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1.
Biol Chem ; 404(11-12): 1123-1136, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37632732

RESUMO

Small non-coding RNAs (sncRNA) are involved in many steps of the gene expression cascade and regulate processing and expression of mRNAs by the formation of ribonucleoprotein complexes (RNP) such as the RNA-induced silencing complex (RISC). By analyzing small RNA Seq data sets, we identified a sncRNA annotated as piR-hsa-1254, which is likely derived from the 3'-end of 7SL RNA2 (RN7SL2), herein referred to as snc7SL RNA. The 7SL RNA is an abundant long non-coding RNA polymerase III transcript and serves as structural component of the cytoplasmic signal recognition particle (SRP). To evaluate a potential functional role of snc7SL RNA, we aimed to define its cellular localization by live cell imaging. Therefore, a Molecular Beacon (MB)-based method was established to compare the subcellular localization of snc7SL RNA with its precursor 7SL RNA. We designed and characterized several MBs in vitro and tested those by live cell fluorescence microscopy. Using a multiplex approach, we show that 7SL RNA localizes mainly to the endoplasmic reticulum (ER), as expected for the SRP, whereas snc7SL RNA predominately localizes to the nucleus. This finding suggests a fundamentally different function of 7SL RNA and its derivate snc7SL RNA.


Assuntos
RNA Citoplasmático Pequeno , Partícula de Reconhecimento de Sinal , Partícula de Reconhecimento de Sinal/genética , RNA , RNA Citoplasmático Pequeno/genética , RNA Citoplasmático Pequeno/metabolismo , RNA Mensageiro
2.
Cancers (Basel) ; 15(6)2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36980635

RESUMO

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.

3.
Hematol Oncol Stem Cell Ther ; 16(3): 217-229, 2023 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-34848216

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/genética
4.
Cancers (Basel) ; 13(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33477794

RESUMO

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.

5.
Int J Med Sci ; 18(1): 137-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33390782

RESUMO

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 & derivados
6.
RNA Biol ; 18(2): 218-236, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32687431

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/metabolismo
7.
Methods Mol Biol ; 2106: 121-136, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31889254

RESUMO

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/química
8.
Mol Cell Biol ; 38(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29084811

RESUMO

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 , Ubiquitinação
9.
PLoS One ; 12(3): e0173246, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28291789

RESUMO

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/metabolismo
10.
PLoS One ; 11(5): e0156365, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27224031

RESUMO

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 , Sumoilação
11.
Oncotarget ; 7(20): 29664-76, 2016 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-27105491

RESUMO

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 Cima
12.
Nucleic Acids Res ; 43(1): 581-94, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25520193

RESUMO

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-B
13.
PLoS One ; 7(9): e45197, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23028842

RESUMO

BACKGROUND: Suppressor of cytokine signaling 3 (SOCS3) is an inducible endogenous negative regulator of signal transduction and activator of transcription 3 (STAT3). Epigenetic silencing of SOCS3 has been shown in head and neck squamous cell carcinoma (HNSCC), which is associated with increased activation of STAT3. There is scarce information on the functional role of the reduction of SOCS3 expression and no information on altered subcellular localization of SOCS3 in HNSCC. METHODOLOGY/PRINCIPAL FINDINGS: We assessed endogenous SOCS3 expression in different HNSCC cell lines by RT-qPCR and western blot. Immunofluorescence and western blot were used to study the subcellular localization of endogenous SOCS3 induced by IL-6. Overexpression of SOCS3 by CMV-driven plasmids and siRNA-mediated inhibition of endogenous SOCS3 were used to verify the role of SOCS3 on tumor cell proliferation, viability, invasion and migration in vitro. In vivo relevance of SOCS3 expression in HNSCC was studied by quantitative immunohistochemistry of commercially-available tissue microarrays. Endogenous expression of SOCS3 was heterogeneous in four HNSCC cell lines and surprisingly preserved in most of these cell lines. Subcellular localization of endogenous SOCS3 in the HNSCC cell lines was predominantly nuclear as opposed to cytoplasmic in non-neoplasic epithelial cells. Overexpression of SOCS3 produced a relative increase of the protein in the cytoplasmic compartment and significantly inhibited proliferation, migration and invasion, whereas inhibition of endogenous nuclear SOCS3 did not affect these events. Analysis of tissue microarrays indicated that loss of SOCS3 is an early event in HNSCC and was correlated with tumor size and histological grade of dysplasia, but a considerable proportion of cases presented detectable expression of SOCS3. CONCLUSION: Our data support a role for SOCS3 as a tumor suppressor gene in HNSCC with relevance on proliferation and invasion processes and suggests that abnormal subcellular localization impairs SOCS3 function in HNSCC cells.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/genética , Invasividade Neoplásica/genética , Fator de Transcrição STAT3/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Proliferação de Células , Citoplasma/genética , Citoplasma/metabolismo , Citoplasma/ultraestrutura , Inativação Gênica , Neoplasias de Cabeça e Pescoço/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Humanos , RNA Interferente Pequeno , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de Citocina/metabolismo
14.
Proc Natl Acad Sci U S A ; 109(34): E2276-83, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22652566

RESUMO

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit(V558Δ) mutation found in a familial case of GIST and the Kit(T669I) (human KIT(T670I)) "gatekeeper" mutation found in imatinib-resistant GIST patients. Similar to Kit(V558/+) mice, Kit(V558;T669I/+) mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant Kit(V558/+) control mice, treatment of the Kit(V558;T669I/+) mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of Kit(V558;T669I/+) mice with sunitinib or sorafenib. Although tumor lesions were smaller in Kit(V558;T669I/+) mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in Kit(V558;T669I/+) mice. Strikingly, the Kit(V558;T669I/+) mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.


Assuntos
Eritrócitos/citologia , Tumores do Estroma Gastrointestinal/genética , Mutação , Piperazinas/farmacologia , Policitemia/genética , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/farmacologia , Animais , Antineoplásicos/farmacologia , Benzamidas , Linhagem da Célula , Dasatinibe , Modelos Animais de Doenças , Resistência a Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Éxons , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Mesilato de Imatinib , Camundongos , Fenótipo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Tiazóis/farmacologia
15.
PLoS One ; 6(10): e25402, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22016766

RESUMO

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-B
16.
Front Biosci ; 13: 5533-47, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18508603

RESUMO

Acute and chronic hepatitis B virus (HBV) infection is the cause of about 1 million deaths each year worldwide. Although a vaccine for HBV is available, new HBV infections are appearing at alarming rates and maternal-fetal transmission is a major cause of viral spread. Thus, new and effective antiviral strategies are necessary for the 300 million chronically HBV-infected individuals to reduce their morbidity and mortality. Precise processing of viral RNAs is essential for the hepatitis viral life cycle, and, to our knowledge, the processing, nuclear export, and stabilization/degradation of viral RNAs are exclusively mediated by host factors. Thus, identification of host factors required for viral RNA metabolism and subsequent molecular analysis of the interactions between viral RNAs and corresponding host factors might represent novel avenues for the development of new antiviral strategies. In this review, we summarize the current knowledge about the posttranscriptional control of HBV RNA metabolism.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Processamento Pós-Transcricional do RNA , RNA Viral/genética , Núcleo Celular/virologia
17.
Proc Natl Acad Sci U S A ; 103(34): 12843-8, 2006 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-16908864

RESUMO

Kit receptor-activating mutations are critical in the pathogenesis of gastrointestinal stromal tumors (GIST). We investigated mechanisms of oncogenic Kit signaling and the consequences of therapeutic intervention in a mouse model of human GIST. Treatment of GIST mice with imatinib decreased cell proliferation and increased apoptosis in the tumor. Analysis of tumor tissue from imatinib-treated mice showed diminished phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) signaling suggesting that oncogenic Kit signaling critically contributes to the translational response in GIST. Treatment with RAD001 (everolimus), an mTOR inhibitor, diminished the translational response and cell proliferation in tumor lesions, pointing to mTOR inhibition as a therapeutic approach for imatinib-resistant GIST. Analysis of RNA expression profiles in GIST lesions with and without imatinib treatment showed changes in expression of IFN-inducible genes and cell cycle regulators. These results convincingly show that KitV558Delta/+ mice represent a unique faithful mouse model of human familial GIST, and they demonstrate the utility of these mice for preclinical investigations and to elucidate oncogenic signaling mechanisms by using genetic approaches and targeted pharmacological intervention.


Assuntos
Modelos Animais de Doenças , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais , Animais , Benzamidas , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo , Everolimo , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Mesilato de Imatinib , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Piperazinas/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Pirimidinas/uso terapêutico , Sirolimo/análogos & derivados , Sirolimo/uso terapêutico , Serina-Treonina Quinases TOR
18.
Nucleic Acids Res ; 34(1): 353-63, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16410615

RESUMO

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.


Assuntos
Regulação Viral da Expressão Gênica , Vírus da Hepatite B/genética , Splicing de RNA , RNA Viral/química , RNA Viral/metabolismo , Sequências Reguladoras de Ácido Ribonucleico , Linhagem Celular Tumoral , Vírus da Hepatite B/metabolismo , Humanos , Fator de Processamento Associado a PTB , Proteínas de Ligação a RNA/metabolismo
19.
Blood ; 106(12): 3958-61, 2005 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-16081693

RESUMO

Multiple genetic alterations are required to induce acute myelogenous leukemia (AML). Mutations in the extracellular domain of the KIT receptor are almost exclusively found in patients with AML carrying translocations or inversions affecting members of the core binding factor (CBF) gene family and correlate with a high risk of relapse. We demonstrate that these complex insertion and deletion mutations lead to constitutive activation of the KIT receptor, which induces factor-independent growth of interleukin-3 (IL-3)-dependent cells. Mutation of the evolutionary conserved amino acid D419 within the extracellular domain was sufficient to constitutively activate the KIT receptor, although high expression levels were required. Dose-dependent growth inhibition and apoptosis were observed using either the protein tyrosine kinase inhibitor imatinib mesylate (STI571, Gleevec) or by blocking the phosphoinositide-3-kinase (PI3K)-AKT pathway. Our data show that the addition of kinase inhibitors to conventional chemotherapy might be a new therapeutic option for CBF-AML expressing mutant KIT.


Assuntos
Antineoplásicos/farmacologia , Fatores de Ligação ao Core/genética , Leucemia Mieloide Aguda/genética , Piperazinas/farmacologia , Pirimidinas/farmacologia , Fator de Células-Tronco/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Benzamidas , Humanos , Mesilato de Imatinib , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Mutagênese Sítio-Dirigida , Mutação
20.
Hepatology ; 42(1): 93-103, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15962285

RESUMO

The biological properties of latent or occult hepatitis B virus (HBV) have been poorly characterized as a result of the extremely low virus concentration. This report describes the phenotype of HBV reactivating in two patients after an HBsAg-negative latency period. One patient had latent HBV infection for at least 12 years without detectable viremia and symptoms of liver disease. Several full-length HBV genomes were cloned at reactivation, sequenced, and functionally tested by transfection into HuH7 cells. Genomes from both patients showed a low replication phenotype. It was caused at the level of RNA encapsidation or HBV DNA synthesis, but was not attributable to uncommon mutations in the terminal protein domain of P protein. A substantial subpopulation ( approximately 50%) of genomes from one patient did not express pre-S2/S mRNA and HBsAg. Site-directed mutagenesis identified a single G-A mutation within the S gene (position 458) to be responsible for this effect. The G458A mutation was also effective if the S gene was placed under control of a heterologous promoter. Furthermore, nuclear run-on transcription showed that the G458A mutation acts at the posttranscriptional level. The mutation affected a 5' splice site and prevented splicing of the pre-S2/S mRNA from position 458 to 1305. In conclusion, HBV latency may be characterized by viruses with reduced replication competence and antigen expression. In one patient, HBsAg expression was terminated by an as yet undescribed posttranscriptional mechanism. A single mutation inactivated a 5' splice site that is obviously essential for pre-S2/S mRNA accumulation. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270-9139/suppmat/index.html).


Assuntos
Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Ativação Viral , Latência Viral , Portador Sadio/imunologia , Células Clonais , Hepatite B/fisiopatologia , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Humanos , Mutação
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