RESUMEN
Access to DNA is the first level of control in regulating gene transcription, a control that is also critical for maintaining DNA integrity. Cellular senescence is characterized by profound transcriptional rearrangements and accumulation of DNA lesions. Here, we discovered an epigenetic complex between HDAC4 and HDAC1/HDAC2 that is involved in the erase of H2BK120 acetylation. The HDAC4/HDAC1/HDAC2 complex modulates the efficiency of DNA repair by homologous recombination, through dynamic deacetylation of H2BK120. Deficiency of HDAC4 leads to accumulation of H2BK120ac, impaired recruitment of BRCA1 and CtIP to the site of lesions, accumulation of damaged DNA and senescence. In senescent cells this complex is disassembled because of increased proteasomal degradation of HDAC4. Forced expression of HDAC4 during RAS-induced senescence reduces the genomic spread of γH2AX. It also affects H2BK120ac levels, which are increased in DNA-damaged regions that accumulate during RAS-induced senescence. In summary, degradation of HDAC4 during senescence causes the accumulation of damaged DNA and contributes to the activation of the transcriptional program controlled by super-enhancers that maintains senescence.
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Senescencia Celular , Daño del ADN , Histona Desacetilasa 1 , Histona Desacetilasa 2 , Histona Desacetilasas , Histonas , Senescencia Celular/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 1/genética , Humanos , Acetilación , Histonas/metabolismo , Histona Desacetilasas/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasa 2/metabolismo , Histona Desacetilasa 2/genética , Proteínas Represoras/metabolismo , Proteínas Represoras/genética , Reparación del ADN por Recombinación , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Línea CelularRESUMEN
In leiomyosarcoma class IIa HDACs (histone deacetylases) bind MEF2 and convert these transcription factors into repressors to sustain proliferation. Disruption of this complex with small molecules should antagonize cancer growth. NKL54, a PAOA (pimeloylanilide o-aminoanilide) derivative, binds a hydrophobic groove of MEF2, which is used as a docking site by class IIa HDACs. However, NKL54 could also act as HDAC inhibitor (HDACI). Therefore, it is unclear which activity is predominant. Here, we show that NKL54 and similar derivatives are unable to release MEF2 from binding to class IIa HDACs. Comparative transcriptomic analysis classifies these molecules as HDACIs strongly related to SAHA/vorinostat. Low expressed genes are upregulated by HDACIs, while abundant genes are repressed. This transcriptional resetting correlates with a reorganization of H3K27 acetylation around the transcription start site (TSS). Among the upregulated genes there are several BH3-only family members, thus explaining the induction of apoptosis. Moreover, NKL54 triggers the upregulation of MEF2 and the downregulation of class IIa HDACs. NKL54 also increases the binding of MEF2D to promoters of genes that are upregulated after treatment. In summary, although NKL54 cannot outcompete MEF2 from binding to class IIa HDACs, it supports MEF2-dependent transcription through several actions, including potentiation of chromatin binding.
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Inhibidores de Histona Desacetilasas , Transcriptoma , Acetilación , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Factores de Transcripción MEF2/genética , Vorinostat/farmacologíaRESUMEN
This study examines 8-hydroxyguanine (8-oxo-Gua) staining in placental tissue samples based on fetal size at birth as well as its relationships with placental histology and other pregnancy variables. This prospective cohort study included women > 18 years with a singleton pregnancy, a live fetus, fluency in Italian, and delivery at term. A total of 165 pregnancies were included in the study. The nuclear syncytiotrophoblast 8-oxo-Gua staining score in LGA was substantially greater than in late FGR (p < 0.05), although the cytoplasm score was lower in SGA and LGA than in AGA (p < 0.05). Furthermore, a sex-specific pattern of 8-oxo-Gua staining was discovered in single-term placentas, with more oxidative damage found in the nuclei of syncytiotrophoblast cells and stromal and endothelial cells in AGA males compared to AGA females (p < 0.05). Second, the histological pattern of late FGR placentae differed by gender. Finally, a significant correlation (p < 0.05) was found between high-intensity 8-oxo-Gua staining in the cytoplasm of syncytiotrophoblast cells and thrombi in the chorionic plate or villi in males. On the other hand, female fetuses demonstrated a significant connection (p < 0.05) between high-intensity 8-oxo-Gua staining in endothelial and stromal cells and high birthweight MoM values. Our findings indicated a significant variation in the oxidative stress pattern between male and female placentae, implying that fetal growth is regulated differently in the two sexes.
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Células Endoteliales , Placenta , Recién Nacido , Femenino , Embarazo , Masculino , Humanos , Estudios Prospectivos , Inmunohistoquímica , Células Endoteliales/patología , Retardo del Crecimiento Fetal/patología , Edad Gestacional , Desarrollo FetalRESUMEN
Transcriptional networks supervising class IIa HDAC expression are poorly defined. Here we demonstrate that MEF2D is the key factor controlling HDAC9 transcription. This control, which is part of a negative feed-back loop during muscle differentiation, is hijacked in cancer. In leiomyosarcomas the MEF2D/HDAC9 vicious circuit sustains proliferation and cell survival, through the repression of the death receptor FAS. Comprehensive genome-wide studies demonstrate that HDAC4 and HDAC9 control different genetic programs and show both specific and common genomic binding sites. Although the number of MEF2-target genes commonly regulated is similar, only HDAC4 represses many additional genes that are not MEF2D targets. As expected, HDAC4-/- and HDAC9-/- cells increase H3K27ac levels around the TSS of the respective repressed genes. However, these genes rarely show binding of the HDACs at their promoters. Frequently HDAC4 and HDAC9 bind intergenic regions. We demonstrate that these regions, recognized by MEF2D/HDAC4/HDAC9 repressive complexes, show the features of active enhancers. In these regions HDAC4 and HDAC9 can differentially influence H3K27 acetylation. Our studies describe new layers of class IIa HDACs regulation, including a dominant positional effect, and can contribute to explain the pleiotropic actions of MEF2 TFs.
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Histona Desacetilasas/genética , Leiomiosarcoma/genética , Proteínas Represoras/genética , Acetilación , Diferenciación Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Leiomiosarcoma/patología , Factores de Transcripción MEF2/genéticaRESUMEN
The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS.
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Histona Desacetilasas/biosíntesis , Leiomiosarcoma/genética , Proteínas Represoras/biosíntesis , Activación Transcripcional/genética , Carcinogénesis/genética , Línea Celular Tumoral , Núcleo Celular/genética , Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/genética , Humanos , Leiomiosarcoma/patología , Factores de Transcripción MEF2/biosíntesis , Factores de Transcripción MEF2/genética , Proteínas Represoras/genéticaRESUMEN
The myocyte enhancer factor 2 and histone deacetylase (MEF2-HDAC) axis is a master regulator of different developmental programs and adaptive responses in adults. In this paper, we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is upregulated during acini formation, which coincides with exit from the proliferative phase. Upregulation of the transcription of MEF2 proteins is coupled to downregulation of HDAC7, which occurs independently from changes in mRNA levels, and proteasome- or autophagy-mediated degradation. During acini formation, the MEF2-HDAC axis contributes to the promotion of cell cycle exit, through the engagement of the CDK inhibitor CDKN1A. Only in proliferating cells can HDAC7 bind to the first intron of the CDKN1A gene, a region characterized by epigenetic markers of active promoters and enhancers. In cells transformed by the oncogene HER2 (ERBB2), acini morphogenesis is altered, MEF2 transcription is repressed and HDAC7 is continuously expressed. Importantly, reactivation of MEF2 transcriptional activity in these cells, through the use of a HER2 inhibitor or by enhancing MEF2 function, corrected the proliferative defect and re-established normal acini morphogenesis.
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Células Acinares/metabolismo , Células Epiteliales/metabolismo , Histona Desacetilasas/metabolismo , Factores de Transcripción MEF2/metabolismo , Línea Celular , Proliferación Celular/genética , Proliferación Celular/fisiología , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente , Histona Desacetilasas/genética , Humanos , Immunoblotting , Factores de Transcripción MEF2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The prospect of intervening, through the use of a specific molecule, with a cellular alteration responsible for a disease, is a fundamental ambition of biomedical science. Epigenetic-based therapies appear as a remarkable opportunity to impact on several disorders, including cancer. Many efforts have been made to develop small molecules acting as inhibitors of histone deacetylases (HDACs). These enzymes are key targets to reset altered genetic programs and thus to restore normal cellular activities, including drug responsiveness. Several classes of HDAC inhibitors (HDACis) have been generated, characterized and, in certain cases, approved for the use in clinic. A new frontier is the generation of subtype-specific inhibitors, to increase selectivity and to manage general toxicity. Here we will discuss about a set of molecules, which can interfere with the activity of a specific subclass of HDACs: the class IIa.
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Enfermedad , Inhibidores de Histona Desacetilasas/uso terapéutico , Histona Desacetilasas/química , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Animales , Humanos , Neoplasias/enzimologíaRESUMEN
MEF2s transcription factors and class IIa HDACs compose a fundamental axis for several differentiation pathways. Functional relationships between this axis and cancer are largely unexplored. We have found that class IIa HDACs are heterogeneously expressed and display redundant activities in breast cancer cells. Applying gene set enrichment analysis to compare the expression profile of a list of putative MEF2 target genes, we have discovered a correlation between the down-regulation of the MEF2 signature and the aggressiveness of ER(+) breast tumors. Kaplan-Meier analysis in ER(+) breast tumors evidenced an association between increased class IIa HDACs expression and reduced survival. The important role of the MEF2-HDAC axis in ER(+) breast cancer was confirmed in cultured cells. MCF7 ER(+) cells were susceptible to silencing of class IIa HDACs in terms of both MEF2-dependent transcription and apoptosis. Conversely, in ER(-) MDA-MB-231 cells, the repressive influence of class IIa HDACs was dispensable. Similarly, a class IIa HDAC-specific inhibitor preferentially promoted the up-regulation of several MEF2 target genes and apoptosis in ER(+) cell lines. The prosurvival function of class IIa HDACs could be explained by the repression of NR4A1/Nur77, a proapoptotic MEF2 target. In summary, our studies underscore a contribution of class IIa HDACs to aggressiveness of ER(+) tumors.-Clocchiatti, A., Di Giorgio, E., Ingrao, S., Meyer-Almes, F.-J., Tripodo, C., Brancolini, C. Class IIa HDACs repressive activities on MEF2-depedent transcription are associated with poor prognosis of ER(+) breast tumors.
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Neoplasias de la Mama/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Histona Desacetilasas/biosíntesis , Factores Reguladores Miogénicos/metabolismo , Proteínas de Neoplasias/metabolismo , Apoptosis/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Femenino , Silenciador del Gen , Histona Desacetilasas/genética , Humanos , Factores Reguladores Miogénicos/genética , Proteínas de Neoplasias/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/biosíntesis , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Pronóstico , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Transcripción Genética/genéticaRESUMEN
Nitric oxide is a pleiotropic free radical produced by three nitric oxide synthases (NOS1-3), of which inducible NOS2 is involved in tumor initiation and progression. In this study, RNA-seq, ChIP-seq and qRT-PCR experiments combined with bioinformatic analyses showed that NRF2 is a repressor of NOS2 gene by maintaining a distal enhancer located 22 kb downstream of TSS in an inactive state. Deletion of NRF2 leads to activation of the enhancer, which exerts a pioneering function before it is fully activated. Specifically, NRF2 controls the expression of NOS2 in response to intracellular oxidative stress and extracellular oxygen pressure. We found that abrogation of NOS2 expression by siRNAs partially reduced the ability of WT Panc-1 cells to form 3D spheroids, but strongly reduced the formation of 3D spheroids by NRF2-depleted Panc-1 cells. Mechanistically, this effect correlates with the finding that NOS2 and nitric oxide stimulate epithelial-to-mesenchymal transition in NRF2-depleted Panc-1 and MIA PaCa-2 cells. We also found that knockdown of NOS2 leads to blockade of 3D matrigel invasion of NRF2-depleted PDAC cells, demonstrating that a short-circuit in the reciprocal regulation of NOS2 and NRF2 attenuates the malignancy of PDAC cells. In summary, we show for the first time that: (i) NRF2 is a suppressor of NOS2 in pancreatic cancer cells; (ii) NRF2 binds to and inactivates an enhancer located 22 kb downstream of TSS of the NOS2 gene; (iii) activation of NOS2 requires suppression of NRF2; (iv) NOS2 is required for NRF2-depleted Panc-1 cells to maintain their malignancy and invasiveness.
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Factor 2 Relacionado con NF-E2 , Neoplasias Pancreáticas , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
An important epigenetic switch marks the onset and maintenance of senescence. This allows transcription of the genetic programs that arrest the cell cycle and alter the microenvironment. Transcription of endogenous retroviruses (ERVs) is also a consequence of this epigenetic switch. In this manuscript, we have identified a group of ERVs that are epigenetically silenced in proliferating cells but are upregulated during replicative senescence or during various forms of oncogene-induced senescence, by RAS and Akt, or after HDAC4 depletion. In a HDAC4 model of senescence, removal of the repressive histone mark H3K27me3 is the plausible mechanism that allows the transcription of intergenic ERVs during senescence. We have shown that ERVs contribute to the accumulation of dsRNAs in senescence, which can initiate the antiviral response via the IFIH1-MAVS signaling pathway and thus contribute to the maintenance of senescence. This pathway, and MAVS in particular, plays an active role in shaping the microenvironment and maintaining growth arrest, two essential features of the senescence program.
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Retrovirus Endógenos , Histonas , Histonas/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Epigénesis Genética , Senescencia Celular/genética , AntiviralesRESUMEN
Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. PDOs derived from two patients with TP53 mutations were tested with two different HDAC inhibitors (SAHA and NKL54). Cell death induction, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and the downregulation of highly expressed genes. A similar differential effect is observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death, which is characterized by the upregulation of BH3-only genes BIK and BMF. Up-regulation of BIK is associated with increased accessibility in an intronic region that has enhancer properties. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In summary, our exploratory study indicates a mechanism of action for SAHA and demonstrate the low efficacy of NKL54 as a single agent for apoptosis induction, using two PDOs. These observations need to be validated in a larger cohort of PDOs.
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Neoplasias del Colon , Inhibidores de Histona Desacetilasas , Humanos , Inhibidores de Histona Desacetilasas/farmacología , Cromatina/genética , Ácidos Hidroxámicos/farmacología , Apoptosis/genética , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Línea Celular Tumoral , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Interaction of transcription factor myocyte enhancer factor-2 (MEF2) family members with class IIa histone deacetylases (HDACs) has been implicated in a wide variety of diseases. Though considerable knowledge on this topic has been accumulated over the years, a high resolution and detailed analysis of the binding mode of multiple class IIa HDAC derived peptides with MEF2D is still lacking. To fulfil this gap, we report here the crystal structure of MEF2D in complex with double strand DNA and four different class IIa HDAC derived peptides, namely HDAC4, HDAC5, HDAC7 and HDAC9. All class IIa HDAC derived peptides form extended amphipathic α-helix structures that fit snugly in the hydrophobic groove of MEF2D domain. Binding mode of class IIa HDAC derived peptides to MEF2D is very similar and occur primarily through nonpolar interactions mediated by highly conserved branched hydrophobic amino acids. Further studies revealed that class IIa HDAC derived peptides are unstructured in solution and appear to adopt a folded α-helix structure only upon binding to MEF2D. Comparison of our peptide-protein complexes with previously characterized structures of MEF2 bound to different co-activators and co-repressors, highlighted both differences and similarities, and revealed the adaptability of MEF2 in protein-protein interactions. The elucidation of the three-dimensional structure of MEF2D in complex with multiple class IIa HDAC derived peptides provide not only a better understanding of the molecular basis of their interactions but also have implications for the development of novel antagonist.
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ADN , Histona Desacetilasas , Factores de Transcripción MEF2 , Péptidos , Humanos , Secuencia de Aminoácidos , Cristalografía por Rayos X , ADN/metabolismo , ADN/química , Histona Desacetilasas/química , Histona Desacetilasas/metabolismo , Factores de Transcripción MEF2/química , Factores de Transcripción MEF2/metabolismo , Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Pliegue de ProteínaRESUMEN
Super-enhancers evolve as elements at the top of the hierarchical control of gene expression. They are important end-gatherers of signaling pathways that control stemness, differentiation or adaptive responses. Many epigenetic regulations focus on these regions, and not surprisingly, during the process of tumorigenesis, various alterations can account for their dysfunction. Super-enhancers are emerging as key drivers of the aberrant gene expression landscape that sustain the aggressiveness of cancer cells. In this review, we will describe and discuss about the structure of super-enhancers, their epigenetic regulation, and the major changes affecting their functionality in cancer.
RESUMEN
The refractoriness of tumor cells to apoptosis represents the main mechanism of resistance to chemotherapy. Smac/DIABLO mimetics proved to be effective in overcoming cancer-acquired resistance to apoptosis as a consequence of overexpression of the anti-apoptotic proteins XIAP, cIAP1, and cIAP2. In this work, we describe a dual-targeting peptide capable of selectively activating apoptosis in cancer cells. The complex consists of a fluorescent periodic mesoporous organosilica nanoparticle that carries the short sequences of Smac/DIABLO bound to the αvß3-integrin ligand. The dual-targeting peptide @PMO shows significantly higher toxicity in αvß3-positive HeLa cells with respect to αvß3-negative Ht29 cells. @PMO exhibited synergistic effects in combination with oxaliplatin in a panel of αvß3-positive cancer cells, while its toxicity is overcome by XIAP overexpression or integrin ß3 silencing. The successful uptake of the molecule by αvß3-positive cells makes @PMO promising for the re-sensitization to apoptosis of many cancer types.
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In pancreatic ductal adenocarcinomas (PDAC), the KRASG12D-NRF2 axis controls cellular functions such as redox homeostasis and metabolism. Disruption of this axis through suppression of NRF2 leads to profound reprogramming of metabolism. Unbiased transcriptome and metabolome analyses showed that PDAC cells with disrupted KRASG12D-NRF2 signaling (NRF2-/- cells) shift from aerobic glycolysis to metabolic pathways fed by amino acids. Metabolome, RNA-seq and qRT-PCR analyses revealed a blockade of the urea cycle, making NRF2-/- cells dependent on exogenous arginine for survival. Arginine is channeled into anabolic pathways, including the synthesis of phosphocreatine, which generates an energy buffer essential for cell growth. A similar switch was observed in tumor clones that had survived FOLFIRINOX therapy or blockade of KRAS signaling. Inhibition of the creatine pathway with cyclocreatine reduced both ATP and invasion rate in 3D spheroids from NRF2-deficient PDAC cells. Our study provides basis for the rational development of combination therapies for pancreatic cancer.
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Bi-directional transcription of Human Endogenous Retroviruses (hERVs) is a common feature of autoimmunity, neurodegeneration and cancer. Higher rates of cancer incidence, neurodegeneration and autoimmunity but a lower prevalence of autoimmune diseases characterize elderly people. Although the re-expression of hERVs is commonly observed in different cellular models of senescence as a result of the loss of their epigenetic transcriptional silencing, the hERVs modulation during aging is more complex, with a peak of activation in the sixties and a decline in the nineties. What is clearly accepted, instead, is the impact of the re-activation of dormant hERV on the maintenance of stemness and tissue self-renewing properties. An innate cellular immunity system, based on the RLR-MAVS circuit, controls the degradation of dsRNAs arising from the transcription of hERV elements, similarly to what happens for the accumulation of cytoplasmic DNA leading to the activation of cGAS/STING pathway. While agonists and inhibitors of the cGAS-STING pathway are considered promising immunomodulatory molecules, the effect of the RLR-MAVS pathway on innate immunity is still largely based on correlations and not on causality. Here we review the most recent evidence regarding the activation of MDA5-RIG1-MAVS pathway as a result of hERV de-repression during aging, immunosenescence, cancer and autoimmunity. We will also deal with the epigenetic mechanisms controlling hERV repression and with the strategies that can be adopted to modulate hERV expression in a therapeutic perspective. Finally, we will discuss if the RLR-MAVS signalling pathway actively modulates physiological and pathological conditions or if it is passively activated by them.
Asunto(s)
Retrovirus Endógenos , Neoplasias , Anciano , Envejecimiento , Retrovirus Endógenos/genética , Humanos , Inmunidad Innata , Neoplasias/genética , Transducción de Señal/fisiologíaRESUMEN
Cationic porphyrins bearing an alkyl side chain of 14 (2b) or 18 (2d) carbons dramatically inhibit proliferation of pancreatic cancer cells following treatment with light. We have compared two different ways of delivering porphyrin 2d: either in free form or engrafted into palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes (L-2d). Cell cytometry shows that while free 2d is taken up by pancreatic cancer cells by active (endocytosis) and passive (membrane fusion) transports, L-2d is internalized solely by endocytosis. Confocal microscopy showed that free 2d co-localizes with the cell membrane and lysosomes, whereas L-2d partly co-localizes with lysosomes and ER. It is found that free 2d inhibits the KRAS-Nrf2-GPX4 axis and strongly triggers lipid peroxidation, resulting in cell death by ferroptosis. By contrast, L-2d does not affect the KRAS-Nrf2-GPX4 axis and activates cell death mainly through apoptosis. Overall, our study demonstrates for the first time that cationic alkyl porphyrins, which have a IC50 ~ 23 nM, activate a dual mechanism of cell death, ferroptosis and apoptosis, where the predominant form depends on the delivery mode.
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Neoplasias Pancreáticas , Porfirinas , Apoptosis , Cationes , Humanos , Liposomas/química , Factor 2 Relacionado con NF-E2 , Neoplasias Pancreáticas/tratamiento farmacológico , Porfirinas/farmacología , Proteínas Proto-Oncogénicas p21(ras) , Neoplasias PancreáticasRESUMEN
BACKGROUND: Histone deacetylase 4 (HDAC4) is a stress-responsive factor that mediates multiple cellular responses. As a member of class IIa HDACs, HDAC4 shuttles between the nucleus and the cytoplasm; however, HDAC4 cytoplasmic functions have never been fully investigated. Duchenne muscular dystrophy (DMD) is a genetic, progressive, incurable disorder, characterized by muscle wasting, which can be treated with the unspecific inhibition of HDACs, despite this approach being only partially effective. More efficient strategies may be proposed for DMD only after the different HDAC members will be characterized. METHODS: To fully understand HDAC4 functions, we generated dystrophic mice carrying a skeletal muscle-specific deletion of HDAC4 (mdx;KO mice). The progression of muscular dystrophy was characterized in mdx and age-matched mdx;KO mice by means of histological, molecular, and functional analyses. Satellite cells (SCs) from these mice were differentiated in vitro, to identify HDAC4 intrinsic functions influencing the myogenic potential of dystrophic SCs. Gain-of-function experiments revealed the cytoplasmic functions of HDAC4 in mdx;KO muscles. RESULTS: Histone deacetylase 4 increased in the skeletal muscles of mdx mice (~3-fold; P < 0.05) and of DMD patients (n = 3, males, mean age 13.3 ± 1.5 years), suggesting that HDAC4 has a role in DMD. Its deletion in skeletal muscles importantly worsens the pathological features of DMD, leading to greater muscle fragility and degeneration over time. Additionally, it impairs SC survival, myogenic potential, and muscle regeneration, ultimately compromising muscle function (P < 0.05-0.001). The impaired membrane repair mechanism in muscles and SCs accounts for the mdx;KO phenotype. Indeed, the ectopic expression of Trim72, a major player in the membrane repair mechanism, prevents SC death (~20%; P < 0.01) and increases myogenic fusion (~40%; P < 0.01) in vitro; in vivo it significantly reduces myofibre damage (~10%; P < 0.005) and improves mdx;KO muscle function (P < 0.05). The mdx;KO phenotype is also fully rescued by restoring cytoplasmic levels of HDAC4, both in vitro and in vivo. The protective role of HDAC4 in the cytoplasm of mdx;KO muscles is, in part, independent of its deacetylase activity. HDAC4 expression correlates with Trim72 mRNA levels; furthermore, Trim72 mRNA decays more rapidly (P < 0.01) in mdx;KO muscle cells, compared with mdx ones. CONCLUSIONS: Histone deacetylase 4 performs crucial functions in the cytoplasm of dystrophic muscles, by mediating the muscle repair response to damage, an important role in ensuring muscle homeostasis, probably by stabilizing Trim72 mRNA. Consequently, the cytoplasmic functions of HDAC4 should be stimulated rather than inhibited in muscular dystrophy treatments, a fact to be considered in future therapeutic approaches.
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Histona Desacetilasas , Distrofia Muscular de Duchenne , Adolescente , Animales , Niño , Citoplasma/metabolismo , Citoplasma/patología , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos mdx , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/genética , Proteínas RepresorasRESUMEN
Understanding how an epigenetic regulator drives different cellular responses can be a tricky task. Very often, their activities are modulated by large multiprotein complexes, the composition of which is context- and time-dependent. As a consequence, experiments aimed to unveil the functions of an epigenetic regulator can provide different outcomes and conclusions, depending on the circumstances. HDAC9 (histone deacetylase), an epigenetic regulator that influences different differentiating and adaptive responses, makes no exception. Since its discovery, different phenotypes and/or dysfunctions have been observed after the artificial manipulation of its expression. The cells and the microenvironment use multiple strategies to control and monitor HDAC9 activities. To date, some of the genes under HDAC9 control have been identified. However, the exact mechanisms through which HDAC9 can achieve all the different tasks so far described, remain mysterious. Whether it can assemble into different multiprotein complexes and how the cells modulate these complexes is not clearly defined. In summary, despite several cellular responses are known to be affected by HDAC9, many aspects of its network of interactions still remain to be defined.
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BACKGROUND: Cellular senescence is a permanent state of replicative arrest defined by a specific pattern of gene expression. The epigenome in senescent cells is sculptured in order to sustain the new transcriptional requirements, particularly at enhancers and super-enhancers. How these distal regulatory elements are dynamically modulated is not completely defined. RESULTS: Enhancer regions are defined by the presence of H3K27 acetylation marks, which can be modulated by class IIa HDACs, as part of multi-protein complexes. Here, we explore the regulation of class IIa HDACs in different models of senescence. We find that HDAC4 is polyubiquitylated and degraded during all types of senescence and it selectively binds and monitors H3K27ac levels at specific enhancers and super-enhancers that supervise the senescent transcriptome. Frequently, these HDAC4-modulated elements are also monitored by AP-1/p300. The deletion of HDAC4 in transformed cells which have bypassed oncogene-induced senescence is coupled to the re-appearance of senescence and the execution of the AP-1/p300 epigenetic program. CONCLUSIONS: Overall, our manuscript highlights a role of HDAC4 as an epigenetic reader and controller of enhancers and super-enhancers that supervise the senescence program. More generally, we unveil an epigenetic checkpoint that has important consequences in aging and cancer.