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1.
Theranostics ; 10(21): 9620-9643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32863950

RESUMO

Long non-coding RNAs (lncRNAs) have emerged as integral components of E2F1-regulated gene regulatory networks (GRNs), but their implication in advanced or treatment-refractory malignancy is unknown. Methods: We combined high-throughput transcriptomic approaches with bioinformatics and structure modeling to search for lncRNAs that participate in E2F1-activated prometastatic GRNs and their phenotypic targets in the highly-relevant case of E2F1-driven aggressive bladder cancer (BC). RNA immunoprecipitation was performed to verify RNA-protein interactions. Functional analyses including qRT-PCR, immunoblotting, luciferase assays and measurement of extracellular fluxes were conducted to validate expression and target gene regulation. Results: We identified E2F1-responsive lncRNA-SLC16A1-AS1 and its associated neighboring protein-coding gene, SLC16A1/MCT1, which both promote cancer invasiveness. Mechanistically, upon E2F1-mediated co-transactivation of the gene pair, SLC16A1-AS1 associates with E2F1 in a structure-dependent manner and forms an RNA-protein complex that enhances SLC16A1/MCT1 expression through binding to a composite SLC16A1-AS1:E2F1-responsive promoter element. Moreover, SLC16A1-AS1 increases aerobic glycolysis and mitochondrial respiration and fuels ATP production by fatty acid ß-oxidation. These metabolic changes are accompanied by alterations in the expression of the SLC16A1-AS1:E2F1-responsive gene PPARA, a key mediator of fatty acid ß-oxidation. Conclusions: Our results unveil a new gene regulatory program by which E2F1-induced lncRNA-SLC16A1-AS1 forms a complex with its transcription factor that promotes cancer metabolic reprogramming towards the acquisition of a hybrid oxidative phosphorylation/glycolysis cell phenotype favoring BC invasiveness.


Assuntos
Reprogramação Celular/fisiologia , Fator de Transcrição E2F1/genética , Transportadores de Ácidos Monocarboxílicos/genética , RNA Longo não Codificante/genética , Simportadores/genética , Neoplasias da Bexiga Urinária/genética , Trifosfato de Adenosina/genética , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica/genética , Glicólise/genética , Humanos , Mitocôndrias/genética , Oxirredução , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Neoplasias da Bexiga Urinária/patologia
2.
J Exp Clin Cancer Res ; 38(1): 292, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287003

RESUMO

BACKGROUND: Bladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways. METHODS: RT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly. RESULTS: Herein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a 'high E2F1/APLF/DCLRE1C' signature, which was found to be particularly favorable for BC patient survival. CONCLUSION: Suppression of the 'out-of-context' activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Fator de Transcrição E2F1/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Metilação de DNA , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/antagonistas & inibidores , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Endonucleases/genética , Endonucleases/metabolismo , Técnicas de Silenciamento de Genes , Recombinação Homóloga , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Neoplasias da Bexiga Urinária/patologia
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