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Carcinogenesis Advance Access originally published online on September 21, 2006
Carcinogenesis 2007 28(2):488-496; doi:10.1093/carcin/bgl176
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Regulation of Cdx2 expression by promoter methylation, and effects of Cdx2 transfection on morphology and gene expression of human esophageal epithelial cells

Tong Liu1,{dagger}, Xinyan Zhang1,2,{dagger}, Chi-Kwong So1,{dagger}, Su Wang1, Peng Wang1, Liying Yan3, Rene Myers3, Zhigang Chen4, Amy P. Patterson4, Chung S. Yang1 and Xiaoxin Chen1,2,*

1 Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854
2 Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute North Carolina Central University, 700 George Street, Durham, NC 27707, USA
3 Biotage Inc., 2 Hampshire Street Suite 100, Foxboro, MA 02035, USA
4 NHLBI, National Institutes of Health Bethesda, MD 20892, USA

*To whom correspondence should be addressed at: Cancer Research Program, Julius L. Chambers Biomedical/Biotechnology Research Institute, North Carolina Central University, 700 George Street, Durham, NC 27707, USA. Tel: +919 530 6425; Fax: +919 530 7998; Email: lchen{at}nccu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Caudal-related homeobox 2 (Cdx2) has been suggested as an early marker of Barrett's esophagus (BE), which is the premalignant lesion of esophageal adenocarcinoma (EAC). However, the mechanism of ectopic Cdx2 expression in the esophageal epithelial cells and its role in the development of BE remained unclear. RT–PCR, pyrosequencing and methylation-specific PCR were used to determine expression and promoter methylation of Cdx2 in human esophageal epithelial cells (HET1A and SEG1) after treatment with 5-aza-2'-deoxycytidine (DAC), acid, bile acids and their combination. HET1A cells with stable transfection of Cdx2 were characterized for morphology and gene expression profiles with Affymetrix array. We found Cdx2 was expressed in most human EAC cell lines, but not in squamous epithelial cell lines. DAC-induced demethylation and expression of Cdx2 in HET1A and SEG1 cells, and treatment with a DNA methylating agent counteracted the effect of DAC. Treatment of HET1A and SEG1 cells with acid, bile acids or both also resulted in promoter demethylation and expression of Cdx2. HET1A cells with stable transfection of human Cdx2 formed crypt-like structures in vitro. Microarray analysis and quantitative real-time PCR showed that stable transfection of Cdx2 up-regulated differentiation markers of intestinal columnar epithelial cells and goblet cells in HET1A cells. This may be partially due to modulation of Notch signaling pathway, as western blotting confirmed down-regulation of Hes1 and up-regulation of Atoh1 and Muc2. Our data suggest that exposure to acid and/or bile acids may activate Cdx2 expression in human esophageal epithelial cells through promoter demethylation, and ectopic Cdx2 expression in esophageal squamous epithelial cells may contribute to intestinal metaplasia of the esophagus.

Abbreviations: BE, Barrett's esophagus; Cdx2, caudal-related homeobox 2; DAC, 5-aza-2'-deoxycytidine; EAC, esophageal adenocarcinoma; GERD, gastroesophageal reflux disease; MMS, methyl methanesulfonate; TSA, trichostatin A


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Barrett's esophagus (BE) is diagnosed when histologic evidence of intestinal metaplasia is present in human esophageal epithelium. This disease has received much attention from both gastroenterologists and basic researchers mainly due to its high risk of developing esophageal adenocarcinoma (EAC), which is the most rapidly increasing cancer in the US and European countries (1). It has been well established that chronic gastroesophageal reflux disease (GERD) is the most important etiological factor (2). However, the molecular mechanism of BE is still not clear.

Transcription factors are known to play causative roles in metaplasia or transdifferentiation. For example, Pdx1 transfection induced transdifferentiation of adult human liver cells into functional insulin-producing cells (3). MyoD1 transfection converted differentiating human keratinocytes to the myogenic pathway (4). Cdx2 is a caudal-related homeobox gene essential for skeletal and intestinal development (5). It has been suggested to play an important role in intestinal metaplasia (e.g. esophagus, stomach and bile duct) and cancers (e.g. colon cancer, leukemia) (6).

Two independent studies have shown that stomach-specific Cdx2 transgenic overexpression induced intestinal metaplasia in the mouse stomach within weeks after birth (7,8). At the age of ~2 years, these mice developed intestinal type gastric adenocarcinoma (9). Homozygous knockout of Cdx2 was embryonically lethal, and heterozygous knockout produced colonic harmatoma with squamous epithelium appearing in the colon (10). These studies suggested that Cdx2 might be a pivotal switch between intestinal columnar epithelium and squamous epithelium in the gastrointestinal tract.

Several lines of evidence have suggested an important role of Cdx2 in the development of human BE. (i) In normal intestinal epithelium, Cdx2 is expressed in most cell lineages with Paneth cells having a lower level of expression than other cells (5). Squamous epithelial cells of normal human esophagus do not express Cdx2, while submucosal glands weakly express Cdx2 protein in the cytoplasm. (ii) In human BE, Cdx2 is expressed in both goblet and non-goblet cells (11). Dysplasia and adenocarcinoma may have decreased levels of Cdx2, or even lose the expression of Cdx2. In EAC, a high level of Cdx2 expression was usually associated with well or moderate differentiation (11–17). (iii) A low level of Cdx2 mRNA was detectable by RT–PCR in biopsy samples of squamous epithelium of GERD patients, even before the appearance of Cdx2 protein and other marker genes of intestinal metaplasia and histological metaplasia (12,14). (iv) Several ‘marker’ genes of BE, such as villin, guanylate cyclase C, sucrase-isomaltase, were known to be regulated by Cdx2 (18–20). (v) Treatment of rodent esophageal squamous epithelial cells with either acid or bile acids, which mimics gastroesophageal reflux, induced expression of Cdx2 (21,22).

However, it is still unclear how Cdx2 is regulated in esophageal squamous epithelial cells, and how its ectopic overexpression may contribute to intestinal metaplasia and esophageal adenocarcinogenesis. In this study, we hypothesized that exposure of esophageal squamous epithelial cells to acid and/or bile acids might induce promoter demethylation and thus activate Cdx2 expression. Changes of gene expression profile and morphology induced by Cdx2 overexpression were then examined by stable transfection of Cdx2 into a human esophageal squamous epithelial cell line. Our data suggested that ectopic Cdx2 expression might contribute to intestinal metaplasia of the esophagus.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell lines and treatments
Human SV40-immortalized esophageal squamous epithelial cell line (HET1A) and human colon adenocarcinoma cells (Caco2) were obtained from the American Type Culture Collection (Manassas, VA). Human EAC cell lines, SEG1, FLO1, SKGT4, BIC1 and BE3, were kind gifts from Dr Xiaochun Xu (MD Anderson Cancer Center) and Dr David G. Beer (University of Michigan). Human EAC cell lines, OE19 and OE33, were obtained from the European Collection of Cell Culture (Salisburg, Wiltshire, UK). HET1A cells were routinely grown in LHC-9 serum-free medium (Biosource, Camarillo, CA), supplemented with 50 U/ml penicillin and 50 µg/ml streptomycin. Caco2, SEG1, FLO1, SKGT4, BIC1 and BE3 cells were grown in modified MEM medium (Mediatech, Herndon, VA) supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 µg/ml streptomycin. OE19 and OE33 were grown in modified RPMI1640 medium (Mediatech) supplemented with 10% FBS, 50 U/ml penicillin and 50 µg/ml streptomycin. All the cell culture experiments were performed in triplicate to ensure reproducibility.

For 5-aza-2'-deoxycytidine (DAC) treatment, cells at ~70% confluence were treated with 5 µM DAC for 72 h. In some experiments, HET1A cells were treated with 1 µM DAC for 7 days. Some cells were treated with tricostatin A (TSA, 300 nM) for 24 h. For the combination experiment, cells were treated with DAC for 72 h and then TSA for an additional 24 h. Methyl methanesulfonate (MMS, 1.5 mM), a potent DNA methylating agent (23), was used to treat the cells for an additional 24 h after DAC exposure for 72 h. As controls, HET1A and Caco2 cells were treated with 1.5 mM MMS for 24 h to determine the net effect of MMS on Cdx2 expression.

Normal culture media for HET1A cells and SEG1 cells were adjusted to the desired pH 4.0 by addition of 0.1 N HCl. Bile acid mixture was a combination of six different forms of bile acids (glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, 20:3:15:3:6:1 in molar concentration). The purpose of using a mixture instead of an individual form was to simulate the bile acids profile in the gastroesophageal refluxate of human BE patients (24). HET1A or SEG1 cells at about 80% confluence were treated by acidified medium (pH 4.0), bile acids (400 µM) or their combination for three times per day and 10 min each time.

Pyrosequencing and methylation-specific PCR of Cdx2 promoter
Promoter region of human Cdx2 genomic sequence (GenBank accession no. AL591024 [GenBank] ) was searched for CpG islands with an online search engine (www.ebi.ac.uk/emboss/cpgplot). One of the CpG islands (–1769 to –1363, or AL591024 [GenBank] nt 28354–28751) was further analyzed for methylation status by pyrosequencing.

Genomic DNA from the SEG1 cells were extracted from cell pellets with the DNeasy Tissue kit (Qiagen, Valencia, CA), according to the manufacturer's instructions. After 1 µg of genomic DNA was treated with bisulfite (EZ DNA Methylation kit; Zymo Research Co., Orange, CA), a 313-bp amplicon containing 35 CpG sites was amplified with two primers, 5'-GTT AAG GGG TTT AGG GTT GGA-3' (forward), and 5'-Biotin-CAA ATA CAA ATC TCC AAA AAT ACC-3' (reverse), at the following conditions: 94°C 15 min, 45 cycles (94°C 30 s, 60.5°C 30 s, 72°C 30 s), 72°C 5 min. This region was then pyrosequenced with the PSQ 96HS system (Biotage AB, Uppsala, Sweden), according the established method (25).

The methylation-specific PCR primers were designed to flank one CpG site (Site 25) in the above-mentioned CpG island. A common reverse primer, 5'-AAA GGA TAT TGG AGA GTA TTT TAG-3' was used for both the methylated and unmethylated alleles. The forward primer for methylated allele was 5'-CGA AAA TAA AAA TCA CTA CGA CG-3' (PCR product size: 200 bp), and the forward primer for unmethylated allele 5'-ATT CAA AAA TAA AAA TCA CTA CAA CA-3' (PCR product size: 204 bp). Amplification was performed with the following conditions: 94°C 10 min, 35 cycles (94°C 30 s, 56°C 30 s, 72°C 45 s), 72°C 7 min. Universally methylated and unmethylated DNA samples (Chemicon International, Temecula, CA) were used as controls in these experiments.

Stable transfection of Cdx2 into HET1A cells
The Cdx2-expressing construct was prepared as described previously (26). The Cdx2 plasmids were transfected into HET1A cells using FuGENE 6 transfection reagent as described by the supplier (Roche Molecular Systems, Alameda, CA). After transient transfection, the cells were analyzed for expression of Cdx2 protein by immunocytochemistry, or lysed to harvest total RNA or protein. To establish stable transfection, the transfected cells were selected by resistance to 20 µg/ml G418 in the culture medium. Surviving colonies were screened with RT–PCR specific for Cdx2. Two clones with the highest expression level of Cdx2 were maintained on G418 for subsequent studies. Cell proliferation was determined with a CellTiter 96 non-radioactive cell proliferation assay based on the MTT method (Promega, Wisconsin, WI).

Reverse transcription–PCR and quantitative real-time reverse transcription PCR
Total RNA was isolated from cells with Trizol (Invitrogen), and reverse transcription performed with the Advantage RT-for-PCR kit (Clontech). Cdx2 RT–PCR was carried out with two primers: 5'-GAG CTG GAG AAG GAG TTT-3' and 5'-GGT GAC GGT GGG GTT TAG-3' (product: 392 bp). Expression of glyceraldehyde-3-phosphate dehydrogenase gene was examined to determine the integrity of RNA samples and to standardize the amount of cDNA added to PCR reactions. Primers from the RT-for-PCR kit were used to amplify a product of 983 bp.

Real-time quantitative reverse transcription–PCR was performed to quantify the expression levels of genes of interest with primers and probes obtained from Applied Biosystems Inc. (Foster City, CA). Briefly, real-time PCR was performed using the qPCR Mastermix (containing HotGoldStar Taq polymerase, dNTPs with dUTP replacing one-third of dTTP, uracil-N-glycosylase, reference dye ROX) and 96-well optical plate on the ABI Prism 7000 sequencing detector in triplicate. The 2–{Delta}{Delta}Ct method was used for quantification with ß-actin or GAPDH as endogenous control. Gene expression in Cdx2-transfected HET1A cells (HET1A-Cdx2) was expressed as fold changes by comparing with that in vector-transfected cells (HET1A-vector).

Immunocytochemistry and western blotting
Cells were seeded in the 8-well chamber slides at a density of 0.5 x 106 cells per ml. After being cultured for two days, the cells were fixed with 100% ethanol overnight. The avidin–biotin–peroxidase complex method (Elite ABC kit; Vector Laboratories, Burlingame, CA), monoclonal mouse anti-Cdx2 antibody (1:100 dilution; Biogenex, San Ramon, CA). For western blotting, cell lysates were loaded on 4–15% acrylamide gel (20 µg/lane) for electrophoresis, and then transferred onto a nitrocellulose membrane. After incubation in blocking buffer [5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20], the membranes were incubated overnight at 4°C with a mouse monoclonal anti-Cdx2 (Biogenex, 1:100), rabbit polyclonal anti-Hes1 (1:500, Chemicon), rabbit polyclonal anti-Atoh1/Math1 (1:500, Chemicon), and rabbit polyclonal anti-Muc2 (1:300, Santa Cruz Biotechnology, Santa Cruz, CA), for detection of Cdx2 (33 kDa), Hes1 (33 kDa), Atoh1/Math1 (45 kDa), and Muc2 (~30 kDa). ß-actin (42 kDa) was also detected with a monoclonal antibody (Sigma, St Louis, MO) as loading controls.

Affymetrix gene array and data analysis
The amplification and labeling procedure was modified on the basis of the procedure described originally by Eberwine et al. (27). GeneChip human genome U133A microarrays (Affymetrix, Santa Clara, CA) were hybridized with 18 µg of fragmented biotinylated cRNA. After removing the hybridization solutions, arrays were first washed and then fluorescence-stained with streptavidin–phycoerythrin. GeneChips were scanned with a 2500 HP GeneArray Scanner for quantification with Affymetrix Microarray Suite Version 5.0 (MAS 5.0). Data analysis was performed using the Affymetrix MAS5 algorithm. Differential expression was determined on the basis of expression ratio with cutoff of >1.5 or <0.5 (HET1A-Cdx2 versus HET1A-vector cells).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Cdx2 expression in human esophageal epithelial cells and its up-regulation by DAC treatment
Using RT–PCR, we found that Cdx2 was expressed in most human EAC cell lines (e.g. BE3, BIC1, FLO1, OE19, OE33, SKGT4 cells), except SEG1 cells. HET1A cells did not express Cdx2, as well as several esophageal squamous cell carcinoma cell lines (KYSE30, KYSE150 and KYSE510 cells obtained from Dr Yutaka Shimada, Kyoto University, Kyoto, Japan)(data not shown). Treatment with DAC (5 µM for 3 days) activated the expression of Cdx2 in SEG1 and HET1A cells, and significantly up-regulated Cdx2 in SKGT4 cells (Figure 1A). To find out whether histone deacetylation might cooperate with promoter methylation in silencing Cdx2, we treated HET1A cells with DAC for 3 days and then TSA (300 nM) for an additional 24 h. Those treated with DAC, or TSA alone, were used as controls. We found that TSA up-regulated Cdx2 expression in synergy with DAC. TSA alone was not effective in inducing Cdx2 expression (Figure 1B). Similar results were obtained in SEG1 cells (data not shown). When we exposed the DAC-treated HET1A cells to MMS (1.5mM), a DNA methylating agent, for 24 h, MMS attenuated Cdx2 expression that had been activated by DAC. Similarly, when Cdx2-positive Caco2 cells were treated with MMS, Cdx2 expression was also down-regulated (Figure 1C). Therefore, promoter methylation seemed to be a major silencing mechanism of Cdx2 expression in human esophageal epithelial cells.


Figure 1
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  		Fig. 1 Expression of Cdx2 in human esophageal epithelial cell lines, and its regulation by promoter methylation. (A) Among a panel of human esophageal epithelial cell lines, HET1A and SEG1 did not express detectable Cdx2 mRNA by RT–PCR. Cdx2 expression was induced in these two cell lines by treatment with 5 µM DAC for 3 days. M, DNA size marker. (B) Cdx2 expression was detected by RT–PCR in HET1A cells after treatment with 5 µM DAC, but not 300 nM TSA alone. However, TSA enhanced the activating effect of DAC; (C) Treatment of HET1A cells with 1.5 mM MMS for 1 day counteracted the effect of DAC (5 µM for 3 days). Similarly, Cdx2 expression in Caco2 cells was also down-regulated by treatment with MMS. (D) Cdx2 protein was not detectable in HET1A cells by immunocytochemistry; (E) High level of Cdx2 protein expression was detected by immunocytochemistry in HET1A after transient transfection of Cdx2; (F) Treatment with 5 µM DAC for 3 days induced expression of Cdx2 protein in HET1A cells; (G) Caco2 cells, which are known to express Cdx2, served as positive control of immunocytochemistry. Both DAC-induced and transfected Cdx2 were expressed and localized in the nuclei of HET1A cells.

 
With immunocytochemistry of Cdx2, it was clear that HET1A cells did not express detectable Cdx2 protein, while DAC treatment induced a moderate amount of Cdx2 in cell nuclei (Figure 1D and F). As controls, Caco2 cells and HET1A cells with transient Cdx2 transfection expressed a high level of Cdx2 (Figure 1E and G).

Promoter methylation as a silencing mechanism of Cdx2 in human esophageal epithelial cells
Pyrosequencing was performed to scan 35 CpG sites of one CpG island (–1769 to –1363) in the promoter region of Cdx2 gene, 24 of which were shown in Figure 2A. As expected, universal methylated DNA sample had ~95% methylation, and universal unmethylated DNA sample ~10% methylation, at all these CpG sites. Untreated SEG1 cells had a methylation level ~90%, and treatment with DAC for 3 days decreased the methylation level to ~50%, indicating a ~50% demethylation at all these CpG sites (Figure 2A). These data correlated with activation of Cdx2 expression. Methylation-specific PCR was then designed to determine the methylation status of the 25th CpG site in this region. Again, in both HET1A and SEG1 cells, DAC treatment induced partial demethylation of Cdx2 promoter (Figure 2B).


Figure 2
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  		Fig. 2 Methylation status of Cdx2 gene promoter in HET1A and SEG1 cells. (A) Pyrosequencing of the promoter region of Cdx2. The methylation percentage of 24 CpG sites of unmethylated control DNA, methylated control DNA, SEG1 cells and SEG1 cells treated with DAC (5 µM for 3 days), were shown as mean and SD (error bars). Only the average percentages of methylation were shown in the table. These CpG sites were localized in the promoter region of Cdx2 gene (nt –1590 to –1411). (B) Methylation status of Cdx2 gene in HET1A and SEG1 cells before and after treatment with DAC by methylation-specific PCR. U, unmethylated allele; M, methylated allele. Universal methylated and unmethylated human DNA samples were used as the positive and negative controls, respectively.

 
Regulation of promoter methylation and expression of Cdx2 in human esophageal epithelial cells by acid and bile acids
In order to determine whether acid and/or bile acids may re-activate Cdx2 expression through promoter demethylation, we pulse-treated HET1A cells and SEG1 cells with acid and/or bile acids to mimic gastroesophageal reflux in BE patients. With methylation-specific PCR, we found treatment with acid, bile acids or their combination for 7 days partially demethylated Cdx2 promoter of HET1A cells (Figure 3A). Corresponding to promoter demethylation, Cdx2 mRNA was activated by acid, bile acids and their combinations (Figure 3B). Such an activating effect was the strongest after the treatment with a combination of acid and bile acids. Similar results were obtained in SEG1 cells (Figure 3C and D).


Figure 3
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  		Fig. 3 Regulation of Cdx2 methylation status and expression in HET1A and SEG1 cells by gastroesophageal refluxate (acid, bile acids or both). (A) Methylation status of Cdx2 in HET1A cells by methylation-specific PCR after treatments as indicated in Materials and methods. U, unmethylated allele; M, methylated allele. (B) Expression of Cdx2 in HET1A cells by RT–PCR; (C) Methylation status of Cdx2 in SEG1 cells by methylation-specific PCR after treatments; (D) Expression of Cdx2 in SEG1 cells by RT–PCR.

 
Morphology of HET1A cells stably transfected with Cdx2
We then established a HET1A cell line stably transfected with Cdx2 and examined its changes in morophology and gene expression.

Using RT–PCR, we screened a series of clones and identified two with the highest level of Cdx2 expression, pIRES/neo-Cdx2-1 and pIRES/neo-Cdx2-2. Western blotting showed that these two clones expressed Cdx2 protein (Figure 4A). These two clones were then maintained with a culture medium containing G418 for subsequent studies. It was interesting that HET1A-Cdx2 cells grew faster than the control and HET1A-vector cells. After 3 days in culture, the difference became statistically significant (Figure 4B). As expected, Cdx2 protein was not detected by immunocytochemistry in HET1A-vector cells (Figure 4C), but expressed strongly in the nuclei of HET1A-Cdx2 cells (Figure 4D).


Figure 4
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  		Fig. 4 Stable transfection of Cdx2 induced metaplastic changes of morphology in HET1A cells. (A) Stable transfection of pIRES/neo-Cdx2 induced Cdx2 expression by RT–PCR. Two representative clones of pIRES/neo or pIRES/neo-Cdx2 transfected HET1A cells were shown. With western blotting, HET1A-Cdx2 cells were shown to overexpress Cdx2, as the positive control cells (Caco2). (B) HET1A-Cdx2 cells grew faster than HET1A-vector and parental HET1A cells, as determined by an MTT-based assay. (C) Cdx2 was not expressed in HET1A-vector cells by immunocytochemistry; (D) Strong nuclear expression of Cdx2 was present in HET1A-Cdx2 cells; (E) HET1A-vector cells under inverted light microscopy; (F) HET1A-Cdx2 cells formed ‘crypt-like structure’ in vitro. (Size bar: 10 µM C–F). (G) Expression of Hes1, Atoh1 and Muc2 in HET1A-vector and HET1A-Cdx2 cells by western blotting.

 
To our surprise, under normal culture condition HET1A-Cdx2 cells formed a ‘crypt-like structure’ in plastic petri dish. Individual cells became bigger and rounder in shape than the HET1A-vector cells (Figure 4E and F).

Gene expression profile of HET1A cells stably transfected with Cdx2
Using Affymetrix arrays, we examined the gene expression profile of HET1A-Cdx2 cells as compared with HET1A-vector cells. Differential expression of most genes was confirmed by real-time RT–PCR. We then categorized these genes into several groups according to their known functions in epithelial cell differentiation and involvement with human BE as follows (Table I): (i) Markers of intestinalized columnar epithelial cells (e.g. villin, ANPEP, GCC, SI, KRT20 and FABP2) and goblet cells (Muc2) were significantly up-regulated by Cdx2. (ii) A set of genes on the Notch pathway were modulated by Cdx2 transfection, with Hes1 down-regulated and Atoh1 and Muc2 up-regulated. (iii) A number of transcription factors important for gastrointestinal development were up-regulated (Cdx1, Cdx4, KLF4, KLF5, HoxA9, HoxA2, HoxA7 and Sox10), or down-regulated (Sox2) by Cdx2 expression. (iv) Some known Cdx2-regulated genes (HEPH, DTR, VDR, FABP6, IL2RB, NPR1, GABRR2, Muc2, GCC, SI, HOXA9, Atoh1, APC, UGT1A10, KLF4 and VIL1) were induced by Cdx2 expression. (v) Genes known to be overexpressed in human BE (PGC, Muc6, AGR2, DNMT3B and TSPAN-1) were induced by Cdx2 expression. (vi) Squamous epithelial cells specific markers (SPRR3, KRT1, KRT9, KRT14, KRT15 and KRT19) were down-regulated by Cdx2 transfection.


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  		Table I Genes up- and down-regulated in HET1A cells by stable transfection of Cdx2

 
To further confirm the involvement of Notch pathway, we performed western blotting of Hes1, Atoh1 and Muc2. Consistent with the microarray data, Hes1 was down-regulated, and Atoh1 and Muc2 up-regulated (Figure 4G). It should be noted that Muc2 is a goblet cell marker, and the presence of goblet cell is diagnostic of human BE.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Mechanism of intestinal metaplasia of the esophagus has been largely unclear. In this study, we found that Cdx2 was expressed in most human EAC cell lines (e.g. BE3, BIC1, FLO1, OE19, OE33, SKGT4 cells), but not in SEG1 cells and squamous epithelial cells studied. Promoter methylation silenced Cdx2 expression in human esophageal epithelial cells (SEG1 and HET1A; Figures 1 and 2). Using the technique of restriction landmark genome scanning, Feltus et al. (28) identified 66 methylation-prone CpG islands in DNMT1-overexpressing cells. One of them was associated with Cdx2. In both gastric cancer and colorectal cancer, promoter hypermethylation was associated with Cdx2 silencing (29,30). However, Cdx2 regulation by promoter methylation may be a tissue- or cell-specific event. Different from the esophageal epithelial cells in our study, some colon cancer cells used silencing mechanisms other than promoter methylation (31).

The pathological relevance of Cdx2 promoter demethylation to BE was established by our data of SEG1 and HET1A cells treated with acid and bile acids (Figure 3). Marchetti et al. reported that chronic acid exposure up-regulated the expression of Cdx2 in primary squamous epithelial cells of mouse esophagus (22). While this manuscript was in preparation, two studies showed that bile acids activated the expression of Cdx2 in cultured rat esophageal keratinocytes and human esophageal epithelial cells. NF{kappa}B pathway played a critical role in this process (21,32). It has been known that acid and bile acids activated the NF{kappa}B and p38 MAPK pathways (33,34), and these pathways might further activate Cdx2 expression to regulate downstream genes (35,36). All these data demonstrated that multiple regulatory mechanisms including promoter methylation contributed to Cdx2 activation in human esophageal epithelial cells due to gastroesophageal reflux.

To examine the functional consequence of Cdx2 in an esophageal squamous epithelial cell line, we established an HET1A cell line with stable transfection of Cdx2. An interesting observation of the HET1A-Cdx2 cells was the ‘crypt-like structure’ formed in vitro (Figure 4). Such a structure has been reported in SW1222 and LIM1863 colon cancer cells (37,38). However, its biological significance is not clear and warrants further studies.

Our microarray data further supported a causative role of Cdx2 in intestinal metaplasia of the esophagus (Table I). Cdx2 up-regulated the differentiation markers of intestinalized columnar epithelial cells and goblet cells in HET1A cells. An interesting observation was that Cdx2 transfection down-regulated Hes1 and up-regulated Atoh1. These are critical genes of the Notch pathway, and are known to regulate the expression of Muc2, a marker of goblet cells. Knockout of Hes1 led to an increased number of goblet cells in mouse small intestine (39), whereas loss of Atoh1 completely prevented the development of goblet cells in mouse intestine (40). Our data were also in line with previous studies showing that Cdx2 transfection up-regulated Atoh1 in IEC6 cells (41), and Muc2 in rat esophageal keratinocytes (21). Since goblet cell is essential for histopathological diagnosis of human BE, all these data suggested that Cdx2 played an important role in differentiation of goblet cells in human BE.

Several differentiation markers of squamous epithelium, such as keratins and small proline-rich protein 3 (SPRR3, also named as esophagin), were down-regulated by Cdx2 transfection. Sox2, a Sry-like high-mobility group gene expressed from the pharynx to the stomach in the gastrointestinal tract (42), was also down-regulated by Cdx2, similar to intestinal metaplasia of human stomach (43). These data suggested that Cdx2 might also contribute to squamous de-differentiation during intestinal metaplasia.

In summary, acid and bile acids activated Cdx2 in esophageal epithelial cells through promoter demethylation. The ectopically expressed Cdx2 may then modulate expression of a series of genes to facilitate intestinal metaplasia. In fact, gene methylation has been reported to play a critical role in the development of mammalian esophagus. When the primitive esophageal columnar epithelium was converted to squamous epithelium during the late stage of embryonic development, de novo DNA methylation was required to silence keratin 8, a marker of columnar epithelium (44). Further studies are needed to study how gene methylation may contribute to the development of BE.


   Footnotes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
Supported by NIH fellowship 5T32ES07148, and grants CA75683, DK63650 and CA092077.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 28, 2006; revised August 11, 2006; accepted September 4, 2006.


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