RESUMO
BACKGROUND: Neuroblastoma is the most common extracranial solid tumour in children. Relapsed or refractory neuroblastoma is associated with a poor outcome. We assessed the combination of irinotecan-temozolomide and dasatinib-rapamycin (RIST) in patients with relapsed or refractory neuroblastoma. METHODS: The multicentre, open-label, randomised, controlled, phase 2, RIST-rNB-2011 trial recruited from 40 paediatric oncology centres in Germany and Austria. Patients aged 1-25 years with high-risk relapsed (defined as recurrence of all stage IV and MYCN amplification stages, after response to treatment) or refractory (progressive disease during primary treatment) neuroblastoma, with Lansky and Karnofsky performance status at least 50%, were assigned (1:1) to RIST (RIST group) or irinotecan-temozolomide (control group) by block randomisation, stratified by MYCN status. We compared RIST (oral rapamycin [loading 3 mg/m2 on day 1, maintenance 1 mg/m2 on days 2-4] and oral dasatinib [2 mg/kg per day] for 4 days with 3 days off, followed by intravenous irinotecan [50 mg/m2 per day] and oral temozolomide [150 mg/m2 per day] for 5 days with 2 days off; one course each of rapamycin-dasatinib and irinotecan-temozolomide for four cycles over 8 weeks, then two courses of rapamycin-dasatinib followed by one course of irinotecan-temozolomide for 12 weeks) with irinotecan-temozolomide alone (with identical dosing as experimental group). The primary endpoint of progression-free survival was analysed in all eligible patients who received at least one course of therapy. The safety population consisted of all patients who received at least one course of therapy and had at least one post-baseline safety assessment. This trial is registered at ClinicalTrials.gov, NCT01467986, and is closed to accrual. FINDINGS: Between Aug 26, 2013, and Sept 21, 2020, 129 patients were randomly assigned to the RIST group (n=63) or control group (n=66). Median age was 5·4 years (IQR 3·7-8·1). 124 patients (78 [63%] male and 46 [37%] female) were included in the efficacy analysis. At a median follow-up of 72 months (IQR 31-88), the median progression-free survival was 11 months (95% CI 7-17) in the RIST group and 5 months (2-8) in the control group (hazard ratio 0·62, one-sided 90% CI 0·81; p=0·019). Median progression-free survival in patients with amplified MYCN (n=48) was 6 months (95% CI 4-24) in the RIST group versus 2 months (2-5) in the control group (HR 0·45 [95% CI 0·24-0·84], p=0·012); median progression-free survival in patients without amplified MYCN (n=76) was 14 months (95% CI 9-7) in the RIST group versus 8 months (4-15) in the control group (HR 0·84 [95% CI 0·51-1·38], p=0·49). The most common grade 3 or worse adverse events were neutropenia (54 [81%] of 67 patients given RIST vs 49 [82%] of 60 patients given control), thrombocytopenia (45 [67%] vs 41 [68%]), and anaemia (39 [58%] vs 38 [63%]). Nine serious treatment-related adverse events were reported (five patients given control and four patients given RIST). There were no treatment-related deaths in the control group and one in the RIST group (multiorgan failure). INTERPRETATION: RIST-rNB-2011 demonstrated that targeting of MYCN-amplified relapsed or refractory neuroblastoma with a pathway-directed metronomic combination of a multkinase inhibitor and an mTOR inhibitor can improve progression-free survival and overall survival. This exclusive efficacy in MYCN-amplified, relapsed neuroblastoma warrants further investigation in the first-line setting. FUNDING: Deutsche Krebshilfe.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Dasatinibe , Irinotecano , Recidiva Local de Neoplasia , Neuroblastoma , Sirolimo , Temozolomida , Humanos , Temozolomida/administração & dosagem , Temozolomida/uso terapêutico , Irinotecano/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Masculino , Feminino , Neuroblastoma/tratamento farmacológico , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Neuroblastoma/genética , Pré-Escolar , Criança , Dasatinibe/administração & dosagem , Dasatinibe/uso terapêutico , Dasatinibe/efeitos adversos , Adolescente , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Lactente , Adulto , Sirolimo/administração & dosagem , Sirolimo/uso terapêutico , Adulto Jovem , Alemanha , Resistencia a Medicamentos Antineoplásicos , Intervalo Livre de ProgressãoRESUMO
BACKGROUND: The outcome for patients with high-risk neuroblastoma remains poor and novel treatment strategies are urgently needed. The RIST protocol represents a novel metronomic and multimodal treatment strategy for high-risk neuroblastoma combining molecular-targeted drugs as 'pre-treatment' with a conventional chemotherapy backbone, currently evaluated in a phase II clinical trial. For preclinical drug testing, cancer cell growth as spheroid compared to mo-nolayer cultures is of advantage since it reproduces a wide range of tumor characteristics, including the three-dimensional architecture and cancer stem cell (CSC) properties. The objective of this study was to establish a neuroblastoma spheroid model for the rigorous assessment of the RIST treatment protocol. METHODS: Evaluation of CSC marker expression was performed by mRNA and protein analysis and spheroid viability by luminescence-based assays. Aberrant expression of RNA-binding protein La in neuroblastoma was assessed by tissue microarray analysis and patients' data mining. RESULTS: Spheroid cultures showed increased expression of a subgroup of CSC-like markers (CXCR4, NANOG and BMI) and higher Thr389 phosphorylation of the neuroblastoma-associated RNA-binding protein La when compared to monolayer cultures. Molecular-targeted 'pre-treatment' of spheroids decreased neoplastic signaling and CSC marker expression. CONCLUSIONS: The RIST treatment protocol efficiently reduced the viability of neuroblastoma spheroids characterized by advanced CSC properties.
RESUMO
Some of the early complications of hematopoietic stem cell transplantation (HSCT) concerning the small vessels can be summarized as transplant-associated systemic vasculopathies (TASV). One enzyme known to play a major role in inflammation, tissue remodeling, and repair processes as well as tumor metastasis is heparanase (HPSE). HPSE genetic variants have recently been associated with significant influence on the risk of developing certain TASV such as a sinusoidal obstruction syndrome. This study aimed to validate the two known HPSE single nucleotide polymorphisms (SNPs)-rs4693608 and rs4364254-as a genetic predictor of TASV in a cohort of 494 patients and were correlated retrospectively with the clinical course post-HSCT. Significant association was revealed for rs4364254, showing that the incidence of TASV (38.0% vs. 57.8%, p = .009) and in particular of acute graft-versus-host disease (aGvHD) (36.3% vs. 54.0%, p = .0138) was lower in wildtype CC carriers than in TC/TT carriers. Moreover, compared with all other genotypes, the allelic combination GG-CC had the lowest incidence of TASV (34.9% vs. 57.4%, p = .0109) and aGvHD in particular (34.9% vs. 53.5%, p = .0315). A competing risk regression analysis confirmed a significantly reduced risk for a TASV in patients with GG (subhazard ratio [SHR] = 0.670, p = .043) and CC (SHR = 0.598, p = .041) compared with the corresponding homozygote SNP as well as for allelic combinations correlated with low HPSE gene expression (SHR = 0.630, p = .016) and in correlation with clinical risk factors. In summary, our study emphasizes an association of HPSE gene SNPs with TASV, in particular with aGvHD, which could be implementable as pre-transplant risk stratification if validated prospectively.
Assuntos
Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Humanos , Estudos Retrospectivos , Incidência , Genótipo , Polimorfismo de Nucleotídeo Único , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Doença Enxerto-Hospedeiro/genéticaRESUMO
The prognosis for patients with relapsed or refractory high-risk neuroblastoma remains dismal and novel therapeutic options are urgently needed. The RIST treatment protocol has a multimodal metronomic therapy design combining molecular-targeted drugs (Rapamycin and Dasatinib) with chemotherapy backbone (Irinotecan and Temozolomide), which is currently verified in a phase II clinical trial (NCT01467986). With the availability of novel and more potent ATP competitive mTOR inhibitors, we expect to improve the RIST combination therapy. By comparing the IC50 values of Torin-1, Torin-2, AZD3147 and PP242 we established that only Torin-2 inhibited cell viability of all three MycN-amplified neuroblastoma cell lines tested at nanomolar concentration. Single treatment of both mTOR inhibitors induced a significant G1 cell cycle arrest and combination treatment with Dasatinib reduced the expression of cell cycle regulator cyclin D1 or increased the expression of cell cycle inhibitor p21. The combinatorial index depicted for both mTOR inhibitors a synergistic effect with Dasatinib. Interestingly, compared to Rapamycin, the combination treatment with Torin-2 resulted in a broader mTOR pathway inhibition as indicated by reduced phosphorylation of AKT (Thr308, Ser473), 4E-BP (Ser65), and S6K (Thr389). Furthermore, substituting Rapamycin in the modified multimodal RIST protocol with Torin-2 reduced cell viability and induced apoptosis despite a significant lower Torin-2 drug concentration applied. The efficacy of nanomolar concentrations may significantly reduce unwanted immunosuppression associated with Rapamycin. However, at this point we cannot rule out that Torin-2 has increased toxicity due to its potency in more complex systems. Nonetheless, our results suggest that including Torin-2 as a substitute for Rapamycin in the RIST protocol may represent a valid option to be evaluated in prospective clinical trials for relapsed or treatment-refractory high-risk neuroblastoma.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Naftiridinas/administração & dosagem , Neuroblastoma/tratamento farmacológico , Sirolimo/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Administração Metronômica , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dasatinibe/administração & dosagem , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Humanos , Indóis/administração & dosagem , Concentração Inibidora 50 , Irinotecano/administração & dosagem , Neuroblastoma/patologia , Purinas/administração & dosagem , Pirimidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Temozolomida/administração & dosagem , Tioureia/administração & dosagem , Tioureia/análogos & derivadosRESUMO
BACKGROUND: the aberrant overexpression of predominantly nuclear localizing RNA-binding protein (RBP) La contributes to proliferation, mobility, and chemoresistance of cancer cells and tumor growth in mice. METHODS: studies included cancer tissue microarrays (TMAs) analyses, cancer tissue data mining, transforming growth factor ß (TGFß)-induced cancer cell plasticity studies, three dimensional sphere growth, epithelial to mesenchymal transition (EMT) assays, analysis of cancer stem cell (CSC) marker expression, and post-translational modification of cancer-associated La protein. RESULTS: we demonstrated that significant overexpression of RBP La in lung and head and neck cancer tissue correlates with poor overall survival. Furthermore, small interfering RNA-mediated depletion of La reduced proliferation and migration of cancer cells, blocked TGFß-induced EMT, and diminished both EMT and CSC marker expression. Rescue experiments with La wildtype but not RNA chaperone domain activity-defective La mutant increased the expression of those cancer progression markers, suggesting a critical role of La's RNA chaperone activity in this process. La depletion in cancer cells also significantly decreased sphere growth in the presence of TGFß. Interestingly, TGFß treatment induced phosphorylation of La at threonine 389 (pLaT389) only in adherents but not in 3D growing cultures. CONCLUSION: our study suggests that the TGFß/AKT/pLaT389 signaling pathway regulates cancer cell plasticity.
RESUMO
RNA-binding proteins are important regulators of RNA metabolism and are of critical importance in all steps of the gene expression cascade. The role of aberrantly expressed RBPs in human disease is an exciting research field and the potential application of RBPs as a therapeutic target or a diagnostic marker represents a fast-growing area of research.Aberrant overexpression of the human RNA-binding protein La has been found in various cancer entities including lung, cervical, head and neck, and chronic myelogenous leukaemia. Cancer-associated La protein supports tumour-promoting processes such as proliferation, mobility, invasiveness and tumour growth. Moreover, the La protein maintains the survival of cancer cells by supporting an anti-apoptotic state that may cause resistance to chemotherapeutic therapy.The human La protein represents a multifunctional post-translationally modified RNA-binding protein with RNA chaperone activity that promotes processing of non-coding precursor RNAs but also stimulates the translation of selective messenger RNAs encoding tumour-promoting and anti-apoptotic factors. In our model, La facilitates the expression of those factors and helps cancer cells to cope with cellular stress. In contrast to oncogenes, able to initiate tumorigenesis, we postulate that the aberrantly elevated expression of the human La protein contributes to the non-oncogenic addiction of cancer cells. In this review, we summarize the current understanding about the implications of the RNA-binding protein La in cancer progression and therapeutic resistance. The concept of exploiting the RBP La as a cancer drug target will be discussed.
Assuntos
Suscetibilidade a Doenças , Neoplasias/etiologia , Neoplasias/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose/genética , Biomarcadores Tumorais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Humanos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosfoproteínas/antagonistas & inibidores , Ligação Proteica , Processamento de Proteína Pós-Traducional , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA de Transferência/genética , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Ribonucleoproteínas/antagonistas & inibidores , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMO
It is well established that the RNA-binding protein La has RNA chaperone activity. Recent work suggests that the La protein has two distinct RNA chaperone domains (RCD-A and RCD-B) assisting structural changes in diverse groups of RNA molecules such as RNA polymerase III transcripts (e.g., pre-tRNA, U6 snRNA), cellular messenger, and viral RNAs. In this protocol we focus on the RNA chaperone domain RCD-B, which is located in the carboxy-terminal domain of La. It has been shown that this RNA chaperone domain assists structural changes in predicted RNA hairpins folded in the 5'-untranslated regions of cyclin D1 and Bcl2 mRNAs. Besides RNA helicases, which are implicated in melting RNA hairpin structures in an ATP-dependent manner, RNA chaperones fulfil a similar function in an ATP-independent manner. Aiming to study the RNA chaperon activity of La, we established a La-dependent molecular beacon-based RNA chaperone assay and systematically tested the various salt conditions. Herein we describe the assay format and design to study the salt dependency of RNA chaperones. This protocol can be easily adapted to test the RNA chaperone activity of other RNA-binding proteins and to optimize assay conditions.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética/métodos , Chaperonas Moleculares/metabolismo , Estabilidade de RNA , Proteínas de Ligação a RNA/metabolismo , Salinidade , Animais , Ciclina D1/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Sequências Repetidas Invertidas , Chaperonas Moleculares/química , Domínios Proteicos , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA/química , RNA/metabolismo , Sondas RNA/química , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/químicaRESUMO
The cancer-associated RNA-binding protein La is posttranslationally modified by phosphorylation and sumoylation. Sumoylation of La regulates not only the trafficking of La in neuronal axons but also its association with specific mRNAs. Depletion of La in various types of cancer cell lines impairs cell proliferation; however, the molecular mechanism whereby La supports cell proliferation is not clearly understood. In this study, we address the question of whether sumoylation of La contributes to cell proliferation of HEK293 cells. We show that HEK293 cells stably expressing green fluorescent protein (GFP)-tagged wild-type La (GFP-LaWT) grow faster than cells expressing a sumoylation-deficient mutant La (GFP-LaSD), suggesting a proproliferative function of La in HEK293 cells. Further, we found that STAT3 protein levels were reduced in GFP-LaSD cells due to an increase in STAT3 ubiquitination and that overexpression of STAT3 partially restored cell proliferation. Finally, we present RNA sequencing data from RNA immunoprecipitations (RIPs) and report that mRNAs associated with the cell cycle and ubiquitination are preferentially bound by GFP-LaWT and are less enriched in GFP-LaSD RIPs. Taken together, results of our study support a novel mechanism whereby sumoylation of La promotes cell proliferation by averting ubiquitination-mediated degradation of the STAT3 protein.
Assuntos
Fosfoproteínas/metabolismo , Fator de Transcrição STAT3/metabolismo , Proliferação de Células , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoprecipitação , Fosfoproteínas/genética , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fator de Transcrição STAT3/genética , Sumoilação , UbiquitinaçãoRESUMO
HuD protein (also known as ELAVL4) has been shown to stabilize mRNAs with AU-rich elements (ARE) in their 3' untranslated regions (UTRs), including Gap43, which has been linked to axon growth. HuD also binds to neuritin (Nrn1) mRNA, whose 3'UTR contains ARE sequences. Although the Nrn1 3'UTR has been shown to mediate its axonal localization in embryonic hippocampal neurons, it is not active in adult dorsal root ganglion (DRG) neurons. Here, we asked why the 3'UTR is not sufficient to mediate the axonal localization of Nrn1 mRNA in DRG neurons. HuD overexpression increases the ability of the Nrn1 3'UTR to mediate axonal localizing in DRG neurons. HuD binds directly to the Nrn1 ARE with about a two-fold higher affinity than to the Gap43 ARE. Although the Nrn1 ARE can displace the Gap43 ARE from HuD binding, HuD binds to the full 3'UTR of Gap43 with higher affinity, such that higher levels of Nrn1 are needed to displace the Gap43 3'UTR. The Nrn1 3'UTR can mediate a higher level of axonal localization when endogenous Gap43 is depleted from DRG neurons. Taken together, our data indicate that endogenous Nrn1 and Gap43 mRNAs compete for binding to HuD for their axonal localization and activity of the Nrn1 3'UTR.
Assuntos
Regiões 3' não Traduzidas , Axônios/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Proteína GAP-43/metabolismo , Neurônios/metabolismo , Neuropeptídeos/metabolismo , Animais , Axônios/ultraestrutura , Sequência de Bases , Ligação Competitiva , Proteína Semelhante a ELAV 4/genética , Proteína GAP-43/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/ultraestrutura , Neuropeptídeos/genética , Cultura Primária de Células , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Elementos de Resposta , Transdução de SinaisRESUMO
The RNA-binding protein La is overexpressed in a number of tumor tissues and is thought to support tumorigenesis by binding to and facilitating the expression of mRNAs encoding tumor-promoting and anti-apoptotic factors. Hence, small molecules able to block the binding of La to specific RNAs could have a therapeutic impact by reducing the expression of tumor-promoting and anti-apoptotic factors. Toward this novel therapeutic strategy, we aimed to develop a high-throughput fluorescence polarization assay to screen small compound libraries for molecules blocking the binding of La to an RNA element derived from cyclin D1 mRNA. Herein, we make use of a robust fluorescence polarization assay and the validation of primary hits by electrophoretic mobility shift assays. We showed recently that La protects cells against cisplatin treatment by stimulating the protein synthesis of the anti-apoptotic factor Bcl2. Here, we show by RNA immunoprecipitation experiments that one small compound specifically impairs the association of La with Bcl2 mRNA in cells and sensitizes cells for cipslatin-induced cell death. In summary, we report the application of a high-throughput fluorescence polarization assay to identify small compounds that impair the binding of La to target RNAs in vitro and in cells.
Assuntos
Polarização de Fluorescência/métodos , Ensaios de Triagem em Larga Escala/métodos , Sondas Moleculares/química , Proteínas de Ligação a RNA/antagonistas & inibidores , RNA/antagonistas & inibidores , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Imunoprecipitação , Técnicas In Vitro , Sondas Moleculares/farmacologia , Ligação Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.
Assuntos
Ciclina D1/genética , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/genética , Regiões 3' não Traduzidas , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Fosfoproteínas/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , SumoilaçãoRESUMO
Up-regulation of anti-apoptotic factors is a critical mechanism of cancer cell resistance and often counteracts the success of chemotherapeutic treatment. Herein, we identified the cancer-associated RNA-binding protein La as novel factor contributing to cisplatin resistance. Our data demonstrate that depletion of the RNA-binding protein La in head and neck squamous cell carcinoma cells (HNSCC) increases the sensitivity toward cisplatin-induced cell death paralleled by reduced expression of the anti-apoptotic factor Bcl2. Furthermore, it is shown that transient expression of Bcl2 in La-depleted cells protects against cisplatin-induced cell death. By dissecting the underlying mechanism we report herein, that the La protein is required for Bcl2 protein synthesis in cisplatin-treated cells. The RNA chaperone La binds in close proximity to the authentic translation start site and unwinds a secondary structure embedding the authentic AUG. Altogether, our data support a novel model, whereby cancer-associated La protein contributes to cisplatin resistance by stimulating the translation of anti-apoptotic factor Bcl2 in HNSCC cells.
Assuntos
Autoantígenos/metabolismo , Carcinoma de Células Escamosas/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias de Cabeça e Pescoço/patologia , Fragmentos de Peptídeos/metabolismo , Biossíntese de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Regulação para CimaRESUMO
The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La's RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.
Assuntos
Autoantígenos/metabolismo , Ciclina D1/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 5' não Traduzidas , Motivos de Aminoácidos , Autoantígenos/química , Sítios de Ligação , Códon de Iniciação , Ciclina D1/biossíntese , Células HEK293 , Humanos , Fosforilação , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Ribonucleoproteínas/química , Treonina/metabolismo , Antígeno SS-BRESUMO
The 5-year survival rate for oral cavity cancer is poorer than for breast, colon or prostate cancer, and has improved only slightly in the last three decades. Hence, new therapeutic strategies are urgently needed. Here we demonstrate by tissue micro array analysis for the first time that RNA-binding protein La is significantly overexpressed in oral squamous cell carcinoma (SCC). Within this study we therefore addressed the question whether siRNA-mediated depletion of the La protein may interfere with known tumor-promoting characteristics of head and neck SCC cells. Our studies demonstrate that the La protein promotes cell proliferation, migration and invasion of lymph node-metastasized hypopharyngeal SCC cells. We also reveal that La is required for the expression of ß-catenin as well as matrix metalloproteinase type 2 (MMP-2) within these cells. Taken together these data suggest a so far unknown function of the RNA-binding protein La in promoting tumor progression of head and neck SCC.
Assuntos
Autoantígenos/metabolismo , Carcinoma de Células Escamosas/patologia , Movimento Celular , Neoplasias Hipofaríngeas/patologia , Ribonucleoproteínas/metabolismo , Autoantígenos/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Ciclo Celular , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Células HeLa , Humanos , Neoplasias Hipofaríngeas/genética , Neoplasias Hipofaríngeas/metabolismo , Metástase Linfática , Metaloproteinase 2 da Matriz/metabolismo , Invasividade Neoplásica , Ribonucleoproteínas/genética , beta Catenina/metabolismo , Antígeno SS-BRESUMO
Chronic hepatitis B virus (HBV) infections may lead to severe diseases like liver cirrhosis or hepatocellular carcinoma (HCC). The HBV post-transcriptional regulatory element (HPRE) facilitates the nuclear export of unspliced viral mRNAs, contains a splicing regulatory element and resides in the 3'-region of all viral transcripts. The HPRE consists of three sub-elements alpha (nucleotides 1151-1346), beta1 (nucleotides 1347-1457) and beta2 (nucleotides 1458-1582), which confer together full export competence. Here, we present the NMR solution structure (pdb 2JYM) of the stem-loop alpha (SLalpha, nucleotides 1292-1321) located in the sub-element alpha. The SLalpha contains a CAGGC pentaloop highly conserved in hepatoviruses, which essentially adopts a CUNG-like tetraloop conformation. Furthermore, the SLalpha harbours a single bulged G residue flanked by A-helical regions. The structure is highly suggestive of serving two functions in the context of export of unspliced viral RNA: binding sterile alpha motif (SAM-) domain containing proteins and/or preventing the utilization of a 3'-splice site contained within SLalpha.
Assuntos
Vírus da Hepatite B/genética , Modelos Moleculares , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Transporte de RNA , RNA Viral/metabolismo , SoluçõesRESUMO
The human RNA-binding protein La, is an essential trans-acting factor in IRES-dependent translation initiation of poliovirus, the prototypic picornavirus. For hepatitis A virus (HAV), an unusual member of this virus family, the role of host proteins in its inefficient translation and slow replication is unclear. Using small interfering RNA in vivo and purified La in vitro, we demonstrate for the first time that La suppresses HAV IRES-mediated translation and replication. We show that La binds specifically to distinct parts of the HAV IRES and that-unlike poliovirus-HAV proteinase 3C does not cleave La. The La-mediated suppression of HAV translation and stimulation of poliovirus translation implies unexpected mechanistic differences between viral IRES elements.
Assuntos
Autoantígenos/fisiologia , Vírus da Hepatite A/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Ribonucleoproteínas/fisiologia , Ribossomos/fisiologia , Proteases Virais 3C , Autoantígenos/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/metabolismo , Humanos , Interferência de RNA , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Antígeno SS-BRESUMO
A surprisingly large population of mRNAs has been shown to localize to sensory axons, but few RNA-binding proteins have been detected in these axons. These axonal mRNAs include several potential binding targets for the La RNA chaperone protein. La is transported into axonal processes in both culture and peripheral nerve. Interestingly, La is posttranslationally modified in sensory neurons by sumoylation. In axons, small ubiquitin-like modifying polypeptides (SUMO)-La interacts with dynein, whereas native La interacts with kinesin. Lysine 41 is required for sumoylation, and sumoylation-incompetent La(K41R) shows only anterograde transport, whereas WT La shows both anterograde and retrograde transport in axons. Thus, sumoylation of La determines the directionality of its transport within the axonal compartment, with SUMO-La likely recycling to the cell body.
Assuntos
Transporte Axonal , Axônios/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína SUMO-1/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Dineínas/metabolismo , Humanos , Cinesinas/metabolismo , Mucoproteínas/genética , Mucoproteínas/metabolismo , Mutação/genética , Ligação Proteica , Transporte Proteico , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/metabolismo , Técnicas de Cultura de TecidosRESUMO
Alternative splicing of the primary transcript plays a key role in retroviral gene expression. In contrast to all known mechanisms that mediate alternative splicing in retroviruses, we found that in murine leukemia virus, distinct elements located upstream of the 5' splice site either inhibited or activated splicing of the genomic RNA. Detailed analysis of the first untranslated exon showed that the primer binding site (PBS) activates splicing, whereas flanking sequences either downstream or upstream of the PBS are inhibitory. This new function of the PBS was independent of its orientation and primer binding but associated with a particular destabilizing role in a proposed secondary structure. On the contrary, all sequences surrounding the PBS that are involved in stem formation of the first exon were found to suppress splicing. Targeted mutations that destabilized the central stem and compensatory mutations of the counter strand clearly validated the concept that murine leukemia virus attenuates its 5' splice site by forming an inhibitory stem-loop in its first exon. Importantly, this mode of splice regulation was conserved in a complete proviral clone. Some of the mutants that increase splicing revealed an opposite effect on translation, implying that the first exon also regulates this process. Together, these findings suggest that sequences upstream of the 5' splice site play an important role in splice regulation of simple retroviruses, directly or indirectly attenuating the efficiency of splicing.
Assuntos
Processamento Alternativo , Vírus da Leucemia Murina/genética , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Linhagem Celular , DNA Viral/genética , Éxons , Regulação Viral da Expressão Gênica , Vetores Genéticos , Vírus da Leucemia Murina/metabolismo , Camundongos , Modelos Moleculares , Conformação de Ácido Nucleico , Plasmídeos/genética , RNA Viral/química , RNA Viral/genética , Deleção de SequênciaRESUMO
BACKGROUND & AIMS: Development of cirrhosis in renal transplant recipients with chronic hepatitis B is associated with the accumulation of complex hepatitis B virus (HBV) variants carrying deletions in the C gene and/or preS region and deletions/insertions in the core promoter. Here, we characterized for the first time the phenotype of these complex HBV variants. METHODS: Representative full-length genomes of the HBV variants that were isolated and cloned from serum and liver of an immunosuppressed renal transplant recipient before and during end-stage liver disease were transfected into the human hepatoma cell line HuH7 and functionally analyzed. RESULTS: The variant genomes showed considerably reduced levels of precore and surface messenger RNA (mRNA) and of the major spliced pregenomic RNA, an increased level of pregenomic RNA, and a partial or complete defect in hepatitis B e antigen, core, and surface protein expression/secretion. Very low amounts of variant core protein with internal deletion were detectable. Reduced hepatitis B surface antigen secretion of some variants correlated with aberrant localization of surface proteins in endoplasmic reticulum. Despite the defects in viral protein expression, enhanced replication and enrichment in competition to wild-type HBV were observed. Enhanced reverse transcription and possibly increased levels of pregenomic RNA seem to be responsible for this effect. CONCLUSIONS: Development of cirrhosis is associated with accumulation of complex variants, which exhibit a drastically altered phenotype combining enhanced replication with defects in protein expression. This phenotype appears to be based on the major mutations in the core promoter and C gene but is considerably influenced by additional mutations throughout the genome.
Assuntos
Genoma Viral , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Cirrose Hepática/etiologia , RNA Viral/genética , Transcrição Gênica , Northern Blotting , Southern Blotting , Ensaio de Imunoadsorção Enzimática , Antígenos E da Hepatite B/imunologia , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/complicações , Hepatite B Crônica/patologia , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Cirrose Hepática/patologia , Mutação , Fenótipo , RNA Viral/fisiologia , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Replicação ViralRESUMO
The posttranscriptional regulatory element (PRE) is considered to enhance hepatitis B virus (HBV) gene expression by facilitating the nuclear export of intronless viral subgenomic RNAs. Its role in the RNA metabolism of the viral pregenomic RNA (pgRNA) is currently unknown. We identified a positively cis-acting splicing regulatory element (SRE-1) and present two lines of evidence for its functionality. Firstly, in a heterologous context SRE-1 functionally substitutes for a retroviral bidirectional exonic splicing enhancer (ESE). As expected, SRE-1 is a splicing enhancer also in its natural viral sequence context, since deletion of SRE-1 reduces splicing of pgRNA in cell culture experiments. Secondly, we show that stimulation of HBV RNA splicing by the splicing factor PSF was repressed by the PRE. Analysis of a variety of PSF mutants indicated that RNA-binding and protein-protein interaction were required to enhance splicing. In addition, we show that the PRE contributed to pgRNA stability, but has little influence on its nuclear export. Herein, we report for the first time that the PRE harbors splicing stimulating and inhibiting regulatory elements controlling processing of the viral pregenome. We discuss a model in which the regulation of pgRNA splicing depends on cellular factors interacting with the PRE.