Hypoxia Induces Genomic DNA Demethylation through the … · Molecular Medicine in Practice Hypoxia Induces Genomic DNA Demethylation through the Activation of HIF-1a and Transcriptional

Molecular Medicine in Practice

Hypoxia Induces Genomic DNA Demethylation throughthe Activation of HIF-1a and Transcriptional Upregulationof MAT2A in Hepatoma Cells

Quanyan Liu1, Li Liu1, Yuhong Zhao3, Jin Zhang1, Dongfeng Wang1, Jiwei Chen1, Yueming He1,Jianguo Wu2, Zhonglin Zhang1, and Zhisu Liu1

AbstractHypoxia-inducible factor 1 (HIF-1) emerges as a crucial player in tumor progression. However, its role in

hepatocellular carcinoma (HCC), especially its relation with global DNAmethylation patterns in HCC under

hypoxic tumor microenvironment is not completely understood. Methionine adenosyltransferase 2A

(MAT2A) maintains the homeostasis of S-adenosylmethionine (SAM), a critical marker of genomic methyla-

tion status. In this study, we investigated the link between HIF-1a and MAT2A as a mechanism responsible

for the change in genomic DNA methylation patterns in liver cancer under hypoxia conditions. Our results

showed that hypoxia induces genomic DNA demethylation in CpG islands by reducing the steady-state SAM

level both in vitro and in vivo. In addition, HIF-1a and MAT2A expression is correlated with tumor size and

TNM stage of liver cancer tissues. We further showed that hypoxia-induced MAT2A expression is HIF-1adependent and requires the recruitment of p300 and HDAC1. We also identified an authentic consensus HIF-

1a binding site in MAT2A promoter by site-directed mutagenesis, electrophoretic mobility shift assay, and

chromatin immunoprecipitation assay. Taken together, we show for the first time that hypoxia induces

genomic DNAdemethylation through the activation of HIF-1a and transcriptional upregulation ofMAT2A in

hepatoma cells. These findings provide new insights into our understanding of the molecular link between

genomic DNA methylation and tumor hypoxia in HCC. Mol Cancer Ther; 10(6); 1113–23. �2011 AACR.

Introduction

The high proliferation of tumor cells induces localhypoxia inside the tumor, recent evidence suggests thathypoxia is crucially involved in tumor progression andangiogenesis (1, 2). Notably, hypoxia modulates themalignant phenotypes of tumor cells via hypoxia-indu-cible factor 1 (HIF-1), a crucial transcription factor thatregulates the expression of numerous target genes (3–5).Nevertheless, it remains unclear whether HIF-1 upregu-lation is involved in the initiation or the progressionstages of hepatocarcinogenesis.Recent studies have shown hypoxia-induced genome-

wide effects in liver tumor (6–8). Such hypoxic changes

may be due to alterations in epigenetic profiles. DNAmethylation is now recognized as a critical epigeneticmark and alterations in DNA methylation is involvedin multistage of hepatocellular carcinoma (HCC), even inthe early precancerous stages (9). Specially, in premalig-nant conditions such as dysplastic nodules or cirrhoticliver, the promoters of tumor suppressor genes includingE-cadherin, glutathione S-transferase P1, and p16Ink4aare frequently hypermethylated (10). In contrast, thegenome-wide hypomethylation in HCC was shown asan ongoing process throughout the lifetime of the tumorcells rather than a historical event occurring in precancerstages, but how this change is associated with genomicinstability or the activation of proto-oncogenes in HCCremains elusive (11).

S-adenosylmethionine (SAM) is a major biologicalmethyl donor. SAM-dependent methylation has beenshown to be central to many biological processes andthe steady-state SAM level has been accepted as a criticalmarker of the genomic methylation status (12, 13). Inhepatocytes, SAM level is related to the differentiationstatus of the cells, being high in quiescent hepatocytes andlow in proliferating hepatocytes (14–16). SAM is synthe-sized frommethionine andATP in a reaction catalyzed bymethionine adenosyltransferase (MAT). In mammals,MAT is encoded by 2 genes, MAT1A and MAT2A.A switch of MAT expression from MAT1A to MAT2A

Authors' Affiliations: 1Department of General Surgery, Zhongnan Hospi-tal, 2State Key Laboratory of Virology and College of Life Sciences, WuhanUniversity, Wuhan; and 3Department of Pharmacology, Guangdong Phar-maceutical University, Guangzhou, China

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Q. Liu and L. Liu contributed equally to this work.

Corresponding Author: Zhisu Liu, Department of General Surgery,Zhongnan Hospital, Wuhan University, Wuhan 430071, China. Phone:86-27-68713007; Fax: 86-27-87330795; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-10-1010

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

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Published OnlineFirst April 1, 2011; DOI: 10.1158/1535-7163.MCT-10-1010

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is frequently observedduringmalignant liver transforma-tion, and this alteration plays an important pathogeneticrole in facilitating liver cancer progression (17–19).

Nevertheless, the influence of the hypoxic tumormicroenvironment on DNA methylation patterns inHCC is not completely understood. Therefore, in thisstudy we investigated the potential interaction betweenHIF-1a and MAT2A as a mechanism responsible for thechange in genomic DNA methylation patterns in livercancer under hypoxia conditions. We identified MAT2Aas a novel target gene that is transcriptionally regulatedby HIF-1a, and provided evidence that hypoxia regulatesgenomic DNA methylation through activation of HIF-1aand transcriptional upregulation of MAT2A in hepatomacells. Thus our data establish a molecular link betweengenomic DNA methylation and hypoxia in liver cancer.

Materials and Methods

Cell culture and transfectionHepatoma cell lines BEL-7404, Hep3B, andHepG2 were

obtained from the Cell Bank of the Chinese Academy ofSciences where they were characterized by mycoplasmadetection, DNA fingerprinting, isozyme detection, andcell-vitality detection. These cell lines were immediatelyexpanded and frozen such that they could be restartedevery 3 to 4 months from a frozen vial of the same batch ofcells. All cells were cultured in the recommended mediasupplemented with 10% (v/v) FBS, 100 units/mL penicil-lin, and streptomycin at 37�C in an incubatorwith 5%CO2.The current authors have not independently tested andauthenticated these cells. Routine testing for Mycoplasmainfection was done using the MycoTect Kit (Invitrogen).For hypoxia experiments, the oxygen partial pressure waslowered to 0.9 kPa (1% O2 by volume). Transfection wascarried out using LipofectAMINE 2000 (Invitrogen).MAT2A promoter region (�2,403/þ102) was PCR ampli-fied from human genomic DNA and cloned into pGL3-Basic vector and cotransfected with HIF-1a expressionvector (pCMV-HIF-1a, OriGene) or pCMV empty vectorand an internal control pRL-SV40 plasmid (Promega). ForMAT2A promoter deletion constructs, pGL3 promotervectors containing different fragments of MAT2A promo-ter (�2,403/þ102,�913/þ102,�411/þ102, or�205/þ102)were transfected. Luciferase activities were measuredusing the Dual-Luciferase system (Promega) and an Infi-nite F200 luminometer (TECAN). For site-directed muta-genesis, the putative hypoxia responsive element (HRE)motif was mutated using the QuickChange Site-DirectedMutagenesis Kit (Stratagene), from 50-ATCCCCCACGTC-TCCTCG-30 to 50-ATCCCCCTAGTCTCCTCG-30.

HCC xenograft modelFemale BALB/c nude mice 4- to 5-week old were

obtained from the Shanghai Experimental Animal Centerof the Chinese Academy of Sciences. All procedures werecarried out according to institutional guidelines. Hepa-toma cells [(1–2)� 106] were subcutaneously injected into

the flank of female nude mice. Tumor growth was mon-itored using Vernier calipers, and the volume was calcu-lated using the standard formula (length�width2 � 0.5).Tissues were harvested once the tumor volume wasapproximately 100 to 200 mm3. Tumors were removedand cut into pieces that were then snap-frozen in cryo-matrix and stored at �80�C.

Endogenous C-5 DNA methyltransferase activityDNA MTase activity was determined as described

previously (20). Twenty microliters reaction mixture con-taining cell homogenates (5 mg protein), poly(dI-dC) (0.25mg) and 11.1 � 1010 mBq of [methyl3H] SAM was incu-bated at 37�C for 2 hours. The DNA was purified usingthe E.Z.N.A. Cycle-Pure Kit, and purified genomic DNAwas spotted onto Whatman GF/C filter disc, dried at80�C for 5 minutes, and counted using a scintillationcounter (1600TR Packard Instrument Comp). Resultswere expressed as dnm/mg protein.

Methylation-dependent restriction analysisThe methyl-accepting capacity of genomic DNA was

measured as the loss of unmethylated cytosine aftergenomic DNA was digested with methylation-sensitiveendonucleases as described previously (21). Five micro-grams purified genomic DNA was digested with HpaIIand BssHII overnight. Samples of undigested genomicDNA served as controls. The digested and undigestedgenomic DNA samples were purified with the E.Z.N.A.Cycle-Pure Kit for the DNA methyl-accepting capacityassay as follows: purified genomicDNA (0.5 mg), bacterialSssI methylase (2 U), and [methyl-3H] SAM (3 Bq persample) were incubated in buffer containing 50 mmol/LNaCl, 10mmol/LTris-HCl, pH8.0, and10mmol/LEDTAfor 2 hours at 37�C. Then, 25 mL aliquots were taken fromeach reaction mixture, applied to GF/C filter discs, andcounted in scintillation counters. The results wereexpressed as [methyl-3H] incorporation/0.5 mg DNA.

Analysis of genomic DNA methylation statusGlobal DNA methylation was determined as pre-

viously described (22, 23). Three micrograms purifiedDNA was incubated with 3 units HpaII or SssI and 2.5mCi of [methyl-3H] SAM in 50 mL buffer. The mixture wasincubated overnight at 4�C, and unlabeled SAMwas thenadded. The total SAM concentration was 160 mmol/L forSssI and 80 mmol/L forHpaII. After incubation at 37�C for3 hours, 5 volumes of ice-cold trichloroacetic acid wereadded to the mixture. The mixture was centrifuged, andthe pellet was washed with trichloroacetic acid andcentrifuged at 10,000 g for 30 minutes. Finally, the pelletwas dissolved in 50 mL 0.1 N NaOH and counted forradioactivity.

Assay of S-adenosylmethionine andS-adenosylhomocysteine

The assay was carried out using reversed-phase high-performance liquid chromatography (HPLC) based on

Liu et al.

Mol Cancer Ther; 10(6) June 2011 Molecular Cancer Therapeutics1114




previously described procedures (18, 24). SAM and S-adenosylhomocysteine (SAH) standards were dissolvedin water at 1 mmol/L and diluted in 0.4 mol/L HClO4 tothe final concentrations used forHPLC analysis. A total of25 mL standard injected solution containing 50 to 11,000pmol were added to the HPLC for making a standardcurve.

Patients and tissue specimensA total of 58 cases of surgically resected HCCs were

collected from Zhongnan Hospital, Wuhan University,between January 2009 and January 2010. No chemother-apy or radiation therapy was carried out before tumorexcision. Both the tumors and the corresponding peritu-moral noncancerous tissues for each case were selected.Matched normal human liver tissues were obtained fromliver trauma patients undergoing partial hepatectomy.Written informed consent was obtained from eachpatient. The study protocol was approval by the localethics committee.

ImmunohistochemistryRepresentative tissues were selected and sectioned in

4 mm thick. The tissue samples were fixed by immersionin buffered formalin and embedded in paraffin accord-ing to standard procedures. All specimens stained forHIF-1a and MAT2A were scored by 2 independentinvestigators who were blinded to the test groups.HIF-1a and MAT2A immunostaining was scored basedon the percentage of cells that had positive staining inthe cytoplasm. Slides were graded as follows: �(0%–10% cells stained), þ(10%–50% cells stained), orþþ(>50% cells stained).

Immunofluorescence experimentsCells were seeded on coverslips in a 6-well plate and

grown to 70% to 80% confluence. After exposure tohypoxia for 24 hours, the medium was aspirated, andcells were washed twice with PBS. Cells were fixed inmethanol for 30 minutes at �20�C, then incubated withHIF-1a antibody (Santa Cruz Biotechnology) for 1 hourat 37�C, followed by incubation with fluorescein isothio-cyanate-labeled secondary antibody (1:2,000 dilution inPBS) for 1 hour at 37�C. Nuclei were counterstained withpropidium iodide. Dual-color fluorescence images werecaptured using a digital camera and confocal microscope(Olympus FluoView FV300).

Western blottingNuclear and cytoplasmic protein extracts were pre-

pared from transfected cells. Protein (30 mg) from eachsample was examined using 10% SDS-PAGE and thenelectrotransferred to nitrocellulose membranes. Themembraneswere subjected toWestern blot analysis usingantibodies against HIF-1a (Santa Cruz), MAT2A,DNMT1, DNMT3a, and DNMT3b (Abcam) followingstandard procedures, and developed using ECL kit(Amersham).

Electrophoresis mobility shift assayOligonucleotide probes were purchased from Life

Technologies (Life Technologies), the sequence (codingstrand) of the wild-type probe was 50-GAGCAATCCCC-CACGTCTCCTCG-30 and that of the mutant probe was50-GAGCAATCCCCCTAGTCTCCTCG-30. Radioactiveoligonucleotides were generated by 50 end labeling usingT4 polynucleotide kinase (Amersham). Binding reactionswere carried out with 5 mg nuclear extracts, 0.1 mgdenatured calf thymusDNA, and 1 ng radiolabeled probe(10,000 cpm). Supershift experiments were carried out inthe presence of a monoclonal HIF-1a antibody (Novus).Electrophoresis was carried out on a 5% nondenaturingPAGE, and the gels were dried for autoradiograph.

Chromatin immunoprecipitation and re-ChIPassays

Chromatin immunoprecipitation (ChIP) assays werecarried out with a rabbit antibody against human HIF-1a (Abcam) using a ChIP assay kit (Millipore), and re-ChIP assay was carried out using a procedure describedby Metivier and colleagues (25). Briefly, HIF-1a ChIPcomplexes were eluted by incubation with 25 mL 10mmol/L dithiothreitol (Caliochem) for 30 minutes at37�C. After centrifugation, the supernatant was dilutedin a re-ChIP buffer (20 mmol/L Tris-HCl, 150 mmol/LNaCl, 2 mmol/L EDTA, and 1% Triton X-100, pH 8.0).The diluted complexes were then subjected to immuno-precipitation using mouse anti-human p300 antibody(BD Pharmingen) or rabbit anti-human HDAC1 antibody(Cell Signaling Technology). The immunoprecipitatedchromatin was analyzed in triplicate by PCR using theprimers AGTGCGCGCCAACGCCG (forward) andAAGTTGGGCGCCGCTTGGA (reverse) for humanMAT2A promoter.

Statistical analysisStatistical analyses were carried out using SPSS 15.0

statistics software (SPSS). Data were expressed as mean� standard deviation (X � SD). Student’s unpaired t testwas used to compare 2 groups. The Spearman rankcorrelation test was used to determine correlationsbetween variables. P < 0.05 was considered significant.

Results

Hypoxia induces genomic DNA hypomethylation inCpG islands in Hep3B cells

We examined the effect of hypoxia on DNA methyla-tion in hepatoma cells. The levels and patterns of DNAmethylation were determined by measuring endogenousC-5 DNAmethyltransferase (C-5 MTase) activity and themethyl-accepting capacity of undigested genomic DNA.The results showed that hypoxia upregulates the activityof C-5 MTase in Hep3B cells compared with normoxiagroup, with maximum activity shown 24 hoursafter hypoxia (P < 0.001, Fig. 1A). In addition, the numberof genomic DNA methylation sites available for SssI

HIF-1a Induces Genomic DNA Demethylation via MAT2A

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methylasewas increased significantly under hypoxic con-ditions compared with normoxia (P < 0.001) and reachedthe peak 24 hours after hypoxia (Fig. 1B). The genomicDNA isolated from the cells exposed to hypoxia exhibited1.38-, 1.59-, and 1.43-fold more methyl acceptance thannormoxia-treated cells 12, 24, and 36 hours after hypoxia,respectively. These data showed that hypoxia induced ademethylation process in genomic DNA.

Because methylation-dependent restriction endonu-cleases can cut specific CG sequences but not methylatedmCG, digestion with HpaII or BssHII results in thedestruction of corresponding CG loci and loss of thepotential methylation-accepting sites. The results showedan increase in the methyl-accepting capacity of genomicDNA that was not digested by restriction endonucleasesin hypoxia groups. However, the digestion of DNA bymethylation-dependent endonucleases led to a decreasein the methyl-accepting capacity of genomic DNA. Agreater decrease in methylation-accepting capacity wasnoted in groups digestedwith BssHII, being 48.1%, 71.4%,and 45.9% comparedwith undigested DNA 12, 24, and 36hours after hypoxia, respectively. The methylation-accepting capacity of the HpaII-digested groups declined

15.9%, 26.1%, and 12.4% comparedwith undigestedDNA12, 24, and 36 hours after hypoxia, respectively. Thus, theresults of the methylation-dependent restriction analysisindicated that hypoxia induced decreased methylation inhepatoma cell genomic DNA with a bias for CpG islands(GC0GCGC sequences) but not C0CGG sequences.

Hypoxia reduces the SAM/SAH ratio in Hep3B cellsThe ratio of SAM/SAH is a predictor of methylation.

Therefore, we examined the changes in SAM, SAH, andMTA levels in Hep3B cells exposed to hypoxia (1%oxygen). As shown in Supplementary Table S1, SAMlevel in cultured Hep3B cells under hypoxic conditionswas decreased significantly compared with normoxiccells (P < 0.001), reaching the lowest point 24 hours afterhypoxia. The ratio of SAM to SAH decreased in parallelwith the SAM level, whereas the SAH and MTA levelsremained relatively unchanged.

Hypoxia induces genomic DNA hypomethylationin vivo

To show the effect of hypoxia on DNA methylationin vivo, we established xenograft tumors in nude mice.

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Figure 1. The effect of hypoxia ongenomic DNA methylation inHep3B cells. A, C-5MTase activityexpressed as the amount ofincorporated [methyl-3H] groupsinto poly(dI-dC) in cellhomogenates. B, the methyl-accepting capacity of undigestedgenomic DNA expressed as theamount of [methyl-3H] groupsincorporated into genomic DNA.C, methylation-dependentrestriction analysis. The amount ofgenomic DNA that wasundigested or digested with HpaIIor BssHII was expressed as thenumber of incorporated methylgroups in DNA. The data werepresented as X � SD for 3independent experiments.*, P < 0.05 versus control.

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All liver cancer cell lines exhibited higher HIF-1a andMAT2A expression in the xenograft tumors than cul-tured in vitro, indicating that xenograft tumor growthcould mimic in vivo hypoxic microenvironment andinduce HIF-1a and MAT2A overexpression (Fig. 2A).Next, we investigated the changes in SAM and SAHlevels after xenograft tumor formation in vivo, andfound that all liver cancer cell lines (Bel-7402, HepG2,and Hep3B) showed a reduction in SAM content whengrown as xenografts compared with cultured in vitro (P< 0.05, Fig. 2B). In contrast, the SAH level observed inxenografts was not significantly different from thecontrol culture (Fig. 2C). Furthermore, DNA methyla-tion status was evaluated by the radioactivity incorpo-rated from labeled SAM in the xenograft tumors andcompared with levels in the same cancer cell linesin vitro. HpaII-mediated incorporation of radioactivity

(CCGG specificity) in DNA isolated from hypoxicxenograft was only slightly higher than that from thecell lines (P > 0.05, Fig. 2D). However, SssI-mediatedincorporation of radioactivity (CpG specificity) inDNA isolated from hypoxic xenografts was higher inxenografts than in the cell lines (Fig. 2E). Takentogether, these results show that hypoxia reduces theSAM level and induces genomic DNA hypomethyla-tion in vivo.

The expression of HIF-1a and MAT2A is correlatedunder hypoxic conditions

To investigate the clinical significance of the linkbetween hypoxia and DNA methylation, we examinedHIF-1a and MAT2A expression in 58 paired liver cancertissues and corresponding peritumoral tissues byimmunohistochemical staining. Representative results

Figure 2. Changes in SAM andSAH levels and [3H]-methylincorporation in xenograft livertumors. A, MAT2A and HIF-1aprotein levels in xenograft tumorsand cells in culture weredetermined by Western blot. SAM(B) and SAH (C) levels in xenografttumors were measured by HPLC.The incorporation of radiolabeled[3H] methyl groups from SAM byDNA isolated from hypoxicxenograft tumors and controlcancer cell lines. D, radioactivityincorporated in the reactionmediated by HpaII. E, radioactivityincorporated in the reactionmediated by SssI. The data werepresented as X � SD for 3independent experiments. *,P < 0.05 versus control.

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showed that HIF-1a expression was high in liver cancertissue but was low in the corresponding peritumoraltissue and undetected in normal liver tissue (Fig. 3A).Similar results were obtained for MAT2A (Fig. 3B). Thepotential correlation between the expression of HIF-1aand MAT2A was further analyzed (SupplementaryTable S2). Spearman analysis showed that the expres-sion of HIF-1awas positively correlated with MAT2A (r¼ 0.752, P < 0.001) in liver cancer tissues. Furthermore,we analyzed the clinicopathologic characteristics ofhuman liver samples and found that the expressionof HIF-1a and MAT2A was correlated with tumor sizeand TNM stage (Supplementary Table S3).

To further confirm the correlation of HIF-1a withMAT2A under hypoxic conditions, we carried out luci-ferase assay and found that MAT2A promoter activitywas notably increased in Hep3B cells under hypoxic

conditions (Fig. 3B). Western blot analysis also revealedthe positive correlation between HIF-1a and MAT2Aexpression in Hep3B cells after hypoxic treatment(Fig. 3C). In addition, we determined whether hypoxiahas an effect on the expression level of the major methyl-transferases DNMT1, DNMT3a, and DNMT3b. Theresults showed that hypoxia upregulated the expressionof DNMT1 and DNMT3a but not DNMT3b in Hep3Bcells (Fig. 3D).

To address the role of HIF-1a in the regulation ofMAT2A expression in hypoxia, we used siRNA toknockdown HIF-1a and found that this could blockMAT2A expression at the protein level induced byeither hypoxia or treatment with CoCl2, a chemicalinducer of HIF-1a (Fig. 3E). Collectively, these datasuggest that HIF-1amediates hypoxia-induced MAT2Aexpression.

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Figure 3. MAT2A is transcriptionally regulated by HIF-1a in Hep3B cells. A, HCC tissues and their adjacent nontumor liver tissues were collected andanalyzed by immunohistochemistry for MAT2A (a–c) and HIF-1a (d–f). Representative results were shown for normal liver tissues (a, d) obtained fromliver trauma patients undergoing partial hepatectomy, liver cancer tissues (b, e), and peritumoral noncancerous tissues (c, f). B, Hep3B cells were transfectedas indicated and exposed to hypoxia; the cell lysate was extracted for luciferase assay. Hep3B cells were exposed to hypoxia, and the protein level of MAT2Aand HIF-1a (C) or DNMT1, DNMT3a, and DNMT3b (D) was determined by Western blot. E, Hep3B cells were transfected as indicated. After 24 hours, the cellswere exposed to hypoxia for 24 hours or treated with 100 mmol/L CoCl2 for 24 hours, and the protein level of MAT2A and HIF-1a was determined by Westernblot. b-actin and histone H3.1 served as loading control for cytoplasmic and nuclear fractions, respectively. Shown were representative blots from 3independent experiments with similar results.

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HIF-1a overexpresson induces MAT2A promoteractivity in normoxic Hep3B cellsTo provide further evidence that MAT2A is a direct

target gene of HIF-1a, we transfected HIF-1a expressionvector into Hep3B cells for the overexpression of HIF-1aunder normoxic condition (Fig. 4A). We located 1 puta-tive HIF-1a binding site at the �281/�261 region inhuman MAT2A promoter (Fig. 4B). Luciferase assayshowed that HIF-1a overexpression significantlyincreased the luciferase activities driven by the pGL3-MAT2A (0.5 kb) promoter but not by the pGL3-MAT2A(0.25 kb) promoter (Fig. 4C). We then truncated thepromoter fragment and showed that �410/�204 frag-ment contains the putative HIF-1a binding motif(Fig. 4D). Mutation of the putative HIF-1a binding motif(ATCCCCCACGTCTCCTCG) abolished the inductionof luciferase activity in HIF-1a overexpressing cells

(Fig. 4E). Taken together, these data suggest that MAT2Ais a direct target of HIF-1a.

HIF-1a binds the consensus HRE in MAT2Apromoter in hypoxic Hep3B cells

By electrophoresis mobility shift assay (EMSA)assay, we observed a supershift obtained by coincuba-tion with monoclonal HIF-1a antibody and the absenceof binding in a 30-fold excess of unlabeled wild-type orlabeled mutant oligonucleotide, showed the specificityof HIF-1a binding. The specificity was further con-firmed by a nonspecific competition assay with a 30-fold excess of unlabeled mutated oligonucleotide,which did not cause any difference in signal intensity(Fig. 5A). ChIP assay showed a significant increase inHIF-1a binding to MAT2A promoter in hypoxic Hep3Bcells (Fig. 5B). In addition, by immunofluorescence

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ATCAAACAAG GAAGAGCAAT CCCCCACGTC TCCTCGCGTC TGCATTTTTC TCCTAGCTCT

Figure 4. Functional characterization of HRE in MAT2A promoter. Hep3B cells were cotransfected with pCMV-HIF-1a and the pGL3 basic vector or differentpGL3 MAT2A promoter reporters under normoxic conditions. The cells were also cotransfected with the pRL-SV40 plasmid to normalize transfectionefficiency. A, at 24 hours after transfection, the protein level of HIF-1a was determined by Western blot. B, the putative HRE binding sites (with the coresequence of ACGT) at the 50-flanking region of the MAT2A gene was underlined. C–E, luciferase activities were measured using the dual-luciferase reporterassay system. Note the significant induction of luciferase activities driven by the 2.5-kb MAT2A promoter in the cells cotransfected with pCMV-HIF-1a (C), thesignificant induction of luciferase activity driven by the 0.25 to 0.5-kb (�410/�204) fragment of the humanMAT2A promoter (D), and the disrupted induction ofluciferase activity by the mutated 0.25 to 0.5-kb fragment of MAT2A promoter. N, normoxia; HP, hypoxia. Results shown were from 3 independentexperiments. *, P < 0.05 versus control.






and cell fractionation we showed that HIF-1a wastranslocated into the nucleus in Hep3B cells underhypoxic conditions (Fig. 5C and D). Taken together,these results suggest that hypoxia induces the nucleartranslocation of HIF-1a, where it could bind directlyto the consensus HRE in MAT2A promoter in Hep3Bcells.

HIF-1a–mediated transcriptional activation ofMAT2A requires the recruitment of coactivatorsHADC1 and p300

To test whether HDAC1 and p300 coactivators areinvolved in MAT2A expression regulated by HIF-1a,we used inhibitors of HDAC (i.e., trichostatin A) orp300 (i.e., chetomin) and found that both treatmentssignificantly inhibited hypoxia-induced MAT2A expres-sion in Hep3B cells (Fig. 6A and B).

To further prove the interaction of HDAC1 and p300with MAT2A promoter under hypoxic conditions, re-ChIP assays were carried out to confirm the binding of

HDAC1 and p300 to HIF-1a transcription complex.Hypoxic Hep3B cells showed a high level of HDAC1and p300 binding to MAT2A promoter compared withthe control. Trichostatin A and chetomin treatment ofhypoxic Hep3B cells caused a significant reduction inHDAC1 and p300 binding to the promoter, respectively,as well as a reduction of the binding of HIF-1a to MAT2Apromoter (Fig. 6C).

Discussion

The liver is one of the organs in which hypoxia regulategene expression under normal physiologic conditionsand in diseases such as cirrhosis and cancer (26–28). Inaddition, hypoxic conditions may disrupt DNAmethyla-tion patterns, providing a potential link between theextracellular environment, epigenetic alterations, andcancer progression (29, 30). Thus the progression ofHCC may be influenced by local epigenetic alterationsunder hypoxic microenvironmental conditions, leading

--+-----Mutated oligonucleotide

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20 μm20 μm20 μm

Overlay

Figure 5. HIF-1a binds the consensus HRE motif in MAT2A promoter in hypoxic hepatoma cells. A, HepG2 cells were cultured under normoxia or hypoxia for12, 24, or 36 hours. Nuclear proteins were harvested, and binding of a consensus HRE oligonucleotide to the MAT2A promoter was analyzed by EMSA. Forsupershift and competition experiments, extracts from cells cultured for 24 hours under hypoxic conditions were used. B, ChIP assay showing the binding ofHIF-1a to MAT2A promoter in hypoxic Hep3B cells (N, normoxia; HP, hypoxia). C, indirect immunofluorescence experiments showing the nucleartranslocation of HIF-1a in hypoxic Hep3B cells. D, Hep3B cells were cultured under normoxia or hypoxia for 12 and 24 hours. Following subcellularfractionation, HIF-1a protein level in the nuclear and cytoplasmic fractions was determined by Western blot.

Liu et al.





to inappropriate silencing and activation of genesinvolved in cancer.In this study, we aimed to examine the linkage between

abnormal DNA methylation in liver cancer and hypoxicmicroenvironmental conditions. We observed thathypoxia upregulated the activity of endogenous C-5MTase activity in Hep3B cells accompanied by thechanges in the methylation status of genomic DNA,showing a hypoxia-induced demethylation process. Toelucidate the mechanism underlying the effect of hypoxiaon DNA methylation in hepatoma cells, we carried outmethylation-dependent restriction analysis and foundthat hypomethylation in hepatoma cell genomic DNAin hypoxia had a sequence bias for the BssHII cuttinglocus, suggesting that hypoxia preferentially inducesdemethylation in CpG islands (GC0GCGC sequences)but not in C0CGG sequences.Because methylation of DNA may be affected by a

limited availability of SAM or an increase in SAH, theratio of SAM/SAH, also termed methylation potential(MP), is often a predictor for methylation (7, 31). Weshowed that SAM level was significantly decreased inHep3B cells cultured under hypoxic conditions com-pared with normoxic cells. The ratio of SAM to SAHwas decreased in parallel, but SAH and MTA levelsremained relatively unchanged. However, Hermes andcolleagues (32) observed that hypoxia leads to increasedSAM and decreased SAH levels in HepG2 cells. This

difference may be due to alterations in the cell densityof cultured cells (33).

To confirm our in vitro results in an in vivo situation, weinvestigated the changes in SAM and SAH levels inxenograft tumors. All liver cancer cell lines showed areduction in their SAM content when grown as xeno-grafts compared with control cultures. However, theSAH level remained relatively unchanged. These resultsare in agreement with the findings of Chawla and col-leagues (24). These results indicate that a limited avail-ability of SAM in hypoxic xenografts probably increasesthe unmethylated sites of DNA.

SAM is an abundant methyl donor in the metabolismand is involved inmore than100methyl transfer reactions,including DNA methylation. It is synthesized frommethionine and ATP by the enzyme MAT. MAT expres-sion is characterized by a switch fromMAT1A toMAT2Aduring malignant liver transformation, which plays animportant pathogenetic role in liver cancer (34–36). There-fore, it is important to study the correlation between theexpression of HIF-1a and MAT2A. In this study, Spear-man analysis showed that the expression of HIF-1a waspositively correlated with MAT2A in liver cancer tissues.Luciferase assay showed that MAT2A expression wasnotably increased under hypoxic conditions and HIF-1amediated the hypoxia-induced expression of MAT2A.

To illustrate the direct binding of HIF-1a to MAT2Apromoter, HIF-1a binding to the consensus HRE

Figure 6. Hypoxia-inducedMAT2A expression is regulated byHDAC1 and p300. Hep3B cellscultured under hypoxia weretreated with trichostatin A (500nmol/L) or chetomin (100 nmol/L)for 24 hours. The cells were lysedfor real-time PCR (A) and Westernblot analysis (B). C, ChIP assayshowing the binding of HIF-1a toMAT2A promoter in hypoxicHep3B cells. The re-ChIP assayshowing the binding of HDAC1and p300 to MAT2A promoter inhypoxic Hep3B cells. Shown arerepresentative results from at least4 independent experiments.*, P < 0.05 versus control.TSA, trichostatin A.

3

4

Hyo

oxia *

*

A

B C

0

1

2

Co

ntr

ol

MAT2A β-Actin

1 2 3 4 5 6 7 8 9

Level of MAT2A mRNA

IP:HIF-1α

P300

MAT2A

HIF-1α Re-Chip:HADC1

Re-Chip:P300

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l

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tom

in

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2010

Hypoxia






sequence at �275 to �271 bp within MAT2A promoterwas examined by EMSA and ChIP assays. On the basis ofthe results, we conclude that MAT2A is a novel targetgene that is transcriptionally regulated by HIF-1a. Wefurther explored the transactivation mechanism of HIF-1a–mediated MAT2A expression and found that thecoactivators HDAC1 and p300 are required for the for-mation of HIF-1a transcription complex to activateMAT2A expression.

The MAT2A-encoded protein is the only SAM-synthe-sizing enzyme in the neoplastic liver because liver-spe-cific MAT1A-encoded isoenzymes that are expressed inhepatocytes are absent in the neoplastic liver. This is thefirst study to show that hypoxia alters DNA methylationpatterns in liver cancer through the reduction of thesteady-state SAM level, although we currently have noinformation about how MAT2A decreases intracellularSAM level. One possible explanation is that SAM isconsumed for polyamine biosynthesis. Another possibi-lity is the known differences in the kinetic parameters ofdifferent MAT isoforms for methionine. Taken together,our study reveals new mechanisms for the regulation ofDNAmethylation patterns in hypoxic tumor microenvir-onments. We propose that in liver cancer, hypoxia

activates MAT2A expression through HIF-1a, whichresults in the increase in MAT II enzyme activity and adecrease in SAM production, thus inducing genomicDNA demethylation.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgment

The authors appreciate the suggestion and editorial assistance ofDr. Yingqun Wang.

Grant Support

This work was supported by Natural Science Foundation of China (30730001and 30872491), National Mega Project on Major Drug Development(2009ZX09301-014), and Natural Science Foundation of Hubei Province(2009CDB292).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received November 6, 2010; revised March 14, 2011; accepted March23, 2011; published OnlineFirst April 1, 2011.

References1. Pouyss�egur J, Dayan F, Mazure NM. Hypoxia signalling in cancer and

approaches to enforce tumour regression. Nature 2006;441:437–43.2. Rosmorduc O, Housset C. Hypoxia: a link between fibrogenesis,

angiogenesis, and carcinogenesis in liver disease. Semin Liver Dis.2010;30:258–70.

3. Huang LE, Bindra RS, Glazer PM, Harris AL. Hypoxia-induced geneticinstability—a calculated mechanism underlying tumor progression.J Mol Med 2007;85:139–48.

4. Kim KW, Bae SK, Lee OH, Bae MH, Lee MJ, Park BC, et al. Insulin-likegrowth factor II induced by hypoxia may contribute to angiogenesis ofhuman hepatocellular carcinoma. Cancer Res 1998;58:348–51.

5. Mylonis I, Lakka A, Tsakalof A, Simos G. The dietary flavonoidkaempferol effectively inhibits HIF-1 activity and liver cancer cellviability under hypoxic conditions. Biochem Biophys Res Commun2010;398:74–8.

6. Menrad H, Werno C, Schmid T, Copanaki E, Deller T, Dehne N, et al.Roles of hypoxia-inducible factor-1alpha (HIF-1alpha) versus HIF-2alpha in the survival of hepatocellular tumor spheroids. Hepatology2010;51:2183–92

7. Dai CX, Gao Q, Qiu SJ, Ju MJ, Cai MY, Xu YF, et al. Hypoxia-induciblefactor-1 alpha, in association with inflammation, angiogenesis andMYC, is a critical prognostic factor in patients with HCC after surgery.BMC Cancer 2009;9:2407–18

8. Van Malenstein H, Gevaert O, Libbrecht L, Daemen A, Allemeersch J,Nevens F, et al. A seven-gene set associated with chronic hypoxia ofprognostic importance in hepatocellular carcinoma. Clin Cancer Res2010;16:4278–88.

9. Kanai Y. Genome-wide DNA methylation profiles in precancerousconditions and cancers. Cancer Sci 2010;101:36–45.

10. Tischoff I, Tannapfe A. DNA methylation in hepatocellular carcinoma.World J Gastroenterol 2008;14:1741–8.

11. Lin CH, Hsieh SY, Sheen IS, Lee WC, Chen TC, Shyu WC, et al.Genome-wide hypomethylation in hepatocellular carcinogenesis.Cancer Res 2001;61:4238–43.

12. Lu SC, Mato JM. S-Adenosylmethionine in cell growth, apoptosis andliver cancer. J Gastroenterol Hepatol 2008;23 Suppl 1:S73–7.

13. Lu SC, Mato JM. Role of methionine adenosyltransferase andS-adenosylmethionine in alcohol-associated liver cancer. Alcohol2005;35:227–32.

14. Pañeda C, Gorospe I, Herrera B, Nakamura T, Fabregat I, Varela-NietoI. Liver cell proliferation requires methionine adenosyltransferase 2AmRNA up-regulation. Hepatology 2002;35:1381–91.

15. García-Trevijano ER, Latasa MU, Carretero MV, Berasain C, Mato JM,Avila MA. S-adenosylmethionine regulates MAT1A and MAT2A geneexpression in cultured rat hepatocytes: a new role for S-adenosyl-methionine in the maintenance of the differentiated status of the liver.FASEB J 2000;14:2511–8.

16. Mato JM, Corrales FJ, Lu SC, Avila MA. S-Adenosylmethionine: acontrol switch that regulates liver function. FASEB J 2002;16:15–26.

17. Wang Q, Liu QY, Liu ZS, Qian Q, Sun Q, Pan DY. Inhibition ofhepatocelluar carcinoma MAT2A and MAT2beta gene expressionsby single and dual small interfering RNA. J Exp Clin Cancer Res2008;27:72–7.

18. Liu Q, Wu K, Zhu Y, He Y, Wu J, Liu Z. Silencing MAT2A gene by RNAinterference inhibited cell growth and induced apoptosis in humanhepatoma cells. Hepatol Res 2007;37:376–88.

19. Martínez-Chantar ML, Latasa MU, Varela-Rey M, Lu SC, García-Trevijano ER, Mato JM, et al. L-methionine availability regulatesexpression of the methionine adenosyltransferase 2A gene in humanhepatocarcinoma cells: role of S-adenosylmethionine. J Biol Chem2003;278:19885–90.

20. Adams RL, Rinaldi A, Seivwright C. Microassay for methyltransferase.J Biochem Biophys Methods 1991;22:19–22.

21. Pogribny I, Yi P, James SJ. A sensitive new method for rapid ofabnormal methylation patterns in global DNA and within CpC islands.Biochem Biophys Res Commun 1999;262:624–8.

22. Chawla RK, Watson WH, Jones DP. Effect of hypoxia on hepatic DNAmethylation and tRNAmethyltransferase in rat: similarities to effects ofmethyl-deficient diets. J Cell Biochem 1996;61:72–80.

23. Wainfan E, Dizik M, Stender M, Christman JK. Rapid appearance ofhypomethylated DNA in livers of rats fed cancer-promoting, methyl-deficient diets. Cancer Res 1989;49:4094–7.

Liu et al.





24. Chawla RK, Jones DP. Abnormal metabolism of S-adenosyl-L-methionine in hypoxic rat liver. Similarities to its abnormal metabolismin alcoholic cirrhosis. Biochim Biophys Acta 1994;1199:45–51.

25. MetivierR,PenotG,HubnerMR,ReidG,BrandH,KosM,etal.Estrogenreceptor-alpha directs ordered, cyclical, and combinatorial recruitmentof cofactors on a natural target promoter. Cell 2003;115:751–63.

26. Shahrzad S, Bertrand K, Minhas K, Coomber BL. Induction of DNAhypomethylation by tumor hypoxia. Epigenetics 2007;2:119–25.

27. Poke FS, Qadi A, Holloway AF. Reversing aberrant methylationpatterns in cancer. Curr Med Chem 2010;17:1246–54.

28. Yao DF, Jiang H, Yao M, Li YM, Gu WJ, Shen YC, et al. Quantitativeanalysis of hepatic hypoxia-inducible factor-1alpha and its abnormalgene expression during the formation of hepatocellular carcinoma.Hepatobiliary Pancreat Dis Int 2009;8:407–13.

29. Zhou J, Schmid T, Schnitzer S, Br€une B. Tumor hypoxia and cancerprogression. Cancer Lett 2006;237:10–21

30. Gwak GY, Yoon JH, Kim KM, Lee HS, Chung JW, Gores GJ. Hypoxiastimulates proliferation of human hepatoma cells through the induc-tion of hexokinase II expression. J Hepatol 2005;42:358–64.

31. Lu SC, Huang ZZ, Yang H, Mato JM, Avila MA, Tsukamoto H.Changes in methionine adenosyltransferase and S-adenosylmethio-

nine homeostasis in alcoholic rat liver. Am J Physiol Gastrointest LiverPhysiol 2000;279:G178–85.

32. Hermes M, Osswald H, Mattar J, Kloor D. Influence of an alteredmethylation potential on mRNA methylation and gene expression inHepG2 cells. Exp Cell Res 2004;294:325–34.

33. Hermes M, von Hippel S, Osswald H, Kloor D. S-adenosylhomocys-teine metabolism in different cell lines: effect of hypoxia and celldensity. Cell Physiol Biochem 2005;15:233–44.

34. Ramani K, Yang H, Xia M, Ara AI, Mato JM, Lu SC. Leptin'smitogenic effect in human liver cancer cells requires induction ofboth methionine adenosyltransferase 2A and 2beta. Hepatology2008;47:521–31.

35. Yang H, Huang ZZ, Wang J, Lu SC. The role of c-Myb and Sp1 in theup-regulation of methionine adenosyltransferase 2A gene expres-sion in human hepatocellular carcinoma. FASEB J 2001;15:1507–16.

36. Martínez-Chantar ML, Latasa MU, Varela-Rey M, Lu SC, García-Trevijano ER, Mato JM, et al. L-methionine availability regulatesexpression of the methionine adenosyltransferase 2A gene in humanhepatocarcinoma cells: role of S-adenosylmethionine. J Biol Chem2003;278:19885–90.






2011;10:1113-1123. Published OnlineFirst April 1, 2011.Mol Cancer Ther Quanyan Liu, Li Liu, Yuhong Zhao, et al. MAT2A in Hepatoma Cells

and Transcriptional Upregulation ofαActivation of HIF-1Hypoxia Induces Genomic DNA Demethylation through the

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