RESUMO
The liver is a metabolic hub characterized by high levels of protein synthesis. Eukaryotic initiation factors, eIFs, control the first phase of translation, initiation. Initiation factors are essential for tumor progression and, since they regulate the translation of specific mRNAs downstream of oncogenic signaling cascades, may be druggable. In this review, we address the issue of whether the massive translational machinery of liver cells contributes to liver pathology and to the progression of hepatocellular carcinoma (HCC); it represents a valuable biomarker and druggable target. First, we observe that the common markers of HCC cells, such as phosphorylated ribosomal protein S6, belong to the ribosomal and translational apparatus. This fact is in agreement with observations that demonstrate a huge amplification of the ribosomal machinery during the progression to HCC. Some translation factors, such as eIF4E and eIF6, are then harnessed by oncogenic signaling. In particular, the action of eIF4E and eIF6 is particularly important in HCC when driven by fatty liver pathologies. Indeed, both eIF4E and eIF6 amplify at the translational level the production and accumulation of fatty acids. As it is evident that abnormal levels of these factors drive cancer, we discuss their therapeutic value.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Neoplasias Hepáticas/metabolismo , Divisão Celular , Ribossomos/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Fatores de Iniciação em Eucariotos/antagonistas & inibidores , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fatores de Iniciação de Peptídeos/antagonistas & inibidores , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismoRESUMO
Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.
Assuntos
Homeostase , Fenótipo , Proteínas/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica , Dano ao DNA , Fibroblastos/metabolismo , Ácido Láctico/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismoRESUMO
Here we discuss the function of eukaryotic initiation factor 6 (eIF6; Tif6 in yeast). eIF6 binds 60S ribosomal subunits and blocks their joining to 40S. In this context, we propose that eIF6 impedes unproductive 80S formation, namely, the formation of 80S subunits without mRNA. Genetic evidence shows that eIF6 has a dual function: in yeast and mammals, nucleolar eIF6 is necessary for the biogenesis of 60S subunits. In mammals, cytoplasmic eIF6 is required for insulin and growth factor-stimulated translation. In contrast to other translation factors, eIF6 activity is not under mTOR control. The physiological significance of eIF6 impacts on cancer and on inherited Shwachman-Bodian-Diamond syndrome. eIF6 is overexpressed in specific human tumors. In a murine model of lymphomagenesis, eIF6 depletion leads to a striking increase of survival, without adverse effects. Shwachman-Bodian-Diamond syndrome is caused by loss of function of SBDS protein. In yeast, point mutations of Tif6, the yeast homolog of eIF6, rescue the quasi-lethal effect due to the loss of the SBDS homolog, Sdo1. We propose that eIF6 is a node regulator of ribosomal function and predict that prioritizing its pharmacological targeting will be of benefit in cancer and Shwachman-Bodian-Diamond syndrome. This article is part of a Special Issue entitled: Translation and Cancer.
Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Linfoma/metabolismo , Proteínas de Neoplasias/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Animais , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/patologia , Fatores de Iniciação em Eucariotos/genética , Insuficiência Pancreática Exócrina/genética , Insuficiência Pancreática Exócrina/metabolismo , Insuficiência Pancreática Exócrina/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipomatose/genética , Lipomatose/metabolismo , Lipomatose/patologia , Linfoma/genética , Linfoma/patologia , Camundongos , Proteínas de Neoplasias/genética , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Fatores de Iniciação de Peptídeos/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Síndrome de Shwachman-Diamond , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , TransativadoresRESUMO
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/metabolismoRESUMO
Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here we show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6(+/-) cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6(+/-) mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6(+/-) cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.
Assuntos
Transformação Celular Neoplásica , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/metabolismo , Fase S , Tecido Adiposo/citologia , Animais , Peso Corporal , Divisão Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Fibroblastos , Fase G1/efeitos dos fármacos , Heterozigoto , Insulina/farmacologia , Fígado/citologia , Fígado/crescimento & desenvolvimento , Camundongos , Células NIH 3T3 , Oncogenes/genética , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fatores de Iniciação de Peptídeos/deficiência , Fatores de Iniciação de Peptídeos/genética , Ribossomos/química , Ribossomos/metabolismo , Fase S/efeitos dos fármacosRESUMO
Ribosomopathies are defined as inherited diseases in which ribosomal factors are mutated. In general, they present multiorgan symptoms. In spite of the fact that in cellular models, ribosomal insufficiency leads to a reduced rate of oncogenic transformation, patients affected by ribosomopathies present a paradoxical increase in cancer incidence. Several hypotheses that explain this paradox have been formulated, mostly on the assumption that altered ribosomes in a stem cell induce compensatory changes that lead to a cancer cell. For instance, the lack of a specific ribosomal protein can lead to the generation of an abnormal ribosome, an oncoribosome, that itself leads to altered translation and increased tumorigenesis. Alternatively, the presence of ribosomal stress may induce compensatory proliferation that in turns selects the loss of tumor suppressors such as p53. However, modern views on cancer have shifted the focus from the cancer cell to the tumor microenvironment. In particular, it is evident that human lymphocytes are able to eliminate mutant cells and contribute to the maintenance of cancer-free tissues. Indeed, many tumors develop in conditions of reduced immune surveillance. In this review, we summarize the current evidence and attempt to explain cancer and ribosomopathies from the perspective of the microenvironment.
RESUMO
Malignant mesothelioma is a type of cancer that affects the mesothelium. It is an aggressive and deadly form of cancer that is often caused by exposure to asbestos. At the molecular level, it is characterized by a low number of genetic mutations and high heterogeneity among patients. In this work, we analyzed the plasticity of gene expression of primary mesothelial cancer cells by comparing their properties on 2D versus 3D surfaces. First, we derived from primary human samples four independent primary cancer cells. Then, we used Nichoids, which are micro-engineered 3D substrates, as three-dimensional structures. Nichoids limit the dimension of adhering cells during expansion by counteracting cell migration between adjacent units of a substrate with their microarchitecture. Tumor cells grow effectively on Nichoids, where they show enhanced proliferation. We performed RNAseq analyses on all the samples and compared the gene expression pattern of Nichoid-grown tumor cells to that of cells grown in a 2D culture. The PCA analysis showed that 3D samples were more transcriptionally similar compared to the 2D ones. The 3D Nichoids induced a transcriptional remodeling that affected mainly genes involved in extracellular matrix assembly. Among these genes responsible for collagen formation, COL1A1 and COL5A1 exhibited elevated expression, suggesting changes in matrix stiffness. Overall, our data show that primary mesothelioma cells can be effectively expanded in Nichoids and that 3D growth affects the cells' tensegrity or the mechanical stability of their structure.
Assuntos
Mesotelioma Maligno , Mesotelioma , Humanos , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma/patologia , Colágeno , Movimento Celular/genéticaRESUMO
BACKGROUND: The COVID-19 pandemic is an infectious disease caused by SARS-CoV-2. The first step of SARS-CoV-2 infection is the recognition of angiotensin-converting enzyme 2 (ACE2) receptors by the receptor-binding domain (RBD) of the viral Spike (S) glycoprotein. Although the molecular and structural bases of the SARS-CoV-2-RBD/hACE2 interaction have been thoroughly investigated in vitro, the relationship between hACE2 expression and in vivo infection is less understood. METHODS: Here, we developed an efficient SARS-CoV-2-RBD binding assay suitable for super resolution microscopy and simultaneous hACE2 immunodetection and mapped the correlation between hACE2 receptor abundance and SARS-CoV-2-RBD binding, both in vitro and in human lung biopsies. Next, we explored the specific proteome of SARS-CoV-2-RBD/hACE2 through a comparative mass spectrometry approach. FINDINGS: We found that only a minority of hACE2 positive spots are actually SARS-CoV-2-RBD binding sites, and that the relationship between SARS-CoV-2-RBD binding and hACE2 presence is variable, suggesting the existence of additional factors. Indeed, we found several interactors that are involved in receptor localization and viral entry and characterized one of them: SLC1A5, an amino acid transporter. High-resolution receptor-binding studies showed that co-expression of membrane-bound SLC1A5 with hACE2 predicted SARS-CoV-2 binding and entry better than hACE2 expression alone. SLC1A5 depletion reduces SARS-CoV-2 binding and entry. Notably, the Omicron variant is more efficient in binding hACE2 sites, but equally sensitive to SLC1A5 downregulation. INTERPRETATION: We propose a method for mapping functional SARS-CoV-2 receptors in vivo. We confirm the existence of hACE2 co-factors that may contribute to differential sensitivity of cells to infection. FUNDING: This work was supported by an unrestricted grant from "Fondazione Romeo ed Enrica Invernizzi" to Stefano Biffo and by AIRC under MFAG 2021 - ID. 26178 project - P.I. Manfrini Nicola.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/metabolismo , Internalização do Vírus , Pandemias , Receptores Virais/química , Receptores Virais/metabolismo , Ligação Proteica , Pulmão/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Sistema ASC de Transporte de Aminoácidos/metabolismoRESUMO
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/genéticaRESUMO
We performed a systematic analysis of the translation rate of tumor-infiltrating lymphocytes (TILs) and the microenvironment inputs affecting it, both in humans and in mice. Measurement of puromycin incorporation, a proxy of protein synthesis, revealed an increase of translating CD4+ and CD8+ cells in tumors, compared to normal tissues. High translation levels are associated with phospho-S6 labeling downstream of mTORC1 activation, whereas low levels correlate with hypoxic areas, in agreement with data showing that T cell receptor stimulation and hypoxia act as translation stimulators and inhibitors, respectively. Additional analyses revealed the specific phenotype of translating TILs. CD8+ translating cells have enriched expression of IFN-γ and CD-39, and reduced SLAMF6, pointing to a cytotoxic phenotype. CD4+ translating cells are mostly regulatory T cells (Tregs) with enriched levels of CTLA-4 and Ki67, suggesting an expanding immunosuppressive phenotype. In conclusion, the majority of translationally active TILs is represented by cytotoxic CD8+ and suppressive CD4+ Tregs, implying that other subsets may be largely composed by inactive bystanders.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Linfócitos do Interstício Tumoral/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level. METHODS: We analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein. RESULTS: We found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in patients with truncating variations. We found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense variations. In this case, we excluded SACS mRNA decay, defective translation, or faster posttranslational degradation as possible causes of protein reduction. Conversely, our results demonstrate that nascent mutant sacsin protein undergoes cotranslational ubiquitination and degradation. DISCUSSION: Our results provide a mechanistic explanation for the lack of genotype-phenotype correlation in ARSACS. We also propose a new and unambiguous criterion for ARSACS diagnosis that is based on the evaluation of sacsin level. Last, we identified preemptive degradation of a mutant protein as a novel cause of a human disease.
Assuntos
Proteínas de Choque Térmico , Ataxias Espinocerebelares , Ataxia/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Espasticidade Muscular/diagnóstico , Espasticidade Muscular/genética , Mutação/genética , Ataxias Espinocerebelares/congênito , Ataxias Espinocerebelares/diagnóstico , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologiaRESUMO
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/metabolismoRESUMO
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 TestesRESUMO
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/metabolismoRESUMO
Ribosomes have been long considered as executors of the translational program. The fact that ribosomes can control the translation of specific mRNAs or entire cellular programs is often neglected. Ribosomopathies, inherited diseases with mutations in ribosomal factors, show tissue specific defects and cancer predisposition. Studies of ribosomopathies have paved the way to the concept that ribosomes may control translation of specific mRNAs. Studies in Drosophila and mice support the existence of heterogeneous ribosomes that differentially translate mRNAs to coordinate cellular programs. Recent studies have now shown that ribosomal activity is not only a critical regulator of growth but also of metabolism. For instance, glycolysis and mitochondrial function have been found to be affected by ribosomal availability. Also, ATP levels drop in models of ribosomopathies. We discuss findings highlighting the relevance of ribosome heterogeneity in physiological and pathological conditions, as well as the possibility that in rate-limiting situations, ribosomes may favor some translational programs. We discuss the effects of ribosome heterogeneity on cellular metabolism, tumorigenesis and aging. We speculate a scenario in which ribosomes are not only executors of a metabolic program but act as modulators.
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 , CicatrizaçãoRESUMO
Breast cancer consists of highly heterogeneous tumors, whose cell of origin and driver oncogenes are difficult to be uniquely defined. Here we report that MYC acts as tumor reprogramming factor in mammary epithelial cells by inducing an alternative epigenetic program, which triggers loss of cell identity and activation of oncogenic pathways. Overexpression of MYC induces transcriptional repression of lineage-specifying transcription factors, causing decommissioning of luminal-specific enhancers. MYC-driven dedifferentiation supports the onset of a stem cell-like state by inducing the activation of de novo enhancers, which drive the transcriptional activation of oncogenic pathways. Furthermore, we demonstrate that the MYC-driven epigenetic reprogramming favors the formation and maintenance of tumor-initiating cells endowed with metastatic capacity. This study supports the notion that MYC-driven tumor initiation relies on cell reprogramming, which is mediated by the activation of MYC-dependent oncogenic enhancers, thus establishing a therapeutic rational for treating basal-like breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Epigênese Genética , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Carcinogênese , Linhagem Celular Tumoral , Reprogramação Celular , Elementos Facilitadores Genéticos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/citologiaRESUMO
The original version of this Article contained an error in the spelling of the author Miriam Gaggianesi, which was incorrectly given as Miriam Giaggianesi. Furthermore, the affiliation details for Gabriella Gaudioso, Valentina Vaira, and Silvano Bosari incorrectly omitted 'Division of Pathology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, 20122, Italy'. Finally, the affiliation details for Alice Turdo, Miriam Gaggianesi, Aurora Chinnici and Elisa Lipari were incorrectly given as 'Dipartimento di Biotecnologie Mediche e Medicina Legale Sezione di Biochimica Medica, Facoltà di Medicina e Chirurgia, Policlinico "P.Giaccone", Università di Palermo, Palermo, 90127, Italy'. The correct affiliation is 'Department of Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, 90127, Italy'. These errors have now been corrected in both the PDF and HTML versions of the Article.
RESUMO
The data described in this article are related to "High levels of eukaryotic Initiation Factor 6 (eIF6) are required for immune system homeostasis and for steering the glycolytic flux of TCR-stimulated CD4+ T cells in both mice and humans" (Manfrini et al., in press) [1]. eIF6 is a translation initiation factor required for ribosomal biogenesis (Sanvito et al., 1999) [2] and for proper translational initiation (Gallo and Manfrini, 2015; Miluzio et al., 2016) [3], [4] whose protein abundance requires tight regulation. Here we analyze by flow cytometry the effects of eIF6 depletion on proportions of specific innate and adaptive immune system subpopulations and on thymocyte maturation in mice.