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[Cancer Research 66, 6665-6674, July 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

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

Greg Foltz1,3, Gi-Yung Ryu1, Jae-Geun Yoon1, Timothy Nelson1, Jessica Fahey1, Amanda Frakes1, Hwahyung Lee1, Lorie Field1, Kaitlin Zander1, Zita Sibenaller1, Timothy C. Ryken1, Rajeev Vibhakar2, Leroy Hood3 and Anup Madan1,3

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

Requests for reprints: Anup Madan, Neurogenomics Research Laboratory, Department of Neurosurgery, University of Iowa Carver College of Medicine, 200 B Eckstein Medical Research Building, Iowa City, IO 52242. Phone: 319-3358491; Fax: 319-3358078; E-mail: anup-madan{at}uiowa.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussions
 References
 
Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2'-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis. (Cancer Res 2006; 66(13): 6665-74)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussions
 References
 
Epigenetic silencing of tumor suppressor genes is a common motif in human cancer (1, 2). Tumor-associated transcriptional silencing is most frequently described in association with a few common underlying epigenetic mechanisms, such as promoter hypermethylation, histone deacetylation, histone methylation, and other histone modifications, which directly or indirectly alter chromatin structure (3–5). When combined with microarray analysis, the pharmacologic reversal of one or more of these common underlying epigenetic mechanisms provides a powerful screening tool for the comprehensive identification of epigenetically silenced genes on a global scale. Several recent studies have validated this large-scale approach using DNA methyltransferase and histone deacetylase (HDAC) inhibitors, either alone or in combination, in a variety of human cancers, including pancreatic, colon, prostate, liver, endometrial, blood, and esophageal cancers (6–12).

The scope of epigenetic gene silencing in malignant glioma has not been well defined. Malignant glioma, the most aggressive and common brain tumor in adults, continues to cause ~13,000 deaths yearly in the United States despite intensive clinical and basic science research. In a recent major advance, promoter hypermethylation and epigenetic silencing of the DNA repair gene O6-methylguanine-DNA methyltransferase were shown to identify a subset of patients with markedly improved survival at 2 years in response to combined treatment with chemotherapy and radiation therapy (13–15). Several other genes critical for cell proliferation, tumor progression, apoptosis, angiogenesis, and astrocyte motility have also been shown to be silenced in association with promoter hypermethylation in malignant glioma (16–18). Despite these important findings, the genome-wide role of epigenetic silencing and the functional significance of individually silenced genes have not been extensively studied.

Abundant evidence supports a synergistic link between promoter hypermethylation and histone deacetylation in the active suppression of gene transcription (19). Importantly, several recent reports suggest that pharmacologic inhibition of histone deacetylation alone markedly reverses transcriptional silencing, even in the absence of promoter hypermethylation. This has led to the hypothesis that histone deacetylation is the primary mechanism that actively modulates epigenetic transcriptional silencing. HDAC inhibitors have subsequently emerged as a powerful new class of chemotherapeutic agents with promising antitumor effects. They 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 HDAC inhibitors are well tolerated, inhibit HDAC activity in tumor cells, and lead to objective tumor regression. In immortalized glioma cell lines, HDAC inhibition results in cell cycle arrest and increased apoptosis (17, 20, 21). To date, few reports exist describing the effect of HDAC inhibition on gene expression in malignant glioma cell lines (18, 22–24). Many HDAC inhibitors are small molecules with pharmacologic properties conducive to effective delivery across the blood brain barrier, a significant therapeutic advantage over traditional chemotherapy agents.

The goal of the present study is to comprehensively identify and confirm epigenetically silenced genes in patient samples of malignant glioma. We used a whole-genome microarray, rigorous statistical analysis, and an experimental paradigm optimized to identify epigenetically silenced genes with tumor suppressor function in primary glioma cultures. We then confirmed transcriptional silencing of these genes in a panel of human tumors with subsequent analysis of individual gene promoter regions for DNA methylation and histone modifications. We characterized the functional consequence of reexpression of two novel epigenetically silenced genes, which exhibit proapoptotic tumor suppressor activity in an in vitro and ex vivo tumor model. This experimental paradigm provides a powerful new approach to the genome-wide identification of epigenetically silenced tumor suppressor genes in malignant glioma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussions
 References
 
Tissue samples and cell lines. Tumor and nontumor brain specimens were obtained from the Central Nervous System Tissue Bank at the University of Iowa Hospitals and Clinics (Iowa City, Iowa). All patients gave informed consent before the collection of specimens according to institutional guidelines. Primary cultures from freshly resected brain tumor specimens were established per standard protocols (25). Each tumor specimen was histologically verified by a board-certified neuropathologist and archived for further DNA, RNA, and protein studies. To generate short-term primary cell cultures, tumor tissue (~200-250 mg) was immersed and incubated in 0.05 mmol/L EDTA solution containing 0.05% trypsin (Sigma, St. Louis, MO) at 4°C for 8 hours. The tissue samples were minced into 0.3-mm3 fragments and suspended in HBSS containing 4 mg DNaseI, 40 mg collagenase IV, and 100 units hyaluronidase type V (all from Sigma). Single-cell suspensions were then passed through no. 100 nylon mesh, washed twice in HBSS, and added to fibronectin-coated tissue culture flasks. Cells were maintained in medium consisting of DMEM/F12, 15% fetal bovine serum, 10 µg/mL insulin, 7.5 ng/mL fibroblast growth factor (FGF), 1 mmol/L pyruvate, 15 mmol/L HEPES buffer (pH 7.2), and 100 units/100 µg/mL penicillin-streptomycin. The immortalized T98 and U87 glioblastoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and maintained in a similar fashion. Normal human astrocytes were maintained in ABG Bullet kit medium (both from Cambrex, Walkersville, MD).

Trichostatin A and 5-aza-2'-deoxycytidine treatment of cells. Cell lines were treated with varying doses of trichostatin A (TSA; Sigma) ranging from 0.2 to 10 µmol/L or a control volume of DMSO (final DMSO concentration not ≤0.1% v/v) for various times (15 minutes-48 hours). For 5-aza-2'-deoxycytidine (5-AzaC) treatment, cell lines were treated with 5 µmol/L 5-AzaC (Sigma) and dissolved in PBS containing 1 µmol/L acetic acid or PBS control for 72 hours.

Western blot analysis. Protein extracts were prepared from T98 and three primary (UI269, UI274, and UI276) glioblastoma cell lines, which had been treated for various times ranging from 15 minutes to 48 hours with 1 µmol/L TSA. Western blot analysis was done as described previously (26) using primary antibodies directed to acetylated histone H3 (Upstate Biotechnology, Lake Placid, NY) and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA) as a control. Secondary horseradish peroxidase–conjugated anti-rabbit antibodies (Santa Cruz Biotechnology) were used at a dilution of 1:5,000.

Proliferation and cell cycle assays. Treated cell lines and mock-treated controls were assessed for growth inhibition using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Immortalized (T98 and U87) and two primary glioblastoma (UI218 and UI260) cells were plated in 96-well culture plates at a density of 5 x 103 per well, allowed to attach overnight, and treated with either DMSO control or TSA (concentrations ranging from 0.2 to 10 µmol/L) for 24, 48, 72, and 96 hours. During the last 4 hours of incubation, the medium was replaced with fresh medium containing MTT at 1 mg/mL. The MTT reaction was stopped after 4 hours by replacing the medium with 100 µL isopropanol, and the cells were incubated for 1 hour at room temperature in the dark. The conversion of soluble MTT to an insoluble blue formazan product was measured with the Thermomax microplate reader (Molecular Devices, Sunnyvale, CA) at 570 nm. Each treatment was done on three independent cultures of each cell line. Within a given experiment, each treatment condition was evaluated in 16 wells of the 96-well plate. Percentage change (mean and SD) is reported for each treatment relative to DMSO control. Statistical significance was determined from the mean and SE across replicate measurements in three independent experiments.

For cell cycle assays, unsynchronized cells were seeded at 1 x 106 per 100-mm dish and exposed to 0.2 to 5 µmol/L TSA for 24 hours. After fixing with 70% ethanol, the nuclei were stained with 50 µg/mL propidium iodide and 10 µg/mL RNase A. The relative DNA content was measured using a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Each experiment was repeated thrice, and data are reported as the percentage of cells (mean and SD) in each phase of the cell cycle. In the cell death commitment assay, T98 cells were initially treated with 1 µmol/L TSA for various times (2-36 hours), then washed to remove any TSA-containing medium, and subsequently cultured in the absence of TSA for a total of 72 hours.

Microarray and real-time PCR analysis. For microarray experiments, T98, U87, patient-derived primary glioma cell lines, and normal human astrocytes were cultured with either 1 µmol/L TSA or DMSO for 24 hours. T98 cells were also treated for a period of 6, 12, 36, and 48 hours to generate a time course of response after drug treatment. To generate gene expression profiles in response to 5-AzaC treatment, cell lines were cultured with 5 µmol/L 5-AzaC for 72 hours. Total RNA was extracted from treated cells using Trizol (Invitrogen, Carlsbad, CA) with an additional purification step using RNeasy (Qiagen, Valencia, CA) before quality assessment with the Agilent Bioanalyzer (Palo Alto, CA). Total RNA (2 µg) was reverse transcribed with the Chemiluminescent RT-IVT Labeling kit (Applied Biosystems, Foster City, CA) and hybridized to a 60-mer whole-genome oligonucleotide microarray (Applied Biosystems) containing 33,202 probes representing 29,098 genes. A total of three microarray hybridizations, one for each biological replicate, were done per treatment and time point for T98 cells. Data were quantile normalized, and a t test was applied to each gene for statistical significance. Correction for multiple testing was done using Storey's q-value method (27). Epigenetically regulated genes were identified using Spotfire software (version 8.2) for data visualization with a threshold cutoff set at two-fold and a false discovery rate of 5%. For the 10 patient-derived primary cell lines, one microarray hybridization per patient per treatment condition was done (a total of 20 microarrays) due to limited tissue samples. For each individual cell line, data were initially normalized, and differential gene expression was tested for statistical significance using the ABI single array error model (Applied Biosystems, reference manual). For more rigorous statistical analysis, each of these 10 different glioma cell lines was treated as a biological replicate of the same experimental condition. Allowing for some variation between individual patient tumors, we set a threshold at 70% (i.e., differential gene expression that was consistent in at least 7 of the 10 glioma cell lines) before passing the gene on for significance testing as outlined above.

Assay-on-Demand gene expression reagents (Applied Biosystems) for 100 selected genes was used to validate microarray results and confirm gene silencing in histologically confirmed glioblastoma specimens relative to nontumor brain specimens. Real-time PCR was done using the ABI PRISM 7900 HT Sequence Detection System (Applied Biosystems) under default conditions: 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute with human glutathione synthetase as control. Confirmed genes were parsed through the Panther pathway analysis program (http://panther.appliedbiosystems.com/pathway/) to identify epigenetically regulated signaling pathways.

Bisulfite sequencing analysis. Genomic DNA was extracted from glioma tissue samples (25 mg) and cell lines (1 x 107) using Wizard Genomic DNA Purification kits (Promega, Madison, WI). Sodium bisulfite modification of 4 µg genomic DNA was carried out using the MethylEasy DNA bisulfite modification kit (Human Genetic Signatures, Sydney, Australia). CpG islands upstream of the start site were identified using the CpG prediction program, NEWCPGREPORT, and PCR primers for nested PCR were designed to yield 400 to 500 bp products. The PCR mixture contained 20 pM of each primer, 40 ng bisulfite-treated DNA (or 2 µL of first round product), 1.25 units Taq DNA Polymerase (Eppendorf, Pittsburgh, PA), 0.2 mmol/L deoxynucleotide triphosphate (dNTP; each), and 2 mmol/L MgSO4 in a final volume of 50 µL. The PCR was done with the following cycling variables: an activation step of 94°C for 3 minutes followed by 50 cycles of 94°C for 2 minutes, 50°C for 2 minutes, and 68°C for 3 minutes, with a final extension step of 68°C for 10 minutes. For BEX1, a CpG-rich region (–174 to +105 relative to transcriptional start site) was amplified using primer pairs F-gggaaaggaagggtatagattttttt and R-crataaacctctactaacctaaccaaaa (first round) and F-gygatgatgttatttgtgggttttt and R-aacrcaaaacaacraaaaactaataacraa (second round). For BEX2, a CpG-rich region (+157 to +408 relative to transcriptional start site) was amplified using primer pairs F-ggtagtggtatttgttttyggtgtttt and R-ctaaccaccattttctacatcaaaaaaaa (first round) and F-ggtaagggattyggagggggtttt and R-aaacaaaaatattaaaaaaacttacrctaata (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 x 106) treated with DMSO or 1 µmol/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 4°C, 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 4°C overnight. The immunoprecipitated complexes were collected by incubation with protein A-Sepharose beads (Upstate Biotechnology) for 1 hour at 4°C. 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 65°C for 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 µL water. The PCR was designed to yield 250 bp products. To ensure that PCR amplification was in linear range, each reaction was set up at different dilutions of DNA for varying amplification cycle numbers, and final PCR conditions were selected accordingly. The PCR mixture contained 20 pM of each primer, 1 µL extracted DNA, 0.5 units Taq DNA Polymerase (Eppendorf), 0.2 mmol/L dNTPs (each), and 2 mmol/L MgSO4 in a final volume of 50 µL. The PCR was done with the following cycling variables: an activation step of 94°C for 3 minutes followed by 30 cycles of 94°C for 2 minutes, 50°C for 2 minutes, and 68°C for 3 minutes, with a final extension step of 68°C for 10 minutes. The promoter region of BEX1 (–284 to –81 relative to transcriptional start site) was amplified using the primer pair F-gaagaggaaagaagaaaaggccaag and R-cctcctcccgtctgtgcgcggtgcc. Primer pair F-ggatgttaaaagggactcccggtga and R-cgacggcggttctgacgccacaacg was used for amplification of the BEX2 promoter (–108 to +62 relative to transcriptional start site). The PCR products were visualized by 1.8% agarose gel electrophoresis and quantitated by densitometry. The assays were done in triplicate.

Construction of expression vectors. Full-length open reading frames for BEX1 and BEX2 were PCR amplified from MGC clones and cloned into the pcDNA3.1D/V5-His-TOPO vector (Invitrogen), and sequence was verified. The PCR products were cloned into pacAD5CMVIRESeGFPpA, and sequence was verified to generate adenoviral vectors. These clones were recombined in HEK293 cells with pacAD5 9.2-100 to produce recombinant adenovirus particles.

Transfection and colony formation assays. Colony formation assays were done in monolayer culture. Cells were plated at 1.5 x 105 per well using six-well plates and transfected with either pcDNA3.1D/V5-His-TOPO/BEX1, pcDNA3.1D/V5-His-TOPO/BEX2, pcDNA3.1D/V5-His-TOPO/lacZ, or pcDNA3.1D/V5-His-TOPO with no insert (mock control) using Trans It-Neural transfection reagents (Mirus, Madison, WI). The cells were selected in G418 (1 mg/mL) supplemented medium at 24 hours after transfection. Some cells were simultaneously harvested to confirm increased expression of the transfected gene by real-time PCR. G418-resistant cells were maintained for 2 weeks in culture. Cells were detached, resuspended in medium containing 0.3% agarose, and overlaid on 0.6% agarose. Medium (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.

Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling. T98 cells infected with adenovirus expressing human BEX1 or BEX2 were treated with either camptothecin (1 µmol/L) and etoposide (3 µmol/L) or DMSO as a control for 40 hours. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) reaction was carried out with the APO-BrdU TUNEL assay kit (Molecular Probes, Raleigh, NC) using Alexa Fluor 647–conjugated monoclonal antibodies and Hoechst 33342 for staining DNA. Cells were recorded with LSRII (Becton Dickinson).

Xenograft mouse assay. All animal experiments were carried out according to approved institutional guidelines following approval of the University of Iowa Animal Care and Use Committee. Four-week-old NCR (nu/nu) mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in the University of Iowa Animal Care Facility. U87 glioma cells were infected with adenovirus vectors encoding either BEX1, BEX2, or GFP (control) ex vivo, and the cells were implanted into mice 24 hours after infection. Tumor cells (2 x 106 per mouse) were injected s.c. as described previously (31). Tumors were monitored twice weekly. At day 28 after injection, animals were sacrificed, and tumors were measured in three dimensions. Tumor volume was calculated using the formula d1 x d2 x d3 x p/6 (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussions
 References
 
TSA/5-AzaC-induced gene expression profiles. We designed an experimental paradigm for microarray analysis optimized to identify epigenetically silenced genes in malignant glioma. First, we determined the optimal dose and timing for gene expression studies by characterizing the time course and dose response of treatment with TSA in two immortalized cell lines (T98 and U87) and 10 primary glioma lines. TSA treatment resulted in marked histone acetylation, growth arrest, cell cycle inhibition, caspase activation, and cell death in T98 cells, U87 cells, and primary glioma cultures when compared with control-treated cells (Supplementary Figs. S1-S3). Ideally, a treatment regimen would optimize the identification of epigenetically silenced genes that are pharmacologically reversed by TSA and that also have tumor suppressor function. To achieve this, we chose a standard dosing regimen of 1 µmol/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 µmol/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-effect". Using the ABI 1700 Human Genome Expression Microarray (Applied Biosystems) and a maximally stringent threshold for statistical significance (q < 0.001), a whole-genome analysis identified a subset of genes in T98 cells whose reexpression closely followed the time course of histone acetylation in response to TSA treatment (Supplementary Fig. S4). Real-time PCR was used to confirm microarray expression data (Supplementary Fig. S5). We then identified 653 genes, which were reexpressed in common in both the immortalized cell lines, T98 and U87, and in a majority (70%) of primary glioma cell lines (Fig. 1A ). A similar comparison identified 170 genes up-regulated in both the immortalized and 70% of the primary glioma cell lines after treatment with 5-AzaC, a significantly smaller cohort compared with the effects of TSA. Taken together, these 823 genes were chosen for further study as the most likely to be epigenetically silenced in the in vivo tumor and directly induced in response to TSA or 5-AzaC, while also being silenced in immortalized cell lines, allowing for further functional analysis. We then confirmed transcriptional silencing of 10 candidate tumor suppressor genes (selection criteria described below) in a panel of histologically confirmed human malignant glioma specimens and nontumor brain specimens using real-time PCR (Fig. 1B). Of note, after TSA or 5-AzaC treatment, a large number of genes were found in common exclusively in the group of primary glioma cell lines, and these genes are of potentially great interest and will be the subject of future studies. An even larger number of silenced genes were found exclusively in the immortalized cell lines, most likely representing the effect of long-term cell culture.


Figure 1
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  		Figure 1. Whole-genome microarray analysis of genes activated in response to HDAC inhibition. A, comparison of genes up-regulated greater than two-fold (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 immortalized and primary glioma cell lines were treated with 1 µmol/L TSA for 24 hours. Total RNA was extracted, purified, and subjected to whole-genome 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, whereas 3,441 genes were found to be up-regulated in T98 glioma cell lines and 2,809 genes were up-regulated in U87 cell line by TSA. B, relative transcriptional silencing of 10 genes in histologically confirmed GBM tissues (n = 10) compared with normal brain tissues (n = 8; P < 0.001). Data from individual tissue. Total RNA was extracted from tissue samples, and relative expression of genes with respect to expression in a normal brain sample was determined using real-time PCR. Y axis, arbitrary expression units used to calculate fold change relative to expression in one nontumor brain sample.

 
Selection criteria for candidate tumor suppressor genes. We developed multiple criteria for narrowing our focus to those epigenetically silenced genes with suspected tumor suppressor function and promoter alterations, which could be used as a potential biomarker. First, it was clear from our microarray results that TSA had a stronger genome-wide effect on gene expression than 5-AzaC. As CpG methylation is associated with HDAC-mediated transcriptional repression, we used bioinformatics methods to identify all genes up-regulated by TSA whose promoter regions contained CpG islands. To increase our yield of TSA-regulated genes with promoter hypermethylation, we included all genes that were activated by both TSA and 5-AzaC. Next, we categorized each gene into a functional pathway (Table 1 ), excluding genes known to be associated with the IFN response, genetic imprinting, or somatic silencing. This analysis revealed that TSA and 5-AzaC induce 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-AzaC treatment. 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-AzaC in glioma cell lines that were found to be silenced in all glioma tumor specimens when compared with nontumor brain specimens (Supplementary Table S1). We confirmed the activation of BEX1 and BEX2 in response to TSA (Fig. 2 ) and 5-AzaC treatment (data not shown) by real-time PCR.


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

 

Figure 2
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  		Figure 2. Activation of epigenetically silenced BEX1 and BEX2 in response to HDAC inhibition by TSA. Total RNA was extracted from 10 primary glioma cell lines treated with either DMSO or 1 µmol/L TSA. Real-time PCR was used to confirm up-regulation of BEX1 or BEX2 in response to TSA treatment using Assay-on-Demand gene expression reagents. Y axis, arbitrary expression units used to calculate fold change.

 
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-AzaC treatment in both immortalized and primary glioma cell lines. Only 120 of 170 genes (72%) that are up-regulated by 5-AzaC contain CpG islands 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-AzaC treatment 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 transcriptional activation, chromatin immunoprecipitation (ChIP) analysis showed a significant increase of acetyl histone H3 binding to the promoters of these genes despite the absence of change in methylation status (Fig. 5 ).


Figure 3
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  		Figure 3. Bisulfite sequence analysis of a CpG island in the promoter region of the BEX1 and BEX2 genes reveals dense methylation of CpG sites in tumor tissues compared with nontumor brain specimens. Bisulfite-treated DNA from histologically confirmed GBM tissue and normal brain specimens were PCR amplified. Methylated PCR products were cloned into TOPO-TA (Invitrogen). 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.

 

Figure 4
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  		Figure 4. Correlation of promoter-associated methylation changes with expression of BEX1 and BEX2 in GBM. The methylation index (MI) was determined by averaging the fractional methylation of CpG sites in the promoter regions of BEX1 and BEX2 in normal (n = 8) and tumor (n = 10) tissue samples. Total RNA was then extracted from the corresponding tissue samples, and the relative expression of BEX1 and BEX2 was determined using real-time PCR. Y axis, arbitrary expression units used to calculate fold change normalized to a reference expression level obtained from one normal brain sample.

 

Figure 5
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  		Figure 5. Confirmation of changes in histone H3-K9 acetylation status with TSA treatment in U87 cells. A, ChIP assay on DNA harvested from the U87 glioblastoma cell line treated with 1 µmol/L TSA or DMSO control. B, antibodies against histone H3-acetylated K9, normal rabbit IgG, or no antibody were used. The assays were done in triplicate, and the relative change in acetylation on TSA treatment was calculated from the amount of histone acetylated with respect to input DNA.

 
Functional analysis of tumor suppressor activity in vitro and in vivo. Using adenoviral transfection constructs, we expressed BEX1 and BEX2 individually in malignant glioma cell lines to determine potential tumor suppressor function. Expression of BEX1 and BEX2 was independently confirmed by real-time PCR. As shown in Fig. 6 , expression of either BEX1 or BEX2 resulted in a marked decrease in colony formation compared with control transfected cells in both T98 and U87 immortalized cell lines.


Figure 6
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  		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.

 
We then examined whether expression of BEX1 or BEX2 could increase the susceptibility of T98 and U87 cells to apoptosis. There was no increase in apoptotic cells after BEX1 or BEX2 transduction 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).


Figure 7
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  		Figure 7. Increased BEX1 or BEX2 expression sensitizes glioblastoma tumor cells to camptothecin- and etoposide-induced apoptosis. A, TUNEL staining of T98 cells transduced with BEX1, BEX2, or control vector, with and without camptothecin/etoposide treatment. Percentage of TUNEL-positive cells. Columns, mean of three independent experiments. B, BEX1 and BEX2 inhibit 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 after injection, animals were sacrificed, and tumors were volumetrically defined and confirmed by histologic analysis. Expression of either BEX1 or BEX2 significantly inhibited tumor growth compared with vector control (P < 0.01).

 
Finally, we injected BEX1- or BEX2-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
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussions
References
 
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 the individual patient in the operating room to a comprehensive genome-wide analysis of epigenetic gene silencing in the patient's tumor, creating a barcode of epigenetic markers outlining individual prognosis and susceptibility to treatment. In this study, we have identified a large cohort of epigenetically silenced genes, each of which may serve as a biomarker for disease detection, progression, prognosis, and susceptibility to treatment. As proof of principle, we have identified and characterized the tumor suppressor function of two of these genes, BEX1 and BEX2, in malignant glioma. We plan further investigations to explore the functional implications of tumor-specific epigenetically silenced genes with expression profiles similar to BEX1 and BEX2, many of which regulate critical pathways in glioma progression.

Epigenetic silencing of tumor suppressor genes in human cancer is widespread, involving signaling pathways controlling angiogenesis, cellular proliferation, migration, apoptosis, and differentiation. We identified components of the Hedgehog, Notch, FGF, and Wnt signaling pathways up-regulated by TSA and 5-AzaC in malignant glioma. Because epigenetic events can be reversed, effectively restoring the normal expression and cellular function of gene, these epigenetic modifications are attractive therapeutic targets for the modulation of key antitumor pathways (12). We initiated our studies with both 5-AzaC and TSA, the most widely used and characterized HDAC inhibitor, to capture the entire spectrum of genes modulated by these two distinct but related epigenetic mechanisms. The promoter region of each reactivated gene can be examined for evidence of histone acetylation, indicating a TSA effect, or CpG methylation, indicating a role for 5-AzaC. In malignant glioma, our results show that TSA has a dominant genome-wide effect, frequently activating genes through chromatin modulation even in the absence of promoter hypermethylation. The complex interaction between promoter hypermethylation, associated histone modifications, and transcriptional activation in response to 5-AzaC has not been fully elucidated (35, 36). As previous studies have shown, robust transcriptional activation of epigenetically silenced genes can occur in response to 5-AzaC treatment before changes in promoter methylation patterns, which usually appear only after chronic treatment (35). For purposes of 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.

In this study, we identified and further characterized the tumor suppressor function of two of these genes, BEX1 and BEX2, in malignant glioma. BEX1 and BEX2 are X-linked uncharacterized cDNAs that have 91% sequence similarity with each other. We did conserved domain analysis using Pfam and PROSITE and found that proteins coded by both these genes contain the BEX domain. 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). NADE has been shown to have diverse effects on the central nervous system, including differentiation and apoptosis (46). BEX2 has an extra casein kinase II phosphorylation site and N-glycosylation site. Taken together with our findings, these results suggest that BEX1 and BEX2 may play an important role in a novel signaling pathway regulating apoptosis in malignant glioma. Further investigation of the functional implications of tumor-specific epigenetically silenced genes should enhance our understanding of other key regulatory pathways altered in malignant glioma and provide potentially reversible targets for therapeutic intervention.


   Acknowledgments
 
Grant support: Department of Neurosurgery, University of Iowa College of Medicine, Carver Foundation grant 99-30 and Seattle Children's Hospital and Regional Medical Center Brain Tumor Research Endowment.

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Beverly Davidson for critical review of the article.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 12/13/05. Revised 4/19/06. Accepted 4/24/06.


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussions
 References
 

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