RESUMO
BACKGROUND: The RNA-binding protein IGF2BP2/IMP2/VICKZ2/p62 is an oncofetal protein that is overexpressed in several cancer entities. Employing IMP2 knockout colorectal cancer cells, we could show the important role of IMP2 in several hallmarks of cancer. This study aimed to functionally characterize IMP2 in lung (A549, LLC1) and hepatocellular carcinoma (HepG2, Huh7) cell lines to assess its role as a potential target for these cancer entities. METHODS: IMP2 knockouts were generated by CRISPR/Cas9 and its variant approach prime editing; the editing efficiency of two single guide RNAs (sgRNAs) was verified via next-generation sequencing. We studied the effect of IMP2 knockout on cell proliferation, colony formation, and migration and employed small-molecule inhibitors of IMP2. RESULTS: Despite multiple attempts, it was not possible to generate IMP2 biallelic knockouts in A549 and Huh7 cells. Both sgRNAs showed good editing efficiency. However, edited cells lost their ability to proliferate. The attempt to generate an IMP2 biallelic knockout in LLC1 cells using CRISPR/Cas9 was successful. Monoallelic knockout cell lines of IMP2 showed a reduction in 2D cell proliferation and reduced migration. In 3D cultures, a change in morphology from compact spheroids to loose aggregates and a distinct reduction in the colony formation ability of the IMP2 knockouts was observed, an effect that was mimicked by previously identified IMP2 inhibitor compounds that also showed an inhibitory effect on colony formation. CONCLUSIONS: Our in vitro target validation supports that IMP2 is essential for tumor cell proliferation, migration, and colony formation in several cancer entities.
Assuntos
Antineoplásicos , Neoplasias Hepáticas , Proteínas de Ligação a RNA , Humanos , Edição de Genes , RNA Guia de Sistemas CRISPR-Cas , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genéticaRESUMO
Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against BRCA1/2-mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired resistance. Therefore, enhancing PARP inhibitor sensitivity and preventing resistance in those cells are an unmet clinical need. Here, we investigated the ability of paraspeckle component 1 (PSPC1), as an additional synthetic lethal partner with BRCA1/2, to enhance olaparib sensitivity in preclinical models of BRCA1/2-mutated breast and ovarian cancers. In vitro, the combined olaparib and PSPC1 small interfering RNA (siRNA) exhibited synergistic anti-proliferative activity in BRCA1/2-mutated breast and ovarian cancer cells. The combination therapy also demonstrated synergistic tumor inhibition in a xenograft mouse model. Mechanistically, olaparib monotherapy increased the expressions of p-ATM and DNA-PKcs, suggesting the activation of a DNA repair pathway, whereas combining PSPC1 siRNA with olaparib decreased the expressions of p-ATM and DNA-PKcs again. As such, the combination increased the formation of γH2AX foci, indicating stronger DNA double-strand breaks. Subsequently, these DNA-damaged cells escaped G2/M checkpoint activation, as indicated by the suppression of p-cdc25C (Ser216) and p-cdc2 (Tyr15) after combination treatment. Finally, these cells entered mitosis, which induced increased apoptosis. Thus, this proves that PSPC1 inhibition enhances olaparib sensitivity by targeting DNA damage response in our preclinical model. The combination of olaparib and PSPC1 inhibition merits further clinical investigation to enhance PARP inhibitor efficacy.
Assuntos
Antineoplásicos , Neoplasias da Mama , Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Antineoplásicos/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Humanos , Feminino , Camundongos , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteína BRCA1/genética , Proteína BRCA2/genética , RNA Interferente Pequeno/genéticaRESUMO
The RNA-binding protein human antigen R (HuR) regulates stability, translation, and nucleus-to-cytoplasm shuttling of its target mRNAs. This protein has been progressively recognized as a relevant therapeutic target for several pathologies, like cancer, neurodegeneration, as well as inflammation. Inhibitors of mRNA binding to HuR might thus be beneficial against a variety of diseases. Here, we present the rational identification of structurally novel HuR inhibitors. In particular, by combining chemoinformatic approaches, high-throughput virtual screening, and RNA-protein pulldown assays, we demonstrate that the 4-(2-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)hydrazineyl)benzoate ligand exhibits a dose-dependent HuR inhibition effect in binding experiments. Importantly, the chemical scaffold is new with respect to the currently known HuR inhibitors, opening up a new avenue for the design of pharmaceutical agents targeting this important protein.
Assuntos
Benzoatos , Bioensaio , Proteína Semelhante a ELAV 1 , Humanos , Núcleo Celular , Peso Molecular , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteína Semelhante a ELAV 1/antagonistas & inibidoresRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a pathological condition which is strongly correlated with fat accumulation in the liver that has become a major health hazard globally. So far, limited treatment options are available for the management of NAFLD and partial agonism of Farnesoid X receptor (FXR) has proven to be one of the most promising strategies for treatment of NAFLD. In present work, a range of validated predictive cheminformatics and molecular modeling studies were performed with a series of 3-benzamidobenzoic acid derivatives in order to recognize their structural requirements for possessing higher potency towards FXR. 2D-QSAR models were able to extract the most significant structural attributes determining the higher activity towards the receptor. Ligand-based pharmacophore model was created with a novel and less-explored open access tool named QPhAR to acquire information regarding important 3D-pharmacophoric features that lead to higher agonistic potential towards the FXR. The alignment of the dataset compounds based on pharmacophore mapping led to 3D-QSAR models that pointed out the most crucial steric and electrostatic influence. Molecular dynamics (MD) simulation performed with the most potent and the least potent derivatives of the current dataset helped us to understand how to link the structural interpretations obtained from 2D-QSAR, 3D-QSAR and pharmacophore models with the involvement of specific amino acid residues in the FXR protein. The current study revealed that hydrogen bond interactions with carboxylate group of the ligands play an important role in the ligand receptor binding but higher stabilization of different helices close to the binding site of FXR (e.g., H5, H6 and H8) through aromatic scaffolds of the ligands should lead to higher activity for these ligands. The present work affords important guidelines towards designing novel FXR partial agonists for new therapeutic options in the management of NAFLD. Moreover, we relied mainly on open-access tools to develop the in-silico models in order to ensure their reproducibility as well as utilization.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Relação Quantitativa Estrutura-Atividade , Reprodutibilidade dos Testes , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismoRESUMO
The RNA-editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) limits the accumulation of endogenous immunostimulatory double-stranded RNA (dsRNA)1. In humans, reduced ADAR1 activity causes the severe inflammatory disease Aicardi-Goutières syndrome (AGS)2. In mice, complete loss of ADAR1 activity is embryonically lethal3-6, and mutations similar to those found in patients with AGS cause autoinflammation7-12. Mechanistically, adenosine-to-inosine (A-to-I) base modification of endogenous dsRNA by ADAR1 prevents chronic overactivation of the dsRNA sensors MDA5 and PKR3,7-10,13,14. Here we show that ADAR1 also inhibits the spontaneous activation of the left-handed Z-nucleic acid sensor ZBP1. Activation of ZBP1 elicits caspase-8-dependent apoptosis and MLKL-mediated necroptosis of ADAR1-deficient cells. ZBP1 contributes to the embryonic lethality of Adar-knockout mice, and it drives early mortality and intestinal cell death in mice deficient in the expression of both ADAR and MAVS. The Z-nucleic-acid-binding Zα domain of ADAR1 is necessary to prevent ZBP1-mediated intestinal cell death and skin inflammation. The Zα domain of ADAR1 promotes A-to-I editing of endogenous Alu elements to prevent dsRNA formation through the pairing of inverted Alu repeats, which can otherwise induce ZBP1 activation. This shows that recognition of Alu duplex RNA by ZBP1 may contribute to the pathological features of AGS that result from the loss of ADAR1 function.
Assuntos
Adenosina Desaminase , Inflamação , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adenosina/metabolismo , Adenosina Desaminase/química , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Animais , Apoptose , Doenças Autoimunes do Sistema Nervoso , Caspase 8/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/prevenção & controle , Inosina/metabolismo , Intestinos/patologia , Camundongos , Necroptose , Malformações do Sistema Nervoso , Edição de RNA , RNA de Cadeia Dupla , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Pele/patologiaRESUMO
Lung cancer is the most malignant form of cancer and has the highest morbidity and mortality worldwide. Due to drug resistance, the current chemotherapy for lung cancer is not effective and has poor therapeutic effects. Tripchlorolide (T4), a natural extract from the plant Tripterygium wilfordii, has powerful immunosuppressive and antitumour effects and may become a potential therapeutic agent for lung cancer. Therefore, this study aimed to investigate the effect of T4 on reducing chemoresistance in lung cancer cells and to explore the mechanism. 1. A549 and A549/DDP cells were separately transfected with AEG-1 overexpression and AEG-1 knockdown plasmids. A549/DDP cells were divided into the A549/DDP empty group, T4 group, and T4 + AEG-1 overexpression group. A CCK-8 assay was used to evaluate the proliferation of cells in each group. RT-qPCR and Western blotting were used to detect the expression of AEG-1 and MDR-1. Expression of AEG-1 in A549 and A549/DDP cells was positively correlated with cisplatin resistance. When the AEG-1 protein was overexpressed in A549 cells, the lethal effect of cisplatin on A549 cells was attenuated (all P < 0.05). After the AEG-1 protein was knocked down in A549/DDP cells, cisplatin was applied. The lethal effect was significantly increased compared to that in the corresponding control cells (all P < 0.05). AEG-1 protein expression gradually decreased with increasing T4 concentration in A549 and A549/DDP cells. Resistance to cisplatin was reduced after the addition of T4 to A549/DDP cells (P < 0.05), and this effect was enhanced after transfection with the AEG-1 knockdown plasmid. T4 plays an important role in increasing the sensitivity of lung cancer cells to cisplatin.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Proteínas de Membrana , Proteínas de Ligação a RNA , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Diterpenos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Fenantrenos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic tumor microenvironment (TME) consisting of abundant activated pancreatic stellate cells (PSCs). PSCs play a key role in the refractory responses of PDAC to immunotherapy and chemotherapy and deactivating PSCs into quiescence through vitamin D receptor (VDR) signaling activation is a promising strategy for PDAC treatment. We observed p62 loss in PSCs hindered the deactivation efficacy of VDR ligands, and hypothesized that reversing p62 levels by inhibiting autophagy processing, which is responsible for p62 loss, could sensitize PSCs toward VDR ligands. Herein, we constructed a PSC deactivator with dual functions of VDR activation and autophagy inhibition, utilizing a pH-buffering micelle (LBM) with an inherent ability to block autophagic flux to encapsulate calcipotriol (Cal), a VDR ligand. This Cal-loaded LBM (C-LBM) could efficiently reprogram PSCs, modulate the fibrotic TME, and alter immunosuppression. In combination with PD-1 antagonists and chemotherapy, C-LBM showed superior antitumor efficacy and significantly prolonged the survival of PDAC mice. These findings suggest that synergistic autophagy blockade and VDR signaling activation are promising therapeutic approaches to reprogram PSCs and improve the PDAC response to immunotherapy.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Células Estreladas do Pâncreas , Receptores de Calcitriol , Animais , Autofagia/efeitos dos fármacos , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reprogramação Celular/efeitos dos fármacos , Humanos , Ligantes , Lisossomos , Camundongos , Micelas , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Células Estreladas do Pâncreas/metabolismo , Células Estreladas do Pâncreas/patologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Receptores de Calcitriol/genética , Microambiente TumoralRESUMO
Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.
Assuntos
Adesão Celular , MAP Quinase Quinase Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Animais , Ligante de CD40/genética , Ligante de CD40/metabolismo , Ligante de CD40/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Quinase 1 de Adesão Focal/antagonistas & inibidores , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Regulator of ribosome synthesis 1 (RRS1) is a key factor in ribosome biosynthesis and other cellular functions. High level of RRS1 in breast cancer cell lines is associated with increased cell proliferation, invasion and migration. RRS1 controls the assembly of the 60s subunit and maturation of 25S rRNA during ribosome biosynthesis. In this study, lentiviral transfection of shRNA was used to knock down the level of RRS1, to detect the effect of RRS1 on cell function and to explore the specific mechanism of RRS1 affecting cell invasion and metastasis by COIP and dualluciferase reporter gene assays. The present study found that RRS1 knockdown reduced the accumulation of ribosome protein L11 (RPL11) in the nucleolus, which then migrated to the nucleoplasm and bound to cMyc. This inhibited transactivation of SNAIL by cMyc and eventually decreased the invasion and metastasis capacity of the human breast cancer cell line BT549. Taken together, RRS1 regulates invasion and metastasis of human breast cancer cells through the RPL11cMycSNAIL axis. The findings are of great significance for exploring the mechanism of breast cancer invasion and metastasis and the corresponding regulatory factors.
Assuntos
Regulação para Baixo/genética , Metástase Neoplásica/genética , Proteínas de Ligação a RNA/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/genética , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Regulação para Baixo/efeitos dos fármacos , Humanos , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/prevenção & controle , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Fatores de Transcrição da Família Snail/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Chemoresistance to cisplatin (DDP) remains a major challenge in advanced gastric cancer (GC) treatment. Although accumulating evidence suggests an association between dysregulation of long non-coding RNAs (lncRNAs) and chemoresistance, the regulatory functions and complexities of lncRNAs in modulating DDP-based chemotherapy in GC remain under-investigated. This study was designed to explore the critical chemoresistance-related lncRNAs in GC and identify novel therapeutic targets for patients with chemoresistant GC. METHODS: Chemoresistance-related lncRNAs were identified through microarray and verified through a quantitative real-time polymerase chain reaction (qRT-PCR). Proteins bound by lncRNAs were identified through a human proteome array and validated through RNA immunoprecipitation (RIP) and RNA pull-down assays. Co-immunoprecipitation and ubiquitination assays were performed to explore the molecular mechanisms of the Musashi2 (MSI2) post-modification. The effects of LINC00942 (LNC942) and MSI2 on DDP-based chemotherapy were investigated through MTS, apoptosis assays and xenograft tumour formation in vivo. RESULTS: LNC942 was found to be up-regulated in chemoresistant GC cells, and its high expression was positively correlated with the poor prognosis of patients with GC. Functional studies indicated that LNC942 confers chemoresistance to GC cells by impairing apoptosis and inducing stemness. Mechanically, LNC942 up-regulated the MSI2 expression by preventing its interaction with SCFß-TRCP E3 ubiquitin ligase, eventually inhibiting ubiquitination. Then, LNC942 stabilized c-Myc mRNA in an N6-methyladenosine (m6 A)-dependent manner. As a potential m6 A recognition protein, MSI2 stabilized c-Myc mRNA with m6 A modifications. Moreover, inhibition of the LNC942-MSI2-c-Myc axis was found to restore chemosensitivity both in vitro and in vivo. CONCLUSIONS: These results uncover a chemoresistant accelerating function of LNC942 in GC, and disrupting the LNC942-MSI2-c-Myc axis could be a novel therapeutic strategy for GC patients undergoing chemoresistance.
Assuntos
Cisplatino/metabolismo , Resistência a Medicamentos/efeitos dos fármacos , Genes myc/efeitos dos fármacos , RNA Longo não Codificante/agonistas , Proteínas de Ligação a RNA/antagonistas & inibidores , Cisplatino/uso terapêutico , Genes myc/fisiologia , Humanos , RNA Longo não Codificante/genética , RNA Longo não Codificante/uso terapêutico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genéticaRESUMO
Inhibition of the RNA-binding protein LIN28 and disruption of the protein-RNA interaction of LIN28-let-7 with small molecules holds great potential to develop new anticancer therapeutics. Herein, we report the LIN28 inhibitory activities of a series of 30 small molecules with a tricyclic tetrahydroquinoline (THQ)-containing scaffold obtained from a Povarov reaction. The THQ molecules were structurally optimized by varying the 2-benzoic acid substituent, the fused ring at 3- and 4-positions, and the substituents at the phenyl moiety of the tetrahydroquinoline core. Among the tested compounds, GG-43 showed dose-dependent inhibition in an EMSA validation assay and low micromolar inhibitory activity in a fluorescence polarization-based assay measuring disruption of LIN28-let-7 interaction. Binding mode between GG-43 and the cold shock domain of LIN28 was proposed via a molecular docking analysis. The study provides one of the first systematic analyses on structural features that are required for LIN28 inhibition, and indicates the necessity to develop small molecules with new scaffolds as LIN28-targeting probes and therapeutic candidates. In parallel, this study demonstrates the polypharmacological nature of tricyclic THQ-containing scaffolds accessible through Povarov reactions.
Assuntos
Antineoplásicos/farmacologia , Quinolinas/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , RNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Quinolinas/síntese química , Quinolinas/química , RNA/química , RNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Igf2bp1 is an oncofetal RNA binding protein whose expression in numerous types of cancers is associated with upregulation of key pro-oncogenic RNAs, poor prognosis, and reduced survival. Importantly, Igf2bp1 synergizes with mutations in Kras to enhance signalling and oncogenic activity, suggesting that molecules inhibiting Igf2bp1 could have therapeutic potential. Here, we isolate a small molecule that interacts with a hydrophobic surface at the boundary of Igf2bp1 KH3 and KH4 domains, and inhibits binding to Kras RNA. In cells, the compound reduces the level of Kras and other Igf2bp1 mRNA targets, lowers Kras protein, and inhibits downstream signalling, wound healing, and growth in soft agar, all in the absence of any toxicity. This work presents an avenue for improving the prognosis of Igf2bp1-expressing tumours in lung, and potentially other, cancer(s).
Assuntos
Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Ligação Proteica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND AND AIMS: Methionine adenosyltransferase 1A (MAT1A) is responsible for S-adenosylmethionine (SAMe) biosynthesis in the liver. Mice lacking Mat1a have hepatic SAMe depletion and develop NASH and HCC spontaneously. Several kinases are activated in Mat1a knockout (KO) mice livers. However, characterizing the phospho-proteome and determining whether they contribute to liver pathology remain open for study. Our study aimed to provide this knowledge. APPROACH AND RESULTS: We performed phospho-proteomics in Mat1a KO mice livers with and without SAMe treatment to identify SAMe-dependent changes that may contribute to liver pathology. Our studies used Mat1a KO mice at different ages treated with and without SAMe, cell lines, in vitro translation and kinase assays, and human liver specimens. We found that the most striking change was hyperphosphorylation and increased content of La-related protein 1 (LARP1), which, in the unphosphorylated form, negatively regulates translation of 5'-terminal oligopyrimidine (TOP)-containing mRNAs. Consistently, multiple TOP proteins are induced in KO livers. Translation of TOP mRNAs ribosomal protein S3 and ribosomal protein L18 was enhanced by LARP1 overexpression in liver cancer cells. We identified LARP1-T449 as a SAMe-sensitive phospho-site of cyclin-dependent kinase 2 (CDK2). Knocking down CDK2 lowered LARP1 phosphorylation and prevented LARP1-overexpression-mediated increase in translation. LARP1-T449 phosphorylation induced global translation, cell growth, migration, invasion, and expression of oncogenic TOP-ribosomal proteins in HCC cells. LARP1 expression is increased in human NASH and HCC. CONCLUSIONS: Our results reveal a SAMe-sensitive mechanism of LARP1 phosphorylation that may be involved in the progression of NASH to HCC.
Assuntos
Autoantígenos/metabolismo , Oligonucleotídeos/genética , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/metabolismo , S-Adenosilmetionina/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/imunologia , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Metionina Adenosiltransferase/genética , Camundongos , Camundongos Knockout , Mutação , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteômica , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , S-Adenosilmetionina/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Antígeno SS-BRESUMO
There has been accumulating evidence that RNA splicing is frequently dysregulated in a variety of cancers and that hotspot mutations affecting key splicing factors, SF3B1, SRSF2 and U2AF1, are commonly enriched across cancers, strongly suggesting that aberrant RNA splicing is a new class of hallmark that contributes to the initiation and/or maintenance of cancers. In parallel, some studies have demonstrated that cancer cells with global splicing alterations are dependent on the transcriptional products derived from wild-type spliceosome for their survival, which potentially creates a therapeutic vulnerability in cancers with a mutant spliceosome. It has been c. 10 y since the frequent mutations affecting splicing factors were reported in cancers. Based on these surprising findings, there has been a growing interest in targeting altered splicing in the treatment of cancers, which has promoted a wide variety of investigations including genetic, molecular and biological studies addressing how altered splicing promotes oncogenesis and how cancers bearing alterations in splicing can be targeted therapeutically. In this mini-review we present a concise trajectory of what has been elucidated regarding the pathogenesis of cancers with aberrant splicing, as well as the development of therapeutic strategies to target global splicing alterations in cancers.
Assuntos
Neoplasias/genética , Splicing de RNA/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Humanos , Mutação , Neoplasias/tratamento farmacológico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Splicing de RNA/efeitos dos fármacos , Fatores de Processamento de RNA/antagonistas & inibidores , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Spliceossomos/efeitos dos fármacos , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Drug re-purposing might be a fast and efficient way of drug development against the novel coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We applied a bioinformatics approach using molecular dynamics and docking to identify FDA-approved drugs that can be re-purposed to potentially inhibit the non-structural protein 9 (Nsp9) replicase and spike proteins in SARS-CoV-2. We performed virtual screening of FDA-approved compounds, including antiviral, anti-malarial, anti-parasitic, anti-fungal, anti-tuberculosis, and active phytochemicals against the Nsp9 replicase and spike proteins. Selected hit compounds were identified based on their highest binding energy and favorable absorption, distribution, metabolism and excretion (ADME) profile. Conivaptan, an arginine vasopressin antagonist drug exhibited the highest binding energy (-8.4 Kcal/mol) and maximum stability with the amino acid residues present at the active site of the Nsp9 replicase. Tegobuvir, a non-nucleoside inhibitor of the hepatitis C virus, also exhibited maximum stability along with the highest binding energy (-8.1 Kcal/mol) at the active site of the spike proteins. Molecular docking scores were further validated by molecular dynamics using Schrodinger, which supported the strong stability of ligands with the proteins at their active sites through water bridges, hydrophobic interactions, and H-bonding. Our findings suggest Conivaptan and Tegobuvir as potential therapeutic agents against SARS-CoV-2. Further in vitro and in vivo validation and evaluation are warranted to establish how these drug compounds target the Nsp9 replicase and spike proteins.
Assuntos
Antivirais/farmacologia , Reposicionamento de Medicamentos , Proteínas de Ligação a RNA/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , COVID-19 , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica MolecularRESUMO
Aptamers offer a great opportunity to develop innovative drug delivery systems that can deliver cargos specifically into targeted cells. In this study, a chimera consisting of two aptamers was developed to deliver doxorubicin into cancer cells and release the drug in cytoplasm in response to adenosine-5'-triphosphate (ATP) binding. The chimera was composed of the AS1411 anti-nucleolin aptamer for cancer cell targeting and the ATP aptamer for loading and triggering the release of doxorubicin in cells. The chimera was first produced by hybridizing the ATP aptamer with its complementary DNA sequence, which is linked with the AS1411 aptamer via a poly-thymine linker. Doxorubicin was then loaded inside the hybridized DNA region of the chimera. Our results show that the AS1411-ATP aptamer chimera was able to release loaded doxorubicin in cells in response to ATP. In addition, selective uptake of the chimera into cancer cells was demonstrated using flow cytometry. Furthermore, confocal laser scanning microscopy showed the successful delivery of the doxorubicin loaded in chimeras to the nuclei of targeted cells. Moreover, the doxorubicin-loaded chimeras effectively inhibited the growth of cancer cell lines and reduced the cytotoxic effect on the normal cells. Overall, the results of this study show that the AS1411-ATP aptamer chimera could be used as an innovative approach for the selective delivery of doxorubicin to cancer cells, which may improve the therapeutic potency and decrease the off-target cytotoxicity of doxorubicin.
Assuntos
Aptâmeros de Nucleotídeos , Doxorrubicina , Sistemas de Liberação de Medicamentos , Neoplasias , Humanos , Trifosfato de Adenosina/metabolismo , Aptâmeros de Nucleotídeos/administração & dosagem , Aptâmeros de Nucleotídeos/sangue , Aptâmeros de Nucleotídeos/genética , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Desenho de Fármacos , Estabilidade de Medicamentos , Técnicas In Vitro , Células MCF-7 , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/sangue , Oligodesoxirribonucleotídeos/genética , Fosfoproteínas/antagonistas & inibidores , Proteínas de Ligação a RNA/antagonistas & inibidores , NucleolinaRESUMO
RNA-binding proteins (RBPs) act as posttranscriptional regulators controlling the fate of target mRNAs. Unraveling how RNAs are recognized by RBPs and in turn are assembled into neuronal RNA granules is therefore key to understanding the underlying mechanism. While RNA sequence elements have been extensively characterized, the functional impact of RNA secondary structures is only recently being explored. Here, we show that Staufen2 binds complex, long-ranged RNA hairpins in the 3'-untranslated region (UTR) of its targets. These structures are involved in the assembly of Staufen2 into RNA granules. Furthermore, we provide direct evidence that a defined Rgs4 RNA duplex regulates Staufen2-dependent RNA localization to distal dendrites. Importantly, disrupting the RNA hairpin impairs the observed effects. Finally, we show that these secondary structures differently affect protein expression in neurons. In conclusion, our data reveal the importance of RNA secondary structure in regulating RNA granule assembly, localization and eventually translation. It is therefore tempting to speculate that secondary structures represent an important code for cells to control the intracellular fate of their mRNAs.
Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/química , Neurônios/metabolismo , Proteínas RGS/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas , Animais , Células Cultivadas , Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Feminino , Neurônios/citologia , Conformação de Ácido Nucleico , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-DawleyRESUMO
Proliferative retinopathies produces an irreversible type of blindness affecting working age and pediatric population of industrialized countries. Despite the good results of anti-VEGF therapy, intraocular and systemic complications are often associated after its intravitreal use, hence novel therapeutic approaches are needed. The aim of the present study is to test the effect of the AS1411, an antiangiogenic nucleolin-binding aptamer, using in vivo, ex vivo and in vitro models of angiogenesis and propose a mechanistic insight. Our results showed that AS1411 significantly inhibited retinal neovascularization in the oxygen induced retinopathy (OIR) in vivo model, as well as inhibited branch formation in the rat aortic ex vivo assay, and, significantly reduced proliferation, cell migration and tube formation in the HUVEC in vitro model. Importantly, phosphorylated NCL protein was significantly abolished in HUVEC in the presence of AS1411 without affecting NFκB phosphorylation and -21 and 221-angiomiRs, suggesting that the antiangiogenic properties of this molecule are partially mediated by a down regulation in NCL phosphorylation. In sum, this new research further supports the NCL role in the molecular etiology of pathological angiogenesis and identifies AS1411 as a novel anti-angiogenic treatment.
Assuntos
Aptâmeros de Nucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/administração & dosagem , Oxigênio/efeitos adversos , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Neovascularização Retiniana/tratamento farmacológico , Animais , Aptâmeros de Nucleotídeos/farmacologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Intravítreas , Camundongos , MicroRNAs/genética , Oligodesoxirribonucleotídeos/farmacologia , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Neovascularização Retiniana/induzido quimicamente , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , NucleolinaRESUMO
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.
Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Grânulos de Ribonucleoproteínas Citoplasmáticas/efeitos dos fármacos , Grânulos de Ribonucleoproteínas Citoplasmáticas/ultraestrutura , Grânulos de Ribonucleoproteínas Citoplasmáticas/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Glicóis/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Haplorrinos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Osmolar , Fosforilação , Propilenoglicol/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/ultraestrutura , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Synaptic targeting with subcellular specificity is essential for neural circuit assembly. Developing neurons use mechanisms to curb promiscuous synaptic connections and to direct synapse formation to defined subcellular compartments. How this selectivity is achieved molecularly remains enigmatic. Here, we discover a link between mRNA poly(A)-tailing and axon collateral branch-specific synaptic connectivity within the CNS. We reveal that the RNA-binding protein Musashi binds to the mRNA encoding the receptor protein tyrosine phosphatase Ptp69D, thereby increasing poly(A) tail length and Ptp69D protein levels. This regulation specifically promotes synaptic connectivity in one axon collateral characterized by a high degree of arborization and strong synaptogenic potential. In a different compartment of the same axon, Musashi prevents ectopic synaptogenesis, revealing antagonistic, compartment-specific functions. Moreover, Musashi-dependent Ptp69D regulation controls synaptic connectivity in the olfactory circuit. Thus, Musashi differentially shapes synaptic connectivity at the level of individual subcellular compartments and within different developmental and neuron type-specific contexts.