RESUMEN
BACKGROUND: The mechanistic target of rapamycin, (mTOR) kinase plays a pivotal role in controlling critical cellular growth and survival pathways, and its aberrant induction is implicated in cancer pathogenesis. Therefore, suppression of active mTOR signaling has been of great interest to researchers; several mTOR inhibitors have been discovered to date. Ethanol (EtOH), similar to pharmacologic mTOR inhibitors, has been shown to suppress the mTOR signaling pathway, though in a non-catalytic manner. Despite population studies showing that the consumption of EtOH has a protective effect against hematological malignancies, the mechanisms behind EtOH's modulation of mTOR activity in cells and its downstream consequences are largely unknown. Here we evaluated the effects of EtOH on the mTOR pathway, in comparison to the active-site mTOR inhibitor INK128, and compared translatome analysis of their downstream effects in diffuse large B-cell lymphoma (DLBCL). RESULTS: Treatment of DLBCL cells with EtOH suppressed mTORC1 complex formation while increasing AKT phosphorylation and mTORC2 complex assembly. INK128 completely abrogated AKT phosphorylation without affecting the structure of mTORC1/2 complexes. Accordingly, EtOH less profoundly suppressed cap-dependent translation and global protein synthesis, compared to a remarkable inhibitory effect of INK128 treatment. Importantly, EtOH treatment induced the formation of stress granules, while INK128 suppressed their formation. Microarray analysis of polysomal RNA revealed that although both agents primarily affected cell growth and survival, EtOH and INK128 regulated the synthesis of mostly distinct genes involved in these processes. Though both EtOH and INK128 inhibited cell cycle, proliferation and autophagy, EtOH, in contrast to INK128, did not induce cell apoptosis. CONCLUSION: Given that EtOH, similar to pharmacologic mTOR inhibitors, inhibits mTOR signaling, we systematically explored the effect of EtOH and INK128 on mTOR signal transduction, components of the mTORC1/2 interaction and their downstream effectors in DLBCL malignancy. We found that EtOH partially inhibits mTOR signaling and protein translation, compared to INK128's complete mTOR inhibition. Translatome analysis of mTOR downstream target genes established that differential inhibition of mTOR by EtOH and INK128 distinctly modulates translation of specific subsets of mRNAs involved in cell growth and survival, leading to differential cellular response and survival.
Asunto(s)
Benzoxazoles/farmacología , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Linfoma de Células B Grandes Difuso/metabolismo , Pirimidinas/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Autofagia/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The phosphorylation of eIF4E1 at serine 209 by MNK1 or MNK2 has been shown to initiate oncogenic mRNA translation, a process that favours cancer development and maintenance. Here, we interrogate the MNK-eIF4E axis in diffuse large B-cell lymphoma (DLBCL) and show a distinct distribution of MNK1 and MNK2 in germinal centre B-cell (GCB) and activated B-cell (ABC) DLBCL. Despite displaying a differential distribution in GCB and ABC, both MNKs functionally complement each other to sustain cell survival. MNK inhibition ablates eIF4E1 phosphorylation and concurrently enhances eIF4E3 expression. Loss of MNK protein itself downregulates total eIF4E1 protein level by reducing eIF4E1 mRNA polysomal loading without affecting total mRNA level or stability. Enhanced eIF4E3 expression marginally suppresses eIF4E1-driven translation but exhibits a unique translatome that unveils a novel role for eIF4E3 in translation initiation. We propose that MNKs can modulate oncogenic translation by regulating eIF4E1-eIF4E3 levels and activity in DLBCL.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Biosíntesis de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/genética , Línea Celular Tumoral , Factor 4E Eucariótico de Iniciación/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/metabolismoRESUMEN
Human diffuse large B-cell lymphomas (DLBCLs) often aberrantly express oncogenes that generally contain complex secondary structures in their 5' untranslated region (UTR). Oncogenes with complex 5'UTRs require enhanced eIF4A RNA helicase activity for translation. PDCD4 inhibits eIF4A, and PDCD4 knockout mice have a high penetrance for B-cell lymphomas. Here, we show that on B-cell receptor (BCR)-mediated p70s6K activation, PDCD4 is degraded, and eIF4A activity is greatly enhanced. We identified a subset of genes involved in BCR signaling, including CARD11, BCL10, and MALT1, that have complex 5'UTRs and encode proteins with short half-lives. Expression of these known oncogenic proteins is enhanced on BCR activation and is attenuated by the eIF4A inhibitor Silvestrol. Antigen-experienced immunoglobulin (Ig)G(+) splenic B cells, from which most DLBCLs are derived, have higher levels of eIF4A cap-binding activity and protein translation than IgM(+) B cells. Our results suggest that eIF4A-mediated enhancement of oncogene translation may be a critical component for lymphoma progression, and specific targeting of eIF4A may be an attractive therapeutic approach in the management of human B-cell lymphomas.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , ARN Helicasas DEAD-box/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Guanilato Ciclasa/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Regiones no Traducidas 5'/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 10 de la LLC-Linfoma de Células B , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Western Blotting , Proteínas Adaptadoras de Señalización CARD/genética , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Células Cultivadas , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/genética , Guanilato Ciclasa/genética , Humanos , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Persona de Mediana Edad , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Triterpenos/farmacologíaRESUMEN
Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. In vitro and in vivo findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.
