockdown of Tet2 significantly inhibited the formation of multinucleated myotubes, which was accompanied by a reduction of relevant pivotal MRFs. Moreover, the level of 5hmC decreased sharply in Tet2 knockdown myoblasts. Attenuated differentiated myoblasts that resulted from reduced Tet2 also demonstrated an increased level of H3K9me2 and H3K27me3. Collectively, these results indicated that Tet2 played an essential role during myogenesis, which affected demethylation of genomic DNA and histone to regulate expression of MRFs and therefore, contributed to myoblast differentiation.

Circular RNA CircMAP3K5 Acts as a MicroRNA-22-3p Sponge to Promote Resolution of Intimal Hyperplasia Via TET2-Mediated Smooth Muscle Cell Differentiation.

Aberrant expression of circular RNA contributes to human diseases. Circular RNAs regulate gene expression by sequestering specific microRNAs. In this study, we investigated whether circMAP3K5 (circular mitogen-activated protein kinase 5) could act as a competing endogenous microRNA-22-3p (miR-22-3p) sponge and regulate neointimal hyperplasia. Circular RNA profiling from genome-wide RNA sequencing data was compared between human coronary artery smooth muscle cells (SMCs) treated with or without platelet-derived growth factor. Expression levels of circMAP3K5 were assessed in human coronary arteries from autopsies on patients with dilated cardiomyopathy or coronary heart disease. The role of circMAP3K5 in intimal hyperplasia was further investigated in mice with adeno-associated virus 9-mediated circMAP3K5 transfection. SMC-specific Tet2 (ten-eleven translocation-2) knockout mice and global miR-22-3p knockout mice were used to delineate the mechanism by which circMAP3K5 attenuated neointimal hyperplasia using the femoral arterial wire injury model. RNA sequencing demonstrated that treatment with platelet-derived growth factor-BB significantly reduced expression of circMAP3K5 in human coronary artery SMCs. Wire-injured mouse femoral arteries and diseased arteries from patients with coronary heart disease (where platelet-derived growth factor-BB is increased) confirmed in vivo downregulation of circMAP3K5 associated with injury and disease. Lentivirus-mediated overexpression of circMAP3K5 inhibited the proliferation of human coronary artery SMCs. In vivo adeno-associated virus 9-mediated transfection of circMap3k5 (mouse circular Map3k5) specifically inhibited SMC proliferation in the wire-injured mouse arteries, resulting in reduced neointima formation. Using a luciferase reporter assay and RNA pull-down, circMAP3K5 (human circular MAP3K5) was found to sequester miR-22-3p, which, in turn, inhibited the expression of TET2. Both in vitro and in vivo results demonstrate that the loss of miR-22-3p recapitulated the antiproliferative effect of circMap3k5 on vascular SMCs. In SMC-specific Tet2 knockout mice, loss of Tet2 abolished the circMap3k5-mediated antiproliferative effect on vascular SMCs. We identify circMAP3K5 as a master regulator of TET2-mediated vascular SMC differentiation. Targeting the circMAP3K5/miR-22-3p/TET2 axis may provide a potential therapeutic strategy for diseases associated with intimal hyperplasia, including restenosis and atherosclerosis.

Cadmium-stimulated invasion of rat liver cells during malignant transformation: Evidence of the involvement of oxidative stress/TET1-sensitive machinery

Cadmium (Cd) is recognized as a highly toxic heavy metal for humans in part because it is a multi-organ carcinogen. To clarify the mechanism of Cd carcinogenicity, we have established an experimental system using rat liver TRL1215 cells exposed to 2.5 μM Cd for 10 weeks and then cultured in Cd-free medium for an additional 4 weeks (total 14 weeks). Recently, we demonstrated, by using this experimental system, that 1) Cd stimulates cell invasion by suppression of apolipoprotein E (ApoE) expression, and 2) Cd induces DNA hypermethylation of the regulatory region of the ApoE gene. However, the underlying mechanism(s) as well as other potential genetic participants in the Cd-stimulated invasion are undefined. In the present work, we found that concurrent with enhanced invasion, Cd induced oxidative stress, coupled with the production of oxidative stress-sensitive metallothionein 2A (MT2A), which lead to down-modulation of ten-eleven translocation methylcytosine dioxygenase 1 (TET1: DNA demethylation) in addition to ApoE, without impacting DNA methyltransferases (DNMTs: DNA methylation) levels. Furthermore, the expression of tissue inhibitor of metalloproteinase 2 and 3 (TIMP2 and TIMP3) that are positively regulated by TET1, were decreased by Cd. The genes (ApoE/TET1/TIMP2/TIMP3) suppressed by Cd were further suppressed by hydroquinone (HQ; a reactive oxygen species [ROS] producer), whereas N-acetyl-l-cysteine (NAC; a ROS scavenger) prevented the suppression of their expression by HQ. In addition, NAC reversed their expression suppressed by Cd. Cd-stimulated cell invasion was clearly dampened by NAC in a concentration-dependent manner. Overall these findings suggest that 1) altered TET1 expression and activity together with ApoE are likely involved in the enhanced invasiveness due to Cd exposure, and 2) Cd down-regulation of TET1 likely evokes a reduction in ApoE expression (possible by DNA hypermethylation), and 3) anti-oxidants are effective in abrogation of the enhanced invasiveness that occurs concurrently with Cd-induced malignant transformation.

NAD+ Metabolism Maintains Inducible PD-L1 Expression to Drive Tumor Immune Evasion

NAD+ metabolism is implicated in aging and cancer. However, its role in immune checkpoint regulation and immune evasion remains unclear. Here, we find nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ biogenesis, drives interferon γ (IFNγ)-induced PD-L1 expression in multiple types of tumors and governs tumor immune evasion in a CD8+ Tcell-dependent manner. Mechanistically, NAD+ metabolism maintains activity and expression of methylcytosine dioxygenase Tet1 via α-ketoglutarate (α-KG). IFNγ-activated Stat1 facilitates Tet1 binding to Irf1 to regulate Irf1 demethylation, leading to downstream PD-L1 expression on tumors. Importantly, high NAMPT-expressing tumors are more sensitive to anti-PD-L1 treatment and NAD+ augmentation enhances the efficacy of anti-PD-L1 antibody in immunotherapy-resistant tumors. Collectively, these data delineate an NAD+ metabolism-dependent epigenetic mechanism contributing to tumor immune evasion, and NAD+ replenishment combined with PD-(L)1 antibody provides a promising therapeutic strategy for immunotherapy-resistant tumors.

STEM-27. miR-10b-5p MODULATES 5hmC EXPRESSION AND THE STEM-LIKE PHENOTYPE IN GBM

Abstract Epigenetic alterations such as DNA methylation and dysregulation of non-coding RNAs (e.g. miRNAs) are found in all types of cancer and are thought to play important roles in tumorigenesis. GBM is characterized by small subsets of cells, referred to as glioma stem cells (GSCs), that display stem-like properties implicated in tumor initiation, therapeutic resistance, and recurrence. DNA methylation patterns are altered in GBM and GSCs and are thought to play critical roles in tumor initiation and propagation. DNA methylation is a reversible process catalyzed, in part, by the ten-eleven translocation (TET) family of enzymes. These enzymes function as deoxygenases that catalyze the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. However, the mechanisms leading to the loss of 5hmC in glioma and the role this phenomenon plays in gliomagenesis remains poorly understood. We found that Sox2 expression decreases TET2 expression and its product 5hmC in GSCs and identified miR-10b-5p as a molecular intermediary of this process. We show that miR-10b-5p expression is high in GBM compared to non-tumor in clinical specimens and high levels of this miRNA correlate with poor patient outcome. Expression of transgenic miR-10b-5p enhanced sphere formation capacity of GSCs and the expression of stem cell markers and drivers. Additionally, using a combination of molecular and biochemical endpoints, we show that miR-10b-5p modifies 5hmC levels by regulating TET2 in GSCs. Finally, we show that repression of miR-10b-5p increases 5hmC levels and inhibits tumor propagation in GBM xenograft models. Taken together, these results present a new molecular mechanism that controls 5hmC and the tumor propagating capacity of GSCs and suggests that miR-10b-5p inhibition and other strategies for enhancing TET2 function can be developed to treat GBM.

Prognostic role of high TET1 expression in patients with solid tumors: A meta-analysis.

Background:Recently, increased expression of TET1 has been shown to inhibit tumor development in many studies. Therefore, a meta-analysis was conducted to assess the prognostic role of TET1 in solid tumors.Methods:PubMed, Embase, and the Web of Science (last updated on June 13, 2019) were searched and 16 eligible studies involving 3100 patients were eventually taken forward into the meta-analysis.Results:Pooled results indicated that higher TET1 expression in cancer tissues was associated with improved overall survival (OS) [hazard ratio (HR) = 0.736, 95% confidence interval (95% CI) = 0.542–0.998, P = .049]. In the subgroup analysis, higher TET1 expression in respiratory tumors (HR = 0.778, 95% CI = 0.639–0.946, P = .012) and breast cancer in Asian patients (HR = 0.326, 95% CI = 0.199–0.533, P < .001) were significantly associated with better OS. In addition, the association between high TET1 expression and prolonged OS was also statistically significant in the following subgroups; data source from samples (HR = 0.561, 95% CI = 0.384–0.819, P = .003), reported in text (HR = 0.539, 95% CI = 0.312–0.931, P = .027), TET1 protein (HR = 0.635, 95% CI = 0.409–0.984, P = .042), Asians (HR = 0.563, 95% CI = 0.376–0.844, P = .005).Conclusion:This meta-analysis displays that high expression levels of TET1 in tissues is significantly associated with better survival in patients with solid tumors. This finding can be used as evidence to the tone that TET1 may be a useful target for the treatment of patients with solid tumors in the future.

DL-propargylglycine administration inhibits TET2 and FOXP3 expression and alleviates symptoms of neonatal Cows’ milk allergy in mouse model

Background Cows’ milk allergy (CMA) is a hypersensitivity immune reaction brought on by specific immunologic mechanisms to cow’s milk proteins. As one of the most common food allergies in infants, the incidence of CMA during the first year of life is estimated to be nearly 7.5%. Due to the limitation in the knowledge of the pathological mechanism underlying CMA, however, the clinical interventions and therapies remain very unsatisfactory. Aim of the study The transcriptional factor FOXP3 possesses crucial roles in CMA, and increased FOXP3 mRNA expression has a predictive function in faster acquisition of tolerance in infants with CMA. But the exact mechanism remains not fully elucidated. Methods For PAG treatment, PAG (dissolved in saline 30 mg/mL, 0, 5, 10, 20 mg/kg BW) was administered daily intraperitoneally (ip) for one week at the time that 6 weeks after the CMP sensitisation. Results In the present study, we revealed that the expression of FOXP3 is significantly up-regulated in PBMCs from CMA patients and CMA mice on mRNA and protein level. Furthermore, a dramatic reduction in the FOXP3 TSDR methylation and a significant increase in the expression of TET2 are observed in CMA patients and CMA mice. More importantly, we found that propargylglycine (PAG) significantly alleviates symptoms of CMA in mice by suppressing the expression of FOXP3 through restoring TET2 expression. Conclusions Our work revealed a novel function of PAG on CMA, which may provide a deeper insight into the pathomechanism of CMA and a novel therapy target for CMA clinical interventions.

Demethylation of the RB1 promoter concomitant with reactivation of TET2 and TET3 impairs gastric carcinogenesis in K19-Wnt1/C2mE transgenic mice

Aberrant methylation of promoter CpG islands (CGIs) can inactivate the expression of many tumor suppressor genes and play an important role in the carcinogenesis of gastric cancer. The tumor suppressor gene RB1, which encodes a cell cycle regulator, is hypermethylated and downregulated in multiple kinds of cancer. Activation of RB1 expression through DNA demethylation is a potential strategy for the treatment of gastric cancer. Herein, we found that the methylation status of the RB1 promoter was negatively related to the development of gastric tumors, while its expression was positively correlated with TET2 and TET3 expression. Further reactivation of RB1 expression by curcumin could inhibit gastric cell viability and carcinogenesis both in vitro and in vivo. Molecular docking and other studies confirmed that curcumin could bind to and upregulate the expression of TET2 and TET3 with hydrogen bonds and arene-H bonds, suggesting that demethylation of RB1 was attributed to reactivation of the demethylation enzymes TET2 and TET3 after curcumin treatment. Thus, our findings reveal a promising therapeutic strategy for gastric cancer prevention and treatment through RB1 demethylation and reactivation.

Phenotypic Switching of Atherosclerotic Smooth Muscle Cells is Regulated by Activated PARP1-Dependent TET1 Expression.

Aim: During the development of atherosclerosis, the vascular smooth muscle cells (SMCs) undergo phenotypic switching from contractile phenotype to synthetic phenotype. This study aimed at examining the role of DNA modification mediated by the oxidative stress dependent ten eleven translocation enzymes (TETs) expression at early stage of phenotypic switching. Methods: Based on the in vitro SMCs calcification model, DNA damage, phenotypic switching and 5-hydroxymethylcytosine (5hmC) were examined by comet assay, alkaline DNA unwinding assay, immunofluorescence staining, Dot blotting and Western blotting. Then Western blotting and qRT-PCR were performed to analyze the TETs expression and the relationship between the activity of poly(ADP-ribose) polymerase 1 (PARP1) and TETs expression. We further alter 5hmC modification by inhibition of TET1 or PARP1 to rescue the phenotypic switching of SMCs using immunofluorescence staining, Dot blotting and qRT-PCR. We performed immunochemistry staining to examine the activated PARP1-TET1 pathway in vivo . Results: The phenotypic switching was observed in the SMCs cultured with calcification medium as the expression of the cell markers of contractile SMCs decreased and cell proliferation increased. In contrast, PAR and 5hmC were markedly increased in SMCs with calcification due to DNA damage. Our study further demonstrated that oxidative stress-activated PARP1, promotes TET1 expression and 5hmC increase during the phenotypic switching. Inhibition of TET1 or PARP1 can rescue the phenotypic switching of SMCs with calcification. Conclusion: Our study demonstrated the important role of PARylation dependent 5hmC, in SMCs phenotypic switching. It raises the possibility to target TET1 and PARP1 for atherosclerosis treatment.

TET2 directs mammary luminal cell differentiation and endocrine response

Epigenetic regulation plays an important role in governing stem cell fate and tumorigenesis. Lost expression of a key DNA demethylation enzyme TET2 is associated with human cancers and has been linked to stem cell traits in vitro; however, whether and how TET2 regulates mammary stem cell fate and mammary tumorigenesis in vivo remains to be determined. Here, using our recently established mammary specific Tet2 deletion mouse model, the data reveals that TET2 plays a pivotal role in mammary gland development and luminal lineage commitment. We show that TET2 and FOXP1 form a chromatin complex that mediates demethylation of ESR1, GATA3, and FOXA1, three key genes that are known to coordinately orchestrate mammary luminal lineage specification and endocrine response, and also are often silenced by DNA methylation in aggressive breast cancers. Furthermore, Tet2 deletion-PyMT breast cancer mouse model exhibits enhanced mammary tumor development with deficient ERα expression that confers tamoxifen resistance in vivo. As a result, this study elucidates a role for TET2 in governing luminal cell differentiation and endocrine response that underlies breast cancer resistance to anti-estrogen treatments.

Inhibition of NF-κB is required for oleanolic acid to downregulate PD-L1 by promoting DNA demethylation in gastric cancer cells.

Gastric cancer is one of the most common causes of cancer-related death worldwide. Immunotherapy via programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade has shown benefits for gastric cancer. Epigenetic DNA methylation critically regulates cancer immune checkpoints. We investigated how the natural compound oleanolic acid (OA) affected PD-L1 expression in gastric cancer cells. Interleukin-1β (IL-1β) at 20 ng/mL was used to stimulate human gastric cancer MKN-45 cells. IL-1β significantly increased PD-L1 expression, which was abolished by OA. Next, OA-treated MKN-45 cells were co-cultured with activated and PD-1-overexpressing Jurkat T cells. OA restored IL-2 levels in the co-culture system and increased T cell killing toward MKN-45 cells. Overexpression of PD-L1 eliminated OA-enhanced T cell killing capacity; however, PD-1 blocking antibody abrogated the cytotoxicity of T cells. Moreover, OA abolished IL-1β-increased DNA demethylase activity in MKN-45 cells. DNA methyltransferase inhibitor 5-azacytidine rescued OA-reduced PD-L1 expression; whereas DNA demethylation inhibitor gemcitabine inhibited PD-L1 expression, and, in combination with OA, provided more potent inhibitory effects. Furthermore, OA selectively reduced the expression of DNA demethylase TET3 in IL-1β-treated MKN-45 cells, and overexpression of TET3 restored OA-reduced PD-L1 expression. Finally, OA disrupted nuclear factor κB (NF-κB) signaling IL-1β-treated MKN-45 cells, and overexpression of NF-κB restored OA downregulation of TET3 and PD-L1. The cytotoxicity of T cells toward MKN-45 cells was also weakened by NF-κB overexpression. Altogether, OA blocked the IL-1β/NF-κB/TET3 axis in gastric cancer cells, leading to DNA hypomethylation and downregulation of PD-L1. Our discoveries suggested OA as an epigenetic modulator for immunotherapy or an adjuvant therapy against gastric cancer.

Endogenous Retrovirus Transcript Levels Are Associated with Immunogenic Signatures in Multiple Metastatic Cancer Types.

Next-generation sequencing of solid tumors has revealed variable signatures of immunogenicity across tumors, but underlying molecular characteristics driving such variation are not fully understood. Although expression of endogenous retrovirus (ERV)-containing transcripts can provide a source of tumor-specific neoantigen in some cancer models, associations between ERV levels and immunogenicity across different types of metastatic cancer are not well established. We performed bioinformatics analysis of genomic, transcriptomic, and clinical data across an integrated cohort of 199 patients with metastatic breast, colorectal, and pancreatic ductal adenocarcinoma tumors. Within each cancer type, we identified a subgroup of viral mimicry tumors in which increased ERV levels were coupled with transcriptional signatures of autonomous antiviral response and immunogenicity. In addition, viral mimicry colorectal and pancreatic tumors showed increased expression of DNA demethylation gene TET2 Taken together, these data demonstrate the existence of an ERV-associated viral mimicry phenotype across three distinct metastatic cancer types, while indicating links between ERV abundance, epigenetic dysregulation, and immunogenicity.

Effect of Perfluorooctanoic Acid on the Epigenetic and Tight Junction Genes of the Mouse Intestine.

Perfluorooctanoic acid (PFOA) has been implicated in various toxicities including neurotoxicity, genotoxicity, nephrotoxicity, epigenetic toxicity, immunotoxicity, reproductive toxicity, and hepatotoxicity. However, information on the accumulation of PFOA in the intestine and its toxic effects on intestinal epigenetics and tight junction (TJ) genes is sparse. CD1 mice were dosed with PFOA (1, 5, 10, or 20 mg/kg/day) for 10 days, and its accumulation and induced alterations in the expression of epigenetic and tight junction genes in the small intestine and colon were evaluated using LC–MS and qPCR techniques. PFOA reduced the expression levels of DNA methyltransferases (Dnmt1, Dnmt3a, Dnmt3b) primarily in the small intestine whereas, in the colon, a decrease was observed only at high concentrations. Moreover, ten-eleven translocation genes (Tet2 and Tet3) expression was dysregulated in the small intestine, whereas in the colon Tets remained unaffected. The tight junction genes Claudins (Cldn), Occludin (Ocln), and Tight Junction Protein (Tjp) were also heavily altered in the small intestine. TJs responded differently across the gut, in proportion to PFOA dosing. Our study reveals that PFOA triggers DNA methylation changes and alters the expression of genes essential for maintaining the physical barrier of intestine, with more profound effects in the small intestine compared to the colon.