RESUMEN
Grade IV glioma, formerly known as glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor, and its treatment remains challenging in part due to extensive interpatient heterogeneity in disease driving mechanisms and lack of prognostic and predictive biomarkers. Using mechanistic inference of node-edge relationship (MINER), we have analyzed multiomics profiles from 516 patients and constructed an atlas of causal and mechanistic drivers of interpatient heterogeneity in GBM (gbmMINER). The atlas has delineated how 30 driver mutations act in a combinatorial scheme to causally influence a network of regulators (306 transcription factors and 73 miRNAs) of 179 transcriptional "programs", influencing disease progression in patients across 23 disease states. Through extensive testing on independent patient cohorts, we share evidence that a machine learning model trained on activity profiles of programs within gbmMINER significantly augments risk stratification, identifying patients who are super-responders to standard of care and those that would benefit from 2 nd line treatments. In addition to providing mechanistic hypotheses regarding disease prognosis, the activity of programs containing targets of 2 nd line treatments accurately predicted efficacy of 28 drugs in killing glioma stem-like cells from 43 patients. Our findings demonstrate that interpatient heterogeneity manifests from differential activities of transcriptional programs, providing actionable strategies for mechanistically characterizing GBM from a systems perspective and developing better prognostic and predictive biomarkers for personalized medicine.
RESUMEN
Poor prognosis and drug resistance in glioblastoma (GBM) can result from cellular heterogeneity and treatment-induced shifts in phenotypic states of tumor cells, including dedifferentiation into glioma stem-like cells (GSCs). This rare tumorigenic cell subpopulation resists temozolomide, undergoes proneural-to-mesenchymal transition (PMT) to evade therapy, and drives recurrence. Through inference of transcriptional regulatory networks (TRNs) of patient-derived GSCs (PD-GSCs) at single-cell resolution, we demonstrate how the topology of transcription factor interaction networks drives distinct trajectories of cell state transitions in PD-GSCs resistant or susceptible to cytotoxic drug treatment. By experimentally testing predictions based on TRN simulations, we show that drug treatment drives surviving PD-GSCs along a trajectory of intermediate states, exposing vulnerability to potentiated killing by siRNA or a second drug targeting treatment-induced transcriptional programs governing non-genetic cell plasticity. Our findings demonstrate an approach to uncover TRN topology and use it to rationally predict combinatorial treatments that disrupts acquired resistance in GBM.
RESUMEN
Poor prognosis and drug resistance in glioblastoma (GBM) can result from cellular heterogeneity and treatment-induced shifts in phenotypic states of tumor cells, including dedifferentiation into glioma stem-like cells (GSCs). This rare tumorigenic cell subpopulation resists temozolomide, undergoes proneural-to-mesenchymal transition (PMT) to evade therapy, and drives recurrence. Through inference of transcriptional regulatory networks (TRNs) of patient-derived GSCs (PD-GSCs) at single-cell resolution, we demonstrate how the topology of transcription factor interaction networks drives distinct trajectories of cell-state transitions in PD-GSCs resistant or susceptible to cytotoxic drug treatment. By experimentally testing predictions based on TRN simulations, we show that drug treatment drives surviving PD-GSCs along a trajectory of intermediate states, exposing vulnerability to potentiated killing by siRNA or a second drug targeting treatment-induced transcriptional programs governing nongenetic cell plasticity. Our findings demonstrate an approach to uncover TRN topology and use it to rationally predict combinatorial treatments that disrupt acquired resistance in GBM.
Asunto(s)
Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Glioma , Células Madre Neoplásicas , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Resistencia a Antineoplásicos/genética , Glioma/genética , Glioma/patología , Glioma/metabolismo , Glioma/tratamiento farmacológico , Temozolomida/farmacología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/patología , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológicoRESUMEN
BACKGROUND: RNA-seq has spurred important gene fusion discoveries in a number of different cancers, including lung, prostate, breast, brain, thyroid and bladder carcinomas. Gene fusion discovery can potentially lead to the development of novel treatments that target the underlying genetic abnormalities. RESULTS: In this study, we provide comprehensive view of gene fusion landscape in 185 glioblastoma multiforme patients from two independent cohorts. Fusions occur in approximately 30-50% of GBM patient samples. In the Ivy Center cohort of 24 patients, 33% of samples harbored fusions that were validated by qPCR and Sanger sequencing. We were able to identify high-confidence gene fusions from RNA-seq data in 53% of the samples in a TCGA cohort of 161 patients. We identified 13 cases (8%) with fusions retaining a tyrosine kinase domain in the TCGA cohort and one case in the Ivy Center cohort. Ours is the first study to describe recurrent fusions involving non-coding genes. Genomic locations 7p11 and 12q14-15 harbor majority of the fusions. Fusions on 7p11 are formed in focally amplified EGFR locus whereas 12q14-15 fusions are formed by complex genomic rearrangements. All the fusions detected in this study can be further visualized and analyzed using our website: http://ivygap.swedish.org/fusions. CONCLUSIONS: Our study highlights the prevalence of gene fusions as one of the major genomic abnormalities in GBM. The majority of the fusions are private fusions, and a minority of these recur with low frequency. A small subset of patients with fusions of receptor tyrosine kinases can benefit from existing FDA approved drugs and drugs available in various clinical trials. Due to the low frequency and rarity of clinically relevant fusions, RNA-seq of GBM patient samples will be a vital tool for the identification of patient-specific fusions that can drive personalized therapy.
Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Glioblastoma/genética , Proteínas de Fusión Oncogénica/genética , Transcriptoma/genética , Perfilación de la Expresión Génica , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Recurrencia Local de Neoplasia/genética , Proteínas de Fusión Oncogénica/clasificación , Proteínas de Fusión Oncogénica/aislamiento & purificaciónRESUMEN
Although the presence of an elevated level of serum amyloid A (SAA) has been regarded as a cardiovascular risk factor, the role of SAA on the progress of atherosclerosis has not been fully elucidated. In the present study, we investigated the effect of SAA on the production of CCL2, an important mediator of monocyte recruitment, and the mechanism underlying the action of SAA in human monocytes. The stimulation of human monocytes with SAA elicited CCL2 production in a concentration-dependent manner. The production of CCL2 by SAA was found to be mediated by the activation of NF-kappaB. Moreover, the signaling events induced by SAA included the activation of ERK and the induction of cyclooxygenase-2, which were required for the production of CCL2. Moreover, SAA-induced CCL2 induction was inhibited by a formyl peptide receptor-like 1 (FPRL1) antagonist. We also found that the stimulation of FPRL1-expressing RBL-2H3 cells induced CCL2 mRNA accumulation, but the vector-expressing RBL-2H3 cells combined with SAA did not. Taken together, our findings suggest that SAA stimulates CCL2 production and, thus, contributes to atherosclerosis. Moreover, FPRL1 was found to be engaged in SAA-induced CCL2 induction, and cyclooxygenase-2 induction was found to be essential for SAA-induced CCL2 expression. These results suggest that SAA and FPRL1 offer a developmental starting point for the treatment of atherosclerosis.
Asunto(s)
Quimiocina CCL2/biosíntesis , Monocitos/inmunología , Monocitos/metabolismo , Receptores de Formil Péptido/fisiología , Receptores de Lipoxina/fisiología , Proteína Amiloide A Sérica/fisiología , Transducción de Señal/inmunología , Adulto , Animales , Línea Celular Tumoral , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Humanos , Ratas , Proteínas Recombinantes/farmacología , Proteína Amiloide A Sérica/genéticaRESUMEN
Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis.
Asunto(s)
Neoplasias Encefálicas/genética , Genes Supresores de Tumor , Glioma/genética , Proteínas del Tejido Nervioso/genética , Azacitidina/análogos & derivados , Azacitidina/farmacología , Neoplasias Encefálicas/patología , Metilación de ADN , Decitabina , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Silenciador del Gen , Genoma Humano , Glioma/patología , Histonas/genética , Histonas/metabolismo , Humanos , Ácidos Hidroxámicos/farmacología , Regiones Promotoras GenéticasRESUMEN
Glioblastoma is an aggressive brain tumor that carries a poor prognosis. The tumor's molecular and cellular landscapes are complex, and their relationships to histologic features routinely used for diagnosis are unclear. We present the Ivy Glioblastoma Atlas, an anatomically based transcriptional atlas of human glioblastoma that aligns individual histologic features with genomic alterations and gene expression patterns, thus assigning molecular information to the most important morphologic hallmarks of the tumor. The atlas and its clinical and genomic database are freely accessible online data resources that will serve as a valuable platform for future investigations of glioblastoma pathogenesis, diagnosis, and treatment.
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , Atlas como Asunto , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Humanos , PronósticoRESUMEN
Medulloblastoma is a heterogeneous pediatric brain tumor with significant therapy-related morbidity, its five-year survival rates ranging from 30% to 70%. Improvement in diagnosis and therapy requires better understanding of medulloblastoma pathology. We used whole-genome microarray analysis to identify putative tumor suppressor genes silenced by epigenetic mechanisms in medulloblastoma. This analysis yielded 714 up-regulated genes in immortalized medulloblastoma cell line D283 on treatment with histone deacetylase (HDAC) inhibitor trichostatin A (TSA). Dickkopf-1 (DKK1), a Wnt antagonist, was found to be up-regulated on HDAC inhibition. We examined DKK1 expression in primary medulloblastoma cells and patient samples by reverse transcriptase PCR and found it to be significantly down-regulated relative to normal cerebellum. Transfection of a DKK1 gene construct into D283 cell lines suppressed medulloblastoma tumor growth in colony focus assays by 60% (P < 0.001). In addition, adenoviral vector-mediated expression of DKK1 in medulloblastoma cells increased apoptosis fourfold (P < 0.001). These data reveal that inappropriate histone modifications might deregulate DKK1 expression in medulloblastoma tumorigenesis and block its tumor-suppressive activity.
Asunto(s)
Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Péptidos y Proteínas de Señalización Intercelular/genética , Meduloblastoma/genética , División Celular/efectos de los fármacos , Neoplasias Cerebelosas/mortalidad , Cromatina/genética , Ensayo de Unidades Formadoras de Colonias , Inhibidores Enzimáticos/farmacología , Silenciador del Gen , Inhibidores de Histona Desacetilasas , Humanos , Ácidos Hidroxámicos/farmacología , Meduloblastoma/mortalidad , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Supervivencia , Células Tumorales CultivadasRESUMEN
Epigenetic changes, including H3K4me3 and H3K27me3 histone modification, play an important role in carcinogenesis. However, no genome-wide histone modification map has been generated for gliomas. Here, we report a genome-wide map of H3K4me3 and H3K27me3 histone modifications for 8 glioma stem cell (GSC) lines, together with the associated gene activation or repression patterns. In addition, we compared the genome-wide histone modification maps of GSC lines to those of astrocytes to identify unique gene activation or repression profiles in GSCs and astrocytes. We also identified a set of bivalent genes, which are genes that are associated with both H3K4me3 and H3K27me3 marks and are poised for action in embryonic stem cells. These bivalent genes are potential targets for inducing differentiation in glioblastoma (GBM) as a therapeutic approach. Finally, we identified SLC17A7 as a bivalent tumor suppressor gene in GBM, as it is down-regulated at both the protein and RNA levels in GBM tissues compared with normal brain tissues, and it inhibits GBM cell proliferation, migration and invasion.
Asunto(s)
Cromatina/genética , Genes Supresores de Tumor , Glioblastoma/genética , Glioblastoma/patología , Histonas/genética , Células Madre Neoplásicas/patología , Proteína 1 de Transporte Vesicular de Glutamato/genética , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Inmunoprecipitación de Cromatina , Epigénesis Genética , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genoma Humano , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Proteína 1 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR â=â5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR â=â3.076, 95% CI [1.301-7.27], pâ=â0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.
Asunto(s)
Metilación de ADN/genética , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioblastoma/genética , Regiones Promotoras Genéticas/genética , Proteínas Supresoras de Tumor/genética , Secuencia de Bases , Islas de CpG , Metilasas de Modificación del ADN/biosíntesis , Enzimas Reparadoras del ADN/biosíntesis , Supervivencia sin Enfermedad , Silenciador del Gen , Glioblastoma/enzimología , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Pronóstico , Resultado del Tratamiento , Proteínas Supresoras de Tumor/biosíntesisRESUMEN
Epigenetic inactivation of tumor suppressor genes is common in human cancer. Using a large-scale whole-genome approach in an earlier study, the authors identified epigenetically silenced genes with potential tumor suppressor function in glioblastoma (GBM). Three genes identified in this analysis-DKK1, SFRP1, and WIF1-are potent inhibitors of the Wnt signal transduction pathway. Here, the authors confirm decreased expression of these genes in GBM tumor tissue samples relative to nontumor brain tissue samples using real-time PCR. They then show that expression of all 3 genes is restored in T98 GBM cells by treatment with the histone deacetylase inhibitor Trichostatin A (TSA), but only DKK1 expression is restored by treatment with the demethylating agent 5-azacytidine. Bisulfite sequencing did not reveal significant methylation in the promoter region of DKK1, whereas histone acetylation and chromatin accessibility increased significantly for all 3 genes after TSA treatment. Ectopic expression of DKK1 significantly reduces colony formation and increases chemotherapy-induced apoptosis in T98 cells. Ectopic expression of the canonical Wnt pathway inhibitors WIF1 and SFRP1 shows a relative lack of response. Chronic Wnt3a stimulation only partially reverses growth suppression after DKK1 reexpression, whereas a specific inhibitor of the JNK pathway significantly reverses the effect of DKK1 reexpression on colony formation and apoptosis in T98 cells. These results support a potential growth-suppressive function for epigenetically silenced DKK1 in GBM and suggest that DKK1 restoration could modulate Wnt signaling through both canonical and noncanonical pathways.
RESUMEN
It is well known that extracts of purple sweet potato (PSP) have potent antioxidant activity. However, it has not been established whether extracts of PSP inhibit oxidation of low-density lipoprotein (LDL) or protein glycation. LDL oxidation and protein glycation are well-known risk factors for chronic metabolic diseases, such as atherosclerosis and diabetes mellitus. Chopped and sliced PSP and yellow sweet potato (YSP) were extracted individually at a concentration of 1 g of PSP tuber/mL using either ethanol or water for 6 hours. The PSP ethanol extract (100-fold diluted) showed stronger radical (2,2-diphenyl-1-picrylhydrazyl radical) scavenging activity than the water extract of PSP and the ethanol extract of YSP (up to a sixfold higher activity). The ethanol extract of PSP also exhibited the highest increase in ferric reducing ability among all extracts. Cupric ion-mediated LDL oxidation was strongly inhibited by the ethanol extract of PSP, with similar potency to vitamin C treatment (final concentration, 10 mM). The PSP extract strongly inhibited fructose-mediated protein glycation as determined by fluorescence spectroscopy. The PSP extract-treated apolipoprotein (apo) A-I showed a decreased multimerization pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas glycated apoA-I showed the strongest multimeric band. PSP extract treatment also inhibited the uptake of oxidized LDL into human macrophage cells with suppression of malondialdehyde production in the cell culture medium. In conclusion, these results suggest that the extract of PSP can be used as a putative anti-atherosclerotic and antidiabetic agent with strong antioxidant functions. This is the first report to show the biological functions of PSP extract to treat hyperlipidemic and hyperglycemic disorders.
Asunto(s)
Antioxidantes/farmacología , Apolipoproteína A-I/metabolismo , Aterosclerosis/prevención & control , Ipomoea batatas/química , Peroxidación de Lípido/efectos de los fármacos , Extractos Vegetales/farmacología , Antioxidantes/uso terapéutico , Ácido Ascórbico , Compuestos de Bifenilo , LDL-Colesterol/metabolismo , Fructosa , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación/efectos de los fármacos , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Malondialdehído/antagonistas & inhibidores , Fitoterapia , Picratos , Extractos Vegetales/uso terapéutico , Raíces de PlantasRESUMEN
Plasma high-density lipoprotein cholesterol (HDL-C) levels are inversely correlated with the risk of cardiovascular disease, and are known to increase with repetitive exercise. In the current study, HDL fractions from athletes' sera were isolated and compared as a function of the type of sport (runners [n = 10], throwers [n = 10], wrestlers [n = 10], and weight lifters [n = 8]), and as an age- and gender-matched reference group (n = 14). Among athletes, HDL from runners had the strongest antioxidant activity. Immunodetection showed that runners and wrestlers had the highest levels of apoA-I and lowest levels of apoA-II in their HDL. Electron microscopy also revealed that HDL(2) of runners and wrestlers were the largest in size. In conclusion, although all athlete groups had significantly better serum lipid/lipoprotein profiles than the reference group, runners and wrestlers had the most desirable lipoprotein function and structure, including antioxidant activity, HDL-associated enzyme activities and increased particle size. [BMB reports 2009; 42(9): 605-610].
Asunto(s)
Rendimiento Atlético , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Carrera , Levantamiento de Peso , Lucha , Adulto , Humanos , Masculino , Adulto JovenRESUMEN
Maspin, a tumour suppressor gene, is differentially expressed in the human placenta. Decreased expression of maspin in the first trimester corresponds with the period of maximum trophoblast invasion, suggesting a role in cell invasion and motility. Although methylation of CpG islands regulates maspin expression in cancer cells, the mechanism of maspin regulation in the human placenta is unknown. Our objectives were to determine the role of epigenetic alterations in the regulation of maspin expression in the placenta. Placental samples obtained from 7 to 40 weeks' gestation were used for bisulphite sequencing and chromatin immunoprecipitation (ChIP) PCR. There was no significant change in the methylation indices in the promoter region of maspin throughout gestation. The levels of histone modifications associated with transcriptionally active chromatin were significantly different in placental tissues from second and third trimester relative to those from first trimester. Addition of trichostatin A (TSA) to placental explants increased the maspin mRNA expression (8- to 20-fold), whereas addition of 5-aza-cytidine (5-AzaC) had no effect on maspin expression. Our data suggest that maspin expression in the human placenta is regulated by changes in histone tail modifications. This is the first report of selective histone modifications associated with differential placental gene expression in human gestation.