RESUMEN
BACKGROUND: Hyperactivation of the mTORC2 signaling pathway has been shown to contribute to the oncogenic properties of gliomas. Moreover, overexpression of the mTORC2 regulatory subunit Rictor has been associated with increased proliferation and invasive character of these tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether Rictor overexpression was sufficient to induce glioma formation in mice, we inserted a Cre-lox-regulated human Rictor transgene into the murine ROSA26 locus. This floxed Rictor strain was crossed with mice expressing the Cre recombinase driven from the glial fibrillary acidic protein (GFAP) promoter whose expression is limited to the glial cell compartment. Double transgenic GFAP-Cre/Rictor(loxP/loxP) mice developed multifocal infiltrating glioma containing elevated mTORC2 activity and typically involved the subventricular zone (SVZ) and lateral ventricle. Analysis of Rictor-dependent signaling in these tumors demonstrated that in addition to elevated mTORC2 activity, an mTORC2-independent marker of cortical actin network function, was also elevated. Upon histological examination of the neoplasms, many displayed oligodendroglioma-like phenotypes and expressed markers associated with oligodendroglial lineage tumors. To determine whether upstream oncogenic EGFRvIII signaling would alter tumor phenotypes observed in the GFAP-Cre/Rictor(loxP/loxP) mice, transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor(loxP/loxP) mice were generated. These mice developed mixed astrocytic-oligodendroglial tumors, however glioma formation was accelerated and correlated with increased mTORC2 activity. Additionally, the subventricular zone within the GFAP-Cre/Rictor(loxP/loxP) mouse brain was markedly expanded, and a further proliferation within this compartment of the brain was observed in transgenic GFAP-EGFRvIII; GFAP-Cre/Rictor(loxP/loxP) mice. CONCLUSION/SIGNIFICANCE: These data collectively establish Rictor as a novel oncoprotein and support the role of dysregulated Rictor expression in gliomagenesis via mTOR-dependent and mTOR-independent mechanisms. Furthermore, oncogenic EGFRvIII signaling appears to potentiate the in vivo proliferative capacity of GFAP-Cre/Rictor(loxP/loxP) gliomas.
Asunto(s)
Proteínas Portadoras , Glioma , Complejos Multiproteicos , Proteínas , Serina-Treonina Quinasas TOR , Animales , Astrocitos/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proliferación Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioma/genética , Glioma/metabolismo , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Transgénicos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN no Traducido , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
The A/U-rich RNA-binding protein tristetraprolin (TTP) is an mRNA destabilizing factor which plays a role in the regulated turnover of many transcripts encoding proteins involved in immune function and cell growth control. TTP also plays a role in stress-induced destabilization of mRNAs. Here we report the interaction of TTP with a component of the mTORC2 kinase, Protor-2 (PRR5-L, protein Q6MZQ0/FLJ14213/CAE45978). Protor-2 is structurally similar to human PRR5 and has been demonstrated to bind mTORC2 via Rictor and/or Sin1 and may signal downstream events promoting apoptosis. Protor-2 dissociates from mTORC2 upon hyperactivation of the kinase and is not required for mTORC2 integrity or activity. We identified Protor-2 in a yeast two-hybrid screen as a TTP interactor using the C-terminal mRNA decay domain of TTP as bait. The interaction of Protor-2 with TTP was also confirmed in vivo in co-immunoprecipitation experiments and Protor-2 was also detected in immunoprecipitates of Rictor. Protor-2 was shown to stimulate TTP-mediated mRNA turnover of several TTP-associated mRNAs (TNF-α, GM-CSF, IL-3 and COX-2) in Jurkat cells when overexpressed while the half-lives of transcripts which do not decay via a TTP-mediated mechanism were unaffected. Knockdown of Protor-2 via RNAi inhibited TTP-mediated mRNA turnover of these TTP-associated mRNAs and inhibited association of TTP with cytoplasmic stress granules (SG) or mRNA processing bodies (P-bodies) following induction of the integrated stress response. These results suggest that Protor-2 associates with TTP to accelerate TTP-mediated mRNA turnover and functionally links the control of TTP-regulated mRNA stability to mTORC2 activity.
Asunto(s)
Proteínas Portadoras/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Estrés Fisiológico , Tristetraprolina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Gránulos Citoplasmáticos/metabolismo , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células Jurkat , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
A variety of mechanisms confer hypersensitivity of tumor cells to the macrolide rapamycin, the prototypic mTORC1 inhibitor. Several studies have shown that the status of the AKT kinase plays a critical role in determining hypersensitivity. Cancer cells in which AKT activity is elevated are exquisitely sensitive to mTORC1 inhibitors while cells in which the kinase is quiescent are relatively resistant. Our previous work has shown that a transcript-specific protein synthesis salvage pathway is operative in cells with quiescent AKT levels, maintaining the translation of crucial mRNAs involved in cell-cycle progression in the face of global eIF-4E-mediated translation inhibition. The activation of this salvage pathway is dependent on SAPK2/p38-mediated activation of IRES-dependent initiation of the cyclin D1 and c-MYC mRNAs, resulting in the maintenance of their protein expression levels. Here, we show that both genetic and pharmacologic inhibition of SAPK2/p38 in glioblastoma multiforme cells significantly reduces rapamycin-induced IRES-mediated translation initiation of cyclin D1 and c-MYC, resulting in increased G(1) arrest in vitro and inhibition of tumor growth in xenografts. Moreover, we observed that the AKT-dependent signaling alterations seen in vitro are also displayed in engrafted tumors cells and were able to show that combined inhibitor treatments markedly reduced the mRNA translational state of cyclin D1 and c-MYC transcripts in tumors isolated from mice. These data support the combined use of SAPK2/p38 and mTORC1 inhibitors to achieve a synergistic antitumor therapeutic response, particularly in rapamycin-resistant quiescent AKT-containing cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes bcl-1/efectos de los fármacos , Genes myc/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones SCID , Proteína Quinasa 11 Activada por Mitógenos/antagonistas & inhibidores , Complejos Multiproteicos , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas/metabolismo , ARN Interferente Pequeño/administración & dosificación , Serina-Treonina Quinasas TOR , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
The relative activity of the AKT kinase has been demonstrated to be a major determinant of sensitivity of tumor cells to mammalian target of rapamycin (mTOR) complex 1 inhibitors. Our previous studies have shown that the multifunctional RNA-binding protein heterogeneous nuclear ribonucleoprotein (hnRNP) A1 regulates a salvage pathway facilitating internal ribosome entry site (IRES)-dependent mRNA translation of critical cellular determinants in an AKT-dependent manner following mTOR inhibitor exposure. This pathway functions by stimulating IRES-dependent translation in cells with relatively quiescent AKT, resulting in resistance to rapamycin. However, the pathway is repressed in cells with elevated AKT activity, rendering them sensitive to rapamycin-induced G(1) arrest as a result of the inhibition of global eIF-4E-mediated translation. AKT phosphorylation of hnRNP A1 at serine 199 has been demonstrated to inhibit IRES-mediated translation initiation. Here we describe a phosphomimetic mutant of hnRNP A1 (S199E) that is capable of binding both the cyclin D1 and c-MYC IRES RNAs in vitro but lacks nucleic acid annealing activity, resulting in inhibition of IRES function in dicistronic mRNA reporter assays. Utilizing cells in which AKT is conditionally active, we demonstrate that overexpression of this mutant renders quiescent AKT-containing cells sensitive to rapamycin in vitro and in xenografts. We also demonstrate that activated AKT is strongly correlated with elevated Ser(P)(199)-hnRNP A1 levels in a panel of 22 glioblastomas. These data demonstrate that the phosphorylation status of hnRNP A1 serine 199 regulates the AKT-dependent sensitivity of cells to rapamycin and functionally links IRES-transacting factor annealing activity to cellular responses to mTOR complex 1 inhibition.
Asunto(s)
Sustitución de Aminoácidos , Antibióticos Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Glioblastoma/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirolimus/farmacología , Animales , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Complejos Multiproteicos , Mutación Missense , Fosforilación/efectos de los fármacos , Fosforilación/genética , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , Proteínas/genética , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina-Treonina Quinasas TORRESUMEN
The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Akt-containing cells sensitive to rapamycin-induced G(1) arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.
Asunto(s)
Ciclina D1/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Animales , Antibióticos Antineoplásicos/farmacología , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Ciclina D1/genética , Ribonucleoproteína Nuclear Heterogénea A1 , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Humanos , Ratones , Fosforilación , Proteínas Proto-Oncogénicas c-myc/genética , Ribosomas/metabolismo , Serina/química , Sirolimus/farmacologíaRESUMEN
mTORC2 is a multiprotein kinase composed of mTOR, mLST8, PRR5, mSIN1 and Rictor. The complex is insensitive to rapamycin and has demonstrated functions controlling cell growth, motility, invasion and cytoskeletal assembly. mTORC2 is the major hydrophobic domain kinase which renders Akt fully active via phosphorylation on serine 473. We isolated Hsp70 as a putative Rictor interacting protein in a yeast two-hybrid assay and confirmed this interaction via co-immunoprecipitation and colocalization experiments. In cells expressing an antisense RNA targeting Hsp70, mTORC2 formation and activity were impaired. Moreover, in cells lacking Hsp70 expression, mTORC2 activity was inhibited following heat shock while controls demonstrated increased mTORC2 activity. These differential effects on mTORC2 activity were specific, in that mTORC1 did not demonstrate Hsp70-dependent alterations under these conditions. These data suggest that Hsp70 is a component of mTORC2 and is required for proper assembly and activity of the kinase both constitutively and following heat shock.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Complejos Multienzimáticos/metabolismo , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Línea Celular , Análisis Mutacional de ADN , Regulación hacia Abajo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Inmunoprecipitación , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN sin Sentido/genética , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Técnicas del Sistema de Dos HíbridosRESUMEN
mTORC2 is a multimeric kinase composed of the mammalian target of rapamycin kinase (mTOR), mLST8, mSin1, and rictor. The complex is insensitive to acute rapamycin exposure and has shown functions in controlling cell growth and actin cytoskeletal assembly. mTORC2 has recently been shown to phosphorylate and activate Akt. Because approximately 70% of gliomas harbor high levels of activated Akt, we investigated whether mTORC2 activity was elevated in gliomas. In this study, we found that mTORC2 activity was elevated in glioma cell lines as well as in primary tumor cells as compared with normal brain tissue (P < 0.05). Moreover, we found that rictor protein and mRNA levels were also elevated and correlated with increased mTORC2 activity. Overexpression of rictor in cell lines led to increased mTORC2 assembly and activity. These lines exhibited increased anchorage-independent growth in soft agar, increased S-phase cell cycle distribution, increased motility, and elevated integrin beta(1) and beta(3) expression. In contrast, small interfering RNA-mediated knockdown of rictor inhibited these oncogenic activities. Protein kinase C alpha (PKC alpha) activity was shown to be elevated in rictor-overexpressing lines but reduced in rictor-knockdown clones, consistent with the known regulation of actin organization by mTORC2 via PKC alpha. Xenograft studies using these cell lines also supported a role for increased mTORC2 activity in tumorigenesis and enhanced tumor growth. In summary, these data suggest that mTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential due to increased complex formation as a result of the overexpression of rictor.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Glioma/genética , Glioma/patología , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Neoplasias Encefálicas , Ciclo Celular , División Celular , Línea Celular Tumoral , Movimiento Celular , Ensayo de Unidades Formadoras de Colonias , Regulación Neoplásica de la Expresión Génica , Glioma/fisiopatología , Humanos , Lentivirus/genética , Invasividad Neoplásica , Plásmidos , Regiones Promotoras Genéticas , Proteína Quinasa C-alfa/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina , Serina-Treonina Quinasas TOR , TransfecciónRESUMEN
GATA-4 is a key member of the GATA family of transcription factors involved in cardiac development and growth as well as in cardiac hypertrophy and heart failure. Our previous studies suggest that GATA-4 protein synthesis may be translationally regulated. We report here that the 518-nt long 5'-untranslated region (5'-UTR) of the GATA-4 mRNA, which is predicted to form stable secondary structures (-65 kcal/mol) such as to be inhibitory to cap-dependent initiation, confers efficient translation to monocistronic reporter mRNAs in cell-free extracts. Moreover, uncapped GATA-4 5'-UTR containing monocistronic reporter mRNAs continue to be well translated while capped reporters are insensitive to the inhibition of initiation by cap-analog, suggesting a cap-independent mechanism of initiation. Utilizing a dicistronic luciferase mRNA reporter containing the GATA-4 5'-UTR within the intercistronic region, we demonstrate that this leader sequence confers functional internal ribosome entry site (IRES) activity. The activity of the GATA-4 IRES is unaffected in trans-differentiating P19CL6 cells, however, is strongly stimulated immediately following arginine-vasopressin exposure of H9c2 ventricular myocytes. IRES activity is then maintained at submaximal levels during hypertrophic growth of these cells. Supraphysiological Ca(2+) levels diminished stimulation of IRES activity immediately following exposure to vasopressin and inhibition of protein kinase C activity utilizing a pseudosubstrate peptide sequence blocked IRES activity during hypertrophy. Thus, our data suggest a mechanism for GATA-4 protein synthesis under conditions of reduced global cap-dependent translation, which is maintained at a submaximal level during hypertrophic growth and point to the regulation of GATA-4 IRES activity by sarco(ER)-reticular Ca(2+) stores and PKC.