epigenetic aberrations and therapeutic implications in gliomas

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Review Article

Epigenetic aberrations and therapeutic implications ingliomasAtsushi Natsume,1,2 Yutaka Kondo,3 Motokazu Ito,1,2 Kazuya Motomura,1 Toshihiko Wakabayashi1

and Jun Yoshida1,4,5

1Department of Neurosurgery and 2Center for Genetic and Regenerative Medicine, Nagoya University School of Medicine, Nagoya; 3Division of MolecularOncology, Aichi Cancer Center Research Institute, Nagoya; 4Higashi-Nagoya National Hospital, Nagoya, Japan

(Received January 25, 2010 ⁄ Revised February 16, 2010 ⁄ Accepted February 17, 2010 ⁄ Accepted manuscript online February 27, 2010 ⁄ Article first published

online April 6, 2010)

Almost all cancer cells have multiple epigenetic abnormalities,

which combine with genetic changes to affect many cellular pro-

cesses, including cell proliferation and invasion, by silencing

tumor-suppressor genes. In this review, we focus on the epige-

netic mechanisms of DNA hypomethylation and CpG island hyper-

methylation in gliomas. Aberrant hypermethylation in promoter

CpG islands has been recognized as a key mechanism involved inthe silencing of cancer-associated genes and occurs at genes with

diverse functions related to tumorigenesis and tumor progression.

Such promoter hypermethylation can modulate the sensitivity of

glioblastomas to drugs and radiotherapy. As an example, the

methylation of the O6-methylguanine DNA methyltransferase

(MGMT) promoter is a specific predictive biomarker of tumor

responsiveness to chemotherapy with alkylating agents. Further,

we reviewed reports on pyrosequencing – a simple technique for

the accurate and quantitative analysis of DNA methylation. We

believe that the quantification of MGMT methylation by pyrose-

quencing might enable the selection of patients who are most

likely to benefit from chemotherapy. Finally, we also evaluated

the potential of de novo NY-ESO-1, the most immunogenic cancer/

testis antigen (CTA) discovered thus far, as an immunotherapy

target. The use of potent epigenetics-based therapy for cancercells might restore the abnormally regulated epigenomes to a

more normal state through epigenetic reprogramming. Thus,

epigenetic therapy may be a promising and potent treatment

for human neoplasia. (Cancer Sci 2010; 101: 1331–1336)

Epigenetic abnormalities in human neoplasia

A berrant epigenetic mechanisms, such as promoter hyper-methylation, histone modifications, or non-coding RNA

expression, are recognized as being important in tumor forma-tion.(1) These epigenetic mechanisms comprise the ‘‘third path-way’’ in Knudson’s model of tumor-suppressor geneinactivation and can affect gene expression without effectinggenetic changes.

(2,3)It is now known that almost all types of

cancer cells have multiple epigenetic abnormalities, which com-bine with genetic changes to affect many cellular processes,including cell proliferation and invasion, by silencing tumor-suppressor genes.

(4)Therefore, research on epigenetic dysregu-

lation is now as commonplace as that on the genetic etiology of human cancers. The fundamental components of cancer epige-netics are the DNA methylation pattern; nucleosome remodel-ing; and a series of acetylation, methylation, and othermodifications at key amino acid residues in histones. Overall,these epigenetic alterations disturb the chromatin structure, lead-ing to abnormal gene expression and tumor formation.

During the early development of mammals, DNA methylationoccurs at the cytosine residues in cytosine-guanine sequences

(CpGs) in the DNA; this methylation pattern is heritable.(5)

In humans, approximately 70% CpG dinucleotides, which aregenerally located in repetitive DNA sequences, are methylated.Other than the methylated CpGs, clusters of unmethylated CpGsare present in the genome, and these clusters are called CpGislands.

(6,7)Around 60% of genes have CpG islands in their 5¢-

promoter regions, and DNA methylation of these islands resultsin irreversible inhibition of gene expression. A recent genome-wide analysis revealed that CpG islands are also found innon-promoter regions: around 50% of the CpG islands are notpresent in association with annotated promoters. In addition,epigenetic abnormalities causing the loss of gene function aremore frequent than genetic abnormalities in cancer cells.

(4,8,9)

Thus, cellular epigenetic inheritance mediated by aberrantDNA methylation that leads to gene silencing, gene imprinting,and ⁄ or activation of cancer-associated genes is now accepted asan important factor defining the transformed phenotype.

DNA hypomethylation and CpG island hypermethylationin gliomas

Hypomethylation has been reported to occur in repetitive ele-ments localized in satellite sequences or pericentromericregions, thus resulting in genomic instability in several cancersincluding glioblastoma.

(10,11)Genome-wide hypomethylation

occurs at a high frequency (80%) in primary glioblasto-mas.

(11–13)The level of hypomethylation ranges from nearly

normal to approximately 50% of the normal level in approxi-mately 20% cases; the latter reflects the massive demethylationof approximately 10 million CpG sites per tumor cell.

In glioblastomas, both repetitive sequences and single-copyloci can be hypomethylated. Glioblastomas with global hypome-thylation show remarkable hypomethylation (22–55% of normalbrain) of the tandem repeat satellite (Sat2) at the pericentromericregions of chromosomes 1, 9, and 16, and moderate hypomethy-lation (71–82% of normal brain) of D4Z4 at the subtelomeric

regions of chromosomes 4q35 and 10q26.(11,13)

Although it isnot as frequent as regional hypermethylation in promoter CpGislands, regional hypomethylation at single-copy loci in glio-blastomas is associated with the activation of some cancer-asso-ciated genes, such as the melanoma antigen gene ( MAGEA1).(14)

Activation of the cancer-testis antigen genes by the epigeneticregulation is described in greater detail in the following section.Another example associated with hypomethylation in glioma isthe loss of imprinting of the insulin-like growth factor 2 ( IGF-2)

5To whom correspondence should be addressed.E-mail: [email protected]

doi: 10.1111/j.1349-7006.2010.01545.x Cancer Sci | June 2010 | vol. 101 | n o. 6 | 1331–1336ªª 2010 Japanese Cancer Association



gene,(15) and it has been known to induce tumor develop-ment.(16)

Locus-specific hypermethylation, which mostly occurs at pro-moter CpG islands, is also frequently observed in gliomas. Ingliomas as well as other malignancies, CpG island hypermethy-lation in promoters occurs in genes with diverse functionsrelated to tumorigenesis and tumor progression, DNA repair,apoptosis, angiogenesis, invasion, and drug resistance. Forexample, promoter hypermethylation in the genes CDKN ⁄ p16 ,

RB, PTEN , TP53, and p14 ARF affects the RB, PI3K, and p53

pathways.

(17–21)

In glioma cells, CpG island hypermethylationmay occur at genes that are not expressed in the brain; this sug-gests that not all instances of CpG island methylation are func-tionally important for tumorigenesis. Loss of heterozygosity inchromosome 19q that is frequently observed in gliomas has sug-gested the presence of a tumor suppressor gene.(22–24) Screensfor promoter hypermethylation in glioma discovered a candidatetumor suppressor, epithelial membrane protein 3 (EMP3).(25)

EMP3 is a myelin-related gene involved in cell proliferation andcell–cell interaction.

Methylation of the MGMT promoter and the resultantresponse to drugs and radiotherapy

Promoter hypermethylation can modulate the sensitivity of

glioblastomas to drugs and radiotherapy. This area has beenintensively investigated. One example is suppressor of cyto-kine signaling 1 (SOCS1), which is silenced by hypermethyla-tion. Re-activation of SOCS1 in glioma sensitized to radiationvia inactivation of the MAPK pathway.

(26)Epigenetic profiling

might shed light on the catalog of glioma and patient-specifictherapy. The best known example is MGMT promoter methyl-ation and the resultant response to DNA alkylating agents. Analkylating agent kills cells by forming cross-links betweenadjacent strands of DNA, thus inhibiting DNA replication.However, the effectiveness of such alkylating agents is fre-quently hampered by inherent or acquired resistance. The maindeterminant of the resistance to alkylating agents is the activ-ity of MGMT, which directly and specifically removes thecytotoxic alkyl adducts formed at the O

6position of guanine

by these alkylating agents.

(27)

Because the MGMT gene is notusually mutated or deleted, DNA methylation – the main epi-genetic modification seen in cancer cell lines and primarytumors – may cause a reduction in the MGMT levels.

(28)

These observations highlight the importance of MGMT meth-ylation as a specific predictive biomarker for the responsive-ness to chemotherapy with alkylating agents. Indeed, MGMT methylation is associated with significantly longer survival inglioblastoma and other gliomas treated with radiation andalkylating agents, such as temozolomide (TMZ).(29,30)

Although it is unclear if this is directly due to reduced MGMTexpression, the predictive value of MGMT methylation is lar-gely confirmed in a number of prospective and retrospectiveclinical investigations. The clinical outcome after TMZ ther-apy depends on the methylation status of the MGMT promoter;

MGMT modification is one of the key factors that couldenhance the clinical benefits of this treatment. In a previousin vitro study on human glioma cells, we found that b-interferonmarkedly enhanced chemosensitivity to TMZ;

(31)this finding

suggested that one of the major mechanisms by which b-inter-feron enhances chemosensitivity is the down-regulation of MGMT transcription via p53 induction. This effect was alsoobserved in an experimental animal model.

(32)The results of

these two studies suggested that compared to chemotherapywith TMZ alone and concomitant radiotherapy, chemotherapywith b-interferon and temozolomide with concomitant radio-therapy might further improve the clinical outcome of malig-nant gliomas.

Pyrosequencing for the quantification of DNAmethylation

MGMT levels vary widely according to the type of tumor andalso among tumors of the same type.

(33)For example, approxi-

mately 30% gliomas have reduced levels of MGMT because of methylation of the corresponding gene, whereas MGMT methyl-ation is rare or absent in other tumor types.

(34–36)Some reports

describe the detection of MGMT methylation based on amodified methylation-specific polymerase chain reaction(MSP).

(37,38)This method enables cost-efficient analysis of

MGMT promoter methylation. However, it is not quantitativeand poses the risk of false-positive or false-negative results,especially when the DNA quality and ⁄ or quantity are low; thisis often the case in clinical settings wherein samples aretypically obtained from formalin-fixed paraffin-embeddedspecimens. Further technical studies are necessary for the devel-opment of an MGMT methylation assay. Mikeska et al. com-pared and optimized three quantitative techniques of MGMT methylation, that is combined bisulfite restriction analysis(COBRA), SNuPE ion pair-reverse phase high-performanceliquid chromatography, and pyrosequencing.(39) They concludedthat pyrosequencing assay provides the most accurate and mostrobust MGMT methylation marker.

Pyrosequencing technology is based on the sequencing-by-

synthesis principle, and it involves a simple technique for accu-rate and quantitative analysis of DNA sequences.(40–42) In thismethod, a sequencing primer is hybridized to a single-stranded,PCR-amplified DNA template and incubated with a DNA poly-merase. Each event of incorporation of a deoxynucleotidetriphosphate (dNTP) is accompanied by the release of pyrophos-phate (PPi) in a quantity equimolar to the amount of nucleotideincorporated. This PPi is quantitatively converted to ATP byATP sulfurylase in the presence of adenosine 5¢-phosphosulfate.The ATP thus obtained drives the luciferase-mediated conver-sion of luciferin to oxyluciferin, thus generating visible light inamounts proportional to the amount of ATP generated. The lightproduced in this luciferase-catalyzed reaction is detected by acharge-coupled device (CCD) camera and seen as a peak in apyrogram. Apyrase – a nucleotide-degrading enzyme – continu-

ously degrades unincorporated dNTPs and excess ATP, duringsynthesis of the complementary DNA, and the nucleotidesequence is determined from the signal peak during pyrogramtracing

(43–45)(Fig. 1). We have shown that the methylation

value obtained using pyrosequencing correlates well with themethylation status and MGMT expression in glioma cell lines(Fig. 2). However, in contrast to cell lines which comprise rela-tively homogenous cells, bulk tumors comprise histologicallyheterogenous cells. Pyrosequencing revealed that one part of thetumor exhibited high MGMT expression with 18% methylationin the nucleus, whereas a different lesion in the same tumorexhibited low MGMT expression with 53% methylation (Fig. 3).However, with MSP, both these areas were found to be unme-thylated; that is, MSP failed to detect the difference betweenthem. The quantification of MGMT methylation by pyrosequenc-

ing might enable the selection of patients who are most likely tobenefit from chemotherapy.

Epigenetic therapy for human glioblastoma multiformes(GBMs)

Malignant glioma represents about 20% of all intracranialtumors. Despite advances in radiation therapy and chemotherapyadministered after the surgical resection, the prognosis of malig-nant glioma remains poor with a median survival of lessthan10 months.(46) In contrast to genetic alterations, epigeneticmodifications such as promoter hypermethylation and histoneacetylation are theoretically reversible by drug treatment. In the

1332 doi: 10.1111/j.1349-7006.2010.01545.xªª 2010 Japanese Cancer Association



context of epigenetic therapy for cancer, while it is necessary tounderstand the precise contribution of epigenetic abnormalitiesto the development of glioblastomas, treatment directed towardrestoring the function of genes that have been silenced by epige-netic changes in glioma cells has the potential of ‘‘normalizing’’

cancer cells. Therefore, DNA methylation has now become atherapeutic target for human cancers.(47,48) DNA methylation isstrongly inhibited by the cytosine analogs 5-azacytidine and5-aza-2¢-deoxycytidine (DAC); these agents are incorporatedinto DNA during cell division, following which they trap DNAmethyltransferases and degrade them.(48–50) Low-dose exposureto these agents induces cell differentiation and represses cellgrowth through demethylation. The use of these demethylatingagents in the treatment of myelodysplastic syndrome hasreceived FDA approval, and clinical trials in this regard arecurrently underway in Japan. But DAC has never been testedin clinical trials for gliomas.

One of the most promising treatments for solid tumors byepigenetic drugs may be the combination of DAC and immuno-therapy. Under normal conditions, a subgroup of tumor-specific

antigens called the cancer ⁄ testis antigens (CTAs) is expressedonly in the tissues of the testis, ovary, and placenta; however,these antigens are also expressed in various types of humantumors.

(51,52)Since normal CTA-expressing tissues do not

express major histocompatibility complex (MHC) class I mole-cules, CD8 T cells cannot recognize the CTAs expressed onthem; this suggests that CTAs are ideal targets for tumor immu-notherapy. The expression of CTAs in tumors results in therecognition of tumor-specific antigens on the cell surface bycytotoxic T lymphocytes (CTLs). Previously, we demonstratedthat the expression of CTA genes such as LAGE-1, CT7 , SCP-1,SSX-1, SSX-2, SSX-4, and NY-ESO-1 was nearly imperceptiblein 30 glioma tissues.

(53)Among these, NY-ESO-1 is the most

3′-CAGTGATGTGATGGAGTG

5′-GTCACTAC

PPi

Apyrase

C

C ATP Luciferase

C

U251nu/nu MGMT methylation: 3%AO2 MGMT methylation: 74%

ATP sulfurylase

Fig. 1. Pyrosequencing technology. A sequencing primer is hybridized to a single-stranded, PCR-amplified DNA template and incubated witha DNA polymerase. Each event of incorporation of a dNTP is accompanied by the release of pyrophosphate (PPi) in a quantity equimolar tothe amount of nucleotide incorporated. This PPi is quantitatively converted to ATP by ATP sulfurylase in the presence of adenosine5¢-phosphosulfate. The ATP thus obtained drives the luciferase-mediated conversion of luciferin to oxyluciferin, thus generating visible light inamounts proportional to the amount of ATP generated. The light produced in this luciferase-catalyzed reaction is detected by a charge-coupleddevice (CCD) camera and seen as a peak in a pyrogram. Apyrase – a nucleotide-degrading enzyme – continuously degrades unincorporateddNTPs and excess ATP, during synthesis of the complementary DNA. Gray columns indicate quantified CpG sites.

RT-PCR

GAPDH

MGMT

UMSP

M

Pyrosequencing

80

100

20

40

60

0NB

Fig. 2. Methylation status and mRNA expression in glioma cell linesand normal brain cells. Normal brain (NB) cells and the U251nu ⁄ nuglioma cells expressed MGMT, as determined by RT-PCR (upperpanel), and contained an unmethylated MGMT promoter, asdetermined by methylation-specific PCR (MSP) (middle panel).Pyrosequencing revealed that the methylation levels in NB andU251nu ⁄ nu cells were 2% and 3%, respectively (lower panel). MGMTwas not expressed in three other glioma cell lines (i.e. AO2, SKMG1,and U251SP), but the level of promoter methylation was very highin these cells (70–85%).

Natsume et al. Cancer Sci | June 2010 | vo l. 101 | no. 6 | 1333ªª 2010 Japanese Cancer Association



immunogenic CTA discovered thus far and is considered to bea very promising immunotherapy target.(54) It has been shownthat NY-ESO-1 is expressed in cancerous cells, probablybecause of the loss of epigenetic regulation that is observedwhen methylated chromatin regions are demethylated ordeacetylated histones are acetylated.(55) We demonstrated thateven though NY-ESO-1 is not expressed in human gliomacells, the prototypic inhibitor of DNA methylation – DAC –markedly reactivates NY-ESO-1 expression in glioma cells butnot in normal human cells. In addition, glioma cells induced toexpress NY-ESO-1 exhibit in vitro and in vivo sensitivity toantigen-specific CTLs.

(56)

It is widely accepted that histone modification and DNAmethylation are intricately interrelated; these mechanisms acttogether to regulate gene expression. The synergistic effect of DNA demethylation and histone deacetylase inhibition hasbeen investigated in detail.(57–59) DNA folds around a core of eight histones to form nucleosomes, which constitute thesmallest structural unit of chromatin. The basic amino-termi-nal tails of histones protrude out of the nucleosomes andundergo posttranslational modifications, including histoneacetylation and methylation. The acetylation status of thelysine residues in histones H3 and H4 is controlled by thebalanced action of histone acetyltransferases and HDACs.

(a) (b)

MGMT methylation; 18%

(c) (d)

MGMT methylation; 53%

Fig. 3. Intratumoral heterogeneity of MGMTexpression and pyrosequencing. One part of thetumor showed high MGMT expression with 18%methylation in the nucleus (a,b), as determined bypyrosequencing, whereas a different lesion in thesame tumor showed low MGMT expression with53% methylation (c,d). However, with methylation-specific PCR (MSP), both these areas were found tobe unmethylated. (a,c) Hematoxylin & Eosin stainedsections of two different lesions in a single tumor.(b,d) MGMT immunohistochemical stained sections.

+

Me

Me Me

Me Me Me MeNo treatment(a)

(b)

(c)

(d)

Transcription H3-K9

Me Me Me Me

Me Me Me Me

H3-K4

Me Me Me MeMe Me

VPA

MeAc Ac

DACMe

Me Me MeMe

Me

CG CG CG CG

Promoter Exon 1

CG CG CG CG

CG CG CG CG

CG CG CG CG

VPA + DAC

Me Me Me ++

Me Me MeMe

AcAc

Me Me Me Me

Me

Fig. 4. A schematic representation of the proposed mechanism underlying histone modifications in the NY-ESO-1 promoter. (a) Methylation oflysine 9, moderate methylation of lysine 4, and hypermethylation of the CpG islands caused the formation of a folded chromatin structure,leading to NY-ESO-1 gene silencing. (b) 5-aza-2¢-deoxycytidine (DAC) dramatically decreases lysine 9 methylation, demethylates the CpG sites,and unfolds the chromatin, thus reactivating gene expression. (c) VPA increases lysine 9 acetylation but has no effect on the methylation oflysine 9 or lysine 4 and does not reverse transcriptional silencing. (d) The combination of DAC and VPA decreases lysine 9 methylation andincreases lysine 9 acetylation, but does not affect lysine 4 methylation. This combination unfolds the chromatin to a considerable extent andreactivates gene expression with high efficiency.




Acetylated histones have often been associated with transcrip-tionally active or open chromatin. In contrast, the deacetylationinduced by HDACs results in chromatin compaction and geneinactivation. Histone methylation, mediated by histone meth-yltransferases, exerts different effects on gene expression,depending on the target residue. In fact, while the methyla-tion of histone H3 lysine 9 (H3-K9) is a marker of transcrip-tionally inactive chromatin, the methylation of H3-K4 isassociated with transcriptionally active chromatin.

(60)We

demonstrated that DAC and the HDAC inhibitor valproic acid

synergistically induced the de novo expression of NY-ESO-1.(61) This synergistic combination decreased the levels of methylated H3-K9 (H3-K9-diMe) and increased those of acet-ylated H3-K9 (H3-K9-Ac), while causing DNA demethylationin the NY-ESO-1 promoter region (Fig. 4). These data areconsistent with the results of many previous intensive studies.We also examined the expression of H3-K4-diMe – the mar-ker of active chromatin – in the same region. However, wedid not find any significant changes in the methylation statusof H3-K4; this finding suggested that compared to H3-K4-diMe, DNA methylation and H3-K9 modification are moredominantly associated with the expression of NY-ESO-1. Areason for the absence of a close correlation between NY-ESO-1 expression and H3-K4 modification might be thatH3K4-diMe is associated with ‘‘permissive’’ chromatin that

is either active or potentially active.

(62)

Nevertheless, our dataare quite consistent with the findings that DAC and valproicacid (VPA) synergistically reactivate NY-ESO-1. Thus, ourresults not only identify a potential epigenetic immunotherapy

but also suggest that the silencing mechanism of NY-ESO-1in gliomas is mediated by both DNA methylation and histonemodification.

Future perspectives

Beyond the issues raised by the clinical trial on the use of deme-thylating agents for the treatment of MDS and the extension of the trial findings to other human malignancies, epigenetic ther-apy may be a promising and potent treatment for human neopla-

sia. However, a large amount of research is still required forunderstanding the role of other components of the epigeneticmachinery in human neoplasia. For example, key amino acidresidues in histones are also targets of aberrant epigenetic pro-cesses such as a series of acetylation, methylation, and othermodifications.(1) Polycomb group proteins can regulate geneexpression independent of DNA methylation via H3-K27 trime-thylation, and early data suggest that this pathway is disruptedin cancer cells; thus, this pathway could represent a promisingtarget for cancer treatment.(63,64)

Overall, it is important to elucidate the precise role of epige-netic abnormalities in human neoplasia. The use of potent epige-netics-based therapy for cancer cells might restore theabnormally regulated epigenomes to a more normal statethrough epigenetic reprogramming. DNA methylation inhibitors

and ⁄

or a combination of agents targeting other epigenetic modi-fications can facilitate this process in vitro,(65)

and the use of such agents might be a promising treatment for cancer.

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epigenetic aberrations and therapeutic implications in gliomas

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