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  1. 1. MOLECULAR CARCINOGENESIS 49:532–544 (2010)Enhanced Expression of Cancer Testis AntigenGenes in Glioma Stem CellsToshio Yawata, Eiichi Nakai, Kae Chang Park, Takahiro Chihara, Ayano Kumazawa, Shinichi Toyonaga,Takanori Masahira, Hiromichi Nakabayashi, Takao Kaji, and Keiji Shimizu*Department of Neurosurgery, Kochi Medical School, Nankoku, Kochi, Japan Cancer stem cells are an important target for effective therapy, since they show tumorigenicity, chemoresistance,and radioresistance. We isolated cancer stem cells from glioma cell lines and tissues and examined the expression ofcancer testis antigen (CTA) genes as potential target molecules for cancer vaccine therapy. CTA genes were highly andfrequently expressed in cancer stem cells compared with differentiated cells. In addition, histone acetylation levels inthe promoter regions of CTA genes were high in cancer stem cells and low in differentiated cells, while DNAmethylation analysis of the promoter regions revealed hypomethylation in cancer stem cells. This epigenetic differencebetween cells leads to heterogeneous expression of CTA genes in the tumor mass, which consists of cells at variouslevels of differentiation. Moreover, the expression level of HLA class I antigens was not affected by the differentiationstatus, suggesting that CTA genes may present as surface antigens in cancer stem cells. Taken together, these findingssuggest that CTA genes may be attractive candidates for targeted vaccine therapy against cancer stem cells in gliomapatients. ß 2010 Wiley-Liss, Inc.Key words: cancer stem cells; epigenetics; cell differentiation INTRODUCTION expression of CTA genes is regulated by epigenetic The cancer stem cell (CSC) hypothesis suggests factors; namely, promoter DNA methylation andthat small populations of stem-like cells exist in the histone modifications [6], which in turn are thoughttumor mass and harbor drug resistance and low to be affected by the differentiation status of tumorradiosensitivity, and thus resulting in tumor recur- cells. We hypothesize that the heterogeneity ofrence after various treatments [1]. In addition, CSCs glioma tissue results from a hierarchical networkin tumors have also been identified as the sole related to the differentiation status of each cell,population with tumorigenicity [2]. These findings yielding nonunique expression of CTA genes.suggest that the eradication of CSCs could lead to a In this article, we demonstrate the transcriptionalcomplete cure of cancer. activation of the CTA gene locus in CSCs. We Glioblastoma multiforme (GBM) is the most investigated the expression of CTA genes and theircommon form of brain tumor. GBM is representative regulation in glioma cells at varying degrees ofof tumors containing populations of CSCs, in- differentiation. One CTA gene was expressed onlydicating potential for differentiation and highly in CSCs, not in differentiated cells, as was HLA class I,heterogeneous tissue. Recently, experimental immu- suggesting that the products of CTA genes may benotherapy using a mouse glioma model was shown immunogenic in CSCs.to be more effective with lysate of CSCs than that ofparent cells [3]; however, CSC-specific antigensremain to be elucidated. Cancer testis antigen (CTA) genes are attractive Additional Supporting Information may be found in the online version of this article.candidates for vaccine therapy, because other than Abbreviations: CSC, cancer stem cell; CTA, cancer testis antigen;in the testis, they are not expressed in normal tissues CTLs, cytotoxic T lymphocytes; NHA, normal human astrocyte; 5-[4]. These antigens are recognized by autologous aza-CdR, 5-aza-20 -deoxycytidine; VPA, valproic acid; ChIP, chroma-cytotoxic T lymphocytes (CTLs), which are restricted tin immunoprecipitation; GFAP, glial fibrillary acidic protein; TS, tumor spheres; HDACi, histone deacetylase inhibitor; NaB, sodiumby HLA class I antigens. Many vaccine therapies butyrate; HDAC, histone deacetylase.based on CTA-derived peptides have been developed *Correspondence to: Kohasu, Okoh-cho, Nankoku-City, Kochifor the treatment of various tumors. However, CTAs 783-8505, Japan.reportedly exhibit focal or heterogeneous expression Received 3 August 2009; Revised 25 November 2009; Accepted 1 December 2009in tumor tissues, and moreover, CTA-negative neo- DOI 10.1002/mc.20614plastic cells within the tumor mass can escape Published online 15 January 2010 in Wiley InterSciencefrom CTA-specific immune recognition [5]. The (www.interscience.wiley.com)ß 2010 WILEY-LISS, INC.
  2. 2. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 533 MATERIALS AND METHODS ing of live cells. After washing, cells were fixed with 0.5% paraformaldehyde in PBS at 48C for 1 h andCulture then incubated with PBS containing 0.1% Triton Human glioma U87MG, T98G, SNB19, and ONS-23 X-100 at room temperature for 5 min. The fixed cellscells were grown in DMEM supplemented with 10% were incubated with rabbit polyclonal anti-CD133fetal bovine serum in a humidified incubator at 378C antibody (1:200; Abcam, Cambridge, UK) diluted inwith 5% CO2. Stem cells were isolated by neurosphere Can Get Signal Immunoreaction Enhancer Solutionculture methods in DMEM/F12 containing N2 sup- (Toyobo, Osaka, Japan) and detected with Alexaplement, 20 ng/mL of bFGF, 20 ng/mL of EGF, and Fluor 488 goat antirabbit IgG antibody. Stained cells10 ng/mL of LIF as described previously [2]. Normal were analyzed by flow cytometry with live cell gatinghuman astrocyte (NHA) cells were purchased and using a FACSCalibur equipped with CellQuest soft-maintained with an Astrocyte Medium Bullet kit ware (Becton Dickinson, San Diego, CA).(Cambrex, Baltimore, MD). For cell differentiation,stem cells were washed and cultured in DMEM Western Blottingcontaining 10% FCS or DMEM/F12 containing N2 Cell lysates from U87MG cells and their der-supplement. For the examination of histone H3 and ivatives were prepared using M-PER reagent (Pierce,H4, U87MG and SNB19 cells were treated with 5 mM Rockford, IL) containing the protease inhibitorsodium butyrate for 24 h. For RT-PCR, U87MG and its cocktail Halt (Pierce). Twenty micrograms of totalstem cells were treated with 0.1 mM sodium butyrate, protein from each sample was subjected to SDS–1 mM 5-aza-20 -deoxycytidine (5-aza-CdR), or 0.3 mM polyacrylamide gel (PAGE) (15%) and then proteinsvalproic acid (VPA) for 5 d. were transferred to a PVDF membrane, which was subsequently probed with rabbit anti-p21 or rabbitLuciferase Assay anti-p27 polyclonal antibody (Santa Cruz Biotech- MAGEA3 promoter was amplified from NHA nology, Inc., Santa Cruz, CA). To detect histonegenomic DNA by PCR using specific primers (see acetylation, total histones were prepared by acidicSupplemental Table 1) and cloned into pGL3 Basic extraction and then resolved on 20% SDS–PAGE as(Promega, Madison, WI). CSCs derived from described previously [7]. The extracted proteins wereU87MG, SNB19, and both parental cells were loaded into each lane and confirmed by Ponceau Scultured in antibiotic-free medium 1 d in advance staining prior to blotting. Antibodies for Westernand then cotransfected with 1 mg of reporter vector blotting were directed against acetylated histone H3and 0.1 mg of Renilla luciferase vector using and H4 (Upstate Biotechnology, Inc., Lake Placid,FUGENE6 (Roche, Mannheim, Germany). Two days NY). Blots were visualized using a ECL-plus kitafter transfection, the cells were harvested and (Amersham Biosciences, Piscataway, NJ) accordingluciferase activity was measured using a Dual to the manufacturer’s instructions.luciferase kit (Promega). Chromatin ImmunoprecipitationRT-PCR Chromatin immunoprecipitation (ChIP) assay RNA was isolated from the cultured cells using an was performed according to the protocols of theRNA isolation kit (Qiagen, Santa Clarita, CA) and Acetyl-Histone H3 and H4 Immunoprecipitationreverse transcribed with an oligo dT primer using the ChIP Assay kits (Upstate Biotechnology, Inc.) usingSuperscript II First Strand cDNA Synthesis kit antibodies for acetylated histone H3 and H4 with(Invitrogen, Carlsbad, CA). cDNA was amplified slight modifications. Briefly, cultured cells (1 Â 106)using AmpliTaq Gold (Applied Biosystems, Foster were subjected to cross-linkage of chromatin by theCity, CA) according to the manufacturer’s instruc- addition of formaldehyde to the medium at a finaltions, and the products were visualized by agarose concentration of 1% and incubation for 5 min atgel electrophoresis. For quantification of CD133, 378C. Cell lysates were sonicated to reduce the DNAMAGED1, MAGED3, and SPANXC mRNA, real-time length to between 200 and 1000 bp, and debris wasPCR was performed with initial denaturation at 948C removed by centrifugation. Sonicated lysates werefor 10 min followed by 45 cycles of 20 s at 948C, 20 s at then diluted to 1.8 mL in ChIP dilution buffer and578C, 20 s at 728C, and 848C at 20 s using the precleared according to the protocol of UpstateLightCycler system (Roche) and Quantitect SYBR Biology. Precleared lysates were incubated with 6 mgGreen PCR kit (Qiagen). Primer sequences, the of antiacetylated H3 antibody or antiacetylatednumber of cycles, and annealing temperatures are H4 (Upstate Biotechnology, Inc.) at 48C for 12 h.listed in Supplemental Table 1. Immunocomplexes were recovered with protein G-Sepharose beads. Each precipitate was washedFlow Cytometry repeatedly with 1 mL of wash buffer and then For flow cytometry, cells were incubated with immunocomplexes were eluted from the beads usingethidium monoazide bromide (final concentration: elution buffer (10 mM Tris–HCl [pH 8.0], 5 mM0.1 mg/mL; Molecular Probes, Eugene, OR) for label- EDTA, 300 mM NaCl, 0.5% SDS); cross-linking wasMolecular Carcinogenesis
  3. 3. 534 YAWATA ET AL.reversed by incubation at 658C for 4 h. The samples sucrose prior to cryosectioning. For immunohisto-were then treated with RNase A (20 mg/mL, 30 min, chemistry, 5-mm sections were prepared and exam-378C) followed by proteinase K (50 mg/mL, 1 h, 558C); ined for CD133, p27 (Kip1), NY-ESO-1, and HLA classphenol/chloroform was then extracted and ethanol I expression using immunohistochemistry. To exam-was precipitated in the presence of carrier glycogen. ine CD133, we carried out antigen retrieval byPellets were resuspended in 50 mL of TE buffer for use heating the sample to 1008C in a microwave ovenas PCR input DNA or 20 mL of TE buffer for use as ChIP for 5 min in 10 mmol/L sodium citrate bufferDNA. Specific DNA sequences in the immunopreci- (pH 6.0). CD133 was stained with rabbit polyclonalpitates were detected by PCR or real-time PCR in anti-CD133 antibody (dilution, 1:200; Abcam), p27which the product yield was dependent on the input (Kip1) with mouse monoclonal anti-p27 antibodyDNA dose. (1:200; BD Bioscience, Pharmingen, San Diego, CA), NY-ESO-1 with mouse monoclonal anti-NY-ESO-1Bisulfite Sequencing antibody (1:500; Invitrogen, Gaithersburg, MD), DNA was isolated from cultured cells according to HLA class I with mouse monoclonal anti-HLA-the standard method. Testis DNA was purchased A,B,C antibody (1:100; MBL, Nagoya, Japan), glialfrom BioChain (Hayward, CA). Bisulfite treatment fibrillary acidic protein (GFAP) with mouse mono-of genomic DNA was performed with a EZ DNA clonal anti-GFAP antibody (1:200; Dako Corp., SantaMethylation kit (Zymo Research, Orange, CA). Barbara, CA), and b-tubulin III with mouse mono-Briefly, 1 mg of genomic DNA was denatured by the clonal anti-b-tubulin III antibody (1:200; Neo-addition of 3 M sodium hydroxide and then incu- markers, Fremont, CA). Bound antibodies werebated for 15 min at 378C. A 6.24 M urea/2 M sodium detected using VECTASTAIN Elite ABC reagentmeta-bisulfite (4 M bisulfite) solution was prepared (Vector, Burlingame, CA) and Envision/AP (Dako,and then 10 mM hydroquinone was added to the Carpinteria, CA) or antimouse IgG conjugated withdenatured DNA. This reaction mixture was heated to Alexa 488 (Invitrogen) and antirabbit IgG conju-558C for 6 h. The modified DNA was purified gated with Alexa 568 (Invitrogen). For immunocy-and amplified with AmpliTaq Gold using specific tochemistry, parental and stem cells were cultured inprimers and then the PCR products were cloned into eight-well chamber slides coated with fibronectina pGEM-T Easy Vector (Promega) and sequenced. (BD Bioscience) containing appropriate medium for 5 d and 3 h, respectively. The differentiated cells wereGlioma Xenograft Model produced by stem cell culture in a coated chamber A total of 1 Â 106 CSCs were suspended with 20 mL slide containing DMEM/10% FCS for a period of 5 d.of PBS and then 2-mL aliquots were stereotactically The cultured cells were fixed with PBS containinginjected into the intracranial space of BALB/c nu/nu 3.7% formaldehyde and permeabilized by treatmentmice. One day or 3 wk after injection, the mice were with PBS containing 0.1% Triton-X. Primary anti-sacrificed and tumorigenicity in the brain was body was applied after washing and blocking andconfirmed by hematoxylin and eosin staining. The then the chamber slide was incubated overnight atanimal experiment protocol was approved by the 48C. CD133, NY-ESO-1, HLA class I, GFAP, b-tubulinEthics Committee of Kochi Medical School. III, and nuclei were visualized with antirabbit IgG conjugated with Alexa 568 or antimouse IgG con-Specimens jugated with Alexa 488 and DAPI staining. For examination of the coexpression of CD133 andNY-ESO-1, a tumor specimen was obtained from a RESULTS57-yr-old female patient with an untreated glioblas-toma. To isolate the tumor sphere, fresh surgical Association Between Heterogeneity of Differentiatedspecimens were also obtained from a 75-yr-old Glioma Cells and Histone Acetylationfemale glioblastoma patient. Tumor tissues were We first set out to investigate the correlationwashed and minced with fine scissors into small between glioma heterogeneity and differentiationfragments. After trypsinization, single tumor cells status in vitro and in vivo. To do so, we isolatedand small clumps were collected with a 100-mm cell tumor spheres (TS) from glioma cell lines by neuro-strainer, and then resuspended in medium for tumor sphere culture, revealing multipotential differentia-sphere culture. The tumor sphere culture was tion. The expression of somatic stem cell markers,performed as described above. Both patients pro- CD133 and SOX2, in the TS was then compared withvided written informed consent and the protocol was that in the original (parent) cells. All cell linesapproved by the Ethics Committee of Kochi Medical indicated strong expression of both genes in the TSSchool, Kochi, Japan. (Figure 1A). When cultured in medium containing serum, the morphology of the TS changed toImmunohisto- and Immunocytochemistry adherent and they were not distinguishable from Tissue specimens were postfixed in 4% parafor- the parent cells (Figure S1). During this culture, themaldehyde overnight and stored at 48C in 30% expression level of CD133 rapidly decreased at day 1Molecular Carcinogenesis
  4. 4. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 535 Figure 1. Tumor heterogeneity reflects differences in differentia- anti-CD133 antibody, and the green and black lines represent thetion status. (A) Tumor spheres (TS) were isolated from glioma cell isotype control-matched antibody and no antibody, respectively. (E)lines by neurosphere culture. Expression levels of somatic stem cell Xenotransplantation of U87-TS cells yielding a heterogeneous cellmarkers, CD133, and SOX2, in adherent (parent) and stem cells were population. The expression of CD133, GFAP, and p27 in U87MGexamined by semiquantitative RT-PCR. (B) Repression of CD133 tumor cells was examined 1 d or 3 wk after injection of 1 Â 105 U87-during in vitro differentiation of U87MG cells. Differentiation of TS cells into nude mice. Tissue sections were stained withU87MG CSCs was induced by culture with medium containing hematoxylin and eosin (HE) and with antibodies to GFAP (brown),serum. Expression levels of CD133 were quantified at 1, 3, and 5 d p27 (brown), and CD133 (blue). The magnified photograph withinafter addition of serum by real-time PCR. (C) Upregulation of CDK the boxed area outlined in CD133/p27 at 3 wk indicates membraneinhibitors, p21 (Cip1) and p27 (Kip1), in differentiated cells was staining of CD133. (F) Differentiation status affects the acetylationexamined by Western blot analysis. (D) Fluorescence-activated cell level of histone H3 and H4. Histone proteins were purified by acidicsorting (FACS) analysis of CD133þ populations in U87-TS cells extraction and analyzed by Western blotting with antiacetyl-histonebefore xenotransplantation. The pink line represents staining with H3 and H4 antibodies.Molecular Carcinogenesis
  5. 5. 536 YAWATA ET AL.(Figure 1B), suggesting the induction of cell differ- histone acetylation is involved with HDAC activityentiation. Differentiation of the CSCs also accom- in glioma cells. Since HDACi effectively induce cellpanied upregulation of cyclin-dependent kinase type-specific differentiation in various cell lines(cdk) inhibitors p21(Cip1) and p27(Kip1), which [15,16], histone H3 and H4 acetylation levels mayare known to be key regulators of cell-cycle pro- be associated with the differentiation status ofgression on days 1 and 3, respectively (Figure 1C). glioma cells. These results indicate that glioma Next, we determined the percentage of CD133- tissue consists of various cells with different histonepositive cells in TS derived from U87MG (U87-TS) H3 and H4 acetylation levels, possibly contributing(Figure 1D) and tumor heterogeneity in the intra- to the heterogeneity of tumor tissue.cranial space of nude mice transplanted with thedissociated U87-TS cells (Figure 1E). Before trans- Expression of CTA and HLA Class I Genes in CSCsplantation, the U87-TS contained a large population The expression of CTA genes is regulated by DNAof CD133-positive cells (approximately 80%), but methylation within the promoter region [5,17] andsome CD133-negative cells also remained. Upregu- could be affected by epigenetic status. Because alation of p27 was accompanied by induction of difference in epigenetic status was observed inGFAP, a well-established marker of mature astro- glioma cells and tumor tissue, we examined thecytes, after treatment with cholera toxin [8], an divergence of CTA gene expression among cellsinhibitor of type IV phosphodiesterase [9] or all-trans of varying differentiation status. To select can-retinoic acid and interferon-gamma [10]. The role of didate CTA and CTA-like genes (melanoma-p27 in cell-cycle exit during cell differentiation of associated antigen genes; MAGE family) significantlyvarious cell types is well known. Here, 1 d after expressed in brain tumors, in silico screening wastransplantation, the expression of CD133 was fre- performed using the NCBI-CGAP EST and SAGEquently observed in injected cells, but that of GFAP database (http://cgap.nci.nih.gov/). Thirty-one ofand p27 was very restricted to a small number of 104 CTA and MAGE family genes were selected forcells, suggesting that most of these cells remained assessment of gene expression by RT-PCR. Surpris-undifferentiated. In the tumor mass formed 3 wk ingly, the analysis revealed that 19 of the 31 (61.3%)after transplantation, GFAP- and p27-positive cells CTA genes were more strongly expressed in TS fromwere abundant compared with day 1. In particular, either cell line than in the parent cells (Figure 2A andCD133-positive and p27-negative cells were inde- B). This increased expression was observed in 36.3%pendently identifiable in the tumor mass, and of the total (45/124). Of these, MAGEB4, MAGED3,therefore, the expression of CD133 and p27 is and TRAG3 indicated increased expression in all TSconsidered an indicator of differentiation status. examined. Additionally, the expression of LAGE-1This observation demonstrates that the transplanta- was detected in CSCs only, indicating a possibletion of CD133-positive cells contained in TS cells candidate CSC-specific antigen gene. Conversely,produced differentiated cells during tumor mass only five (16.1%) genes indicated decreased expres-formation, yielding tumor heterogeneity. sion in the TS. Neuronal lineage progression is associated with the To determine whether this culture model based ongenome-wide acetylation levels of histone H3 and H4 glioma cell lines preserves the nature of CSCs in[11]. Oligodendrocyte differentiation is also affected patient tissue, TS were isolated from a glioblastomaby histone acetylation [12,13]. Histone H3 is more surgical specimen. The expression of CD133 wasacetylated in glioblastomas than normal brain tissue observed in the TS culture, while that of GFAP and b-[14]. However, this has yet to be reported in tumor tubulin III was obvious 10 d after the induction ofcells with varying degrees of differentiation. We differentiation (Figure 2C). Semiquantitative RT-PCRtherefore examined the acetylation status of histone was performed to examine the enhanced expression ofH3 and H4 in parent cells, stem cells, differentiated CTA and MAGE family genes observed in the TS usingcells, and NHA cells as the normal counterparts of a cell line model. Five genes, IL13RA, MAGED1,glioma cells (Figure 1F). The acetylation level was MAGED3, SPANXA, and SPANXC, showed highhighest in stem cells and decreased with differ- expression levels in the TS compared with the differ-entiation, consistent with a previous report [11] entiation-induced cells (Table 1). Quantitative RT-PCRindicating histone H3 and H4 hyperacetylation in analysis revealed that the expression of CD133 wasneural stem cells and hypoacetylation in astrocyte decreased 2.86-fold during differentiation (Figure 2D).cells. In addition, the acetylation level of histone H3 Similar to this downregulation, the expressions ofand H4 in both parent U87MG and SNB19 cells was MAGED1, MAGED3, and SPANXC were also decreasedhigher than in NHA cells. This increase in acetylation 2.32-, 2.68-, 4.13-fold, respectively, during differ-might have resulted from the existence of stem cells entiation (Figure 2D). CTA genes strongly expressedin the glioma cells. Furthermore, treatment of parent in the primary culture model were not always identicalcells with an histone deacetylase inhibitor (HDACi), to the genes identified in the cell line model; however,sodium butyrate (NaB), caused an accumulation of MAGED3 was also strongly expressed in both models.acetylated histone H3 and H4, suggesting that This result indicates that a subset of CTA and MAGEMolecular Carcinogenesis
  6. 6. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 537 Figure 2. Differential expression of CTA genes in stem and medium lacking bFGF, EGF, and LIF for 10 d, the differentiateddifferentiated cells. (A) Representative photograph of semiquantita- tumor spheres expressed GFAP and b-tubulin III (bottom left andtive RT-PCR analysis of CTA and HLA class I genes in glioma cancer bottom right). (D) Quantification of CD133, MAGED1, MAGED3,stem and parent cells. (B) Summary of CTA gene expression. Closed and SPANXC mRNA in primary tumor spheres (GB3-TS) andrectangles indicate strong expression in TS. Open and dashed their differentiated cells (GB3-Diff.) by quantitative RT-PCR. Therectangles indicate unchanged and weak expression, respectively. vertical axis shows the expression ratios for tested genes/b-actin(C) Tumor spheres were isolated from glioblastoma tissues (left gene. (E) Expression level of HLA class I was quantified in TS andupper). Expression of CD133 was observed in the tumor spheres by parent U87MG cells by real-time PCR.immunocytochemical staining (right upper). After culture withfamily genes shows enhanced expression in CSCs Products of CTA genes are thought to be degradedderived from glioma cell lines and tissues. and presented by HLA class I molecules on the cell Immunocyto- and immunohistochemical stain- surface, leading to cell lysis through CTL recogni-ing was also performed to examine the expression of tion. To assess this possibility, expression of the HLANY-ESO-1 in U87MG cultured cells, a U87-TS class I gene was examined in vitro and in gliomaxenograft model, and tissues from a glioblastoma tissues. Compared with the U87MG cultured cells,patient. In vitro, the expression of NY-ESO-1 was U87-TS showed no significant reduction in HLAstrongest in stem cells compared with parent cells class I expression in quantitative RT-PCR analysisand differentiated cells (Figure S2). We also found (Figure 2E). In addition, immunocytochemical anal-that most CD133-positive cells also expressed NY- ysis of U87MG, SNB19, and their TS indicated noESO-1 in the U87-TS xenograft and glioblastoma heterogeneous or differential expression of thetissue (Figure 3). HLA class I gene, reflecting the results of quantitativeMolecular Carcinogenesis
  7. 7. 538 YAWATA ET AL. Table 1. CTA Genes With Altered Expression Levels in CSCs. HDACs are recruited to the promoter, chang-Tumor Sphere (TS) and Differentiated Cells Derived From ing the chromatin conformation and regulating the a Glioblastoma Surgical Specimen accessibility of transcription factors [19]. Moreover, HDACi markedly enhance NY-ESO-1 expressionUpregulated in TS Downregulated in TS in lung cancer cells [20]. We therefore examinedIL13RA BAGE whether enhanced expression in TS is involved inMAGED1 CSAGE histone acetylation within the promoter regions ofMAGED3 SYCP1 NY-ESO-1, TRAG3, MAGEA3, and LAGE-1. ChIPSPANXA analysis revealed high levels of histone H3 and H4SPANXC acetylation in U87-TS, but not in parent or differ- entiated cells (Figure 4A and B). These promoterDifferentiation of TS was induced by culture in medium lacking regions therefore appear to be accessible for regu-bFGF, EGF, and LIF in laminin-coated dishes for 10 d. Theexpression levels of 34 CTA genes were observed in the TS and latory factors involved in CTA gene activation in thedifferentiated cells by semiquantitative RT-PCR. No differences TS. Additionally, the histone acetylation levels inwere found in CTA genes not described in this table. the MAGEA3 promoter were unchanged in SNB19 expressing the same gene level. Thus, the histoneRT-PCR (Figure S3). These results suggest the possi- acetylation level of the MAGEA3 promoter region isbility that CTA expressed in CSCs could be recog- correlated with the expression level. These resultsnized by CTL in the context of specific HLA class I suggest that the promoter regions are regulated in amolecules. histone acetylation-dependent manner, resulting in formation of a transcriptionally active chromatinDifferential Histone Acetylation of the Promoter Region domain in CSCs included in TS.of CTA Genes in Stem and Differentiated Cells Acetylation of histone H3 and H4 in the promoter DNA Hypomethylation of the Promoter Regions of CTAregion is generally accepted as a hallmark of an active Genes in CSCspromoter [18], suggesting that the promoter region DNA hypermethylation in the promoter regionof CTA genes might be histone hyperacetylated in is known to silence CTA genes [5,17], but our Figure 3. Expression of NY-ESO-1 and HLA class I (HLA-A, B, C) in U87-TS xenograft and glioblastoma tissues. (A) Double-staining with CD133 and NY-ESO-1 in a U87MG secondary tumor. Tissue sections were stained with anti-CD133 (Blue) and anti-NY-ESO-1 (brown) antibodies. Tissue obtained from a patient with glioblastoma was stained with H&E (B), anti-NY-ESO-1 (C), anti-CD133 (D and H), anti-HLA-A, B, C (G), and DAPI (E and I). The photograph was merged for visualization of cancer stem cells expressing NY-ESO-1 and HLA class I (F and J).Molecular Carcinogenesis
  8. 8. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 539 Figure 4. Hyperacetylation of histone H3 and H4 at CTA gene promoters. (A) Acetylation levels of histone H3 and H4 surrounding NY-ESO-1 and MAGEA3 promoter regions were examined at different degrees of differentiation in U87MG and SNB19 cells by ChIP assay. (B) Real-time PCR was used to quantify the enrichment of NY-ESO-1, TRAG3, and LAGE-1 promoters in acetylated histone H3 and H4 ChIP DNA using the input DNA as a reference.understanding of the relationship between the meth- expression of these genes. The methylation level inylation status and differentiation level remains poor. parent cells was slightly lower than that in NHA cellsWe therefore examined DNA methylation levels in but still indicated a hypermethylated status, reflectingthe promoter region of CTA genes in parent cells, TS no or low expression of these genes in the former.cells, differentiated cells, NHA cells, and testis by Conversely, a hypomethylation status was detected inbisulfite sequencing. Methylation of CpG dinucleo- the TS, consistent with the high expression of CTAtides located in LAGE-1, MAGEA3, and NY-ESO-1 was genes. In differentiated cells, the LAGE-1 and NY-ESO-also evaluated to assess whether differential promoter 1 regions were more methylated than those in the TS,methylation could account for the enhanced expres- but the methylation level of MAGEA3 was similar tosion in CSCs (Figure 5A–C). In testis, but not NHA that in the TS. The expression of MAGEA3 wascells, the promoter regions of these genes were downregulated in U87MG-differentiated cells, accom-hypomethylated, consistent with the testis-specific panied by hypoacetylation of histones H3 and H4;Molecular Carcinogenesis
  9. 9. 540 YAWATA ET AL. Figure 5. Hypomethylation of CTA gene promoters in cancer stem cells. Summary of sodium bisulfite sequencing analysis of CpG islands associated with NY-ESO-1 (A; À199 to þ7 and þ110 to þ293), LAGE-1 (B; À123 to þ123), and MAGEA3 (C; À320 to þ162) in parent, TS, and differentiated cells. Ten clones were sequenced for each sample, and each circle displays the percent methylation of all clones for a single CpG dinucleotide (open circle, 100% unmethylated; filled circle, 100% methylated). The average percent methylation across all CpG sites for each cell is shown next to each row of circles (vertical bars, CpG dinucleotides; arrow, position of the transcriptional start site).however, DNA of the promoter region was hypome- results reveal that DNA hypomethylation of thethylated as in the testis expressing MAGEA3. Thus, promoter regions of CTA genes is a hallmark of CSCshistone deacetylation may be dominant over DNA in tumors as well as necessary but not sufficient formethylation in the repression of MAGEA3. These CTA gene expression.Molecular Carcinogenesis
  10. 10. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 541Inhibition of MAGEA3 Repression During Differentiation entiated cells (Figures 1 and 2D). However, it has alsoof CSCs by 5-aza-CdR and HDAC Inhibitors been reported that CD133-negative glioma cells have To determine the influence of epigenetic factors tumorigenic potential when implanted into the raton the repression of CTA genes during differentia- brain [29]. In fact, not all glioblastoma specimenstion, differentiating cells were treated with an contain CD133-positive cells [30]. Thus, CD133 isinhibitor of DNA methylation, 5-aza-CdR, or HDACi, not an absolute marker of stem cells in glioma,VPA, and NaB. Addition of these inhibitors obscured although the present study suggests that some CTArepression of the CTA genes (Figure 6A). Moreover, genes might be useful for the identification of CSCstreatment of parent cells with 5-aza-CdR or VPA/NaB in CD133-negative tumors.did not induce the same expression level as that in In this study, MAGED3 preserved the enhancedthe differentiating cells (data not shown). In addi- expression in TS in both primary cultured and cell-tion, the induction of robust CTA gene expression by line-based models (Figure 2B and D). However,treatment with both HDACi and 5-aza-CdR has also which CTA genes are activated seems to be depend-been reported [6], suggesting that multiple factors ent on the tumor tissue, because TS derived from ainvolved in acetylation of histones and suppression surgical specimen strongly expressed those genes notof DNA methyltransferase are required for CTA gene showing enhanced expression in the cell line model.expression. To identify the CTA genes strongly expressed at a To confirm epigenetic regulation of the MAGEA3 high level in CSCs, further studies with more primarypromoter, we introduced a reporter construct of the cultured TS or glioblastoma specimens are needed.promoter into parent and stem cells. Promoter The mechanisms inducing the transcriptionalactivity of MAGEA3 in the CSCs did not significantly activation of CTA genes remain unclear. Most CTAdiffer from that in the parent cells (Figure 6B), genes are mapped on the X chromosome; however,suggesting that the strong expression of CTA genes in the present study, the chromosomal location ofin CSCs is driven by the epigenetic status, including CTA genes indicating altered expression was nothistone acetylation and DNA methylation, not by associated with any specific region (Figure S4). Thistrans-acting factors within the stem cells. suggests that there is no correlation between this expression pattern and an activated chromosomal region or specific chromosomal abnormality. Hence, DISCUSSION a certain factor(s) affecting the whole genome may A poorly differentiated tumor is generally more be responsible for the expression pattern observed.malignant than a well-differentiated tumor. Histo- In fact, we found that genome-wide changes inpathological studies have shown that GFAP-positive histone acetylation levels were dependent on thecells decrease with increasing malignancy of glial differentiation status of glioma cells as well as neuraltumors [21–23]. In this study, we showed an lineage cells (Figure 1F). We further revealed thatincreased frequency of GFAP and p27(Kip1) expres- DNA hypomethylation of the promoter region ofsion in U87-TS cells 3 wk after transplantation. CTA genes in CSCs and the promoter regions of NY-Moreover, the population of p27(Kip1)-positive cells ESO-1 and LAGE-1 were methylated during differ-was abundant compared with that of GFAP-positive entiation (Figure 5A and B). Methylation of thecells. This may have resulted from the fact that MAGEA3 promoter was not observed on day 5 afterp27(Kip1)-positive cells also contain immature dif- induction of differentiation (Figure 5C), suggestingferentiating cells; however, here p27(Kip1) expres- that the modification of histone H3 and H4 occurssion was used to identify the differentiation status of first, followed by DNA methylation, thus stabilizingvarious tumor cells. the differentiation status. Accordingly, the MAGEA3 The population of CSCs in a tumor mass is also promoter may require more time to establish de novoassociated with the differentiation level of the methylation. This may be one reason why thetumor. A high content of CSCs has been observed methylation status was clearly correlated within higher grade tumors [24] but the influence on the expression even though analysis was performed onprognosis has yet to be determined. The expression TS cells, which contain both stem and differentiatedof CD133 was previously shown to be upregulated in cells, a nonpurified population. Thus, in addition torecurrent glioblastoma tissues compared with autol- stem and differentiated cells TS cells also appear toogous primary tumor tissue [25], while the frequency contain differentiating cells, which are not fullyof CTA gene expression is known to be correlated methylated at their CTA gene promoters. The testiswith tumor grade and prognosis [26–28]. In our DNA was used as the control in this analysis since itobservations, CSCs showed strong and frequent was expected to show hypomethylation. It is possibleexpression of CTA genes, suggesting that popula- that the methylation seen at the NY-ESO-1 (Region 1),tions of CSCs affect tumor grade and prognosis of LAGE-1, and MAGEA3 promoters (30.7–37.1%,astrocytic tumors. Figure 5A–C) was caused by contamination of testis All TS isolated in this study strongly expressed cells not expressing these CTA genes. That is, CTACD133 compared with parental cell lines and differ- gene promoters may be more tightly regulated by theMolecular Carcinogenesis
  11. 11. 542 YAWATA ET AL. Figure 6. Epigenetic regulation of CTA gene repression during differentiation. (A) Inhibition of DNA methyltransferase or HDAC block differentiation-inducing repression of CTA genes. Differentiation of U87-TS was induced for 5 d with 5-aza-CdR, VPA, or NaB or with no addition. (B) Activity of the MAGEA3 promoter was independent of cell differentiation status in the promoter assay. A fragment of the MAGEA3 gene extending from À52 to þ2980 was cloned in a luciferase reporter vector (pGL3 basic). The transcription-directed MAGEA3 promoter was then assessed by measuring the amount of luciferase activity in the transfectants.methylation status than initially suspected. It is not cells, with no specific direction of differentiation. Onknown why methylation levels in these promoter the other hand, a quick reaction of deacetylatingregions were low in CSCs, but CSCs are known to histones may be required for appropriate differ-have the multipotential to differentiate into various entiation. Thus, the chromatin status of stem cellsMolecular Carcinogenesis
  12. 12. EXPRESSION OF CANCER TESTIS ANTIGEN GENES 543could make them competent for differentiation. Grants-in-Aid (No. 19591685) from the Ministry ofExactly how modification of epigenetic factors Education, Science, Sports and Culture, Japan.during differentiation regulates genome-wide geneexpression requires further study. Brother of regulator of imprinted sites (BORIS), REFERENCESa paralog of zinc finger protein CTCF, upregulates 1. Singh SK, Clarke ID, Hide T, Dirks PB. Cancer stem cells in nervous system tumors. Oncogene 2004;23:7267–NY-ESO-1 by recruitment at the promoter region 7273.[31]. However, BORIS is not involved with the 2. Singh SK, Hawkins C, Clarke ID, et al. Identification of humaninduction of CTA gene expression in CSCs since no brain tumour initiating cells. Nature 2004;432:396–401.expression was found in the glioma cell lines used 3. Pellegatta S, Poliani PL, Corno D, et al. Neurosphereshere (Figure 2B). 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