Genome-Wide Analysis of Epigenetic Silencing Identifies BEX1 and BEX2 As Candidate Tumor Suppressor Genes in Malignant Glioma

Research Article

Genome-Wide Analysis of Epigenetic Silencing Identifies BEX1 and BEX2 as Candidate Tumor Suppressor Genes in Malignant Glioma

Greg Foltz,1,3 Gi-Yung Ryu,1 Jae-Geun Yoon,1 Timothy Nelson,1 Jessica Fahey,1 Amanda Frakes,1 Hwahyung Lee,1 Lorie Field,1 Kaitlin Zander,1 Zita Sibenaller,1 Timothy C. Ryken,1 Rajeev Vibhakar,2 Leroy Hood,3 and Anup Madan1,3

1Neurogenomic Research Laboratory, Department of Neurosurgery; 2Department of Pediatrics, University of Iowa Carver College of Medicine, Iowa City, Iowa; and 3Institute for Systems Biology, Seattle, Washington

Abstract common underlying epigenetic mechanisms, such as promoter hypermethylation, histone deacetylation, histone methylation, Promoter hypermethylation and histone deacetylation are and other histone modifications, which directly or indirectly common epigenetic mechanisms implicated in the transcrip- alter chromatin structure (3–5). When combined with microarray tional silencing of tumor suppressor genes in human cancer. analysis, the pharmacologic reversal of one or more of these We treated two immortalized glioma cell lines, T98 and U87, common underlying epigenetic mechanisms provides a powerful and 10patient-derived primary glioma cell lines with screening tool for the comprehensive identification of epigenet- trichostatin A (TSA), a histone deacetylase inhibitor, or 5- ically silenced genes on a global scale. Several recent studies aza-2¶-deoxycytidine (5-AzaC), a DNA methyltransferase in- have validated this large-scale approach using DNA methyltrans- hibitor, to comprehensively identify the cohort of genes ferase and histone deacetylase (HDAC) inhibitors, either alone or reactivated through the pharmacologic reversal of these in combination, in a variety of human cancers, including distinct but related epigenetic processes. Whole-genome pancreatic,colon,prostate,liver,endometrial,blood,and microarray analysis identified genes induced by TSA (653) or esophageal cancers (6–12). 5-AzaC treatment (170). We selected a subset of reactivated The scope of epigenetic gene silencing in malignant glioma has genes that were markedly induced (greater than two-fold) not been well defined. Malignant glioma, the most aggressive and after treatment with either TSA or 5-AzaC in a majority of common brain tumor in adults, continues to cause f13,000 deaths glioma cell lines but not in cultured normal astrocytes. We yearly in the United States despite intensive clinical and basic then characterized the degree of promoter methylation and science research. In a recent major advance, promoter hyper- transcriptional silencing of selected genes in histologically methylation and epigenetic silencing of the DNA repair gene confirmed human tumor and nontumor brain specimens. We O6-methylguanine-DNA methyltransferase were shown to identify a identified two novel brain expressed genes, BEX1 and BEX2, subset of patients with markedly improved survival at 2 years in which were silenced in all tumor specimens and exhibited response to combined treatment with chemotherapy and radiation extensive promoter hypermethylation. Viral-mediated reex- therapy (13–15). Several other genes critical for cell proliferation, pression of either BEX1 or BEX2 led to increased sensitivity to tumor progression, apoptosis, angiogenesis, and astrocyte motility chemotherapy-induced apoptosis and potent tumor suppres- in vitro have also been shown to be silenced in association with promoter sor effects and in a xenograft mouse model. Using an hypermethylation in malignant glioma (16–18). Despite these integrated approach, we have established a novel platform for important findings, the genome-wide role of epigenetic silencing the genome-wide screening of epigenetically silenced genes in and the functional significance of individually silenced genes have malignant glioma. This experimental paradigm provides a not been extensively studied. powerful new method for the identification of epigenetically Abundant evidence supports a synergistic link between pro- silenced genes with potential function as tumor suppressors, moter hypermethylation and histone deacetylation in the active biomarkers for disease diagnosis and detection, and thera- suppression of gene transcription (19). Importantly, several recent peutically reversible modulators of critical regulatory path- reports suggest that pharmacologic inhibition of histone deacety- ways important in glioma pathogenesis. (Cancer Res 2006; lation alone markedly reverses transcriptional silencing, even in the 66(13): 6665-74) absence of promoter hypermethylation. This has led to the hypothesis that histone deacetylation is the primary mechanism Introduction that actively modulates epigenetic transcriptional silencing. HDAC Epigenetic silencing of tumor suppressor genes is a common inhibitors have subsequently emerged as a powerful new class of motif in human cancer (1, 2). Tumor-associated transcriptional chemotherapeutic agents with promising antitumor effects. They silencing is most frequently described in association with a few are potent inducers of growth arrest, differentiation, and apoptotic cell death in a variety of malignant cells in vitro and in vivo. Recent human clinical trials have shown that HDACinhibitors are well tolerated, inhibit HDACactivity in tumor cells, and lead to Note: Supplementary data for this article are available at Cancer Research Online objective tumor regression. In immortalized glioma cell lines, (http://cancerres.aacrjournals.org/). Requests for reprints: Anup Madan, Neurogenomics Research Laboratory, HDACinhibition results in cell cycle arrest and increased apoptosis Department of Neurosurgery, University of Iowa Carver College of Medicine, 200 B (17, 20, 21). To date, few reports exist describing the effect of Eckstein Medical Research Building, Iowa City, IO 52242. Phone: 319-3358491; Fax: 319- HDACinhibition on gene expression in malignant glioma cell lines 3358078; E-mail: [email protected]. I2006 American Association for Cancer Research. (18, 22–24). Many HDACinhibitors are small molecules with doi:10.1158/0008-5472.CAN-05-4453 pharmacologic properties conducive to effective delivery across the www.aacrjournals.org 6665 Cancer Res 2006; 66: (13). July 1, 2006

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research blood brain barrier, a significant therapeutic advantage over hours. During the last 4 hours of incubation, the medium was replaced with traditional chemotherapy agents. fresh medium containing MTT at 1 mg/mL. The MTT reaction was stopped A The goal of the present study is to comprehensively identify and after 4 hours by replacing the medium with 100 L isopropanol, and the confirm epigenetically silenced genes in patient samples of cells were incubated for 1 hour at room temperature in the dark. The conversion of soluble MTT to an insoluble blue formazan product was malignant glioma. We used a whole-genome microarray, rigorous measured with the Thermomax microplate reader (Molecular Devices, statistical analysis, and an experimental paradigm optimized to Sunnyvale, CA) at 570 nm. Each treatment was done on three independent identify epigenetically silenced genes with tumor suppressor cultures of each cell line. Within a given experiment, each treatment function in primary glioma cultures. We then confirmed transcrip- condition was evaluated in 16 wells of the 96-well plate. Percentage change tional silencing of these genes in a panel of human tumors with (mean and SD) is reported for each treatment relative to DMSO control. subsequent analysis of individual gene promoter regions for DNA Statistical significance was determined from the mean and SE across methylation and histone modifications. We characterized the replicate measurements in three independent experiments. 6 functional consequence of reexpression of two novel epigenetically For cell cycle assays, unsynchronized cells were seeded at 1 Â 10 per 100- A silenced genes, which exhibit proapoptotic tumor suppressor mm dish and exposed to 0.2 to 5 mol/L TSA for 24 hours. After fixing with A activity in an in vitro and ex vivo tumor model. This experimental 70% ethanol, the nuclei were stained with 50 g/mL propidium iodide and 10 Ag/mL RNase A. The relative DNA content was measured using a FACScan paradigm provides a powerful new approach to the genome-wide flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Each experiment identification of epigenetically silenced tumor suppressor genes in was repeated thrice, and data are reported as the percentage of cells (mean malignant glioma. and SD) in each phase of the cell cycle. In the cell death commitment assay, T98 cells were initially treated with 1 Amol/L TSA for various times (2-36 hours), then washed to remove any TSA-containing medium, and Materials and Methods subsequently cultured in the absence of TSA for a total of 72 hours. Tissue samples and cell lines. Tumor and nontumor brain specimens Microarray and real-time PCR analysis. For microarray experiments, were obtained from the Central Nervous System Tissue Bank at the T98, U87, patient-derived primary glioma cell lines, and normal human University of Iowa Hospitals and Clinics (Iowa City, Iowa). All patients gave astrocytes were cultured with either 1 Amol/L TSA or DMSO for 24 hours. informed consent before the collection of specimens according to T98 cells were also treated for a period of 6, 12, 36, and 48 hours to generate institutional guidelines. Primary cultures from freshly resected brain tumor a time course of response after drug treatment. To generate gene expression specimens were established per standard protocols (25). Each tumor profiles in response to 5-AzaCtreatment, cell lines were cultured with specimen was histologically verified by a board-certified neuropathologist 5 Amol/L 5-AzaCfor 72 hours. Total RNA was extracted from treated cells and archived for further DNA, RNA, and protein studies. To generate short- using Trizol (Invitrogen, Carlsbad, CA) with an additional purification step term primary cell cultures, tumor tissue (f200-250 mg) was immersed and using RNeasy (Qiagen, Valencia, CA) before quality assessment with the incubated in 0.05 mmol/L EDTA solution containing 0.05% trypsin (Sigma, Agilent Bioanalyzer (Palo Alto, CA). Total RNA (2 Ag) was reverse St. Louis, MO) at 4jCfor 8 hours. The tissue samples were minced into transcribed with the Chemiluminescent RT-IVT Labeling kit (Applied 0.3-mm3 fragments and suspended in HBSS containing 4 mg DNaseI, 40 mg Biosystems, Foster City, CA) and hybridized to a 60-mer whole-genome collagenase IV, and 100 units hyaluronidase type V (all from Sigma). Single- oligonucleotide microarray (Applied Biosystems) containing 33,202 probes cell suspensions were then passed through no. 100 nylon mesh, washed twice representing 29,098 genes. A total of three microarray hybridizations, one in HBSS, and added to fibronectin-coated tissue culture flasks. Cells were for each biological replicate, were done per treatment and time point for maintained in medium consisting of DMEM/F12, 15% fetal bovine serum, T98 cells. Data were quantile normalized, and a t test was applied to each 10 Ag/mL insulin, 7.5 ng/mL fibroblast growth factor (FGF), 1 mmol/L gene for statistical significance. Correction for multiple testing was done pyruvate, 15 mmol/L HEPES buffer (pH 7.2), and 100 units/100 Ag/mL using Storey’s q-value method (27). Epigenetically regulated genes were penicillin-streptomycin. The immortalized T98 and U87 glioblastoma cell identified using Spotfire software (version 8.2) for data visualization with a lines were obtained from American Type Culture Collection (Manassas, VA) threshold cutoff set at two-fold and a false discovery rate of 5%. For the and maintained in a similar fashion. Normal human astrocytes were main- 10 patient-derived primary cell lines, one microarray hybridization per tained in ABG Bullet kit medium (both from Cambrex, Walkersville, MD). patient per treatment condition was done (a total of 20 microarrays) due to Trichostatin A and 5-aza-2¶-deoxycytidine treatment of cells. Cell limited tissue samples. For each individual cell line, data were initially lines were treated with varying doses of trichostatin A (TSA; Sigma) ranging normalized, and differential gene expression was tested for statistical from 0.2 to 10 Amol/L or a control volume of DMSO (final DMSO significance using the ABI single array error model (Applied Biosystems, concentration not V0.1% v/v) for various times (15 minutes-48 hours). For reference manual). For more rigorous statistical analysis, each of these 5-aza-2¶-deoxycytidine (5-AzaC) treatment, cell lines were treated with 10 different glioma cell lines was treated as a biological replicate of the 5 Amol/L 5-AzaC(Sigma) and dissolved in PBS containing 1 Amol/L acetic same experimental condition. Allowing for some variation between acid or PBS control for 72 hours. individual patient tumors, we set a threshold at 70% (i.e., differential gene Western blot analysis. Protein extracts were prepared from T98 and expression that was consistent in at least 7 of the 10 glioma cell lines) before three primary (UI269, UI274, and UI276) glioblastoma cell lines, which had passing the gene on for significance testing as outlined above. been treated for various times ranging from 15 minutes to 48 hours with Assay-on-Demand gene expression reagents (Applied Biosystems) for 100 1 Amol/L TSA. Western blot analysis was done as described previously (26) selected genes was used to validate microarray results and confirm gene using primary antibodies directed to acetylated histone H3 (Upstate silencing in histologically confirmed glioblastoma specimens relative to Biotechnology, Lake Placid, NY) and glyceraldehyde-3-phosphate dehydro- nontumor brain specimens. Real-time PCR was done using the ABI PRISM genase (Santa Cruz Biotechnology, Santa Cruz, CA) as a control. Secondary 7900 HT Sequence Detection System (Applied Biosystems) under default horseradish peroxidase–conjugated anti-rabbit antibodies (Santa Cruz conditions: 95jCfor 10 minutes, 40 cycles of 95 jCfor 15 seconds, and 60 jC Biotechnology) were used at a dilution of 1:5,000. for 1 minute with human glutathione synthetase as control. Confirmed Proliferation and cell cycle assays. Treated cell lines and mock-treated genes were parsed through the Panther pathway analysis program (http:// controls were assessed for growth inhibition using the 3-(4,5-dimethylth- panther.appliedbiosystems.com/pathway/) to identify epigenetically regu- iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immortalized lated signaling pathways. (T98 and U87) and two primary glioblastoma (UI218 and UI260) cells were Bisulfite sequencing analysis. Genomic DNA was extracted from plated in 96-well culture plates at a density of 5 Â 103 per well, allowed to glioma tissue samples (25 mg) and cell lines (1 Â 107) using Wizard attach overnight, and treated with either DMSO control or TSA Genomic DNA Purification kits (Promega, Madison, WI). Sodium bisulfite (concentrations ranging from 0.2 to 10 Amol/L) for 24, 48, 72, and 96 modification of 4 Ag genomic DNA was carried out using the MethylEasy

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DNA bisulfite modification kit (Human Genetic Signatures, Sydney, R-cgacggcggttctgacgccacaacg was used for amplification of the BEX2 Australia). CpG islands upstream of the start site were identified using promoter (À108 to +62 relative to transcriptional start site). The PCR the CpG prediction program, NEWCPGREPORT, and PCR primers for products were visualized by 1.8% agarose gel electrophoresis and nested PCR were designed to yield 400 to 500 bp products. The PCR mixture quantitated by densitometry. The assays were done in triplicate. contained 20 pM of each primer, 40 ng bisulfite-treated DNA (or 2 AL of first Construction of expression vectors. Full-length open reading frames round product), 1.25 units Taq DNA Polymerase (Eppendorf, Pittsburgh, for BEX1 and BEX2 were PCR amplified from MGC clones and cloned into PA), 0.2 mmol/L deoxynucleotide triphosphate (dNTP; each), and 2 mmol/L the pcDNA3.1D/V5-His-TOPO vector (Invitrogen), and sequence was MgSO4 in a final volume of 50 AL. The PCR was done with the following verified. The PCR products were cloned into pacAD5CMVIRESeGFPpA, cycling variables: an activation step of 94jCfor 3 minutes followed by 50 and sequence was verified to generate adenoviral vectors. These clones were cycles of 94jCfor 2 minutes, 50 jCfor 2 minutes, and 68 jCfor 3 minutes, recombined in HEK293 cells with pacAD5 9.2-100 to produce recombinant with a final extension step of 68jCfor 10 minutes. For BEX1, a CpG-rich adenovirus particles. region (À174 to +105 relative to transcriptional start site) was amplified Transfection and colony formation assays. Colony formation assays using primer pairs F-gggaaaggaagggtatagattttttt and R-crataaacctctactaacc- were done in monolayer culture. Cells were plated at 1.5 Â 105 per well taaccaaaa (first round) and F-gygatgatgttatttgtgggttttt and R-aacrcaaaacaa- using six-well plates and transfected with either pcDNA3.1D/V5-His-TOPO/ craaaaactaataacraa (second round). For BEX2, a CpG-rich region (+157 to BEX1, pcDNA3.1D/V5-His-TOPO/BEX2, pcDNA3.1D/V5-His-TOPO/lacZ,or +408 relative to transcriptional start site) was amplified using primer pairs pcDNA3.1D/V5-His-TOPO with no insert (mock control) using Trans It- F-ggtagtggtatttgttttyggtgtttt and R-ctaaccaccattttctacatcaaaaaaaa ( first Neural transfection reagents (Mirus, Madison, WI). The cells were selected round) and F-ggtaagggattyggagggggtttt and R-aaacaaaaatattaaaaaaacttacrc- taata (second round). PCR products were cloned using the TOPO-TA cloning kit (Invitrogen). The transformed bacterial colonies (16 from each sample) were inoculated in 96-well plates in LB medium with appropriate antibiotic. Plasmid DNA was prepared using Sprint Prep (Agencourt Biosciences, Beverly, MA) on a Biomek robot. The DNA templates were sequenced using BIG Dye terminators (version 3.1). Sequences were resolved on a PE Biosystems 3730-XL capillary sequencer (Applied Biosystems), and data were assembled using Phred-Phrap and edited using Consed (28–30). In-house software written in Perl was used to calculate and visualize the fractional methylation at each CpG site. In brief, cytosine residues at non-CpG sites are converted to uracils by bisulfite treatment, whereas those at CpG sites remain as cytosines (if methylated) or are converted to uracil (if unmethylated). The fractional methylation was determined by calculating the percentage of methylated cytosines at each CpG site. The total methylation for each sample, represented as the methylation index, was determined by averaging the fractional methylation of CpG sites over the entire PCR product. Chromatin immunoprecipitation assays. T98 cells (1 Â 106) treated with DMSO or 1 Amol/L TSA were incubated with 1% formaldehyde for 10 minutes, washed with cold PBS, resuspended in lysis buffer (Upstate Biotechnology), and sonicated for 10 seconds with continuous output using a Branson sonifier (Branson Ultrasonics Corporation, Danbury, CT). The lysate was centrifuged for 10 minutes at 13,200 rpm at 4jC, after which the supernatant was incubated with protein A-agarose beads (Upstate Biotechnology) for 2 hours. The slurry was removed by centrifugation at 1,000 rpm for 1 minute, and the supernatant was divided into four parts. The first part was used as input control, and the other three parts were incubated with either anti-K9-acetylated H3 (Upstate Biotechnology), normal rabbit IgG (Santa Cruz Biotechnology, Lake Placid, NY), or no antibody (negative control) at 4jCovernight. The immunoprecipitated complexes were collected by incubation with protein A-Sepharose beads (Upstate Biotechnology) for 1 hour at 4jC. After washing the beads with buffers (low salt, high salt, LiCl, and TE; Upstate Biotechnology), the cross links were reversed by heating the samples at 65jCfor 4 hours with NaCl. The samples were then treated with proteinase K overnight, and DNA was extracted by the phenol chloroform method, ethanol precipitated, and resuspended in 50 AL water. The PCR was designed to yield 250 bp Figure 1. Whole-genome microarray analysis of genes activated in response to products. To ensure that PCR amplification was in linear range, each HDAC inhibition. A, comparison of genes up-regulated greater than two-fold reaction was set up at different dilutions of DNA for varying amplification (false discovery rate of 5%)in the immortalized glioma cell lines U87 and T98 and in 70% of the primary glioma cell lines after HDAC inhibition with TSA. Both cycle numbers, and final PCR conditions were selected accordingly. immortalized and primary glioma cell lines were treated with 1 Amol/L TSA for The PCR mixture contained 20 pM of each primer, 1 AL extracted DNA, 24 hours. Total RNA was extracted, purified, and subjected to whole-genome 0.5 units Taq DNA Polymerase (Eppendorf), 0.2 mmol/L dNTPs (each), and microarray analysis using the ABI 1700 microarray platform. A total of 2,736 genes were found to be up-regulated in 70% of the primary glioma cell lines, 2 mmol/L MgSO4 in a final volume of 50 AL. The PCR was done with the j whereas 3,441 genes were found to be up-regulated in T98 glioma cell lines following cycling variables: an activation step of 94 Cfor 3 minutes and 2,809 genes were up-regulated in U87 cell line by TSA. B, relative followed by 30 cycles of 94jCfor 2 minutes, 50 jCfor 2 minutes, and 68 jC transcriptional silencing of 10 genes in histologically confirmed GBM tissues for 3 minutes, with a final extension step of 68jCfor 10 minutes. The (n = 10)compared with normal brain tissues ( n =8;P < 0.001). Data from promoter region of BEX1 (À284 to À81 relative to transcriptional start site) individual tissue. Total RNA was extracted from tissue samples, and relative expression of genes with respect to expression in a normal brain sample was was amplified using the primer pair F-gaagaggaaagaagaaaaggccaag and determined using real-time PCR. Y axis, arbitrary expression units used to R-cctcctcccgtctgtgcgcggtgcc. Primer pair F-ggatgttaaaagggactcccggtga and calculate fold change relative to expression in one nontumor brain sample. www.aacrjournals.org 6667 Cancer Res 2006; 66: (13). July 1, 2006

Table 1. TSA or 5-AzaC treatment induces widespread up-regulation of genes across several important regulatory pathways

Gene Activated by TSA Activated by 5-AzaC Panther pathway

ACAA2 + Nicotinic acetylcholine receptor signaling pathway ADRB2 + Heterotrimeric G-protein signaling pathway—Gia- and Gsa-mediated pathway ARNTL + Circadian clock system—brain and muscle arylhydrocarbon receptor nuclear translocator-like 1 BAI2 + P53 pathway BCL2 + Apoptosis signaling pathway, oxidative stress response BDKRB1 + Heterotrimeric G-protein signaling pathway—Gqa- and Goa-mediated pathway BIRC3 + + Apoptosis signaling pathway C12orf5 + Glycolysis CCL26 + + Inflammation mediated by chemokine and cytokine signaling pathway CDKN1A + IL signaling pathway, p53 pathway CITED2 + TGF-h signaling pathway CREB-H + Heterotrimeric G-protein signaling pathway—Gia- and Gsa-mediated pathway CXCR4 + Axon guidance mediated by Slit/Robo, inflammation mediated by chemokine and cytokine signaling pathway DEFCAP + FAS signaling pathway DKFZp434C0631 + Blood coagulation DKK1 + Wnt signaling pathway DUSP1 + Oxidative stress response DUSP5 + Oxidative stress response FGFR3 + Angiogenesis, FGF signaling pathway FKBP1B + TGF-h signaling pathway FOS + Angiogenesis, apoptosis signaling pathway, insulin/IGF pathway-MAPK kinase/MAPK cascade, IL signaling pathway, PDGF signaling pathway FOXO1A + PI3K pathway FZD4 + Alzheimer’s disease-presenilin pathway, angiogenesis, cadherin signaling pathway, Wnt signaling pathway GDF11 + TGF-h signaling pathway GFPT2 + O-antigen biosynthesis GNAO1 + Heterotrimeric G-protein signaling pathway—Gqa- and Goa-mediated pathway, metabotropic glutamate receptor group II pathway, muscarinic acetylcholine receptor 2 and 4 signaling pathway, D2/D3/D4 dopamine receptor–mediated signaling pathway, enkephalin release HDAC3 + Wnt signaling pathway, p53 pathway HTR4 + Heterotrimeric G-protein signaling pathway—Gia- and Gsa-mediated pathway IGF2 + Insulin/IGF pathway-MAPK kinase/MAPK cascade, insulin/IGF pathway-protein kinase B signaling cascade IL13RA2 + IL signaling pathway IL23A + IL signaling pathway INHBE + TGF-h signaling pathway ITGA6 + Inflammation mediated by chemokine and cytokine signaling pathway, integrin signaling pathway ITGB4 + Integrin signaling pathway JAG1 + Angiogenesis, Notch signaling pathway KCNQ2 + Muscarinic acetylcholine receptor 1 and 3 signaling pathway LOC90701 + Endothelin signaling pathway LTC4S + Inflammation mediated by chemokine and cytokine signaling pathway MAP2K6 + Angiogenesis, apoptosis signaling pathway, EGFR signaling pathway, FGF signaling pathway, Huntington’s disease, insulin/IGF pathway-MAPK kinase/MAPK cascade, oxidative stress response, Toll receptor signaling pathway, Ras pathway MAP3K5 + Apoptosis, EGFR signaling pathway, FGF signaling pathway, integrin signaling pathway MGC45474 + + Nicotinic acetylcholine receptor signaling pathway

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GeneTable 1. TSA or 5-AzaCActivated treatment by TSA induces Activated widespread by 5-AzaCPanther up-regulation pathway of genes across several important regulatory pathways (Cont’d)

Gene Activated by TSA Activated by 5-AzaC Panther pathway

MKNK1 + IL signaling pathway, PDGF signaling pathway MKNK2 + IL signaling pathway, PDGF signaling pathway NEDD4L + Ubiquitin proteasome pathway PAK6 + Cytoskeletal regulation by Rho GTPase, inflammation mediated by chemokine and cytokine signaling pathway PCDH10 + Cadherin signaling pathway, Wnt signaling pathway PDGFRL + PDGF signaling pathway—PDGF receptor B PIK3R3 + Angiogenesis, axon guidance mediated by netrin, hypoxia response via hypoxia-inducible factor activation, insulin/IGF pathway-protein kinase B signaling cascade, integrin signaling pathway, PDGF signaling pathway, PI3K pathway, T-cell activation, VEGF signaling pathway, p53 pathway, p53 pathway feedback loops PLA2G4A + Angiogenesis, endothelin signaling pathway, inflammation mediated by chemokine and cytokine signaling pathway, oxidative stress response, VEGF signaling pathway PLAT + + Blood coagulation, insulin/IGF pathway-MAPK kinase/MAPK cascade, plasminogen-activating cascade PLCD1 + a-adrenergic receptor signaling pathway, angiogenesis, axon guidance mediated by netrin, B-cell activation, EGFR signaling pathway, endothelin signaling pathway, FGF signaling pathway, heterotrimeric G-protein signaling pathway—Gqa and Go, inflammation mediated by chemokine and cytokine signaling pathway, metabotropic glutamate receptor group I pathway, muscarinic acetylcholine receptor 1 and 3 signaling pathway, PDGF signaling pathway, T-cell activation, VEGF signaling pathway, Wnt signaling pathway PLCL1 + a-adrenergic receptor signaling pathway, angiogenesis, axon guidance mediated by netrin, B-cell activation, EGFR signaling pathway, endothelin signaling pathway, FGF signaling pathway, heterotrimeric G-protein signaling pathway—Gq-a- and Goa-mediated pathway, inflammation mediated by chemokine, and cytokine signaling pathway, metabotropic glutamate receptor group I pathway, muscarinic acetylcholine receptor 1 and 3 signaling pathway, PDGF signaling pathway, T-cell activation, VEGF signaling pathway, Wnt signaling pathway PNUTL2 + Parkinson’s disease RaLP + Angiogenesis, EGFR signaling pathway, FGF signaling pathway, inflammation mediated by chemokine and cytokine signaling pathway, integrin signaling pathway, IL signaling pathway, PDGF signaling pathway, Ras pathway RGS10 + Heterotrimeric G-protein signaling pathway—Gia- and Gsa-mediated pathway, heterotrimeric G-protein signaling pathway—Gqa- and Goa-mediated pathway S100A2 + Inflammation mediated by chemokine and cytokine signaling pathway SERPINB2 + Blood coagulation, plasminogen-activating cascade SFRP1 + Angiogenesis SIRT4 + P53 pathway SLC1A3 + Ionotropic glutamate receptor pathway, metabotropic glutamate receptor group III pathway SNCB + Parkinson’s disease STAT4 + EGFR signaling pathway, inflammation mediated by chemokine and cytokine signaling pathway, IL signaling pathway, Janus-activated kinase/signal transducer and activator of transcription signaling pathway, PDGF signaling pathway STMN4 + Cytoskeletal regulation by Rho GTPase TLN2 + Integrin signaling pathway

NOTE: Genes that are regulated in both immortalized and primary glioma cell lines were categorized into different functional pathways using the 60 primary signaling Panther pathways for analysis (http://panther.appliedbiosystems.com/pathway/). Abbreviations: IL, interleukin; TGF-h, transforming growth factor-h; IGF, insulin-like growth factor; MAPK, mitogen-activated protein kinase; PDGF, platelet-derived growth factor; PI3K, phosphatidylinositol 3-kinase; EGFR, epidermal growth factor receptor.

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research in G418 (1 mg/mL) supplemented medium at 24 hours after transfection. followed the time course of histone acetylation in response to TSA Some cells were simultaneously harvested to confirm increased expression treatment (Supplementary Fig. S4). Real-time PCR was used to of the transfected gene by real-time PCR. G418-resistant cells were confirm microarray expression data (Supplementary Fig. S5). We maintained for 2 weeks in culture. Cells were detached, resuspended in then identified 653 genes, which were reexpressed in common in medium containing 0.3% agarose, and overlaid on 0.6% agarose. Medium both the immortalized cell lines, T98 and U87, and in a majority (0.5 mL) was added to the plates every 4 days, and colony formation was quantitated after fixation and staining with methylene blue after 3 weeks. (70%) of primary glioma cell lines (Fig. 1A). A similar comparison Terminal deoxynucleotidyl transferase–mediated dUTP nick end identified 170 genes up-regulated in both the immortalized and labeling. T98 cells infected with adenovirus expressing human BEX1 or 70% of the primary glioma cell lines after treatment with 5-AzaC, a BEX2 were treated with either camptothecin (1 Amol/L) and etoposide significantly smaller cohort compared with the effects of TSA. (3 Amol/L) or DMSO as a control for 40 hours. Terminal deoxynucleotidyl Taken together, these 823 genes were chosen for further study as transferase–mediated dUTP nick end labeling (TUNEL) reaction was carried the most likely to be epigenetically silenced in the in vivo tumor out with the APO-BrdU TUNEL assay kit (Molecular Probes, Raleigh, NC) and directly induced in response to TSA or 5-AzaC, while also being using Alexa Fluor 647–conjugated monoclonal antibodies and Hoechst silenced in immortalized cell lines, allowing for further functional 33342 for staining DNA. Cells were recorded with LSRII (Becton Dickinson). analysis. We then confirmed transcriptional silencing of 10 Xenograft mouse assay. All animal experiments were carried out candidate tumor suppressor genes (selection criteria described according to approved institutional guidelines following approval of the University of Iowa Animal Care and Use Committee. Four-week-old NCR below) in a panel of histologically confirmed human malignant (nu/nu) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) glioma specimens and nontumor brain specimens using real-time and maintained in the University of Iowa Animal Care Facility. U87 glioma cells PCR (Fig. 1B). Of note, after TSA or 5-AzaCtreatment, a large were infected with adenovirus vectors encoding either BEX1, BEX2, or GFP number of genes were found in common exclusively in the group of (control) ex vivo, and the cells were implanted into mice 24 hours after primary glioma cell lines, and these genes are of potentially great infection. Tumor cells (2 Â 106 per mouse) were injected s.c. as described interest and will be the subject of future studies. An even larger previously (31). Tumors were monitored twice weekly. At day 28 after injection, number of silenced genes were found exclusively in the immortal- animals were sacrificed, and tumors were measured in three dimensions. ized cell lines, most likely representing the effect of long-term cell Â Â Â Tumor volume was calculated using the formula d1 d2 d3 p/6 (32). culture. Selection criteria for candidate tumor suppressor genes. Results We developed multiple criteria for narrowing our focus to those TSA/5-AzaC-induced gene expression profiles. We designed epigenetically silenced genes with suspected tumor suppressor an experimental paradigm for microarray analysis optimized to function and promoter alterations, which could be used as a identify epigenetically silenced genes in malignant glioma. First, we potential biomarker. First, it was clear from our microarray determined the optimal dose and timing for gene expression results that TSA had a stronger genome-wide effect on gene studies by characterizing the time course and dose response of expression than 5-AzaC. As CpG methylation is associated with treatment with TSA in two immortalized cell lines (T98 and U87) HDAC-mediated transcriptional repression, we used bioinfor- and 10 primary glioma lines. TSA treatment resulted in marked matics methods to identify all genes up-regulated by TSA whose histone acetylation, growth arrest, cell cycle inhibition, caspase promoter regions contained CpG islands. To increase our yield activation, and cell death in T98 cells, U87 cells, and primary of TSA-regulated genes with promoter hypermethylation, we glioma cultures when compared with control-treated cells included all genes that were activated by both TSA and 5-AzaC. (Supplementary Figs. S1-S3). Ideally, a treatment regimen would Next, we categorized each gene into a functional pathway optimize the identification of epigenetically silenced genes that are (Table 1), excluding genes known to be associated with the IFN pharmacologically reversed by TSA and that also have tumor suppressor function. To achieve this, we chose a standard dosing regimen of 1 Amol/L TSA for 24 hours. At this dose and time point, cell viability is 100% at the time of sampling (24 hours) with >90% of the cells already committed to cell death at 72 hours. A similar paradigm was used to determine the optimal dose and timing of treatment with 5-AzaC, an inhibitor of DNA methyltransferase. A standard dosage of 5 Amol/L for 72 hours was selected for 5-AzaC based on a similar set of experiments (data not shown). Next, we designed an integrated microarray experiment (a total of 62 independent microarrays) to determine which subset of reexpressed genes most likely represent a primary effect of TSA treatment in primary glioma cell lines. Immortalized cell lines are known to have widespread epigenetic silencing but have the advantage of being well characterized and widely available for further study (33). We hypothesized that using early passage primary glioma cell lines from freshly resected surgical specimens represented the best approximation of the in vivo tumor while minimizing any significant epigenetic silencing due to ‘‘culture- Figure 2. Activation of epigenetically silenced BEX1 and BEX2 in response to effect’’. Using the ABI 1700 Human Genome Expression Microarray HDAC inhibition by TSA. Total RNA was extracted from 10 primary glioma cell (Applied Biosystems) and a maximally stringent threshold for lines treated with either DMSO or 1 Amol/L TSA. Real-time PCR was used to confirm up-regulation of BEX1 or BEX2 in response to TSA treatment using statistical significance (q < 0.001), a whole-genome analysis Assay-on-Demand gene expression reagents. Y axis, arbitrary expression units identified a subset of genes in T98 cells whose reexpression closely used to calculate fold change.

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Confirmation of epigenetic markers: promoter methylation and histone modification. We sought to further characterize the mechanism of transcriptional silencing in our candidate tumor suppressor genes through analysis of promoter methylation in a panel of histologically confirmed human tumor and nontumor brain specimens. First, we looked for the presence of CpG islands in the promoter region of all genes that were up-regulated greater than two-fold (false discovery rate of 5%) by 5-AzaCtreatment in both immortalized and primary glioma cell lines. Only 120 of 170 genes (72%) that are up-regulated by 5-AzaCcontain CpGislands in their promoter region. We then analyzed the methylation status of CpG islands in the promoter regions of 40 genes using bisulfite DNA sequencing. Interestingly, we found that only 25% of genes have significant methylation in their promoter region in tumor specimens compared with nontumor brain specimens. Consistent with previous studies, these results suggest that activation of a large fraction of these genes is possibly due to indirect effects, arising from the activation of other upstream regulatory genes. Analysis of CpG islands in the promoter region of BEX1 and BEX2 (Fig. 3) shows that both genes have differentially methylated CpG sites in tumor samples compared with normal brain samples (M:F ratio 1:1; Supplementary Table S1). The increased methylation of the promoter-associated CpG islands correlated with the decreased expression of BEX1 (P < 0.05) and BEX2 (P < 0.05) in the tumor tissues (Fig. 4). We characterized BEX1 and BEX2 promoter methylation in the T98 and U87 cell lines before and after treatment with TSA and 5-AzaC. BEX1 is densely methylated in both T98 and U87 cells, whereas BEX2 is only methylated in T98 cells. Neither TSA nor 5-AzaCtreatment has any effect on methylation patterns during the specified treatment interval (Supplementary Fig. S6). To further dissect the complex epigenetic alterations associated with the promoter region of BEX1 and BEX2, we investigated the acetylation of histone H3 at the lysine-9 position in U87 and T98 cells. Consistent with the TSA-induced Figure 3. Bisulfite sequence analysis of a CpG island in the promoter region of transcriptional activation, chromatin immunoprecipitation (ChIP) the BEX1 and BEX2 genes reveals dense methylation of CpG sites in tumor analysis showed a significant increase of acetyl histone H3 binding tissues compared with nontumor brain specimens. Bisulfite-treated DNA from histologically confirmed GBM tissue and normal brain specimens were PCR to the promoters of these genes despite the absence of change in amplified. Methylated PCR products were cloned into TOPO-TA (Invitrogen). methylation status (Fig. 5). Plasmid DNA from 16 independent clones was prepared and sequenced from both ends with BIG Dye terminators (version 3.1). Square, individual CpG site with the degree of shading representing the fractional methylation. response, genetic imprinting, or somatic silencing. This analysis revealed that TSA and 5-AzaCinduce widespread regulation of genes across several signaling pathways of interest in glioma biology. Using microarray analysis, we identified 345 genes that are highly expressed in normal astrocytes, have negligible expression in malignant glioma cells, but are strongly induced in response to TSA or 5-AzaCtreatment. Finally, we compared expression levels of each individual candidate tumor suppressor gene in cultured normal human astrocytes and malignant glioma cell lines with real-time PCR. The goal was to identify genes with high expression levels in normal astrocytes and negligible expression in malignant glioma cells. This comparative analysis led to the identification of two novel brain expressed genes, BEX1 and BEX2, both strongly induced by TSA and 5-AzaCin glioma Figure 4. Correlation of promoter-associated methylation changes with expression of BEX1 and BEX2 in GBM. The methylation index (MI)was cell lines that were found to be silenced in all glioma tumor determined by averaging the fractional methylation of CpG sites in the promoter specimens when compared with nontumor brain specimens regions of BEX1 and BEX2 in normal (n = 8)and tumor ( n = 10)tissue samples. (Supplementary Table S1). We confirmed the activation of BEX1 Total RNA was then extracted from the corresponding tissue samples, and the relative expression of BEX1 and BEX2 was determined using real-time PCR. and BEX2 in response to TSA (Fig. 2) and 5-AzaCtreatment Y axis, arbitrary expression units used to calculate fold change normalized to a (data not shown) by real-time PCR. reference expression level obtained from one normal brain sample. www.aacrjournals.org 6671 Cancer Res 2006; 66: (13). July 1, 2006

alone as measured by TUNEL staining (Fig. 7A). After treatment with a subtherapeutic dose of camptothecin and etoposide, two chemotherapeutic agents known to induce apoptosis in glioma cells at higher doses (34), there was a striking increase in the number of cells undergoing apoptosis in the BEX1 and BEX2 transduced population when compared with control vector-treated cells (Fig. 7A). Finally, we injected BEX1-orBEX2-transduced U87 cells in a nude mouse model to measure in vivo tumor growth. As seen in Fig. 7B, there is marked suppression of tumor growth in BEX1-or BEX2-transduced cells when compared with control vector- transduced cells (P < 0.01). These results support a tumor suppressor role for both BEX1 and BEX2 in malignant glioma. Reexpression of either BEX1 or BEX2 suppresses tumor growth and chemosensitizes malignant glioma cells to camptothecin-induced apoptosis.

Discussions We are entering a new era of patient-centered genomic medicine, which will allow for rapid advancement in our understanding of disease progression and treatment. We envision an experimental paradigm, which will successfully translate from Figure 5. Confirmation of changes in histone H3-K9 acetylation status with TSA the individual patient in the operating room to a comprehensive treatment in U87 cells. A, ChIP assay on DNA harvested from the U87 genome-wide analysis of epigenetic gene silencing in the patient’s glioblastoma cell line treated with 1 Amol/L TSA or DMSO control. B, antibodies against histone H3-acetylated K9, normal rabbit IgG, or no antibody were used. tumor, creating a barcode of epigenetic markers outlining The assays were done in triplicate, and the relative change in acetylation on TSA individual prognosis and susceptibility to treatment. In this treatment was calculated from the amount of histone acetylated with respect to study, we have identified a large cohort of epigenetically silenced input DNA. genes, each of which may serve as a biomarker for disease detection, progression, prognosis, and susceptibility to treatment. Functional analysis of tumor suppressor activity in vitro and As proof of principle, we have identified and characterized the in vivo. Using adenoviral transfection constructs, we expressed tumor suppressor function of two of these genes, BEX1 and BEX2, BEX1 and BEX2 individually in malignant glioma cell lines to in malignant glioma. We plan further investigations to explore the determine potential tumor suppressor function. Expression of functional implications of tumor-specific epigenetically silenced BEX1 and BEX2 was independently confirmed by real-time PCR. As genes with expression profiles similar to BEX1 and BEX2, many of shown in Fig. 6, expression of either BEX1 or BEX2 resulted in a which regulate critical pathways in glioma progression. marked decrease in colony formation compared with control Epigenetic silencing of tumor suppressor genes in human cancer transfected cells in both T98 and U87 immortalized cell lines. is widespread, involving signaling pathways controlling angiogen- We then examined whether expression of BEX1 or BEX2 could esis, cellular proliferation, migration, apoptosis, and differentiation. increase the susceptibility of T98 and U87 cells to apoptosis. There We identified components of the Hedgehog, Notch, FGF, and Wnt was no increase in apoptotic cells after BEX1 or BEX2 transduction signaling pathways up-regulated by TSA and 5-AzaCin malignant

Figure 6. Effect of expression of the BEX gene on the growth of glioblastoma cell lines. A, colony focus assays in T98 and U87 glioblastoma cell lines. B, quantification of the number of G418-selected colonies in three independent experiments was determined relative to empty vector control.

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this study, we narrowed our selection criteria to those genes that were activated in both primary and immortalized glioma cell lines. A larger cohort of genes, activated only in the primary cell lines, offers a novel opportunity to gain insight into the mechanism of action of this class of drug, further defines the role of epigenetically regulated pathways in individual patient tumors, and correlate these changes with individual patient outcomes. Epigenetic changes in cancer cells also offer unique prospects for cancer diagnostics (37–44). Densely methylated DNA is associated with deacetylated histones and compacted chromatin, which is refractory to transcription. Several members of a family of methyl- binding domain proteins have been shown to associate with large protein complexes containing HDAC, histone methylase, and chromatin-remodeling enzymes. These protein complexes can also contain transcription factors, which target them to specific gene promoters. The tightly controlled interaction between these different mechanisms of epigenetic regulation (DNA methylation, histone methylation and acetylation, and chromatin remodeling) provides a mechanism by which densely methylated DNA is associated with hypoacetylated histones and transcriptionally repressive chromatin. Promoter methylation as a biomarker for disease detection and prognosis has been validated in prostate, lung, and bladder cancer. We have established a patient-centered database of whole-genome microarray and bisulfite-sequencing data with a focus on epigenetically silenced genes, which are capable of being pharmacologically induced by either HDAC inhibitors or 5-AzaC. Recent reports indicate that many of these methylation markers can be detected in serum and other body fluids with methylation-specific PCR techniques. The correlation of specific epigenetic signatures with disease state, progression, and response to treatment holds great promise for the development of therapeutic advances targeted to genetically stratified patients. Figure 7. Increased BEX1 or BEX2 expression sensitizes glioblastoma tumor In this study, we identified and further characterized the tumor cells to camptothecin- and etoposide-induced apoptosis. A, TUNEL staining of T98 cells transduced with BEX1, BEX2, or control vector, with and without suppressor function of two of these genes, BEX1 and BEX2,in camptothecin/etoposide treatment. Percentage of TUNEL-positive cells. malignant glioma. BEX1 and BEX2 are X-linked uncharacterized Columns, mean of three independent experiments. B, BEX1 and BEX2 inhibit cDNAs that have 91% sequence similarity with each other. We did tumor growth in vivo. U87 glioma cells were transduced with adenovirus vectors ex vivo and implanted into mice (n = 3)24 hours after infection. At 28 days conserved domain analysis using Pfam and PROSITE and found after injection, animals were sacrificed, and tumors were volumetrically defined that proteins coded by both these genes contain the BEX domain. and confirmed by histologic analysis. Expression of either BEX1 or BEX2 significantly inhibited tumor growth compared with vector control (P < 0.01). Interestingly, this domain is also present in human p75NTR- associated cell death executor (NADE) protein, which has been implicated in the p75-mediated signal transduction pathway (45). glioma. Because epigenetic events can be reversed, effectively NADE has been shown to have diverse effects on the central restoring the normal expression and cellular function of gene, these nervous system, including differentiation and apoptosis (46). BEX2 epigenetic modifications are attractive therapeutic targets for the has an extra casein kinase II phosphorylation site and N- modulation of key antitumor pathways (12). We initiated our glycosylation site. Taken together with our findings, these results studies with both 5-AzaCand TSA, the most widely used and suggest that BEX1 and BEX2 may play an important role in a novel characterized HDACinhibitor, to capture the entire spectrum of signaling pathway regulating apoptosis in malignant glioma. genes modulated by these two distinct but related epigenetic Further investigation of the functional implications of tumor- mechanisms. The promoter region of each reactivated gene can be specific epigenetically silenced genes should enhance our under- examined for evidence of histone acetylation, indicating a TSA standing of other key regulatory pathways altered in malignant effect, or CpG methylation, indicating a role for 5-AzaC. In glioma and provide potentially reversible targets for therapeutic malignant glioma, our results show that TSA has a dominant intervention. genome-wide effect, frequently activating genes through chromatin modulation even in the absence of promoter hypermethylation. The complex interaction between promoter hypermethylation, Acknowledgments associated histone modifications, and transcriptional activation in Received 12/13/2005; revised 4/19/2006; accepted 4/24/2006. response to 5-AzaChas not been fully elucidated (35, 36). As Grant support: Department of Neurosurgery, University of Iowa College of Medicine, Carver Foundation grant 99-30 and Seattle Children’s Hospital and Regional previous studies have shown, robust transcriptional activation of Medical Center Brain Tumor Research Endowment. epigenetically silenced genes can occur in response to 5-AzaC The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance treatment before changes in promoter methylation patterns, which with 18 U.S.C. Section 1734 solely to indicate this fact. usually appear only after chronic treatment (35). For purposes of We thank Beverly Davidson for critical review of the article. www.aacrjournals.org 6673 Cancer Res 2006; 66: (13). July 1, 2006

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Genome-Wide Analysis of Epigenetic Silencing Identifies BEX1 and BEX2 as Candidate Tumor Suppressor Genes in Malignant Glioma

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