RESUMO
INTRODUCTION: HGF/c-MET signaling is a significant driver of glioblastoma (GBM) growth and disease progression. Unfortunately, c-MET targeted therapies have been found to be largely ineffective suggesting additional redundant mechanisms of c-MET activation. METHODS: Utilizing RNA-sequencing (RNA-seq) and ribosome profiling analyses of circular RNAs, circ-HGF (hsa_circ_0080914) was identified as markedly upregulated in primary GBM and found to potentially encode an HGF protein variant (C-HGF) 119 amino acids in length. This candidate HGF variant was characterized and evaluated for its ability to mediate c-MET activation and regulate PDX GBM cell growth, motility and invasive potential in vitro and tumor burden in intracranial xenografts in mice. RESULTS: An internal ribosome entry site (IRES) was identified within the circ-HGF RNA which mediated translation of the cross-junctional ORF encoding C-HGF and was observed to be highly expressed in GBM relative to normal brain tissue. C-HGF was also found to be secreted from GBM cells and concentrated cell culture supernatants or recombinant C-HGF activated known signaling cascades downstream of c-MET. C-HGF was shown to interact directly with the c-MET receptor resulting in its autophosphorylation and activation in PDX GBM lines. Knockdown of C-HGF resulted in suppression of c-MET signaling and marked inhibition of cell growth, motility and invasiveness, whereas overexpression of C-HGF displayed the opposite effects. Additionally, modulation of C-HGF expression regulated tumor growth in intracranial xenografted PDX GBM models. CONCLUSIONS: These results reveal an alternative mechanism of c-MET activation via a circular RNA encoded HGF protein variant which is relevant in GBM biology. Targeting C-HGF may offer a promising approach for GBM clinical management.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Animais , Humanos , Camundongos , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/patologia , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , RNA , Transdução de Sinais , Proteínas Proto-Oncogênicas c-met/genética , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
Heat shock proteins (HSPs), especially Hsp70 (HSPA1), have been associated with cellular protection from various cellular stresses including heat, hypoxia-ischemia, neurodegeneration, toxins, and trauma. Endogenous HSPs are often synthesized in direct response to these stresses but in many situations are inadequate in protecting cells. The present study addresses the transduction of Hsp70 into cells providing protection from acute oxidative stress by H2O2. The recombinant Fv-Hsp70 protein and two mutant Fv-Hsp70 proteins minus the ATPase domain and minus the ATPase and terminal lid domains were tested at 0.5 and 1.0 µM concentrations after two different concentrations of H2O2 treatment. All three recombinant proteins protected SH-SY5Y cells from acute H2O2 toxicity. This data indicated that the protein binding domain was responsible for cellular protection. In addition, experiments pretreating cells with inhibitors of antioxidant proteins catalase and gamma-glutamylcysteine synthase (GGCS) before H2O2 resulted in cell death despite treatment with Fv-Hsp70, implying that both enzymes were protected from acute oxidative stress after treatment with Fv-Hsp70. This study demonstrates that Fv-Hsp70 is protective in our experiments primarily by the protein-binding domain. The Hsp70 terminal lid domain was also not necessary for protection.
Assuntos
Peróxido de Hidrogênio , Neuroblastoma , Humanos , Peróxido de Hidrogênio/toxicidade , Cisteína Sintase , Catalase , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Recombinantes , Adenosina TrifosfatasesRESUMO
A major mechanism conferring resistance to mTOR inhibitors is activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated mRNA translation, driving the synthesis of proteins promoting resistance of glioblastoma (GBM). Previously, we found this pathway is stimulated by the requisite IRES-trans-acting factor (ITAF) hnRNP A1, which itself is subject to phosphorylation and methylation events regulating cyclin D1 and c-myc IRES activity. Here we describe the requirement for m6A-modification of IRES RNAs for efficient translation and resistance to mTOR inhibition. DRACH-motifs within these IRES RNAs upon m6A modification resulted in enhanced IRES activity via increased hnRNP A1-binding following mTOR inhibitor exposure. Inhibitor exposure stimulated the expression of m6A-methylosome components resulting in increased activity in GBM. Silencing of METTL3-14 complexes reduced IRES activity upon inhibitor exposure and sensitized resistant GBM lines. YTHDF3 associates with m6A-modified cyclin D1 or c-myc IRESs, regulating IRES activity, and mTOR inhibitor sensitivity in vitro and in xenograft experiments. YTHDF3 interacted directly with hnRNP A1 and together stimulated hnRNP A1-dependent nucleic acid strand annealing activity. These data demonstrate that m6A-methylation of IRES RNAs regulate GBM responses to this class of inhibitors.
Assuntos
Ciclina D1 , Glioblastoma , Humanos , Ciclina D1/genética , Ciclina D1/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Sítios Internos de Entrada Ribossomal , Metiltransferases/metabolismo , Biossíntese de Proteínas , Serina-Treonina Quinases TOR/metabolismo , Genes mycRESUMO
DEPTOR is a 48 kDa protein upregulated in multiple myeloma (MM) cells. DEPTOR inhibits mTOR and, by repressing a negative feedback loop, promotes AKT activation. We previously identified a compound that binds to DEPTOR in MM cells and induces its proteasomal degradation. To identify the mechanism of degradation, here, we screened for drug-induced posttranslational modifications and identified reduced phosphorylation of DEPTOR on serine 235 (S235). We show that an S235 phosphomimetic DEPTOR mutant was resistant to degradation, confirming the importance of this posttranslational modification. In addition, a DEPTOR mutant with a serine-to-alanine substitution at S235 could only be expressed upon concurrent proteasome inhibition. Thus, S235 phosphorylation regulates DEPTOR stability. Screening the DEPTOR interactome identified that the association of USP-7 deubiquitinase with DEPTOR was dependent upon S235 phosphorylation. Inhibition of USP-7 activity resulted in DEPTOR polyubiquitination and degradation. A scansite search suggested that ERK1 may be responsible for S235 phosphorylation, which was confirmed through the use of inhibitors, ERK1 knockdown, and an in vitro kinase assay. Inhibition of ERK1 also downregulated AKT phosphorylation. To test if DEPTOR phosphorylation mediated this crosstalk, MM cells were transfected with WT or phosphomimetic DEPTOR and exposed to ERK inhibitors. Although WT DEPTOR had no effect on the inhibition of AKT phosphorylation, the phosphomimetic DEPTOR prevented inhibition. These results indicate that ERK1 maintains AKT activity in MM cells via phosphorylation of DEPTOR. We propose that DEPTOR-dependent crosstalk provides MM cells with a viability-promoting signal (through AKT) when proliferation is stimulated (through ERK).
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Mieloma Múltiplo , Proteínas Proto-Oncogênicas c-akt , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Inibidores de MTOR/farmacologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina/metabolismo , Transdução de SinaisRESUMO
Dysregulated c-myc is a determinant of multiple myeloma progression. Translation of c-myc can be achieved by an mTOR-mediated, cap-dependent mechanism or a cap-independent mechanism where a sequence in the 5'UTR of mRNA, termed the internal ribosome entry site (IRES), recruits the 40S ribosomal subunit. This mechanism requires the RNA-binding factor hnRNP A1 (A1) and becomes critical when cap-dependent translation is inhibited during endoplasmic reticulum (ER) stress. Thus, we studied the role of A1 and the myc IRES in myeloma biology. A1 expression correlated with enhanced c-myc expression in patient samples. Expression of A1 in multiple myeloma lines was mediated by c-myc itself, suggesting a positive feedback circuit where myc induces A1 and A1 enhances myc translation. We then deleted the A1 gene in a myc-driven murine myeloma model. A1-deleted multiple myeloma cells demonstrated downregulated myc expression and were inhibited in their growth in vivo. Decreased myc expression was due to reduced translational efficiency and depressed IRES activity. We also studied the J007 inhibitor, which prevents A1's interaction with the myc IRES. J007 inhibited myc translation and IRES activity and diminished myc expression in murine and human multiple myeloma lines as well as primary samples. J007 also inhibited tumor outgrowth in mice after subcutaneous or intravenous challenge and prevented osteolytic bone disease. When c-myc was ectopically reexpressed in A1-deleted multiple myeloma cells, tumor growth was reestablished. These results support the critical role of A1-dependent myc IRES translation in myeloma.
Assuntos
Ribonucleoproteína Nuclear Heterogênea A1 , Camundongos , Mieloma Múltiplo , Proteínas Proto-Oncogênicas c-myc , Animais , Genes myc , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
We previously found that human heroin addicts and mice chronically exposed to morphine exhibit a significant increase in the number of detected hypocretin/orexin (Hcrt)-producing neurons. However, it remains unknown how this increase affects target areas of the hypocretin system involved in opioid withdrawal, including norepinephrine containing structures locus coeruleus (LC) and A1/A2 medullary regions. Using a combination of immunohistochemical, biochemical, imaging, and behavioral techniques, we now show that the increase in detected hypocretin cell number translates into a significant increase in hypocretin innervation and tyrosine hydroxylase (TH) levels in the LC without affecting norepinephrine-containing neuronal cell number. We show that the increase in TH is completely dependent on Hcrt innervation. The A1/A2 regions were unaffected by morphine treatment. Manipulation of the Hcrt system may affect opioid addiction and withdrawal.SIGNIFICANCE STATEMENT Previously, we have shown that the hypothalamic hypocretin system undergoes profound anatomic changes in human heroin addicts and in mice exposed to morphine, suggesting a role of this system in the development of addictive behaviors. The locus coeruleus plays a key role in opioid addiction. Here we report that the hypothalamic hypocretin innervation of the locus coeruleus increases dramatically with morphine administration to mice. This increase is correlated with a massive increase in tyrosine hydroxylase expression in locus coeruleus. Elimination of hypocretin neurons prevents the tyrosine hydroxylase increase in locus coeruleus and dampens the somatic and affective components of opioid withdrawal.
Assuntos
Morfina/efeitos adversos , Neurônios/metabolismo , Norepinefrina/metabolismo , Alcaloides Opiáceos/efeitos adversos , Orexinas/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Animais , Locus Cerúleo/metabolismo , Camundongos , Atividade Motora/fisiologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Carga Tumoral/fisiologia , Proteínas de Sinalização YAP/metabolismo , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Via de Sinalização Hippo/fisiologia , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Camundongos , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fosforilação/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Proteínas de Sinalização YAP/genéticaRESUMO
INTRODUCTION: Recent studies have suggested that dysregulated Hippo pathway signaling may contribute to glioblastoma proliferation and invasive characteristics. The downstream effector of the pathway, the Yes-associated protein (YAP) oncoprotein, has emerged as a promising target in glioblastoma multiforme (GBM). METHODS: Utilizing a high-throughput yeast two-hybrid based screen, a small molecule was identified which inhibits the association of the co-transcriptional activator YAP1 and the TEA domain family member 1 (TEAD1) transcription factor protein-protein interaction interface. This candidate inhibitor, NSC682769, a novel benzazepine compound, was evaluated for its ability to affect Hippo/YAP axis signaling and potential anti-glioblastoma properties. RESULTS: NSC682769 potently blocked association of YAP and TEAD in vitro and in GBM cells treated with submicromolar concentrations. Moreover, inhibitor-coupled bead pull down and surface plasmon resonance analyses demonstrate that NSC682769 binds to YAP. NSC682769 treatment of GBM lines and patient derived cells resulted in downregulation of YAP expression levels resulting in curtailed YAP-TEAD transcriptional activity. In GBM cell models, NSC682769 inhibited proliferation, colony formation, migration, invasiveness and enhanced apoptosis. In tumor xenograft and genetically engineered mouse models, NSC682769 exhibited marked anti-tumor responses and resulted in increased overall survival and displayed significant blood-brain barrier penetration. CONCLUSIONS: These results demonstrate that blockade of YAP-TEAD association is a viable therapeutic strategy for glioblastoma. On the basis of these favorable preclinical studies further clinical studies are warranted.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Antineoplásicos/farmacologia , Neoplasias Encefálicas , Proteínas de Ligação a DNA/antagonistas & inibidores , Glioblastoma , Proteínas Nucleares/antagonistas & inibidores , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Fatores de Transcrição de Domínio TEA , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Internal ribosome entry site (IRES)-mediated protein synthesis has been demonstrated to play an important role in resistance to mechanistic target of rapamycin (mTOR) targeted therapies. Previously, we have demonstrated that the IRES trans-acting factor (ITAF), hnRNP A1 is required to promote IRES activity and small molecule inhibitors which bind specifically to this ITAF and curtail IRES activity, leading to mTOR inhibitor sensitivity. Here we report the identification of riluzole (Rilutek®), an FDA-approved drug for amyotrophic lateral sclerosis (ALS), via an in silico docking analysis of FDA-approved compounds, as an inhibitor of hnRNP A1. In a riluzole-bead coupled binding assay and in surface plasmon resonance imaging analyses, riluzole was found to directly bind to hnRNP A1 and inhibited IRES activity via effects on ITAF/RNA-binding. Riluzole also demonstrated synergistic anti-glioblastoma (GBM) affects with mTOR inhibitors in vitro and in GBM xenografts in mice. These data suggest that repurposing riluzole, used in conjunction with mTOR inhibitors, may serve as an effective therapeutic option in glioblastoma.
Assuntos
Antineoplásicos/farmacologia , Glioblastoma/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/antagonistas & inibidores , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Riluzol/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Reposicionamento de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Sítios Internos de Entrada Ribossomal/fisiologia , Camundongos , Camundongos SCID , Simulação de Acoplamento Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Riluzol/química , Riluzol/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
INTRODUCTION: Clinical trials directed at mechanistic target of rapamycin (mTOR) inhibition have yielded disappointing results in glioblastoma (GBM). A major mechanism of resistance involves the activation of a salvage pathway stimulating internal ribosome entry site (IRES)-mediated protein synthesis. PRMT5 activity has been implicated in the enhancement of IRES activity. METHODS: We analyzed the expression and activity of PRMT5 in response to mTOR inhibition in GBM cell lines and short-term patient cultures. To determine whether PRMT5 conferred resistance we used genetic and pharmacological approaches to ablate PRMT5 activity and assessed the effects on in vitro and in vivo sensitivity. Mutational analyses of the requisite IRES-trans-acting factor (ITAF), hnRNP A1 determined whether PRMT5-mediated methylation was necessary for ITAF RNA binding and IRES activity. RESULTS: PRMT5 activity is stimulated in response to mTOR inhibitors. Knockdown or treatment with a PRMT5 inhibitor blocked IRES activity and sensitizes GBM cells. Ectopic expression of non-methylatable hnRNP A1 mutants demonstrated that methylation of either arginine residues 218 or 225 was sufficient to maintain IRES binding and hnRNP A1-dependent cyclin D1 or c-MYC IRES activity, however a double R218K/R225K mutant was unable to do so. The PRMT5 inhibitor EPZ015666 displayed synergistic anti-GBM effects in vitro and in a xenograft mouse model in combination with PP242. CONCLUSIONS: These results demonstrate that PRMT5 activity is stimulated upon mTOR inhibition in GBM. Our data further support a signaling cascade in which PRMT5-mediated methylation of hnRNP A1 promotes IRES RNA binding and activation of IRES-mediated protein synthesis and resultant mTOR inhibitor resistance.
Assuntos
Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Apoptose , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Indóis/farmacologia , Sítios Internos de Entrada Ribossomal , Isoquinolinas/farmacologia , Camundongos , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Purinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Prior work indicates DEPTOR expression in multiple myeloma cells could be a therapeutic target. DEPTOR binds to mTOR via its PDZ domain and inhibits mTOR kinase activity. We previously identified a drug, which prevented mTOR-DEPTOR binding (NSC126405) and induced multiple myeloma cytotoxicity. We now report on a related therapeutic, drug 3g, which induces proteasomal degradation of DEPTOR. DEPTOR degradation followed drug 3g binding to its PDZ domain and was not due to caspase activation or enhanced mTOR phosphorylation of DEPTOR. Drug 3g enhanced mTOR activity, and engaged the IRS-1/PI3K/AKT feedback loop with reduced phosphorylation of AKT on T308. Activation of TORC1, in part, mediated multiple myeloma cytotoxicity. Drug 3g was more effective than NSC126405 in preventing binding of recombinant DEPTOR to mTOR, preventing binding of DEPTOR to mTOR inside multiple myeloma cells, in activating mTOR and inducing apoptosis in multiple myeloma cells. In vivo, drug 3g injected daily abrogated DEPTOR expression in xenograft tumors and induced an antitumor effect although modest weight loss was seen. Every-other-day treatment, however, was equally effective without weight loss. Drug 3g also reduced DEPTOR expression in normal tissues. Although no potential toxicity was identified in hematopoietic or hepatic function, moderate cardiac enlargement and glomerular mesangial hypertrophy was seen. DEPTOR protected multiple myeloma cells against bortezomib suggesting anti-DEPTOR drugs could synergize with proteasome inhibitors (PI). Indeed, combinations of drug NSC126405 + bortezomib were synergistic. In contrast, drug 3g was not and was even antagonistic. This antagonism was probably due to prevention of proteasomal DEPTOR degradation.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Proteólise , Animais , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Mieloma Múltiplo/metabolismo , Proteólise/efeitos dos fármacos , Resultado do TratamentoRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0176599.].
RESUMO
DEPTOR is a 48kDa protein that binds to mTOR and inhibits this kinase within mTORC1 and mTORC2 complexes. Over-expression of DEPTOR specifically occurs in the multiple myeloma (MM) tumor model and DEPTOR knockdown is cytotoxic to MM cells, suggesting it is a potential therapeutic target. Since mTORC1 paralysis protects MM cells against DEPTOR knockdown, it indicates that the protein-protein interaction between DEPTOR and mTOR is key to MM viability vs death. In a previous study, we used a yeast two-hybrid screen of a small inhibitor library to identify a compound that inhibited DEPTOR/mTOR binding in yeast. This therapeutic (compound B) also prevented DEPTOR/mTOR binding in MM cells and was selectively cytotoxic to MM cells. We now present a structure-activity relationship (SAR) study around this compound as a follow-up report of this previous work. This study has led to the discovery of five new leads - namely compounds 3g, 3k, 4d, 4e and 4g - all of which have anti-myeloma cytotoxic properties superior to compound B. Due to their targeting of DEPTOR, these compounds activate mTORC1 and selectively induce MM cell apoptosis and cell cycle arrest.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Concentração Inibidora 50 , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Regulatória Associada a mTOR , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/antagonistas & inibidoresRESUMO
A small molecule which specifically blocks the interaction of Rictor and mTOR was identified utilizing a high-throughput yeast two-hybrid screen and evaluated as a potential inhibitor of mTORC2 activity in glioblastoma multiforme (GBM). In vitro, CID613034 inhibited mTORC2 kinase activity at submicromolar concentrations and in cellular assays specifically inhibited phosphorylation of mTORC2 substrates, including AKT (Ser-473), NDRG1 (Thr-346) and PKCα (Ser-657), while having no appreciable effects on the phosphorylation status of the mTORC1 substrate S6K (Thr-389) or mTORC1-dependent negative feedback loops. CID613034 demonstrated significant inhibitory effects on cell growth, motility and invasiveness in GBM cell lines and sensitivity correlated with relative Rictor or SIN1 expression. Structure-activity relationship analyses afforded an inhibitor, JR-AB2-011, with improved anti-GBM properties and blocked mTORC2 signaling and Rictor association with mTOR at lower effective concentrations. In GBM xenograft studies, JR-AB2-011 demonstrated significant anti-tumor properties. These data support mTORC2 as a viable therapeutic target in GBM and suggest that targeting protein-protein interactions critical for mTORC2 function is an effective strategy to achieve therapeutic responses.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Transporte/metabolismo , Glioblastoma/patologia , Complexos Multiproteicos/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Complexos Multiproteicos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêutico , Relação Estrutura-Atividade , Serina-Treonina Quinases TOR/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
DEPTOR is a 48 kDa protein that binds to mTOR and inhibits this kinase in TORC1 and TORC2 complexes. Overexpression of DEPTOR specifically occurs in a model of multiple myeloma. Its silencing in multiple myeloma cells is sufficient to induce cytotoxicity, suggesting that DEPTOR is a potential therapeutic target. mTORC1 paralysis protects multiple myeloma cells against DEPTOR silencing, implicating mTORC1 in the critical role of DEPTOR in multiple myeloma cell viability. Building on this foundation, we interrogated a small-molecule library for compounds that prevent DEPTOR binding to mTOR in a yeast-two-hybrid assay. One compound was identified that also prevented DEPTOR-mTOR binding in human myeloma cells, with subsequent activation of mTORC1 and mTORC2. In a surface plasmon resonance (SPR) assay, the compound bound to recombinant DEPTOR but not to mTOR. The drug also prevented binding of recombinant DEPTOR to mTOR in the SPR assay. Remarkably, although activating TORC1 and TORC2, the compound induced apoptosis and cell-cycle arrest in multiple myeloma cell lines and prevented outgrowth of human multiple myeloma cells in immunodeficient mice. In vitro cytotoxicity against multiple myeloma cell lines was directly correlated with DEPTOR protein expression and was mediated, in part, by the activation of TORC1 and induction of p21 expression. Additional cytotoxicity was seen against primary multiple myeloma cells, whereas normal hematopoietic colony formation was unaffected. These results further support DEPTOR as a viable therapeutic target in multiple myeloma and suggest an effective strategy of preventing binding of DEPTOR to mTOR. Cancer Res; 76(19); 5822-31. ©2016 AACR.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Mieloma Múltiplo/tratamento farmacológico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Mieloma Múltiplo/patologia , Complexos Multiproteicos/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
Our previous work has demonstrated an intrinsic mRNA-specific protein synthesis salvage pathway operative in glioblastoma (GBM) tumor cells that is resistant to mechanistic target of rapamycin (mTOR) inhibitors. The activation of this internal ribosome entry site (IRES)-dependent mRNA translation initiation pathway results in continued translation of critical transcripts involved in cell cycle progression in the face of global eIF-4E-mediated translation inhibition. Recently we identified compound 11 (C11), a small molecule capable of inhibiting c-MYC IRES translation as a consequence of blocking the interaction of a requisite c-MYC IRES trans-acting factor, heterogeneous nuclear ribonucleoprotein A1, with its IRES. Here we demonstrate that C11 also blocks cyclin D1 IRES-dependent initiation and demonstrates synergistic anti-GBM properties when combined with the mechanistic target of rapamycin kinase inhibitor PP242. The structure-activity relationship of C11 was investigated and resulted in the identification of IRES-J007, which displayed improved IRES-dependent initiation blockade and synergistic anti-GBM effects with PP242. Mechanistic studies with C11 and IRES-J007 revealed binding of the inhibitors within the UP1 fragment of heterogeneous nuclear ribonucleoprotein A1, and docking analysis suggested a small pocket within close proximity to RRM2 as the potential binding site. We further demonstrate that co-therapy with IRES-J007 and PP242 significantly reduces tumor growth of GBM xenografts in mice and that combined inhibitor treatments markedly reduce the mRNA translational state of cyclin D1 and c-MYC transcripts in these tumors. These data support the combined use of IRES-J007 and PP242 to achieve synergistic antitumor responses in GBM.
Assuntos
Neoplasias Encefálicas/terapia , Ciclina D1/genética , Genes myc , Glioblastoma/terapia , Sítios Internos de Entrada Ribossomal , Biossíntese de Proteínas , RNA Mensageiro/genética , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Feminino , Glioblastoma/metabolismo , Xenoenxertos , Humanos , CamundongosRESUMO
The mechanistic target of rapamycin (mTOR) and Hippo signaling pathways are two major signaling cascades that coordinately regulate cell growth and proliferation. Dysregulation of these pathways plays a critical role in gliomagenesis. Recent reports have provided evidence of cross-talk between the mTOR and Hippo pathways; however, a complete description of the signaling relationships between these pathways remains to be elucidated. Utilizing a gene-trapping strategy in a mouse glioma model, we report the identification of AMOTL2 as a candidate substrate for mTORC2. AMOTL2 is phosphorylated at serine 760 by mTORC2. Mutation of AMOTL2 mimicking constitutive Ser(760) phosphorylation blocks its ability to bind and repress YAP leading to increased relative expression of known YAP gene targets. Moreover, overexpression of AMOTL2 or a nonphosphorylatable AMOTL2-S760A mutant inhibited YAP-induced transcription, foci formation, growth, and metastatic properties, whereas overexpression of a phosphomimetic AMOTL2-S760E mutant negated these repressive effects of AMOTL2 in glioblastoma (GBM) cells in vitro. Similar effects on xenograft growth were observed in GBM cells expressing these AMOTL2 Ser(760) mutants. YAP was also shown to be required for Rictor-mediated GBM growth and survival. Finally, an analysis of mTORC2/AMOTL2/YAP activities in primary GBM samples supported the clinical relevance of this signaling cascade, and we propose that pharmacological agents cotargeting these regulatory circuits may hold therapeutic potential.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Encefálicas/metabolismo , Proteínas de Transporte/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Complexos Multiproteicos/metabolismo , Fosfoproteínas/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Angiomotinas , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Feminino , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos/genética , Mutação , Invasividade Neoplásica , Transplante de Neoplasias , Fosfoproteínas/genética , Fosforilação , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Fatores de Transcrição , Transplante Heterólogo , Proteínas de Sinalização YAPRESUMO
Because multiple myeloma (MM) cells are at risk for endoplasmic reticulum (ER) stress, they require a carefully regulated mechanism to promote protein translation of selected transcripts when proliferation is stimulated. MAPK-interacting kinases (MNKs) may provide this mechanism by enhancing cap-dependent translation of a small number of critical transcripts. We, thus, tested whether MNKs played a role in MM responses to the myeloma growth factor interleukin-6 (IL-6). IL-6 activated MNK1 phosphorylation and induced phosphorylation of its substrate, eIF-4E, in MM lines and primary specimens. MNK paralysis, achieved pharmacologically or by shRNA, prevented MM expansion stimulated by IL-6. A phosphodefective eIF-4E mutant also prevented the IL-6 response, supporting the notion that MNK's role was via phosphorylation of eIF-4E. Both pharmacological MNK inhibition and expression of the phosphodefective eIF-4E mutant inhibited MM growth in mice. Although critical for IL-6-induced expansion, eIF-4E phosphorylation had no significant effect on global translation or Ig expression. Deep sequencing of ribosome-protected mRNAs revealed a repertoire of genes involved in metabolic processes and ER stress modulation whose translation was regulated by eIF-4E phosphorylation. These data indicate MM cells exploit the MNK/eIF-4E pathway for selective mRNA translation without enhancing global translation and risking ER stress.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Interleucina-6/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático , Humanos , Camundongos , Fosforilação , Biossíntese de ProteínasRESUMO
Dual-specificity phosphatases (DUSPs) dephosphorylate MAP kinases (MAPKs) resulting in their inactivation. Activation of MAPK signaling leads to enhanced DUSP expression, thus establishing feedback regulation of the MAPK pathway. The DUSPs are subject to regulation at the post-translational level via phosphorylation resulting in alterations of protein stability. Here we report that mTORC2 function leads to stabilization of the p38 MAPK phosphatase, DUSP10, thereby inhibiting p38 activity. We demonstrate that mTORC2 binds DUSP10 and phosphorylates DUSP10 on serine residues 224 and 230. These phosphorylation events block DUSP10 turnover resulting in inactivation of p38 signaling. We further show that insulin-stimulated PI3K/mTORC2 signaling regulates DUSP10 stability and p38 activity. Importantly, knockdown of DUSP10 or ectopic overexpression of nonphosphorylatable or phosphomimetic DUSP10 mutants was sufficient to confer differential mTOR kinase inhibitor responses to GBM cells in vitro and in murine xenografts. Finally, DUSP10 was shown to be overexpressed in a significant number of GBM patients. These data demonstrate the ability of the mTORC2 pathway to exert regulatory effects on the DUSP10/p38 feedback loop to control the cellular effects of mTOR kinase inhibitors in GBM and support the use of DUSP10 expression as a surrogate biomarker to predict responsiveness.
RESUMO
We investigated the mechanism by which gene silencing of the mTOR inhibitor, DEPTOR, induces cytoreductive effects on multiple myeloma (MM) cells. DEPTOR knockdown resulted in anti-MM effects in several MM cell lines. Using an inducible shRNA to silence DEPTOR, 8226 MM cells underwent TORC1 activation, downregulation of AKT/SGK activity, apoptosis, cell cycle arrest and senescence. These latter cytotoxic effects were prevented by TORC1 paralysis (Raptor knockdown) but not by over-expression of AKT activity. In addition, DEPTOR knockdown-induced MM death was not associated with activation of the unfolded protein response, suggesting that enhanced ER stress did not play a role. In contrast, DEPTOR knockdown in 8226 cells induced p21 expression, independent of p53, and p21 knockdown prevented all of the cytotoxic effects following DEPTOR silencing. DEPTOR silencing resulted in p21 upregulation in additional MM cell lines. Furthermore, DEPTOR silencing in a murine xenograft model resulted in anti-MM effects associated with p21 upregulation. DEPTOR knockdown also resulted in a decreased expression of p21-targeting miRNAs and transfection of miRNA mimics prevented p21 upregulation and apoptosis following DEPTOR silencing. Use of a shRNA-resistant DEPTOR construct ruled out off-target effects of the shRNA. These results indicate that DEPTOR regulates growth and survival of MM cells via a TORC1/p21 pathway and suggest an involvement of p21-targeted miRNAs.