RESUMO
During the integrated stress response (ISR), global translation initiation is attenuated; however, noncanonical mechanisms allow for the continued translation of specific transcripts. Eukaryotic initiation factor 5B (eIF5B) has been shown to play a critical role in canonical translation as well as in noncanonical mechanisms involving internal ribosome entry site (IRES) and upstream open reading frame (uORF) elements. The uORF-mediated translation regulation of activating transcription factor 4 (ATF4) mRNA plays a pivotal role in the cellular ISR. Our recent study confirmed that eIF5B depletion removes uORF2-mediated repression of ATF4 translation, which results in the upregulation of growth arrest and DNA damage-inducible protein 34 (GADD34) transcription. Accordingly, we hypothesized that eIF5B depletion may reprogram the transcriptome profile of the cell. Here, we employed genome-wide transcriptional analysis on eIF5B-depleted cells. Further, we validate the up- and downregulation of several transcripts from our RNA-seq data using RT-qPCR. We identified upregulated pathways including cellular response to endoplasmic reticulum (ER) stress, and mucin-type O-glycan biosynthesis, as well as downregulated pathways of transcriptional misregulation in cancer and T cell receptor signaling. We also confirm that depletion of eIF5B leads to activation of the c-Jun N-terminal kinase (JNK) arm of the mitogen-activated protein kinase (MAPK) pathway. This data suggests that depletion of eIF5B reprograms the cellular transcriptome and influences critical cellular processes such as ER stress and ISR.
Assuntos
Estresse do Retículo Endoplasmático , Fatores de Iniciação em Eucariotos/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Interferência de RNA , Transcriptoma , Ativação Enzimática , Células HEK293 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , RNA Interferente Pequeno/genéticaRESUMO
Glioblastoma multiforme (GBM) is among the deadliest cancers, owing in part to complex inter- and intra-tumor heterogeneity and the presence of a population of stem-like cells called brain tumour stem cells (BTSCs/BTICs). These cancer stem cells survive treatment and confer resistance to the current therapies - namely, radiation and the chemotherapeutic, temozolomide (TMZ). TMZ induces cell death by alkylating DNA, and BTSCs resist this mechanism via a robust DNA damage response. Hence, recent studies aimed to sensitize BTSCs to TMZ using combination therapy, such as inhibition of DNA repair machinery. We have previously demonstrated in established GBM cell lines that eukaryotic initiation factor 5B (eIF5B) promotes the translation of pro-survival and anti-apoptotic proteins. Consequently, silencing eIF5B sensitizes these cells to TRAIL-induced apoptosis. However, established cell lines do not always recapitulate the features of human glioma. Therefore, we investigated this mechanism in patient-derived BTSCs. We show that silencing eIF5B leads to increased TMZ sensitivity in two BTSC lines: BT25 and BT48. Depletion of eIF5B decreases the levels of anti-apoptotic proteins in BT48 and sensitizes these cells to TMZ-induced activation of caspase-3, cleavage of PARP, and apoptosis. We suggest that eIF5B represents a rational target to sensitize GBM tumors to the current standard-of-care.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Temozolomida/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/genética , Humanos , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologiaRESUMO
Physiological stress conditions attenuate global mRNA translation via modifications of key eukaryotic initiation factors. However, non-canonical translation initiation mechanisms allow cap-independent translation of certain mRNAs. We have previously demonstrated that eIF5B promotes cap-independent translation of the mRNA encoding the antiapoptotic factor, XIAP, during cellular stress. Here, we show that depletion of eIF5B sensitizes glioblastoma multiforme cells to TRAIL-induced apoptosis by a pathway involving caspases-8, -9, and -7, with no significant effect on cell cycle progression. eIF5B promotes evasion of apoptosis by promoting the translation of several IRES-containing mRNAs, encoding the antiapoptotic proteins XIAP, Bcl-xL, cIAP1, and c-FLIPS. We also show that eIF5B promotes translation of nuclear factor erythroid 2-related factor 2 and suggest that reactive oxygen species contribute to increased apoptosis under conditions of eIF5B depletion. Finally, eIF5B depletion leads to decreased activation of the canonical NF-κB pathway. Taken together, our data suggest that eIF5B represents a regulatory node, allowing cancer cells to evade apoptosis by promoting the translation of pro-survival proteins from IRES-containing mRNAs.
Assuntos
Fatores de Iniciação em Eucariotos/genética , Glioblastoma/genética , Glioblastoma/patologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Resistencia a Medicamentos Antineoplásicos , Fatores de Iniciação em Eucariotos/deficiência , Fatores de Iniciação em Eucariotos/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Células HEK293 , Humanos , Proteínas Inibidoras de Apoptose/biossíntese , Proteínas Inibidoras de Apoptose/genética , Fator 2 Relacionado a NF-E2/biossíntese , Fator 2 Relacionado a NF-E2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Regulação para Cima , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteína bcl-X/biossíntese , Proteína bcl-X/genéticaRESUMO
A variety of cellular stresses lead to global translation attenuation due to phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2), which decreases the availability of the eIF2-GTP-Met-tRNAi ternary complex. However, a subset of mRNAs continues to be translated by non-canonical mechanisms under these conditions. In fact, although translation initiation of activating transcription factor 4 (ATF4) is normally repressed by an upstream open reading frame (uORF), a decreased availability of ternary complex leads to increased translation of the main ATF4-coding ORF. We show here that siRNA-mediated depletion of eIF5B-which can substitute for eIF2 in delivering Met-tRNAi-leads to increased levels of ATF4 protein in mammalian cells. This de-repression is not due to phosphorylation of eIF2α under conditions of eIF5B depletion. Although eIF5B depletion leads to a modest increase in the steady-state levels of ATF4 mRNA, we show by polysome profiling that the depletion of eIF5B enhances ATF4 expression primarily at the level of translation. Moreover, eIF5B silencing increases the expression of an ATF4-luciferase translational reporter by a mechanism requiring the repressive uORF2. Further experiments suggest that eIF5B cooperates with eIF1A and eIF5, but not eIF2A, to facilitate the uORF2-mediated repression of ATF4 translation.
