Epigenetic Strategies Research Articles

Inhibition of DNA Methylation in Picochlorum soloecismus Alters Algae Productivity.

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism’s molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (∼1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 μM 5-aza-2′-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (∼66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

Therapeutic Potential of Endothelial Colony-Forming Cells in Ischemic Disease: Strategies to Improve their Regenerative Efficacy.

Cardiovascular disease (CVD) comprises a range of major clinical cardiac and circulatory diseases, which produce immense health and economic burdens worldwide. Currently, vascular regenerative surgery represents the most employed therapeutic option to treat ischemic disorders, even though not all the patients are amenable to surgical revascularization. Therefore, more efficient therapeutic approaches are urgently required to promote neovascularization. Therapeutic angiogenesis represents an emerging strategy that aims at reconstructing the damaged vascular network by stimulating local angiogenesis and/or promoting de novo blood vessel formation according to a process known as vasculogenesis. In turn, circulating endothelial colony-forming cells (ECFCs) represent truly endothelial precursors, which display high clonogenic potential and have the documented ability to originate de novo blood vessels in vivo. Therefore, ECFCs are regarded as the most promising cellular candidate to promote therapeutic angiogenesis in patients suffering from CVD. The current briefly summarizes the available information about the origin and characterization of ECFCs and then widely illustrates the preclinical studies that assessed their regenerative efficacy in a variety of ischemic disorders, including acute myocardial infarction, peripheral artery disease, ischemic brain disease, and retinopathy. Then, we describe the most common pharmacological, genetic, and epigenetic strategies employed to enhance the vasoreparative potential of autologous ECFCs by manipulating crucial pro-angiogenic signaling pathways, e.g., extracellular-signal regulated kinase/Akt, phosphoinositide 3-kinase, and Ca2+ signaling. We conclude by discussing the possibility of targeting circulating ECFCs to rescue their dysfunctional phenotype and promote neovascularization in the presence of CVD.

Abstract IA14: Immunosensitivity and resistance in advanced uveal melanoma

Abstract Despite the ability to effectively achieve control of uveal melanoma at the site of origin, patient outcomes have remained suboptimal due to the ultimate development of recurrent distant disease in a significant proportion of those diagnosed, together with the limited efficacy of currently available therapies in this setting. Initial insights into the tumorigenic effects of the functionally activating, mutually exclusive, early event mutations in GNAQ, GNA11, PLCB4, and CYSLTR2 led to the development of a generation of trials evaluating the effects of inhibition of single or multiple downstream pathways including the MAPK and PI3K pathways. Given the dramatic efficacy of targeted therapy in cutaneous melanoma harboring a BRAF mutation, there was initial hope that a similar strategy might demonstrate comparable effects in uveal melanoma. Unfortunately, the completed and reported clinical trials targeting these pathways have demonstrated minimal, if any, efficacy. Likewise, despite the initial enthusiasm for immunologic checkpoint blockade for the treatment of metastatic uveal melanoma given the durable antitumor responses and cures achieved in cutaneous melanoma, the activity observed thus far has been limited. Nevertheless, with the critical and clinically significant advances made over that past 10 years in precision oncology and immuno-oncology, coupled with rapidly developing advances in our understanding of the biology of uveal melanoma, there is now a new cause for hope for our patients. Trials evaluating the inhibition of alternative nodes in activated pathways alone or in preclinically synergistic combinations are ongoing, new epigenetic strategies are being explored, and preliminary promising data from novel immunologic treatment strategies including adoptive cellular therapy and T-cell redirection all provide new optimism that these innovative treatment options may meaningfully impact the course of the disease. Citation Format: Richard D. Carvajal. Immunosensitivity and resistance in advanced uveal melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA14.

An integrated multi-omics approach identifies epigenetic alterations associated with Alzheimer's disease.

Protein aggregation is the hallmark of neurodegeneration but the molecular mechanisms underlying late-onset Alzheimer’s disease (AD) remain unclear. Here we integrated transcriptomic, proteomic and epigenomic analyses of post-mortem human brains to identify molecular pathways involved in AD. RNA-seq analysis revealed upregulation of transcription- and chromatin-related genes, including the histone acetyltransferases for H3K27ac and H3K9ac. An unbiased proteomic screening singled out H3K27ac and H3K9ac as main enrichments specific to AD. In turn, epigenomic profiling revealed gains of H3K27ac and H3K9ac linked to transcription, chromatin, and disease pathways in AD. Increasing genome-wide H3K27ac and H3K9ac in a fly model of AD exacerbated amyloid-β42-driven neurodegeneration. Together, these findings suggest that AD involves a reconfiguration of the epigenome, where H3K27ac and H3K9ac impact disease pathways by dysregulating transcription- and chromatin-gene feedback loops. The identification of this process highlights potential epigenetic strategies for early-stage disease treatment.

Plasma-Based Longitudinal Evaluation of ESR1 Epigenetic Status in Hormone Receptor-Positive HER2-Negative Metastatic Breast Cancer.

BackgroundEndocrine therapy (ET) is the mainstay of treatment for hormone receptor-positive human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer; however, adaptive mechanisms emerge in about 25–30% of cases through alterations in the estrogen receptor ligand-binding domain, with a consequent ligand-independent estrogen receptor activity. Epigenetic-mediated events are less known and potentially involved in alternative mechanisms of resistance. The aim of this study was to test the feasibility of estrogen receptor 1 (ESR1) epigenetic characterization through liquid biopsy and to show its potential longitudinal application for an early ET sensitivity assessment.MethodsA cohort of 49 women with hormone receptor-positive HER2-negative MBC was prospectively enrolled and characterized through circulating tumor DNA using methylation-specific droplet digital PCR (MS-ddPCR) before treatment start (BL) and after 3 months concomitantly with computed tomography (CT) scan restaging (EV1). ESR1 epigenetic status was defined by assessing the methylation of its main promoters (promA and promB). The most established cell-free tumor DNA (ctDNA) factors associated with ET resistance [ESR1 and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations] were assessed through next-generation sequencing. Associations were tested through Mann–Whitney U test, matched pairs variations through Wilcoxon signed rank test, and survival was analyzed by log-rank test.ResultsThe ET backbone was mainly based on aromatase inhibitors (AIs) (70.83%) in association with CDK4/6 inhibitors (93.75%). Significantly lower promA levels at baseline were observed in patients with liver metastases (P = 0.0212) and in patients with ESR1 mutations (P = 0.0091). No significant impact on PFS was observed for promA (P = 0.3777) and promB (P = 0.7455) dichotomized at the median while a ≥2-fold increase in promB or in either promA or promB at EV1 resulted in a significantly worse prognosis (respectively P = 0.0189, P = 0.0294). A significant increase at EV1 was observed for promB among patients with PIK3CA mutation (P = 0.0173). A trend was observed for promB in ESR1 wild-type patients and for promA in the ESR1 mutant subgroup.ConclusionThe study proofed the concept of an epigenetic characterization strategy based on ctDNA and is capable of being integrated in the current clinical workflow to give useful insights on treatment sensitivity.

Abstract 417: Developing mouse models to dissect the function of a driver gene epimutation in lung tumorigenesis

Abstract Background: Cancer is a complex disease caused by the accumulation of both genetic and epigenetic alterations in oncogenes and tumor suppressor genes. Although an extensive catalog of epigenetic changes has been generated in patients with lung cancer, we have only a fragmentary understanding of their functional roles, particularly for the initiation and maintenance of tumors. The use of genetically engineered mouse models definitively demonstrate that mutations at specific genes cause cancer. An analogous “epigenetically engineered mouse models” should not only lead to important insights about how cancer develop but could provide in vivo platforms in which to screen and validate novel epigenetic therapies. Methods and Results: We published the first epigenetically engineered mouse cancer model based on a driver gene epimutation of p16 (also known as cyclin-dependent kinase inhibitor 2A). In human lung cancers, p16 epimutation is common, occurring in ~50% of cases. Importantly, p16 epimutation is frequently detected in lung pre-neoplastic lesions and p16 is of the best documented gene that controls K-RAS oncogene-induced senescence. Therefore, to elucidate the functional role of p16 epimutation for lung cancer establishment and progression, we developed a mouse model that combines oncogenic K-ras with p16 epimutation. We found that mice with combined K-ras mutation and p16 epimutation (K-ras+/G12D; p16meth/meth) displayed substantially increased tumor numbers and sizes, with tumor progression to adenocarcinoma. In addition, to investigate the therapeutic effects of reversing p16 epimutation, we developed a mouse model that enables conditional and inducible CRISPR based epigenetic gene editing. Our system utilizes deactivated Cas9 (dCas9) fused to a repetitive protein scaffold known as SunTag, which recruits multiple copies of antibody-fused demethylase TET1. We showed that this system is robust and reproducible to achieve targeted DNA demethylation at CpGs within 100 bp from the sgRNA target site of p16 promoter and to reactivate p16. Conclusion: We have established clinically relevant and immunocompetent mouse models based on defined genetic and epigenetic alterations to drive benign lung tumor growths towards the ultimate malignant phenotype. Our model should prove useful as an in vivo preclinical platform for testing epigenetic therapeutic strategies. Citation Format: Li Yang, Anusha Mandala, Yi Chen, Lanlan Shen. Developing mouse models to dissect the function of a driver gene epimutation in lung tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 417.

Abstract 3645: Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer

Abstract Since androgen receptor (AR) is a master transcription factor of prostate gland in both normal development and carcinogenesis, androgen deprivation therapy (ADT) is quite effective for primary prostate cancer (PC) even in advanced disease. During ADT, however, PC tumors generally acquire resistance through oncogenic activation independent from androgen-AR signaling, and progress to lethal castration-resistant PC (CRPC). Transcription factors (TFs) such as AR regulate gene expression through binding regulatory elements of genome called as enhancers. Aberrantly activated enhancers regulating oncogenes could be one of the mechanisms of cancer therapy resistance. In this study, to clarify how aberrant enhancer activation contribute to genesis of CRPC, we performed comprehensive transcriptome and epigenome analyses by RNA-seq and ChIP-seq for H3K4me1, H3K4me3, H3K27ac, H3K79me2, BRD4, AR, FOXA1 and RNA polymerase 2 (RNApol2). Using androgen dependent PC cells LNCaP and its derivative CRPC cells LNCaP95, we extracted BRD4(+)/AR(+)/FOXA1(+) enhancers and super enhancers (SEs) defined by cluster of H3K27ac regardless of BRD4, AR, FOXA1 binding. SE neighboring genes were mostly targeted by BRD4(+)/AR(+)/FOXA1(+) enhancers in LNCaP (57.5%), but markedly less in LNCaP95 (25.3%), suggesting a shift from AR-dependent to AR-independent regulation. In contrast, SE neighboring genes were mostly epigenetically modified with H3K79me2 in both LNCaP (77.4%) and LNCaP95 (76.2%). In addition, the mRNA expression levels of H3K79methyltansferase DOT1L was upregulated in CRPC clinical tissues. While BRD4 inhibition was quite effective for suppressing cell growth in LNCaP, but less effective in LNCaP95, DOT1L inhibition was markedly effective to both cells, inducing marked cell cycle arrest and apoptosis. Furthermore, precise analyses of RNApol2 behavior and transcriptome alteration revealed that BRD4 and DOT1L regulated different genes in a different manner. Mechanistically, while BRD4 inhibition suppressed AR target genes through reducing initiation of transcriptional elongation, DOT1L inhibition suppressed SE neighboring genes through reducing termination of transcriptional elongation. In conclusion, our results indicated that BRD4 and DOT1L played critical role in regulating transcription mediated by aberrantly activated enhancers, and could be targeted as an epigenetic therapeutic strategy for CRPC. Citation Format: Hiroaki Sato, Masahiro Sugiura, Manato Kanesaka, Atsushi Okabe, Masaki Fukuyo, Shinichi Sakamoto, Akira Komiya, Tomohiko Ichikawa, Atsushi Kaneda. Epigenetic regulation of oncogenic transcription mediated by aberrantly activated enhancers in prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3645.

Epigenetic Mechanisms in Gastric Cancer: Potential New Therapeutic Opportunities.

Gastric cancer (GC) is one of the deadliest malignancies worldwide. Complex disease heterogeneity, late diagnosis, and suboptimal therapies result in the poor prognosis of patients. Besides genetic alterations and environmental factors, it has been demonstrated that alterations of the epigenetic machinery guide cancer onset and progression, representing a hallmark of gastric malignancies. Moreover, epigenetic mechanisms undergo an intricate crosstalk, and distinct epigenomic profiles can be shaped under different microenvironmental contexts. In this scenario, targeting epigenetic mechanisms could be an interesting therapeutic strategy to overcome gastric cancer heterogeneity, and the efforts conducted to date are delivering promising results. In this review, we summarize the key epigenetic events involved in gastric cancer development. We conclude with a discussion of new promising epigenetic strategies for gastric cancer treatment.

Augmenting engineered T-cell strategies in solid cancers through epigenetic priming.

T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-based adoptive cell transfer (ACT) has shown promising results in hematological malignancies, but remains immature in solid cancers. The challenges associated with identification of tumor-specific targets, the heterogenic antigen expression, limited T-cell trafficking to tumor sites and the hostile tumor microenvironment (TME), are all factors contributing to the limited efficacy of ACT therapies against solid tumors. Epigenetic priming of tumor cells and the microenvironment may be a way of overcoming these obstacles and improving the clinical efficacy of adoptive T-cell therapies in the future. Here, we review the current literature and suggest combining epigenetic modulators and ACT strategies as a way of augmenting the efficacy of TCR- and CAR-engineered T cells against solid tumors.

Epigenetic regulation of white adipose tissue in the onset of obesity and metabolic diseases.

Obesity and metabolic syndrome are among the most prevalent health problems in developed countries. The impairment of adipose tissue (AT) function is partially responsible for the aetiology of these conditions. Epigenetics refers to several processes that add modifications to either the DNA or chromatin architectural proteins (histones). These processes can regulate gene expression, chromatin compaction and DNA repair. Epigenetics includes mechanisms by which the cell can adapt the cellular response to the environmental conditions. Here, we review the role of epigenetics in the onset of obesity and related metabolic disorders, with special focus on AT. We highlight the importance of nutrients and lifestyle in the regulation of the epigenetic mechanisms and how they can impact on AT plasticity and function in obesity and metabolic diseases. Thus, the epigenetic landscape emerges as a fine-tune regulator of the cellular responses according to the energetic, metabolic and physiological conditions of the cell. Alterations in metabolic pathways deregulated during obesity and metabolic syndrome could in part explain the disturbances in the epigenetic marks of the AT in these disorders. The understanding of how this epigenetic deregulation may affect AT biology and function could lead to new therapeutic approaches based on epigenetic strategies.

Maternal Epigenetic Regulation Contributes to Prevention of Estrogen Receptor-negative Mammary Cancer with Broccoli Sprout Consumption.

Cruciferous vegetables have been of special interest due to the rich presence of bioactive compounds such as sulforaphane which show promising potential on cancer prevention and therapy as an epigenetic dietary strategy. Abnormal epigenetic alteration as one of the primary contributors to tumor development is closely related to breast cancer initiation and progression. In the present study, we investigated the effect of dietary broccoli sprouts (BSp), a common cruciferous vegetable, on prevention of estrogen receptor (ER)-negative mammary tumors at three different temporal exposure windows using a spontaneous breast cancer mouse model. Our findings indicate that maternal BSp treatment exhibited profound inhibitory and preventive effects on mammary cancer formation in the nontreated mouse offspring. The BSp diet administered to adult mice also showed suppressive effects on mammary cancer but was not as profound as the maternal BSp preventive effects. Moreover, such protective effects were linked with differentially expressed tumor- and epigenetic-related genes, as well as altered global histone acetylation, DNA methylation, and DNA hydroxymethylation levels. We also found that the expression changes of tumor-related genes were associated with the levels of histone methylation of H3K4 and H3K9 in the gene promoter regions. In addition, BSp-enriched sulforaphane was shown to increase protein expression of tumor suppressor genes such as p16 and p53 and inhibit the protein levels of Bmi1, DNA methyltransferases, and histone deacetylases in ERα-negative breast cancer cell lines. Collectively, these results suggest that maternal exposure to key phytochemicals may contribute to ER-negative mammary tumor prevention in their offspring through epigenetic regulations.

Methylation of Exogenous Promoters Regulates Soybean Isoflavone Synthase (GmIFS) Transgene in T0 Transgenic Wheat (Triticum aestivum)

DNA methylation is one epigenetic strategy for gene regulation in living organisms. In this study, the expression of soybean isoflavone synthase (GmIFS) transgene in T0 transgenic wheat plants was investigated at the RNA and the final product genestin level. T0 plants showed variations in the GmIFS expression. Methylation status of the exogenous promoters (35S or Oleocin (OL)) proximal sequence was investigated in T0 plants using bisulphite sequencing to disclose their methylation in parallel with the GmIFS level of expression. Results concluded that the high GmIFS expressers of T0 plants exhibited high methylation of exogenous promoter proximal sequences as well as low expression of DNA methyltransferases (Mets). Variation in GmIFS was associated with the level of methylation in the 35S or OL promoters. High expression of GmIFS was negatively correlated with methylation level of 35S and OL promoter proximal regions. In 35S promoter, methylation level of the CpG sites –56 and –88 is strongly linked to GmIFS expression and is involved in the regulation of GmIFS gene. In OL promoter, the CpG site could be involved in the regulation of the GmIFS. Wheat Met3 expression varied among T0 transgenic plants. Its expression profile could explain the regulation of GmIFS transgene by methylation.

Muscle Disuse Atrophy Caused by Discord of Intracellular Signaling.

Significance: Regular contractile activity plays a critical role in maintaining skeletal muscle morphological integrity and physiological function. If the muscle is forced to stop contraction, such as during limb immobilization (IM), the IGF/Akt/mTOR signaling pathway that normally stimulates protein synthesis and inhibits proteolysis will be suppressed, whereas the FoxO-controlled catabolic pathways such as ubiquitin-proteolysis and autophagy/mitophagy will be activated and dominate, resulting in muscle fiber atrophy. Recent Advances: Mitochondria occupy a central position in the regulation of both protein synthesis and degradation through several redox-sensitive pathways, including peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), mitochondrial fusion and fission proteins, mitophagy, and sirtuins. Prolonged IM downregulates PGC-1α due to AMPK (5'-AMP-activated protein kinase) and FoxO activation, thus decreasing mitochondrial biogenesis and causing oxidative damage. Decrease of mitochondrial inner membrane potential and increase of mitochondrial fission can trigger cascades of mitophagy leading to loss of mitochondrial homeostasis (mitostasis), inflammation, and apoptosis. The phenotypic outcomes of these disorders are compromised muscle function and fiber atrophy. Critical Issues: Given the molecular mechanism of the pathogenesis, it is imperative that the integrity of intracellular signaling be restored to prevent the deterioration. So far, overexpression of PGC-1α via transgene and in vivo DNA transfection has been found to be effective in ameliorating mitostasis and reduces IM-induced muscle atrophy. Nutritional supplementation of select amino acids and phytochemicals also provides mechanistic and practical insights into the prevention of muscle disuse atrophy. Future Directions: In light of the importance of mitochondria in regulating the various critical signaling pathways, future work should focus on exploring new epigenetic strategies to restore mitostasis and redox balance.

Transient DNMT3L Expression Reinforces Chromatin Surveillance to Halt Senescence Progression in Mouse Embryonic Fibroblast.

Global heterochromatin reduction, which is one of the hallmarks of senescent cells, is associated with reduced transposable element repression and increased risk of chromatin instability. To ensure genomic integrity, the irreparable cells in a population exit permanently from the cell cycle, and this process is termed “senescence.” However, senescence only blocks the expansion of unwanted cells, and the aberrant chromatin of senescent cells remains unstable. Serendipitously, we found that the transient ectopic expression of a repressive epigenetic modulator, DNA methyltransferase 3-like (DNMT3L) was sufficient to delay the premature senescence progression of late-passage mouse embryonic fibroblasts (MEFs) associated with a tightened global chromatin structure. DNMT3L induces more repressive H3K9 methylation on endogenous retroviruses and downregulates the derepressed transposons in aging MEFs. In addition, we found that a pulse of ectopic DNMT3L resulted in the reestablishment of H3K27me3 on polycomb repressive complex 2 (PRC2)-target genes that were derepressed in old MEFs. We demonstrated that ectopic DNMT3L interacted with PRC2 in MEFs. Our data also suggested that ectopic DNMT3L might guide PRC2 to redress deregulated chromatin regions in cells undergoing senescence. This study might lead to an epigenetic reinforcement strategy for overcoming aging-associated epimutation and senescence.

Discovery of specific HDAC6 inhibitor with anti-metastatic effects in pancreatic cancer cells through virtual screening and biological evaluation.

Histone deacetylase 6 (HDAC6) has been demonstrated to play a major role in cell motility and aggresome formation, and HDAC6 inhibition is therefore considered as a promising epigenetic strategy for cancer treatment. At present, only a minority of compounds have been reported as HDAC6 inhibitors, so specific HDAC6 inhibitors with safety profile need to be discovered urgently. In this paper, HDAC6 inhibitors with diverse structures were used to generate the pharmacophore model by ligand-based method, which contained two hydrogen bond acceptors and two hydrophobic groups. A combined virtual screening based on pharmacophore model and molecular docking was adopted to screen potential HDAC6 inhibitors. Subsequently, the HDAC6 inhibitory activity of the hit compounds were evaluated using an in vitro enzyme binding inhibition assay. The experimental results illustrated that cefoperazone sodium had the strongest inhibitory effect on HDAC6 among the six screened compounds, and its IC50 value was 8.59±1.06μM. Cefoperazone sodium significantly catalyzed the hyperacetylation of α-tubulin but not histone H3, proving that cefoperazone sodium was a selective inhibitor of HDAC6. Since the expression of HDAC6 plays an important role in cancer metastasis, the effects of cefoperazone sodium on migration and invasion of human pancreatic cancer PANC-1 cells were further investigated by wound healing and transwell chamber assays. It was found that cefoperazone sodium could evidently inhibit the migration and invasion of PANC-1 cells. Furthermore, the binding pattern of inhibitor at the active site of the crystal structure was revealed by molecular docking, providing a reference value for the structural design and optimization of HDAC6 inhibitors. This study provides a systematic virtual screening approach for discovering HDAC6 active inhibitors, and by which the specific effect of cefoperazone sodium against HDAC6 was found, suggesting its potential application on cancer therapy.

Plasma microRNA as a noninvasive biomarker for asthma

There are few validated diagnostic and predictive noninvasive biomarkers for asthma in clinical use. Extracellular miRNAs may offer promise for biomarkers and therapeutics strategies for asthma, particularly after environmental exposures. We want to search for epigenetic biomarkers (miRNAs) to diagnose or predict asthma in children. Plasma samples from asthma children and healthy controls were analyzed by miRNAs arries for expression profiling. The association between miRNA fold changes and asthma were assessed by logistic regression. Asthma subjects were further classified by severity and atopic status. We also created ROC curves to see how well miRNA can predict asthma. Top 3 miRNAs (miR15a-1,miR101-3, and miR-19-b with fold change 6.01, 5.38, and 5.32) upregulated in asthma patients from arraies were further confirmed by quantitative PCR in another larger sample of 124 children. In this sample, only miR15a-1was significantly associated with asthma (OR=5.53,95%CI=1.48-20.58). Furthermore, miR15a-1 displayed higher area under the ROC curve than miR-19-b and miR101-3 (miR15a-1=0.712 vs. miR19-b:0.654 and mir101-3:0.620). Three combined miRNA panel did not show higher area under ROCcurve than miR15a-1 alone. miR15a-1 offers promise for noninvasive biomarkers for asthma with sensitivity 0.587 and specificity 0.875. In addition, asthma subjects with high severity by GINA guideline had a significantly increased miR15a-1 fold change (OR=1.34,95% CI,1.02-1.76). When futher categorized children into atopic and non-atopic asthma, we found only atopic asthma was significantly correlated with miR15a-1(OR=5.91,95%CI=1.57-22.26). miR15a-1 may offer promise for a noninvasive epigenetic biomarker and therapeutics strategy for asthma, especially for atopic asthma.

Methylation-mediated LINC00261 suppresses pancreatic cancer progression by epigenetically inhibiting c-Myc transcription.

Background: Due to the limitations of strategies for its early diagnosis and treatment, pancreatic cancer (PC) remains a substantial human health threat. We previously discovered a methylation-mediated lncRNA, LINC00261, which is downregulated in PC tissues. However, the underlying role of LINC00261 in PC remains largely unknown.Methods: Quantitative real-time PCR and in situ hybridization were performed to evaluate the expression levels of LINC00261 in PC, adjacent nontumor and normal pancreas tissues. The clinical significance of LINC00261 was assessed in multicenter PC samples. The functions of LINC00261 in PC were investigated by gain- and loss-of-function assays in vitro and in vivo. Potential downstream pathways and mechanisms were explored via RNA sequencing and bioinformatic analyses. RNA immunoprecipitation and chromatin immunoprecipitation assays were used to validate the underlying mechanisms. Pyrosequencing and targeted demethylation of the LINC00261 promoter were performed to explore the upstream epigenetic mechanisms and therapeutic potential.Results: LINC00261 was significantly downregulated in PC tissues, and its expression was positively associated with the prognosis of PC patients. Phenotypic studies indicated that LINC00261 overexpression significantly suppressed PC cell proliferation, migration and metastasis in vitro and in vivo. c-Myc was identified as a downstream target of LINC00261. LINC00261 repressed c-Myc transcription by physically interacting and binding with the bromo domain of p300/CBP, preventing the recruitment of p300/CBP to the promoter region of c-Myc and decreasing the H3K27Ac level. Moreover, the methylation level of the LINC00261 promoter was high in PC tissues and was correlated with poor prognosis. Targeted demethylation of the LINC00261 promoter inhibited PC progression both in vitro and in vivo.Conclusions: Our findings indicate that methylation-mediated LINC00261 suppresses PC progression by epigenetically repressing c-Myc expression. These findings expand the therapeutic potential of LINC00261, possibly providing evidence to support the development of epigenetic drugs or therapeutic strategies. This research adds further insights into the etiology of PC and indicates that LINC00261 may be a prognostic and therapeutic target in PC.

Genetic, epigenetic, and lineage-directed mechanisms in benzene-induced malignancies and hematotoxicity targeting hematopoietic stem cells niche.

Benzene is a known hematotoxic and leukemogenic agent with hematopoietic stem cells (HSCs) niche being the potential target. Occupational and environmental exposure to benzene has been linked to the incidences of hematological disorders and malignancies. Previous studies have shown that benzene may act via multiple modes of action targeting HSCs niche, which include induction of chromosomal and micro RNA aberrations, leading to genetic and epigenetic modification of stem cells and probable carcinogenesis. However, understanding the mechanism linking benzene to the HSCs niche dysregulation is challenging due to complexity of its microenvironment. The niche is known to comprise of cell populations accounted for HSCs and their committed progenitors of lymphoid, erythroid, and myeloid lineages. Thus, it is fundamental to address novel approaches via lineage-directed strategy to elucidate precise mechanism involved in benzene-induced toxicity targeting HSCs and progenitors of different lineages. Here, we review the key genetic and epigenetic factors that mediate hematotoxicological effects by benzene and its metabolites in targeting HSCs niche. Overall, the use of combined genetic, epigenetic, and lineage-directed strategies targeting the HSCs niche is fundamental to uncover the key mechanisms in benzene-induced hematological disorders and malignancies.

BET bromodomains' functions in bone-related pathologies.

Throughout life, bones are subjected to the so-called 'bone-remodeling' process, which is a balanced mechanism between the apposition and the resorption of bone. This remodeling process depends on the activities of bone-specialized cells, namely the osteoblasts and the osteoclasts. Any deregulation in this process results in bone-related pathologies, classified as either metabolic nonmalignant diseases (such as osteoporosis) or malignant primary bone sarcomas. As these pathologies are not characterized by common targetable genetic alterations, epigenetic strategies could be relevant and promising options. Recently, targeting epigenetic regulators such as the bromodomains and extraterminal domains (BET) readers have achieved success in numerous other pathologies, including cancers. In this review, we highlight the current state of the art in terms of the diverse implications of BET bromodomain proteins in the bone's biology and its defects. Consequently, their role in bone-related pathologies will also be developed, especially in the context of the primary bone sarcomas.

Bromodomain and Extraterminal Proteins as Novel Epigenetic Targets for Renal Diseases.

Epigenetic mechanisms, especially DNA methylation and histone modifications, are dynamic processes that regulate the gene expression transcriptional program in normal and diseased states. The bromodomain and extraterminal (BET) protein family (BRD2, BRD3, BRD4, and BRDT) are epigenetic readers that, via bromodomains, regulate gene transcription by binding to acetylated lysine residues on histones and master transcriptional factors. Experimental data have demonstrated the involvement of some BET proteins in many pathological conditions, including tumor development, infections, autoimmunity, and inflammation. Selective bromodomain inhibitors are epigenetic drugs that block the interaction between BET proteins and acetylated proteins, thus exerting beneficial effects. Recent data have described the beneficial effect of BET inhibition on experimental renal diseases. Emerging evidence underscores the importance of environmental modifications in the origin of pathological features in chronic kidney diseases (CKD). Several cellular processes such as oxidation, metabolic disorders, cytokines, inflammation, or accumulated uremic toxins may induce epigenetic modifications that regulate key processes involved in renal damage and in other pathological conditions observed in CKD patients. Here, we review how targeting bromodomains in BET proteins may regulate essential processes involved in renal diseases and in associated complications found in CKD patients, such as cardiovascular damage, highlighting the potential of epigenetic therapeutic strategies against BET proteins for CKD treatment and associated risks.