RESUMO
In this study, we aimed to address the current limitations of therapies for macro-metastatic triple-negative breast cancer (TNBC) and provide a therapeutic lead that overcomes the high degree of heterogeneity associated with this disease. Specifically, we focused on well-documented but clinically underexploited cancer-fueling perturbations in mRNA translation as a potential therapeutic vulnerability. We therefore developed an orally bioavailable rocaglate-based molecule, MG-002, which hinders ribosome recruitment and scanning via unscheduled and non-productive RNA clamping by the eukaryotic translation initiation factor (eIF) 4A RNA helicase. We demonstrate that MG-002 potently inhibits mRNA translation and primary TNBC tumor growth without causing overt toxicity in mice. Importantly, given that metastatic spread is a major cause of mortality in TNBC, we show that MG-002 attenuates metastasis in pre-clinical models. We report on MG-002, a rocaglate that shows superior properties relative to existing eIF4A inhibitors in pre-clinical models. Our study also paves the way for future clinical trials exploring the potential of MG-002 in TNBC and other oncological indications.
Assuntos
RNA Helicases , Neoplasias de Mama Triplo Negativas , Humanos , Animais , Camundongos , RNA Helicases/genética , RNA Helicases/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Biossíntese de Proteínas , Fator de Iniciação 4A em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Ribossomos/metabolismoRESUMO
The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.
Assuntos
Estresse do Retículo Endoplasmático , Fator de Iniciação 3 em Eucariotos/metabolismo , Fibroblastos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/biossíntese , Transcrição Gênica , eIF-2 Quinase/metabolismo , Animais , Reprogramação Celular , Fator de Iniciação 3 em Eucariotos/genética , Fibroblastos/patologia , Células HEK293 , Humanos , Camundongos , Fases de Leitura Aberta , Fenótipo , Proteostase , Interferência de RNA , RNA Mensageiro/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , eIF-2 Quinase/genéticaRESUMO
The integrated stress response (ISR) is a key determinant of tumorigenesis in response to oncogenic forms of stress like genotoxic, proteotoxic and metabolic stress. ISR relies on the phosphorylation of the translation initiation factor eIF2 to promote the translational and transcriptional reprogramming of gene expression in stressed cells. While ISR promotes tumor survival under stress, its hyperactivation above a level of tolerance can also cause tumor death. The tumorigenic function of ISR has been recently demonstrated for lung adenocarcinomas (LUAD) with KRAS mutations. ISR mediates the translational repression of the dual-specificity phosphatase DUSP6 to stimulate ERK activity and LUAD growth. The significance of this finding is highlighted by the strong anti-tumor responses of ISR inhibitors in pre-clinical LUAD models. Elucidation of the mechanisms of ISR action in LUAD progression via cell-autonomous and immune regulatory mechanisms will provide a better understanding of its tumorigenic role to fully exploit its therapeutic potential in the treatment of a deadly form of cancer.
Assuntos
Neoplasias Pulmonares , Proteínas Proto-Oncogênicas p21(ras) , Carcinogênese/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Estresse Fisiológico/genéticaRESUMO
Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/biossíntese , Autofagia/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Fator de Transcrição STAT1/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Linhagem Celular Tumoral , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas/fisiologia , Fator de Transcrição STAT1/genética , Sirolimo/farmacologiaRESUMO
Vascular calcification increases the risk of cardiovascular disease and death in patients with chronic kidney disease (CKD). Increased activity of mammalian target of rapamycin complex 1 (mTORC1) and endoplasmic reticulum (ER) stress-unfolded protein response (UPR) are independently reported to partake in the pathogenesis of vascular calcification in CKD. However, the association between mTORC1 activity and ER stress-UPR remains unknown. We report here that components of the uremic state [activation of the receptor for advanced glycation end products (RAGE) and hyperphosphatemia] potentiate vascular smooth muscle cell (VSMC) calcification by inducing persistent and exaggerated activity of mTORC1. This gives rise to prolonged and excessive ER stress-UPR as well as attenuated levels of sestrin 1 ( Sesn1) and Sesn3 feeding back to inhibit mTORC1 activity. Activating transcription factor 4 arising from the UPR mediates cell death via expression of CCAAT/enhancer-binding protein (c/EBP) homologous protein (CHOP), impairs the generation of pyrophosphate, a potent inhibitor of mineralization, and potentiates VSMC transdifferentiation to the osteochondrocytic phenotype. Short-term treatment of CKD mice with rapamycin, an inhibitor of mTORC1, or tauroursodeoxycholic acid, a bile acid that restores ER homeostasis, normalized mTORC1 activity, molecular markers of UPR, and calcium content of aortas. Collectively, these data highlight that increased and/or protracted mTORC1 activity arising from the uremic state leads to dysregulated ER stress-UPR and VSMC calcification. Manipulation of the mTORC1-ER stress-UPR pathway opens up new therapeutic strategies for the prevention and treatment of vascular calcification in CKD.
Assuntos
Doenças da Aorta/enzimologia , Estresse do Retículo Endoplasmático , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Músculo Liso Vascular/enzimologia , Resposta a Proteínas não Dobradas , Uremia/complicações , Calcificação Vascular/enzimologia , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/enzimologia , Aorta/patologia , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Morte Celular , Proliferação de Células , Transdiferenciação Celular , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Camundongos Mutantes , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Osteogênese , Fosforilação , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Proteínas S100/genética , Proteínas S100/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Ácido Tauroquenodesoxicólico/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Calcificação Vascular/tratamento farmacológico , Calcificação Vascular/etiologia , Calcificação Vascular/patologiaRESUMO
The signal transducer and activator of transcription 1 (Stat1) functions as a tumor suppressor via immune regulatory and cell-autonomous pathways. Herein, we report a previously unidentified cell-autonomous Stat1 function, which is its ability to exhibit both antiproliferative and prosurvival properties by facilitating translation of mRNAs encoding for the cyclin-dependent kinase inhibitor p27(Kip1) and antiapoptotic proteins X-linked inhibitor of apoptosis and B-cell lymphoma xl. Translation of the select mRNAs requires the transcriptional function of Stat1, resulting in the up-regulation of the p110γ subunit of phosphoinositide 3-kinase (PI3K) class IB and increased expression of the translational repressor translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). Increased PI3Kγ signaling promotes the degradation of the eIF4A inhibitor programmed cell death protein 4, which favors the cap-independent translation of the select mRNAs under conditions of general inhibition of protein synthesis by up-regulated eIF4E-binding protein 1. As such, Stat1 inhibits cell proliferation but also renders cells increasingly resistant to antiproliferative effects of pharmacological inhibitors of PI3K and/or mammalian target of rapamycin. Stat1 also protects Ras-transformed cells from the genotoxic effects of doxorubicin in culture and immune-deficient mice. Our findings demonstrate an important role of mRNA translation in the cell-autonomous Stat1 functions, with implications in tumor growth and treatment with chemotherapeutic drugs.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Doxorrubicina/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Capuzes de RNA/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Camundongos , Camundongos Knockout , Iniciação Traducional da Cadeia Peptídica/fisiologia , Capuzes de RNA/genética , Fator de Transcrição STAT1/genéticaRESUMO
Eukaryotic cells assemble stress granules (SGs) when translation initiation is inhibited. Different cell signaling pathways regulate SG production. Particularly relevant to this process is 5'-AMP-activated protein kinase (AMPK), which functions as a stress sensor and is transiently activated by adverse physiologic conditions. Here, we dissected the role of AMPK for oxidant-induced SG formation. Our studies identified multiple steps of de novo SG assembly that are controlled by the kinase. Single-cell analyses demonstrated that pharmacological AMPK activation prior to stress exposure changed SG properties, because the granules became more abundant and smaller in size. These altered SG characteristics correlated with specific changes in cell survival, cell signaling, cytoskeletal organization, and the abundance of translation initiation factors. Specifically, AMPK activation increased stress-induced eukaryotic initiation factor (eIF) 2α phosphorylation and reduced the concentration of eIF4F complex subunits eIF4G and eIF4E. At the same time, the abundance of histone deacetylase 6 (HDAC6) was diminished. This loss of HDAC6 was accompanied by increased acetylation of α-tubulin on Lys40. Pharmacological studies further confirmed this novel AMPK-HDAC6 interplay and its importance for SG biology. Taken together, we provide mechanistic insights into the regulation of SG formation. We propose that AMPK activation stimulates oxidant-induced SG formation but limits their fusion into larger granules.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Grânulos Citoplasmáticos/metabolismo , Microtúbulos/metabolismo , Oxidantes/toxicidade , Transdução de Sinais/efeitos dos fármacos , Animais , Compostos de Bifenilo , Sobrevivência Celular/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Células HeLa , Histona Desacetilases/metabolismo , Humanos , Maleatos/farmacologia , Camundongos , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Fosforilação , Pironas/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Tiofenos/farmacologia , Quinases Associadas a rho/metabolismoRESUMO
Cells respond to various forms of stress by activating anti-proliferative pathways, which allow them to correct the damage caused by stress before re-entering proliferation. If the damage, however, is beyond repair, stressed cells are eliminated from the host by undergoing death. The balance between cell survival and death is essential for cancer formation and is determined by several key pathways that impact on different stages of gene expression. In recent years, it has become evident that phosphorylation of the alpha (α) subunit of the translation initiation factor eIF2 at serine 51 (eIF2αS51P) is an important determinant of cell fate in response to stress. Induction of eIF2αS51P is mediated by a family of four kinases namely, HRI, PKR, PERK and GCN2, each of which responds to distinct forms of stress. Increased eIF2αS51P results in a global inhibition of protein synthesis but at the same time enhances the translation of select mRNAs encoding for proteins that control cell adaptation to stress. Short-term induction of eIF2αS51P has been associated with cell survival whereas long-term induction with cell death. Studies in mouse and human models of cancer have provided compelling evidence that eIF2αS51P plays an essential role in stress-induced tumorigenesis. Increased eIF2αS51P exhibits cell autonomous as well as immune regulatory effects, which can influence tumor growth and the efficacy of anti-tumor therapies. The findings suggest that eIF2αS51P may be of prognostic value and a suitable target for the design and implementation of effective anti-tumor therapies. This article is part of a Special Issue entitled: Translation and Cancer.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Fator de Iniciação 2 em Eucariotos/genética , Humanos , Camundongos , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismoRESUMO
The accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) causes stress to which an unfolded protein response is activated to render cell survival or apoptosis (chronic stress). Transcriptional and translational reprogramming is tightly regulated during the unfolded protein response to ensure specific gene expression. The master regulator of this response is the PERK/eIF2α/ATF4 signaling where eIF2α is phosphorylated (eIF2α-P) by the kinase PERK. This signal leads to global translational shutdown, but it also enables translation of the transcription factor ATF4 mRNA. We showed recently that ATF4 induces an anabolic program through the up-regulation of selected amino acid transporters and aminoacyl-tRNA synthetases. Paradoxically, this anabolic program led cells to apoptosis during chronic ER stress in a manner that involved recovery from stress-induced protein synthesis inhibition. By using eIF2α-P-deficient cells as an experimental system, we identified a communicating network of signaling pathways that contribute to the inhibition of protein synthesis during chronic ER stress. This eIF2α-P-independent network includes (i) inhibition of mammalian target of rapamycin kinase protein complex 1 (mTORC1)-targeted protein phosphorylation, (ii) inhibited translation of a selective group of 5'-terminal oligopyrimidine mRNAs (encoding proteins involved in the translation machinery and translationally controlled by mTORC1 signaling), and (iii) inhibited translation of non-5'-terminal oligopyrimidine ribosomal protein mRNAs and ribosomal RNA biogenesis. We propose that the PERK/eIF2α-P/ATF4 signaling acts as a brake in the decline of protein synthesis during chronic ER stress by positively regulating signaling downstream of the mTORC1 activity. These studies advance our knowledge on the complexity of the communicating signaling pathways in controlling protein synthesis rates during chronic stress.
Assuntos
Estresse do Retículo Endoplasmático , Fator de Iniciação 2 em Eucariotos/metabolismo , Fibroblastos/metabolismo , Biossíntese de Proteínas , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Animais , Proteína 5 Relacionada à Autofagia , Western Blotting , ATPases Transportadoras de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , Embrião de Mamíferos/citologia , Fator de Iniciação 2 em Eucariotos/genética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Fosforilação , Polirribossomos/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Tapsigargina/farmacologia , Fatores de Tempo , eIF-2 Quinase/metabolismoRESUMO
Endoplasmic reticulum (ER) stress-induced responses are associated with the loss of insulin-producing ß-cells in type 2 diabetes mellitus. ß-Cell survival during ER stress is believed to depend on decreased protein synthesis rates that are mediated via phosphorylation of the translation initiation factor eIF2α. It is reported here that chronic ER stress correlated with increased islet protein synthesis and apoptosis in ß-cells in vivo. Paradoxically, chronic ER stress in ß-cells induced an anabolic transcription program to overcome translational repression by eIF2α phosphorylation. This program included expression of amino acid transporter and aminoacyl-tRNA synthetase genes downstream of the stress-induced ATF4-mediated transcription program. The anabolic response was associated with increased amino acid flux and charging of tRNAs for branched chain and aromatic amino acids (e.g. leucine and tryptophan), the levels of which are early serum indicators of diabetes. We conclude that regulation of amino acid transport in ß-cells during ER stress involves responses leading to increased protein synthesis, which can be protective during acute stress but can lead to apoptosis during chronic stress. These studies suggest that the increased expression of amino acid transporters in islets can serve as early diagnostic biomarkers for the development of diabetes.
Assuntos
Aminoácidos/metabolismo , Apoptose , Diabetes Mellitus Tipo 2/metabolismo , Estresse do Retículo Endoplasmático , Células Secretoras de Insulina/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Sobrevivência Celular , Diabetes Mellitus Tipo 2/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , RNA de Transferência/metabolismo , Ativação TranscricionalRESUMO
Ferritin stores and detoxifies an excess of intracellular iron, and thereby plays an important role in the metabolism of this metal. As unshielded iron promotes oxidative stress, ferritin is crucial in maintaining cellular redox balance and may also modulate cell growth, survival, and apoptosis. The expression of ferritin is controlled by transcriptional and post-transcriptional mechanisms. In light of the well-established transcriptional induction of ferritin by inflammatory signals, we examined how ferritin mRNA translation responds to stress conditions. We first used HT1080 fibrosarcoma cells engineered for coumermycin-inducible expression of PKR, a stress kinase that inhibits protein synthesis in virus-infected cells by phosphorylating eIF2α. In this setting, iron triggered partial ferritin mRNA translation despite a PKR-induced global shutdown in protein synthesis. Moreover, iron-mediated ferritin synthesis was evident in cells infected with an attenuated strain of poliovirus; when eIF4GI was cleaved, eIF2α was phosphorylated, and host protein synthesis was inhibited. Under global inhibition of protein synthesis or specific inhibition of ferritin mRNA translation in cells overexpressing PKR or IRP1, respectively, we demonstrate association of ferritin mRNA with heavy polysomes. Further experiments revealed that the 5' untranslated region (5' UTR) of ferritin mRNA contains a bona fide internal ribosomal entry site (IRES). Our data are consistent with the existence of an alternative, noncanonical mechanism for ferritin mRNA translation, which may primarily operate under stress conditions to protect cells from oxidative stress.
Assuntos
Ferritinas/genética , Iniciação Traducional da Cadeia Peptídica , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Fator de Iniciação 2 em Eucariotos/metabolismo , Ferritinas/biossíntese , Regulação da Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos , Humanos , Fosforilação , Polirribossomos/metabolismo , RNA Mensageiro/metabolismoRESUMO
mRNA translation plays an important role in tumor development and represents a valid target of pharmaceutical intervention in cancer. A key step in mRNA translation involves the regulation of initiation by the eukaryotic initiation factor eIF2. Eukaryotic cells respond to various forms of stress by inducing the phosphorylation of the α-subunit of eIF2 at S51, a modification that leads to protein synthesis inhibition. Phosphorylated eIF2α can act either as a promoter of cell survival or an inducer of cell death in response to distinct stimuli. Increased eIF2α phosphorylation has a cytoprotective function in response to genetic or pharmacological inhibition of the PI3K-Akt pathway but also exhibits a proapoptotic function downstream of the PTEN tumor suppressor, independent of PI3K-Akt signaling inhibition. The functional interplay between the PI3K-Akt and eIF2α phosphorylation pathways may have important implications in the design of anti-tumor therapies that depend on the cell fate decisions of phosphorylated eIF2α.
Assuntos
Carcinogênese/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Apoptose , Carcinogênese/genética , Sobrevivência Celular , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fosforilação , Biossíntese de Proteínas , Transdução de SinaisRESUMO
The Hippo pathway nuclear effector Yes-associated protein (YAP) potentiates the progression of polycystic kidney disease (PKD) arising from ciliopathies. The mechanisms underlying the increase in YAP expression and transcriptional activity in PKD remain obscure. We observed that in kidneys from mice with juvenile cystic kidney (jck) ciliopathy, the aberrant hyperactivity of mechanistic target of rapamycin complex 1 (mTORC1), driven by ERK1/2 and PI3K/AKT cascades, induced ER proteotoxic stress. To reduce this stress by reprogramming translation, the protein kinase R-like ER kinase-eukaryotic initiation factor 2α (PERK/eIF2α) arm of the integrated stress response (ISR) was activated. PERK-mediated phosphorylation of eIF2α drove the selective translation of activating transcription factor 4 (ATF4), potentiating YAP expression. In parallel, YAP underwent K63-linked polyubiquitination by SCF S-phase kinase-associated protein 2 (SKP2) E3 ubiquitin ligase, a Hippo-independent, nonproteolytic ubiquitination that enhances YAP nuclear trafficking and transcriptional activity in cancer cells. Defective ISR cellular adaptation to ER stress in eIF2α phosphorylation-deficient jck mice further augmented YAP-mediated transcriptional activity and renal cyst growth. Conversely, pharmacological tuning down of ER stress/ISR activity and SKP2 expression in jck mice by administration of tauroursodeoxycholic acid (TUDCA) or tolvaptan impeded these processes. Restoring ER homeostasis and/or interfering with the SKP2-YAP interaction represent potential therapeutic avenues for stemming the progression of renal cystogenesis.
Assuntos
Proteínas Quinases Associadas a Fase S , Ubiquitina-Proteína Ligases , Camundongos , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator 4 Ativador da Transcrição/metabolismo , Fosforilação , Rim/metabolismoRESUMO
Phosphorylation of the alpha (α) subunit of the eukaryotic initiation factor 2 (eIF2) at serine 51 is an important mechanism of translational control in response to various forms of environmental stress. In metazoans, eIF2α phosphorylation is mediated by four kinases each of which becomes activated by distinct stimuli. Previous work established that expression of a chimera protein comprising of the bacteria Gyrase B N-terminal (GyrB) domain fused to the kinase domain (KD) of the eIF2α kinase PKR is capable of inducing eIF2α phosphorylation in cultured cells after treatment with the antibiotic coumermycin. Herein, we report the development of transgenic mice expressing the fusion protein GyrB.PKR ubiquitously. Treatment of mice with coumermycin induces eIF2α phosphorylation in vivo as demonstrated by immunoblotting and immunoshistochemistry of mouse tissues. The GyrB.PKR transgene represents a useful model system to investigate the biological effects of the conditional induction of eIF2α phosphorylation in vivo in the absence of parallel signaling pathways that are elicited in response to stress.
Assuntos
DNA Girase/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Modelos Animais , Inibidores da Topoisomerase II , eIF-2 Quinase/metabolismo , Aminocumarinas/farmacologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , DNA Girase/genética , Fator de Iniciação 2 em Eucariotos/genética , Expressão Gênica , Genótipo , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Estresse Fisiológico , Transgenes/genética , eIF-2 Quinase/genéticaRESUMO
Regulation of cell volume is of great importance because persistent swelling or shrinkage leads to cell death. Tissues experience hypertonicity in both physiological (kidney medullar cells) and pathological states (hypernatremia). Hypertonicity induces an adaptive gene expression program that leads to cell volume recovery or apoptosis under persistent stress. We show that the commitment to apoptosis is controlled by phosphorylation of the translation initiation factor eIF2alpha, the master regulator of the stress response. Studies with cultured mouse fibroblasts and cortical neurons show that mutants deficient in eIF2alpha phosphorylation are protected from hypertonicity-induced apoptosis. A novel link is revealed between eIF2alpha phosphorylation and the subcellular distribution of the RNA-binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). Stress-induced phosphorylation of eIF2alpha promotes apoptosis by inducing the cytoplasmic accumulation of hnRNP A1, which attenuates internal ribosome entry site-mediated translation of anti-apoptotic mRNAs, including Bcl-xL that was studied here. Hypertonic stress induced the eIF2alpha phosphorylation-independent formation of cytoplasmic stress granules (SGs, structures that harbor translationally arrested mRNAs) and the eIF2alpha phosphorylation-dependent accumulation of hnRNP A1 in SGs. The importance of hnRNP A1 was demonstrated by induction of apoptosis in eIF2alpha phosphorylation-deficient cells that express exogenous cytoplasmic hnRNP A1. We propose that eIF2alpha phosphorylation during hypertonic stress promotes apoptosis by sequestration of specific mRNAs in SGs in a process mediated by the cytoplasmic accumulation of hnRNP A1.
Assuntos
Apoptose , Fator de Iniciação 2 em Eucariotos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/metabolismo , Osmose , Animais , Citoplasma/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1 , Heterozigoto , Camundongos , Microscopia de Fluorescência/métodos , Modelos Biológicos , Pressão Osmótica , Fosforilação , Plasmídeos/metabolismo , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
Exposure of cells to hormones, mitogens and growth factors can lead to the activation of PI3K and serine-threonine kinase Akt/PKB. Akt is recruited to the plasma membrane, a process that facilitates its phosphorylation at threonine 308 and serine (S)473 mediated by the PI3K-dependent kinase 1 and mTORC2 kinase, respectively. Active Akt has several targets, one of the most important being mTORC1, which is essential for stimulation of protein synthesis and cell growth. Active Akt phosphorylates the α- and ß-isoforms of glycogen synthase kinase 3 (GSK-3), leading to the inhibition of GSK-3 activity and phosphorylation of proteins with key roles in cell proliferation and metabolism. Akt activity is induced in response to various forms of stress, mainly to facilitate cell survival and maintain cell proliferation. Recent work by Chen and colleagues has placed GSK-3ß in a negative regulatory loop resulting in Akt inactivation. GSK-3ß carries out this function via the phosphorylation of the mTORC2 component protein rictor at S1235, a modification that compromises the ability of mTORC2 to induce Akt activity by phosphorylation at S473. Rictor phosphorylation at S1235 can be detected in proliferating cells, but it can be particularly induced in cells exposed to endoplasmic reticulum stress. Rictor S1235 phosphorylation decreases the tumorigenic properties of activated Ras, providing evidence for a link between the GSK-3ß-mTORC2 axis and tumorigenesis. A part of its potential role in regulating metabolic processes associated with endoplasmic reticulum stress, an interesting question is whether disruption of the GSK-3ß-mTORC2 arm would have any implications in tumor formation and treatment.
RESUMO
The integrated stress response (ISR) is an essential stress-support pathway increasingly recognized as a determinant of tumorigenesis. Here we demonstrate that ISR is pivotal in lung adenocarcinoma (LUAD) development, the most common histological type of lung cancer and a leading cause of cancer death worldwide. Increased phosphorylation of the translation initiation factor eIF2 (p-eIF2α), the focal point of ISR, is related to invasiveness, increased growth, and poor outcome in 928 LUAD patients. Dissection of ISR mechanisms in KRAS-driven lung tumorigenesis in mice demonstrated that p-eIF2α causes the translational repression of dual specificity phosphatase 6 (DUSP6), resulting in increased phosphorylation of the extracellular signal-regulated kinase (p-ERK). Treatments with ISR inhibitors, including a memory-enhancing drug with limited toxicity, provides a suitable therapeutic option for KRAS-driven lung cancer insofar as they substantially reduce tumor growth and prolong mouse survival. Our data provide a rationale for the implementation of ISR-based regimens in LUAD treatment.
Assuntos
Adenocarcinoma/metabolismo , Fosfatase 6 de Especificidade Dupla/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Animais , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Linhagem Celular Tumoral , Feminino , Humanos , Indóis/farmacologia , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Masculino , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Estresse Fisiológico/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.
Assuntos
Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , eIF-2 Quinase/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Ativação Enzimática , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos , Humanos , Camundongos , Modelos Biológicos , Fosfoproteínas/metabolismo , Fosforilação , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismoRESUMO
With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.
Assuntos
Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/patologia , Ultrassonografia , Animais , Modelos Animais de Doenças , Progressão da Doença , Neoplasias Pulmonares/genética , Camundongos , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Carga TumoralRESUMO
Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5' Untranslated Regions (5'UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and "fate-switching" toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.