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Histamine, a monoamine implicated in stress-related arousal states, is synthesized in neurons exclusively located in the hypothalamic tuberomammillary nucleus (TMN) from where they diffusely innervate striatal and mesolimbic networks including the nucleus accumbens (NAc), a vital node in the limbic loop. Since histaminecontaining TMN neuron output increases during stress, we hypothesized that exposure of mice to acute restrain stress (ARS) recruits endogenous histamine type 2 receptor (H2R) signaling in the NAc, whose activation increases medium spiny neurons (MSNs) intrinsic excitability via downregulation of A-type K+ currents. We employed an ARS paradigm in which mice were restrained for 120 min, followed by a 20-min recovery period, after which brain slices were prepared for ex vivo electrophysiology. Using whole-cell patch-clamp recordings, we found that pharmacological activation of H2R failed to affect MSN excitability and A-type K+ currents in mice that underwent ARS. Interestingly, in mice treated with H2R-antagonist prior to ARS paradigm, H2R activation increased evoked firing and decreased A-type K+ currents similarly to what observed in control mice. Furthermore, H2R-antagonist treatment ameliorated anxiety-like behavior in ARS mice. Together, our findings indicate that ARS paradigm recruits endogenous H2R signaling in MSNs and suggest the involvement of H2R signaling in stress-related motivational states.

Simple Summary Despite the rapid advance in cancer therapies, treatment-resistant relapse remains a significant challenge in cancer treatment. Acquired resistance arises during or after treatment administration, and is usually the main contributor to relapse. For example, prostate cancer, the most frequent type of cancer in the elderly male population, frequently develops into aggressive forms resistant to chemical and hormonal therapies. In this condition, the so-called "cholinic phenotype" that is characterized by the overexpression of choline kinase alpha (CHKA) and increased phosphocholine levels leads to aberrant lipid metabolism. Our work demonstrates that CHKA, which is necessary for membrane phospholipid synthesis, is a target of the long non-coding RNA MALAT1. This study helps to further decipher how MALAT1 affects the regulation of crucial phospholipid/sphingolipid metabolic enzymes, as well as how the androgen receptor pathway is involved in MALAT1-dependent transcriptional regulation. Background. Choline kinase alpha (CHKA), an essential gene in phospholipid metabolism, is among the modulated MALAT1-targeted transcripts in advanced and metastatic prostate cancer (PCa). Methods. We analyzed CHKA mRNA by qPCR upon MALAT1 targeting in PCa cells, which is characterized by high dose-responsiveness to the androgen receptor (AR) and its variants. Metabolome analysis of MALAT1-depleted cells was performed by quantitative High-resolution 1 H-Nuclear Magnetic Resonance (NMR) spectroscopy. In addition, CHKA genomic regions were evaluated by chromatin immunoprecipitation (ChIP) in order to assess MALAT1-dependent histone-tail modifications and AR recruitment. Results. In MALAT1-depleted cells, the decrease of CHKA gene expression was associated with reduced total choline-containing metabolites compared to controls, particularly phosphocholine (PCho). Upon MALAT1 targeting a significant increase in repressive histone modifications was observed at the CHKA intron-2, e

BackgroundThe present study investigates whether epigenetic differences emerge in the heart of patients undergoing cardiac surgery for an aortic valvular replacement (AVR) or coronary artery bypass graft (CABG). An algorithm is also established to determine how the pathophysiological condition might influence the human biological cardiac age.ResultsBlood samples and cardiac auricles were collected from patients who underwent cardiac procedures: 94 AVR and 289 CABG. The CpGs from three independent blood-derived biological clocks were selected to design a new blood- and the first cardiac-specific clocks. Specifically, 31 CpGs from six age-related genes, ELOVL2, EDARADD, ITGA2B, ASPA, PDE4C, and FHL2, were used to construct the tissue-tailored clocks. The best-fitting variables were combined to define new cardiac- and blood-tailored clocks validated through neural network analysis and elastic regression. In addition, telomere length (TL) was measured by qPCR. These new methods revealed a similarity between chronological and biological age in the blood and heart; the average TL was significantly higher in the heart than in the blood. In addition, the cardiac clock discriminated well between AVR and CABG and was sensitive to cardiovascular risk factors such as obesity and smoking. Moreover, the cardiac-specific clock identified an AVR patient's subgroup whose accelerated bioage correlated with the altered ventricular parameters, including left ventricular diastolic and systolic volume.ConclusionThis study reports on applying a method to evaluate the cardiac biological age revealing epigenetic features that separate subgroups of AVR and CABG.

The lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes growth and progression in prostate cancer (PCa); however, little is known about its possible impact in PCa metabolism. The aim of this work has been the assessment of the metabolic reprogramming associated with MALAT1 silencing in human PCa cells and in an ex vivo model of organotypic slice cultures (OSCs). Cultured cells and OSCs derived from primary tumors were transfected with MALAT1 specific gapmers. Cell growth and survival, gene profiling, and evaluation of targeted metabolites and metabolic enzymes were assessed. Computational analysis was made considering expression changes occurring in metabolic markers following MALAT1 targeting in cultured OSCs. MALAT1 silencing reduced expression of some metabolic enzymes, including malic enzyme 3, pyruvate dehydrogenase kinases 1 and 3, and choline kinase A. Consequently, PCa metabolism switched toward a glycolytic phenotype characterized by increased lactate production paralleled by growth arrest and cell death. Conversely, the function of mitochondrial succinate dehydrogenase and the expression of oxidative phosphorylation enzymes were markedly reduced. A similar effect was observed in OSCs. Based on this, a predictive algorithm was developed aimed to predict tumor recurrence in a subset of patients. MALAT1 targeting by gapmer delivery restored normal metabolic energy pathway in PCa cells and OSCs.

Purpose: In presence of indeterminate lesions by fine needle aspiration (FNA), thyroid cancer cannot always be easily diagnosed by conventional cytology. As a consequence, unnecessary removal of thyroid gland is performed in patients without cancer based on the lack of optimized diagnostic criteria. Aim of this study is identifying a molecular profile based on long noncoding RNAs (lncRNAs) expression capable to discriminate between benign and malignant nodules. Methods: Patients were subjected to surgery (n = 19) for cytologic suspicious thyroid nodules or to FNA biopsy (n = 135) for thyroid nodules suspicious at ultrasound. Three thyroid-specific genes (TG, TPO, and NIS), six cancer-associated lncRNAs (MALAT1, NEAT1, HOTAIR, H19, PVT1, MEG3), and two housekeeping genes (GAPDH and P0) were analyzed using Droplet Digital PCR (ddPCR). Results: Based on higher co-expression in malignant (n = 11) but not in benign (n = 8) nodules after surgery, MALAT1, PVT1 and HOTAIR were selected as putative cancer biomarkers to analyze 135 FNA samples. Cytological and histopathological data from a subset of FNA patients (n = 34) were used to define a predictive algorithm based on a Naïve Bayes classifier using co-expression of MALAT1, PVT1, HOTAIR, and cytological class. This classifier exhibited a significant separation capability between malignant and benign nodules (P < 0.0001) as well as both rule in and rule out test potential with an accuracy of 94.12% and a negative predictive value (NPV) of 100% and a positive predictive value (PPV) of 91.67%. Conclusions: ddPCR analysis of selected lncRNAs in FNA biopsies appears a suitable molecular tool with the potential of improving diagnostic accuracy.

Although a significant subset of prostate tumors remain indolent during the entire life, the advanced forms are still one of the leading cause of cancer-related death. There are not reliable markers distinguishing indolent from aggressive forms. Here we highlighted a new molecular circuitry involving microRNA and coding genes promoting cancer progression and castration resistance. Our preclinical and clinical data demonstrated that c-Met activation increases miR-130b levels, inhibits androgen receptor expression, promotes cancer spreading and resistance to hormone ablation therapy. The relevance of these findings was confirmed on patients’ samples and by in silico analysis on an independent patient cohort from Taylor’s platform. Data suggest c-Met/miR-130b axis as a new prognostic marker for patients’ risk assessment and as an indicator of therapy resistance. Our results propose new biomarkers for therapy decision-making in all phases of the pathology. Data may help identify high-risk patients to be treated with adjuvant therapy together with alternative cure for castration-resistant forms while facilitating the identification of possible patients candidates for anti-Met therapy. In addition, we demonstrated that it is possible to evaluate Met/miR-130b axis expression in exosomes isolated from peripheral blood of surgery candidates and advanced patients offering a new non-invasive tool for active surveillance and therapy monitoring.Oncogene advance online publication, 13 February 2017; doi:10.1038/onc.2016.505.

This study aims at investigating the epigenetic landscape of cardiomyocytes exposed to long elevated glucose levels in vitro and in vivo Exposure of mouse HL-1 and rat differentiated H9C2 cardiomyocyte cell lines to high glucose (30 mM) for 72 hours determined some epigenetic changes including upregulation of class I and III HDAC protein levels and activity, inhibition of histone acetylase p300 activity, increase in histone H3 lysine (K) 27 trimethylation (H3K27me3) and reduction in H3K9 acetylation (H3K9Ac). Gene expression analysis focused on cardiotoxicity, revealed that high glucose induced markers associated with tissue damage, fibrosis and cardiac remodelling such as Nexilin, Versican, CREM and ADRA2. Notably, the transcription factor CREM was found to be important in the regulation of cardiotoxicity-associated genes as assessed by specific siRNA and chromatin immunoprecipitation experiments. In CD1 mice, made hyperglycaemic by STZ injection, cardiac structural alterations were evident at six months after STZ treatment and were associated with a significant increase of H3K27me3 and reduction of H3K9Ac. Consistently, Nexilin, CREM and ADRA2 expression was significantly induced at RNA and protein level. Confocal microscopy analysis of Nexilin localization showed this protein irregularly distributed along the sarcomeres in the heart of hyperglycaemic mice. This evidence suggested for a structural alteration of cardiac Z-disk with potential consequences on contractility. In conclusion, high glucose may alter the epigenetic landscape of cardiac cells. Sildenafil, restoring cGMP levels, counteracted the increase of CREM and Nexilin providing a protective effect in the presence of hyperglycemia.

Estrogen and hypoxia promote an aggressive phenotype in prostate cancer (PCa), driving transcription of progression-associated genes. Here, we molecularly dissect the contribution of long non-coding RNA H19 to PCa metastatic potential under combined stimuli, a topic largely uncovered. The effects of estrogen and hypoxia on H19 and cell adhesion molecules' expression were investigated in PCa cells and PCa-derived organotypic slice cultures (OSCs) by qPCR and Western blot. The molecular mechanism was addressed by chromatin immunoprecipitations, overexpression, and silencing assays. PCa cells' metastatic potential was analyzed by in vitro cell-cell adhesion, motility test, and trans-well invasion assay. We found that combined treatment caused a significant H19 down-regulation as compared with hypoxia. In turn, H19 acts as a transcriptional repressor of cell adhesion molecules, as revealed by up-regulation of both beta 3 and beta 4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and beta integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of beta integrins and invasion was achieved through epigenetic modulation of H19-dependent transcription. Our study revealed that estrogen and hypoxia transcriptionally regulate, via H19, cell adhesion molecules redirecting metastatic dissemination from EMT to a beta integrin-mediated invasion.

Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM.

Aims Beyond the control of nuclear-cytoplasmic trafficking nucleoporins regulate gene expression and are involved in cardiac diseases. Notably, a number of cardiovascular disorders have been linked to alterations in epigenetic mechanisms. Here we aimed to determine the contribution of Nup153 to the epigenetic alterations occurring in cardiomyopathy of dystrophin-deficient mdx mice (C57BL/10ScSn-Dmdmdx/J). Methods and results Nup153 was lysine-acetylated and its expression was significantly increased at protein level in mdx hearts compared with controls. Accordingly, lysine acetyl transferase (KAT) activity associated with Nup153 was higher in mdx hearts paralleling increased binding with the lysine acetylases P300/CBP-associated factor (PCAF) and p300. Interestingly, Nup153 silencing in mdx organotypic heart tissue slices caused a reduction in PCAF- and p300-specific activities. Remarkably, the level of nitric oxide (NO), which is reduced in mdx mice, was important for KAT-dependent regulation of Nup153. In fact, treatment of mdx heart tissue with an NO donor or the KAT inhibitor anacardic acid normalized Nup153 protein expression. Nup153 was recruited to chromatin and regulated the transcription of genes involved in cardiac remodelling, including the actin-binding protein nexilin. Accordingly, nexilin protein expression was abrogated by Nup153 silencing in mdx organotypic cultures. Electrophysiological and molecular experiments revealed that Nup153 overexpression in normal cardiomyocytes increases Cav1.2 calcium channel expression and function. Alterations in Nup153 protein expression and intracellular localization were also found in dystrophic cardiomyocytes derived from patient-specific induced pluripotent stem cells. Importantly, Nup153 up-regulation and increased acetylation were also found in the heart of Duchenne muscular dystrophy patients. Conclusions Our data indicate that Nup153 is an epigenetic regulator which, upon altered NO signalling, mediates the ac

Purpose The aim of this study was to generate immortalized human anterior pituitary adenoma cells. Reliable cell models for the study of human pituitary adenomas are as yet lacking and studies performed so far used repeated passaging of freshly excised adenomas, with the attendant limitations due to limited survival in culture, early senescence, and poor reproducibility. Methods & Results We devised a technique based upon repeated co-transfections of two retroviral vectors, one carrying the catalytic subunit of human telomerase, hTERT, the other SV40 large T antigen. This approach extended the lifespan of cells derived from a human growth hormone-secreting adenoma up to 18 months while retaining morphology of primary cells, growth hormone synthesis and growth hormone secretion. Conclusions Our attempt represents the first demonstration of successful lifespan extension of human growth hormonesecreting pituitary adenoma cells via co-transfection of hTERT and SV40T and paves the way to future attempts to obtain stable cell lines.

Nucleoporin 153 (Nup153), key regulator of nuclear import/export, has been recently associated to oncogenic properties in pancreatic and breast tumour cells modulating either cell motility and migration or gene expression by chromatin association. In the present work, we have characterized the role of Nup153 in a cellular model of prostate cancer (PCa). The analysis of several immortalized cell lines derived from freshly explants of prostate cancer specimens showed that Nup153 protein was higher and present in multimeric complexes with eNOS and ERß as compared to normal/hyperplastic prostate epithelial cells. This phenomenon was enhanced in the presence of 17ß-estradiol (E2, 10-7M). Further experiments revealed that eNOS and ERß were present in a DNA binding complexes associated with Nup153 promoter as demonstrated by ChIPs. Notably, after Nup153 depletion (siNup153), a reduction of migration capacity and colony formation in primary tumor-derived and metastatic PCa cells was observed. In addition, eNOS and ERß nuclear localization was lost upon siNup153 regardless of E2 treatment, suggesting that Nup153 is a key regulator of prostate cancer cell function and of the nuclear translocation of these proteins in response to hormone stimulus. Taken altogether our findings indicate that in PCa cells: i. the expression and function of Nup153 is modulated by estrogen signaling; ii. Nup153 contributes to cell migration and proliferation; iii. Nup153 regulates the nuclear translocation of eNOS and ERß by forming a multimeric complex. Our findings unveil Nup153 as a novel component of the estrogen-dependent multimeric complex, thus representing a potential therapeutic candidate in prostate cancer.

The epigenetics of early commitment to embryonal cardiomyocyte is poorly understood. In this work, we compared the effect of thyroid hormone and that of anacardic acid, a naturally occurring histone acetylase inhibitor, or both in combination, on mouse embryonic stem cells (mES) differentiating into embryonal cardiomyocyte by embryoid bodies (EBs) formation. Although the results indicated that anacardic acid (AA) and thyroid hormone were both efficient in promoting cardiomyocyte differentiation, we noticed that a transient exposure of mES to AA alone was sufficient to enlarge the beating areas of EBs compared to those of untreated controls. This effect was associated with changes in the chromatin structure at the promoters of specific cardiomyogenic genes. Among them, a rapid induction of the transcription factor Castor 1 (CASZ1), important for cardiomyocytes differentiation and maturation during embryonic development, was observed in the presence of AA. In contrast, thyroid hormone (T 3) was more effective in stimulating spontaneous firing, thus suggesting a role in the production of a population of cardiomyocyte with pacemaker properties. In conclusion, AA and thyroid hormone both enhanced cardiomyocyte formation along in apparently distinct pathways.

The c-Myc protein is dysregulated in many human cancers and its function has not been fully elucitated yet. The c-Myc inhibitor Omomyc displays potent anticancer properties in animal models. It perturbs the c-Myc protein network, impairs c-Myc binding to the E-boxes, retaining transrepressive properties and inducing histone deacetylation. Here we have employed Omomyc to further analyse c-Myc activity at the epigenetic level. We show that both Myc and Omomyc stimulate histone H4 symmetric dimethylation of arginine (R) 3 (H4R3me2s), in human glioblastoma and HEK293T cells. Consistently, both associated with protein Arginine Methyltransferase 5 (PRMT5)-the catalyst of the reaction-and its co-factor Methylosome Protein 50 (MEP50). Confocal experiments showed that Omomyc co-localized with c-Myc, PRMT5 and H4R3me2s-enriched chromatin domains. Finally, interfering with PRMT5 activity impaired target gene activation by Myc whereas it restrained Omomyc-dependent repression. The identification of a histone-modifying complex associated with Omomyc represents the first demonstration of an active role of this miniprotein in modifying chromatin structure and adds new information regarding its action on c-Myc targets. More importantly, the observation that c-Myc may recruit PRMT5-MEP50, inducing H4R3 symmetric di-methylation, suggests previously unpredictable roles for c-Myc in gene expression regulation and new potential targets for therapy.