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1.
Nucleic Acids Res ; 43(10): 5182-93, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25925570

RESUMO

Multiple myeloma, the second most frequent hematologic tumor after lymphomas, is an incurable cancer. Recent sequencing efforts have identified the ribonuclease DIS3 as one of the most frequently mutated genes in this disease. DIS3 represents the catalytic subunit of the exosome, a macromolecular complex central to the processing, maturation and surveillance of various RNAs. miRNAs are an evolutionarily conserved class of small noncoding RNAs, regulating gene expression at post-transcriptional level. Ribonucleases, including Drosha, Dicer and XRN2, are involved in the processing and stability of miRNAs. However, the role of DIS3 on the regulation of miRNAs remains largely unknown. Here we found that DIS3 regulates the levels of the tumor suppressor let-7 miRNAs without affecting other miRNA families. DIS3 facilitates the maturation of let-7 miRNAs by reducing in the cytoplasm the RNA stability of the pluripotency factor LIN28B, a inhibitor of let-7 processing. DIS3 inactivation, through the increase of LIN28B and the reduction of mature let-7, enhances the translation of let-7 targets such as MYC and RAS leading to enhanced tumorigenesis. Our study establishes that the ribonuclease DIS3, targeting LIN28B, sustains the maturation of let-7 miRNAs and suggests the increased translation of critical oncogenes as one of the biological outcomes of DIS3 inactivation.


Assuntos
Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/genética , Animais , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo
2.
Cell Mol Life Sci ; 70(8): 1439-50, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23212600

RESUMO

The receptor for activated C-kinase 1 (RACK1) is a conserved structural protein of 40S ribosomes. Strikingly, deletion of RACK1 in yeast homolog Asc1 is not lethal. Mammalian RACK1 also interacts with many nonribosomal proteins, hinting at several extraribosomal functions. A knockout mouse for RACK1 has not previously been described. We produced the first RACK1 mutant mouse, in which both alleles of RACK1 gene are defective in RACK1 expression (ΔF/ΔF), in a pure C57 Black/6 background. In a sample of 287 pups, we observed no ΔF/ΔF mice (72 expected). Dissection and genotyping of embryos at various stages showed that lethality occurs at gastrulation. Heterozygotes (ΔF/+) have skin pigmentation defects with a white belly spot and hypopigmented tail and paws. ΔF/+ have a transient growth deficit (shown by measuring pup size at P11). The pigmentation deficit is partly reverted by p53 deletion, whereas the lethality is not. ΔF/+ livers have mild accumulation of inactive 80S ribosomal subunits by polysomal profile analysis. In ΔF/+ fibroblasts, protein synthesis response to extracellular and pharmacological stimuli is reduced. These results highlight the role of RACK1 as a ribosomal protein converging signaling to the translational apparatus.


Assuntos
Neuropeptídeos/genética , Pigmentação , Biossíntese de Proteínas , Animais , Células Cultivadas , Perda do Embrião/genética , Embrião de Mamíferos/metabolismo , Embrião de Mamíferos/ultraestrutura , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuropeptídeos/metabolismo , Fenótipo , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores de Quinase C Ativada , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
3.
EMBO Rep ; 10(5): 459-65, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19373251

RESUMO

Eukaryotic ribosome biogenesis and translation are linked processes that limit the rate of cell growth. Although ribosome biogenesis and translation are mainly controlled by distinct factors, eukaryotic initiation factor 6 (eIF6) has been found to regulate both processes. eIF6 is a necessary protein with a unique anti-association activity, which prevents the interaction of 40S ribosomal subunits with 60S subunits through its binding to 60S ribosomes. In the nucleolus, eIF6 is a component of the pre-ribosomal particles and is required for the biogenesis of 60S subunits, whereas in the cytoplasm it mediates translation downstream from growth factors. The translational activity of eIF6 could be due to its anti-association properties, which are regulated by post-translational modifications; whether this anti-association activity is required for the biogenesis and nuclear export of ribosomes is unknown. eIF6 is necessary for tissue-specific growth and oncogene-driven transformation, and could be a new rate-limiting step for the initiation of translation.


Assuntos
Fatores de Iniciação de Peptídeos/fisiologia , Biossíntese de Proteínas/fisiologia , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Animais , Humanos , Modelos Biológicos , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas/genética
4.
Nat Commun ; 12(1): 6979, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34848685

RESUMO

Regulatory T cells (Treg cells) inhibit effector T cells and maintain immune system homeostasis. Treg cell maturation in peripheral sites requires inhibition of protein kinase mTORC1 and TGF-beta-1 (TGF-beta). While Treg cell maturation requires protein synthesis, mTORC1 inhibition downregulates it, leaving unanswered how Treg cells achieve essential mRNA translation for development and immune suppression activity. Using human CD4+ T cells differentiated in culture and genome-wide transcription and translation profiling, here we report that TGF-beta transcriptionally reprograms naive T cells to express Treg cell differentiation and immune suppression mRNAs, while mTORC1 inhibition impairs translation of T cell mRNAs but not those induced by TGF-beta. Rather than canonical mTORC1/eIF4E/eIF4G translation, Treg cell mRNAs utilize the eIF4G homolog DAP5 and initiation factor eIF3d in a non-canonical translation mechanism that requires cap-dependent binding by eIF3d directed by Treg cell mRNA 5' noncoding regions. Silencing DAP5 in isolated human naive CD4+ T cells impairs their differentiation into Treg cells. Treg cell differentiation is mediated by mTORC1 downregulation and TGF-beta transcriptional reprogramming that establishes a DAP5/eIF3d-selective mechanism of mRNA translation.


Assuntos
Diferenciação Celular , Fator de Iniciação 3 em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Terapia de Imunossupressão , Biossíntese de Proteínas , Linfócitos T Reguladores/metabolismo , Regulação para Baixo , Fator de Iniciação 3 em Eucariotos/genética , Fator de Iniciação Eucariótico 4G/genética , Regulação da Expressão Gênica , Células HEK293 , Homeostase , Humanos , Ativação Linfocitária , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , RNA Mensageiro , Fator de Crescimento Transformador beta1/metabolismo
5.
Biochem Biophys Res Commun ; 386(4): 598-601, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19540199

RESUMO

Protein homeostasis relies on a balance between protein synthesis and protein degradation. The ubiquitin-proteasome system is a major catabolic pathway for protein degradation. In this respect, proteasome inhibition has been used therapeutically for the treatment of cancer. Whether inhibition of protein degradation by proteasome inhibitor can repress protein translation via a negative feedback mechanism, however, is unknown. In this study, proteasome inhibitor MG-132 lowered the proliferation of colon cancer cells HT-29 and SW1116. In this connection, MG-132 reduced the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448 and Ser2481 and the phosphorylation of its downstream targets 4E-BP1 and p70/p85 S6 kinases. Further analysis revealed that MG-132 inhibited protein translation as evidenced by the reductions of (35)S-methionine incorporation and polysomes/80S ratio. Knockdown of raptor, a structural component of mTOR complex 1, mimicked the anti-proliferative effect of MG-132. To conclude, we demonstrate that the inhibition of protein degradation by proteasome inhibitor represses mTOR signaling and protein translation in colon cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/enzimologia , Inibidores de Cisteína Proteinase/farmacologia , Leupeptinas/farmacologia , Proteínas Quinases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Fosforilação/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Inibidores de Proteassoma , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas/genética , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR
6.
Biochem J ; 415(1): 77-85, 2008 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-18557705

RESUMO

RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.


Assuntos
Biossíntese de Proteínas/efeitos dos fármacos , Proteína Quinase C/fisiologia , Proteínas Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Células HeLa , Humanos , Masculino , Camundongos , Polirribossomos/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C beta , Pironas/farmacologia , Receptores de Quinase C Ativada , Ribossomos/metabolismo , Serina-Treonina Quinases TOR
7.
Cancer Res ; 79(13): 3360-3371, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043378

RESUMO

Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer that displays profound cancer stem cell (CSC) and mesenchymal features that promote rapid metastasis. Another hallmark of IBC is high infiltration of M2 tumor-associated (immune-suppressing) macrophages. The molecular mechanism that drives these IBC phenotypes is not well understood. Using patient breast tumor specimens, breast cancer cell lines, and a patient-derived xenograft model of IBC, we demonstrate that IBC strongly expresses IL8 and growth-regulated oncogene (GRO) chemokines that activate STAT3, which promotes development of high levels of CSC-like cells and a mesenchymal phenotype. We also show that IBC expresses high levels of many monocyte recruitment and macrophage polarization factors that attract and differentiate monocytes into tumor-promoting, immune-suppressing M2-like macrophages. The M2 macrophages in turn were found to secrete high levels of IL8 and GRO chemokines, thereby creating a feed-forward chemokine loop that further drives an IBC epithelial-to-mesenchymal transition. Our study uncovers an intricate IBC-initiated autocrine-paracrine signaling network between IBC cells and monocytes that facilitates development of this highly aggressive form of breast cancer. SIGNIFICANCE: This study uncovers a signaling network in which IBC cells commandeer macrophages to become tumor-promoting, and they in turn drive IBC cells to be more cancer stem-like, mesenchymal, and aggressive.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/13/3360/F1.large.jpg.


Assuntos
Quimiocinas/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Inflamatórias Mamárias/patologia , Macrófagos/patologia , Células-Tronco Mesenquimais/patologia , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral , Apoptose , Comunicação Autócrina , Proliferação de Células , Quimiocinas/genética , Feminino , Humanos , Neoplasias Inflamatórias Mamárias/genética , Neoplasias Inflamatórias Mamárias/metabolismo , Macrófagos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Neoplásicas/metabolismo , Comunicação Parácrina , Prognóstico , Transdução de Sinais , Células Tumorais Cultivadas
8.
Biochem Biophys Res Commun ; 376(1): 65-9, 2008 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-18768137

RESUMO

PKC isoform betaII modulates translation and can be recruited on ribosomes via its scaffold RACK1 (receptor for activated protein kinase C 1), which resides on the 40S ribosomal subunit. However, whether a PKC activity exists on the ribosome is not yet demonstrated. We purified native ribosomes by two different techniques, which avoid stripping of initiation factors and other associated proteins. In both cases, purified ribosomes are able to phosphorylate a specific PKC substrate, MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate). MARCKS phosphorylation is switched on by treatment with PKC agonist PMA (Phorbol 12-Myristate 13-Acetate). Consistently, the broad PKC inhibitor BMI (Bisindolyl Maleimide I) abrogates MARCKS phosphorylation. These data show that native ribosomes host active PKC and hence allow the phosphorylation of ribosome-associated substrates like initiation factors and mRNA binding proteins.


Assuntos
Proteína Quinase C/metabolismo , Ribossomos/enzimologia , Linhagem Celular , Proteínas de Fluorescência Verde/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Isoenzimas/metabolismo , Proteínas de Membrana/metabolismo , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C beta , Proteínas de Ligação a RNA/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
9.
Mol Cell Biol ; 37(5)2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27956700

RESUMO

mTOR coordinates growth signals with metabolic pathways and protein synthesis and is hyperactivated in many human cancers. mTOR exists in two complexes: mTORC1, which stimulates protein, lipid, and ribosome biosynthesis, and mTORC2, which regulates cytoskeleton functions. While mTOR is known to be involved in the DNA damage response, little is actually known regarding the functions of mTORC1 compared to mTORC2 in this regard or the respective impacts on transcriptional versus translational regulation. We show that mTORC1 and mTORC2 are both required to enact DNA damage repair and cell survival, resulting in increased cancer cell survival during DNA damage. Together mTORC1 and -2 enact coordinated transcription and translation of protective cell cycle and DNA replication, recombination, and repair genes. This coordinated transcriptional-translational response to DNA damage was not impaired by rapalog inhibition of mTORC1 or independent inhibition of mTORC1 or mTORC2 but was blocked by inhibition of mTORC1/2. Only mTORC1/2 inhibition reversed cancer cell resistance to DNA damage and replicative stress and increased tumor cell killing and tumor control by DNA damage therapies in animal models. When combined with DNA damage, inhibition of mTORC1/2 blocked transcriptional induction more strongly than translation of DNA replication, survival, and DNA damage response mRNAs.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , Replicação do DNA/genética , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Transcrição Gênica , Animais , Pontos de Checagem do Ciclo Celular/genética , DNA/metabolismo , Reparo do DNA/genética , Feminino , Fase G1/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fase S/genética , Estresse Fisiológico/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto
10.
PLoS One ; 8(3): e58051, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23526965

RESUMO

Malignant pleural mesothelioma (MPM) is a poor prognosis disease lacking adequate therapy. We have previously shown that ascorbic acid administration is toxic to MPM cells. Here we evaluated a new combined therapy consisting of ascorbate/epigallocatechin-3-gallate/gemcitabine mixture (called AND, for Active Nutrients/Drug). In vitro effects of AND therapy on various MPM cell lines revealed a synergistic cytotoxic mechanism. In vivo experiments on a xenograft mouse model for MPM, obtained by REN cells injection in immunocompromised mice, showed that AND strongly reduced the size of primary tumor as well as the number and size of metastases, and prevented abdominal hemorrhage. Kaplan Meier curves and the log-rank test indicated a marked increase in the survival of AND-treated animals. Histochemical analysis of dissected tumors showed that AND induced a shift from cell proliferation to apoptosis in cancer cells. Lysates of tumors from AND-treated mice, analyzed with an antibody array, revealed decreased TIMP-1 and -2 expressions and no effects on angiogenesis regulating factors. Multiplex analysis for signaling protein phosphorylation exhibited inactivation of cell proliferation pathways. The complex of data showed that the AND treatment is synergistic in vitro on MPM cells, and blocks in vivo tumor progression and metastasization in REN-based xenografts. Hence, the AND combination is proposed as a new treatment for MPM.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Mesotelioma/tratamento farmacológico , Neoplasias Pleurais/tratamento farmacológico , Animais , Ácido Ascórbico/administração & dosagem , Catequina/administração & dosagem , Catequina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/metabolismo , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Neoplasias Pleurais/metabolismo , Neoplasias Pleurais/patologia , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Gencitabina
11.
Biochem Biophys Res Commun ; 337(1): 89-94, 2005 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-16188229

RESUMO

Tif6p (eIF6) is necessary for 60S biogenesis, rRNA maturation and must be released from 60S to permit 80S assembly and translation. We characterized Tif6p interactors. Tif6p is mostly on 66S-60S pre-ribosomes, partly free. Tif6p complex(es) contain nucleo-ribosomal factors and Asc1p. Surprisingly, Tif6p particle contains the low-abundance endonuclease Sen34p. We analyzed Sen34p role on rRNA/tRNA synthesis, in vivo. Sen34p depletion impairs tRNA splicing and causes unexpected 80S accumulation. Accordingly, Sen34p overexpression causes 80S decrease and increased polysomes which suggest increased translational efficiency. With delayed kinetics, Sen34p depletion impairs rRNA processing. We conclude that Sen34p is absolutely required for tRNA splicing and that it is a rate-limiting element for efficient translation. Finally, we confirm that Tif6p accompanies 27S pre-rRNA maturation to 25S rRNA and we suggest that Sen34p endonuclease in Tif6p complex may affect also rRNA maturation.


Assuntos
Endorribonucleases/fisiologia , Processamento Pós-Transcricional do RNA , Splicing de RNA , RNA Ribossômico/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Transporte/metabolismo , Endorribonucleases/antagonistas & inibidores , Endorribonucleases/genética , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Proteínas de Filamentos Intermediários/metabolismo , Cinética , Fosfoproteínas/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/biossíntese , Proteínas Ribossômicas , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
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