RESUMO
The relevance of cysteine metabolism in cancer has gained considerable interest in recent years, largely focusing on its role in generating the antioxidant glutathione. Through metabolomic profiling using a combination of high-throughput liquid and gas chromatography-based mass spectrometry on a total of 69 patient-derived glioma specimens, this report documents the discovery of a parallel pathway involving cysteine catabolism that results in the accumulation of cysteine sulfinic acid (CSA) in glioblastoma. These studies identified CSA to rank as one of the top metabolites differentiating glioblastoma from low-grade glioma. There was strong intratumoral concordance of CSA levels with expression of its biosynthetic enzyme cysteine dioxygenase 1 (CDO1). Studies designed to determine the biologic consequence of this metabolic pathway identified its capacity to inhibit oxidative phosphorylation in glioblastoma cells, which was determined by decreased cellular respiration, decreased ATP production, and increased mitochondrial membrane potential following pathway activation. CSA-induced attenuation of oxidative phosphorylation was attributed to inhibition of the regulatory enzyme pyruvate dehydrogenase. Studies performed in vivo abrogating the CDO1/CSA axis using a lentiviral-mediated short hairpin RNA approach resulted in significant tumor growth inhibition in a glioblastoma mouse model, supporting the potential for this metabolic pathway to serve as a therapeutic target. Collectively, we identified a novel, targetable metabolic pathway involving cysteine catabolism contributing to the growth of aggressive high-grade gliomas. These findings serve as a framework for future investigations designed to more comprehensively determine the clinical application of this metabolic pathway and its contributory role in tumorigenesis.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Cisteína/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Redes e Vias Metabólicas , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Cisteína/análogos & derivados , Cisteína/farmacologia , Cisteína Dioxigenase/antagonistas & inibidores , Cisteína Dioxigenase/genética , Cisteína Dioxigenase/metabolismo , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Glioblastoma/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Gradação de Tumores , Complexo Piruvato Desidrogenase/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy.
Assuntos
Glioblastoma/metabolismo , Glioma/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glioblastoma/genética , Glioblastoma/patologia , Glioma/genética , Glioma/patologia , Humanos , Mesoderma/metabolismo , Mesoderma/patologia , Metabolômica , Gradação de Tumores , Fenótipo , Fosfoenolpiruvato/metabolismo , Piruvato Quinase/metabolismo , Transdução de SinaisRESUMO
Post-translational modification through protein acetylation is emerging as an important mode of cellular regulation. We have previously demonstrated the role that glucose-regulated protein 78 kDa (GRP78) acetylation and subsequent activation of the unfolded protein response (UPR) play in the antitumor activity of class I histone deacetylase (HDAC) inhibitors, which primarily target class I HDACs. In this study, we explored the contributory role these class I HDACs may play in UPR regulation. Binding studies were performed using immunoprecipitation/immunoblotting following dual-transfection with HA-tagged GRP78 and FLAG-tagged HDACs. Subcellular localization was performed using Western blot of fractionated cell lysates and confocal microscopy. Individual HDACs were inhibited using RNA interference. We identified the potential of HDACs 1, 2, and 3 to bind to GRP78. These HDACs colocalized with GRP78 in the endoplasmic reticulum (ER). Inhibition of individual HDACs resulted in GRP78 acetylation and selective activation of the UPR. Although traditionally viewed as nuclear enzymes, we demonstrate that Class I HDACs localize to the ER, bind to GRP78, and selectively activate the UPR, representing a novel mode of UPR regulation and mechanism of action of HDAC inhibitors.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Histona Desacetilases/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Inibidores de Histona Desacetilases , Humanos , Interferência de RNARESUMO
Rapidly growing tumors require efficient means to allow them to adapt to fluctuating microenvironments consisting of hypoxia, nutrient deprivation, and acidosis. The unfolded protein response (UPR) represents a defense mechanism allowing cells to respond to these adverse conditions. The chaperone protein GRP78 serves as a master UPR regulator that is aberrantly expressed in a variety of cancers, including glioma. Therefore, cancer cells may be particularly reliant upon the adaptive mechanisms offered by the UPR and targeting GRP78 may represent a unique therapeutic strategy. Here we report that diffuse expression of GRP78 protein is present in Grade III-IV, but not Grade I-II glioma. To determine the role GRP78 plays in glioblastoma tumorigenesis, we explored the anti-tumor activity of the novel fusion protein EGF-SubA, which combines EGF with the cytotoxin SubA that has been recently shown to selectively cleave GRP78. EGF-SubA demonstrated potent tumor-specific proteolytic activity and cytotoxicity in glioblastoma lines and potentiated the anti-tumor activity of both temozolomide and ionizing radiation. To determine if the tumor microenvironment influences EGF-SubA activity, we maintained cells in acidic conditions that led to both UPR activation and increased EGF-SubA induced cytotoxicity. EGF-SubA was well tolerated in mice and led to a significant tumor growth delay in a glioma xenograft mouse model. The UPR is emerging as an important adaptive pathway contributing to glioma tumorigenesis. Targeting its primary mediator, the chaperone protein GRP78, through specific, proteolytic cleavage with the immunotoxin EGF-SubA represents a novel and promising multi-targeted approach to cancer therapy.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Glioblastoma/metabolismo , Subtilisinas/metabolismo , Subtilisinas/farmacologia , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/metabolismo , Glioblastoma/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Immunoblotting , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subtilisinas/genética , Análise Serial de Tecidos , Resposta a Proteínas não Dobradas/genéticaRESUMO
A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of vorinostat with bevacizumab and CPT-11 in recurrent glioblastoma. Vorinostat was combined with bevacizumab and CPT-11 and was escalated using a standard 3 + 3 design. Vorinostat was escalated up to 2 actively investigated doses of this compound or until the MTD was identified on the basis of DLTs. Correlative science involving proteomic profiling of serial patient plasma samples was performed. Nineteen patients were treated. The MTD of vorinostat was established at 400 mg on days 1-7 and 15-21 every 28 days when combined with bevacizumab and CPT-11. Common toxicities were fatigue and diarrhea. DLTs included fatigue, hypertension/hypotension, and central nervous system ischemia. Although the MTD was established, CPT-11 dose reductions were common early in therapy. High-dose vorinostat had an improved progression-free survival and overall survival when compared with low-dose vorinostat. Serum proteomic profiling identified IGFBP-5 and PDGF-AA as markers for improved PFS and recurrence, respectively. A MTD for the combination of vorinostat with bevacizumab and CPT-11 has been established, although it has poor long-term tolerability. With the increased toxicities associated with CPT-11 coupled with its unclear clinical significance, investigating the efficacy of vorinostat combined with bevacizumab alone may represent a more promising strategy to evaluate in the context of a phase II clinical trial.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/toxicidade , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidade , Bevacizumab , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Camptotecina/toxicidade , Intervalo Livre de Doença , Feminino , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/toxicidade , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Irinotecano , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/tratamento farmacológico , Fator de Crescimento Derivado de Plaquetas/análise , Proteômica , VorinostatRESUMO
The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G(2) checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G(2) checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G(2) checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Glioblastoma/tratamento farmacológico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacocinética , Pirazóis/farmacologia , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Pirimidinonas , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacocinética , Radiossensibilizantes/farmacologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although the function of histone deacetylases (HDACs) have primarily been associated with influencing transcription through chromatin remodeling, the capacity of these enzymes to interface with a diverse array of biologic processes by modulating a growing list of nonhistone substrates has gained recent attention. Recent investigations have demonstrated the potential of HDACs to directly regulate the unfolded protein response (UPR) through acetylation of its central regulatory protein, Grp78. Further, this appears to be an important mechanism underlying the anti-tumor activity of HDAC inhibitors. Herein, we provide a summary of the literature supporting the role HDACs play in regulating the UPR and a detailed description of methods to allow for the study of both acetylation of nonhistone proteins and UPR pathway activation following HDAC inhibition.
Assuntos
Histona Desacetilases/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Acetilação , Animais , Morte Celular , Linhagem Celular , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/fisiologia , eIF-2 Quinase/metabolismoRESUMO
Histone deacetylase (HDAC) inhibitors represent a promising class of anti-cancer agents that are actively being evaluated in the context of clinical trials in solid tumors, including glioblastoma. What makes these agents particularly attractive is their capacity to enhance the activity of commonly used cytotoxics in cancer therapy, including both chemotherapy and ionizing radiation. As recent investigations suggest HDAC inhibitors may potentiate the cytotoxicity of topoisomerase inhibitors, which continue to be a commonly used class of agents in the treatment of glioblastoma, we performed preclinical studies to determine if this combination may be a promising strategy in glioblastoma. The effects of the HDAC inhibitor vorinostat and SN38, which is the active metabolite of the topoisomerase I inhibitor CPT-11, was evaluated using the clonogenic assay. Various treatment schedules were tested to determine optimum drug sequencing. Induction of DNA double strand breaks (DSBs) with the combination of vorinostat and SN38 was evaluated using the neutral comet assay, and their subsequent repair was evaluated by gammaH2AX foci kinetics using immunofluorescent cytochemistry. Vorinostat enhanced the cytotoxicity of SN38 in glioblastoma cell lines. Optimal treatment schedules involved maximal concurrent administration of agents. Pretreatment with either agent did not enhance cytotoxicity. Vorinostat potentiated SN38-induced DNA DSBs and attenuated their subsequent repair. These results indicate vorinostat enhances the cytotoxicity of SN38 in glioblastoma cell lines, suggesting this combination may be a worthwhile strategy to test in the context of a clinical trial.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Proliferação de Células/efeitos dos fármacos , Ensaio Cometa , DNA Topoisomerases Tipo I/química , Sinergismo Farmacológico , Imunofluorescência , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Irinotecano , Células Tumorais Cultivadas , Ensaio Tumoral de Célula-Tronco , VorinostatRESUMO
Histone deacetylase (HDAC) inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP) 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78) is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR). Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK). Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2alpha phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.