Expression of PGC1? in glioblastoma multiforme patients.
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ABSTRACT:
SUBMITTER: Cho SY
PROVIDER: S-EPMC5453058 | biostudies-literature | 2017 Jun
REPOSITORIES: biostudies-literature
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Expression of PGC1α in glioblastoma multiforme patients.
Cho Sang Yeon SY Kim Seon-Hwan SH Yi Min-Hee MH Zhang Enji E Kim Eunjee E Park Jisoo J Jo Eun-Kyeong EK Lee Young Ho YH Park Min Soo MS Kim Yonghyun Y Park Jongsun J Kim Dong Woon DW
Oncology letters 20170403 6
Peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) is a key modulator of mitochondrial biogenesis. It is a coactivator of multiple transcription factors and regulates metabolic processes. However, little is known about the expression and function of PGC1α in glioblastoma multiforme (GBM), the most prevalent and invasive type of brain tumor. The purpose of the present study was to investigate the biological function, localization and expression of PGC1α in GBM. It was observed th ...[more]
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Project description:Background: Even though much progress has been made in the understanding of the molecular nature of glioma, the survival rates of patients affected of this tumour have not changed significantly during these years. Thus, a deeper understanding of this malignancy is still needed in order to predict its outcome and improve patient treatment. Here, we report that VAV1, a GDP/GTP exchange factor for Rho/Rac proteins with oncogenic potential that is involved in the regulation of cytoskeletal dynamics and cell migration. Methodology/Principal Findings: VAV1 is overexpressed in 32 patients diagnosed with high-grade glioma. Such overexpression is linked to the parallel upregulation of a number of genes coding for proteins also involved in cell invasion- and migration-related processes. Unexpectedly, immunohistochemical experiments revealed that VAV1 is not expressed in glioma cells. Instead, VAV1 is found in non-tumoral astrocyte-like cells that are located either peritumoraly or perivascularly, suggesting that its expression is linked to synergistic signalling cross-talk between cancer and infiltrating cells. Conclusions/Significance: Interestingly, we show that the pattern of expression of VAV1 is a good prognostic factor to unveil populations of high-grade glioma patients with different survival and progression free survival rates. 1. Oligonucleotide microarray analyses Total RNAs were extracted using the Triazol reagent (Life Technologies, Gaithersburg, MD, USA) and purified with the RNeasy Mini kit (Qiagen, Valencia, CA, USA). The integrity of RNA samples obtained was assessed using the 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). Double-stranded cDNAs and biotinylated cRNAs were synthesized using a T7-polyT primer and the BioArray RNA labelling Kit (Enzo Farningdale, NY, USA), respectively. Labelled RNAs were then fragmented and hybridised to HU-133A oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) according to standard Affymetrix protocols. After hybridization and washes, arrays were scanned using the Gene Array Scanner (Affymetrix), and the expression value for each probe set calculated using the MAS 5.0 software (Affymetrix). All examples had a scaling factor lower than threefold and 3’/5’ of GAPDH probe set <2.5. Gene levels were transformed to base two logarithms. A median normalization approach was applied. Only genes with al least three “present” calls across all samples were selected. All these steps were done at the Genomics and Proteomics Unit of the “Centro de Investigación del Cáncer, Salamanca”. 2. Microarray data analyses To visualize clusters of genes with similar expression patterns, we used a hierarchical clustering method (Cluster and TreeView software) based on the average-linkage method with the centred correlation metric [26]. A multidimensional scaling method (BRB Arrays Tools version 3.0) was also utilized by using Euclidean distance criteria [27]. Supervised learning was used to identify genes with statistically significant changes in expression among different classes by using the Significant Analysis of Microarrays (SAM) algorithm [28]. All data were permuted over 100 cycles by using the two-class (unpaired) and multi-class response format. Significant genes were selected based on the lowest false discovery ratio (between 0.6 and 0.9). In addition, nonparametric tests such as Wilcoxon rank sum test and Kruskal-Wallis test to compare more than two unpaired group were also used (SPSS 18, SPSS Inc). 3. Functional annotation of microarray data Probe sets showing significant expression change were functionally annotated and grouped according to biological function criteria using GeneOntology biological process descriptions. The functional analysis to identify the most relevant biological mechanism, pathways and functional categories in gene dada sets was generated using the Ingenuity Pathway software (Ingenuity Systems, Mountain View, CA, USA) available in the web (www.ingenuity.com) [29]. A functional network was considered significant when it fulfilled the following criteria: i) to have a minimal score of 15; ii) to have a minimum of 20 direct functional interactions among the network members. 4. Quantitative reverse transcription-PCR Total RNA was quantified in a RNA 6000 Nano Chip (Agilent Technologies) and quantitative PCR performed using the QuantiTect SYBR Green RT–PCR kit (Qiagen). To quantify VAV1 mRNA levels, we used two different sets of probes: PAIR A (5’-AAC AAC GGG AGG TTC ACC CT-3’ and 5’-GGT CCC TCA TGG CAT CCA-3’) and PAIR B (5’-AGC CAT TGG ACC CTT TCT ACG-3’ and 5’-GCC ATG GAC ATA GGG CTT CA-3’). Amplifications were performed using the iCycler apparatus (Bio-Rad Laboratories, California, USA). Analyses of data were done using the iCycler iQ Optical System Software, version 3.0a (Bio-Rad Laboratories). Primers to GAPDH were used as intersample normalizing controls. Variations in expression of VAV1 mRNA were represented as the mean value of the fold change respect the VAV1 expression levels detected in sample #19209 with both pairs of oligonucleotide primers. 5. Immunohistochemical analyses The VAV1 antibody was generated in rabbits using a synthetic peptide and purified by affinity chromatography in Bustelo’s laboratory. This antibody recognizes VAV1 proteins from humans and mice but it does not recognize other VAV family members (unpublished data). For immunostaining, tissue sections were washed thrice with Xylene and once with 100% ethanol, rehydrated by sequential changes in 80%, 70%, and 50% ethanol and a final incubation in phosphate-buffered saline (PBS). Each rehydrating step involved 3 min incubations with the indicated solutions. Endogenous peroxidases were quenched by the addition of a 3% H2O2 solution in methanol for 30 min at room temperature (RT). Tissue sections were subsequently washed twice with PBS. Antigen retrieval was performed by incubation in 1 mM EDTA for 30 min at 37°C. The slides were washed twice in PBS and blocked in blocking buffer (Zymed, CA, USA) for 30 min at RT. Specimens were then incubated with the primary antibody (1:250 dilution) in blocking buffer. After an 1 hr incubation at 37°C, slides were washed three times in PBS, incubated with a biotinylated secondary antibody for 30 min at 37°C, washed thrice in PBS, incubated with horseradish peroxidase-streptavidin for 30 min at 37°C, washed three times in PBS, and developed using the AEC substrate (Zymed). Slides were then washed twice in water, counterstained with hematoxilin (Zymed), washed again in water, and mounted with GVA (Zymed). Samples were analyzed by light microscopy and images acquired suing an Axiophot imaging system (Zeiss, Munich, Germany). 6. Fluorescence in situ hybridization analyses FISH experiments were carried out in 40 cases of glioblastoma multiforme (grade IV) positive for VAV1 expression. For this purpose, we performed dual-colour FISH analyses with locus-specific probes for centromere 7 (Abbott Molecualr, Des Plaines) exactly as previously described.[30] Polysomies were defined when more than 10% of the nuclei surveyed contained three or more CEP signals (chromosome-specific FISH probes that hybridize to highly repetitive human satellite DNA sequences, usually located near centromeres). 7. Immunohistochemistry and fluorescence in situ hybridization (FISH) in paraffin-embedded tumours Four um sections were cut from routinely processed paraffin blocks and mounted onto glass slides with a charged coating. Sections were dewaxed in Xylene and then rehydrated using increasing concentrations of alcohol before being rinsed briefly in water. Slides were heated 2 min in 1 mM EDTA (pH 9.0) in a microwavable pressure cooker. After antigen retrieval, slides were incubated 1 h at RT in a moist chamber with a primary antibody diluted in PBS supplemented with 10% foetal calf serum. Slides were incubated for 1 h with fluorochrome-conjugated antibodies to the appropriate IgG isotypes in a moist chamber in the dark. Finally, slides were washed thrice in PBS containing 0.5% Tween 20 three before FISH analysis. 8. Degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR) analyses After the staining of tissue sections with VAV1 antibodies (see above), the regions of the tumour were identified, microdissected, and collected using the PALM® microscope system (P.A.L.M. Microlaser Technologies, Munich, Germany). The genomic DNA was extracted as indicated by Isola et al [31] with modifications to small DNA amounts. Those included the resuspension of the microdissected sections in extraction buffer followed by a digestion with proteinase K (0.6 mg/ml). All samples were resuspended in 10 ul of 10 mM Tris-HCl (pH 7.4) and 0.1 mM EDTA. DOP-PCR amplification was performed in two steps. For the first, low-stringency step, 1 ul of sample was added to 4 ul of buffer A (2.5 ul of 600 uM dNTPs (Roche, Pleasanton, CA), 0.5 ul of 10 uM DOP primer 5’-CCGACTCGAGNNNNNNNATGTGG-3’, where N= A, C, G, or T) [32] and 1 ul of 5x Sequenase Reaction Buffer (Amersham, Cleveland, OH). Reactions were performed using 5 cycles of 30ºC for 5 min, 37ºC for 2 min, and 96ºC for 2 min, adding 0.65 units of Sequenase in each 30ºC step. The first phase product was then subjected to the second step usin
| S-ECPF-GEOD-24072 | biostudies-other
Project description:One of the most common type of primary brain tumors in adults is the glioblastoma multiforme (GBM) (World Health Organization grade IV astrocytoma). It is the most common malignant and aggressive form of glioma and it is among the most lethal ones. Poly (ADP-ribose) polymerase 1 (PARP-1) gene, located to 1q42, plays an important role for the efficient maintenance of genome integrity. PARP-1 protein is required for the apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus. PARP-1 is proteolytically cleaved at the onset of apoptosis by caspase-3. Microarray analysis of PARP-1 gene expression in more than 8,000 samples revealed that PARP-1 is more highly expressed in several types of cancer compared with the equivalent normal tissues. Overall, the most differences in PARP-1 gene expression have been observed in breast, ovarian, endometrial, lung, and skin cancers, and non-Hodgkin's lymphoma. We evaluated the expression of PARP-1 protein in normal brain tissues and primary GBM by immunohistochemistry. Positive nuclear PARP-1 staining was found in all samples with GBM, but not in normal neurons from controls (n=4) and GBM patients (n=27). No cytoplasmic staining was observed in any sample. In conclusion, PARP-1 gene is expressed in GBM. This finding may be envisioned as an attempt to trigger apoptosis in this tumor, as well as in many other malignancies. The presence of the protein exclusively at the nucleus further support the function played by this gene in genome integrity maintenance and apoptosis. Finally, PARP-1 staining may be used as GBM cell marker.
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Project description:Glioblastoma multiforme (GBM) is the most lethal adult brain cancer. Temozolomide (TMZ), the standard chemotherapeutic drug used in GBM, has limited benefit and alternate therapies are needed to improve GBM treatment. Nerve growth factor (NGF) and its precursor proNGF are increasingly recognized as stimulators of human tumor progression. The expression and stimulatory effect of NGF on GBM cell growth has previously been reported, but the status of proNGF in GBM is unreported. In this study, we have investigated proNGF expression and biological activity in GBM. A clinical cohort of GBM (n = 72) and low-grade glioma (n = 20) was analyzed by immunohistochemistry for proNGF and digital quantification. ProNGF expression was significantly increased in GBM compared to low grade gliomas and proNGF was also detected in patient plasma samples. ProNGF was also detected in most GBM cell lines by Western blotting. Although anti-proNGF blocking antibodies inhibited cell growth in GBM cells with methylated MGMT gene promoter, targeting proNGF could not potentiate the efficacy of TMZ. In subcutaneous xenograft of human GBM cells, anti-proNGF antibodies slightly reduced tumor volume but had no impact on TMZ efficacy. In conclusion, this data reveals that proNGF is overexpressed in GBM and can stimulate cancer cell growth. The potential of proNGF as a clinical biomarker and therapeutic target warrants further investigations.
| S-EPMC9863529 | biostudies-literature
Project description:Comparative expression analysis of RNA isolated from microvesicles from serum from glioblastoma multiforme (GBM) patients and normal healthy individuals. Blood samples from 9 patients diagnosed with de-novo primary GBM were collected immediately prior to surgery. Blood from 7 normal healthy controls was collected from volunteers recruited at the MGH blood bank. From each sample, RNA was isolated from microvesicles isolated from 1 mL of serum, and the RNA was linearly amplified and labeled with Cy3. The amplified and labeled RNA was hybridized to 16 individual Agilent Arrays.
| S-ECPF-GEOD-24084 | biostudies-other
Project description:Human Glioblastoma Multiforme tumors taken before dendritic cell vaccination, the recurrent tumors taken after vaccination and control GBM tumors from non vaccinated patients. Keywords: Disease State Analysis
2009-04-03 | GSE11100 | GEO
Project description:RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients.Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups).Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production.Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.
| S-EPMC3329625 | biostudies-literature
Project description:Glioblastoma multiforme are highly malignant brain tumours with frequent genetic and epigenetic alterations. The poor clinical outcome of these tumours necessitates the development of new treatment options. Immunotherapies for glioblastoma multiforme including PD1/PD-L1 inhibition are currently tested in ongoing clinical trials. The purpose of this study was to investigate the molecular background of PD-L1 expression in glioblastoma multiforme and to find associated pathway activation and genetic alterations. We show that PD-L1 is up-regulated in IDH1/2 wildtype glioblastoma multiforme compared to lower-grade gliomas. In addition, a strong association of PD-L1 with the mesenchymal expression subgroup was observed. Consistent with that, NF1 mutation and corresponding activation of the MAPK pathway was strongly connected to PD-L1 expression. Our findings may explain different response to PD-L1 inhibition of patients in ongoing trials and may help to select patients that may profit of immunotherapy in the future.
| S-EPMC5522061 | biostudies-literature
Project description:The Polycomb group (PcG) proteins play a critical role in histone mediated epigenetics which has been implicated in the malignant evolution of glioblastoma multiforme (GBM). By systematically interrogating The Cancer Genome Atlas (TCGA), we discovered widespread aberrant expression of the PcG members in GBM samples compared to normal brain. The most striking differences were upregulation of EZH2, PHF19, CBX8 and PHC2 and downregulation of CBX7, CBX6, EZH1 and RYBP. Interestingly, changes in EZH2, PHF19, CBX7, CBX6 and EZH1 occurred progressively as astrocytoma grade increased. We validated the aberrant expression of CBX6, CBX7, CBX8 and EZH2 in GBM cell lines by Western blotting and qRT-PCR, and further the aberrant expression of CBX6 in GBM tissue samples by immunohistochemical staining. To determine if there was functional significance to the diminished CBX6 levels in GBM, CBX6 was overexpressed in GBM cells resulting in decreased proliferative capacity. In conclusion, aberrant expression of PcG proteins in GBMs may play a role in the development or maintenance of the malignancy.
| S-EPMC3829908 | biostudies-literature
Project description:The purpose of this study is to determine whether the combination of two agents, INC280 and bevacizumab, is safe and effective when administered to patients with Glioblastoma Multiforme (GBM) who have progressed after receiving prior therapy or who have unresectable GBM.
| 2185625 | ecrin-mdr-crc
Project description:BackgroundAs the most aggressive brain tumor, patients with glioblastoma multiforme (GBM) have a poor prognosis. Our purpose was to explore prognostic value of Polo-like kinase 2 (PLK2) in GBM, a member of the PLKs family.MethodsThe expression profile of PLK2 in GBM was obtained from The Cancer Genome Atlas database. The PLK2 expression in GBM was tested. Kaplan-Meier curves were generated to assess the association between PLK2 expression and overall survival (OS) in patients with GBM. Furthermore, to assess its prognostic significance in patients with primary GBM, we constructed univariate and multivariate Cox regression models. The association between PLK2 expression and its methylation was then performed. Differentially expressed genes correlated with PLK2 were identified by Pearson test and functional enrichment analysis was performed.ResultsOverall survival results showed that low PLK2 expression had a favorable prognosis of patients with GBM (P-value = 0.0022). Furthermore, PLK2 (HR = 0.449, 95% CI [0.243-0.830], P-value = 0.011) was positively associated with OS by multivariate Cox regression analysis. In cluster 5, DNA methylated PLK2 had the lowest expression, which implied that PLK2 expression might be affected by its DNA methylation status in GBM. PLK2 in CpG island methylation phenotype (G-CIMP) had lower expression than non G-CIMP group (P = 0.0077). Regression analysis showed that PLK2 expression was negatively correlated with its DNA methylation (P = 0.0062, Pearson r = -0.3855). Among all differentially expressed genes of GBM, CYGB (r = 0.5551; P < 0.0001), ISLR2 (r = 0.5126; P < 0.0001), RPP25 (r = 0.5333; P < 0.0001) and SOX2 (r = -0.4838; P < 0.0001) were strongly correlated with PLK2. Functional enrichment analysis results showed that these genes were enriched several biological processes or pathways that were associated with GBM.ConclusionPolo-like kinase 2 expression is regulated by DNA methylation in GBM, and its low expression or hypermethylation could be considered to predict a favorable prognosis for patients with GBM.
| S-EPMC6873877 | biostudies-literature