RESUMO
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.
Assuntos
Senescência Celular , Dano ao DNA , Histona Desacetilase 1 , Histona Desacetilase 2 , Histona Desacetilases , Histonas , Senescência Celular/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Humanos , Acetilação , Histonas/metabolismo , Histona Desacetilases/metabolismo , Histona Desacetilases/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Reparo de DNA por Recombinação , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Linhagem CelularRESUMO
Single-strand selective uracil-DNA glycosylase 1 (SMUG1) initiates base excision repair (BER) of uracil and oxidized pyrimidines. SMUG1 status has been associated with cancer risk and therapeutic response in breast carcinomas and other cancer types. However, SMUG1 is a multifunctional protein involved, not only, in BER but also in RNA quality control, and its function in cancer cells is unclear. Here we identify several novel SMUG1 interaction partners that functions in many biological processes relevant for cancer development and treatment response. Based on this, we hypothesized that the dominating function of SMUG1 in cancer might be ascribed to functions other than BER. We define a bad prognosis signature for SMUG1 by mapping out the SMUG1 interaction network and found that high expression of genes in the bad prognosis network correlated with lower survival probability in ER+ breast cancer. Interestingly, we identified hsa-let-7b-5p microRNA as an upstream regulator of the SMUG1 interactome. Expression of SMUG1 and hsa-let-7b-5p were negatively correlated in breast cancer and we found an inhibitory auto-regulatory loop between SMUG1 and hsa-let-7b-5p in the MCF7 breast cancer cells. We conclude that SMUG1 functions in a gene regulatory network that influence the survival and treatment response in several cancers.
Assuntos
Neoplasias da Mama , MicroRNAs , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Humanos , MicroRNAs/genética , Prognóstico , Uracila/metabolismo , Uracila-DNA Glicosidase/genéticaRESUMO
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.
Assuntos
Inibidores de Histona Desacetilases , Transcriptoma , Acetilação , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Fatores de Transcrição MEF2/genética , Vorinostat/farmacologiaRESUMO
Increasing evidence suggests different, not completely understood roles of microRNA biogenesis in the development and progression of lung cancer. The overexpression of the DNA repair protein apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) is an important cause of poor chemotherapeutic response in lung cancer and its involvement in onco-miRNAs biogenesis has been recently described. Whether APE1 regulates miRNAs acting as prognostic biomarkers of lung cancer has not been investigated, yet. In this study, we analyzed miRNAs differential expression upon APE1 depletion in the A549 lung cancer cell line using high-throughput methods. We defined a signature of 13 miRNAs that strongly correlate with APE1 expression in human lung cancer: miR-1246, miR-4488, miR-24, miR-183, miR-660, miR-130b, miR-543, miR-200c, miR-376c, miR-218, miR-146a, miR-92b and miR-33a. Functional enrichment analysis of this signature revealed its biological relevance in cancer cell proliferation and survival. We validated DICER1 as a direct functional target of the APE1-regulated miRNA-33a-5p and miR-130b-3p. Importantly, IHC analyses of different human tumors confirmed a negative correlation existing between APE1 and Dicer1 protein levels. DICER1 downregulation represents a prognostic marker of cancer development but the mechanisms at the basis of this phenomenon are still completely unknown. Our findings, suggesting that APE1 modulates DICER1 expression via miR-33a and miR-130b, reveal new mechanistic insights on DICER1 regulation, which are of relevance in lung cancer chemoresistance and cancer invasiveness.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismoRESUMO
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.
Assuntos
Histona Desacetilases/genética , Leiomiossarcoma/genética , Proteínas Repressoras/genética , Acetilação , Diferenciação Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Leiomiossarcoma/patologia , Fatores de Transcrição MEF2/genéticaRESUMO
The persistence of long-term coronavirus-induced disease 2019 (COVID-19) sequelae demands better insights into its natural history. Therefore, it is crucial to discover the biomarkers of disease outcome to improve clinical practice. In this study, 160 COVID-19 patients were enrolled, of whom 80 had a "non-severe" and 80 had a "severe" outcome. Sera were analyzed by proximity extension assay (PEA) to assess 274 unique proteins associated with inflammation, cardiometabolic, and neurologic diseases. The main clinical and hematochemical data associated with disease outcome were grouped with serological data to form a dataset for the supervised machine learning techniques. We identified nine proteins (i.e., CD200R1, MCP1, MCP3, IL6, LTBP2, MATN3, TRANCE, α2-MRAP, and KIT) that contributed to the correct classification of COVID-19 disease severity when combined with relative neutrophil and lymphocyte counts. By analyzing PEA, clinical and hematochemical data with statistical methods that were able to handle many variables in the presence of a relatively small sample size, we identified nine potential serum biomarkers of a "severe" outcome. Most of these were confirmed by literature data. Importantly, we found three biomarkers associated with central nervous system pathologies and protective factors, which were downregulated in the most severe cases.
Assuntos
COVID-19 , Proteômica , Biomarcadores/sangue , COVID-19/diagnóstico , Humanos , Contagem de Linfócitos , Aprendizado de MáquinaRESUMO
The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically.
Assuntos
Insuficiência Cardíaca/patologia , Mecanotransdução Celular , Miocárdio/patologia , Pericitos/metabolismo , Pericitos/patologia , Actomiosina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fenômenos Biomecânicos , Caveolina 1/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Vasos Coronários/patologia , Vasos Coronários/fisiopatologia , Citoesqueleto/metabolismo , Adesões Focais , Humanos , Microvasos/patologia , Microvasos/fisiopatologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Transporte Proteico , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas de Sinalização YAPRESUMO
In hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients, HIV enhances HCV replication and liver damage. Several microRNAs (miRNAs), active in pro-fibrotic and inflammatory pathways, have been implicated in the pathogenesis of this phenomenon. However, these miRNAs have been tested only in explanted cirrhotic livers, when the liver damage has become chronic and irreversible. No data are available on the early phase of viral infection, such as early after liver transplantation (LT). In the present study, the expression of miR-101, miR-122, miR-155, miR-192, miR-200c, miR-338, and miR-532 was determined by quantitative real-time polymerase chain reaction in liver biopsies of HCV (n = 19) and HCV/HIV-infected (n = 20) LT recipients, as well as in a control group (n = 18) of noninfected patients, transplanted for alcoholic cirrhosis. The timing of liver biopsy was 6 months post-LT. None of the patients was treated with direct-acting anti-HCV drugs. All co-infected recipients had suppressed HIV viral load. Grading and staging were assessed according to the Ishak Classification. HCV and HIV viral load were measured in the sera. miR-101 (p = .03), miR-122 (p = .012), and miR-192 (p = .038) were significantly downregulated in HCV/HIV co-infected and HCV mono-infected recipients when compared with noninfected recipients, and such downregulation was more pronounced in co-infected ones. Moreover, in co-infected recipients but not in mono-infected ones, miR-101 inversely correlated with the peripheral HCV-RNA levels (r = .41, p = .04) and miR-122 inversely correlated with peripheral HCV-RNA levels (r = .49, p = .03) and with the histological grading (r = .51, p = .02). In conclusion, as early as 6 months after LT, the presence of HIV-HCV co-infection enhanced a significant downregulation of certain miRNAs that showed a direct correlation with HCV viral load and liver inflammation.
Assuntos
Coinfecção/terapia , Infecções por HIV/terapia , Hepatite C/terapia , Transplante de Fígado , Fígado/metabolismo , MicroRNAs/metabolismo , Adulto , Aloenxertos/metabolismo , Aloenxertos/patologia , Aloenxertos/virologia , Coinfecção/genética , Coinfecção/patologia , Coinfecção/virologia , Feminino , HIV/fisiologia , Infecções por HIV/genética , Infecções por HIV/patologia , Infecções por HIV/virologia , Hepacivirus/fisiologia , Hepatite C/genética , Hepatite C/patologia , Hepatite C/virologia , Humanos , Fígado/patologia , Fígado/virologia , Cirrose Hepática Alcoólica/genética , Cirrose Hepática Alcoólica/patologia , Cirrose Hepática Alcoólica/terapia , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , RNA Viral/genética , RNA Viral/metabolismo , Carga ViralRESUMO
Among the family of regulatory B cells, the subset able to produce interleukin-10 (IL-10) is the most studied, yet its biology is still a matter of investigation. The DNA methylation profiling of the il-10 gene locus revealed a novel epigenetic signature characterizing murine B cells ready to respond through IL-10 synthesis: a demethylated region located 4.5 kb from the transcription starting site (TSS), that we named early IL10 regulatory region (eIL10rr). This feature allows to distinguish B cells that are immediately prone and developmentally committed to IL-10 production from those that require a persistent stimulation to exert an IL-10-mediated regulatory function. These late IL-10 producers are instead characterized by a delayed IL10 regulatory region (dIL10rr), a partially demethylated DNA portion located 9 kb upstream from the TSS. A demethylated region was also found in human IL-10-producing B cells and, very interestingly, in some B-cell malignancies, such as chronic lymphocytic leukemia and mantle cell lymphoma, characterized by an immunosuppressive microenvironment. Our findings define murine and human regulatory B cells as an epigenetically controlled functional state of mature B cell subsets and open a new perspective on IL-10 regulation in B cells in homeostasis and disease.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B Reguladores/fisiologia , Interleucina-10/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Linfoma de Célula do Manto/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Diferenciação Celular , Metilação de DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Tolerância Imunológica , Imunidade Humoral , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microambiente TumoralRESUMO
Alterations of DNA repair enzymes and consequential triggering of aberrant DNA damage response (DDR) pathways are thought to play a pivotal role in genomic instabilities associated with cancer development, and are further thought to be important predictive biomarkers for therapy using the synthetic lethality paradigm. However, novel unpredicted perspectives are emerging from the identification of several non-canonical roles of DNA repair enzymes, particularly in gene expression regulation, by different molecular mechanisms, such as (i) non-coding RNA regulation of tumour suppressors, (ii) epigenetic and transcriptional regulation of genes involved in genotoxic responses and (iii) paracrine effects of secreted DNA repair enzymes triggering the cell senescence phenotype. The base excision repair (BER) pathway, canonically involved in the repair of non-distorting DNA lesions generated by oxidative stress, ionising radiation, alkylation damage and spontaneous or enzymatic deamination of nucleotide bases, represents a paradigm for the multifaceted roles of complex DDR in human cells. This review will focus on what is known about the canonical and non-canonical functions of BER enzymes related to cancer development, highlighting novel opportunities to understand the biology of cancer and representing future perspectives for designing new anticancer strategies. We will specifically focus on APE1 as an example of a pleiotropic and multifunctional BER protein.
Assuntos
Enzimas Reparadoras do DNA/fisiologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Neoplasias/enzimologia , DNA/metabolismo , Dano ao DNA , Enzimas Reparadoras do DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
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.
Assuntos
Retrovirus Endógenos , Histonas , Histonas/metabolismo , Retrovirus Endógenos/genética , Retrovirus Endógenos/metabolismo , Epigênese Genética , Senescência Celular/genética , AntiviraisRESUMO
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.
Assuntos
Neoplasias do Colo , Inibidores de Histona Desacetilases , Humanos , Inibidores de Histona Desacetilases/farmacologia , Cromatina/genética , Ácidos Hidroxâmicos/farmacologia , Apoptose/genética , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Linhagem Celular Tumoral , Proteína Supressora de Tumor p53/genéticaRESUMO
The base excision repair (BER) Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme is endowed with several non-repair activities including miRNAs processing. APE1 is overexpressed in many cancers but its causal role in the tumorigenic processes is largely unknown. We recently described that APE1 can be actively secreted by mammalian cells through exosomes. However, APE1 role in EVs or exosomes is still unknown, especially regarding a putative regulatory function on vesicular small non-coding RNAs. Through dedicated transcriptomic analysis on cellular and vesicular small RNAs of different APE1-depleted cancer cell lines, we found that miRNAs loading into EVs is a regulated process, dependent on APE1, distinctly conveying RNA subsets into vesicles. We identified APE1-dependent secreted miRNAs characterized by enriched sequence motifs and possible binding sites for APE1. In 33 out of 34 APE1-dependent-miRNA precursors, we surprisingly found EXO-motifs and proved that APE1 cooperates with hnRNPA2B1 for the EV-sorting of a subset of miRNAs, including miR-1246, through direct binding to GGAG stretches. Using TCGA-datasets, we showed that these miRNAs identify a signature with high prognostic significance in cancer. In summary, we provided evidence that the ubiquitous DNA-repair enzyme APE1 is part of the EV protein cargo with a novel post-transcriptional role for this ubiquitous DNA-repair enzyme that could explain its role in cancer progression. These findings could open new translational perspectives in cancer biology.
Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , MicroRNAs , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Prognóstico , Reparo do DNA/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Exossomos/metabolismo , Exossomos/genética , Linhagem Celular Tumoral , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Regulação Neoplásica da Expressão GênicaRESUMO
Clear cell renal cell carcinoma (ccRCC) is the most common kidney cancer in the adult population. Late diagnosis, resistance to therapeutics and recurrence of metastatic lesions account for the highest mortality rate among kidney cancer patients. Identifying novel biomarkers for early cancer detection and elucidating the mechanisms underlying ccRCC will provide clues to treat this aggressive malignant tumor. Here, we report that the ubiquitin ligase praja2 forms a complex with-and ubiquitylates the AP2 adapter complex, contributing to receptor endocytosis and clearance. In human RCC tissues and cells, downregulation of praja2 by oncogenic miRNAs (oncomiRs) and the proteasome markedly impairs endocytosis and clearance of the epidermal growth factor receptor (EGFR), and amplifies downstream mitogenic and proliferative signaling. Restoring praja2 levels in RCC cells downregulates EGFR, rewires cancer cell metabolism and ultimately inhibits tumor cell growth and metastasis. Accordingly, genetic ablation of praja2 in mice upregulates RTKs (i.e. EGFR and VEGFR) and induces epithelial and vascular alterations in the kidney tissue.In summary, our findings identify a regulatory loop between oncomiRs and the ubiquitin proteasome system that finely controls RTKs endocytosis and clearance, positively impacting mitogenic signaling and kidney cancer growth.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Adulto , Animais , Humanos , Camundongos , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Regulação para Baixo , Endocitose , Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Renais/genética , Neoplasias Renais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores Proteína Tirosina Quinases/genética , Ubiquitina/metabolismoRESUMO
Aims: The existence of modified ribonucleotide monophosphates embedded in genomic DNA, as a consequence of oxidative stress conditions, including 8-oxo-guanosine and ribose monophosphate abasic site (rAP), has been recently highlighted by several works and associated with oxidative stress conditions. Although human apurinic-apyrimidinic endodeoxyribonuclease 1 (APE1), a key enzyme of the base-excision repair pathway, repairs rAP sites and canonical deoxyribose monophosphate abasic sites with similar efficiency, its incision-repairing activity on 8-oxo-guanosine is very weak. The aims of this work were to: (i) identify proteins able to specifically bind 8-oxo-guanosine embedded in DNA and promote APE1 endoribonuclease activity on this lesion, and (ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. Results: By using an unbiased proteomic approach, we discovered that the AU-rich element RNA-binding protein 1 (AUF1) actively recognizes 8-oxo-guanosine and stimulates the APE1 enzymatic activity on this DNA lesion. By using orthogonal approaches, we found that: (i) the interaction between AUF1 and APE1 is modulated by H2O2-treatment; (ii) depletion of APE1 and AUF1 causes the accumulation of single- and double- strand breaks; and (iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. Innovation: These results establish unexpected functions of AUF1 in modulating genome stability and improve our knowledge of APE1 biology with respect to 8-oxo-guanosine embedded in DNA. Conclusion: By showing a novel function of AUF1, our findings shed new light on the process of genome stability in mammalian cells toward oxidative stress-related damages. Antioxid. Redox Signal. 39, 411-431.
Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Animais , Humanos , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Peróxido de Hidrogênio , Proteômica , DNA/metabolismo , Dano ao DNA , Endorribonucleases/metabolismo , Instabilidade Genômica , Mamíferos/metabolismoRESUMO
Background and Aims: Identification of prognostic factors for hepatocellular carcinoma (HCC) opens new perspectives for therapy. Circulating and cellular onco-miRNAs are noncoding RNAs which can control the expression of genes involved in oncogenesis through post-transcriptional mechanisms. These microRNAs (miRNAs) are considered novel prognostic and predictive factors in HCC. The apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) contributes to the quality control and processing of specific onco-miRNAs and is a negative prognostic factor in several tumors. The present work aims to: a) define APE1 prognostic value in HCC; b) identify miRNAs regulated by APE1 and their relative target genes and c) study their prognostic value. Methods: We used The Cancer Genome Atlas (commonly known as TCGA) data analysis to evaluate the expression of APE1 in HCC. To identify differentially-expressed miRNAs (DEmiRNAs) upon APE1 depletion through specific small interfering RNA, we used NGS and nanostring approaches in the JHH-6 HCC tumor cell line. Bioinformatics analyses were performed to identify signaling pathways involving APE1-regulated miRNAs. Microarray analysis was performed to identify miRNAs correlating with serum APE1 expression. Results: APE1 is considerably overexpressed in HCC tissues compared to normal liver, according to the TCGA-liver HCC (known as LIHC) dataset. Enrichment analyses showed that APE1-regulated miRNAs are implicated in signaling and metabolic pathways linked to cell proliferation, transformation, and angiogenesis, identifying Cyclin Dependent Kinase 6 and Lysosomal Associated Membrane Protein 2 as targets. miR-33a-5p, miR-769, and miR-877 are related to lower overall survival in HCC patients. Through array profiling, we identified eight circulating DE-miRNAs associated with APE1 overexpression. A training phase identified positive association between sAPE1 and miR-3180-3p and miR-769. Conclusions: APE1 regulates specific miRNAs having prognostic value in HCC.
RESUMO
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.
RESUMO
Exosomes are well established effectors of cell-cell communication. Their role on maturation of embryonic cells located in hippocampus, seat of memory, is unknown. Here we show that ceramide facilitates release of exosomes from HN9.10e cells extending information for cell differentiation to neighboring cells. We found only 38 miRNAs differentially expressed in exosomes derived from ceramide-treated cells in comparison with control cells (including 10 up-regulated and 28 down-regulated). Some overexpressed miRNAs (mmu-let-7f-1-3p, mmu-let-7a-1-3p, mmu-let-7b-3p, mmu-let-7b-5p, mmu-miR-330-3p) regulate genes encoding for protein involved in biological, homeostatic, biosynthetic and small molecule metabolic processes, embryo development and cell differentiation, all phenomena relevant for HN9.10e cell differentiation. Notably, the overexpressed mmu-let-7b-5p miRNA appears to be important for our study based on its ability to regulate thirty-five gene targets involved in many processes including sphingolipid metabolism, sphingolipid-related stimulation of cellular functions and neuronal development. Furthermore, we showed that by incubating embryonic cells with exosomes released under ceramide treatment, some cells acquired an astrocytic phenotype and others a neuronal phenotype. We anticipate our study to be a start point for innovative therapeutic strategies to regulate the release of exosomes useful to stimulate delayed brain development in the newborn and to improve the cognitive decline in neurodegenerative disorders.
Assuntos
Exossomos , MicroRNAs , Exossomos/genética , Exossomos/metabolismo , Ceramidas/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Comunicação Celular , Diferenciação Celular/genéticaRESUMO
Despite the state-of-the-art treatment, patients diagnosed with glioblastoma (GBM) have a median overall survival (OS) of 14 months. The insertion of carmustine wafers (CWs) into the resection cavity as adjuvant treatment represents a promising option, although its use has been limited due to contrasting clinical results. Our retrospective evaluation of CW efficacy showed a significant improvement in terms of OS in a subgroup of patients. Given the crucial role of the tumor microenvironment (TME) in GBM progression and response to therapy, we hypothesized that the TME of patients who benefited from CW could have different properties compared to that of patients who did not show any advantage. Using an in vitro model of the glioma microenvironment, represented by glioma-associated-stem cells (GASC), we performed a transcriptomic analysis of GASC isolated from tumors of patients responsive and not responsive to CW to identify differentially expressed genes. We found different transcriptomic profiles, and we identified four genes, specifically down-regulated in GASC isolated from long-term survivors, correlated with clinical data deposited in the TCGA-GBM dataset. Our results highlight that studying the in vitro properties of patient-specific glioma microenvironments can help to identify molecular determinants potentially prognostic for patients treated with CW.
RESUMO
BACKGROUND: After liver transplantation, HCV/HIV co-infected patients present, compared to the HCV mono-infected ones, increased HCV viral load, rapid progression to liver fibrosis and higher mortality. Liver biopsies (LB), obtained routinely 6 months after transplantation, represent a unique model to assess the early events related to graft re-infection. Here, we used miRNA sequencing of LB obtained from both HCV-and HCV/HIV-infected recipients, to identify transcriptional profiles able to explain the more severe outcome of these latter. METHODS: miRNAs of 3 healthy livers, 3 HCV-LB and 3 HCV/HIV-LB were sequenced by Illumina HiSeq2500 platform. The DIANA-miRPath v3.0 webserver and DIANA-microT-CDS algorithm (v5.0) were used to characterize the functions of differentially expressed (DE-) miRNAs, querying the KEGG and Gene Ontology-Biological Process databases. RESULTS: LB obtained from infected patients were characterized, with respect to controls, by a miRNA profile related to viral infection, immune system signaling and DNA damage in HCV-induced carcinogenesis. Instead, HCV-LB and HCV/HIV-LB differed in the expression of miRNAs involved in immunological and apoptotic processes and in extracellular matrix remodeling. CONCLUSIONS: liver reinfection processes are associated with early miRNA changes. Further studies are necessary to establish their prognostic role and possible actionability.