RESUMEN
Electromagnetic fields (EMF) in the radio frequency energy (RFE) range can affect cells at the molecular level. Here we report a technology that can record the specific RFE signal of a given molecule, in this case the siRNA of epidermal growth factor receptor (EGFR). We demonstrate that cells exposed to this EGFR siRNA RFE signal have a 30-70% reduction of EGFR mRNA expression and ~60% reduction in EGFR protein expression vs. control treated cells. Specificity for EGFR siRNA effect was confirmed via RNA microarray and antibody dot blot array. The EGFR siRNA RFE decreased cell viability, as measured by Calcein-AM measures, LDH release and Caspase 3 cleavage, and increased orthotopic xenograft survival. The outcomes of this study demonstrate that an RFE signal can induce a specific siRNA-like effect on cells. This technology opens vast possibilities of targeting a broader range of molecules with applications in medicine, agriculture and other areas.
Asunto(s)
Radiación Electromagnética , Receptores ErbB/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioma/metabolismo , Apoptosis/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Receptores ErbB/genética , Glioma/genética , Humanos , Antígeno Ki-67/metabolismo , Interferencia de ARN/fisiología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Cancer cells are heterogeneous and, it has been proposed, fall into at least two classes: the tumor-initiating cancer stem cells (CSC) and the more differentiated tumor cells. The transmembrane protein CD133 has been widely used to isolate putative CSC populations in several cancer types, but its validity as a CSC marker and hence its clinical ramifications remain controversial. Here, we conducted transcriptomic profiling of sorted CD133(+) and CD133(-) cells from human glioblastoma multiforme (GBM) and, by subtractive analysis, established a CD133 gene expression signature composed of 214 differentially expressed genes. Extensive computational comparisons with a compendium of published gene expression profiles reveal that the CD133 gene signature transcriptionally resembles human ES cells and in vitro cultured GBM stem cells, and this signature successfully distinguishes GBM from lower-grade gliomas. More importantly, the CD133 gene signature identifies an aggressive subtype of GBM seen in younger patients with shorter survival who bear excessive genomic mutations as surveyed through the Cancer Genome Atlas Network GBM mutation spectrum. Furthermore, the CD133 gene signature distinguishes higher-grade breast and bladder cancers from their lower-grade counterparts. Our systematic analysis provides molecular and genetic support for the stem cell-like nature of CD133(+) cells and an objective means for evaluating cancer aggressiveness.
Asunto(s)
Antígenos CD/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glicoproteínas/metabolismo , Péptidos/metabolismo , Antígeno AC133 , Análisis por Conglomerados , Células Madre Embrionarias/metabolismo , Citometría de Flujo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Inmunohistoquímica , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Tumorales CultivadasRESUMEN
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
SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells that appears to re-activate in several human cancers including glioblastoma multiforme. Using immunoprecipitation (IP)/MS/MS, we identified 144 proteins that are putative SOX2 interacting proteins. Of note, SOX2 was found to interact with several heterogeneous nuclear ribonucleoprotein family proteins, including HNRNPA2B1, HNRNPA3, HNRNPC, HNRNPK, HNRNPL, HNRNPM, HNRNPR, HNRNPU, as well as other ribonucleoproteins, DNA repair proteins and helicases. Gene ontology (GO) analysis revealed that the SOX2 interactome was enriched for GO terms GO:0030529 ribonucleoprotein complex and GO:0004386 helicase activity. These findings indicate that SOX2 associates with the heterogeneous nuclear ribonucleoprotein complex, suggesting a possible role for SOX2 in post-transcriptional regulation in addition to its function as a transcription factor.
Asunto(s)
Regulación de la Expresión Génica , Glioblastoma/metabolismo , Neoplasias de Tejido Nervioso/metabolismo , Mapeo de Interacción de Proteínas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Adulto , Animales , Sitios de Unión , Línea Celular Tumoral , ADN Helicasas/metabolismo , Reparación del ADN/fisiología , Células Madre Embrionarias/fisiología , Elementos de Facilitación Genéticos , Glioblastoma/genética , Humanos , Inmunoprecipitación , Espectrometría de Masas , Ratones , Neoplasias de Tejido Nervioso/genética , Células Madre Pluripotentes/fisiología , Unión Proteica , Procesamiento Postranscripcional del ARN , Ratas , Ribonucleoproteínas/metabolismoRESUMEN
BACKGROUND: SOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined. RESULTS: We show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells. CONCLUSIONS: We present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.
Asunto(s)
Glioblastoma/genética , MicroARNs/genética , Factores de Transcripción SOXB1/metabolismo , Inmunoprecipitación de Cromatina , Humanos , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis de Secuencia de ADNRESUMEN
Motor imagery-based brain-computer interfaces (MI-BCIs) send commands to a computer using the brain activity registered when a subject imagines-but does not perform-a given movement. However, inconsistent MI-BCI performance occurs in variations of brain signals across subjects and experiments; this is considered to be a significant problem in practical BCI. Moreover, some subjects exhibit a phenomenon referred to as "BCI-inefficiency," in which they are unable to generate brain signals for BCI control. These subjects have significant difficulties in using BCI. The primary goal of this study is to identify the connections of the resting-state network that affect MI performance and predict MI performance using these connections. We used a public database of MI, which includes the results of psychological questionnaires and pre-experimental resting-state taken over two sessions on different days. A dynamic causal model was used to calculate the coupling strengths between brain regions with directionality. Specifically, we investigated the motor network in resting-state, including the dorsolateral prefrontal cortex, which performs motor planning. As a result, we observed a significant difference in the connectivity strength from the supplementary motor area to the right dorsolateral prefrontal cortex between the low- and high-MI performance groups. This coupling, measured in the resting-state, is significantly stronger in the high-MI performance group than the low-MI performance group. The connection strength is positively correlated with MI-BCI performance (Session 1: r = 0.54; Session 2: r = 0.42). We also predicted MI performance using linear regression based on this connection (r-squared = 0.31). The proposed predictors, based on dynamic causal modeling, can develop new strategies for improving BCI performance. These findings can further our understanding of BCI-inefficiency and help BCI users to lower costs and save time.
RESUMEN
Otosclerosis is a complex disease that results in a common form of conductive hearing loss due to impaired mobility of the stapes. Stapedial motion becomes compromised secondary to invasion of otosclerotic foci into the stapedio-vestibular joint. Although environmental factors and genetic causes have been implicated in this process, the pathogenesis of otosclerosis remains poorly understood. To identify molecular contributors to otosclerosis we completed a microarray study of otosclerotic stapedial footplates. Stapes footplate samples from otosclerosis and control patients were used in the analysis. One-hundred-and-ten genes were found to be differentially expressed in otosclerosis samples. Ontological analysis of differentially expressed genes in otosclerosis provides evidence for the involvement of a number of pathways in the disease process that include interleukin signaling, inflammation and signal transduction, suggesting that aberrant regulation of these pathways leads to abnormal bone remodeling. Functional analyses of genes from this study will enhance our understanding of the pathogenesis of this disease.
Asunto(s)
Remodelación Ósea/genética , Perfilación de la Expresión Génica , Osteosclerosis/genética , Estribo/fisiopatología , Perfilación de la Expresión Génica/métodos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteosclerosis/metabolismo , Osteosclerosis/fisiopatología , ARN/análisis , Estribo/químicaRESUMEN
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
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
MicroRNAs (miRNAs) are small non-coding RNAs of 20-25 nucleotides in length that are capable of modulating gene expression post-transcriptionally. The potential roles of miRNAs in the tumorigenesis of glioblastoma (GBM) have been under intensive studies in the past few years. In the present study, we found a positive correlation between the levels of miR-127-3p and the cell migration and invasion abilities in several human GBM cell lines. We showed that miR-127-3p promoted cell migration and invasion of GBM cells using in vitro cell lines and in vivo mouse models. We identified SEPT7, a known tumor-suppressor gene that has been reported to suppress GBM cell migration and invasion, as a direct target of miR-127-3p. SEPT7 was able to partially abrogate the effect of miR-127-3p on cell migration and invasion. In addition, microarray analysis revealed that miR-127-3p regulated a number of migration and invasion-related genes. Finally, we verified that miR-127-3p affected the remodeling of the actin cytoskeleton mediated by SEPT7 in GBM cells.
Asunto(s)
Proteínas de Ciclo Celular/genética , Movimiento Celular/genética , Glioblastoma/patología , MicroARNs/genética , Septinas/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , Invasividad Neoplásica/genética , Invasividad Neoplásica/patologíaRESUMEN
Glioblastoma (GBM) proliferation is a multistep process during which the expression levels of many genes that control cell proliferation, cell death, and genetic stability are altered. MicroRNAs (miRNAs) are emerging as important modulators of cellular signaling, including cell proliferation in cancer. In this study, using next generation sequencing analysis of miRNAs, we found that miR-127-3p was downregulated in GBM tissues compared with normal brain tissues; we validated this result by RT-PCR. We further showed that DNA demethylation and histone deacetylase inhibition resulted in downregulation of miR-127-3p. We demonstrated that miR-127-3p overexpression inhibited GBM cell growth by inducing G1-phase arrest both in vitro and in vivo. We showed that miR-127-3p targeted SKI (v-ski sarcoma viral oncogene homolog [avian]), RGMA (RGM domain family, member A), ZWINT (ZW10 interactor, kinetochore protein), SERPINB9 (serpin peptidase inhibitor, clade B [ovalbumin], member 9), and SFRP1 (secreted frizzled-related protein 1). Finally, we found that miR-127-3p suppressed GBM cell growth by inhibiting tumor-promoting SKI and activating the tumor suppression effect of transforming growth factor-ß (TGF-ß) signaling. This study showed, for the first time, that miR-127-3p and its targeted gene SKI, play important roles in GBM and may serve as potential targets for GBM therapy.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genómica , Glioblastoma/genética , MicroARNs/genética , Línea Celular Tumoral , Proliferación Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
NDRG4 is a member of the N-myc downregulated gene family (NDRG) belonging to the alpha/beta hydrolase superfamily. We have previously documented discrepancy between our analysis of the expression and function of NDRG4 in glioblastoma multiforme (GBM) and a recent publication by Schilling et al., who reported that NDRG4 is upregulated in GBM compared to human cortex tissues and knock down of NDRG4 reduced the viability of GBM cells. In the present study, we found that NDRG4 is indeed downregulated, at both RNA and protein levels, by quantitative RT-PCR and Western blot analysis, in GBM compared to normal tissues, and that over expression of NDRG4 inhibited proliferation of GBM cells. These new observations can inform the selection of lead molecular compounds for drug discovery as well as novel diagnostics for GBM. They also lend evidence to NDRG4 a role of tumor suppressor.
Asunto(s)
Neoplasias Encefálicas/genética , Glioblastoma/genética , Proteínas Musculares/genética , Proteínas del Tejido Nervioso/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas Musculares/biosíntesis , Proteínas del Tejido Nervioso/biosíntesisRESUMEN
Glioblastoma Multiforme (GBM) continues to have a poor patient prognosis despite optimal standard of care. Glioma stem cells (GSCs) have been implicated as the presumed cause of tumor recurrence and resistance to therapy. With this in mind, we screened a diverse chemical library of 2,000 compounds to identify therapeutic agents that inhibit GSC proliferation and therefore have the potential to extend patient survival. High-throughput screens (HTS) identified 78 compounds that repeatedly inhibited cellular proliferation, of which 47 are clinically approved for other indications and 31 are experimental drugs. Several compounds (such as digitoxin, deguelin, patulin and phenethyl caffeate) exhibited high cytotoxicity, with half maximal inhibitory concentrations (IC50) in the low nanomolar range. In particular, the FDA approved drug for the treatment of alcoholism, disulfiram (DSF), was significantly potent across multiple patient samples (IC50 of 31.1 nM). The activity of DSF was potentiated by copper (Cu), which markedly increased GSC death. DSF-Cu inhibited the chymotrypsin-like proteasomal activity in cultured GSCs, consistent with inactivation of the ubiquitin-proteasome pathway and the subsequent induction of tumor cell death. Given that DSF is a relatively non-toxic drug that can penetrate the blood-brain barrier, we suggest that DSF should be tested (as either a monotherapy or as an adjuvant) in pre-clinical models of human GBM. Data also support targeting of the ubiquitin-proteasome pathway as a therapeutic approach in the treatment of GBM.
Asunto(s)
Disuasivos de Alcohol/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Disulfiram/farmacología , Glioblastoma/tratamiento farmacológico , Ensayos Analíticos de Alto Rendimiento , Células Madre Neoplásicas/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Citometría de Flujo , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Células Tumorales CultivadasRESUMEN
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
BACKGROUND: A comprehensive network-based understanding of molecular pathways abnormally altered in glioblastoma multiforme (GBM) is essential for developing effective therapeutic approaches for this deadly disease. METHODOLOGY/PRINCIPAL FINDINGS: Applying a next generation sequencing technology, massively parallel signature sequencing (MPSS), we identified a total of 4535 genes that are differentially expressed between normal brain and GBM tissue. The expression changes of three up-regulated genes, CHI3L1, CHI3L2, and FOXM1, and two down-regulated genes, neurogranin and L1CAM, were confirmed by quantitative PCR. Pathway analysis revealed that TGF- beta pathway related genes were significantly up-regulated in GBM tumor samples. An integrative pathway analysis of the TGF beta signaling network identified two alternative TGF-beta signaling pathways mediated by SOX4 (sex determining region Y-box 4) and TGFBI (Transforming growth factor beta induced). Quantitative RT-PCR and immunohistochemistry staining demonstrated that SOX4 and TGFBI expression is elevated in GBM tissues compared with normal brain tissues at both the RNA and protein levels. In vitro functional studies confirmed that TGFBI and SOX4 expression is increased by TGF-beta stimulation and decreased by a specific inhibitor of TGF-beta receptor 1 kinase. CONCLUSIONS/SIGNIFICANCE: Our MPSS database for GBM and normal brain tissues provides a useful resource for the scientific community. The identification of non-SMAD mediated TGF-beta signaling pathways acting through SOX4 and TGFBI (GENE ID:7045) in GBM indicates that these alternative pathways should be considered, in addition to the canonical SMAD mediated pathway, in the development of new therapeutic strategies targeting TGF-beta signaling in GBM. Finally, the construction of an extended TGF-beta signaling network with overlaid gene expression changes between GBM and normal brain extends our understanding of the biology of GBM.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Factores de Transcripción SOXC/genética , Factor de Crecimiento Transformador beta/genética , Química Encefálica , Biología Computacional , Perfilación de la Expresión Génica , Humanos , Transducción de Señal , Regulación hacia ArribaRESUMEN
In attempts to isolate genetic vulnerability factors for panic disorder (PD), a number of investigators have used genome-wide linkage or association analyses. But these attempts have been only modestly successful which suggests that alternative approaches may be needed to define the biology of PD. Therefore, using recently developed genome-wide gene expression profiling, we explored whether transcriptional signatures associated with PD are present in lymphoblast cell line. The expression of 2,469 transcripts in lymphoblast cell lines from 16 subjects was arithmetically increased in every line and significantly increased overall and 354 transcripts was arithmetically decreased in every cell line and significantly decreased overall as compared to those lymphoblast lines from 17 subjects without a history of behavioral illness. Further sex specific analyses showed that in those 10 lines derived from female probands, the expression of a further 67 transcripts was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line and significantly decreased. Conversely, in cell lines from the six male probands, the expression of an additional 212 was arithmetically increased in every line and significantly increased overall and a further 332 transcripts was arithmetically decreased in every cell line. We conclude that lymphoblast cell lines derived from subjects with PD have significant, partially sex dependent changes in gene transcription. Further studies are necessary to correlate these changes in these hemopoetically derived cells with those changes postulated to occur in the CNS in association with PD.
Asunto(s)
Perfilación de la Expresión Génica , Linfocitos/metabolismo , Trastorno de Pánico/genética , Adulto , Línea Celular , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Humanos , Entrevista Psicológica , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Caracteres SexualesRESUMEN
Transcriptional profiling has been used to identify gene expression patterns indicative of general medical illnesses such as atherosclerosis. However, whether these methods can identify common psychiatric disorders has not been established. To answer this question with respect to nicotine use, we used genome-wide expression profiling lymphoblast cell lines from six actively smoking Iowa Adoption Studies (IAS) subjects and nine "clean" control subjects, followed by real-time PCR (RT-PCR) of gene expression patterns in lymphoblast derived RNA from 94 subjects in the IAS. As compared to those from controls without a history of smoking (n = 9), the expression levels of 579 of 29,098 genes were significantly up-regulated and expression levels of 584 of 29,098 genes were significantly down-regulated in lymphoblast lines from currently smoking subjects (n = 6). RT-PCR confirmation of four select RNA levels confirmed the validity of the overall profile and revealed highly significant relationships between the expression of some of these transcripts and (1) major depression, (2) antisocial personality, (3) nicotine dependence, and (4) cannabis dependence. We conclude that the use of expression profiling may contribute significant insights into the biology of complex behavioral disorders.
Asunto(s)
Alcoholismo/genética , Trastorno Depresivo/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Trastornos Relacionados con Sustancias/genética , Tabaquismo/genética , Línea Celular , Trastorno Depresivo/psicología , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Humanos , Entrevistas como Asunto , Iowa , Linfocitos , Masculino , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
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.