RESUMEN
Glioblastoma (GBM) is a lethal primary brain tumor characterized by treatment resistance and inevitable tumor recurrence, both of which are driven by a subpopulation of GBM cancer stem-like cells (GSC) with tumorigenic and self-renewal properties. Despite having broad implications for understanding GSC phenotype, the determinants of upregulated DNA-damage response (DDR) and subsequent radiation resistance in GSC are unknown and represent a significant barrier to developing effective GBM treatments. In this study, we show that constitutive DDR activation and radiation resistance are driven by high levels of DNA replication stress (RS). CD133+ GSC exhibited reduced DNA replication velocity and a higher frequency of stalled replication forks than CD133- non-GSC in vitro; immunofluorescence studies confirmed these observations in a panel of orthotopic xenografts and human GBM specimens. Exposure of non-GSC to low-level exogenous RS generated radiation resistance in vitro, confirming RS as a novel determinant of radiation resistance in tumor cells. GSC exhibited DNA double-strand breaks, which colocalized with "replication factories" and RNA: DNA hybrids. GSC also demonstrated increased expression of long neural genes (>1 Mbp) containing common fragile sites, supporting the hypothesis that replication/transcription collisions are the likely cause of RS in GSC. Targeting RS by combined inhibition of ATR and PARP (CAiPi) provided GSC-specific cytotoxicity and complete abrogation of GSC radiation resistance in vitro These data identify RS as a cancer stem cell-specific target with significant clinical potential.Significance: These findings shed new light on cancer stem cell biology and reveal novel therapeutics with the potential to improve clinical outcomes by overcoming inherent radioresistance in GBM. Cancer Res; 78(17); 5060-71. ©2018 AACR.
Asunto(s)
Carcinogénesis , Glioblastoma/genética , Recurrencia Local de Neoplasia/genética , Células Madre Neoplásicas , Tolerancia a Radiación/genética , Antígeno AC133/genética , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Roturas del ADN de Doble Cadena/efectos de la radiación , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Replicación del ADN/efectos de los fármacos , Replicación del ADN/efectos de la radiación , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/radioterapia , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacologíaRESUMEN
Glioblastoma multiforme lacks effective therapy options. Although deregulated kinase pathways are drivers of malignant progression in glioblastoma multiforme, glioma cells exhibit intrinsic resistance toward many kinase inhibitors, and the molecular basis of this resistance remains poorly understood. Here, we show that overexpression of the protein phosphatase 2A (PP2A) inhibitor protein PME-1 drives resistance of glioma cells to various multikinase inhibitors. The PME-1-elicited resistance was dependent on specific PP2A complexes and was mediated by a decrease in cytoplasmic HDAC4 activity. Importantly, both PME-1 and HDAC4 associated with human glioma progression, supporting clinical relevance of the identified mechanism. Synthetic lethality induced by both PME-1 and HDAC4 inhibition was dependent on the coexpression of proapoptotic protein BAD. Thus, PME-1-mediated PP2A inhibition is a novel mechanistic explanation for multikinase inhibitor resistance in glioma cells. Clinically, these results may inform patient stratification strategies for future clinical trials with selected kinase inhibitors in glioblastoma multiforme. Cancer Res; 76(23); 7001-11. ©2016 AACR.
Asunto(s)
Glioma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteína Fosfatasa 2/metabolismo , Animales , Glioma/patología , Humanos , Ratones , TransfecciónRESUMEN
L-Glutamine (Gln) functions physiologically to balance the carbon and nitrogen requirements of tissues. It has been proposed that in cancer cells undergoing aerobic glycolysis, accelerated anabolism is sustained by Gln-derived carbons, which replenish the tricarboxylic acid (TCA) cycle (anaplerosis). However, it is shown here that in glioblastoma (GBM) cells, almost half of the Gln-derived glutamate (Glu) is secreted and does not enter the TCA cycle, and that inhibiting glutaminolysis does not affect cell proliferation. Moreover, Gln-starved cells are not rescued by TCA cycle replenishment. Instead, the conversion of Glu to Gln by glutamine synthetase (GS; cataplerosis) confers Gln prototrophy, and fuels de novo purine biosynthesis. In both orthotopic GBM models and in patients, (13)C-glucose tracing showed that GS produces Gln from TCA-cycle-derived carbons. Finally, the Gln required for the growth of GBM tumours is contributed only marginally by the circulation, and is mainly either autonomously synthesized by GS-positive glioma cells, or supplied by astrocytes.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Proliferación Celular , Glioblastoma/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glutamina/metabolismo , Nucleótidos/biosíntesis , Animales , Astrocitos/citología , Astrocitos/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Células Cultivadas , Ciclo del Ácido Cítrico , Técnicas de Cocultivo , Femenino , Glioblastoma/genética , Glioblastoma/patología , Glutamato-Amoníaco Ligasa/genética , Ácido Glutámico/metabolismo , Humanos , Masculino , Ratones Endogámicos NOD , Ratones SCID , Modelos Biológicos , Células Madre Neoplásicas/metabolismo , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante HeterólogoRESUMEN
Glioblastoma is the most common form of primary brain tumor in adults and is essentially incurable. Despite aggressive treatment regimens centered on radiotherapy, tumor recurrence is inevitable and is thought to be driven by glioblastoma stem-like cells (GSC) that are highly radioresistant. DNA damage response pathways are key determinants of radiosensitivity but the extent to which these overlapping and parallel signaling components contribute to GSC radioresistance is unclear. Using a panel of primary patient-derived glioblastoma cell lines, we confirmed by clonogenic survival assays that GSCs were significantly more radioresistant than paired tumor bulk populations. DNA damage response targets ATM, ATR, CHK1, and PARP1 were upregulated in GSCs, and CHK1 was preferentially activated following irradiation. Consequently, GSCs exhibit rapid G2-M cell-cycle checkpoint activation and enhanced DNA repair. Inhibition of CHK1 or ATR successfully abrogated G2-M checkpoint function, leading to increased mitotic catastrophe and a modest increase in radiation sensitivity. Inhibition of ATM had dual effects on cell-cycle checkpoint regulation and DNA repair that were associated with greater radiosensitizing effects on GSCs than inhibition of CHK1, ATR, or PARP alone. Combined inhibition of PARP and ATR resulted in a profound radiosensitization of GSCs, which was of greater magnitude than in bulk populations and also exceeded the effect of ATM inhibition. These data demonstrate that multiple, parallel DNA damage signaling pathways contribute to GSC radioresistance and that combined inhibition of cell-cycle checkpoint and DNA repair targets provides the most effective means to overcome radioresistance of GSC.
Asunto(s)
Daño del ADN , Glioblastoma/genética , Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Tolerancia a Radiación/genética , Transducción de Señal/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Reparación del ADN , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Humanos , Mitosis/efectos de los fármacos , Mitosis/genética , Mitosis/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Radiación Ionizante , Transducción de Señal/efectos de los fármacosRESUMEN
During apoptosis, the mitochondrial outer membrane is permeabilized, leading to the release of cytochrome c that activates downstream caspases. Mitochondrial outer membrane permeabilization (MOMP) has historically been thought to occur synchronously and completely throughout a cell, leading to rapid caspase activation and apoptosis. Using a new imaging approach, we demonstrate that MOMP is not an all-or-nothing event. Rather, we find that a minority of mitochondria can undergo MOMP in a stress-regulated manner, a phenomenon we term "minority MOMP." Crucially, minority MOMP leads to limited caspase activation, which is insufficient to trigger cell death. Instead, this caspase activity leads to DNA damage that, in turn, promotes genomic instability, cellular transformation, and tumorigenesis. Our data demonstrate that, in contrast to its well-established tumor suppressor function, apoptosis also has oncogenic potential that is regulated by the extent of MOMP. These findings have important implications for oncogenesis following either physiological or therapeutic engagement of apoptosis.
Asunto(s)
Apoptosis/fisiología , Daño del ADN , Inestabilidad Genómica , Membranas Mitocondriales/fisiología , Animales , Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Western Blotting , Caspasas/metabolismo , Línea Celular Tumoral , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p19 de las Quinasas Dependientes de la Ciclina/genética , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Células HCT116 , Células HeLa , Histonas/metabolismo , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Nitrofenoles/farmacología , Permeabilidad , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Estaurosporina/farmacología , Sulfonamidas/farmacología , Factores de TiempoRESUMEN
Resistance to radiotherapy in glioblastoma (GBM) is an important clinical problem and several authors have attributed this to a subpopulation of GBM cancer stem cells (CSCs) which may be responsible for tumour recurrence following treatment. It is hypothesised that GBM CSCs exhibit upregulated DNA damage responses and are resistant to radiation but the current literature is conflicting. We investigated radioresistance of primary GBM cells grown in stem cell conditions (CSC) compared to paired differentiated tumour cell populations and explored the radiosensitising effects of the ATM inhibitor KU-55933. We report that GBM CSCs are radioresistant compared to paired differentiated tumour cells as measured by clonogenic assay. GBM CSC's display upregulated phosphorylated DNA damage response proteins and enhanced activation of the G2/M checkpoint following irradiation and repair DNA double strand breaks (DSBs) more efficiently than their differentiated tumour cell counterparts following radiation. Inhibition of ATM kinase by KU-55933 produced potent radiosensitisation of GBM CSCs (sensitiser enhancement ratios 2.6-3.5) and effectively abrogated the enhanced DSB repair proficiency observed in GBM CSCs at 24 h post irradiation. G2/M checkpoint activation was reduced but not abolished by KU-55933 in GBM CSCs. ATM kinase inhibition overcomes radioresistance of GBM CSCs and, in combination with conventional therapy, has potential to improve outcomes for patients with GBM.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Glioblastoma/radioterapia , Morfolinas/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Células Madre Neoplásicas/enzimología , Pironas/farmacología , Tolerancia a Radiación/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Femenino , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de la radiación , Rayos gamma , Glioblastoma/enzimología , Glioblastoma/patología , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/efectos de la radiación , Masculino , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/patología , Tolerancia a Radiación/efectos de la radiación , Células Tumorales CultivadasRESUMEN
SIRT1 is an NAD-dependent deacetylase and epigenetic regulator essential for normal mammalian development and homeostasis. Here we describe a human SIRT1 splice variant, designated SIRT1-Δ2/9, in which the deacetylase coding sequence is lost due to splicing between exons 2 and 9. This work aimed to determine if SIRT1-Δ2/9 is a novel functional product of the SIRT1 gene. Endogenous SIRT1-Δ2/9 protein was identified in human cell lysate by immunoblotting and splice variant-specific RNA interference (RNAi). SIRT1-Δ2/9 mRNA is bound by CUGBP2, which downregulates its translation. Using pulldown assays, we demonstrate that SIRT1-Δ2/9 binds p53 protein. SIRT1-Δ2/9 maintains basal p53 protein levels and supports p53 function in response to DNA damage, as evidenced by RNAi-mediated depletion of SIRT1-Δ2/9 prior to damage. In turn, basal p53 downregulates SIRT1-Δ2/9 RNA levels, while stress-activated p53 eliminates SIRT1-Δ2/9. Loss of wild-type (wt) p53 has been correlated with overexpression of SIRT1-Δ2/9 in a range of human cancers. Exogenous SIRT1-Δ2/9 protein associates with specific promoters in chromatin and can regulate cancer-related gene expression, as evidenced by chromatin immunoprecipitation analysis and RNAi/genomic array data. SIRT1 is of major therapeutic importance, and potential therapeutic drugs are screened against SIRT1 deacetylase activity. Our discovery of SIRT1-Δ2/9 identifies a new, deacetylase-independent therapeutic target for SIRT1-related diseases, including cancer.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Sirtuina 1/genética , Proteína p53 Supresora de Tumor/metabolismo , Empalme Alternativo , Línea Celular Tumoral , Daño del ADN , Eliminación de Gen , Humanos , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/metabolismo , Sirtuina 1/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 µM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 µM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.
Asunto(s)
Antineoplásicos/síntesis química , Isoindoles/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Isoindoles/química , Isoindoles/farmacología , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Unión Proteica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
BACKGROUND: The NAD-dependent deacetylase SIRT1 is a nutrient-sensitive coordinator of stress-tolerance, multiple homeostatic processes and healthspan, while p53 is a stress-responsive transcription factor and our paramount tumour suppressor. Thus, SIRT1-mediated inhibition of p53 has been identified as a key node in the common biology of cancer, metabolism, development and ageing. However, precisely how SIRT1 integrates such diverse processes remains to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that SIRT1 is alternatively spliced in mammals, generating a novel SIRT1 isoform: SIRT1-ΔExon8. We show that SIRT1-ΔExon8 is expressed widely throughout normal human and mouse tissues, suggesting evolutionary conservation and critical function. Further studies demonstrate that the SIRT1-ΔExon8 isoform retains minimal deacetylase activity and exhibits distinct stress sensitivity, RNA/protein stability, and protein-protein interactions compared to classical SIRT1-Full-Length (SIRT1-FL). We also identify an auto-regulatory loop whereby SIRT1-ΔExon8 can regulate p53, while in reciprocal p53 can influence SIRT1 splice variation. CONCLUSIONS/SIGNIFICANCE: We characterize the first alternative isoform of SIRT1 and demonstrate its evolutionary conservation in mammalian tissues. The results also reveal a new level of inter-dependency between p53 and SIRT1, two master regulators of multiple phenomena. Thus, previously-attributed SIRT1 functions may in fact be distributed between SIRT1 isoforms, with important implications for SIRT1 functional studies and the current search for SIRT1-activating therapeutics to combat age-related decline.
Asunto(s)
Empalme Alternativo , Sirtuina 1/fisiología , Proteína p53 Supresora de Tumor/fisiología , Acetilación , Animales , Exones , Humanos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genéticaRESUMEN
From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.
Asunto(s)
Antineoplásicos/síntesis química , Indoles/síntesis química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Técnicas Químicas Combinatorias , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indoles/química , Indoles/farmacología , Modelos Moleculares , Unión Proteica , Estereoisomerismo , Relación Estructura-Actividad , Transcripción Genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
A series of 2-N-alkyl-3-aryl-3-alkoxyisoindolinones has been synthesised and evaluated as inhibitors of the MDM2-p53 interaction. The most potent compound, 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (NU8231), exhibited an IC50 of 5.3 +/- 0.9 microM in an ELISA assay, and induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line.