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1.
Nat Commun ; 12(1): 4878, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34385447

RESUMO

A postprandial increase of translation mediated by eukaryotic Initiation Factor 6 (eIF6) occurs in the liver. Its contribution to steatosis and disease is unknown. In this study we address whether eIF6-driven translation contributes to disease progression. eIF6 levels increase throughout the progression from Non-Alcoholic Fatty Liver Disease (NAFLD) to hepatocellular carcinoma. Reduction of eIF6 levels protects the liver from disease progression. eIF6 depletion blunts lipid accumulation, increases fatty acid oxidation (FAO) and reduces oncogenic transformation in vitro. In addition, eIF6 depletion delays the progression from NAFLD to hepatocellular carcinoma, in vivo. Mechanistically, eIF6 depletion reduces the translation of transcription factor C/EBPß, leading to a drop in biomarkers associated with NAFLD progression to hepatocellular carcinoma and preserves mitochondrial respiration due to the maintenance of an alternative mTORC1-eIF4F translational branch that increases the expression of transcription factor YY1. We provide proof-of-concept that in vitro pharmacological inhibition of eIF6 activity recapitulates the protective effects of eIF6 depletion. We hypothesize the existence of a targetable, evolutionarily conserved translation circuit optimized for lipid accumulation and tumor progression.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Hepatopatia Gordurosa não Alcoólica/genética , Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas/genética , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Clofazimina/farmacologia , Dieta Hiperlipídica/efeitos adversos , Progressão da Doença , Inativação Gênica , Humanos , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/etiologia , Obesidade/genética , Obesidade/metabolismo , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/metabolismo
2.
Nat Immunol ; 22(6): 735-745, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34017124

RESUMO

Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Hematopoiese Clonal/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma Pulmonar de Células não Pequenas/terapia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Quimioterapia Adjuvante/métodos , Quitinases/metabolismo , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Conjuntos de Dados como Assunto , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Granzimas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismo
3.
Curr Biol ; 31(4): 892-899.e3, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33275890

RESUMO

The plant hormone auxin is a fundamental regulator of organ patterning and development that regulates gene expression via the canonical AUXIN RESPONSE FACTOR (ARF) and AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) combinatorial system. ARF and Aux/IAA factors interact, but at high auxin concentrations, the Aux/IAA transcriptional repressor is degraded, allowing ARF-containing complexes to activate gene expression. ARF5/MONOPTEROS (MP) is an important integrator of auxin signaling in Arabidopsis development and activates gene transcription in cells with elevated auxin levels. Here, we show that in ovules, MP is expressed in cells with low levels of auxin and can activate the expression of direct target genes. We identified and characterized a splice variant of MP that encodes a biologically functional isoform that lacks the Aux/IAA interaction domain. This MP11ir isoform was able to complement inflorescence, floral, and ovule developmental defects in mp mutants, suggesting that it was fully functional. Our findings describe a novel scenario in which ARF post-transcriptional regulation controls the formation of an isoform that can function as a transcriptional activator in regions of subthreshold auxin concentration.

4.
Cancer Res ; 80(21): 4693-4706, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32963011

RESUMO

Multiple myeloma is a plasma cell neoplasm characterized by the production of unfolded immunoglobulins, which cause endoplasmic reticulum (ER) stress and sensitivity to proteasome inhibition. The genomic landscape of multiple myeloma is characterized by the loss of several genes rarely mutated in other cancers that may underline specific weaknesses of multiple myeloma cells. One of these is FAM46C that is lost in more than 10% of patients with multiple myeloma. We show here that FAM46C is part of a new complex containing the ER-associated protein FNDC3A, which regulates trafficking and secretion and, by impairing autophagy, exacerbates proteostatic stress. Reconstitution of FAM46C in multiple myeloma cells that had lost it induced apoptosis and ER stress. Apoptosis was preceded by an increase of intracellular aggregates, which was not linked to increased translation of IgG mRNA, but rather to impairment of autophagy. Biochemical analysis showed that FAM46C requires interaction with ER bound protein FNDC3A to reside in the cytoplasmic side of the ER. FNDC3A was lost in some multiple myeloma cell lines. Importantly, depletion of FNDC3A increased the fitness of FAM46C-expressing cells and expression of FNDC3A in cells that had lost it recapitulated the effects of FAM46C, inducing aggregates and apoptosis. FAM46C and FNDC3A formed a complex that modulates secretion routes, increasing lysosome exocytosis. The cellular landscape generated by FAM46C/FNDC3A expression predicted sensitivity to sphingosine kinase inhibition. These results suggest that multiple myeloma cells remodel their trafficking machinery to cope with ER stress. SIGNIFICANCE: This study identifies a new multiple myeloma-specific tumor suppressor complex that regulates autophagy and unconventional secretion, highlighting the sensitivity of multiple myeloma cells to the accumulation of protein aggregates.


Assuntos
Fibronectinas/metabolismo , Mieloma Múltiplo/patologia , Nucleotidiltransferases/metabolismo , Agregação Patológica de Proteínas/metabolismo , Animais , Autofagia/fisiologia , Genes Supressores de Tumor , Xenoenxertos , Humanos , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Nucleotidiltransferases/genética , Agregados Proteicos/fisiologia , Transporte Proteico/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
5.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
7.
Mol Cell Biol ; 38(23)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30201806

RESUMO

The translational capability of ribosomes deprived of specific nonfundamental ribosomal proteins may be altered. Physiological mechanisms are scanty, and it is unclear whether free ribosomal proteins can cross talk with the signaling machinery. RACK1 (receptor for activated C kinase 1) is a highly conserved scaffold protein, located on the 40S subunit near the mRNA exit channel. RACK1 is involved in a variety of intracellular contexts, both on and off the ribosomes, acting as a receptor for proteins in signaling, such as the protein kinase C (PKC) family. Here we show that the binding of RACK1 to ribosomes is essential for full translation of capped mRNAs and efficient recruitment of eukaryotic initiation factor 4E (eIF4E). In vitro, when RACK1 is partially depleted, supplementing the ribosome machinery with wild-type RACK1 restores the translational capability, whereas the addition of a RACK1 mutant that is unable to bind ribosomes does not. Outside the ribosome, RACK1 has a reduced half-life. By accumulating in living cells, free RACK1 exerts an inhibitory phenotype, impairing cell cycle progression and repressing global translation. Here we present RACK1 binding to ribosomes as a crucial way to regulate translation, possibly through interaction with known partners on or off the ribosome that are involved in signaling.

8.
Cancer Res ; 78(20): 5741-5753, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30072395

RESUMO

The expression of miRNAs in cancer has been widely studied and has allowed the definition of oncomirs and oncosuppressors. We note that it is often underestimated that many mRNAs are expressed, but translationally silent. In spite of this, systematic identification of miRNAs in equilibrium with their target mRNAs on polysomes has not been widely exploited. To identify biologically active oncomirs, we performed a screen for miRNAs acting on the polysomes of malignant mesothelioma (MPM) cells. Only a small percentage of expressed miRNAs physically associated with polysomes. On polysomes, we identified miRNAs already characterized in MPM, as well as novel ones like miR-24-3p, which acted as a promigratory miRNA in all cancer cells tested. miR-24-3p positively regulated Rho-GTP activity, and inhibition of miR-24-3p reduced growth in MPM cells. Analysis of miR-24-3p common targets, in two mesothelioma cell lines, identified a common subset of downregulated genes. These same genes were downregulated during the progression of multiple cancer types. Among the specific targets of miR-24-3p was cingulin, a tight junction protein that inhibits Rho-GTP activity. Overexpression of miR-24-3p only partially abrogated cingulin mRNA, but completely abrogated cingulin protein, confirming its action via translational repression. We suggest that miR-24-3p is an oncomir and speculate that identification of polysome-associated miRNAs efficiently sorts out biologically active miRNAs from inactive ones.Significance: Subcellular localization of miRNAs may predict their role in cancer and identify novel oncogenic miRNAs involved in cancer progression.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/20/5741/F1.large.jpg Cancer Res; 78(20); 5741-53. ©2018 AACR.


Assuntos
Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , MicroRNAs/genética , Neoplasias/genética , Polirribossomos/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Progressão da Doença , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Proteínas de Membrana/metabolismo , Mesotelioma/genética , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas dos Microfilamentos/metabolismo , Metástase Neoplásica , Ribossomos/metabolismo , Análise de Sequência de RNA , Cicatrização
9.
World J Biol Chem ; 8(1): 45-56, 2017 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-28289518

RESUMO

MicroRNAs (miRNAs) are pervasively expressed and regulate most biological functions. They function by modulating transcriptional and translational programs and therefore they orchestrate both physiological and pathological processes, such as development, cell differentiation, proliferation, apoptosis and tumor growth. miRNAs work as small guide molecules in RNA silencing, by negatively regulating the expression of several genes both at mRNA and protein level, by degrading their mRNA target and/or by silencing translation. One of the most recent advances in the field is the comprehension of their role in oncogenesis. The number of miRNA genes is increasing and an alteration in the level of miRNAs is involved in the initiation, progression and metastases formation of several tumors. Some tumor types show a distinct miRNA signature that distinguishes them from normal tissues and from other cancer types. Genetic and biochemical evidence supports the essential role of miRNAs in tumor development. Although the abnormal expression of miRNAs in cancer cells is a widely accepted phenomenon, the cause of this dysregulation is still unknown. Here, we discuss the biogenesis of miRNAs, focusing on the mechanisms by which they regulate protein synthesis. In addition we debate on their role in cancer, highlighting their potential to become therapeutic targets.

10.
Hepatology ; 65(5): 1512-1525, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28027576

RESUMO

Ferroportin (FPN1) is the sole iron exporter in mammals, but its cell-specific function and regulation are still elusive. This study examined FPN1 expression in human macrophages, the cells that are primarily responsible on a daily basis for plasma iron turnover and are central in the pathogenesis of ferroportin disease (FD), the disease attributed to lack-of-function FPN1 mutations. We characterized FPN1 protein expression and traffic by confocal microscopy, western blotting, gel filtration, and immunoprecipitation studies in macrophages from control blood donors (donor) and patients with either FPN1 p.A77D, p.G80S, and p.Val162del lack-of-function or p.A69T gain-of-function mutations. We found that in normal macrophages, FPN1 cycles in the early endocytic compartment does not multimerize and is promptly degraded by hepcidin (Hepc), its physiological inhibitor, within 3-6 hours. In FD macrophages, endogenous FPN1 showed a similar localization, except for greater accumulation in lysosomes. However, in contrast with previous studies using overexpressed mutant protein in cell lines, FPN1 could still reach the cell surface and be normally internalized and degraded upon exposure to Hepc. However, when FD macrophages were exposed to large amounts of heme iron, in contrast to donor and p.A69T macrophages, FPN1 could no longer reach the cell surface, leading to intracellular iron retention. CONCLUSION: FPN1 cycles as a monomer within the endocytic/plasma membrane compartment and responds to its physiological inhibitor, Hepc, in both control and FD cells. However, in FD, FPN1 fails to reach the cell surface when cells undergo high iron turnover. Our findings provide a basis for the FD characterized by a preserved iron transfer in the enterocytes (i.e., cells with low iron turnover) and iron retention in cells exposed to high iron flux, such as liver and spleen macrophages. (Hepatology 2017;65:1512-1525).


Assuntos
Proteínas de Transporte de Cátions/deficiência , Macrófagos/metabolismo , Animais , Estudos de Casos e Controles , Células Hep G2 , Hepcidinas/metabolismo , Humanos , Ferro/metabolismo , Camundongos
11.
Biochem Soc Trans ; 44(6): 1667-1673, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27913676

RESUMO

Over the past few years, there has been a growing interest in the interconnection between translation and metabolism. Important oncogenic pathways, like those elicited by c-Myc transcription factor and mTOR kinase, couple the activation of the translational machinery with glycolysis and fatty acid synthesis. Eukaryotic initiation factor 6 (eIF6) is a factor necessary for 60S ribosome maturation. eIF6 acts also as a cytoplasmic translation initiation factor, downstream of growth factor stimulation. eIF6 is up-regulated in several tumor types. Data on mice models have demonstrated that eIF6 cytoplasmic activity is rate-limiting for Myc-induced lymphomagenesis. In spite of this, eIF6 is neither transcriptionally regulated by Myc, nor post-transcriptionally regulated by mTOR. eIF6 stimulates a glycolytic and fatty acid synthesis program necessary for tumor growth. eIF6 increases the translation of transcription factors necessary for lipogenesis, such as CEBP/ß, ATF4 and CEBP/δ. Insulin stimulation leads to an increase in translation and fat synthesis blunted by eIF6 deficiency. Paradoxycally, long-term inhibition of eIF6 activity increases insulin sensitivity, suggesting that the translational activation observed upon insulin and growth factors stimulation acts as a feed-forward mechanism regulating lipid synthesis. The data on the role that eIF6 plays in cancer and in insulin sensitivity make it a tempting pharmacological target for cancers and metabolic diseases. We speculate that eIF6 inhibition will be particularly effective especially when mTOR sensitivity to rapamycin is abrogated by RAS mutations.


Assuntos
Fatores de Iniciação de Peptídeos/genética , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição/genética , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Lipogênese/efeitos dos fármacos , Lipogênese/genética , Doenças Metabólicas/genética , Doenças Metabólicas/metabolismo , Camundongos , Modelos Genéticos , Neoplasias/genética , Neoplasias/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/metabolismo
12.
Oncotarget ; 6(35): 37471-85, 2015 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-26462016

RESUMO

eIF6 is an antiassociation factor that regulates the availability of active 80S. Its activation is driven by the RACK1/PKCß axis, in a mTORc1 independent manner. We previously described that eIF6 haploinsufficiency causes a striking survival in the Eµ-Myc mouse lymphoma model, with lifespans extended up to 18 months. Here we screen for eIF6 expression in human cancers. We show that Malignant Pleural Mesothelioma tumors (MPM) and a MPM cell line (REN cells) contain high levels of hyperphosphorylated eIF6. Enzastaurin is a PKC beta inhibitor used in clinical trials. We prove that Enzastaurin treatment decreases eIF6 phosphorylation rate, but not eIF6 protein stability. The growth of REN, in vivo, and metastasis are reduced by either Enzastaurin treatment or eIF6 shRNA. Molecular analysis reveals that eIF6 manipulation affects the metabolic status of malignant mesothelioma cells. Less glycolysis and less ATP content are evident in REN cells depleted for eIF6 or treated with Enzastaurin (Anti-Warburg effect). We propose that eIF6 is necessary for malignant mesothelioma growth, in vivo, and can be targeted by kinase inhibitors.


Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Fatores de Iniciação em Eucariotos/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Indóis/farmacologia , Mesotelioma/genética , Mesotelioma/patologia , Mesotelioma/terapia , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Neoplasias Pleurais/terapia , Proteína Quinase C beta/antagonistas & inibidores , Proteína Quinase C beta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica , Interferência de RNA , Terapêutica com RNAi , Transdução de Sinais , Fatores de Tempo , Transfecção , Carga Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
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