RESUMO
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
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
Prostate cancer is the second leading cause of cancer-related death in men. Despite having a relatively lower tumor mutational burden than most tumor types, multiple gene fusions such as TMPRSS2:ERG have been characterized and linked to more aggressive disease. Individual tumor samples have been found to contain multiple fusions, and it remains unknown whether these fusions increase tumor immunogenicity. Here, we investigated the role of fusion burden on the prevalence and expression of key molecular and immune effectors in prostate cancer tissue specimens that represented the different stages of disease progression and androgen sensitivity, including hormone-sensitive and castration-resistant prostate cancer. We found that tumor fusion burden was inversely correlated with tumor mutational burden and not associated with disease stage. High fusion burden correlated with high immune infiltration, PD-L1 expression on immune cells, and immune signatures, representing activation of T cells and M1 macrophages. High fusion burden inversely correlated with immune-suppressive signatures. Our findings suggest that high tumor fusion burden may be a more appropriate biomarker than tumor mutational burden in prostate cancer, as it more closely associates with immunogenicity, and suggests that tumors with high fusion burden could be potential candidates for immunotherapeutic agents.
Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Mutação , Fusão Oncogênica , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Antígeno B7-H1/imunologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/imunologia , Masculino , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias da Próstata/patologia , RNA-Seq/métodosRESUMO
Regulatory T cells (Treg) are immunosuppressive and negatively impact response to cancer immunotherapies. CREB-binding protein (CBP) and p300 are closely related acetyltransferases and transcriptional coactivators. Here, we evaluate the mechanisms by which CBP/p300 regulate Treg differentiation and the consequences of CBP/p300 loss-of-function mutations in follicular lymphoma. Transcriptional and epigenetic profiling identified a cascade of transcription factors essential for Treg differentiation. Mass spectrometry analysis showed that CBP/p300 acetylates prostacyclin synthase, which regulates Treg differentiation by altering proinflammatory cytokine secretion by T and B cells. Reduced Treg presence in tissues harboring CBP/p300 loss-of-function mutations was observed in follicular lymphoma. Our findings provide novel insights into the regulation of Treg differentiation by CBP/p300, with potential clinical implications on alteration of the immune landscape. SIGNIFICANCE: This study provides insights into the dynamic role of CBP/p300 in the differentiation of Tregs, with potential clinical implications in the alteration of the immune landscape in follicular lymphoma.
Assuntos
Proteína de Ligação a CREB/imunologia , Proteína p300 Associada a E1A/imunologia , Linfoma Folicular/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Acetilação , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Diferenciação Celular/fisiologia , Regulação para Baixo , Proteína p300 Associada a E1A/antagonistas & inibidores , Proteína p300 Associada a E1A/genética , Histonas/metabolismo , Humanos , Linfoma Folicular/genética , Linfoma Folicular/metabolismo , Linfoma Folicular/patologia , Mutação , Pirazóis/farmacologia , Piridinas/farmacologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Transcrição Gênica , TranscriptomaRESUMO
KRAS- and BRAF-mutant tumors are often dependent on MAPK signaling for proliferation and survival and thus sensitive to MAPK pathway inhibitors. However, clinical studies have shown that MEK inhibitors are not uniformly effective in these cancers indicating that mutational status of these oncogenes does not accurately capture MAPK pathway activity. A number of transcripts are regulated by this pathway and are recurrently identified in genome-based MAPK transcriptional signatures. To test whether the transcriptional output of only 10 of these targets could quantify MAPK pathway activity with potential predictive or prognostic clinical utility, we created a MAPK Pathway Activity Score (MPAS) derived from aggregated gene expression. In vitro, MPAS predicted sensitivity to MAPK inhibitors in multiple cell lines, comparable to or better than larger genome-based statistical models. Bridging in vitro studies and clinical samples, median MPAS from a given tumor type correlated with cobimetinib (MEK inhibitor) sensitivity of cancer cell lines originating from the same tissue type. Retrospective analyses of clinical datasets showed that MPAS was associated with the sensitivity of melanomas to vemurafenib (HR: 0.596) and negatively prognostic of overall or progression-free survival in both adjuvant and metastatic CRC (HR: 1.5 and 1.4), adrenal cancer (HR: 1.7), and HER2+ breast cancer (HR: 1.6). MPAS thus demonstrates potential clinical utility that warrants further exploration.
RESUMO
The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR's ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.
Assuntos
Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/genética , Proteína-Arginina N-Metiltransferases/genética , Receptores Androgênicos/genética , Serina Endopeptidases/genética , Sequência de Bases , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/patologia , Humanos , Masculino , Metilação , Modelos Moleculares , Mutação , Proteínas de Fusão Oncogênica/metabolismo , Próstata/metabolismo , Próstata/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Secundária de Proteína , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Serina Endopeptidases/metabolismo , Transdução de Sinais , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
UNLABELLED: The mTOR nucleates two complexes, namely mTOR complex 1 and 2 (mTORC1 and mTORC2), which are implicated in cell growth, survival, metabolism, and cancer. Phosphorylation of the α-subunit of translation initiation factor eIF2 at serine 51 (eIF2αS51P) is a key event of mRNA translation initiation and a master regulator of cell fate during cellular stress. Recent studies have implicated mTOR signaling in the stress response, but its connection to eIF2αS51P has remained unclear. Herein, we report that genetic as well as catalytic inhibition of mTORC2 induces eIF2αS51P. On the other hand, the allosteric inhibitor rapamycin induces eIF2αS51P through pathways that are independent of mTORC1 inactivation. Increased eIF2αS51P by impaired mTORC2 depends on the inactivation of AKT, which primes the activation of the endoplasmic reticulum (ER)-resident kinase PERK/PEK. The biologic function of eIF2αS51P was characterized in tuberous sclerosis complex (TSC)-mutant cells, which are defective in mTORC2 and AKT activity. TSC-mutant cells exhibit increased PERK activity, which is downregulated by the reconstitution of the cells with an activated form of AKT1. Also, TSC-mutant cells are increasingly susceptible to ER stress, which is reversed by AKT1 reconstitution. The susceptibility of TSC-mutant cells to ER stress is further enhanced by the pharmacologic inhibition of PERK or genetic inactivation of eIF2αS51P. Thus, the PERK/eIF2αS51P arm is an important compensatory prosurvival mechanism, which substitutes for the loss of AKT under ER stress. IMPLICATIONS: A novel mechanistic link between mTOR function and protein synthesis is identified in TSC-null tumor cells under stress and reveals potential for the development of antitumor treatments with stress-inducing chemotherapeutics.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/antagonistas & inibidores , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Transfecção , Regulação para Cima , eIF-2 Quinase/metabolismoRESUMO
Metazoans respond to various forms of environmental stress by inducing the phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α) at serine-51, a modification that leads to global inhibition of mRNA translation. We demonstrate induction of the phosphorylation of eIF2α in mammalian cells after either pharmacological inhibition of the phosphoinositide 3-kinase (PI3K)-Akt pathway or genetic or small interfering RNA-mediated ablation of Akt. This increase in the extent of eIF2α phosphorylation also occurred in Drosophila cells and depended on the endoplasmic reticulum (ER)-resident protein kinase PERK, which was inhibited by Akt-dependent phosphorylation at threonine-799. The activity of PERK and the abundance of phosphorylated eIF2α (eIF2αP) were reduced in mouse mammary gland tumors that contained activated Akt, as well as in cells exposed to ER stress or oxidative stress. In unstressed cells, the PERK-eIF2αP pathway mediated survival and facilitated adaptation to the deleterious effects of the inactivation of PI3K or Akt. Inactivation of the PERK-eIF2αP pathway increased the susceptibility of tumor cells to death by pharmacological inhibitors of PI3K or Akt. Thus, we suggest that the PERK-eIF2αP pathway provides a link between Akt signaling and translational control, which has implications for tumor formation and treatment.
Assuntos
Morte Celular/fisiologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , eIF-2 Quinase/metabolismo , Análise de Variância , Animais , Western Blotting , Linhagem Celular Tumoral , Drosophila , Estresse do Retículo Endoplasmático/fisiologia , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Imunoprecipitação , Camundongos , Estresse Oxidativo/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Inhibition of protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2) at Ser(51) occurs as a result of the activation of a family of kinases in response to various forms of stress. Although some consequences of eIF2alpha phosphorylation are cytoprotective, phosphorylation of eIF2alpha by RNA-dependent protein kinase (PKR) is largely proapoptotic and tumor suppressing. Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is a tumor suppressor protein that is mutated or deleted in various human cancers, with functions that are mediated through phosphatase-dependent and -independent pathways. Here, we demonstrate that the eIF2alpha phosphorylation pathway is downstream of PTEN. Inactivation of PTEN in human melanoma cells reduced eIF2alpha phosphorylation, whereas reconstitution of PTEN-null human glioblastoma or prostate cancer cells with either wild-type PTEN or phosphatase-defective mutants of PTEN induced PKR activity and eIF2alpha phosphorylation. The antiproliferative and proapoptotic effects of PTEN were compromised in mouse embryonic fibroblasts that lacked PKR or contained a phosphorylation-defective variant of eIF2alpha. Induction of the pathway leading to phosphorylation of eIF2alpha required an intact PDZ-binding motif in PTEN. These findings establish a link between tumor suppression by PTEN and inhibition of protein synthesis that is independent of PTEN's effects on phosphoinositide 3'-kinase signaling.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Inibidores da Síntese de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/metabolismo , eIF-2 Quinase/metabolismo , Análise de Variância , Animais , Western Blotting , Linhagem Celular Tumoral , Ensaio de Unidades Formadoras de Colônias , Imunofluorescência , Humanos , Imunoprecipitação , Camundongos , Microscopia Confocal , PTEN Fosfo-Hidrolase/genética , Fosforilação , Interferência de RNA , Proteínas Supressoras de Tumor/genéticaRESUMO
The eIF2alpha kinases have been involved in the inhibition of vesicular stomatatis virus replication but the contribution of each kinase to this process has not been fully investigated. Using mouse embryonic fibroblasts (MEFs) from knock-out mice we show that PKR and HRI have no effects on VSV replication as opposed to PERK and GCN2, which exhibit strong inhibitory effects. When MEFs containing the serine 51 to alanine mutation of eIF2alpha were used, we found that VSV replication is independent of eIF2alpha phosphorylation. Nevertheless, the kinase domain of the eIF2alpha kinases is both necessary and sufficient to inhibit VSV replication in cultured cells. Induction of PI3K-Akt/PKB pathway by eIF2alpha kinase activation plays no role in the inhibition of VSV replication. Our data provide strong evidence that VSV replication is not affected by eIF2alpha phosphorylation or downstream effector pathways such as the PI3K-Akt/PKB pathway. Thus, the anti-viral properties of eIF2alpha kinases are not always related to their inhibitory effects on host protein synthesis as previously thought and are possibly mediated by phosphorylation of proteins other than eIF2alpha.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Vesiculovirus/fisiologia , Replicação Viral/fisiologia , eIF-2 Quinase/fisiologia , Animais , Antivirais/metabolismo , Células Cultivadas , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Camundongos , Camundongos Knockout , Fosforilação , Estomatite Vesicular/enzimologia , Estomatite Vesicular/metabolismo , eIF-2 Quinase/química , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress.
Assuntos
Ciclina D1/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Regulação da Expressão Gênica , eIF-2 Quinase/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Retículo Endoplasmático/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Fosforilação , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Serina/química , Ubiquitina/químicaRESUMO
BACKGROUND/AIMS: Infection with hepatitis C virus (HCV) is associated with alterations in body iron homeostasis by poorly defined mechanisms. To seek for molecular links, we employed an established cell culture model for viral replication, and assessed how the expression of an HCV subgenomic replicon affects iron metabolism in host Huh7 hepatoma cells. METHODS: The expression of iron metabolism genes and parameters defining the cellular iron status were analyzed and compared between parent and replicon Huh7 cells. RESULTS: By using the IronChip microarray platform, we observed replicon-induced changes in expression profiles of iron metabolism genes. Notably, ceruloplasmin mRNA and protein expression were decreased in replicon cells. In addition, transferrin receptor 1 (TfR1) was also downregulated, while ferroportin levels were elevated, resulting in reduced iron uptake and increased iron release capacity of replicon cells. These responses were associated with an iron-deficient phenotype, manifested in decreased levels of the "labile iron pool" and concomitant induction of IRE-binding activity and IRP2 expression. Furthermore, hemin-treated replicon cells exhibited a defect in retaining iron. The clearance of the replicon by prolonged treatment with interferon-alpha only partially reversed the iron-deficient phenotype but almost completely restored the capacity of cured cells to retain iron. CONCLUSIONS: We propose that Huh7 cells undergo genetic reprogramming to permit subgenomic viral replication that results in reduction of intracellular iron levels. This response may provide a mechanism to bypass iron-mediated inactivation of the viral RNA polymerase NS5B.