Non-neoplastic Brain Research Articles

Nuclear factor-kappa B1 inhibits early apoptosis of glioma cells by promoting the expression of Bcl-2

Glioma is one of the most common types of adult primary brain tumors, and the underlying molecular mechanisms still remain unclear. Nuclear factor-kappa B1 (NF-κB1) is involved in a variety of malignancies and is widely expressed in malignant tumors. However, the expression of NF-κB1 in different grades of glioma, the correlation between NF-κB1 and Bcl-2 expressions in gliomas, and the research between NF-κB1 and early apoptosis of glioma cells have not been reported so far. In this study, the expression level of NF-κB1 in 31 human glioma tissues and six nonneoplastic brain tissues was determined using quantitative real-time polymerase chain reaction. Results showed that the expression of NF-κB1 in human glioma tissues and glioma cell lines, SHG44 and U87, was significantly higher compared to noncancerous brain tissues and that the expression increased with increasing degrees of tumor malignancy. Similar results were demonstrated with the expression of Bcl-2 in the same human glioma specimens. Flow cytometry results showed that inhibition of NF-κB1 expression significantly promoted apoptosis of SHG44 and U87 in human glioma cells. Western blot analysis further confirmed decreased expression of Bcl-2 protein after inhibition of NF-κB1 protein expression. Taken together, NF-κB1 overexpression inhibits early apoptosis of glioma cells and high expression of NF-κB1 promotes the expression of antiapoptotic gene Bcl-2. Therefore, our study results provide a theoretical basis for antiapoptotic mechanism of tumor cells in association with NF-κB1.

Overexpression of mitochondrial serine hydroxyl-methyltransferase 2 is associated with poor prognosis and promotes cell proliferation and invasion in gliomas.

Mitochondrial serine hydroxyl-methyltransferase 2 (SHMT2), participating in the synthesis of mitochondrial thymidine monophosphate, has been reported to drive glioma cell survival in ischemia. However, its clinical relevance in gliomas remains unclear. In the current study, immunohistochemistry was performed to examine subcellular localization and expression levels of SHMT2 protein in glioma and non-neoplastic brain tissue specimens. Then, the associations of SHMT2 expression with various clinicopathological features and patients’ prognosis were statistically evaluated. The roles of SHMT2 in the proliferation and invasion of glioma cells after siRNA-SHMT2 vector transfection were also detected by cell counting kit-8 and transwell assays, respectively. Results showed that SHMT2 immunostaining was predominantly localized in the cellular cytoplasm of tumor cells in glioma tissues but weakly in non-neoplastic brain tissues. Statistically, SHMT2 protein expression was significantly higher in glioma tissues than in non-neoplastic brain tissues (P<0.001). In addition, SHMT2 overexpression more frequently occurred in glioma patients with an advanced grade of malignancy (P<0.001) and poor prognosis (P=0.001). Notably, multivariate analysis based on a Cox regression model identified SHMT2 expression as an independent prognostic factor for glioma patients (P=0.01). Functionally, SHMT2 knockdown efficiently suppressed the proliferation (P=0.02) and invasion (P<0.001) of glioma cells in vitro. In conclusion, our findings suggest that SHMT2 may function as an oncogene in glioma development and progression. Clinically, SHMT2 may serve as a prognostic factor and as a potential therapeutic target for human gliomas.

PROGNOSTIC VALUE OF COMBINED IMMUNOHISTOCHEMICAL EXPRESSION OF BMI1 AND EZH2 IN ASTROCYTOMA

Background: Although current improvements in surgery, chemotherapy and radiotherapy, the still survival of astrocytoma’s patient is poor. It is assumed that the combined expression of BMI1 and EZH2 may be associated with malignant transformation of astrocytomas and also it may reveal the biological aggressiveness of this disease. Aim of work: assessment the value of the combined immunohistochemical expression of BMI1 and EZH2 and their correlation with the clinicopathological characters and prognosis in astrocytoma patient. Subjects & Methods: BMI1 and EZH2 expressions were evaluated using immunohistochemical staining in 70 patients 40 cases with astrocytomas and 30 cases of non-neoplastic brain tissue. The relationship between their expressions and clinicopathological factors were analyzed. Results: A significant difference (P 0.05). The spearman correlation analysis revealed a significant positive association betweenBMI1 and EZH2 expressions (r = 0.311; P=0.05) revealing direct relationship betweenBMI1 and EZH2. Conclusions: BMI1 and EZH2 were involved in astrocytoma malignant transformation and poor prognosis in astrocytoma particularly glioblastoma. Keywords: BMI1, EZH2, astrocytoma, PcG, glioblastoma and GSCs.

ATRT-09. CHROMATIN SEGMENTATION ANALYSES IN ATYPICAL TERATOID/RHABDOID TUMORS REVEAL H3K27ME3-MEDIATED EPIGENETIC SILENCING OF TUMOR SUPPRESSOR GENES AND IDENTIFY A NOVEL ROLE OF EZH2, ASSOCIATED WITH ACTIVE CHROMATIN

AbstractAlthough SMARCB1 mutations represent the only genetic aberrations in atypical teratoid / rhabdoid tumors (ATRT), a comprehensive assessment of the epigenetic effect of SMARCB1 loss has not been achieved so far. We performed ChIP-sequencing for six active and repressive histone marks (H3K4me1, H3K4me3, H3K27me3, H3K27Ac, H3K9me3, H3K36me3) and EZH2 in 11 primary ATRTs, encompassing all three molecular subgroups of ATRT. Chromatin segmentation analyses revealed that across all three subgroups a high percentage of ATRT genomes resides in a quiescent state and active enhancer states are significantly underrepresented as compared to non-neoplastic brain tissues. These data show that loss of SMARCB1 generally results in a subgroup independent repression of genes In addition, we observed gain of the repressive mark H3K27me3 in ATRTs at genes bound by SMARCB1 in normal brain, in line with the known antagonistic role between SMARCB1 and EZH2. Tumor suppressor genes and neuronal transcription factors (such as NEUROD2) were particularly affected by this mechanism of epigenetic silencing. Interestingly, by comparing the genome wide distribution of EZH2 and H3K27me3, we also identified classes of ATRT-specific active genes bound by EZH2, but lackingH3K27me3, pointing at a non-canonical role of EZH2 in gene activation. In subgroup ATRT-TYR, which displayed the most striking pattern of EZH2 associated gene activation, this pattern could for instance explain the overexpression of previously identified subgroup specific signature genes such as BMP4. The comprehensive study of histone marks in this genomically simple but epigenetically heterogeneous disease enabled us to not only shed light on the chromatin landscape of these tumors but also on the potentially tumorigenic consequences of SMARCB1 loss. In addition, the dual role of EZH2 in these tumors further enhances the importance of this molecule as a therapeutic target.

MiR-424 functions as a tumor suppressor in glioma cells and is down-regulated by DNA methylation.

Glioma is one of the most lethal malignancies, and increasing reports revealed that microRNAs (miRNAs), a class of small non-coding RNAs, play a critical role in the development and pathology of human gliomas. MiR-424 has been found to be dysregulated in many different types of human cancers. However, the clinical significance and function of miR-424 in glioma remains unclear. Here, based on RTq-PCR analysis in 148 clinical specimens, we found miR-424 expression was significantly decreased in glioma tumor tissues than in adjacent non-neoplastic brain tissues, and decreased miR-424 expression was associated with glioma KPS (P = 0.009) and high grades (P = 0.029). In vitro cellular function assays further revealed that miR-424 inhibited cell invasion and migration, and promoted cell apoptosis. In addition, based on DNA methylation analysis on clinical specimens and cell lines, we found miR-424 promoter CpG island was frequently methylated and correlated with glioma high grades (P = 0.035) and IDH mutation status (P = 0.042). Moreover, the promoter CpG island was demethylated by 5-aza-2'-deoxycytidine treatment in a time-dependent manner and the expression levels of miR-424 were gradually induced and increased. Taken together, our data suggest that the promoter region CpG island methylation is associated with tumor suppressive miR-424 silencing and the pathology of human gliomas.

OS01.1 Identification of the pre-malignant cell of origin in Human Glioblastoma Multiforme

AbstractOne of the major difficulties in identifying the cell of origin for glioblastoma is the complex cellular composition of this disease. It is now hypothesized that the different subtypes of high-grade glioma may have different cell of origin and in mouse models it has been shown that the cell of origin resides within the progenitor populations. To identify the cell of origin in human GBM we decided to first dissect out the various stages of CNS stem cell differentiation in fetal and adult human brain. After a functional high throughput screen, we identified a panel of four antibodies (i.e CD15, Notch1, EGFR, and CD90) that in various combinations can separate long-term self-renewing multi-potent neural stem cells (NSCs: LinCD15Notch1EGFRCD90) from multi-potent progenitors with limited self-renewal capacity (NPC: LinCD15Notch1EGFRCD90) from non-neoplastic human fetal and adult Brain (sub-ventricular zone). FACS sorted cells were analyzed for single cell lineage multi-potentiality, lineage bias and self-renewal to trace and characterize their lineage relationships. In surgical human glioblastoma samples, however we observed in-vivo tumor initiating and in-vitro tumorsphere-forming frequency was predominant in the progenitor cells and not in the neural stem cells. Interestingly we did observe a small frequency of NSC cells forming distinct lesions in the brain, which on further analysis showed the presence of both the neural stem cells and Glioma stem cells, where as the tumor initiating enriched population (GSC: LinCD15Notch1EGFRCD90) did not show the presence of neural stem cells but only tumor initiating NPCs. limiting dilution transplantation and serial transplantation confirmed the LinCD15Notch1EGFRCD90 to have the highest frequency of self renewing cells compared to other populations. Taken together our data suggests the presence of “normal” or pre-malignant stem cells in glioblastoma, which suggests a possible mechanism of relapse. Using Transcriptome analysis and targeted sequencing of known mutations we identify the signal transduction and genomic events (PTEN point mutations vs Deletions) which distinguish pre-malignant neural stem cells from the tumor initiating glioma stem cells. We further show a significant enrichment of the protein translational machinery as cells transition from pre-malignant to tumor initiating cells. These pre-malignant cells comprise a cellular reservoir that needs to be targeted to prevent tumor relapse. SSM: Seibel Fellowship and Ludwig Cancer Center, ILW: National Institutes of Health, Ludwig Cancer Center, SHC: Stanford Child Health Research Institute, St Baldrick’s Foundation and Ludwig Cancer Center

Thyroid transcription factor-1 distinguishes subependymal giant cell astrocytoma from its mimics and supports its cell origin from the progenitor cells in the medial ganglionic eminence

Subependymal giant cell astrocytoma is a benign brain tumor mostly associated with tuberous sclerosis complex. However, it may be misinterpreted as other high-grade brain tumors due to the presence of large tumor cells with conspicuous pleomorphism and occasional atypical features, such as tumor necrosis and endothelial proliferation. In this study, we first investigated thyroid transcription factor-1 (TTF-1) expression in a large series of subependymal giant cell astrocytomas and other histologic and locational mimics to validate the diagnostic utility of this marker. We then examined TTF-1 expression in non-neoplastic brain tissue to determine the cell origin of subependymal giant cell astrocytoma. Twenty-four subependymal giant cell astrocytoma specimens were subjected to tissue microarray construction. For comparison, a selection of tumors, including histologic mimics (21 gemistocytic astrocytomas and 24 gangliogliomas), tumors predominantly occurring at the ventricular system (50 ependymomas, 19 neurocytomas, and 7 subependymomas), and 134 astrocytomas (3 pleomorphic xanthoastrocytomas, 45 diffuse astrocytomas, 46 anaplastic astrocytomas, and 40 glioblastomas) were used. Immunohistochemical stain for TTF-1 was positive in all 24 subependymal giant cell astrocytomas, whereas negative in all astrocytomas, gangliogliomas, ependymomas, and subependymomas. Neurocytomas were positive for TTF-1 in 4/19 (21%) of cases using clone 8G7G3/1 and in 9/19 (47%) of cases using clone SPT24. In the three fetal brains that we examined, TTF-1 expression was seen in the medial ganglionic eminence, a transient fetal structure between the caudate nucleus and the thalami. There was no BRAFV600E mutation identified by direct sequencing in the 20 subependymal giant cell astrocytomas that we studied. In conclusion, TTF-1 is a useful marker in distinguishing subependymal giant cell astrocytoma from its mimics. Expression of TTF-1 in the fetal medial ganglionic eminence indicates that subependymal giant cell astrocytoma may originate from the progenitor cells in this region.

Downregulation of LKB1 promotes tumor progression and predicts unfavorable prognosis in patients with glioma.

The liver kinase B1 (LKB1)/5′-adenosine monophosphate-activated protein kinase pathway has been reported to facilitate glioma cell growth by improving growth conditions. To investigate the clinical significance of LKB1 in human gliomas western blot analysis and quantitative polymerase chain reaction experiments were performed. The present study demonstrated that LKB1 expression was markedly decreased at the messenger RNA and protein levels in 30 freshly prepared glioma tissues, compared with non-neoplastic brain tissues (P<0.001). Subsequently, immunohistochemical analysis demonstrated that LKB1 immunostaining in 180 glioma tissues was significantly decreased compared with that in the corresponding non-neoplastic brain tissues (P<0.001). Notably, this downregulation frequently occurred in high-grade gliomas, and statistical analysis revealed that low LKB1 expression was significantly associated with large tumor size (P=0.02), advanced World Health Organization grade (P=0.006) and low Karnofsky performance scale (P=0.01). The prognostic value of LKB1 expression in patients with glioma was additionally evaluated using Kaplan-Meier survival curves and Cox proportional hazards regression models. As a result, the overall survival time of patients with glioma with low LKB1 expression was shorter compared with that of patients with high LKB1 expression (P<0.001), and low LKB1 expression also indicated decreased survival time in patients with high-grade glioma (P<0.001). Collectively, the present data indicated that the downregulation of LKB1 was closely associated with the malignant degree of human gliomas, exhibiting lower expression at a higher grade. Notably, LKB1 may serve as a potential prognostic biomarker for patients with glioma following surgery.

Evaluation of miR-362 Expression in Astrocytoma of Human Brain Tumors.

Background:Patients affected by gliomas have a poor prognosis. Astrocytoma is a subtype of glioma. Identification of biomarkers could be an effective way to an early diagnosis of tumor or to distinguish more aggressive tumors that need more intensive therapy. In this study, we investigated whether the expression of miR-362 was increased or decreased in patients with different grades of astrocytoma.Materials and Methods:miR-362 expression was compared in 25 patients with astrocytoma with that of 4 normal nonneoplastic brain tissues.Results:In all tumor tissues, the expression of miR-362 was significantly decreased relative to its expression in normal brain tissues. However, there was no significant difference between miR-362 expressions in high and low grades of astrocytoma.Conclusions:In conclusion, miR-362 showed a down-regulation pattern in astrocytoma tissues that was different from the pattern obtained from previously published microarray studies.

MicroRNA-215 enhances invasion and migration by targeting retinoblastoma tumor suppressor gene 1 in high-grade glioma.

To elucidate the molecular mechanism of microRNA-215 (miR-215) in the migration and invasion of high grade glioma. 42 Patients were analysed for clinicopathological characteristics. qRT-PCR showed that miR-215 was up-regulated in glioma tissues compared with non-neoplastic brain tissues (P<0.05). The up-regulated miR-215 was closely associated with high grade glioma (P<0.01) and poor overall survival (P<0.01). Transwell assay showed that re-expression of miR-215 enhanced migration and invasion of glioma cells. miR-215 also down-regulated retinoblastoma tumor suppressor gene 1 (RB1) expression by targeting its 3'-UTR. Reversely, re-expression of RB1 inhibited partial effect of miR-215 on migration and invasion in vitro. Re-expression of miR-215 promoted cell migration and invasion of glioma by targeting RB1. miR-215 can thus be used as a biomarker for tumor progression and prognosis in human high grade glioma.

Inhibition of histone deacetylases sensitizes glioblastoma cells to lomustine.

Glioblastoma (GBM) ranks among the deadliest solid cancers worldwide and its prognosis has remained dismal, despite the use of aggressive chemo-irradiation treatment regimens. Limited drug delivery into the brain parenchyma and frequent resistance to currently available therapies are problems that call for a prompt development of novel therapeutic strategies. While only displaying modest efficacies as mono-therapy in pre-clinical settings, histone deacetylase inhibitors (HDACi) have shown promising sensitizing effects to a number of cytotoxic agents. Here, we sought to investigate the sensitizing effect of the HDACi trichostatin A (TSA) to the alkylating agent lomustine (CCNU), which is used in the clinic for the treatment of GBM. Twelve primary GBM cell cultures grown as neurospheres were used in this study, as well as one established GBM-derived cell line (U87 MG). Histone deacetylase (HDAC) expression levels were determined using quantitative real-time PCR and Western blotting. The efficacy of either CCNU alone or its combination with TSA was assessed using various assays, i.e., cell viability assays (MTT), cell cycle assays (flow cytometry, FACS), double-strand DNA break (DSB) quantification assays (microscopy/immunofluorescence) and expression profiling assays of proteins involved in apoptosis and cell stress (Western blotting and protein array). We found that the HDAC1, 3 and 6 expression levels were significantly increased in GBM samples compared to non-neoplastic brain control samples. Additionally, we found that pre-treatment of GBM cells with TSA resulted in an enhancement of their sensitivity to CCNU, possibly via the accumulation of DSBs, decreased cell proliferation and viability rates, and an increased apoptotic rate. From our data we conclude that the combined administration of TSA and CCNU eradicates GBM cells with a higher efficacy than either drug alone, thereby opening a novel avenue for the treatment of GBM.

Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1): A molecular predictor of poor survival in glioblastoma multiforme in Egyptian patients

BackgroundLong noncoding RNAs (lncRNAs) are a recently discovered class of transcribed RNA molecules with a length of more than 200 nucleotides. Recent studies have shown that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) could play an important role in carcinogenesis and cancer progression in several types of malignancies. ObjectiveAs little is known about the role and clinical significance of lncRNA MALAT1 in glioblastoma multiform (GBM) patients in Egyptian population, this study aimed to investigate the expressions of lncRNA-MALAT1 in human GBM samples and to correlate these expressions with the available clinicopathological features including patient survival data. Subjects and methodsThe relative expression of MALAT1 was determined in 37 human glioblastoma formalin-fixed paraffin embedded (FFPE) tissue samples and 10 FFPE non-neoplastic brain tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology. ResultsThe current results revealed that lncRNA MALAT1 expression was down-regulated in all tumor specimens compared to normal tissues. A receiver operating characteristic (ROC) curve analysis showed high diagnostic performance; area under curve (AUC)=0.925±0.038 (P<0.001), 95% CI=0.850–1.00, with 94.6% sensitivity, and 72.7% specificity. Lower MALAT1 expression was associated with poor prognosis; higher frequency of recurrence (P<0.044), lower overall survival (P<0.005), and shorter disease-free survival (P<0.004). ConclusionTaken together, we could postulate that MALAT1 might have a tumor-suppressive function in GBM in Egyptian population and this specific type of lncRNAs may be included in the lists of both potential prognostic biomarkers and the future therapeutic targets for glioblastomas.

P08.68 The Ran signaling pathway is upregulated dependent from glioma grade and radiochemotherapy

AbstractIntroduction:Numerous studies on different non-brain cancers indicate Ran (Ras-related nuclear protein), a key protein in the nucleoplasmic transport during interphase and the regulation of mitotic spindle formation, to be involved in tumorigenesis, cancer progression and tumor cell survival. It further is predictive for poor outcome.Overexpression of Ran has been reported in glioblastoma [GBM] tissue. To further evaluate the role of the Ran signaling pathway in astrocytoma, we measured the expression of its key components Ran, RCC1, RANBP1 and RANBP2 in different types of brain tumor tissue.Materials and Methods:Using qRT-PCR and Western blot analysis the gene and protein expression was analyzed in 80 tissue samples. The tissue subtypes were astrocytomas WHO II°, anaplastic astrocytomas, primary and secondary glioblastomas and tissue samples after recurrence subsequent to radiochemotherapy. Peritumoral brain tissue was used as a control. For PCR analysis at least 10 glioma probes and for Western blot analysis at least 6 glioma probes were used per group.Results:Ran protein overexpression compared to non-neoplastic brain was detected in anaplastic astrocytomas (90%), primary glioblastoma (140%) and secondary glioblastoma (170%). With increasing WHO grade there was an increase of Ran mRNA expression and chemo-radiation therapy led to further enhanced expression in primary and secondary glioblastoma (from 1.36 to 2.25 and from 2.08 to 3.08 arbitrary unites [AU], respectively). There was also an increase in gene expression of RCC1, RANBP1 and RANBP2 analogue to that of Ran: showing a steady increase with higher WHO grading. Correspondingly to Ran, gene expression of RCC1 increased in primary and secondary glioblastoma recurrent after radiochemotherapy from 3.19 to 3.87 AU and from 4.14 to 10.11 AU respectively as well as that of RANBP2 from 2.36 to 5.14 AU and from 2.77 to 4.74 AU respectively.Discussion:Our data clearly show a grade dependent upregulation of key components of the Ran pathway in astrocytomas and a further increase after radiochemotherapy. Therefore, this pathway may represent a future target for glioblastoma therapy.

Acylglycerol kinase functions as an oncogene and an unfavorable prognostic marker of human gliomas

Acylglycerol kinase (AGK) regulates various cellular processes involved into tumorigenesis and tumor progression. To investigate involvement of AGK in human gliomas, here, quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry analyses were performed to respectively detect the expression of AGK mRNA and protein in glioma and nonneoplastic brain tissue specimens. Then, the associations of AGK expression with various clinicopathological characteristics and patients' prognosis were statistically evaluated. Moreover, the effects of siRNA-mediated AGK knockdown on glioma cell proliferation, migration, and invasion were respectively assessed via Cell Counting Kit-8 and Transwell assays in vitro. As a result, AGK expression, at both mRNA and protein levels, were markedly up-regulated in glioma tissues compared with nonneoplastic brain tissues (both P < .001). In addition, high AGK expression was significantly associated with the grade of malignancy and poor prognosis in glioma patients (all P < .05). Importantly, Cox regression model of multivariate analysis identified AGK expression as an independent prognostic factor for glioma patients (P = .03). Furthermore, silencing the expression of AGK dramatically suppressed cell proliferation, migration, and invasion of glioma cells in vitro (all P < .05). In conclusion, AGK up-regulation may be involved into glioma development and progression, highlighting its prognostic value for the treatment of patients with this malignancy. Further loss-of-function experiments suggest that AGK might play an important role in the viability and motility of glioma cells, implying its potentials as an attractive therapeutic target for this tumor.

Reduced expression of microRNA-497 is associated with greater angiogenesis and poor prognosis in human gliomas

MicroRNA (miR)-497 plays a tumor-suppressive role in several malignancies and is involved in glioma invasiveness and resistance to chemotherapy. To add to the knowledge of the clinical significance of miR-497 in human gliomas, quantitative real-time polymerase chain reaction was performed to detect the expression of miR-497 in 110 pairs of freshly prepared glioma and nonneoplastic brain tissues. Then the associations of miR-497 expression with various clinicopathological characteristics and overall survival of glioma patients were estimated statistically. Gain-of-function assays were also performed to examine the role of miR-497 in glioma angiogenesis. The expression of miR-497 in human glioma tissues was significantly lower than in nonneoplastic brain tissues (P<.001). In addition, low miR-497 expression was significantly associated with advanced World Health Organization grade (P<.001) and low Karnofsky performance scores (P=.02). Moreover, the survival of glioma patients with low miR-497 expression was dramatically shorter than that of patients with high miR-497 expression (P=.001). Forced expression of miR-497 in glioma cells inhibited tube formation by cocultured human brain microvascular endothelial cells. We also found that miR-497 overexpression in glioma cells led to decreased expression of vascular endothelial growth factor. In conclusion, miR-497 may be a favorable prognostic marker in human gliomas, in part by being a negative regulator of angiogenesis, implying its potential as a therapeutic target for this cancer.

MiRNA regulation of Sirtuin-1 expression in human astrocytoma.

Sirtuins are a family of 7 histone deacetylases largely involved in the regulation of cell proliferation, survival and death. The role of sirtuins in tumorigenesis and cancer progression has been previously studied in certain cancer types. Few studies have investigated sirtuin expression in gliomas, with controversial results. The aim of the present study was to investigate the expression of sirtuin-1 (Sirt-1) in diffuse astrocytoma [low grade astrocytoma (LGA)], anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) and in primary glioma cell lines: PLGAC (primary LGA cells); PAAC (primary AA cells); and PGBMC (primary GBM cells). Tumor samples were obtained from patients who underwent craniotomy for microsurgical tumor resection at the Neurosurgery Unit of the University of Messina between 2011 and 2014. Sirt-1 expression was qualitatively analyzed in 30 human glial tumor samples and 5 non-neoplastic brain tissue (NBT) specimens using immunohistochemistry and western blotting techniques. Sirt-1 expression was quantitatively analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In addition, Sirt-1 expression in primary cell lines was investigated by immunoblotting and RT-qPCR. Sirt-1 expression was downregulated in gliomas compared to NBTs. Sirt-1 levels also varied among different tumor grades, with more evident downregulation in high-grade (P<0.001) than low-grade tumors (P<0.01). These data were confirmed in cell lines, with the exception of upregulation of protein level in the highest malignancy grade cell lines. The present results suggest a role for miRNA-34a, miRNA-132 and miRNA-217 in the epigenetic control of Sirt-1 during gliomagenesis and progression, and demonstrate the different implications of Sirt-1 in human tissues and cell lines. Furthermore, the present results reveal that Sirt-1 may be an intrinsic regulator of tumor progression and the regulation of Sirt-1 involves complex molecular pathways. However, the biological functions of Sirt-1 in gliomagenesis require additional investigation.

332 Molecular Characteristics of Tumor Infiltrating Front in Glioblastoma

INTRODUCTION: Molecular heterogeneity in glioblastoma (GBM) is a novel area of research recently. Because tumor infiltrating front in GBM is often left behind following tumor decompression, knowledge of its genetic makeup can improve the rationale of potential molecular targets. METHODS: MRI localized biopsies of the tumor core and peritumoral brain zone (PBZ) were obtained from 25 GBM patients. The PBZ samples containing 15% to 30% tumor cells and core samples with predominantly tumor cells along with 8 nonneoplastic brain tissues were selected for a whole Genome Gene Expression Microarray. The list of differentially expressed genes between the tumor core and PBZ compared with nonneoplastic brain tissue was identified using R Bioconductor. Selected genes were validated using quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry (IHC). RESULTS: Unsupervised hierarchical clustering of the genes revealed that the tumor core and PBZ samples showed a varied gene expression profile and clustered into 2 distinct groups. Approximately 7123, 5188, and 991 genes were differentially expressed in Core vs Normal, PBZ vs Normal and PBZ vs Core. Novel genes like PBK (role in cell cycle), MELK (stem cell marker), and TOP2A (proliferation marker) were upregulated in PBZ and tumor core compared with normal brain and robustly validated on IHC. Pathways involved in cell cycle, immune response, cell-cell adhesion, cell-matrix interaction, etc, were commonly enriched in periphery and tumor core compared with normal brain. Some of the novel markers were validated by real-time PCR and IHC. CONCLUSION: We demonstrate that the key genes involved in tumor cell proliferation, invasion, migration, response to immune system, and stemness markers are highly enriched in the PBZ. These genes, probably contribute to the resistance of PBZ cells to standard adjuvant therapy, resulting in tumor recurrence.

ARID4B is a good biomarker to predict tumour behaviour and decide WHO grades in gliomas and meningiomas

AimsAlthough ARID4B is known to promote tumour metastasis in breast cancer and inhibit transformation and progression in leukaemia, the possible effect of ARID4B on primary brain tumours (PBTs) is not...

Downregulation of miR-154 in human glioma and its clinicopathological and prognostic significance.

Objective MicroRNA-154 (miR-154) was previously reported to be downregulated in several types of human cancers and may act as a tumour suppressor. This study aimed to measure miR-154 levels and determine its clinical significance in human glioma.Methods This retrospective study analysed fresh human glioma specimens and non-neoplastic brain tissues using real-time quantitative reverse transcription–polymerase chain reaction to determine the relative levels of miR-154. The association between miR-154 levels and various clinicopathological characteristics and survival was analysed.Results A total of 115 patients with gliomas and 115 non-neoplastic brain tissues were examined. MiR-154 levels were significantly downregulated in gliomas compared with non-neoplastic brain tissues. Low levels of miR-154 were associated with high World Health Organization grade, large tumour size (≥ 5 cm), a low Karnofsky performance status score (< 80), and a shorter overall survival. Multivariate analyses using the Cox proportional hazards regression model confirmed that decreased miR-154 level was an independent predictor of a poor prognosis.Conclusions These results suggest that miR-154 downregulation may be involved in glioma formation and progression, and that miR-154 might serve as a potential prognostic biomarker for patients with this disease.

Abstract 1458: Toll-like receptors 1, 2, 4 and 6 expression levels in diffusely infiltrative astrocytomas

Abstract Toll-like receptors (TLRs) are the first receptors of the immune system that identify disturbances in homeostasis, capable of recognizing molecules associated to cell and tissue damage. TLRs signaling pathway activates inflammatory transcription factors, mainly Nuclear Factor-kappa B (NF-kB) and Interferon Regulatory Factors (IRF3/7). Due to their importance in inflammatory process, TLRs are research hot targets for therapy in several diseases, including cancer. This study aims to analyze TLRs (1, 2, 4 and 6) expressions in human diffusely infiltrative astrocytomas, WHO grades II to IV of malignancy (AGII-AGIV), and correlate their expressions among them and with the clinical outcome of AGIV (glioblastoma, GBM) patients. TLRs mRNA levels were evaluated by qRT-PCR in 143 astrocytoma samples and 22 non-neoplastic (NN) brain specimens from epilepsy surgery. Upregulated mRNA levels of all studied TLRs were observed in astrocytomas compared to NN cases (p&amp;lt;0.05), except for TLR2 in AGII. TLR1 and TLR2 median expression values increased according to astrocytoma malignancy. TLRs mRNA expressions presented positive correlation with significance (p&amp;lt;0.05) in all grades of astrocytoma, except for TLR2 and TLR4 in AGIII. Strong associations (r&amp;lt;0.7) were detected with TLR1-TLR2 mRNA expressions among AGII, AGIII and AGIV, TLR1-TLR4 in AGIII, and TLR6-TLR1 in AGII. In GBM patients, TLR2 upregulation was associated to better outcome with longer overall survival (p&amp;lt;0.007). The same was verified for concomitant upregulation of TLR1, 2 and 4 (p&amp;lt;0.05). Aiming to analyze the distribution of these receptors in different cells present in the tumor, we performed immunofluorescence staining of GBM cell lines (A172 and U87MG). All TLRs were detected in both cell lines, indicating that their expression were not limited to immune cells. The current literature has described controversial roles for the TLRs in cancer, either activating an anti-inflammatory condition leading to tumor progression or a pro-inflammatory state leading to tumor abrogation. TLRs distribution among different cells in the tumor environment might be relevant for the inflammatory process. Our findings of TLRs on tumor cells may suggest a tendency towards an anti-inflammatory state. The comprehension of TLRs role in each cell type, that compose the tumor, may help to determine the signaling pathways involved in diffusely infiltrative astrocytoma progression, and potential new therapeutic strategies. Citation Format: Isabele F. Moretti, Daiane Gil Franco, Roseli Silva, Thais F. Galatro, Sueli M. Oba-Shinjo, Suely K.N. Marie. Toll-like receptors 1, 2, 4 and 6 expression levels in diffusely infiltrative astrocytomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1458.