Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives.

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

Development of Stable Amino-Pyrimidine-Curcumin Analogs: Synthesis, Equilibria in Solution, and Potential Anti-Proliferative Activity.

With the clear need for better cancer treatment, naturally occurring molecules represent a powerful inspiration. Recently, curcumin has attracted attention for its pleiotropic anticancer activity in vitro, especially against colorectal and prostate cancer cells. Unfortunately, these encouraging results were disappointing in vivo due to curcumin's low stability and poor bioavailability. To overcome these issues, herein, the synthesis of eight new pyrimidine-curcumin derivatives is reported. The compounds were fully characterized (1H/13C NMR (Nuclear Magnetic Resonance), LC-MS (Liquid Chromatography-Mass Spectrometri), UV-Vis spectroscopy), particularly their acid/base behavior; overall protonation constants were estimated, and species distribution, as a function of pH, was predicted, suggesting that all the compounds are in their neutral form at pH 7.4. All the compounds were extremely stable in simulated physiological media (phosphate-buffered saline and simulated plasma). The compounds were tested in vitro (48 h incubation treatment) to assess their effect on cell viability in prostate cancer (LNCaP and PC3) and colorectal cancer (HT29 and HCT116) cell lines. Two compounds showed the same anti-proliferative activity as curcumin against HCT116 cells and improved cytotoxicity against PC3 cells.

BS148 Reduces the Aggressiveness of Metastatic Melanoma via Sigma-2 Receptor Targeting.

The management of advanced-stage melanoma is clinically challenging, mainly because of its resistance to the currently available therapies. Therefore, it is important to develop alternative therapeutic strategies. The sigma-2 receptor (S2R) is overexpressed in proliferating tumor cells and represents a promising vulnerability to target. Indeed, we have recently identified a potent S2R modulator (BS148) that is effective in melanoma. To elucidate its mechanism of action, we designed and synthesized a BS148 fluorescent probe that enters SK-MEL-2 melanoma cells as assessed using confocal microscopy analysis. We show that S2R knockdown significantly reduces the anti-proliferative effect induced by BS148 administration, indicating the engagement of S2R in BS148-mediated cytotoxicity. Interestingly, BS148 treatment showed similar molecular effects to S2R RNA interference-mediated knockdown. We demonstrate that BS148 administration activates the endoplasmic reticulum stress response through the upregulation of protein kinase R-like ER kinase (PERK), activating transcription factor 4 (ATF4) genes, and C/EBP homologous protein (CHOP). Furthermore, we show that BS148 treatment downregulates genes related to the cholesterol pathway and activates the MAPK signaling pathway. Finally, we translate our results into patient-derived xenograft (PDX) cells, proving that BS148 treatment reduces melanoma cell viability and migration. These results demonstrate that BS148 is able to inhibit metastatic melanoma cell proliferation and migration through its interaction with the S2R and confirm its role as a promising target to treat cancer.

Histone Marks-Dependent Effect on Alternative Splicing: New Perspectives for Targeted Splicing Modulation in Cancer?

Alternative splicing (AS) is a tightly regulated mechanism that generates the complex human proteome from a small number of genes. Cis-regulatory RNA motifs in exons and introns control AS, recruiting positive and negative trans-acting splicing regulators. At a higher level, chromatin affects splicing events. Growing evidence indicates that the popular histone code hypothesis can be extended to RNA-level processes, such as AS. In addition to nucleosome positioning, which can generate transcriptional barriers to shape the final splicing outcome, histone post-translational modifications can contribute to the detailed regulation of single exon inclusion/exclusion. A histone-based system can identify alternatively spliced chromatin stretches, affecting RNAPII elongation locally or recruiting splicing components via adaptor complexes. In tumor cells, several mechanisms trigger misregulated AS events and produce cancer-associated transcripts. On a genome-wide level, aberrant AS can be the consequence of dysfunctional epigenetic splicing code, including altered enrichment in histone post-translational modifications. This review describes the main findings related to the effect of histone modifications and variants on splicing outcome and how a dysfunctional epigenetic splicing code triggers aberrant AS in cancer. In addition, it highlights recent advances in programmable DNA-targeting technologies and their possible application for AS targeted epigenetic modulation.

Inhibitors of histone deacetylase 6 based on a novel 3-hydroxy-isoxazole zinc binding group.

Histone deacetylase 6 (HDAC6) is an established drug target for cancer treatment. Inhibitors of HDAC6 based on a hydroxamic acid zinc binding group (ZBG) are often associated with undesirable side effects. Herein, we describe the identification of HDAC6 inhibitors based on a completely new 3-hydroxy-isoxazole ZBG. A series of derivatives decorated with different aromatic or heteroaromatic linkers, and various cap groups were synthesised and biologically tested. In vitro tests demonstrated that some compounds are able to inhibit HDAC6 with good potency, the best candidate reaching an IC50 of 700 nM. Such good potency obtained with a completely new ZBG make these compounds particularly attractive. The effect of the most active inhibitors on the acetylation levels of histone H3 and α- tubulin and their anti-proliferative activity of DU145 cells were also investigated. Docking studies were performed to evaluate the binding mode of these new derivatives and discuss structure-activity relationships.

The 1,10-Phenanthroline Ligand Enhances the Antiproliferative Activity of DNA-Intercalating Thiourea-Pd(II) and -Pt(II) Complexes Against Cisplatin-Sensitive and -Resistant Human Ovarian Cancer Cell Lines.

Ovarian cancer is the most lethal gynecological malignancy, often because of the frequent insurgence of chemoresistance to the drugs currently used. Thus, new therapeutical agents are needed. We tested the toxicity of 16 new DNA-intercalating agents to cisplatin (cDDP)-sensitive human ovarian carcinoma cell lines and their resistant counterparts. The compounds were the complexes of Pt(II) or Pd(II) with bipyridyl (bipy) and phenanthrolyl (phen) and with four different thiourea ancillary ligands. Within each of the four series of complexes characterized by the same thiourea ligand, the Pd(phen) drugs invariably showed the highest anti-proliferative efficacy. This paralleled both a higher intracellular drug accumulation and a more efficient DNA intercalation than all the other metal-bidentate ligand combinations. The consequent inhibition of topoisomerase II activity led to the greatest inhibition of DNA metabolism, evidenced by the inhibition of the expression of the folate cycle enzymes and a marked perturbation of cell-cycle distribution in both cell lines. These findings indicate that the particular interaction of Pd(II) with phenanthroline confers the best pharmacokinetic and pharmacodynamic properties that make this class of DNA intercalators remarkable inhibitors, even of the resistant cell growth.

Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy.

The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases. Stem Cells 2017;35:725-738.

Curcumin derivatives and Aß-fibrillar aggregates: An interactions' study for diagnostic/therapeutic purposes in neurodegenerative diseases.

Several neurodegenerative diseases, like Alzheimer's (AD), are characterized by amyloid fibrillar deposition of misfolded proteins, and this feature can be exploited for both diagnosis and therapy design. In this paper, structural modifications of curcumin scaffold were examined in order to improve its bioavailability and stability in physiological conditions, as well as its ability to interfere with ß-amyloid fibrils and aggregates. The acid-base behaviour of curcumin derivatives, their pharmacokinetic stability in physiological conditions, and in vitro ability to interfere with Aß fibrils at different incubation time were investigated. The mechanisms governing these phenomena have been studied at atomic level by means of molecular docking and dynamic simulations. Finally, biological activity of selected curcuminoids has been investigated in vitro to evaluate their safety and efficiency in oxidative stress protection on hippocampal HT-22 mouse cells. Two aromatic rings, π-conjugated structure and H-donor/acceptor substituents on the aromatic rings showed to be the sine qua non structural features to provide interaction and disaggregation activity even at very low incubation time (2h). Computational simulations proved that upon binding the ligands modify the conformational dynamics and/or interact with the amyloidogenic region of the protofibril facilitating disaggregation. Significantly, in vitro results on hippocampal cells pointed out protection against glutamate toxicity and safety when administered at low concentrations (1 µM). On the overall, in view of its higher stability in physiological conditions with respect to curcumin, of his rapid binding to fibrillar aggregates and strong depolymerizing activity, phtalimmide derivative K2F21 appeared a good candidate for both AD diagnostic and therapeutic purposes.

Potent Anti-Cancer Properties of Phthalimide-Based Curcumin Derivatives on Prostate Tumor Cells.

Metastatic castration-resistant prostate cancer is commonly treated with chemotherapy, whose effect is less than satisfactory. This raised the need for novel agents for the treatment of prostate cancer. In the present study, five phthalimide-based curcumin derivatives were synthesized and completely characterized to assess improved stability, pharmacodynamics, and radical scavenging ability. To investigate the potential application in anti-cancer therapy, the anti-proliferative activity of the synthesized molecules was determined on aggressive prostate tumor cells. We demonstrated that the K3F21 derivative has increased potency compared to curcumin, in terms of GI50, anti-proliferative and anti-migrating activities. K3F21 inhibits anchorage-dependent and -independent growth of prostate cancer cells by altering the expression of key genes controlling cell proliferation, such as Cylins D1, B1 and B2, and apoptosis, among which Puma, Noxa, and Bcl-2 family members. Finally, the anti-cancer activity of K3F21 was demonstrated by the analysis of cancer-associated PI3K/AKT, ERK, and p38 signaling pathways.

NF-YA splice variants have different roles on muscle differentiation.

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

Direct non transcriptional role of NF-Y in DNA replication.

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors.

NF-Y, a trimeric transcription factor (TF) composed of two histone-like subunits (NF-YB and NF-YC) and a sequence-specific subunit (NF-YA), binds to the CCAAT motif, a common promoter element. Genome-wide mapping reveals 5000-15,000 NF-Y binding sites depending on the cell type, with the NF-YA and NF-YB subunits binding asymmetrically with respect to the CCAAT motif. Despite being characterized as a proximal promoter TF, only 25% of NF-Y sites map to promoters. A comparable number of NF-Y sites are located at enhancers, many of which are tissue specific, and nearly half of the NF-Y sites are in select subclasses of HERV LTR repeats. Unlike most TFs, NF-Y can access its target DNA motif in inactive (nonmodified) or polycomb-repressed chromatin domains. Unexpectedly, NF-Y extensively colocalizes with FOS in all genomic contexts, and this often occurs in the absence of JUN and the AP-1 motif. NF-Y also coassociates with a select cluster of growth-controlling and oncogenic TFs, consistent with the abundance of CCAAT motifs in the promoters of genes overexpressed in cancer. Interestingly, NF-Y and several growth-controlling TFs bind in a stereo-specific manner, suggesting a mechanism for cooperative action at promoters and enhancers. Our results indicate that NF-Y is not merely a commonly used proximal promoter TF, but rather performs a more diverse set of biological functions, many of which are likely to involve coassociation with FOS.

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells.

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

The NF-Y/p53 liaison: well beyond repression.

NF-Y is a sequence-specific transcription factor - TF - targeting the common CCAAT promoter element. p53 is a master TF controlling the response to stress signals endangering genome integrity, often mutated in human cancers. The NF-Y/p53 - and p63, p73 - interaction results in transcriptional repression of a subset of genes within the vast NF-Y regulome under DNA-damage conditions. Recent data shows that NF-Y is also involved in pro-apoptotic activities, either directly, by mediating p53 transcriptional activation, or indirectly, by being targeted by a non coding RNA, PANDA. The picture is subverted in cells carrying Gain-of-function mutant p53, through interactions with TopBP1, a protein also involved in DNA repair and replication. In summary, the connection between p53 and NF-Y is crucial in determining cell survival or death.

Specific inhibition of NF-Y subunits triggers different cell proliferation defects.

Regulated gene expression is essential for a proper progression through the cell cycle. The transcription factor NF-Y has a fundamental function in transcriptional regulation of cell cycle genes, particularly of G2/M genes. In order to investigate common and distinct functions of NF-Y subunits in cell cycle regulation, NF-YA, NF-YB and NF-YC have been silenced by shRNAs in HCT116 cells. NF-YA loss led to a delay in S-phase progression, DNA damage and apoptosis: we showed the activation of the replication checkpoint, through the recruitment of Δp53 and of the replication proteins PCNA and Mcm7 to chromatin. Differently, NF-YB depletion impaired cells from exiting G2/M, but did not interfere with S-phase progression. Gene expression analysis of NF-YA and NF-YB inactivated cells highlighted a common set of hit genes, as well as a plethora of uncommon genes, unveiling a different effect of NF-Y subunits loss on NF-Y binding to its target genes. Chromatin extracts and ChIP analysis showed that NF-YA depletion was more effective than NF-YB in hitting NF-Y recruitment to CCAAT-promoters. Our data suggest a critical role of NF-Y expression, highlighting that the lack of the single subunits are differently perceived by the cells, which activate diverse cell cycle blocks and signaling pathways.

Nuclear Estrogen Receptors in Prostate Cancer: From Genes to Function.

Estrogens are almost ubiquitous steroid hormones that are essential for development, metabolism, and reproduction. They exert both genomic and non-genomic action through two nuclear receptors (ERα and ERß), which are transcription factors with disregulated functions and/or expression in pathological processes. In the 1990s, the discovery of an additional membrane estrogen G-protein-coupled receptor augmented the complexity of this picture. Increasing evidence elucidating the specific molecular mechanisms of action and opposing effects of ERα and Erß was reported in the context of prostate cancer treatment, where these issues are increasingly investigated. Although new approaches improved the efficacy of clinical therapies thanks to the development of new molecules targeting specifically estrogen receptors and used in combination with immunotherapy, more efforts are needed to overcome the main drawbacks, and resistance events will be a challenge in the coming years. This review summarizes the state-of-the-art on ERα and ERß mechanisms of action in prostate cancer and promising future therapies.

Discovery of potent pyrrolo-pyrimidine and purine HDAC inhibitors for the treatment of advanced prostate cancer.

The development of drugs for the treatment of advanced prostate cancer (PCA) remains a challenging task. In this study we have designed, synthesized and tested twenty-nine novel HDAC inhibitors based on three different zinc binding groups (trifluoromethyloxadiazole, hydroxamic acid, and 2-mercaptoacetamide). These warheads were conveniently tethered to variously substituted phenyl linkers and decorated with differently substituted pyrrolo-pyrimidine and purine cap groups. Remarkably, most of the compounds showed nanomolar inhibitory activity against HDAC6. To provide structural insights into the Structure-Activity Relationships (SAR) of the investigated compounds, docking of representative inhibitors and molecular dynamics of HDAC6-inhibitor complexes were performed. Compounds of the trifluoromethyloxadiazole and hydroxamic acid series exhibited promising anti-proliferative activities, HDAC6 targeting in PCA cells, and in vitro tumor selectivity. Representative compounds of the two series were tested for solubility, cell permeability and metabolic stability, demonstrating favorable in vitro drug-like properties. The more interesting compounds were subjected to migration assays, which revealed that compound 13 and, to a lesser extent, compound 15 inhibited the invasive behaviour of androgen-sensitive and -insensitive advanced prostate cancer cells. Compound 13 was profiled against all HDACs and found to inhibit all members of class II HDACs (except for HDAC10) and to be selective with respect to class I and class IV HDACs. Overall, compound 13 combines potent inhibitory activity and class II selectivity with favorable drug-like properties, an excellent anti-proliferative activity and marked anti-migration properties on PCA cells, making it an excellent lead candidate for further optimization.

Corrigendum: Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

[This corrects the article DOI: 10.3389/fmolb.2023.1130183.].

Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

Skeletal muscle is a highly adaptive organ that sustains continuous metabolic changes in response to different functional demands. Healthy skeletal muscle can adjust fuel utilization to the intensity of muscle activity, the availability of nutrients and the intrinsic characteristics of muscle fibers. This property is defined as metabolic flexibility. Importantly, impaired metabolic flexibility has been associated with, and likely contributes to the onset and progression of numerous pathologies, including sarcopenia and type 2 diabetes. Numerous studies involving genetic and pharmacological manipulations of histone deacetylases (HDACs) in vitro and in vivo have elucidated their multiple functions in regulating adult skeletal muscle metabolism and adaptation. Here, we briefly review HDAC classification and skeletal muscle metabolism in physiological conditions and upon metabolic stimuli. We then discuss HDAC functions in regulating skeletal muscle metabolism at baseline and following exercise. Finally, we give an overview of the literature regarding the activity of HDACs in skeletal muscle aging and their potential as therapeutic targets for the treatment of insulin resistance.

The NF-Y splicing signature controls hybrid EMT and ECM-related pathways to promote aggressiveness of colon cancer.

Aberrant splicing events are associated with colorectal cancer (CRC) and provide new opportunities for tumor diagnosis and treatment. The expression of the splice variants of NF-YA, the DNA binding subunit of the transcription factor NF-Y, is deregulated in multiple cancer types compared to healthy tissues. NF-YAs and NF-YAl isoforms differ in the transactivation domain, which may result in distinct transcriptional programs. In this study, we demonstrated that the NF-YAl transcript is higher in aggressive mesenchymal CRCs and predicts shorter patients' survival. In 2D and 3D conditions, CRC cells overexpressing NF-YAl (NF-YAlhigh) exhibit reduced cell proliferation, rapid single cell amoeboid-like migration, and form irregular spheroids with poor cell-to-cell adhesion. Compared to NF-YAshigh, NF-YAlhigh cells show changes in the transcription of genes involved in epithelial-mesenchymal transition, extracellular matrix and cell adhesion. NF-YAl and NF-YAs bind similarly to the promoter of the E-cadherin gene, but oppositely regulate its transcription. The increased metastatic potential of NF-YAlhigh cells in vivo was confirmed in zebrafish xenografts. These results suggest that the NF-YAl splice variant could be a new CRC prognostic factor and that splice-switching strategies may reduce metastatic CRC progression.