Cancer Investigation, 25:685–690, 2007ISSN: 0735-7907 print / 1532-4192 onlineCopyright c© Informa Healthcare USA, Inc.DOI: 10.1080/07357900701561131

ORIGINAL ARTICLECellular and Molecular Biology

Promoter Hypermethylation of the RUNX3 Genein Esophageal Squamous Cell CarcinomaChaozhong Long, M.D.,1 Bangliang Yin, M.D., Ph.D.,1 Qianjin Lu, M.D., Ph.D.,2 Xinmin Zhou, M.D., Ph.D.,1

Jianguo Hu, M.D.,1 Yifeng Yang, M.D., Ph.D.,1 Fenglei Yu, M.D., Ph.D.,1 and Yunchang Yuan, M.D.1

Department of Cardiothoracic Surgery1 and Epigenetic Research Center2, The Second Xiangya Hospital of Central South University, Changsha,Hunan, China.

ABSTRACT

Alteration in transforming growth factor-β (TGF-β) signaling pathway is one of the maincauses of esophageal squamous cell carcinoma (ESCC). The human runt-related transcriptionfactor 3 (RUNX3), an important component of TGF-β pathway which is located at 1p36, is com-monly deleted in a variety of human cancers, including ESCC. Hypermethylation of RUNX3 pro-moter was frequently found in gastrointestinal cancers, including those of stomach, liver, colonand pancreas. However, RUNX3 promoter methylation status in ESCC has not been studied. Theaim of this study was to determine whether promoter methylation of the RUNX3 gene correlateswith ESCC tumor progression.Accordingly, we first determined RUNX3 mRNA expression andmethylation status of its promoter region in 42 primary tumors with ESCC and Eca-109, an ESCCcell line. Loss of RUNX3 mRNA expression was detected by RT-PCR in 23 out of 42 (54.8%)ESCC specimens and Eca-109 cells. The Promoter hypermethylation was detected by Methy-lation Specific Polymerase Chain Reaction (MS-PCR) in 27 out of 42 (64.3%) ESCC specimenand Eca-109 cells.Importantly, we found positive correlations, not only between the promoterhypermethylation and tumor clinical pathologic stages (P = 0.003), but also between the loss ofRUNX3 mRNA expression and the tumor progression (P = 0.016). Finally, we observed that theloss of RUNX3 mRNA expression is statistically correlated with the promoter hypermethylationin these tumors (P < 0.001). Our results suggest that epigenetic silencing of RUNX3 geneexpression by promoter hypermethylation may play an important role in ESCC development.

INTRODUCTION

Esophageal carcinoma is the sixth most frequent causeof cancer death in the world, and esophageal squamouscell carcinoma (ESCC) accounts for 90% of the esophagealcarcinoma in Asian countries (1). Although surgical techniques

Keywords: DNA methylation, RUNX3, and esophageal squamouscell carcinoma.This study was supported in part by the Hunan Natural ScienceFoundation of China (06JJ5028).Correspondence to:Bangliang Yin, M.D., Ph.DDepartment of Cardiothoracic SurgeryThe Second Xiangya Hospital of Central South UniversityChangsha 410011, HunanChinaemail: Bangliangyin1@yahoo.com.cn

and preoperative management have made certain progresses,the prognosis for patients with ESCC remains poor. Findingmolecular therapeutic targets for ESCC treatment is one of themost promising avenues of research that may help oncologiststo improve the survival of patients with ESCC. Althoughmultiple genetic and epigenetic changes have been reportedin ESCC (2–4), the precise molecular mechanisms governingESCC development remain unknown.

Transforming growth factor-β (TGF-β) signaling is a well-established tumor suppressor pathway in terms of carcinogene-sis (5). As such, TGF-β receptors and their downstream signaltransducer Smads are frequently inactivated in various cancers(6). It has been demonstrated that modulation of the expressionof molecules in TGF-β signaling is involved in the progressionof esophageal cancer (7). Specifically, runt-related transcrip-tion factor (RUNX) proteins, including RUNX3, were reportedto interact with Smads through their C-terminal segment and,thus, recruit Smads to subnuclear sites of active transcription,

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where they exert their biological control function (8). SinceRUNX3 is an integral component of the TGF-β-induced sig-naling pathway, it is plausible that RUNX3 may also functionas a tumor suppressor gene in different types of cancers thatTGF-β-signaling is impaired.Comparative genomic hybridiza-tion of ESCC has revealed the frequent occurrence of multiplechromosomal deletions at 1p, 3p, 4p, 18q, 19p, 19q, etc (9, 10).The region of 1p commonly observed to be deleted in ESCCwas mapped in 1p36 (9). Interestingly, the chromosome 1p36,where RUNX3 resides, is a commonly deleted region in varioustypes of human cancers (11–13). Loss of RUNX3 expressionthrough high frequent hemizygous deletion and hypermethy-lation was found in 25–75% of breast, gastric and pancreaticcancers (11–13). Furthermore, the gastric mucosa of RUNX3knocked out mice exhibited a phenotype of hyperplasia, sup-pressed apoptosis, and growth inhibition induced by TGF-β inepithelial cells (11). However, RUNX3 promoter methylationstatus in ESCC has not been studied.We hypothesized that epi-genetic alterations in RUNX3 may play an important role inESCC development.

In this study, we first determined the RUNX3 DNA methy-lation status by methylation-specific polymerase chain reaction(MS-PCR) and the mRNA expression by reverse transcriptionpolymerase chain reaction (RT-PCR) in 42 primary ESCC tis-sues, the corresponding normal tissues, and in Eca-109, an ESCCcell line.We further evaluated the correlations among RUNX3promoter hypermethylation, loss of RUNX3 mRNA expression,and the tumor clinical pathologic stages.

MATERIALS AND METHODS

Specimens

Tumor specimens and the corresponding normal tissues wereobtained from 42 patients who underwent surgery for ESCC (34male and 8 female; median age, 56.3 years; and range, 46–67years) from June to November of 2005 in the clinics at the Sec-ond Xiangya Hospital of Central South University, Changsha,Hunan. None of these patients received preoperative treatment,such as chemotherapy or radiotherapy. All of the tumors werepathologically confirmed as ESCC. Written informed consent,as required by the institutional review board, was obtained fromall patients. One patient was at clinical pathological stage I; 17were at stage II; 23 were were at stage III; and 1 was at stageIV, according to the Tumor-Node-Metastasis classification (14).The tissue samples were excised and immediately stored in liq-uid nitrogen.

METHODS

Cell culture

Eca-109, the esophageal carcinoma cell line was purchasedfrom the Cell Bank of Shanghai Institute for Cell Biology, Chi-nese Academy of Sciences (Shanghai, China).The cells werecultured in RPMI-1640 supplemented with 10% fetal bovineserum (FBS) and incubated in 5% CO2 at 37◦C.

RT-PCR

Total RNA was isolated from primary ESCC tissues, thecorresponding normal tissues, and Eca-109 cells using TrizolReagent (Invitrogen, Carlsbad, California, USA), and reversetranscribed into cDNA (1.2 µg) with a reverse transcriptasesystem Kit (Promega, Madison, Wisconsin, USA). PCRwas performed by using primers for RUNX3 (15): sense5′-ACTGTGATGGCAGGCAATGAC-3′ and antisense 5′-AATGGGTTCAGTTCCGAGGTG-3′, which amplify a 404 bpproduct.The β-actin mRNA was also amplified in the same PCRreactions as an internal control using the following primers:sense 5′- CGCGAGAAGATGACCCAGAT-3′ and antisense5′-GCACTGTGTTGGCGTACAGG-3′, which amplify a 550bp product.The PCR amplification cycles consisted of denatu-ration at 95◦C for 3 min, 34 cycles of denaturation at 94◦C for30 sec, annealing at 60◦C for 30 sec, extension at 72◦C for 30sec, and a final elongation at 72◦C for 7 min. Ten microliters ofPCR production was loaded onto 1.5% agarose gels containingethidium bromide (EB) and visualized under UV illumination.

MS-PCR

Genomic DNA was obtained from above specimens andEca-109 cells by digestion with proteinase K, followed byphenol/chloroform extraction. DNA samples were treated withsodium bisulfite (16). The methylation status in the promoterregion of the RUNX3 was determined by MS-PCR as de-scribed previously (16–18). Briefly, 5 µg of genomic DNAwas denatured by 3M NaOH, and then incubated in 3.12 Msodium bisulfite and 10 mM hydroquinone for 17 h at 55◦C.Bisulfate-treated DNA was extracted using a genomic DNAcleanup kit (Promega).After purification, the samples weretreated with NaOH at a final concentration of 0.3 M in avolume of 50 µL for 15 min at 37◦C, then neutralized with50 µL 6M NH4OAc. The DNA was ethanol-precipitated andre-suspended in 50 µL ddH2O for MS-PCR analysis. Modi-fied DNA was amplified by two different sets of primers spe-cific to unmethylated and methylated RUNX3 sequences, asdescribed previously (18). The primer pairs for the unmethy-lated detection were in the RUNX3 promoter region near Exon1: sense 5′-GTGGGTGGTTGTTGGGTTAGT-3′ and antisense5′-TCCTCAACCACCACTACCACA-3′, which amplify a 138bp product; for the methylated detection the primers were:sense 5′-CGTCGGGTTAGCGAGGTTTC-3′ and antisense 5′-GCCGCTACCGCGAAAAACGA-3′, which amplify a 120 bpproduct.The PCR amplification consisted of 35 cycles at 94◦Cfor 20 s; 60◦C for 20 s; and 72◦C for 15 s after the initial denat-uration step (94◦C for 5 min). A final extension was at 72◦C for10 min. The PCR was performed with an ABI Gene Amp PCRsystem 9700. Ten microliters of PCR products were analyzed on2% agarose gels with EB and visualized under UV illumination.

Demethylating treatment with 5-azaxycytidine(5-Aza)

Eca-109 cells were cultured at low density (5 × 105 per T-25cm2 flask) for 24 hrs in cell growth medium. Then, the cells

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Figure 1. Representative RUNX3 RT-PCR analysis in primaryesophageal squamous cell carcinomas.T: tumor, N: correspond-ing normal tissues.β-actin: amplification served as a control forcDNA quality (550bp), RUNX3 amplify product: 404bpH2O: nega-tive control.

were treated with 5-Aza (Sigma-Aldrich, St. Louis, Missouri,USA) at a concentration of 0 µM, 10 µM, 20 µM, and 50 µMfor 72 hrs to achieve demethylation.

Total RNA was extracted and RT-PCR was performed as de-scribed above.

Statistical analysis

The correlation between the RUNX3 promoter methylationstatus and clinical pathological stages in ESCC specimens, thecorrelation between RUNX3 mRNA expression and clinicalpathological stages in ESCC specimens, and the correlation be-tween RUNX3 promoter methylation status and RUNX3 mRNAexpression were analyzed by using x2 test or Fisher’s exact test.P values less than 0.05 were considered as significant.

RESULTS

To determine whether the promoter methylation of theRUNX3 gene correlates with ESCC tumor progression, we firstdetermined RUNX3 mRNA expression and methylation statusof RUNX3 promoter region in 42 ESCC primary tumors andthe corresponding normal tissues by RT-PCR and MS-PCR,respectively. Loss of RUNX3 mRNA expression was detectedby RT-PCR in 23 out of 42 (54.8%) ESCC specimens, whereasall the corresponding normal tissues expressed RUNX3 mRNA(Figure 1 and Table 1). To examine whether the RUNX3 genesilencing (loss of mRNA expression) in ESCC specimenswas due to the RUNX3 promoter hypermethylation, MS-PCRwas performed on the above tissue specimens.Methylation

of the RUNX3 promoter region was detected in 27 out of 42(64.3%) primary ESCC tissues (Table 1). Twenty-seven primaryESCC tissues showed both methylated and unmethylated DNAPCR product. However, the methylated DNA PCR productswere not able to be detected in all the corresponding normaltissues.Fifteen out of 42 primary ESCC tissues (35.7%) and all42 corresponding noncancerous tissues showed unmethylatedbands.As expected, in primary tumors, the contaminatedstromal cells might contribute to the presence of unmethylatedbands (Figure 2). To further investigate whether RUNX3mRNA expression could be re-activated after the treatment with5-Aza, a commonly utilized demethylation reagent, Eca-109cells were used as a model system and treated with a 5-Aza.The expression of the RUNX3 gene was measured by RT-PCR.Interestingly, 5-Aza re-activated the silenced RUNX3 genein the Eca-109 cell line and restored it to greater levels in adose-dependent manner (Figure 3).

Finally, we evaluated the correlation between RUNX3 pro-moter methylation in the ESCC tissues with clinical pathologicalstages.We found that in ESCC tissues from clinical stages I andII, frequency of RNNX3 promoter hypermethylation was 7/18(38.9%). In contrast, in tissues from stages III and IV, frequencyof RNNX3 promoter hypermethylation was 20/24 (83.3%).

The frequency of RUNX3 promoter hypermethylation ap-peared greater in stages III and IV than that of in stages I and II. Asignificant correlation between RUNX3 promoter hypermethy-lation and clinical pathological stages was observed (P = 0.003)(Table 1). There was no statistical correlation between RUNX3promoter hypermethylation with any of parameters such as age,sex, tumor size or histological differentiation.

The correlation between RUNX3 mRNA expression andclinical pathological stages in ESCC specimens and thecorrelation between RUNX3 promoter methylation status andRUNX3 mRNA expression were further analyzed. The loss ofRUNX3 mRNA expression in ESCC tissues correlated withpathological stages (Table 1). Importantly, RUNX3 mRNAwas not expressed in any of the 23 tumors with RUNX3promoter hypermethylation. The remaining 4 tumors with the

Table 1. Association of RUNX3 hypermethylation and loss of RUNX3 gene expression with various clinicopathologic characteristics in ESCC

RUNX3 MS-PCR RUNX3 RT-PCRClinic pathologic factor Subgroups No. + − p value∗ No. + − p value∗

Sex Male 34 22 12 0.907 34 18 16 0.625Female 8 5 3 8 5 3

Age <60 y 29 21 8 0.101 29 19 10 0.101≥ 60y 13 6 7 13 5 8

Tumor size <5cm 19 12 7 0.890 19 10 9 0.801≥ 5cm 23 15 8 23 13 10

Differentiation Well 3 1 2 0.326 3 1 2 0.141Moderate 26 16 10 26 12 14Low 13 10 3 13 10 3

Clinical stage# I/II 18 7 11 0.003 18 6 12 0.016III/IV 24 20 4 24 17 7

P values were obtained after correction for multiple comparisons within each prognostic group. P < 0.05 was considered statistically significant.Clinical stage was determined by combining T, N, and M (distant metastasis) stages.

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Figure 2. Representative MS-PCR analysis of RUNX3 promoter in ESCC specimens. Representative MS-PCR of RUNX3 promoter by using bothunmethylated (U) and methylated (M)-specific primers in ESCC.Product in “U” lanes indicates unmethylated DNA; product in “M” lanes indicatemethylated DNA. Corresponding normal tissue showed only unmethylated, whereas ESCC demonstrated both unmethylated and methylatedPCR products. T: tumor; N: corresponding normal tissue; U: results of unmethylated primer; M: results of methylated primer.

RUNX3 promoter hypermethylation and all 42 noncanceroustissues showed RUNX3 expression. We also observed thatthere was a positive correlation between RUNX3 promoterhypermethylation and the loss of RUNX3 mRNA expression inthese tumor specimens (P = 0.000; Fisher’s exact test).

DISCUSSION

Hypermethylation is a regional event that occurs frequentlyin a GC-rich sequence called CpG islands.These islands areoften located within the 5′-regulatory nontranscribed regions ofgenes.It has been recognized that aberrant hypermethylation ofCpG islands in the promoter region of certain tumor suppressorgenes is associated with gene transcriptional inactivation andloss of the gene function. In cancer, the promoter hypermethyla-tion occurs as an early event in the multi-steps ofcarcinogenesis(19). In ESCC, methylation associated gene silencing has beenreported in certain genes, such as p16, FHIT, MGMT and E-cadherin, which are involved in ESCC tumorigenesis (20–24).Although hypermethylation of RUNX3 gene promoter wasreported in gastrointestinal cancers including those of stomach,liver, colon and pancreas (12–15, 18, 25, 26), however, RUNX3promoter methylation status in ESCC has not been studied.To our knowledge, this is the first report that demonstratedepigenetic silencing of RUNX3 gene expression by promoterhypermethylation in ESCC development.

TGF-β is a multifunctional cytokine known to be a potentgrowth inhibitor of most epithelial cells (27, 28). TGF-β signal-ing pathways, consisted of TGF-β type I and type II receptorsand Smad proteins, function as a negative regulator of cell prolif-eration. TGF-β is usually transducted by forming a heteromericcomplex with its type I and type II transmembrane receptors. Ac-tivated type I receptors activate cytoplasmic Smad 2 and Smad3

Figure 3. RT-PCR analysis of RUNX3 mRNA for Eca-109 treatedby various concentrations of 5-Aza. β-actin amplification served asa control for cDNA quality (550 bp), RUNX3 amplify product: 404bp. H2O: negative control.0: 0 µmol/L 5-Aza, 10 µM:10 µmol/L5-Aza, 20 µM: 20 µmol/L 5-Aza, 50 µM: 50 µmol/L 5-Aza.

by phosphorylation, and allow them to form a hetermeric com-plex with Smad 4. This Smad complex can activate TGF-β re-sponsive gene transcription only after it is translocated into thenucleus and bound to specific site of target genes (28). The al-tered expression of the TGF-β receptors, Smad2 and Smad4,has been shown to correlate with tumor progression and poorprognosis in esophageal carcinomas.It is commonly acceptedthat reduced expression of these factors, rather than gene muta-tion, is more likely to be a guilty mechanism acting through theTGF-β signaling pathway (29). The functional loss of this path-way may increase cell proliferation, decreased cell apoptosis,and increase tumorigenesis (5, 29).

The 1p36 region is believed to harbor tumor suppressor genesbecause previous studies have identified frequent allelic imbal-ance at 1p36 in various types of human cancers (30). Recently,several studies have demonstrated the expression of RUNX3 inthe gastrointestinal organs of a developing embryo.In addition,the silencing of this gene was reported to be contributed by lossof heterozygosity (LOH), point mutation, or hypermethylationof the promoter region (31, 32). In the present study, we foundsimilar incidences of promoter hypermethylation in 64.3%ESCC tissues. Statistically, we did not observe correlationbetween hypermethylation with any of the following clinicalparameters, such as age, sex, tumor size and differentiation;however, we found a statistically significant correlation betweenthe presence of RUNX3 promoter hypermethylation and theESCC clinical pathological stages (P = 0.003). In addition,there was a significant concordance between the RUNX3promoter methylation status and RUNX3 mRNA expression.These findings indicate that hypermethylation of RUNX3promoter may represent a common mechanism of inactivationof this candidate tumor suppressor gene in human primary

Table 2. Methylation status of RUNX3 and expression of RUNX3 inESCC

RUNX3 methylation statusRUNX3 expression + −− 23 0+ 4 15

∗P = 0.000 RUNX3 expression (−): loss or decrease, RUNX3expression (+): normal; RUNX3 methylation status (+): methylatedband, RUNX3 methylation status (−): no methylated band.

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cancers. Finally, we showed that 5-Aza, a demethylating drug,was able to re-activate the expression of the RUNX3 gene inESCC cell lines (Figure 3). Taken together, the high prevalenceof RUNX3 promoter hypermethylation observed in this studyand reactivation of the expression of RUNX3 by a demethy-lating drug provide a strong evidence that hypermethylationof RUNX3 promoter appears to be a dominant mode forinactivating RUNX3 gene expression in human ESCC.

Recently, Maruyama et al reported that promoter hyperme-thylation of RASSF1A was significantly correlated to severalparameters for the poor prognosis of bladder cancers (33). Here,we demonstrated that RUNX3 hypermethylation correlates tothe ESCC tumor progression. These findings suggest that, inmany cases, RUNX3 hypermethylation may also occur as a lateevent during ESCC progression. Therefore, RUNX3 hyperme-thylation may become a novel prognostic marker for patientswith ESCC.

In summary, we have demonstrated a higher frequency of pro-moter hypermethylation of the RUNX3 gene in ESCC tissuesand cell lines. These observations provide evidence that pro-moter hypermethylation may be one of the major mechanismsfor inactivating RUNX3 gene in ESCC. Inactivation of RUNX3gene function may result in impairment of TGF-β-Smads sig-naling pathway and thus contribute to the development of ESCC.

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