RESUMO
The invasive behavior of glioblastoma, the most aggressive primary brain tumor, is considered highly relevant for tumor recurrence. However, the invasion zone is difficult to visualize by Magnetic Resonance Imaging (MRI) and is protected by the blood brain barrier, posing a particular challenge for treatment. We report biological features of invasive growth accompanying tumor progression and invasion based on associated metabolic and transcriptomic changes observed in patient derived orthotopic xenografts (PDOX) in the mouse and the corresponding patients' tumors. The evolution of metabolic changes, followed in vivo longitudinally by 1H Magnetic Resonance Spectroscopy (1H MRS) at ultra-high field, reflected growth and the invasive properties of the human glioblastoma transplanted into the brains of mice (PDOX). Comparison of MRS derived metabolite signatures, reflecting temporal changes of tumor development and invasion in PDOX, revealed high similarity to spatial metabolite signatures of combined multi-voxel MRS analyses sampled across different areas of the patients' tumors. Pathway analyses of the transcriptome associated with the metabolite profiles of the PDOX, identified molecular signatures of invasion, comprising extracellular matrix degradation and reorganization, growth factor binding, and vascular remodeling. Specific analysis of expression signatures from the invaded mouse brain, revealed extent of invasion dependent induction of immune response, recapitulating respective signatures observed in glioblastoma. Integrating metabolic profiles and gene expression of highly invasive PDOX provided insights into progression and invasion associated mechanisms of extracellular matrix remodeling that is essential for cell-cell communication and regulation of cellular processes. Structural changes and biochemical properties of the extracellular matrix are of importance for the biological behavior of tumors and may be druggable. Ultra-high field MRS reveals to be suitable for in vivo monitoring of progression in the non-enhancing infiltration zone of glioblastoma.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Adulto , Idoso , Animais , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Metaboloma , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Transplante de Neoplasias , Espectroscopia de Prótons por Ressonância Magnética , TranscriptomaRESUMO
Glioblastoma (GBM) is the most aggressive brain tumor type in adults. GBM is heterogeneous, with a compact core lesion surrounded by an invasive tumor front. This front is highly relevant for tumor recurrence but is generally non-detectable using standard imaging techniques. Recent studies demonstrated distinct metabolic profiles of the invasive phenotype in GBM. Magnetic resonance (MR) of hyperpolarized 13C-labeled probes is a rapidly advancing field that provides real-time metabolic information. Here, we applied hyperpolarized 13C-glucose MR to mouse GBM models. Compared to controls, the amount of lactate produced from hyperpolarized glucose was higher in the compact GBM model, consistent with the accepted "Warburg effect". However, the opposite response was observed in models reflecting the invasive zone, with less lactate produced than in controls, implying a reduction in aerobic glycolysis. These striking differences could be used to map the metabolic heterogeneity in GBM and to visualize the infiltrative front of GBM.
Assuntos
Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Isótopos de Carbono/química , Glioblastoma/diagnóstico por imagem , Glioblastoma/metabolismo , Glucose/metabolismo , Glicólise , Imageamento por Ressonância Magnética , Aerobiose , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Linhagem Celular Tumoral , Humanos , Ácido Láctico/metabolismo , Metabolômica , Camundongos SCID , Ácido Pirúvico/metabolismoRESUMO
Glioblastoma are notorious for their highly invasive growth, diffusely infiltrating adjacent brain structures that precludes complete resection, and is a major obstacle for cure. To characterize this "invisible" tumor part, we designed a high resolution multimodal imaging approach assessing in vivo the metabolism of invasively growing glioma xenografts in the mouse brain. Animals were subjected longitudinally to magnetic resonance imaging (MRI) and 1 H spectroscopy (MRS) at ultra high field (14.1 Tesla) that allowed the measurement of 16 metabolic biomarkers to characterize the metabolic profiles. As expected, the neuronal functionality was progressively compromised as indicated by decreasing N-acetyl aspartate, glutamate and gamma-aminobutyric acid and reduced neuronal TCA cycle (-58%) and neurotransmission (-50%). The dynamic metabolic changes observed, captured differences in invasive growth that was modulated by re-expression of the tumor suppressor gene WNT inhibitory factor 1 (WIF1) in the orthotopic xenografts that attenuates invasion. At late stage mice were subjected to 13 C MRS with infusion of [1,6-13 C]glucose and 18 FDG positron emission tomography (PET) to quantify cell-specific metabolic fluxes involved in glucose metabolism. Most interestingly, this provided the first in vivo evidence for significant glucose oxidation in glioma cells. This suggests that the infiltrative front of glioma does not undergo the glycolytic switch per se, but that environmental triggers may induce metabolic reprograming of tumor cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Encefálicas/diagnóstico por imagem , Encéfalo/metabolismo , Fluordesoxiglucose F18/metabolismo , Glioma/diagnóstico por imagem , Glucose/metabolismo , Proteínas Repressoras/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Encéfalo/diagnóstico por imagem , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Glioma/genética , Glioma/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Oxirredução , Tomografia por Emissão de Pósitrons/métodos , Espectroscopia de Prótons por Ressonância Magnética/métodos , Proteínas Repressoras/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
PURPOSE: EORTC 26082 assessed the activity of temsirolimus in patients with newly diagnosed glioblastoma harboring an unmethylated O6 methylguanine-DNA-methyltransferase (MGMT) promoter. EXPERIMENTAL DESIGN: Patients (n = 257) fulfilling eligibility criteria underwent central MGMT testing. Patients with MGMT unmethylated glioblastoma (n = 111) were randomized 1:1 between standard chemo-radiotherapy with temozolomide or radiotherapy plus weekly temsirolimus (25 mg). Primary endpoint was overall survival at 12 months (OS12). A positive signal was considered >38 patients alive at 12 months in the per protocol population. A noncomparative reference arm of 54 patients evaluated the assumptions on OS12 in a standard-treated cohort of patients. Prespecified post hoc analyses of markers reflecting target activation were performed. RESULTS: Both therapies were administered per protocol with a median of 13 cycles of maintenance temsirolimus. Median age was 55 and 58 years in the temsirolimus and standard arms, the WHO performance status 0 or 1 for most patients (95.5%). In the per protocol population, 38 of 54 patients treated with temsirolimus reached OS12. The actuarial 1-year survival was 72.2% [95% confidence interval (CI), 58.2-82.2] in the temozolomide arm and 69.6% (95% CI, 55.8-79.9) in the temsirolimus arm [hazard ratio (HR) 1.16; 95% CI, 0.77-1.76; P = 0.47]. In multivariable prognostic analyses of clinical and molecular factors, phosphorylation of mTORSer2448 in tumor tissue (HR 0.13; 95% CI, 0.04-0.47; P = 0.002), detected in 37.6%, was associated with benefit from temsirolimus. CONCLUSIONS: Temsirolimus was not superior to temozolomide in patients with an unmethylated MGMT promoter. Phosphorylation of mTORSer2448 in the pretreatment tumor tissue may define a subgroup benefitting from mTOR inhibition. Clin Cancer Res; 22(19); 4797-806. ©2016 AACR.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Quimiorradioterapia/métodos , Glioblastoma/tratamento farmacológico , Sirolimo/análogos & derivados , Adulto , Idoso , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Dacarbazina/administração & dosagem , Dacarbazina/análogos & derivados , Feminino , Glioblastoma/mortalidade , Glioblastoma/radioterapia , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Modelos de Riscos Proporcionais , Sirolimo/administração & dosagem , Temozolomida , Proteínas Supressoras de Tumor/genética , Adulto JovemRESUMO
Glioma cell lines are an important tool for research in basic and translational neuro-oncology. Documentation of their genetic identity has become a requirement for scientific journals and grant applications to exclude cross-contamination and misidentification that lead to misinterpretation of results. Here, we report the standard 16 marker short tandem repeat (STR) DNA fingerprints for a panel of 39 widely used glioma cell lines as reference. Comparison of the fingerprints among themselves and with the large DSMZ database comprising 9 marker STRs for 2278 cell lines uncovered 3 misidentified cell lines and confirmed previously known cross-contaminations. Furthermore, 2 glioma cell lines exhibited identity scores of 0.8, which is proposed as the cutoff for detecting cross-contamination. Additional characteristics, comprising lack of a B-raf mutation in one line and a similarity score of 1 with the original tumor tissue in the other, excluded a cross-contamination. Subsequent simulation procedures suggested that, when using DNA fingerprints comprising only 9 STR markers, the commonly used similarity score of 0.8 is not sufficiently stringent to unambiguously differentiate the origin. DNA fingerprints are confounded by frequent genetic alterations in cancer cell lines, particularly loss of heterozygosity, that reduce the informativeness of STR markers and, thereby, the overall power for distinction. The similarity score depends on the number of markers measured; thus, more markers or additional cell line characteristics, such as information on specific mutations, may be necessary to clarify the origin.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Impressões Digitais de DNA , Glioma/genética , Repetições de Microssatélites/genética , DNA de Neoplasias/genética , Humanos , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Glioblastoma (GBM) is a morphologically heterogeneous tumor type with a median survival of only 15 months in clinical trial populations. However, survival varies greatly among patients. As part of a central pathology review, we addressed the question if patients with GBM displaying distinct morphologic features respond differently to combined chemo-radiotherapy with temozolomide. Morphologic features were systematically recorded for 360 cases with particular focus on the presence of an oligodendroglioma-like component and respective correlations with outcome and relevant molecular markers. GBM with an oligodendroglioma-like component (GBM-O) represented 15% of all confirmed GBM (52/339) and was not associated with a more favorable outcome. GBM-O encompassed a pathogenetically heterogeneous group, significantly enriched for IDH1 mutations (19 vs. 3%, p = 0.003) and EGFR amplifications (71 vs. 48%, p = 0.04) compared with other GBM, while co-deletion of 1p/19q was found in only one case and the MGMT methylation frequency was alike (47 vs. 46%). Expression profiles classified most of the GBM-O into two subtypes, 36% (5/14 evaluable) as proneural and 43% as classical GBM. The detection of pseudo-palisading necrosis (PPN) was associated with benefit from chemotherapy (p = 0.0002), while no such effect was present in the absence of PPN (p = 0.86). In the adjusted interaction model including clinical prognostic factors and MGMT status, PPN was borderline nonsignificant (p = 0.063). Taken together, recognition of an oligodendroglioma-like component in an otherwise classic GBM identifies a pathogenetically mixed group without prognostic significance. However, the presence of PPN may indicate biological features of clinical relevance for further improvement of therapy.
Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Oligodendroglioma/patologia , Adolescente , Adulto , Idoso , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Quimiorradioterapia , Ensaios Clínicos Fase III como Assunto , Metilação de DNA , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Oligodendroglioma/genética , Oligodendroglioma/terapia , Prognóstico , Análise de Sobrevida , Temozolomida , Resultado do Tratamento , Adulto JovemRESUMO
Gene expression-based prediction of genomic copy number aberrations in the chromosomal region 12q13 to 12q15 that is flanked by MDM2 and CDK4 identified Wnt inhibitory factor 1 (WIF1) as a candidate tumor suppressor gene in glioblastoma. WIF1 encodes a secreted Wnt antagonist and was strongly downregulated in most glioblastomas as compared with normal brain, implying deregulation of Wnt signaling, which is associated with cancer. WIF1 silencing was mediated by deletion (7/69, 10%) or epigenetic silencing by promoter hypermethylation (29/110, 26%). Co-amplification of MDM2 and CDK4 that is present in 10% of glioblastomas was associated in most cases with deletion of the whole genomic region enclosed, including the WIF1 locus. This interesting pathogenetic constellation targets the RB and p53 tumor suppressor pathways in tandem, while simultaneously activating oncogenic Wnt signaling. Ectopic expression of WIF1 in glioblastoma cell lines revealed a dose-dependent decrease of Wnt pathway activity. Furthermore, WIF1 expression inhibited cell proliferation in vitro, reduced anchorage-independent growth in soft agar, and completely abolished tumorigenicity in vivo. Interestingly, WIF1 overexpression in glioblastoma cells induced a senescence-like phenotype that was dose dependent. These results provide evidence that WIF1 has tumor suppressing properties. Downregulation of WIF1 in 75% of glioblastomas indicates frequent involvement of aberrant Wnt signaling and, hence, may render glioblastomas sensitive to inhibitors of Wnt signaling, potentially by diverting the tumor cells into a senescence-like state.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Envelhecimento , Neoplasias Encefálicas/prevenção & controle , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Glioblastoma/prevenção & controle , Proteínas Repressoras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Metilação de DNA , Regulação para Baixo , Epigênese Genética , Feminino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Amplification of the epidermal growth factor receptor (EGFR) gene is one of the most common oncogenic alterations in glioblastoma (45%) making it a prime target for therapy. However, small molecule inhibitors of the EGFR tyrosine kinase showed disappointing efficacy in clinical trials for glioblastoma. Here we aimed at investigating the molecular effects of the tyrosine kinase inhibitor gefitinib on the EGFR signaling pathway in human glioblastoma. Twenty-two patients selected for reoperation of recurrent glioblastoma were treated within a phase II trial for 5 days with 500 mg gefitinib before surgery followed by postoperative gefitinib until recurrence. Resected glioblastoma tissues exhibited high concentrations of gefitinib (median, 4.1 µg/g), 20 times higher than respective plasma. EGFR-pathway activity was evaluated with phosphorylation-specific assays. The EGFR was efficiently dephosphorylated in treated patients as compared to a control cohort of 12 patients. However, no significant effect on 12 pathway constituents was detected. In contrast, in vitro treatment of a glioblastoma cell line, BS-153, with endogenous EGFRwt amplification and EGFRvIII expression resulted not only in dephosphorylation of the EGFR, but also of key regulators in the pathway such as AKT. Treating established xenografts of the same cell line as an in vivo model showed dephosphorylation of the EGFR without affecting downstream signal transductors, similar to the human glioblastoma. Taken together, gefitinib reaches high concentrations in the tumor tissue and efficiently dephosphorylates its target. However, regulation of downstream signal transducers in the EGFR pathway seems to be dominated by regulatory circuits independent of EGFR phosphorylation.
Assuntos
Antineoplásicos/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Quinazolinas/uso terapêutico , Adulto , Idoso , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Gefitinibe , Glioblastoma/enzimologia , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto JovemRESUMO
PURPOSE: Quantitative methylation-specific tests suggest that not all cells in a glioblastoma with detectable promoter methylation of the O6-methylguanine DNA methyltransferase (MGMT) gene carry a methylated MGMT allele. This observation may indicate cell subpopulations with distinct MGMT status, raising the question of the clinically relevant cutoff of MGMT methylation therapy. Epigenetic silencing of the MGMT gene by promoter methylation blunts repair of O6-methyl guanine and has been shown to be a predictive factor for benefit from alkylating agent therapy in glioblastoma. EXPERIMENTAL DESIGN: Ten paired samples of glioblastoma and respective glioblastoma-derived spheres (GS), cultured under stem cell conditions, were analyzed for the degree and pattern of MGMT promoter methylation by methylation-specific clone sequencing, MGMT gene dosage, chromatin status, and respective effects on MGMT expression and MGMT activity. RESULTS: In glioblastoma, MGMT-methylated alleles ranged from 10% to 90%. In contrast, methylated alleles were highly enriched (100% of clones) in respective GS, even when 2 MGMT alleles were present, with 1 exception (<50%). The CpG methylation patterns were characteristic for each glioblastoma exhibiting 25% to 90% methylated CpGs of 28 sites interrogated. Furthermore, MGMT promoter methylation was associated with a nonpermissive chromatin status in accordance with very low MGMT transcript levels and undetectable MGMT activity. CONCLUSIONS: In MGMT-methylated glioblastoma, MGMT promoter methylation is highly enriched in GS that supposedly comprise glioma-initiating cells. Thus, even a low percentage of MGMT methylation measured in a glioblastoma sample may be relevant and predict benefit from an alkylating agent therapy.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Glioblastoma/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatina/ultraestrutura , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Tumor-initiating cells with stem cell properties are believed to sustain the growth of gliomas, but proposed markers such as CD133 cannot be used to identify these cells with sufficient specificity. We report an alternative isolation method purely based on phenotypic qualities of glioma-initiating cells (GICs), avoiding the use of molecular markers. We exploited intrinsic autofluorescence properties and a distinctive morphology to isolate a subpopulation of cells (FL1(+)) from human glioma or glioma cultures. FL1(+) cells are capable of self-renewal in vitro, tumorigenesis in vivo and preferentially express stem cell genes. The FL1(+) phenotype did not correlate with the expression of proposed GIC markers. Our data propose an alternative approach to investigate tumor-initiating potential in gliomas and to advance the development of new therapies and diagnostics.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Encefálicas/patologia , Glioma/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Animais , Antígenos CD/análise , Diferenciação Celular , Células Cultivadas , Fluorescência , Perfilação da Expressão Gênica , Glicoproteínas/análise , Humanos , Camundongos , Peptídeos/análiseRESUMO
For patients with brain tumors identification of diagnostic and prognostic markers in easy accessible biological material, such as plasma or cerebrospinal fluid (CSF), would greatly facilitate patient management. MIC-1/GDF15 (growth differentiation factor 15) is a secreted protein of the TGF-beta superfamily and emerged as a candidate marker exhibiting increasing mRNA expression during malignant progression of glioma. Determination of MIC-1/GDF15 protein levels by ELISA in the CSF of a cohort of 94 patients with intracranial tumors including gliomas, meningioma and metastasis revealed significantly increased concentrations in glioblastoma patients (median, 229 pg/ml) when compared with control cohort of patients treated for non-neoplastic diseases (median below limit of detection of 156 pg/ml, p < 0.0001, Mann-Whitney test). However, plasma MIC-1/GDF15 levels were not elevated in the matching plasma samples from these patients. Most interestingly, patients with glioblastoma and increased CSF MIC-1/GDF15 had a shorter survival (p = 0.007, log-rank test). In conclusion, MIC-1/GDF15 protein measured in the CSF may have diagnostic and prognostic value in patients with intracranial tumors.
Assuntos
Biomarcadores Tumorais/líquido cefalorraquidiano , Neoplasias Encefálicas/líquido cefalorraquidiano , Glioblastoma/líquido cefalorraquidiano , Fator 15 de Diferenciação de Crescimento/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/cirurgia , Estudos de Casos e Controles , Estudos de Coortes , Ensaio de Imunoadsorção Enzimática , Feminino , Glioblastoma/sangue , Glioblastoma/cirurgia , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Prognóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Resultado do Tratamento , Adulto JovemRESUMO
Glioblastoma are rapidly proliferating brain tumors in which hypoxia is readily recognizable, as indicated by focal or extensive necrosis and vascular proliferation, two independent diagnostic criteria for glioblastoma. Gene expression profiling of glioblastoma revealed a gene expression signature associated with hypoxia-regulated genes. The correlated gene set emerging from unsupervised analysis comprised known hypoxia-inducible genes involved in angiogenesis and inflammation such as VEGF and BIRC3, respectively. The relationship between hypoxia-modulated angiogenic genes and inflammatory genes was associated with outcome in our cohort of glioblastoma patients treated within prospective clinical trials of combined chemoradiotherapy. The hypoxia regulation of several new genes comprised in this cluster including ZNF395, TNFAIP3, and TREM1 was experimentally confirmed in glioma cell lines and primary monocytes exposed to hypoxia in vitro. Interestingly, the cluster seems to characterize differential response of tumor cells, stromal cells and the macrophage/microglia compartment to hypoxic conditions. Most genes classically associated with the inflammatory compartment are part of the NF-kappaB signaling pathway including TNFAIP3 and BIRC3 that have been shown to be involved in resistance to chemotherapy.Our results associate hypoxia-driven tumor response with inflammation in glioblastoma, hence underlining the importance of tumor-host interaction involving the inflammatory compartment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Hipóxia , Neovascularização Patológica , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Humanos , Inflamação , Monócitos/metabolismo , Família Multigênica , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Tenascin-C (TNC) expression is known to correlate with malignancy in glioblastoma (GBM), a highly invasive and aggressive brain tumor that shows limited response to conventional therapies. In these malignant gliomas as well as in GBM cell lines, we found Notch2 protein to be strongly expressed. In a GBM tumor tissue microarray, RBPJk protein, a Notch2 cofactor for transcription, was found to be significantly coexpressed with TNC. We show that the TNC gene is transactivated by Notch2 in an RBPJk-dependent manner mediated by an RBPJk binding element in the TNC promoter. The transactivation is abrogated by a Notch2 mutation, which we detected in the glioma cell line Hs683 that does not express TNC. This L1711M mutation resides in the RAM domain, the site of interaction between Notch2 and RBPJk. In addition, transfection of constructs encoding activated Notch2 or Notch1 increased endogenous TNC expression identifying TNC as a novel Notch target gene. Overexpression of a dominant negative form of the transcriptional coactivator MAML1 or knocking down RBPJk in LN319 cells led to a dramatic decrease in TNC protein levels accompanied by a significant reduction of cell migration. Because addition of purified TNC stimulated glioma cell migration, this represents a mechanism for the invasive properties of glioma cells controlled by Notch signaling and defines a novel oncogenic pathway in gliomagenesis that may be targeted for therapeutic intervention in GBM patients.
Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/biossíntese , Receptor Notch2/biossíntese , Tenascina/genética , Sequência de Aminoácidos , Sequência de Bases , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Movimento Celular/fisiologia , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Imuno-Histoquímica , Dados de Sequência Molecular , Oligodendroglioma/genética , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Regiões Promotoras Genéticas , Receptor Notch2/genética , Receptor Notch2/metabolismo , Elementos de Resposta , Transdução de Sinais , Tenascina/biossíntese , Ativação TranscricionalRESUMO
Uveal melanoma is associated with a high mortality rate once metastases occur, with over >90% of metastatic patients dying within less than 1 year from metastases to the liver. The intraarterial hepatic (iah) administration of the alkylating agent fotemustine holds some promise with response rates of 36% and median survival of 15 months. Here, we investigated whether the DNA-repair-protein MGMT may be involved in the variability of response to fotemustine and temozolomide in uveal melanoma. Epigenetic inactivation of MGMT has been demonstrated to be a predictive marker for benefit from alkylating agent therapy in glioblastoma. We found a methylated MGMT promoter in 6% of liver metastases from 34 uveal melanoma patients. The mean MGMT activity measured in liver metastases with negligible liver tissue content was significantly lower than in liver tissue (146 versus 523 fmol/mg protein, p = 0.002). Expression of the MGMT protein was detectable in 50% of 88 metastases by immunohistochemistry on a tissue microarray. Expression was heterogeneous, and in accordance with MGMT activity data, usually lower than in the surrounding liver. Differential MGMT activity/expression between metastasis and liver tissue and more efficient depletion of MGMT with higher doses of alkylating agent therapy using iah delivery may provide the pharmacologic window for the higher response rate. However, these results do not support MGMT methylation status or protein expression as predictive markers for treatment outcome to iah chemotherapy with alkylating agents.
Assuntos
Biomarcadores Tumorais/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Melanoma/genética , Melanoma/secundário , Proteínas Supressoras de Tumor/genética , Neoplasias Uveais/patologia , Antineoplásicos/uso terapêutico , Metilação de DNA , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/tratamento farmacológico , Melanoma/tratamento farmacológico , Valor Preditivo dos Testes , Regiões Promotoras Genéticas , Análise Serial de ProteínasRESUMO
PURPOSE: Glioblastomas are notorious for resistance to therapy, which has been attributed to DNA-repair proficiency, a multitude of deregulated molecular pathways, and, more recently, to the particular biologic behavior of tumor stem-like cells. Here, we aimed to identify molecular profiles specific for treatment resistance to the current standard of care of concomitant chemoradiotherapy with the alkylating agent temozolomide. PATIENTS AND METHODS: Gene expression profiles of 80 glioblastomas were interrogated for associations with resistance to therapy. Patients were treated within clinical trials testing the addition of concomitant and adjuvant temozolomide to radiotherapy. RESULTS: An expression signature dominated by HOX genes, which comprises Prominin-1 (CD133), emerged as a predictor for poor survival in patients treated with concomitant chemoradiotherapy (n = 42; hazard ratio = 2.69; 95% CI, 1.38 to 5.26; P = .004). This association could be validated in an independent data set. Provocatively, the HOX cluster was reminiscent of a "self-renewal" signature (P = .008; Gene Set Enrichment Analysis) recently characterized in a mouse leukemia model. The HOX signature and EGFR expression were independent prognostic factors in multivariate analysis, adjusted for the O-6-methylguanine-DNA methyltransferase (MGMT) methylation status, a known predictive factor for benefit from temozolomide, and age. Better outcome was associated with gene clusters characterizing features of tumor-host interaction including tumor vascularization and cell adhesion, and innate immune response. CONCLUSION: This study provides first clinical evidence for the implication of a "glioma stem cell" or "self-renewal" phenotype in treatment resistance of glioblastoma. Biologic mechanisms identified here to be relevant for resistance will guide future targeted therapies and respective marker development for individualized treatment and patient selection.
Assuntos
Células-Tronco Adultas/patologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Receptores ErbB/biossíntese , Glioblastoma/patologia , Glioblastoma/terapia , Adulto , Idoso , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Terapia Combinada , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/genética , Perfilação da Expressão Gênica , Genes Homeobox , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , Pessoa de Meia-Idade , Família Multigênica , Tolerância a Radiação , TemozolomidaRESUMO
Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.
Assuntos
Metilação de DNA , Glioma/diagnóstico , O(6)-Metilguanina-DNA Metiltransferase/genética , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas/genética , Glioma/genética , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Silencing of O6-methylguanine-DNA methyltransferase (MGMT) protein expression because of MGMT gene promoter hypermethylation is considered to be associated with postoperative chemoradiotherapy benefits in glioblastoma multiforme (GBM) patients. The objective of this study was to clarify the usability of MGMT immunohistochemistry (IHC) as a clinical biomarker. We immunostained a tissue microarray containing biopsy samples of 164 GBM patients from the European Organization for Research and Treatment of Cancer and the National Cancer Institute of Canada (EORTC/NCIC) trial 26981/22981 using two commercial anti-MGMT antibodies (clones MT3.1 and MT23.2). Immunostaining results were semiquantitatively evaluated by four observers from three neuropathological laboratories using a predefined algorithm. We analyzed (i) inter- and intraobserver agreement on MGMT expression (kappa statistics); (ii) correlation of MGMT expression with MGMT promoter methylation status (kappa statistics); and (iii) correlation of MGMT expression with patient outcome (log-rank test). Interobserver agreement on MGMT expression varied from slight to almost perfect, whereas intraobserver agreement ranged from substantial to almost perfect. MGMT expression showed poor to moderate correlation with MGMT promoter methylation status. We found no significant association of MGMT expression with patient outcome. In our hands, observer variability as well as lack of association with the MGMT promoter methylation status and patient survival impeded the use of anti-MGMT immunohistochemistry as a clinical biomarker for routine diagnostic purposes.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/enzimologia , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/diagnóstico , Glioblastoma/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Anticorpos , Especificidade de Anticorpos , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/fisiopatologia , Estudos de Coortes , Metilação de DNA , Metilases de Modificação do DNA/análise , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/análise , Enzimas Reparadoras do DNA/genética , Diagnóstico Diferencial , Glioblastoma/fisiopatologia , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Regiões Promotoras Genéticas/genética , Estudos Prospectivos , Reprodutibilidade dos Testes , Taxa de Sobrevida , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Epigenetic silencing of the MGMT (O6-methylguanine-DNA methyltransferase) DNA-repair gene by promoter methylation compromises DNA repair and has been associated with longer survival in patients with glioblastoma who receive alkylating agents. METHODS: We tested the relationship between MGMT silencing in the tumor and the survival of patients who were enrolled in a randomized trial comparing radiotherapy alone with radiotherapy combined with concomitant and adjuvant treatment with temozolomide. The methylation status of the MGMT promoter was determined by methylation-specific polymerase-chain-reaction analysis. RESULTS: The MGMT promoter was methylated in 45 percent of 206 assessable cases. Irrespective of treatment, MGMT promoter methylation was an independent favorable prognostic factor (P<0.001 by the log-rank test; hazard ratio, 0.45; 95 percent confidence interval, 0.32 to 0.61). Among patients whose tumor contained a methylated MGMT promoter, a survival benefit was observed in patients treated with temozolomide and radiotherapy; their median survival was 21.7 months (95 percent confidence interval, 17.4 to 30.4), as compared with 15.3 months (95 percent confidence interval, 13.0 to 20.9) among those who were assigned to only radiotherapy (P=0.007 by the log-rank test). In the absence of methylation of the MGMT promoter, there was a smaller and statistically insignificant difference in survival between the treatment groups. CONCLUSIONS: Patients with glioblastoma containing a methylated MGMT promoter benefited from temozolomide, whereas those who did not have a methylated MGMT promoter did not have such a benefit.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/genética , Metilação de DNA , Dacarbazina/análogos & derivados , Dacarbazina/uso terapêutico , Inativação Gênica , Glioblastoma/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/radioterapia , Quimioterapia Adjuvante , Intervalo Livre de Doença , Glioblastoma/tratamento farmacológico , Glioblastoma/mortalidade , Glioblastoma/radioterapia , Humanos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sobrevida , TemozolomidaRESUMO
Tenascin-C is an adhesion-modulating extracellular matrix molecule that is highly expressed in tumor stroma and stimulates tumor cell proliferation. Adhesion of T98G glioblastoma cells to a fibronectin substratum is inhibited by tenascin-C. To address the mechanism of action, we performed a RNA expression analysis of T89G cells grown in the presence or absence of tenascin-C and found that tenascin-C down-regulates tropomyosin-1. Upon overexpression of tropomyosin-1, cell spreading on a fibronectin/tenascin-C substratum was restored, indicating that tenascin-C destabilizes actin stress fibers through down-regulation of tropomyosin-1. Tenascin-C also increased the expression of the endothelin receptor type A and stimulated the corresponding mitogen-activated protein kinase signaling pathway, which triggers extracellular signal-regulated kinase 1/2 phosphorylation and c-Fos expression. Tenascin-C additionally caused down-regulation of the Wnt inhibitor Dickkopf 1. In consequence, Wnt signaling was enhanced through stabilization of beta-catenin and stimulated the expression of the beta-catenin target Id2. Finally, our in vivo data derived from astrocytoma tissue arrays link increased tenascin-C and Id2 expression with high malignancy. Because increased endothelin and Wnt signaling, as well as reduced tropomyosin-1 expression, are closely linked to transformation and tumorigenesis, we suggest that tenascin-C specifically modulates these signaling pathways to enhance proliferation of glioma cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Tenascina/farmacologia , Actinas/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Regulação para Baixo/efeitos dos fármacos , Perfilação da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Proteína 2 Inibidora de Diferenciação , Proteínas Proto-Oncogênicas/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Tropomiosina/biossíntese , Proteínas WntRESUMO
Amplification of the epidermal growth factor receptor (EGFR) or expression of its constitutively activated mutant, DeltaEGFR(2-7), in association with the inactivation of the INK4a/Arf gene locus is a frequent alteration in human glioblastoma. The notion of a cooperative effect between these two alterations has been demonstrated in respective mouse brain tumor models including our own. Here, we investigated underlying molecular mechanisms in early passage cortical astrocytes deficient for p16(INK4a)/p19(Arf) or p53, respectively, with or without ectopic expression of DeltaEGFR(2-7). Targeting these cells with the specific EGFR inhibitor tyrphostin AG1478 revealed that phosphorylation of ERK was only abrogated in the presence of an intact INK4a/Arf gene locus. The sensitivity to inhibit ERK phosphorylation was independent of ectopic expression of DeltaEGFR(2-7) and independent of the TP53 status. This resistance to downregulate the MAPK pathway in the absence of INK4a/Arf was confirmed in cell lines derived from our mouse glioma models with the respective initial genetic alterations. Thus, deletion of INK4a/Arf appears to keep ERK in its active, phosphorylated state insensitive to an upstream inhibitor specifically targeting EGFR/DeltaEGFR(2-7). This resistance may contribute to the cooperative tumorigenic effect selected for in human glioblastoma that may be of crucial clinical relevance for treatments specifically targeting EGFR/DeltaEGFR(2-7) in glioblastoma patients.