Agnese Re

Silencing of GSTP1, a prostate cancer prognostic gene, by the estrogen receptor-β and endothelial nitric oxide synthase complex.

We recently identified in prostate tumors (PCa) a transcriptional prognostic signature comprising a significant number of genes differentially regulated in patients with worse clinical outcome. Induction of up-regulated genes was due to chromatin remodeling by a combinatorial complex between estrogen receptor (ER)-β and endothelial nitric oxide synthase (eNOS). Here we show that this complex can also repress transcription of prognostic genes that are down-regulated in PCa, such as the glutathione transferase gene GSTP1. Silencing of GSTP1 is a common early event in prostate carcinogenesis, frequently caused by promoter hypermethylation. We validated loss of glutathione transferase (GST) P1-1 expression in vivo, in tissue microarrays from a retrospective cohort of patients, and correlated it with decreased disease-specific survival. Furthermore, we show that in PCa cultured cells ERβ/eNOS causes GSTP1 repression by being recruited at estrogen responsive elements in the gene promoter with consequential remodeling of local chromatin. Treatment with ERβ antagonist or its natural ligand 5α-androstane-3β,17β-diol, eNOS inhibitors or ERβ small interference RNA abrogated the binding and reversed GSTP1 silencing, demonstrating the direct involvement of the complex. In vitro, GSTP1 silencing by ERβ/eNOS was specific for cells from patients with worse clinical outcome where it appeared the sole mechanism regulating GSTP1 expression because no promoter hypermethylation was present. However, in vivo chromatin immunoprecipitation assays on fresh PCa tissues demonstrated that silencing by ERβ/eNOS can coexist with promoter hypermethylation. Our findings reveal that the ERβ/eNOS complex can exert transcriptional repression and suggest that this may represent an epigenetic event favoring inactivation of the GSTP1 locus by methylation. Moreover, abrogation of ERβ/eNOS function by 3β-adiol emphasizes the significance of circulating or locally produced sex steroid hormones or their metabolites in PCa biology with relevant clinical/therapeutic implications.

MALAT1 and HOTAIR Long Non-Coding RNAs Play Opposite Role in Estrogen-Mediated Transcriptional Regulation in Prostate Cancer Cells

In the complex network of nuclear hormone receptors, the long non-coding RNAs (lncRNAs) are emerging as critical determinants of hormone action. Here we investigated the involvement of selected cancer-associated lncRNAs in Estrogen Receptor (ER) signaling. Prior studies by Chromatin Immunoprecipitation (ChIP) Sequencing showed that in prostate cancer cells ERs form a complex with the endothelial nitric oxide synthase (eNOS) and that in turn these complexes associate with chromatin in an estrogen-dependent fashion. Among these associations (peaks) we focused our attention on those proximal to the regulatory region of HOTAIR and MALAT1. These transcripts appeared regulated by estrogens and able to control ERs function by interacting with ERα/ERβ as indicated by RNA-ChIP. Further studies performed by ChIRP revealed that in unstimulated condition, HOTAIR and MALAT1 were present on pS2, hTERT and HOTAIR promoters at the ERE/eNOS peaks. Interestingly, upon treatment with17β-estradiol HOTAIR recruitment to chromatin increased significantly while that of MALAT1 was reduced, suggesting an opposite regulation and function for these lncRNAs. Similar results were obtained in cells and in an ex vivo prostate organotypic slice cultures. Overall, our data provide evidence of a crosstalk between lncRNAs, estrogens and estrogen receptors in prostate cancer with important consequences on gene expression regulation.

Estrogen-Dependent Dynamic Profile of eNOS-DNA Associations in Prostate Cancer

In previous work we have documented the nuclear translocation of endothelial NOS (eNOS) and its participation in combinatorial complexes with Estrogen Receptor Beta (ERβ) and Hypoxia Inducible Factors (HIFs) that determine localized chromatin remodeling in response to estrogen (E2) and hypoxia stimuli, resulting in transcriptional regulation of genes associated with adverse prognosis in prostate cancer (PCa). To explore the role of nuclear eNOS in the acquisition of aggressive phenotype in PCa, we performed ChIP-Sequencing on chromatin-associated eNOS from cells from a primary tumor with poor outcome and from metastatic LNCaP cells. We found that: 1. the eNOS-bound regions (peaks) are widely distributed across the genome encompassing multiple transcription factors binding sites, including Estrogen Response Elements. 2. E2 increased the number of peaks, indicating hormone-dependent eNOS re-localization. 3. Peak distribution was similar with/without E2 with ≈ 55% of them in extragenic DNA regions and an intriguing involvement of the 5′ domain of several miRs deregulated in PCa. Numerous potentially novel eNOS-targeted genes have been identified suggesting that eNOS participates in the regulation of large gene sets. The parallel finding of downregulation of a cluster of miRs, including miR-34a, in PCa cells associated with poor outcome led us to unveil a molecular link between eNOS and SIRT1, an epigenetic regulator of aging and tumorigenicity, negatively regulated by miR-34a and in turn activating eNOS. E2 potentiates miR-34a downregulation thus enhancing SIRT1 expression, depicting a novel eNOS/SIRT1 interplay fine-tuned by E2-activated ER signaling, and suggesting that eNOS may play an important role in aggressive PCa.

The nuclear pore protein Nup153 associates with chromatin and regulates cardiac gene expression in dystrophic mdx hearts

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-Dmd mdx /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 Ca v 1.2 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 activation of genes potentially associated with early dystrophic cardiac remodelling.

Anacardic acid and thyroid hormone enhance cardiomyocytes production from undifferentiated mouse ES cells along functionally distinct pathways

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 (T3) 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 role of nuclear endothelial nitric oxide synthase in the endothelial and prostate microenvironments

Abstract This review is based on novel observations from our laboratory on the nuclear translocation and functional role of endothelial nitric oxide synthase (eNOS) in endothelial and prostate cancer (PCa) epithelial cells. Nitric oxide (NO), the product of eNOS, is a free radical involved in the physiology and pathophysiology of living organisms and in a variety of biological processes including the maintenance of vascular homeostasis. Of relevance in this context is the role that estrogens play in the apoptotic process and the migration of endothelial cells through the regulation of target genes such as eNOS itself. It has been shown that both estrogen and NO signaling, mediated respectively by the estrogen receptors (ERs) and eNOS, can strongly counteract endothelial senescence through a common effector, the catalytic subunit of human telomerase. Therefore, this protein has been identified as a key molecule in the aging process which, intriguingly, is considered the only risk factor in the development of PCa and one of the major determinants of cardiovascular diseases. Indeed, in both these contexts we have defined a molecular mechanism involving activation of eNOS and hypoxia-inducible factors in association with ERβ that characterizes the most aggressive form of PCa or influences endothelial cell differentiation. Altogether these data led us to postulate that activation of eNOS is a crucial requirement for the delaying of endothelial senescence as well as for the acquisition of androgen-independence and for tumor progression in the prostate microenvironment.

Nucleoporin 153 regulates estrogen-dependent nuclear translocation of endothelial nitric oxide synthase and estrogen receptor beta in prostate cancer

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 ERβ as compared to normal/hyperplastic prostate epithelial cells. This phenomenon was enhanced in the presence of 17β-estradiol (E2, 10-7M). Further experiments revealed that eNOS and ERβ 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 ERβ nuclear localization was lost upon siNup 153 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 ERβ 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.

Zeb1-Hdac2-eNOS circuitry identifies early cardiovascular precursors in naive mouse embryonic stem cells

Nitric oxide (NO) synthesis is a late event during differentiation of mouse embryonic stem cells (mESC) and occurs after release from serum and leukemia inhibitory factor (LIF). Here we show that after release from pluripotency, a subpopulation of mESC, kept in the naive state by 2i/LIF, expresses endothelial nitric oxide synthase (eNOS) and endogenously synthesizes NO. This eNOS/NO-positive subpopulation (ESNO+) expresses mesendodermal markers and is more efficient in the generation of cardiovascular precursors than eNOS/NO-negative cells. Mechanistically, production of endogenous NO triggers rapid Hdac2 S-nitrosylation, which reduces association of Hdac2 with the transcriptional repression factor Zeb1, allowing mesendodermal gene expression. In conclusion, our results suggest that the interaction between Zeb1, Hdac2, and eNOS is required for early mesendodermal differentiation of naive mESC.The production of nitric oxide (NO) is required for early stage embryo implantation into the uterus. Here the authors show that during differentiation of naive mouse ESCs, early production of endogenous NO leads to a mesendoderm differentiation commitment pathway by inhibiting the action of the transcriptional repressor Zeb1.

GSK3β Modulates Timing-Dependent Long-Term Depression Through Direct Phosphorylation of Kv4.2 Channels

Spike timing-dependent plasticity (STDP) is a form of activity-dependent remodeling of synaptic strength that underlies memory formation. Despite its key role in dictating learning rules in the brain circuits, the molecular mechanisms mediating STDP are still poorly understood. Here, we show that spike timing-dependent long-term depression (tLTD) and A-type K+ currents are modulated by pharmacological agents affecting the levels of active glycogen-synthase kinase 3 (GSK3) and by GSK3β knockdown in layer 2/3 of the mouse somatosensory cortex. Moreover, the blockade of A-type K+ currents mimics the effects of GSK3 up-regulation on tLTD and occludes further changes in synaptic strength. Pharmacological, immunohistochemical and biochemical experiments revealed that GSK3β influence over tLTD induction is mediated by direct phosphorylation at Ser-616 of the Kv4.2 subunit, a molecular determinant of A-type K+ currents. Collectively, these results identify the functional interaction between GSK3β and Kv4.2 channel as a novel mechanism for tLTD modulation providing exciting insight into the understanding of GSK3β role in synaptic plasticity.