Glycine-induced activation of GPR158 increases the intrinsic excitability of medium spiny neurons in the nucleus accumbens.

It has been recently established that GPR158, a class C orphan G protein-coupled receptor, serves as a metabotropic glycine receptor. GPR158 is highly expressed in the nucleus accumbens (NAc), a major input structure of the basal ganglia that integrates information from cortical and subcortical structures to mediate goal-directed behaviors. However, whether glycine modulates neuronal activity in the NAc through GPR158 activation has not been investigated yet. Using whole-cell patch-clamp recordings, we found that glycine-dependent activation of GPR158 increased the firing rate of NAc medium spiny neurons (MSNs) while it failed to significantly affect the excitability of cholinergic interneurons (CIN). In MSNs GPR158 activation reduced the latency to fire, increased the action potential half-width, and reduced action potential afterhyperpolarization, effects that are all consistent with negative modulation of potassium M-currents, that in the central nervous system are mainly carried out by Kv7/KCNQ-channels. Indeed, we found that the GPR158-induced increase in MSN excitability was associated with decreased M-current amplitude, and selective pharmacological inhibition of the M-current mimicked and occluded the effects of GPR158 activation. In addition, when the protein kinase A (PKA) or extracellular signal-regulated kinase (ERK) signaling was pharmacologically blocked, modulation of MSN excitability by GPR158 activation was suppressed. Moreover, GPR158 activation increased the phosphorylation of ERK and Kv7.2 serine residues. Collectively, our findings suggest that GPR158/PKA/ERK signaling controls MSN excitability via Kv7.2 modulation. Glycine-dependent activation of GPR158 may significantly affect MSN firing in vivo, thus potentially mediating specific aspects of goal-induced behaviors.

Targeting of H19/cell adhesion molecules circuitry by GSK-J4 epidrug inhibits metastatic progression in prostate cancer.

BACKGROUND: About 30% of Prostate cancer (PCa) patients progress to metastatic PCa that remains largely incurable. This evidence underlines the need for the development of innovative therapies. In this direction, the potential research focus might be on long non-coding RNAs (lncRNAs) like H19, which serve critical biological functions and show significant dysregulation in cancer. Previously, we showed a transcriptional down-regulation of H19 under combined pro-tumoral estrogen and hypoxia treatment in PCa cells that, in turn, induced both E-cadherin and ß4 integrin expression. H19, indeed, acts as transcriptional repressor of cell adhesion molecules affecting the PCa metastatic properties. Here, we investigated the role of H19/cell adhesion molecules circuitry on in vivo PCa experimental tumor growth and metastatic dissemination models. METHODS: H19 was silenced in luciferase-positive PC-3 and 22Rv1 cells and in vitro effect was evaluated by gene expression, proliferation and invasion assays before and after treatment with the histone lysine demethylase inhibitor, GSK-J4. In vivo tumor growth and metastasis dissemination, in the presence or absence of GSK-J4, were analyzed in two models of human tumor in immunodeficient mice by in vivo bioluminescent imaging and immunohistochemistry (IHC) on explanted tissues. Organotypic Slice Cultures (OSCs) from fresh PCa-explant were used as ex vivo model to test GSK-J4 effects. RESULTS: H19 silencing in both PC-3 and 22Rv1 cells increased: i) E-cadherin and ß4 integrin expression as well as proliferation and invasion, ii) in vivo tumor growth, and iii) metastasis formation at bone, lung, and liver. Of note, treatment with GSK-J4 reduced lesions. In parallel, GSK-J4 efficiently induced cell death in PCa-derived OSCs. CONCLUSIONS: Our findings underscore the potential of the H19/cell adhesion molecules circuitry as a targeted approach in PCa treatment. Modulating this interaction has proven effective in inhibiting tumor growth and metastasis, presenting a logical foundation for targeted therapy.

Activation of histamine type 2 receptors enhances intrinsic excitability of medium spiny neurons in the nucleus accumbens.

Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole-cell patch-clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half-width. The increased excitability was protein kinase A-dependent and associated with decreased A-type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A-type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R-dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal-induced behaviours. KEY POINTS: Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole-brain activity including the nucleus accumbens. Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A. Whole-cell patch-clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A-dependent mechanisms. HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after-hyperpolarization and increased the action potential half-width. HR2 activation also reduced A-type potassium current. Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.

Acute restraint stress impairs histamine type 2 receptor ability to increase the excitability of medium spiny neurons in the nucleus accumbens.

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 histamine-containing 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.

Evidence for Biological Age Acceleration and Telomere Shortening in COVID-19 Survivors.

The SARS-CoV-2 infection determines the COVID-19 syndrome characterized, in the worst cases, by severe respiratory distress, pulmonary and cardiac fibrosis, inflammatory cytokine release, and immunosuppression. This condition has led to the death of about 2.15% of the total infected world population so far. Among survivors, the presence of the so-called persistent post-COVID-19 syndrome (PPCS) is a common finding. In COVID-19 survivors, PPCS presents one or more symptoms: fatigue, dyspnea, memory loss, sleep disorders, and difficulty concentrating. In this study, a cohort of 117 COVID-19 survivors (post-COVID-19) and 144 non-infected volunteers (COVID-19-free) was analyzed using pyrosequencing of defined CpG islands previously identified as suitable for biological age determination. The results show a consistent biological age increase in the post-COVID-19 population, determining a DeltaAge acceleration of 10.45 ± 7.29 years (+5.25 years above the range of normality) compared with 3.68 ± 8.17 years for the COVID-19-free population (p < 0.0001). A significant telomere shortening parallels this finding in the post-COVID-19 cohort compared with COVID-19-free subjects (p < 0.0001). Additionally, ACE2 expression was decreased in post-COVID-19 patients, compared with the COVID-19-free population, while DPP-4 did not change. In light of these observations, we hypothesize that some epigenetic alterations are associated with the post-COVID-19 condition, particularly in younger patients (< 60 years).

Stable Oxidative Cytosine Modifications Accumulate in Cardiac Mesenchymal Cells From Type2 Diabetes Patients: Rescue by α-Ketoglutarate and TET-TDG Functional Reactivation.

RATIONALE: Human cardiac mesenchymal cells (CMSCs) are a therapeutically relevant primary cell population. Diabetes mellitus compromises CMSC function as consequence of metabolic alterations and incorporation of stable epigenetic changes. OBJECTIVE: To investigate the role of α-ketoglutarate (αKG) in the epimetabolic control of DNA demethylation in CMSCs. METHODS AND RESULTS: Quantitative global analysis, methylated and hydroxymethylated DNA sequencing, and gene-specific GC methylation detection revealed an accumulation of 5-methylcytosine, 5-hydroxymethylcytosine, and 5-formylcytosine in the genomic DNA of human CMSCs isolated from diabetic donors. Whole heart genomic DNA analysis revealed iterative oxidative cytosine modification accumulation in mice exposed to high-fat diet (HFD), injected with streptozotocin, or both in combination (streptozotocin/HFD). In this context, untargeted and targeted metabolomics indicated an intracellular reduction of αKG synthesis in diabetic CMSCs and in the whole heart of HFD mice. This observation was paralleled by a compromised TDG (thymine DNA glycosylase) and TET1 (ten-eleven translocation protein 1) association and function with TET1 relocating out of the nucleus. Molecular dynamics and mutational analyses showed that αKG binds TDG on Arg275 providing an enzymatic allosteric activation. As a consequence, the enzyme significantly increased its capacity to remove G/T nucleotide mismatches or 5-formylcytosine. Accordingly, an exogenous source of αKG restored the DNA demethylation cycle by promoting TDG function, TET1 nuclear localization, and TET/TDG association. TDG inactivation by CRISPR/Cas9 knockout or TET/TDG siRNA knockdown induced 5-formylcytosine accumulation, thus partially mimicking the diabetic epigenetic landscape in cells of nondiabetic origin. The novel compound (S)-2-[(2,6-dichlorobenzoyl)amino]succinic acid (AA6), identified as an inhibitor of αKG dehydrogenase, increased the αKG level in diabetic CMSCs and in the heart of HFD and streptozotocin mice eliciting, in HFD, DNA demethylation, glucose uptake, and insulin response. CONCLUSIONS: Restoring the epimetabolic control of DNA demethylation cycle promises beneficial effects on cells compromised by environmental metabolic changes.

H19-Dependent Transcriptional Regulation of ß3 and ß4 Integrins Upon Estrogen and Hypoxia Favors Metastatic Potential in Prostate Cancer.

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 ß3 and ß4 integrins and E-cadherin upon H19 silencing or combined treatment. Importantly, H19 down-regulation and ß integrins induction were also observed in treated OSCs. Combined treatment increased both cell motility and invasion of PCa cells. Lastly, reduction of ß 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 ß integrin-mediated invasion.

A nitric oxide-dependent cross-talk between class I and III histone deacetylases accelerates skin repair.

In a mouse model of skin repair we found that the class I-IIa histone deacetylase inhibitor trichostatin A accelerated tissue regeneration. Unexpectedly, this effect was suppressed by Sirtinol, a class III histone deacetylase (HDAC) (sirtuin)-selective inhibitor. The role of sirtuins (SIRTs) was then investigated by using resveratrol and a novel SIRT1-2-3 activator, the MC2562 compound we synthesized recently. Both resveratrol and MC2562 were effective in accelerating wound repair. The local administration of natural or synthetic SIRT activators, in fact, significantly accelerated skin regeneration by increasing keratinocyte proliferation. In vitro experiments revealed that the activation of SIRTs stimulated keratinocyte proliferation via endothelial NO synthase phosphorylation and NO production. In this condition, the class I member HDAC2 was found S-nitrosylated on cysteine, a post-transduction modification associated with loss of activity and DNA binding capacity. After deacetylase inhibitor or SIRT activator treatment, ChIP showed, in fact, a significant HDAC2 detachment from the promoter region of insulin growth factor I (IGF-I), fibroblast growth factor 10 (FGF-10), and Epithelial Growth Factor (EGF), which may be the final recipients and effectors of the SIRT-NO-HDAC signaling cascade. Consistently, the effect of SIRT activators was reduced in the presence of NG-nitro-L-arginine methyl ester (L-NAME), a general inhibitor of NO synthesis. In conclusion, the NO-dependent cross-talk among class III and I histone deacetylases suggests an unprecedented signaling pathway important for skin repair.

Detrimental effect of class-selective histone deacetylase inhibitors during tissue regeneration following hindlimb ischemia.

Histone deacetylase inhibitors (DIs) are promising drugs for the treatment of several pathologies including ischemic and failing heart where they demonstrated efficacy. However, adverse side effects and cardiotoxicity have also been reported. Remarkably, no information is available about the effect of DIs during tissue regeneration following acute peripheral ischemia. In this study, mice made ischemic by femoral artery excision were injected with the DIs MS275 and MC1568, selective for class I and IIa histone deacetylases (HDACs), respectively. In untreated mice, soon after damage, class IIa HDAC phosphorylation and nuclear export occurred, paralleled by dystrophin and neuronal nitric-oxide synthase (nNOS) down-regulation and decreased protein phosphatase 2A activity. Between 14 and 21 days after ischemia, dystrophin and nNOS levels recovered, and class IIa HDACs relocalized to the nucleus. In this condition, the MC1568 compound increased the number of newly formed muscle fibers but delayed their terminal differentiation, whereas MS275 abolished the early onset of the regeneration process determining atrophy and fibrosis. The selective DIs had differential effects on the vascular compartment: MC1568 increased arteriogenesis whereas MS275 inhibited it. Capillarogenesis did not change. Chromatin immunoprecipitations revealed that class IIa HDAC complexes bind promoters of proliferation-associated genes and of class I HDAC1 and 2, highlighting a hierarchical control between class II and I HDACs during tissue regeneration. Our findings indicate that class-selective DIs interfere with normal mouse ischemic hindlimb regeneration and suggest that their use could be limited by alteration of the regeneration process in peripheral ischemic tissues.

Smad-interacting protein-1 and microRNA 200 family define a nitric oxide-dependent molecular circuitry involved in embryonic stem cell mesendoderm differentiation.

OBJECTIVE: Smad-interacting protein-1 (Sip1/ZEB2) is a transcriptional repressor of the telomerase reverse transcriptase catalytic subunit (Tert) and has recently been identified as a key regulator of embryonic cell fate with a phenotypic effect similar, in our opinion, to that reported for nitric oxide (NO). Remarkably, SIP1/ZEB2 is a known target of the microRNA 200 (miR-200) family. In this light, we postulated that Sip1/ZEB2 and the miR-200 family could play a role during the NO-dependent differentiation of mES. METHODS AND RESULTS: The results of the present study show that Sip1/ZEB2 expression is downregulated during the NO-dependent expression of mesendoderm and early cardiovascular precursor markers, including Flk1 and CXCR4 in mES. Coincidently, members of the miR-200 family, namely miR-429, -200a, -200b, and -200c, were transcriptionally induced in parallel to mouse Tert. This regulation occurred at the level of chromatin. Remarkably, miR-429/miR-200a overexpression or Sip1/ZEB2 knockdown by short hairpin RNA interference elicited a gene expression pattern similar to that of NO regardless of the presence of leukemia inhibitory factor. CONCLUSIONS: These results are the first demonstrating that the miR-200 family and Sip1/ZEB2 transcription factor are regulated by NO, indicating an unprecedented molecular circuitry important for telomerase regulation and early differentiation of mES.

MALAT1 as a Regulator of the Androgen-Dependent Choline Kinase A Gene in the Metabolic Rewiring of Prostate Cancer.

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, encompassing relevant AR binding sites. Combining of MALAT1 targeting with androgen treatment prevented MALAT1-dependent CHKA silencing in androgen-responsive (LNCaP) cells, while it did not in hormone-refractory cells (22RV1 cells). Moreover, AR nuclear translocation and its activation were detected by confocal microscopy analysis and ChIP upon MALAT1 targeting or androgen treatment. CONCLUSIONS: These findings support the role of MALAT1 as a CHKA activator through putative association with the liganded or unliganded AR, unveiling its targeting as a therapeutic option from a metabolic rewiring perspective.

Nitric oxide determines mesodermic differentiation of mouse embryonic stem cells by activating class IIa histone deacetylases: potential therapeutic implications in a mouse model of hindlimb ischemia.

In human endothelial cells, nitric oxide (NO) results in class IIa histone deacetylases (HDACs) activation and marked histone deacetylation. It is unknown whether similar epigenetic events occur in embryonic stem cells (ESC) exposed to NO and how this treatment could influence ESC therapeutic potential during tissue regeneration.This study reports that the NO-dependent class IIa HDACs subcellular localization and activity decreases the global acetylation level of H3 histones in ESC and that this phenomenon is associated with the inhibition of Oct4, Nanog, and KLF4 expression. Further, a NO-induced formation of macromolecular complexes including HDAC3, 4, 7, and protein phosphatase 2A (PP2A) have been detected. These processes correlated with the expression of the mesodermal-specific protein brachyury (Bry) and the appearance of several vascular and skeletal muscle differentiation markers. These events were abolished by the class IIa-specific inhibitor MC1568 and by HDAC4 or HDAC7 short interfering RNA (siRNA). The ability of NO to induce mesodermic/cardiovascular gene expression prompted us to evaluate the regenerative potential of these cells in a mouse model of hindlimb ischemia. We found that NO-treated ESCs injected into the cardiac left ventricle selectively localized in the ischemic hindlimb and contributed to the regeneration of muscular and vascular structures. These findings establish a key role for NO and class IIa HDACs modulation in ESC mesodermal commitment and enhanced regenerative potential in vivo.

Diagnostic and prognostic potential of the proteomic profiling of serum-derived extracellular vesicles in prostate cancer.

Extracellular vesicles (EVs) and their cargo represent an intriguing source of cancer biomarkers for developing robust and sensitive molecular tests by liquid biopsy. Prostate cancer (PCa) is still one of the most frequent and deadly tumor in men and analysis of EVs from biological fluids of PCa patients has proven the feasibility and the unprecedented potential of such an approach. Here, we exploited an antibody-based proteomic technology, i.e. the Reverse-Phase Protein microArrays (RPPA), to measure key antigens and activated signaling in EVs isolated from sera of PCa patients. Notably, we found tumor-specific protein profiles associated with clinical settings as well as candidate markers for EV-based tumor diagnosis. Among others, PD-L1, ERG, Integrin-ß5, Survivin, TGF-ß, phosphorylated-TSC2 as well as partners of the MAP-kinase and mTOR pathways emerged as differentially expressed endpoints in tumor-derived EVs. In addition, the retrospective analysis of EVs from a 15-year follow-up cohort generated a protein signature with prognostic significance. Our results confirm that serum-derived EV cargo may be exploited to improve the current diagnostic procedures while providing potential prognostic and predictive information. The approach proposed here has been already applied to tumor entities other than PCa, thus proving its value in translational medicine and paving the way to innovative, clinically meaningful tools.

Estrogen receptor-alpha and endothelial nitric oxide synthase nuclear complex regulates transcription of human telomerase.

We report that in endothelial cells, the angiogenic effect of 17beta-estradiol (E2) is inhibited by the estrogen receptor (ER) antagonist ICI or the NO synthase (NOS) inhibitor 7-nitroindazole via downregulation of hTERT, the telomerase catalytic subunit, suggesting that E2 and NO are involved in controlling hTERT transcription. Quantitative Real-Time PCR and chromatin immunoprecipitations in E2-treated human umbilical vein endothelial cells, showed recruitment of ERs on the hTERT promoter and concomitant enrichment in histone 3 methylation at Lysine 79, a modification associated with transcription-competent chromatin. Confocal microscopy and re-chromatin immunoprecipitations revealed that on E2 induction, endothelial (e)NOS rapidly localized into the nucleus and associated with ERalpha on the hTERT promoter. Transfections of a constitutively active eNOS mutant (S1177D) strongly induced the hTERT promoter, indicating a direct role of the protein in hTERT transcriptional regulation. Mutation of the estrogen response element in the promoter abolished response to both ERs and active eNOS, demonstrating that the estrogen response element integrity is required for hTERT regulation by these factors. To investigate this novel regulation in a reduced NO environment, pulmonary endothelial cells were isolated from eNOS(-/-) mice and grown with/without E2. In wild-type cells, E2 significantly increased telomerase activity. In eNOS(-/-) cells, basal telomerase activity was rescued by exogenous eNOS or an NO donor, whereas responsiveness to E2 demanded the active protein. In conclusion, we document the novel findings of a combinatorial eNOS/ERalpha complex at the hTERT estrogen response element site and that active eNOS and ligand-activated ERs cooperate in regulating hTERT expression in the endothelium.

Process Control Charts in Falls Prevention: The Experience of the Local Healthcare Authority of Romagna, Italy.

Patient safety is a great concern of healthcare institutions and the correct reporting and management of adverse events is a key element for supporting safety improvement efforts. Patient falls are the most frequent adverse event in hospitals and often cause serious patient outcomes. In this work, we describe the experience of the Local Healthcare Authority of Romagna, Italy that, within the framework of a quality and safety improvement programme, designed, developed and implemented a desktop application for monitoring inpatient falls. A multidisciplinary team created a software tool based on R, an open source software for statistical computing that, appropriately combined with the existing hospital information system, is used to obtain Shewhart u-control charts for monitoring the monthly fall rates. The tool had been implemented in twenty nine hospital units. The results indicate that the proposed application gave a valuable contribution in the safety improvement activities. Its usefulness extended beyond the "safety problem" as it also enabled hospital managers to identify a number of critical issues in data collection. As a result, where necessary, improvement actions had been implemented. Furthermore, the use of open source software led to a considerable cost reduction and facilitated customization of the software tool.

Signaling through estrogen receptors modulates long non-coding RNAs in prostate cancer.

Prostate cancer (PCa) is a sex-steroid hormone-dependent cancer in which estrogens play a critical role in both initiation and progression. Recently, several long non-coding RNAs (lncRNAs) have been associated with PCa and are supposedly playing a pivotal role in the biology and progression of this type of cancer. In this review, we focused on some lncRNAs that are known for their androgen and estrogen transcriptional responsiveness in PCa. Specifically, we summarized recent pieces of evidence about lncRNAs NEAT1, H19, MALAT1, and HOTAIR, in estrogen signaling, emphasizing their role in PCa progression and the acquisition of a castration-resistant phenotype. Here, the reader will find information about lncRNAs present in estrogen-dependent transcriptional complexes. The potential role of lncRNA/estrogen signaling as a novel pathway for PCa treatment will be discussed.

CPEB1 orchestrates a fine-tuning of miR-145-5p tumor-suppressive activity on TWIST1 translation in prostate cancer cells.

TWIST1 is a basic helix-loop-helix transcription factor, and one of the master Epithelial-to-Mesenchymal Transition (EMT) regulators. We show that tumor suppressor miR-145-5p controls TWIST1 expression in an immortalized prostate epithelial cell line and in a tumorigenic prostate cancer-derived cell line. Indeed, shRNA-mediated miR-145-5p silencing enhanced TWIST1 expression and induced EMT-associated malignant properties in these cells. However, we discovered that the translational inhibitory effect of miR-145-5p on TWIST1 is lost in 22Rv1, another prostate cancer cell line that intrinsically expresses high levels of the CPEB1 cytoplasmic polyadenylation element binding protein. This translational regulator typically reduces TWIST1 translation efficiency by shortening the TWIST1 mRNA polyA tail. However, our results indicate that the presence of CPEB1 also interferes with the binding of miR-145-5p to the TWIST1 mRNA 3'UTR. Mechanistically, CPEB1 binding to its first cognate site either directly hampers the access to the miR-145-5p response element or redirects the cleavage/polyadenylation machinery to an intermediate polyadenylation site, resulting in the elimination of the miR-145-5p binding site. Taken together, our data support the notion that the tumor suppressive activity of miR-145-5p on TWIST1 translation, consequently on EMT, self-renewal, and migration, depends on the CPEB1 expression status of the cancer cell. A preliminary prospective study using clinical samples suggests that reconsidering the relative status of miR-145-5p/TWIST1 and CPEB1 in the tumors of prostate cancer patients may bear prognostic value.

Metabolic Reprogramming by Malat1 Depletion in Prostate Cancer.

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.

Wild-type but not mutant androgen receptor inhibits expression of the hTERT telomerase subunit: a novel role of AR mutation for prostate cancer development.

Androgens play a central role in prostate development and prostate cancer proliferation. Induction of telomerase is an early event in prostate carcinogenesis and is considered as a marker for both primary tumors and metastases. Interestingly, several reports suggest that telomerase activity is regulated by androgens in vivo. Here, we show that the wild-type (WT) human androgen receptor (AR) inhibits the expression of the human telomerase reverse transcriptase (hTERT) and telomerase activity via inhibition of hTERT promoter activity in the presence of androgen receptor agonists. However, pure androgen antagonists failed to repress hTERT transcription. The androgen-mediated repression of hTERT is abrogated in a human prostate cancer cell line exhibiting hormone-dependent growth, which expresses a mutant AR (T877A) frequently occurring in prostate cancer. We reveal that this single amino acid exchange is sufficient for the lack of transrepression. Interestingly, chromatin immunoprecipitation data suggest that, in contrast to the WT AR, the mutant AR is recruited less efficiently to the hTERT promoter in vivo, indicating that loss of transrepression results from reduced chromatin recruitment. Thus, our findings suggest that the WT AR inhibits expression of hTERT, which is indicative of a protective mechanism, whereas the T877A mutation of AR not only broadens the ligand spectrum of the receptor but abrogates this inhibitory mechanism in prostate cancer cells. This novel role of AR mutations in prostate cancer development suggests the benefit to a search for new AR antagonists that inhibit transactivation but allow transrepression.

The Protein Arginine Methyltransferases 1 and 5 affect Myc properties in glioblastoma stem cells.

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.