Aberrant Protein Glycosylation in Brain Cancers, with Emphasis on Glioblastoma.

Aberrant glycosylation has been associated with several processes of tumorigenesis from cell signaling, migration and invasion, to immune regulation and metastasis formation. The biosynthesis of glycoconjugates is regulated through concerted and finely tuned enzymatic reactions. This includes the levels and activity of glycosyltransferases and glycosidases, nucleotide sugar metabolism, substrate availability, epigenetic condition, and cellular functional state. Glioblastoma (GBM) is the most aggressive brain tumor, frequently occurring in adults with overall survival not surpassing 17 months after diagnosis. GBM has been classified by the World Health Organization (WHO) as a grade 4 astrocytoma and stratified into G-CIMP, proneural, classical, and mesenchymal subtypes. Several biomolecular features associated with GBM aggressiveness have been elucidated; however, more studies are needed to elucidate the role of glycosylation in GBM pathology, looking at their potential as cancer targets. Here, we focus on the alteration of genes involved in protein N- and O-linked glycosylation in GBM. Specifically, the mRNA levels of glycogenes were analyzed using astrocytoma-TCGA-RNAseq datasets from public repositories. A total of 68 genes were differentially regulated in the most aggressive, mesenchymal subtype of GBM compared to the proneural and classical subtypes, and the expression of these genes was compared to normal brain tissues. Among them, we focused on 38 genes coding for proteins that belong to: 1) asparagine glycosylation (ALG); 2) glycosyltransferases (B3T, B4T); 3) fucosyltransferase (FUT); 4) acetylgalactosaminyltransferases (GALNT); 5) hexosaminidase (HEX); 6) mannosidase (MAN); 7) acetylglucosaminyltransferase (MGAT); 8) sialidase or neuraminidase (NEU); 9) solute carrier 35 family (SLC); and 10) sialyltransferase (ST). The differential expression of some genes was already reported in several solid tumors; however, several of them were found to be dysregulated in GBM for the first time. These data represent an important starting point to perform further orthogonal and functional validations to pinpoint the role of these glycogenes in GBM as diagnostic and therapeutic targets.

Correlation of Matrisome-Associatted Gene Expressions with LOX Family Members in Astrocytomas Stratified by IDH Mutation Status.

Tumor cell infiltrative ability into surrounding brain tissue is a characteristic of diffusely infiltrative astrocytoma and is strongly associated with extracellular matrix (ECM) stiffness. Collagens are the most abundant ECM scaffolding proteins and contribute to matrix organization and stiffness. LOX family members, copper-dependent amine oxidases, participate in the collagen and elastin crosslinking that determine ECM tensile strength. Common IDH mutations in lower-grade gliomas (LGG) impact prognosis and have been associated with ECM stiffness. We analyzed the expression levels of LOX family members and matrisome-associated genes in astrocytoma stratified by malignancy grade and IDH mutation status. A progressive increase in expression of all five LOX family members according to malignancy grade was found. LOX, LOXL1, and LOXL3 expression correlated with matrisome gene expressions. LOXL1 correlations were detected in LGG with IDH mutation (IDHmut), LOXL3 correlations in LGG with IDH wild type (IDHwt) and strong LOX correlations in glioblastoma (GBM) were found. These increasing correlations may explain the increment of ECM stiffness and tumor aggressiveness from LGG-IDHmut and LGG-IDHwt through to GBM. The expression of the mechanosensitive transcription factor, ß-catenin, also increased with malignancy grade and was correlated with LOXL1 and LOXL3 expression, suggesting involvement of this factor in the outside-in signaling pathway.

Cellular Model of Malignant Transformation of Primary Human Astrocytes Induced by Deadhesion/Readhesion Cycles.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG-a human glioblastoma-derived cell lineage-reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/ß-catenin signaling pathways, in addition to upregulation of genes related to epithelial-mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

Extracellular Matrix Proteome Remodeling in Human Glioblastoma and Medulloblastoma.

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.

Exercise Training Attenuates Ubiquitin-Proteasome Pathway and Increases the Genes Related to Autophagy on the Skeletal Muscle of Patients With Inflammatory Myopathies.

BACKGROUND/OBJECTIVE: The aim of this study was to evaluate the effects of exercise training on the ubiquitin-proteasome system (UPS) and genes related to autophagy on the skeletal muscle of patients with dermatomyositis (DM) and immune-mediated necrotizing myopathies (IMNMs). METHODS: Seven DM patients and 6 IMNM patients were treated for 12 weeks with a twice-weekly aerobic and resistance training exercise program. Aerobic capacity, muscle strength, and expression of genes in the skeletal muscle related to UPS and to autophagy were evaluated at the baseline and after the intervention. Moreover, only at the baseline, 10 healthy control individuals were also evaluated. RESULTS: The age of DM and IMNM patients was 49.8 and 58.5 years, respectively. Genes related to UPS were upregulated, whereas genes related to autophagy and antioxidative systems were downregulated only in the DM group when compared with control group. After completion of the exercise training program, several genes related to UPS were downregulated, whereas genes related to autophagy, mitochondrial pathways, and antioxidative systems were upregulated in both the DM and IMNM groups. CONCLUSIONS: Exercise training can increase genes related to autophagy, mitophagy, and lysosomal biogenesis in the skeletal muscle of patients. These results suggest an increase in the recycling of damaged proteins and organelles, which may also contribute to the performance and endurance of skeletal muscles in these patients. Furthermore, in patients with myositis, exercise training led to a decrease in genes related to UPS and an increase in genes related to antioxidative capacity. Therefore, this may also contribute to an attenuation of skeletal muscle loss and of the deleterious effects of oxidative stress on the skeletal muscle of these patients.

The TP53 p.R337H mutation is uncommon in a Brazilian cohort of pediatric patients diagnosed with ependymoma.

BACKGROUND: Ependymoma (EPN) is the third most common childhood cancer of the central nervous system. RELA fusion-positive EPN accounts for approximately 70% of all childhood supratentorial tumors and shows the worst prognosis among the supratentorial EPNs. TP53 mutation is infrequent in RELA fusions EPNs. In the population from the Southern region of Brazil, there is a high incidence of the germline TP53 p.R337H mutation that predisposes carriers to develop early-onset tumors. However, despite this high incidence, the frequency of this mutation among EPN patients remains to be determined. Here, we investigated the presence of the TP53 p.R337H mutation in a larger cohort of pediatric EPNs of three institutions located in the state of São Paulo, Brazil. METHODS: The TP53 p.R337H mutation was screened by conventional RT-PCR and Sanger sequencing in 49 pediatric EPNs diagnosed during the period from 1995 to 2016. RESULTS: We described for the first time a case of a 5-year-old girl with RELA fusion EPN with a heterozygous TP53 p.R337H mutation. CONCLUSIONS: The present finding indicates that the TP53 p.R337H germline mutation is uncommon in patients with EPN in Brazil and screening of pediatric patients RELA fusion EPN may be informative to better understand the role of TP53 germline mutations in the development and prognosis of these tumors.

Serum interleukin-17A level is associated with disease activity of adult patients with dermatomyositis and polymyositis.

OBJECTIVES: To assess serum interleukin (IL)-17A levels in patients with dermatomyositis (DM) and polymyositis (PM) and correlate them with the demographic, clinical, laboratory and therapeutic data of these diseases. METHODS: This was a cross-sectional, single-centre study that included defined DM and PM patients who were age-, gender- and ethnicity-matched to healthy individuals. Serum IL-17A analysis, as well as analysis for other cytokines (IL-6, TNFα and IFNγ), was performed by multiplex immunoassay. The disease status parameters were based on the International Myositis Assessment and Clinical Studies Group (IMACS) set scores. RESULTS: Eighty DM, 32 PM patients and 104 healthy individuals were enrolled. Mean age of patients with DM and PM was 46.0 and 47.7, respectively, with a predominance of women and white ethnicity in both groups. Overall, clinical, laboratory, therapeutic, and current disease status were similar among patients with DM and PM. Median serum IL-17A level was higher in patients with PM and DM than the control group (0.73 vs. 0.49 vs. 0.35 pg/mL, respectively; p<0.050) and higher in PM when compared to DM (p<0.001). In DM, serum IL-17A levels were associated with cumulative cutaneous lesions, IMACS parameters, and serum IL-6 and IFNγ levels. In PM, serum IL-17A levels correlated with patients' current age, IMACS parameters and serum TNFα and IFNγ levels. CONCLUSIONS: Serum IL-17A levels are not only increased, but also associated with disease activity in patients with DM and PM. Our data strongly suggest that IL-17A may be a biomarker of disease activity for these systemic autoimmune myopathies.

A novel type of C11orf95-LOC-RELA fusion in a grade II supratentorial ependymoma: report of a case with literature review.

BACKGROUND: Ependymoma (EPN) is the third most common central nervous system tumor in childhood. Recent advances in the molecular classification of EPN revealed a supratentorial (ST) ependymoma subgroup characterized by C11orf95-RELA fusion. CASE REPORT: We describe a novel RELA-fusion composed by a chimeric transcript C11orf95-LOC-RELA in a supratentorial WHO grade II EPN occurring in a 4-year-old child. Metastatic loci at the brain, leptomeningeal involvement, and pulmonary nodules were identified at tumor recurrence. The child eventually died before 1 year after recurrence. CONCLUSION: This index case showed aggressive behavior and nuclear accumulation of p65/RELA.

LOXL3 Function Beyond Amino Oxidase and Role in Pathologies, Including Cancer.

Lysyl oxidase like 3 (LOXL3) is a copper-dependent amine oxidase responsible for the crosslinking of collagen and elastin in the extracellular matrix. LOXL3 belongs to a family including other members: LOX, LOXL1, LOXL2, and LOXL4. Autosomal recessive mutations are rare and described in patients with Stickler syndrome, early-onset myopia and non-syndromic cleft palate. Along with an essential function in embryonic development, multiple biological functions have been attributed to LOXL3 in various pathologies related to amino oxidase activity. Additionally, various novel roles have been described for LOXL3, such as the oxidation of fibronectin in myotendinous junction formation, and of deacetylation and deacetylimination activities of STAT3 to control of inflammatory response. In tumors, three distinct roles were described: (1) LOXL3 interacts with SNAIL and contributes to proliferation and metastasis by inducing epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma cells; (2) LOXL3 is localized predominantly in the nucleus associated with invasion and poor gastric cancer prognosis; (3) LOXL3 interacts with proteins involved in DNA stability and mitosis completion, contributing to melanoma progression and sustained proliferation. Here we review the structure, function and activity of LOXL3 in normal and pathological conditions and discuss the potential of LOXL3 as a therapeutic target in various diseases.

CD99 Expression in Glioblastoma Molecular Subtypes and Role in Migration and Invasion.

Glioblastoma (GBM) is the most aggressive type of brain tumor, with an overall survival of 17 months under the current standard of care therapy. CD99, an over-expressed transmembrane protein in several malignancies, has been considered a potential target for immunotherapy. To further understand this potentiality, we analyzed the differential expression of its two isoforms in human astrocytoma specimens, and the CD99 involved signaling pathways in glioma model U87MG cell line. CD99 was also analyzed in GBM molecular subtypes. Whole transcriptomes by RNA-Seq of CD99-siRNA, and functional in vitro assays in CD99-shRNA, that are found in U87MG cells, were performed. Astrocytoma of different malignant grades and U87MG cells only expressed CD99 isoform 1, which was higher in mesenchymal and classical than in proneural GBM subtypes. Genes related to actin dynamics, predominantly to focal adhesion, and lamellipodia/filopodia formation were down-regulated in the transcriptome analysis, when CD99 was silenced. A decrease in tumor cell migration/invasion, and dysfunction of focal adhesion, were observed in functional assays. In addition, a striking morphological change was detected in CD99-silenced U87MG cells, further corroborating CD99 involvement in actin cytoskeleton rearrangement. Inhibiting the overexpressed CD99 may improve resectability and decrease the recurrence rate of GBM by decreasing tumor cells migration and invasion.

Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord.

Cell-free DNA shed by cancer cells has been shown to be a rich source of putative tumor-specific biomarkers. Because cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we studied whether the cerebrospinal fluid (CSF) that bathes the CNS is enriched for tumor DNA, here termed CSF-tDNA. We analyzed 35 primary CNS malignancies and found at least one mutation in each tumor using targeted or genome-wide sequencing. Using these patient-specific mutations as biomarkers, we identified detectable levels of CSF-tDNA in 74% [95% confidence interval (95% CI) = 57-88%] of cases. All medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space were detectable (100% of 21 cases; 95% CI = 88-100%), whereas no CSF-tDNA was detected in patients whose tumors were not directly adjacent to a CSF reservoir (P < 0.0001, Fisher's exact test). These results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.

Serum amyloid A1 is upregulated in human glioblastoma.

Serum amyloid A1 (SAA1) is a sensitive acute phase reactant primarily produced by the liver in response to acute inflammation. We have recently shown that SAA affects proliferation, migration, and invasion of glioblastoma cell lines, which suggest its participation in the malignant process. Consistently, levels of SAA have been used as a non-invasive biomarker for the prognosis of many cancers. In this study, we aimed to investigate SAA serum levels and expression of SAA genes in human astrocytomas tissues. Serum and tissue samples were obtained from patients with astrocytoma grades I to III and glioblastoma (GBM or grade IV). Levels of circulating SAA were significantly higher in the serum of patients with AGII-IV when compared to non-neoplastic samples derived from non-neoplastic patients (NN) (p > 0.0001). Quantitative real time PCR (qRT-PCR) of 148 astrocytomas samples (grades I-IV) showed that SAA1 mRNA was significantly higher in GBM when compared to AGI-III and NN samples (p < 0.0001). Immunohistochemistry analysis revealed cytoplasmic positivity for SAA in GBM. There was no correlation of SAA1 with clinical end-point of overall survival among GBM patients. However, it was found a positive correlation between SAA1 and genes involved in tumor progression, such as: HIF1A (r = 0.50; p < 0.00001), CD163 (r = 0.52; p < 0.00001), CXCR4 (r = 0.42; p < 0.00001) and CXCR7 (r = 0.33; p = 0.002). In conclusions, we show that astrocytoma patients have increased levels of serum SAA and SAA1 is expressed and secreted in GBM, and its co-expression with tumor-related genes supports its involvement in GBM angiogenesis and progression.

Stathmin involvement in the maternal embryonic leucine zipper kinase pathway in glioblastoma.

BACKGROUND: Maternal Embryonic Leucine Zipper Kinase (MELK) is a serine/threonine kinase involved in cell cycle, differentiation, proliferation, and apoptosis. These multiple features are consistent with it being a potential anticancer target. Nevertheless, the MELK pathway in tumorigenesis is not yet completely understood. This study aims to identify proteins associated with MELK pathway in astrocytomas. To this end, proteomic data of the human glioma cell line U87MG transfected with siRNA for MELK were compared with non-target transfected control cells and compared with oligonucleotide microarray data. RESULTS: In both assays, we identified stathmin/oncoprotein 18 (STMN1), involved in cell cycle. STMN1 gene expression was further assessed in a series of 154 astrocytomas and 22 non-neoplastic brain samples by qRT-PCR. STMN1 expression was significantly increased in malignant diffusely infiltrative astrocytomas compared with pilocytic astrocytoma (p < 0.0001). A strong correlation between MELK and STMN1 expressions was observed (r = 0.741, p < 0.0001) in glioblastoma (GBM) samples. However, no difference on survival times was found when compared GBM cases with upregulated and downregulated STMN1 (Breslow = 0.092, median survival time: 11 and 13 months, respectively). Functional assays knocking down MELK by siRNA in GBM cell line showed that gene and protein expression of both MELK and stathmin were diminished. On the other hand, when the same analysis was performed for STMN1, only stathmin gene and protein was silenced. CONCLUSIONS: The results presented herein point stahtmin as a downstream target in the MELK pathway that plays a role in malignant progression of astrocytomas.

Melatonergic system-based two-gene index is prognostic in human gliomas.

Gliomas, the most common primary brain tumors in adults, are classified into four malignancy grades according to morphological features. Recent studies have shown that melatonin treatment induces cytotoxicity in glioma-initiating cells and reduces the invasion and migration of glioma cell lines, inhibiting the nuclear factor κB (NFκB) oncopathway. Given that C6 rat glioma cells produce melatonin, we investigated the correlation between the capacity of gliomas to synthesize/metabolize melatonin and their overall malignancy. We first characterized the melatonergic system of human gliomas cell lines with different grades of aggressiveness (HOG, T98G, and U87MG) and demonstrated that glioma-synthesized melatonin exerts an autocrine antiproliferative effect. Accordingly, the sensitivity to exogenous melatonin was higher for the most aggressive cell line, U87MG, which synthesized/accumulated less melatonin. Using The Cancer Genome Atlas RNAseq data of 351 glioma patients, we designed a predictive model of the content of melatonin in the tumor microenvironment, the ASMT:CYP1B1 index, combining the gene expression levels of melatonin synthesis and metabolism enzymes. The ASMT:CYP1B1 index negatively correlated with tumor grade, as well as with the expression of pro-proliferation and anti-apoptotic NFκB target genes. More importantly, the index was a grade- and histological type-independent prognostic factor. Even when considering only high-grade glioma patients, a low ASMT:CYP1B1 value, which suggests decreased melatonin and enhanced aggressiveness, was strongly associated with poor survival. Overall, our data reveal the prognostic value of the melatonergic system of gliomas and provide insights into the therapeutic role of melatonin.

Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma.

BACKGROUND: Gliomas account for more than 60 % of all primary central nervous system neoplasms. Low-grade gliomas display a tendency to progress to more malignant phenotypes and the most frequent and malignant gliomas are glioblastomas (GBM). Another type of glioma, oligodendroglioma originates from oligodendrocytes and glial precursor cells and represents 2-5 % of gliomas. The discrimination between these two types of glioma is actually controversial, thus, a molecular distinction is necessary for better diagnosis. METHODS: iTRAQ-based quantitative proteomic analysis was performed on non-neoplastic brain tissue, on astrocytoma grade II, glioblastoma with short and long survival and oligodendrogliomas. RESULTS: We found that expression of nucleophosmin (NPM1), glucose regulated protein 78 kDa (GRP78), nucleolin (NCL) and heat shock protein 90 kDa (HSP90B1) were increased, Raf kinase inhibitor protein (RKIP/PEBP1) was decreased in glioblastoma and they were associated with a network related to tumor progression. Expression level of heat shock protein 27 (HSPB1/HSP27) discriminated glioblastoma presenting short (6 ± 4 months, n = 4) and long survival (43 ± 15 months, n = 4) (p = 0.00045). Expression level of RNA binding protein nova 1 (NOVA1) differentiated low-grade oligodendroglioma and astrocytoma grade II (p = 0.0082). Validation were done by Western blot, qRT-PCR and immunohistochemistry in a larger casuistry. CONCLUSION: Taken together, our quantitative proteomic analysis detected the molecular triad, NPM1, GRP78 and RKIP participating together with NCL and HSP27/HSPB1 in a network related to tumor progression. Additionally, two new important targets were uncovered: NOVA1 useful for diagnostic refinement differentiating astrocytoma from oligodendroglioma, and HSPB1/HSP27, as a predictive factor of poor prognosis for GBM.

Immunohistochemical expression of cyclin D1 is higher in supratentorial ependymomas and predicts relapses in gross total resection cases.

Ependymomas are tumors of the CNS. Although cyclin D1 overexpression has been related to several cancers, its prognostic value in ependymomas has not yet been fully established. We evaluated cyclin D1 expression by an immunohistochemistry analysis of 149 samples of ependymomas, including some relapses, corresponding to 121 patients. Eighty-one patients were adults, 60 were intracranial cases and 92 tumors were grade II. Gross total resection (GTR) was achieved in 62% of cases, and relapse was confirmed in 41.4% of cases. Cyclin D1 protein expression was analyzed by immunohistochemistry and scored with a labeling index (LI) calculated as the percentage of positively stained cells by intensity. We also analyzed expression of CCND1 and NOTCH1 in 33 samples of ependymoma by quantitative real-time PCR. A correlation between cyclin D1 LI score and anaplastic cases (P < 0.001), supratentorial location (P < 0.001) and age (P = 0.001) were observed. A stratified analysis demonstrated that cyclin D1 protein expression was strong in tumors with a supratentorial location, independent of the histological grade or age. Relapse was more frequent in cases with a higher cyclin D1 LI score (P = 0.046), and correlation with progression-free survival was observed in cases with GTR (P = 0.002). Only spinal canal tumor location and GTR were suggestive markers of PFS in multivarite analyses. Higher expression levels were observed in anaplastic cases for CCND1 (P = 0.002), in supratentorial cases for CCND1 (P = 0.008) and NOTCH1 (P = 0.011). There were correlations between the cyclin D1 mRNA and protein expression levels (P < 0.0001) and between CCND1 and NOTCH1 expression levels (P = 0.003). Higher cyclin D1 LI was predominant in supratentorial location and predict relapse in GTR cases. Cyclin D1 could be used as an immunohistochemical marker to guide follow-up and treatment in these cases.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240.

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

CD99 is upregulated in placenta and astrocytomas with a differential subcellular distribution according to the malignancy stage.

In the present study, we searched for genes highly expressed in placenta and that could contribute to the establishment and maintenance of a malignant phenotype in different types of tumours, and in astrocytomas in particular. We employed a strategy based on the integration of in silico data from previously generated massively parallel signature sequencing and public serial analysis of gene expression databases. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both cell lines. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

Angiogenesis and expression of PDGF-C, VEGF, CD105 and HIF-1α in human glioblastoma.

Glioblastoma (GBM), the most frequent and aggressive brain tumor, is characterized by marked angiogenesis directly related to invasiveness and poor prognosis. Hypoxia is considered to be an important stimulus for angiogenesis by inducing hypoxia-inducible factor 1-alpha (HIF-1α) overexpression that activates platelet-derived growth factor (PDGF) and VEGF. The aim of this study is to analyze the expression of PDGF-C, VEGF in endothelial and tumor cells of GBM and their relation to HIF-1α expression. Two hundred and eight GBM cases were studied by tissue microarray immunohistochemical preparation. Expression of HIF-1α, VEGF and PDGF-C was observed in 184 (88.5%), 131 (63%) and 160 (76.9%) tumor cases, respectively. The numbers of vessels were quantified by CD34, PDGF-C, VEGF and CD105 staining, and were in median 20, 16, 5 and 6, respectively. The GBMs that showed positive or negative expression for HIF-1α showed a median vascular density of 30 and 14, respectively, for CD34 (P < 0.015). Positive expression for HIF-1α was correlated with VEGF and PDGF-C expression in tumors (P < 0.001). There was a significant correlation between VEGF and PDGF-C expression in the cytoplasm of GBM tumor cells (P < 0.0001). We showed that VEGF expression in tumor cells was correlated with its expression in blood vessels (P < 0.0001). Endothelial cells with PDGF-C and VEGF positive expression were also positive for CD105 and their nuclei for Ki-67, confirming the neoangiogenic and proliferative influence of VEGF and PDGF-C. VEGF nuclear staining in tumor cells (P = 0.002) as well as nuclear staining for HIF-1α and VEGF (P = 0.005) correlated with survival. In summary, our present findings of the concomitant upregulation of PDGF-C with VEGF in GBM tumor cells and vessels further reinforce the benefit of using combined anti-angiogenic approaches to potentially improve the therapeutic response for GBM.