Genome-Wide Hypomethylation in Human Glioblastomas Associated

Research Article

Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation

Benoıˆt Cadieux,1 Tsui-Ting Ching,1 Scott R. VandenBerg,1,2 and Joseph F. Costello1

The Brain Tumor Research Center, Departments of 1Neurological Surgery and 2Pathology, University of California, San Francisco, California

Abstract mechanisms in oncogene activation and tumor suppressor Genome-wide reduction in 5-methylcytosine is an epigenetic inactivation in GBM is well documented (2), very little is known hallmark of human tumorigenesis. Experimentally induced about the epigenetic component of this disease. Localized hyper- hypomethylation in mice promotes genomic instability and is methylation of gene-associated CpG islands and a more extensive sufficient to initiate tumorigenesis. Here, we report that global genome-wide reduction in 5-methylcytosine are epigenetic alter- hypomethylation is common in primary human glioblastomas ations that typify many cancers (3–5). [glioblastoma multiforme (GBM)] and can affect up to an Methylation is the only known covalent modification of genomic DNA in humans and occurs at cytosines followed by guanines estimated 10 million CpG dinucleotides per haploid tumor f genome. Demethylation involves satellite 2 (Sat2) pericentro- (CpG). CpG islands are 1 kb on average and contain a 5-fold meric DNA at chromosomes 1 and 16, the subtelomeric repeat excess of CpGs relative to the rest of the genome. The estimated f sequence D4Z4 at chromosomes 4q and 10q, and interspersed 30,000 CpG islands comprise 7% of all CpGs (6), most of which Alu elements. Severe hypomethylation of Sat2 sequences is are unmethylated in normal tissues. Normally methylated sequen- associated with copy number alterations of the adjacent ces include CpG islands associated with the inactive X chromosome euchromatin, suggesting that hypomethylation may be one and some imprinted and tissue-specific genes, as well as non-CpG factor predisposing to specific genetic alterations commonly island sequences such as juxtacentromeric DNA, intragenic regions, occurring in GBMs. An additional apparent consequence of and transposon sequences. Aberrant methylation of some CpG global hypomethylation is reactivation of the cancer-testis islands in cancer has been associated with silencing of tumor- suppressor genes (7). In GBMs, aberrant methylation in the antigen MAGEA1 via promoter demethylation, but only in 6 GBMs and GBM cell lines exhibiting a 5-methylcytosine promoter of the O -methylguanine-DNA methyltransferase gene has content below a threshold of f50%. Primary GBMs with been associated with significantly longer survival in patients treated significant hypomethylation tended to be heterozygous or with radiation and the alkylating agent temozolomide (8). This and homozygous for the low-functioning Val allele of the rate- other studies underscore the importance of addressing the changes limiting methyl group metabolism gene methylenetetrahydro- in the DNA methylation landscape in GBM and their effect on brain folate reductase (MTHFR), or had a deletion encompassing tumorigenesis. this gene at 1p36. Tumors with severe genomic hypomethyla- Global genomic hypomethylation has been documented for tion also had an elevated proliferation index and deletion multiple malignancies compared with matching normal tissues of the MTHFR gene. These data suggest a model whereby (9–11), and is associated with tumor progression in a mouse model either excessive cell proliferation in the context of inadequate (12). Three mechanisms by which hypomethylation contributes to methyl donor production from MTHFR deficiency promotes malignancy have been proposed, including oncogene activation, genomic hypomethylation and further genomic instability, loss of imprinting, and promoting genomic instability via unmask- or that MTHFR deficiency–associated demethylation leads to ing of repetitive elements. Experimental support for a role of increased proliferative activity in GBM. (Cancer Res 2006; hypomethylation in driving tumorigenesis via genomic instability 66(17): 8469-76) and an elevated rate of mitotic recombination is derived from studies of mice carrying hypomorphic alleles of DNA methyltransferase 1 (DNMT1; ref. 13). These mice develop aggressive Introduction lymphomas with high penetrance, and the tumors are commonly Glioblastoma multiforme (GBM) is the most common primary characterized by trisomy of chromosome 15. Similarly, global DNA brain tumor in adults and is characterized by a median survival of hypomethylation achieved through reduced DNMT1 function f 1 year for newly diagnosed cases (1). Although the role of genetic accelerates the onset of tumor formation in a p53 and NF1 mutant mouse model of sarcoma (14). Also, the removal of DNMT1 in murine embryonic stem cells increases mutation rates and the incidence of aneuploidy at reporter genes (15), whereas double knockout of DNMT1 and DNMT3b in the near diploid HCT116 Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). colorectal cancer cell line results in dramatic hypomethylation and Current address for T-T. Ching: Department of Internal Medicine and Geriatrics genomic instability manifested by chromosomal translocations Center, University of Michigan, Ann Arbor, MI 48109-0940. (16). Loss of imprinting generated through transient deficiency of Requests for reprints: Joseph F. Costello, The Brain Tumor Research Center, Department of Neurological Surgery, University of California San Francisco, 2340 DNMT1 activity during murine development leads to formation of Sutter, Room N225, San Francisco, CA 94143-0875. Phone: 415-514-1183; Fax: 415-502- a spectrum of solid tumors and hematologic cancer (17). These 6779; E-mail: [email protected]. I2006 American Association for Cancer Research. studies indicate that hypomethylation can cause genomic instabil- doi:10.1158/0008-5472.CAN-06-1547 ity and initiate tumorigenesis. www.aacrjournals.org 8469 Cancer Res 2006; 66: (17).September 1, 2006

The demethylation of repetitive elements, which comprise cancer development in elderly men, possibly through insufficient f45% of the human genome, accounts for the majority of DNA methylation-induced genomic instability (30). Homozygosity 5-methylcytosine loss in human cancers (9). Repetitive elements of the low-functioning MTHFR allele may predispose to an consist of tandem repeats of simple or complex sequences as well increased risk of gastric and breast cancer (31), but may be as interspersed repeats derived from transposable elements, such protective against the development other cancers (32). Thus, as the short interspersed nucleotide element Alu and the long decreased function of MTHFR could contribute to DNA hypo- interspersed nucleotide element LINE-1, which comprise f10% methylation, possibly in a cell and tissue type–dependent manner. and f20% of the human genome, respectively (18). The tandem This possibility has not yet been explored in GBM, although it is repeat satellite 2 (Sat2) DNA located at the juxtacentromeric region intriguing to note that the 1p36 locus containing the MTHFR gene of chromosomes 1 and 16 (Chr1 and Chr16), as well as the is deleted in a subset of these tumors. nonsatellite D4Z4 repeats located at the subtelomeric region of Chr4q35 and Chr10q26, are also targets for pronounced demethy- Materials and Methods lation in several cancers (19–21), in addition to lymphoblast and Tissues and cell lines. Human tumor and nontumor brain samples were fibroblast cell lines derived from patients with the immunodefi- obtained from the Neurosurgery Tissue Bank at the University of California- ciency-centromeric instability-facial anomalies (ICF) syndrome, San Francisco (UCSF), including 10 WHO grade 4 astrocytomas, one which usually involves germ-line mutations in DNMT3B (22). surgical sample from an adult individual with epilepsy (normal brain1, Breakage events at Chr1 also occur in the ICFcells, but only after NB1), and one autopsy sample (normal brain2, NB2). Human peripheral stimulating the cells to divide. This suggests a possible model blood lymphocytes (PBL) were isolated from two healthy adult donors. All whereby the hypomethylated state followed by cell division lead to samples were obtained with informed consent, and their usage was chromosome breakage in ICFsyndrome. In GBM, however, the approved by the Committee on Human Research at the UCSF. Six human genomic locations affected and the potential contribution of glioma cell lines, A172, LN18, LN229, LN319, LN443, and U373, were grown hypomethylation to the genetic alterations characteristic of GBMs in DME H21 supplemented with 10% fetal bovine serum. Genomic 5-methylcytosine quantitation. The genome-wide 5-methyl- are unknown. cytosine content was measured indirectly by methyl acceptance as previously There is also correlative evidence from human tumors, suggest- described (26, 33). Essentially, following restriction digestion with XbaI and ing that repeat-specific DNA demethylation may cause structural subsequent phenol/chloroform extraction, purified DNA was subjected 3 alterations of adjacent chromosomal regions. For instance, to radiolabeling with a methyl donor, S-adenosyl-L-(methyl- H)methionine chromosome 1 pericentromeric rearrangements and Sat2 DNA (15.0 Ci/mmol; GE Healthcare, Piscataway. NJ) using either M.SssI or dam hypomethylation are frequent in ovarian epithelial carcinomas (23). methylases, respectively. M.SssI methylase catalyzes the transfer of a methyl These studies suggest hypomethylation may lead to, or be caused group from SAM to cytosine residues within CpG dinucleotides; whereas dam by breakage events. Likewise, q-arm gain of Chr1 in hepatocellular methylates the adenine residue within GATC sequences. The percentage of methylated CpG was calculated from the ratio between M.SssI and dam carcinoma correlates with satellite DNA hypomethylation (24). 3 Taken together with the data from ICFsyndrome and DNMT- methylase H incorporation (SssI/dam ratio) as follows: percentage of methylated CpG = [1 À (SssI/dam ratio / 3.2)] Â 100, where 3.2 corresponds deficient cells, however, it seems most likely that hypomethylation to the estimated frequency of the dam recognition site (1 of 256), compared precedes rearrangements. with that of the M.SssI recognition site (1 of 80; ref. 26). Cancer-associated hypomethylation affecting single-copy genes Immunohistochemistry. Five-micrometer paraffin sections of formalin- was first reported in the gene body of Ras oncogenes (4). Recent fixed tissues were mounted on poly-L-lysine–coated glass slides. Tissues examples of genes marked by aberrant promoter hypomethylation were deparaffinized, fully hydrated through graded ethanol, and incubated and up-regulated expression that might contribute to tumorigen- in methanol with 0.5% H2O2 for 30 minutes at 22jC to block endogenous esis have also been reported (25). MAGEA1, which belongs to a peroxidase activity. The sections were then processed by heating with a broad category of genes commonly called cancer-testis antigens, is 750-W microwave for 25-minute intervals in 10 mmol/L citrate buffer also demethylated and its expression is activated in tumors. (pH 6). Primary mouse monoclonal antibodies (Ki-67, MIB-1 clone: Dako A/ MAGEA1 activation correlates with genome-wide hypomethylation S Carpinteria, CA) were applied (1:100) and allowed to react for 1 hour at 37jC. Visualization of the primary antibody used SuperPicture-Plus Polymer in tumors and tumor cell lines (26). Whether this mechanism also Mouse Detection horseradish peroxidase–3,3¶-diaminobenzidine (Zymed, underlies MAGEA1 expression in GBM has not been reported. South San Francisco, CA) with a Mayer’s hematoxylin counterstain. To The cause of hypomethylation in cancer is unknown. Because no calculate the nuclear labeling index for Ki-67 f900 to 1,700 total nuclei mutation in DNMT genes has been reported in cancer, impaired were examined for each tissue section in several regions with the highest DNMT function and altered DNA methylation levels may occur labeling. The labeled nuclei were scored as a percentage (labeling index) of through perturbations in the production of the universal donor of the total nuclei examined. Additional methods are available at Cancer methyl groups, S-adenosylmethionine (SAM). For instance, methyl- Research online (Supplementary Data). deficient diets, such as reduced choline and methionine, or inadequate supplies of coenzymes for methyl group metabolism, such as folate and vitamins B6 and B12, cause reduced intracellular SAM Results production, leading to genomic hypomethylation (27). Cooperating Global DNA hypomethylation in human primary GBMs and with these dietary factors are key enzymatic components of the glioma cell lines. The genomic 5-methylcytosine content of 10 methyl-group metabolism, such as the methylenetetrahydrofolate primary GBMs (WHO grade 4 astrocytoma) and six glioma cell reductase (MTHFR) gene. A common C667T polymorphism in lines was measured by methyl acceptance and compared with MTHFR, leading to an Ala to Val amino acid change, renders this values obtained for normal adult brain. The presence of slightly enzyme thermolabile and reduces activity by f50% (28). The Val/ more elevated 5-methylcytosine levels in brain relative to PBLs Val genotype was shown to be associated with decreased genomic (mean brain = 76.5%, mean PBLs = 72.8%) served as an indication DNA methylation in subjects with a low folate status (29). More- of the reliability of this assay, because earlier 5-methylcytosine over, the Val/Val genotype has been found to be a risk factor for measurements using the sensitive high-performance liquid

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Hypomethylation in GBM chromatography technique revealed a similar relationship between between genome-wide DNA methylation levels and the patterns of the DNA methylation content of these tissues (34). repeat-specific 5-methylcytosine content was observed. For in- Genome-wide hypomethylation was observed in 8 of 10 primary stance, GBMs that exhibited the most extensive genome-wide tumors, two of which (GBM28 and GBM30) had a more dramatic hypomethylation (GBM28 and GBM30; Fig. 1A) also had a dramatic reduction in 5-methylcytosine levels with values <50% (Fig. 1A). reduction in Sat2 DNA methylation relative to normal brain (50% Assuming 30 million CpG per haploid genome (6), we estimate that and 78%, respectively; Fig. 2A). In contrast, D4Z4 methylation was 10.3 million CpGs are demethylated in GBM28 and 8.2 million reduced by only 18% (GBM28) and 29% (GBM30) in these same CpGs were demethylated in GBM30. On average, the global 5- tumors (Fig. 2C). This percentage reduction was calculated by methylcytosine content of cultured glioma cell lines was more subtracting the methylation level of each GBM from the value of reduced than that of hypomethylated primary GBMs (mean GBMs NB2, then dividing that number by the value for NB2. It is possible = 59.8%, mean glioma cell lines = 48.4%; t test P = 0.01; Fig. 1B), a that the juxtacentromeric satellite DNA region of Chr1 and/or pattern that was previously noted for tumor cell lines of the breast, Chr16 is more prone to undergoing demethylation than the colon, and lung relative to their primary tumor counterpart (10). subtelomeric repeats of Chr4q and Chr10q (D4Z4) in GBMs. In fact, Hypomethylation of juxtacentromeric, subtelomeric, and Sat2 but not D4Z4 methylation was significantly reduced in GBM19 interspersed repetitive sequences in GBMs. More than one third and GBM27 relative to normal brain (12% and 16% reduction, of DNA methylation in normal tissues occurs in repetitive elements respectively; Fig. 2A and C), despite the normal global 5-methyl- (6). The juxtacentromeric Sat2 DNA region of Chr1 and Chr16, the cytosine content of these tumors (Fig. 1A). It is not surprising that nonsatellite D4Z4 subtelomeric repeats predominantly located at the contribution of Sat2 demethylation to global hypomethylation Chr4q35 and Chr10q26, and interspersed Alu elements have been could go undetected using a genome-wide assessment of 5- shown to be demethylated in several cancers, but their methylation methylcytosine content because it is estimated that only f4% of status in gliomas or normal brain is not known. Using bisulfite genomic CpGs are derived from classic satellite repeats (6). sequencing analyses, we found that DNA methylation content of In contrast to satellite DNA, extensive demethylation of the Sat2 was higher than that of D4Z4 repeats in brain (range %5mC: interspersed Alu element paralleled severe genomic hypomethyla- 90-91% and 72-79%, respectively), whereas interspersed Alu tion in one of two GBMs (51% in GBM30; 10% in GBM28; Fig. 2C). It elements collectively showed a 5-methylcytosine content interme- is of interest to consider the effect of Alu demethylation on the global diate to juxtacentromeric and subtelomeric satellite end regions, levels of 5-methylcytosine in light of its considerable abundance with values ranging from 82% to 84% (Fig. 2A). Two GBMs with in the human genome. On average, 36.7% of CpG dinucleotides have highest and two with lowest 5-methylcytosine content were not undergone sequence drift or spontaneous deamination accord- selected for analysis and, among these, a general correspondence ing to our bisulfite sequencing analyses. If we take into account that that there are 22 CpG dinucleotides in the consensus Alu repeat (Genbank accession no. U14567) and there are f1 to 1.4 million copies of Alu per haploid genome, then it can be estimated that Alu repeats contribute f8 to 11 million CpGs to DNA methylation in the human genome. A reduction of 51% in GBM30 would be estimated to include loss of methylation at 4 to 5 million CpGs. Demethylation of other abundant interspersed repetitive elements, such as the long interspersed nucleotide elements LINE-1, may also contribute extensively to genomic hypomethylation in some GBMs. Sat2 demethylation is associated with copy number alterations of adjacent euchromatin. We investigated whether satellite repeat DNA hypomethylation was associated with copy number alteration of nearby genomic sequences using array comparative genomic hybridization (aCGH) analysis. Concomitant with Sat2 hypomethylation, a gain in copy number spanning z1.6 Mb (RP11-29M22 to RP11-192J8; 1p11-12, UCSC May 2004 freeze), and a deletion of z35.3 Mb (RP11-29M22 to RP11-254E16; 1p11-22, UCSC May 2004 freeze) were noted in the pericentromeric region of chromosome 1p for severely hypomethylated GBM28 and GBM30, respectively (Fig. 2B). Additionally, a Chr1q copy gain with a 1q12 breakpoint was found in GBM28. For GBM30, copy number in the 1q12 region was not interpretable. Interestingly, no alteration was detected in the pericentromeric region of Chr1 for GBM19 and GBM27, which had only small decreases in Sat2 methylation (Fig. 2A). Cumulatively, these observations lend support to the Figure 1. Global hypomethylation is a common event in primary GBMs and established glioma cell lines. A, 8 of 10 primary GBMs (numbers on the X-axis) proposal that classic satellite DNA hypomethylation predisposes to exhibit significant loss of genomic 5-methylcytosine content (*) compared with chromosomal breakage and specific copy number alteration at this normal brain controls, as determined by the methyl acceptor assay. The status of ‘‘genomic hypomethylation’’ was attributed to GBMs that exhibited a percentage region of chromosome 1 in GBMs. 5-methylcytosine value 2 SDs below the mean of triplicate measurements of MAGEA1 promoter demethylation and gene expression NB2. B, established glioma cell lines exhibit hypomethylation relative to in extensively hypomethylated GBMs and glioma cell lines. uncultured normal brain and lymphocyte samples. NB1, normal brain 1 from adult gray matter; NB2, normal brain 2 from adult gray and white matter. PBL We examined whether a relationship exists between MAGEA1 values represent the combined measurements from two individuals. expression and the extent of genomic hypomethylation in primary www.aacrjournals.org 8471 Cancer Res 2006; 66: (17).September 1, 2006

Figure 2. Demethylation of juxtacentromeric DNA contributes to global reduction in 5-methylcytosine and is associated with copy number changes in GBMs. A, bisulfite sequencing of representative Sat2 repeats (chromosomes 1 and 16 juxtacentromeric DNA) reveals important contributions to global hypomethylation in GBMs. o, unmethylated CpG; ., methylated CpG; Â, CpG site lost from consensus sequence, including TpA sequences representing possible spontaneous deamination of methylated CpGs on the opposite DNA strand. Each row of circles, sequence results of an individual clone of the bisulfite-PCR product. The number of methylated CpGs divided by total number of true CpGs analyzed is given as percentages below each bisulfite result. GBM28 and GBM30 exhibit global hypomethylation relative to normal brain, whereas GBM19 and GBM27 do not (Fig. 1). B, Sat2 juxtacentromeric hypomethylation is associated with copy number changes in the pericentromeric region of chromosome 1. Vertical bars, gains (right of the chromosome ideogram) and losses (left of the chromosome ideogram). Arrows, position of Sat2 repeats and MTHFR (1p36.22). C, bisulfite sequencing of D4Z4 (chromosomes 4q and 10q subtelomeric DNA) and Alu elements reveal important contributions of these repetitive sequences to global hypomethylation in GBMs. o, unmethylated CpG; ., methylated CpG; Â, CpG site lost from consensus sequence, including TpA sequences representing possible spontaneous deamination of methylated CpGs on the opposite DNA strand. Each row of circles, sequence results of an individual clone of the bisulfite-PCR product. The number of methylated CpGs divided by total number of true CpGs analyzed is given as a percentage below each bisulfite result. GBM28 and GBM30 exhibit global hypomethylation relative to normal brain, whereas GBM19 and GBM27 do not (see Fig. 1).

GBMs and cultured glioma cell lines. MAGEA1 expression was treated cells (Fig. 3C). Because promoter methylation of MAGEA1 silenced in normal brain, but found to be reactivated in two tumors is a primary mechanism silencing this gene in somatic tissues (35), that exhibited the most dramatic genome-wide demethylation we next evaluated whether MAGEA1 reactivation in GBM was (Fig. 3A). Also, MAGEA1 was reactivated at a higher frequency in coupled with demethylation of its promoter. Although MAGEA1 glioma cell lines relative to their primary tumor counterparts silencing in normal brain and GBMs was associated with significant (Fig. 3B), coinciding with the more common occurrence of global methylation at critical CpG sites (35), the extent of demethylation at hypomethylation in glioma cell lines (Fig. 1B). Interestingly, both these CpG sites was paralleled by the degree of MAGEA1 expression primary GBMs and cultured glioma cells that were positive for in hypomethylated GBMs (Fig. 3D). For example, the more MAGEA1 expression were segregated from nonexpressing tumors abundant expression of MAGEA1 detected in GBM30 relative to by exhibiting a global 5-methylcytosine threshold of f50% or less GBM28 correlated with a more extensive demethylation of the (the mean of MAGEA1+ and MAGEA1À samples were significantly MAGEA1 promoter in GBM30 (Fig. 3A and D). Similarly, MAGEA1 different, P = 0.003; Fig. 4). expression was higher and promoter demethylation more pro- To determine whether DNA methylation exerted a negative nounced in glioma cell lines LN229 and LN18 relative to LN319 influence on MAGEA1 expression, nonexpressing glioma cell lines (Fig. 3B and D). were exposed to the demethylating agent 5-aza-2¶-deoxycytidine. Reduced global 5-methylcytosine content is associated with In each cell line examined, MAGEA1 expression was induced upon the presence of the low-functioning Val allele of MTHFR. drug treatment, but remained silenced in corresponding mock- We screened for the low-functioning C667T variant of MTHFR,a

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Hypomethylation in GBM rate-limiting enzyme in the production of the universal methyl GBMs with severe genomic hypomethylation and LOH of donor, SAM, in primary GBMs and glioma cell lines using PCR MTHFR exhibit a very high proliferation index. A distinguishing followed by RFLP analysis (28). In primary GBMs, genotype feature of GBMs is their high rate of proliferation relative to lower frequencies were 30% for C/C homozygous (Ala/Ala), 30% for grade gliomas. Using MIB-1 immunoreactivity as an indicator of C/T heterozygous (Ala/Val), and 40% for T/T homozygous (Val/Val) the proliferative index of GBM, all 10 GBM samples exhibited a MTHFR (Fig. 5A). A similar distribution of the MTHFR genotypes significant fraction of mitotically active cells. Relative to the was observed among glioma cell lines (Fig. 5B). The MTHFR gene is two GBMs without hypomethylation and the six with moderate located on chromosome 1p36, in a region previously shown to hypomethylation, the two severely hypomethylated GBMs had a exhibit deletion in a proportion of GBMs (2). Among the 10 GBMs, much higher mitotic index (33.9% for GBM28 and 69.1% for GBM24, GBM28, GBM30, and GBM31 displayed a clear allelic loss GBM30; Fig. 6). These two highly proliferative GBMs also had of the region encompassing MTHFR (Supplementary Fig. S1). LOH for MTHFR. These data suggest a model whereby reduced Normal tissue from these patients was not available, precluding the MTHFR activity could cooperate with enhanced proliferation to determination of the original MTHFR genotype. The frequency of exacerbate genomic hypomethylation and chromosome breakage the Val/Val genotype for MTHFR is f10% in the general population events. (36). The higher than expected prevalence (40%) of apparent homozygosity for the low-functioning allele of MTHFR in primary GBMs results in part from loss of heterozygosity (LOH) at Chr1p36 Discussion in GBM24 and GBM28. Genome-wide hypomethylation is a hallmark of many cancers Notably, MTHFR allelic loss occurred in GBMs displaying and is thought to contribute to tumorigenesis independently of significant degrees of genomic hypomethylation, including the CpG island hypermethylation (9, 33, 37). Expanding on early two most extensively hypomethylated tumors, highlighting a observations from a very limited number of gliomas (5, 38), we potentially important requirement of normal MTHFR activity in found that genomic hypomethylation occurs at a high frequency the maintenance of normal DNA methylation levels. This is in GBM and GBM cell lines. Interestingly, global hypomethylation reinforced by the observation that the only tumor with an Ala/ was not observed in two primary GBMs, indicating that a small Ala genotype (GBM19) showed no reduction in total 5-methyl- proportion of GBMs may arise in the absence of extensive cytosine, whereas hypomethylated tumors tended to carry at least demethylation, although these two tumors exhibit clear differences one copy of the lower functioning (Val) allele for MTHFR or LOH in in genetic and proliferative characteristics relative to severely the MTHFR region. Similarly, the presence of the Val allele was hypomethylated tumors. However, the whole-genome approach to associated with a more profound reduction in global 5-methyl- quantify DNA methylation is not sufficiently sensitive to detect cytosine content in three of four glioma cell lines (Figs. 1B and 5B). potentially important regional reductions in 5-methylcytosine that These results suggest that the constitutional MTHFR genotype, and can contribute to genomic instability. The high prevalence of modifications to it via distal Chr1p loss in the tumor may be one genomic hypomethylation in GBM supports an important role for cause of hypomethylation in GBM. 5-methylcytosine reduction in GBM pathology.

Figure 3. MAGEA1 is expressed in the most extensively hypomethylated primary GBMs and glioma cell lines. A, reverse transcription-PCR (RT-PCR) analysis of the normally repressed cancer-testis antigen MAGEA1 uncovers its reactivation in the two GBMs that exhibit the most extensive global hypomethylation. B, reactivation of MAGEA1 occurs in the three glioma cell lines that exhibit an extent of global 5-methylcytosine reduction comparable with MAGEA1-positive primary GBMs. C, MAGEA1 expression is induced in MAGEA1-negative glioma cells undergoing experimental hypomethylation through exposure with the demethylating agent 5¶-aza-2¶-deoxycytidine. D, bisulfite sequence analysis of the MAGEA1 promoter region reveals that demethylation at CpG sites 3 and 4, which are each within Ets transcription factor binding sites whose methylation abrogates expression, as well as a stretch of CpG sites (7-11) overlapping the transcription start site, correlates with gene reactivation as previously reported (35). o, unmethylated CpG; ., methylated CpG.

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defective p53 or RB pathways, that themselves drive additional chromosomal instability. Unlike Sat2, demethylation of the D4Z4 repeat did not occur in GBMs with normal global DNA methylation levels, and was manifested to a lesser extent than Sat2 in severely hypomethylated GBMs. The distinct behavior of Sat2 and D4Z4 methylation patterns in GBM suggests that either regional or sequence-specific determinants influence the propensity for demethylation of repeat regions in cancer. It will be interesting to determine whether differential methylation between distinct classes of repetitive elements occurs in other tissues, and whether these are under the influence of sequence-specific determinants because of the important consequences these differences could bear on the manifestation of cell type–specific patterns of genomic instability resulting from genomic hypomethylation in cancer. The level of MAGEA1 expression in GBM correlated with the level of demethylation at key CpG sites within its promoter, indicating that promoter methylation is a primary mechanism for silencing MAGEA1 in brain. It was recently shown that Brother of Figure 4. Aberrant MAGEA1 expression in primary GBMs and glioma cell lines segregates with a global hypomethylation threshold below f50%. Conventional the Regulator of Imprinted Sites (BORIS) actively promotes RT-PCR analysis reveals that the normally silenced MAGEA1 gene is demethylation/derepression of MAGEA1 (41) by competing with reactivated in primary GBMs and established glioma cell lines that contain an its paralog CTCFfor binding in a methylation-insensitive manner f50% or less 5-methylcytosine content, suggesting a global hypomethylation threshold associated with the derepression of this cancer-testis antigen. Note in the promoter region of this gene. Like MAGEA1, BORIS that the percentage of 5-methylcytosine content of normal brain was not included expression is restricted to hypomethylated germ cells during in the calculation of the mean value (horizontal bar) in MAGEA1-negative spermatogenesis in conjunction with CTCFsilencing, and is samples. aberrantly reactivated in various cancers (42). It is, therefore, possible that MAGEA1 expression in GBM is not a random Global decreases in methylation correlate with advancing age consequence of severe genome-wide hypomethylation, but may (39) as does overall cancer incidence. The extent of the age-related result from epigenetic modifications targeted by CTCF/BORIS– decrease seems to be significantly less than that observed in binding sequences. It will be interesting to test whether aberrant tumors. The extent of global DNA methylation changes in aging BORIS expression occurs in GBM in association with progressive brain, if any, is not known. We did not observe a relationship genome-wide hypomethylation or itself leads to genome-wide between the extent of genomic hypomethylation and age of the hypomethylation in brain cancer. This proposed scenario might patients with GBM, suggesting that hypomethylation is primarily a explain a threshold effect for the reactivation of the downstream feature of the GBM rather than aging. target MAGEA1, although this remains to be determined. Several observations lend support to the proposal that regional The expression of cancer-testis antigens are being exploited for chromosomal rearrangements may be attributable to repeat- the development of immunologically based therapy in cancer, specific hypomethylation in GBM. First, there was a trend between the methylation status of the juxtacentromeric Sat2 DNA repeat region and global DNA methylation levels in GBM, similar to that reported in cancers of the breast (20), liver (24), ovary (37), and in Wilms’ tumors (9). Second, severe demethylation of Sat2 in two GBMs was accompanied by copy number alterations defined by apparent breaks precisely next to the centromere-adjacent region of Chr1p, whereas this region was of normal copy number in tumors with marginal decreases in Sat2 methylation. Last, similar to that previously noted in human hepatocellular carcinoma (24), a Chr1q copy gain with a 1q12 breakpoint was also found in one GBM, which correlated with Sat2 hypomethylation in this tumor. In interpretation of the Sat2 methylation status and Chr1 copy number alterations, it should be noted that we cannot distinguish the Sat2 elements of Chr1 from those of Chr16 using the bisulfite assay. Other categories of chromosomal rearrangements might be relevant to hypomethylation-induced chromosome breakage, but are not detectable by aCGH, such as inversions and translocations. In contrast to Sat2 demethylation, alteration of the size and/or methylation status of D4Z4 repeats are thought to give rise to Figure 5. Genotype of the MTHFR C667T polymorphism in primary GBMs and altered gene expression of nearby genes, rather than leading to glioma cell lines. Restriction enzyme analysis of the 198 bp PCR product from chromosome breakage events (40). One must also consider the genomic DNA amplification containing the C667T polymorphism that leads to the Ala222 to Val change in MTHFR. The C667T substitution creates a HinfI proposed model of hypomethylation-induced chromosome break- recognition sequence, which digests the 198 bp PCR product into 175 and 23 bp age in the larger context of know genetic alterations, such as fragments; the latter fragment has been run off the gel.

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enzymatic components of the methyl group metabolism pathway, such as methionine synthase and cystathionine b-synthase (12), which could synergize with reduced MTHFR activity. Alternatively, chronic exposure to exaggerated levels of micronutrients—for example, the >1,000-fold excess of folic acid in GBM culture medium relative to normal serum levels—may lead to a down- regulation of genes that enable the capacity of the cell to adequately use these micronutrients. Interestingly, a more elevated mitotic index observed in the two severely hypomethylated GBMs was accompanied by Chr1p deletion spanning MTHFR in these tumors. A clear inverse Figure 6. Elevated proliferation index in primary GBM is associated with severe relationship was observed between elevated proliferation and genomic hypomethylation and LOH of MTHFR. MIB-1 immunohistochemical genomic hypomethylation in cultured PBL (33). Similarly, a high staining reveals that the primary GBMs with severe genomic hypomethylation exhibit a more elevated rate of proliferation (GBM28 and GBM30). These frequency of Sat2 hypomethylation and Chr1 pericentromeric proliferative tumors are also marked by LOH of MTHFR (indicated below). GBMs rearrangements are also observed in mitogen-stimulated lympho- are plotted in order of increasing genomic hypomethylation. cytes isolated from ICFpatients with DNMT3B mutations (22), and increased proliferative activity and DNA hypomethylation co-occur including MAGEA1 in GBM (43). However, despite its expression in in the rectal mucosa of patients with ulcerative colitis (46). f40% of primary cultures of human GBM and glioma cell lines Whether genomic hypomethylation leads to a deregulated cell (43), MAGEA1 mRNA was not detected in 20 noncultured primary cycle in cancer or tumor-associated proliferation drives hypome- GBMs previously (44) nor in 8 of 10 primary GBMs in our study. thylation is not known. We hypothesize that cells that are deficient Our study now shows that promoter hypomethylation is one likely for MTHFR activity are unable to sustain adequate levels of SAM mechanism of MAGEA1 reactivation in GBM. These findings necessary for maintaining normal genomic DNA methylation in reinforce the proposal that a DNA demethylating agent may allow instances of increased cellular proliferation, and that these cells for enhanced sensitivity to immunotherapy against MAGEA1 and gradually accrue genomic instability as a consequence of be of benefit for the clinical management of GBM (43). In fact, a progressive hypomethylation. This model predicts that genomic similar strategy was recently shown to confer responsiveness of hypomethylation will be less frequently encountered in lower grade GBM cells to tumor-necrosis factor-related apoptosis-inducing gliomas, which are characterized by a lower proliferative index ligand–induced death through reactivation of caspase-8 (45). than GBMs despite the high incidence of Chr1p deletion There seems to be an important contribution of MTHFR allelic encompassing MTHFR in oligodendrogliomas. status to genomic hypomethylation in GBM. First, the low Experimental hypomethylation shifts the spectrum of tumor functioning (Val) allele of MTHFR tended to be present in primary types induced by genetic events in mice. Pioneering studies showed GBMs and glioma cell lines that displayed varying degrees of that DNA hypomethylation strongly suppresses overall tumorigen- genomic hypomethylation. Conversely, there was no global esis in the intestine of ApcMin/+ mice (47). Similarly, induced demethylation in a GBM that was homozygous for the normal hypomethylation has recently been shown to be protective against (Ala) MTHFR allele. Second, aCGH indicated a reduction in copy the onset of tumorigenesis in a mouse model of prostate cancer number of Chr1p in a region that encompasses MTHFR in four (48). In contrast, DNMT1 deficiency alone is sufficient to induce hypomethylated GBMs, including two tumors with the most severe sarcomas in mice (13). Therefore, given the high frequency of reduction in total 5-methylcytosine, supporting a protective role genomic hypomethylation of GBM, it will be of considerable for normal MTHFR activity against genomic demethylation. It is interest to determine whether genome-wide demethylation exerts a important to note that the deletions involving MTHFR are not promoting or protective influence on the progression of this contiguous with the copy number alterations near Sat2 on the disease. same chromosome, suggesting they arise from at least two different Due to the limited number of cases analyzed, it was not possible breakage events. Similarly, the presence of either the Val/Val and to determine whether genomic hypomethylation, or lack thereof, Ala/Val genotypes of MTHFR were found to be strongly associated has prognostic value in GBM. In fact, the coordinate expression of a with a low content of 5-methylcytosine in the DNA of both tumors group of cancer-testis antigens has recently been shown to be and adjacent normal tissue of the patients with either colorectal, indicative of advance disease and predict poor outcome in patients breast, or lung cancer (12). Taken together, these observations with non–small cell lung carcinoma (49). In this respect, it will be suggest an early influence of MTHFR status on susceptibility to pertinent to address whether severe genomic hypomethylation or progressive hypomethylation that may lead to or exacerbate MAGEA1 expression is associated with expression patterns that chromosomal instability in GBM. define recently recognized subsets of GBM, in particular those A manifestation of the Val allelotype of MTHFR upon genomic characterized by a proliferative gene expression pattern and poor hypomethylation is presumed to occur through reduced SAM prognosis (50). production and is contingent upon limited nutritional folate and vitamin B6 bioavailability (27). Interestingly, all cultured glioma cell lines exhibited significant hypomethylation irrespective of MTHFR Acknowledgments status despite their growth in supraphysiologic levels of all methyl Received 4/26/2006; revised 6/13/2006; accepted 7/6/2006. group donors. Unlike normal fibroblasts, genome-wide demethy- Grant support: NIH R01 CA094971 (J.F. Costello), NIH Specialized Programs of lation in GBM may not be reversible by nutritional supplementa- Research Excellence grant in brain tumors at UCSF(B. Cadieux and S.R. VandenBerg). The costs of publication of this article were defrayed in part by the payment of page tion either due to the presence of genetic alterations that disrupt charges. This article must therefore be hereby marked advertisement in accordance the normal activity of DNMTs or polymorphisms in other key with 18 U.S.C. Section 1734 solely to indicate this fact. www.aacrjournals.org 8475 Cancer Res 2006; 66: (17).September 1, 2006

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2006 American Association for Cancer Research. Genome-wide Hypomethylation in Human Glioblastomas Associated with Specific Copy Number Alteration, Methylenetetrahydrofolate Reductase Allele Status, and Increased Proliferation

Benoît Cadieux, Tsui-Ting Ching, Scott R. VandenBerg, et al.

Cancer Res 2006;66:8469-8476.

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