Novel Proteins Regulated by mTOR in Subependymal Giant Cell Astrocytomas of Patients with Tuberous Sclerosis Complex and New Therapeutic Implications
2010; Elsevier BV; Volume: 176; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.090950
ISSN1525-2191
AutoresMagdalena E. Tyburczy, Katarzyna Kotulska, Piotr Pokarowski, Jakub Mieczkowski, Joanna Kucharska, Wiesława Grajkowska, Marcin Roszkowski, Sergiusz Jóźwiak, Bożena Kamińska,
Tópico(s)Neurofibromatosis and Schwannoma Cases
ResumoSubependymal giant cell astrocytomas (SEGAs) are rare brain tumors associated with tuberous sclerosis complex (TSC), a disease caused by mutations in TSC1 or TSC2, resulting in enhancement of mammalian target of rapamycin (mTOR) activity, dysregulation of cell growth, and tumorigenesis. Signaling via mTOR plays a role in multifaceted genomic responses, but its effectors in the brain are largely unknown. Therefore, gene expression profiling on four SEGAs was performed with Affymetrix Human Genome arrays. Of the genes differentially expressed in TSC, 11 were validated by real-time PCR on independent tumor samples and 3 SEGA-derived cultures. Expression of several proteins was confirmed by immunohistochemistry. The differentially-regulated proteins were mainly involved in tumorigenesis and nervous system development. ANXA1, GPNMB, LTF, RND3, S100A11, SFRP4, and NPTX1 genes were likely to be mTOR effector genes in SEGA, as their expression was modulated by an mTOR inhibitor, rapamycin, in SEGA-derived cells. Inhibition of mTOR signaling affected size of cultured SEGA cells but had no influence on their proliferation, morphology, or migration, whereas inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways led to significant alterations of these processes. For the first time, we identified genes related to the occurrence of SEGA and regulated by mTOR and demonstrated an effective modulation of SEGA growth by pharmacological inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways, which could represent a novel therapeutic approach. Subependymal giant cell astrocytomas (SEGAs) are rare brain tumors associated with tuberous sclerosis complex (TSC), a disease caused by mutations in TSC1 or TSC2, resulting in enhancement of mammalian target of rapamycin (mTOR) activity, dysregulation of cell growth, and tumorigenesis. Signaling via mTOR plays a role in multifaceted genomic responses, but its effectors in the brain are largely unknown. Therefore, gene expression profiling on four SEGAs was performed with Affymetrix Human Genome arrays. Of the genes differentially expressed in TSC, 11 were validated by real-time PCR on independent tumor samples and 3 SEGA-derived cultures. Expression of several proteins was confirmed by immunohistochemistry. The differentially-regulated proteins were mainly involved in tumorigenesis and nervous system development. ANXA1, GPNMB, LTF, RND3, S100A11, SFRP4, and NPTX1 genes were likely to be mTOR effector genes in SEGA, as their expression was modulated by an mTOR inhibitor, rapamycin, in SEGA-derived cells. Inhibition of mTOR signaling affected size of cultured SEGA cells but had no influence on their proliferation, morphology, or migration, whereas inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways led to significant alterations of these processes. For the first time, we identified genes related to the occurrence of SEGA and regulated by mTOR and demonstrated an effective modulation of SEGA growth by pharmacological inhibition of both mTOR and extracellular signal-regulated kinase signaling pathways, which could represent a novel therapeutic approach. Subependymal giant cell astrocytomas (SEGAs) are rare, low-grade brain tumors (World Health Organization Grade I) of a mixed glioneuronal lineage.1Hirose T Scheithauer BW Lopes MB Gerber HA Altermatt HJ Hukee MJ VandenBerg SR Charlesworth JC Tuber and subependymal giant cell astrocytoma associated with tuberous sclerosis: an immunohistochemical, ultrastructural, and immunoelectron and microscopic study.Acta Neuropathol. 1995; 90: 387-399Crossref PubMed Scopus (131) Google Scholar, 2Buccoliero AM Franchi A Castiglione F Gheri CF Mussa F Giordano F Genitori L Taddei GL Subependymal giant cell astrocytoma (SEGA): Is it an astrocytoma? Morphological, immunohistochemical and ultrastructural study.Neuropathology. 2009; 29: 25-30Crossref PubMed Scopus (51) Google Scholar They are observed in 10% to 20% of patients with tuberous sclerosis complex (TSC) and are the major cause of morbidity in children and young adults with TSC.3Kim SK Wang KC Cho BK Jung HW Lee YJ Chung YS Lee JY Park SH Kim YM Choe G Chi JG Biological behavior and tumorigenesis of subependymal giant cell astrocytomas.J Neurooncol. 2001; 52: 217-225Crossref PubMed Scopus (64) Google Scholar The disease affects about one in 6000 people, is characterized by the formation of benign tumors in multiple organs (mainly brain, heart, kidneys, skin, or lungs), and is often associated with epilepsy, mental retardation, and autism.4Jozwiak S Schwartz RA Janniger CK Bielicka-Cymerman J Usefulness of diagnostic criteria of tuberous sclerosis complex in pediatric patients.J Child Neurol. 2000; 15: 652-659Crossref PubMed Scopus (191) Google Scholar, 5Jozwiak S Goodman M Lamm SH Poor mental development in patients with tuberous sclerosis complex: clinical risk factors.Arch Neurol. 1998; 55: 379-384Crossref PubMed Scopus (86) Google Scholar Tuberous sclerosis complex is caused by mutation in one of two tumor suppressor genes, TSC1 and TSC2, which encode Hamartin and Tuberin, respectively.6European Chromosome 16 Tuberous Sclerosis Consortium Identification and characterization of the tuberous sclerosis gene on chromosome 16.Cell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1508) Google Scholar, 7van Slegtenhorst M de Hoogt R Hermans C Nellist M Janssen B Verhoef S Lindhout D van den Ouweland A Halley D Young J Burley M Jeremiah S Woodward K Nahmias J Fox M Ekong R Osborne J Wolfe J Povey S Snell RG Cheadle JP Jones AC Tachataki M Ravine D Sampson JR Reeve MP Richardson P Wilmer F Munro C Hawkins TL Sepp T Ali JB Ward S Green AJ Yates JR Kwiatkowska J Henske EP Short MP Haines JH Jozwiak S Kwiatkowski DJ Identification of the tuberous sclerosis gene TSC1 on chromosome 9q34.Science. 1997; 277: 805-808Crossref PubMed Scopus (1392) Google Scholar Both proteins form the TSC complex that inhibits the mammalian target of rapamycin complex 1 (mTORC1). Within the TSC complex, TSC1 stabilizes TSC2, whereas TSC2 acts as a GTPase-activating protein for the small GTPase RHEB (Ras homolog enriched in brain).8Benvenuto G Li S Brown SJ Braverman R Vass WC Cheadle JP Halley DJ Sampson JR Wienecke R DeClue JE The tuberous sclerosis-1 (TSC1) gene product hamartin suppresses cell growth and augments the expression of the TSC2 product tuberin by inhibiting its ubiquitination.Oncogene. 2000; 19: 6306-6316Crossref PubMed Scopus (207) Google Scholar, 9Chong-Kopera H Inoki K Li Y Zhu T Garcia-Gonzalo FR Rosa JL Guan KL TSC1 stabilizes TSC2 by inhibiting the interaction between TSC2 and the HERC1 ubiquitin ligase.J Biol Chem. 2006; 281: 8313-8316Crossref PubMed Scopus (183) Google Scholar, 10Zhang Y Gao X Saucedo LJ Ru B Edgar BA Pan D Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins.Nat Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (715) Google Scholar Mutation in one of the genes leads to elevated RHEB-GTP levels and activation of mTORC1, which further triggers a downstream kinase signaling cascade, including phosphorylation of eukaryotic translation initiation factor 4E-binding proteins and p70 S6 kinases, proteins involved in translation initiation and ribosome biogenesis.11Burnett PE Barrow RK Cohen NA Snyder SH Sabatini DM RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1.Proc Natl Acad Sci USA. 1998; 95: 1432-1437Crossref PubMed Scopus (935) Google Scholar Increased activation of mTOR kinase results in disorganized cellular overgrowth, abnormal differentiation, and formation of neoplasms.12Fingar DC Blenis J Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression.Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1056) Google Scholar, 13Wullschleger S Loewith R Hall MN TOR signaling in growth and metabolism.Cell. 2006; 124: 471-484Abstract Full Text Full Text PDF PubMed Scopus (4684) Google Scholar Clinical trials with rapamycin (sirolimus), a specific inhibitor of mTORC1, have been initiated and showed a regression of SEGAs after a systemic administration of the drug.14Franz DN Leonard J Tudor C Chuck G Care M Sethuraman G Dinopoulos A Thomas G Crone KR Rapamycin causes regression of astrocytomas in tuberous sclerosis complex.Ann Neurol. 2006; 59: 490-498Crossref PubMed Scopus (510) Google Scholar, 15Sampson JR Therapeutic targeting of mTOR in tuberous sclerosis.Biochem Soc Trans. 2009; 37: 259-264Crossref PubMed Scopus (75) Google Scholar However, molecular mechanisms underlying a regression of SEGAs in TSC patients are still poorly understood. By combining the use of rapamycin, transcriptional profiling, and RNA interference, more than 400 genes in yeast and 90 genes in D. melanogaster were identified to be up- or down-regulated by mTOR inhibition.16Hardwick JS Kuruvilla FG Tong JK Shamji AF Schreiber SL Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins.Proc Natl Acad Sci USA. 1999; 96: 14866-14870Crossref PubMed Scopus (466) Google Scholar, 17Chan TF Carvalho J Riles L Zheng XF A chemical genomics approach toward understanding the global functions of the target of rapamycin protein (TOR).Proc Natl Acad Sci USA. 2000; 97: 13227-13232Crossref PubMed Scopus (157) Google Scholar, 18Xie MW Jin F Hwang H Hwang S Anand V Duncan MC Huang J Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method.Proc Natl Acad Sci USA. 2005; 102: 7215-7220Crossref PubMed Scopus (117) Google Scholar, 19Guertin DA Guntur KV Bell GW Thoreen CC Sabatini DM Functional genomics identifies TOR-regulated genes that control growth and division.Curr Biol. 2006; 16: 958-970Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar Moreover, the gene expression analysis in Tsc2 null murine neuroepithelial progenitor cells revealed altered expression of many genes encoding proteins involved in cell growth, adhesion, and neuronal transmission.20Onda H Crino PB Zhang H Murphey RD Rastelli L Gould Rothberg BE Kwiatkowski DJ Tsc2 null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway.Mol Cell Neurosci. 2002; 21: 561-574Crossref PubMed Scopus (108) Google Scholar However, understanding of mTOR signaling and its downstream targets in the human brain remains far from complete. In the current study, gene expression profiling on SEGA samples was performed and we identified specific genes involved in tumorigenesis (up-regulated) and the nervous system development (down-regulated) in SEGAs or SEGA-derived cell cultures when compared with the normal brain or cultured human astrocytes. Immunohistochemistry on paraffin-embedded sections confirmed up-regulated levels of several identified proteins in SEGAs. Rapamycin affected the expression of selected genes in SEGA-derived cell cultures showing their dependence on mTOR signaling. Moreover, pharmacological inhibition of mTOR and extracellular signal-regulated kinase (ERK) signaling pathways in cultured SEGA cells affected their proliferation, size, morphology, and migration. Specific expression of the identified genes in the pathological brain and the influence of mTOR and ERK signaling on biology of SEGA cells may provide explanation of how these pathways contribute to the pathogenesis of SEGA and neurological alterations associated with tuberous sclerosis complex. Ten SEGA samples and three control brain tissues were accessed from the Department of Pathology and Department of Pediatric Neurology, The Children's Memorial Health Institute, Warsaw, Poland. SEGA specimens were originally obtained from tumors immediately after resection from TSC patients diagnosed clinically according to the criteria of Roach. A genetic analysis proved that four of five analyzed patients had mutations in TSC2. Control tissues consisted of periventricular regions of non-TSC patients. Two additional controls were: FirstChoice® Human Brain Reference RNA pooled from 23 donors (Applied Biosystems, Darmstadt, Germany) and Human Brain Total RNA pooled from 2 donors (Clontech, Saint-Germain-en-Laye, France). Total RNA was prepared by Tri-Reagent (Sigma-Aldrich, Munich, Germany) extraction from snap-frozen tissues. RNA was cleaned up using RNeasy Mini Kit (Qiagen, Hilden, Germany) which was also used to isolate total RNA from harvested cells. The quality and quantity of total RNA were verified using the Agilent bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA (5 μg) was converted to double-stranded cDNA. Biotin-labeled cRNA was generated after an in vitro transcription reaction. The cRNA was fragmented and then hybridized to a control microarray (Test3) and then, after sample quality evaluation, to the arrays HG-U133 Plus 2.0 (Affymetrix, Santa Clara, CA). Immediately after hybridization, the arrays underwent automated washing and staining steps. Finally, they were scanned and the software computed intensities for each cell. Samples hybridization was done in the Department of Nuclear Medicine and Endocrine Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice, Poland, using a standard protocol provided by Affymetrix. Microarray data were analyzed using five popular preprocessing methods: RMA,21Irizarry RA Bolstad BM Collin F Cope LM Hobbs B Speed TP Summaries of Affymetrix GeneChip probe level data.Nucleic Acids Res. 2003; 31: e15Crossref PubMed Scopus (4021) Google Scholar MAS5.0 (Affymetrix Inc. 2002,), GC-RMA,22Wu Z Irizarry RA Preprocessing of oligonucleotide array data.Nature Biotechnol. 2004; 22: 656-658; author reply 658Crossref Scopus (273) Google Scholar MBEI pmonly,23Li C Hung Wong W Model-based analysis of oligonucleotide arrays: model validation, design issues and standard error application.Genome Biol. 2001; 2 (RESEARCH0032)Google Scholar and PDNN.24Zhang L Miles MF Aldape KD A model of molecular interactions on short oligonucleotide microarrays.Nature Biotechnol. 2003; 21: 818-821Crossref Scopus (268) Google Scholar This was done to identify changes in gene expression robust to a particular choice of a preprocessing method. Probe set measurements were transformed into measurements for genes using annotation provided in the Ensembl database. SEGA gene expression profiling data were deposited at ArrayExpress, accession: E-MEXP-2351. Additionally, to remove a possible cross-hybridization effect, all probe sets with annotation to more than one gene were excluded from further analysis. Furthermore, expression measurements computed for probe sets annotated explicitly to the same gene were averaged using robust Tukey biweight function. Changes in gene expression were examined separately for each preprocessing algorithm using Welsh t test. Next, to obtain a robust estimator of P values, five values of t test computed for each gene were averaged with Tukey biweight function, and the mean values were used to obtain P values. Finally, we computed q values for all analyzed genes. That allowed us to select a set of differentially expressed genes in which false discovery rate was at 5% level. Most of preprocessing and all statistical computations were done with the R programming environment and Bioconductor packages.25Gentleman RC Carey VJ Bates DM Bolstad B Dettling M Dudoit S Ellis B Gautier L Ge Y Gentry J Hornik K Hothorn T Huber W Iacus S Irizarry R Leisch F Li C Maechler M Rossini AJ Sawitzki G Smith C Smyth G Tierney L Yang JY Zhang J Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar Only the PDNN expression measure was computed with the original PerfectMatch software.24Zhang L Miles MF Aldape KD A model of molecular interactions on short oligonucleotide microarrays.Nature Biotechnol. 2003; 21: 818-821Crossref Scopus (268) Google Scholar Total RNA (1 μg) was used to synthesize cDNA by extension of oligo(dT)15 primers (2.5 mmol/L) with 200 units of M-MLV reverse transcriptase (Sigma-Aldrich, Munich, Germany). Real-time PCR amplifications were performed in duplicate on cDNA equivalent to 25 ng RNA in 20-μl reaction volume containing 1xSYBR GREEN PCR Master Mix (Part. No. 4309155, Applied Biosystems, Darmstadt, Germany) and the primer sets QuantiTect Primer Assays (200; Qiagen, Hilden, Germany): Hs_ANXA1_1_SG, Hs_CNDP1_ 1_SG, Hs_GPNMB_1_SG, Hs_KIAA1189_1_SG, Hs_LTF_ 2_SG, Hs_MBP_1_SG, Hs_NEUROD1_1_SG, Hs_NPTX1_ 1_SG, Hs_RND3_1_SG, Hs_S100A11_1_SG, and Hs_SFRP4_2_SG. 18SrRNA was used as an internal standard reference. 18SrRNA primers were designed with the Primer Express Software (Applied Biosystems, Foster City, CA): forward 5′-CGGACATCTAAGGGCATCAC-3′ and reverse 5′-AACGAACGAGACTCTGGCAT-3′. The thermal cycling conditions were as follows: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C for denaturation, and 1 minute at 60°C for annealing and extension. The relative quantification of gene expression was determined with ABI PRISM 7700 Sequence Detection System using the comparative CT method. Immunostaining was performed on 5-μm sections of paraffin-embedded brain tissues of five SEGAs and five control brains obtained from the Department of Pathology, The Children's Memorial Health Institute, Warsaw, Poland. The sections were deparaffinized in xylene, hydrated with a descending ethanol series, and rinsed with deionized water. For each washing 0.05 mol/L Tris-buffered saline, pH 7.4 was used. To retrieve the antigen, sections were boiled for 10 minutes in citrate buffer, pH 6.0. Endogenous peroxidase activity was blocked for 30 minutes with 0.3% hydrogen peroxide. Sections were then incubated overnight at 4°C with the following antibodies: anti-human-Annexin A1 (Abcam, Cambridge, UK, diluted 1:300), anti-human-GPNMB (Abcam, Cambridge, UK, diluted 1:300), and anti-human-S100A11 (ProteinTech Group, Manchester, UK, diluted 1:50) in 1% Swine Serum (Dako, Hamburg, Germany) in Tris buffered saline. Sections were incubated sequentially with biotinylated secondary anti-rabbit or anti-mouse antibodies and extravidin- or streptavidin-peroxidase conjugate (Sigma-Aldrich, Munich, Germany). Peroxidase activity was revealed by 3,3′-diaminobenzidine (DAB, 10 minutes), and then counterstaining was performed with hematoxylin (Sigma-Aldrich, Munich, Germany). Finally, sections were dehydrated through ethanols, cleared in xylene, and mounted. Sections without primary antibodies were used as negative controls. Freshly resected SEGA samples from three patients were collected, cut into small fragments, and trypsinized for 20 minutes in 37°C. Afterward, cell suspension was centrifuged and suspended in Dulbecco Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen, Karlsruhe, Germany) and antibiotics (50 U/ml penicillin, 50 μg/ml streptomycin; Sigma-Aldrich, Munich, Germany). Cells were maintained for about 2 weeks before they were used in experiments. Normal Human Astrocytes (Clonetics® NHA) were cultured in Astrocyte Medium (Lonza, Walkersville, MD). SEGA and NHA were treated with rapamycin (LC Laboratories, Woburn, MA), U0126 (Cell Signaling Technology, Danvers, MA), or dimethyl sulfoxide (DMSO; Sigma-Aldrich, Munich, Germany), a solvent for both drugs. During the treatment cells were grown in DMEM with 2% FBS. Whole-cell protein extracts from SEGA and NHA cells were prepared as described,26Ciechomska I Pyrzynska B Kazmierczak P Kaminska B Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells.Oncogene. 2003; 22: 7617-7627Crossref PubMed Scopus (74) Google Scholar loaded onto 10% polyacrylamide gel, electrophorezed, and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Piscataway, NJ). Membranes were incubated overnight at 4°C with primary antibodies: anti–phospho-p70 S6 Kinase (T389), anti-p70 S6 Kinase (clone 49D7), anti–phospho-ERK1/2 (T202/Y204), and anti-ERK1/2 (1:1000, Cell Signaling Technology, Danvers, MA). After blocking in 5% low-fat milk in TBS-T (0.1% Tween 20/Tris-buffered saline, pH 7.6), the membranes were incubated overnight with primary antibodies diluted in a blocking buffer and then, for one hour, with a secondary anti-rabbit antibody linked to horseradish peroxidase (1:2000, Cell Signaling Technology, Danvers, MA). Membranes were reprobed with a monoclonal anti–β-Actin–Peroxidase conjugated antibody (1:50000, Sigma-Aldrich, Munich, Germany) that served as a loading control. Immunocomplexes were visualized by the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Piscataway, NJ). Cells were cultured in 24-well plates with the addition of rapamycin, U0126, or DMSO as a control. After treatment, MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, Munich, Germany) was added to each well to a final concentration of 0.5 mg/ml. After 3 hours, formazan crystals that formed from MTT in actively metabolizing cells were dissolved in lysis buffer containing 20% SDS and 50% DMF. Optical densities were measured at 570 nm using a scanning multiwell spectrophotometer (Thermo labsystem Multiscan EX). Cells were cultured in 24-well plates with the addition of rapamycin, U0126, or DMSO as a control. Cell Proliferation ELISA BrdU assay (Roche Diagnostics GmbH, Mannheim, Germany) was used to estimate cell proliferation rate. Briefly, BrdU labeling solution was added to each well 6 hours before fixing and incubating with a mouse monoclonal anti-BrdU antibody conjugated with peroxidase. Next, 1 mol/L H2SO4 was added and optical densities were measured at 450 nm using a scanning multiwell spectrophotometer (Thermo labsystem Multiscan EX). Cells were cultured on glass slides with the addition of rapamycin, U0126, or DMSO as a control. Medium with the inhibitors was changed every second day. After 6 days cells were fixed with 4% paraformaldehyde, stained with Phalloidin-TRITC (0.1 μg/ml, Sigma-Aldrich, Munich, Germany) for 1 hour, and mounted. Cells were viewed with a Leica SP5 laser confocal microscope (Leica, Mannheim, Germany) using the 561 nm line of the DPSS laser and a 20×, 0.7 an PlanApo oil-immersion objective. Optical sections were collected at 0.16-μm increments, and 2D projections were made with the LAS AF software. Image stacks were processed using the 3D Constructor plug-in to Image-Pro Plus 6.3 software. Individual cells were analyzed for cell volume and surface area. Cells were cultured in 6-well plates and pre-incubated with rapamycin, U0126, or DMSO as a control for 3 hours before wounding the cells. Next, a scratch was created in the center of the cell monolayer using a pipette tip. Immediately thereafter, cells were washed with PBS to remove cellular debris and incubated with the inhibitors for additional 27 hours. The ability of cells to migrate into the scratch area was measured by counting of DAPI-stained nuclei along the scratch at 0 time point and after 30 hours of cell exposure to the inhibitors. Gene expression profiling was performed on four SEGA samples from patients with TSC (S1-4), one control brain tissue (C1), and two commercial control brain RNA (C2 [from two donors] and C3 [from 23 donors]) using Affymetrix HG-U133 Plus 2.0 array sets. Figure 1 shows the results of microarray analysis obtained for probe sets called Present after the filtration as described in Materials and Methods. We first identified genes significantly altered in SEGAs versus control brain using false discovery rate of <5%, then we selected 50 differentially expressed genes with the biggest difference in expression level between SEGAs and control brain samples, and the highest similarity within these groups using the MultiExperiment Viewer 4.0 software (Figure 1A). A list of genes differentially expressed in SEGAs is shown in Table 1.Table 1Summary of Genes with Highest Up- or Down-Regulation Scores in SEGAs Compared with Control BrainsUnigeneGene symbolGene nameFold change over controlsTumorigenesis Hs.658169SFRP4secreted frizzled-related protein 42.514 Hs.610567LTFlactotransferrin2.197 Hs.190495GPNMBglycoprotein (transmembrane) nmb2.139 Hs.494173ANXA1annexin A11.791 Hs.489142COL1A2collagen, type I, alpha 21.757 Hs.6838RND3Rho family GTPase 31.690 Hs.148641CTSHcathepsin H1.451 Hs.593414S100A11s100 calcium binding protein a111.390 Hs.702229APODapolipoprotein D0.686 Hs.525205NDRG2NDRG family member 20.668 Hs.444212VSNL1visinin-like 10.632 Hs.287518SEPT4septin 40.580 Hs.591255FAT2FAT tumor suppressor homolog 2 (Drosophila)0.423 Hs.655499ST18suppression of tumorigenicity 180.386Nervous system development Hs.551713MBPmyelin basic protein0.757 Hs.75061MARCKSL1MARCKS-like 10.682 Hs.167317SNAP25synaptosomal-associated protein, 25kDa0.660 Hs.149035SHANK3SH3 and multiple ankyrin repeat domains 30.632 Hs.270055SH3GL3SH3-domain GRB2-like 30.608 Hs.478153SERPINI1neuroserpin0.607 Hs.518267TFtransferrin0.573 Hs.702002NPTX1neuronal pentraxin I0.540 Hs.80395MALmal, T-cell differentiation protein0.525 Hs.708214CADPS2Ca2+-dependent activator protein for secretion 20.521 Hs.79361KLK6kallikrein-60.514 Hs.175934GABRA1gamma-aminobutyric acid (GABA) A receptor, alpha 10.506 Hs.647962ZIC1Zic family member 1 (odd-paired homolog, Drosophila)0.506 Hs.655654RELNreelin0.504 Hs.121333MOBPmyelin-associated oligodendrocyte basic protein0.463 Hs.653700ZIC2Zic family member 2 (odd-paired homolog, Drosophila)0.451 Hs.400613CNDP1carnosine dipeptidase 1 (metallopeptidase M20 family)0.444 Hs.443894ERMNermin, ERM-like protein0.431 Hs.591255FAT2FAT tumor suppressor homolog 2 (Drosophila)0.423 Hs.295449PVALBparvalbumin0.412 Hs.458423CBLN1cerebellin 1 precursor0.388 Hs.709709NEUROD1neurogenic differentiation 10.342 Hs.90791GABRA6gamma-aminobutyric acid (GABA) A receptor, alpha 60.311Others and unknown Hs.334629SLNsarcolipin2.021 Hs.643005SLC40A1solute carrier family 40, member 11.719 Hs.628678VIMvimentin1.314 Hs.155247ALDOCaldolase C, fructose-bisphosphate0.708 Hs.40808TMEM178transmembrane protein 1780.634 Hs.528335FAM123Afamily with sequence similarity 123A0.555 Hs.592182AMPHamphiphysin0.538 Hs.208544KCNK1potassium channel, subfamily K, member 10.519 Hs.201083MAL2mal, T-cell differentiation protein 20.473 Hs.207603CBLN3cerebellin 3 precursor0.463 Hs.106576AGXT2L1alanine-glyoxylate aminotransferase 2-like 10.422 Hs.591707FSTL5fstl50.408 Hs.159523CRTAMcytotoxic and regulatory T cell molecule0.407 Hs.511757GJB6gap junction beta-6 protein0.381Genes used for further studies are printed in bold type. Open table in a new tab Genes used for further studies are printed in bold type. The 50 selected genes (11 up-regulated and 39 down-regulated in SEGAs) were categorized into functional groups using the Ingenuity Pathway Analysis software. Based on their known biological functions, the genes were mainly involved in tumorigenesis (14 genes) and the nervous system development (23 genes). Interestingly, all genes related to the nervous system development were down-regulated in SEGAs when compared with control brain samples (Table 1). Of the 50 genes, we chose 11 differentially expressed genes for further studies by a systematic PubMed search for genes with potential relevance to TSC biology and pathology. The expression pattern of these genes: six up- and five down-regulated in SEGAs showed evident differences between SEGAs and control brain samples (Figure 1, B and C). The selected genes up-regulated in SEGA: ANXA1, GPNMB, LTF, RND3, S100A11, and SFRP4 are involved in tumorigenesis, and genes down-regulated in SEGA: CNDP1, ERMIN, MBP, NEUROD1, and NPTX1 are potentially related to the nervous system development. Among them ANXA1, GPNMB, and NPTX1 were previously shown to be differentially expressed in Tsc2 null murine neuroepithelial progenitor cells compared with wild-type neuroepithelial progenitor cells.20Onda H Crino PB Zhang H Murphey RD Rastelli L Gould Rothberg BE Kwiatkowski DJ Tsc2 null murine neuroepithelial cells are a model for human tuber giant cells, and show activation of an mTOR pathway.Mol Cell Neurosci. 2002; 21: 561-574Crossref PubMed Scopus (108) Google Scholar To verify the results of microarray analysis, we performed real-time PCR and immunohistochemical validation, wherever antibody was available, mostly on an independent set of tumor samples. We selected 11 differentially expressed genes for a quantitative validation using real-time PCR. Analysis was performed on 10 SEGAs and 5 control brain samples and included tumor samples used for microarray analysis and additional samples from different individuals: six SEGAs and two control brain samples. As shown in Figure 2, real-time PCR confirmed the findings of the microarray experiment, with ANXA1, GPNMB, LTF, RND3, S100A11, and SFRP4 mRNAs up-regulated in SEGAs and CNDP1, ERMIN, MBP, NEUROD1, and NPTX1 mRNAs down-regulated in SEGAs. Differences in the levels of expression between SEGAs and control brains were significant for all tested genes (P < 0.05, Figure 2, A and B) and paralleled those observed in the microarray study. We evaluated ANXA1, GPNMB, and S100A11 expression immunohistochemically on a panel of SEGA tissue sections with commercially available antibodies. A staining was performed on paraffin-embedded SEGAs from five TSC patients (distinct from those used in the microarray and real-time PCR experiments) and five non-tumoral brain tissue samples. Representative micrographs in Figure 3 show that all tested SEGA tissues were immunoreactive for ANXA1, GPNMB, and S100A11, and the levels of studied proteins were higher in SEGA relative to the non-tumoral brain. Cell membrane and cytoplasmic ANXA1 and
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