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Clinical Cancer Research Vol. 9, 5257-5263, November 1, 2003
© 2003 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Hypermethylation-Associated Inactivation of Retinoic Acid Receptor ß in Human Esophageal Squamous Cell Carcinoma

Yimin Wang1, Ming Zhu Fang1, Jie Liao, Guang-Yu Yang, Yan Nie, Yunlong Song, Chi So, Xiaochun Xu, Li-Dong Wang and Chung S. Yang2

Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey [Y. W., M. Z. F., J. L., G-Y. Y., Y. N., Y. S., C. S., C. S. Y.]; Department of Clinical Cancer Prevention, University of Texas, M. D. Anderson Cancer Center, Houston, Texas [X. X.]; and Laboratory for Cancer Research, Zhengzhou University, Henan, China [L-D. W.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: The purpose of this study was to investigate the mechanism of altered retinoic acid receptor ß (RARß) expression during esophageal squamous carcinogenesis.

Experimental Design: Samples were collected from Linzhou, China. The hypermethylation of CpG islands in the promoter region of the RARß gene was examined by methylation-specific PCR in human esophageal squamous cell carcinoma (ESCC) samples, as well as in neighboring tissues with normal epithelium, basal cell hyperplasia, and dysplasia. RARß mRNA expression was determined by in situ hybridization. The DNA methyltransferase inhibitor 2'-deoxy-5-azacytidine was used to treat the ESCC cell line, and the DNA hypermethylation status and mRNA expression level were examined.

Results: Two of 17 (12%) normal, 9 of 21 basal cell hyperplasia (43%), 7 of 12 dysplasia (58%), and 14 of 20 ESCC (70%) samples had hypermethylation of the RARß promoter region. The loss of RARß mRNA expression was highly concordant with RARß promoter CpG island hypermethylation when individual samples were considered in the correlation analysis. Good statistical correlation between hypermethylation and loss of RARß expression was revealed. Frequencies of hypermethylation appeared to increase with the progression of carcinogenesis. In samples from the same patients, if hypermethylation was detected in earlier lesions, it was usually observed in more severe lesions. In the ESCC cell line KYSE 510, 2'-deoxy-5-azacytidine partially reversed CpG island hypermethylation and restored RARß mRNA expression.

Conclusions: The results suggest that hypermethylation of RARß promoter region is an important mechanism for RARß gene silencing in esophageal squamous carcinogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid and its analogues can modulate cell growth and differentiation in vitro and in vivo (1 , 2) . Retinoids have been used successfully in the treatment of preneoplastic diseases, such as oral leukoplakia (3) , cervical dysplasia (4) , and xeroderma pigmentosum (5) . These compounds have also been reported to reduce second malignancies in the liver, aerodigestive tract, and breast (6, 7, 8, 9) . Retinoids exert their biological effects by binding to the specific nuclear retinoid receptors RARs3 and retinoid X receptors, each of which consists of three subtypes ({alpha}, ß, and {gamma}). RARs are differentially expressed during development and in adult life, and there is strong evidence that RARß plays a central role in growth regulation of epithelial cells and in tumorigenesis (10, 11, 12) . RARß- and retinoid X receptor-dependent pathways, but not RAR{alpha}- or RAR{gamma}-dependent pathways, lead to cyclin D1 degradation and blockage of cell cycle progress (13) .

RARß expression is frequently down-regulated in human cancers, including those of the head and neck, lung, breast, pancreas, and esophagus (14) . In a study of esophageal cancer, RARß expression was lost in 54% of ESCC and 57% of dysplastic lesions (15) . RARß loss of expression was not correlated with loss of heterozygosity at the RARß chromosome location 3p24 in esophageal cancer (16) . The molecular mechanism for down-regulation or loss of RARß expression in ESCC is still unknown.

Hypermethylation of CpG islands is an important epigenetic mechanism for the transcriptional silencing of many genes (17, 18, 19) . DNA hypermethylation may directly affect the basal transcriptional machinery by altering the DNA secondary structure and inducing chromosome remodeling through the methyl-group-binding proteins and histone deacetylase, which leads to transcriptional repression (20) . Treatment with DAC, a DNA methyltransferase inhibitor, causes the reactivation of many genes that have been inactivated by hypermethylation (21, 22, 23, 24, 25) . Hypermethylation of CpG islands in the RARß promoter region has been reported in many human cancers, including breast, lung, colon, stomach, cervical, bladder, and prostate cancers (26, 27, 28, 29, 30, 31) . In lung and breast cancers, hypermethylation of the RARß promoter region was found to be the mechanism for RARß gene silencing (30 , 32 , 33) , but there was no correlation between hypermethylation and loss of expression of this gene in prostate cancer (31) . The effect of hypermethylation of the RARß promoter region on the expression of the RARß gene in esophageal squamous carcinogenesis still needs to be examined.

We have been studying the molecular alterations in human ESCC samples from a well-recognized high-risk area, Linzhou City (formerly named Linxian), in the Henan province of China. p53 mutations, loss of heterozygosity, and microsatellite instability of the tumor suppressor gene cluster 9p21 (p14ARF, p15INK4b, and p16INK4a) and the Rb gene on 13q14, and hypermethylation of p16INK4a and HLA class I genes have been observed as frequent early events in esophageal carcinogenesis (34, 35, 36, 37, 38, 39) .

In the present study, we examined hypermethylation of RARß promoter CpG islands in ESCC and precancerous lesions and correlated the results with the expression of RARß in the same samples, determined by in situ hybridization. The effect of a DNA methyltransferase inhibitor, DAC, on the expression of RARß was also studied in an ESCC cell line.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Sample Preparation and DNA Extraction.
Primary ESCC specimens containing neighboring nontumorous epithelial tissues were collected from patients in the Linzhou City People Hospital in Linzhou City. The use of the human specimens was approved by Rutgers Human Subject Protocol 94-138. The samples were frozen in liquid nitrogen within 1 h after surgical resection and stored in liquid nitrogen, on dry ice, or at -80°C until use. For each specimen, two pieces of tissue, one from the tumor mass and the other from the nontumorous region, were dissected and embedded with tissue freeze medium (OTC). Samples with normal epithelia, BCH, DYS, or ESCC were confirmed histopathologically on slides stained with H&E. OTC-embedded frozen tissues were cut to a thickness of 10 µm and placed on RNase-free slides. After air-drying, these slides were stored at -80°C before in situ hybridization.

Genomic DNA was extracted from ESCC tissues and adjacent nontumorous tissues with use of the DNeasy Tissue Kit (Qiagen, Valencia, CA) and stored at -80°C before analysis. In another set of studies, cells from normal and precancerous lesions were microdissected with use of a laser capture microdissection system (Arcturus, Mountain View, CA). For methylation analysis of the RARß promoter region, DNA was extracted from ~500 cells dissected from each sample.

MSP.
MSP takes advantage of the differences between methylated and unmethylated sequences after bisulfite modification (36 , 40) . To verify the specificity of the PCR reaction, we used placenta DNA as a negative control and SssI-methylated placenta DNA as a positive control for hypermethylation. A first round of PCR was performed using primers specific for bisulfite-modified DNA (Ref. 32 ; Table 1Citation ). PCR was performed under the conditions as described by Bovenzi and Momparler (32) . All reactions contained 5–10 ng of bisulfite-modified DNA and 1.2 units of Qiagen Hotstart Taq polymerase in a 25-µl reaction volume. The DNA fragments resulting from the first PCR were used for MSP. Methylation- and unmethylation-specific primer pairs for the RARß promoter region (RARß-M and RARß-U) were the same as described by Cote et al. (29) . These primers were used to amplify a 146-bp fragment of the RARß promoter region. PCR products were analyzed on a 3% agarose gel with ethidium bromide staining.


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  		Table 1 Primer sequences, annealing temperatures, and PCR product sizes used for MSP and reverse transcription-PCR

 
Samples with only the methylation-specific band were designated as M, possibly reflecting homozygous hypermethylation; samples with only the unmethylation-specific band were designated as U. If the sample had both methylation- and unmethylation-specific bands, a densitometry visual comparison system was used to determine the band intensities, and the ratio of the methylation-specific and unmethylation-specific band intensities was calculated. We use the designations MU to indicate the presence of both of these bands with an intensity ratio <=2, M/U to indicate that the intensity ratio of M/U is >2, and U/M to indicate that the intensity ratio of U/M is >2.

In Situ Hybridization.
The presence of RARß mRNA in human ESCC samples was analyzed by use of nonradioactive in situ hybridization with a digoxigenin-labeled RARß cRNA probe as described previously (41 , 42) . The sense and antisense probes were prepared, and the quality and specificity of the probes were determined by Northern blotting as described previously (43) . Either sense probe or probe vehicle was used as a negative control to verify the specificity of the antisense cRNA probe. The stained sections were reviewed under a Nikon microscope. For each case, two pathologists (G-Y. Y. and J. L.) examined the adjacent histological section stained with H&E to identify normal, BCH, DYS, and ESCC samples. In situ hybridization staining intensity was graded as -, -/+, +, or ++.

Studies with ESCC Cell Lines.
The ESCC cell lines KYSE 510, 150, and 450 were maintained in RPMI 1640 and Ham F12 mixed (1:1) medium containing 5% fetal bovine serum. KYSE 150 and 450 cells were harvested on day 3 for total RNA and genomic DNA extraction. For treatment of KYSE 510 cell with DAC, after seeding, medium containing DAC (8.7 µM) was added to the cells on days 1, 3, and 5. Cells were harvested on day 7 for extraction of total RNA and genomic DNA. RARß mRNA was analyzed with RARß-specific primers, using the Advantage RT for PCR kit and Advantage PCR kit (Clontech, Palo Alto, CA; Table 1Citation ). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was used as an internal control. PCR products were analyzed on a 2% agarose gel. In these cell samples, methylation status was also determined by MSP assay as described above.

Classification and Statistical Analysis.
For methylation status classification, we considered samples with M, M/U, and MU as methylation positive, and those with U/M and U as methylation negative. For in situ hybridization, intense purple staining (+ and ++) was classified as positive; no staining (-) and background or trace faint staining (-/+) were classified as negative. Fisher’s exact test was used to analyze the correlation between DNA methylation and lost expression of the RARß gene. Differences with P < 0.05 were regarded as significant.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hypermethylation of 5' CpG Islands in the RARß Gene Locus.
The methylation status of the RARß CpG islands was investigated by MSP in ESCC samples together with their adjacent normal epithelial. Examples of esophageal specimens producing methylation-specific bands, unmethylation-specific bands, or both bands are shown in Fig. 1Citation . The CpG islands in the promoter region of the RARß gene were methylated in 14 of 20 ESCC samples (70%). Of these 14 samples, 9 showed only the methylation-specific bands (M), and 4 (samples 20, 26, 32, and 40) showed both methylation- and unmethylation-specific bands with similar intensities (MU). One (sample 13) showed a methylation-specific band and a weak unmethylation-specific band (M/U). In the 17 adjacent nontumorous epithelial tissue samples, 14 showed only the unmethylation-specific bands (U), 1 showed a stronger unmethylation-specific band and a weaker methylation-specific band (U/M; sample 22), and 2 showed only the methylation-specific bands (samples 28 and 30).



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  		Fig. 1. Determination of the methylation of the RARß promoter region. Methylation- or unmethylation-specific PCR was performed with sodium bisulfite-modified DNA samples. A, tumor samples showing methylation-specific bands only. B, tumor samples showing both methylation- and unmethylation-specific bands. C, methylation status in microdissected cells from different stages of esophageal carcinogenesis. N, normal epithelium; T, tumor; MS, methylation-specific PCR band; US, unmethylation-specific band.

 
To further analyze the methylation status, we analyzed laser-capture-microdissected cells from morphologically normal regions of the esophageal epithelium. Of the five normal samples analyzed, three (samples 17, 21, and 26) had only unmethylation-specific bands, one (sample 22) was U/M, and one (sample 30) had only a methylation-specific band (Fig. 1Citation and Table 3Citation ). Microdissected samples with precancerous lesions were also used for the methylation analysis. Of the 21 BCH samples, CpG islands were hypermethylated in 9 (43%), of which sample 22 showed M/U status. Of the 12 DYS samples, 7 (58%) were hypermethylated, of which 3 showed both methylation- and unmethylation-specific bands: sample 26 was classified as MU, and samples 13 and 22 as M/U.


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  		Table 3 RARß gene methylation status and corresponding mRNA expression in sample with different lesions

 
Expression of RARß mRNA in Human ESCC Tissues and Precancerous Lesions.
We determined RARß mRNA expression in human esophageal frozen sections containing different lesions, using in situ hybridization. RARß was expressed in the cytoplasm and around the cell nucleus, and positive cells were distributed in the entire epithelium layer (Fig. 2)Citation . RARß mRNA was expressed in 76% (13 of 17) of normal epithelium samples, 81% (17 of 21) of BCH samples, 50% (4 of 8) of DYS samples, and 42% (10 of 24) of ESCC samples (Table 2)Citation . In BCH, the positive staining intensity was generally lower than in normal epithelium (Table 2)Citation . The results indicated that in patients with ESCC, RARß expression was lost in ~20% of samples containing normal epithelium or BCH, but in ~50% of the samples with DYS and ESCC.



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  		Fig. 2. Expression of RARß in normal esophageal epithelium, DYS, and ESCC as determined with in situ hybridization. Digoxigenin-labeled RARß cRNA probe was hybridized with human esophageal frozen sections. A and B, normal esophageal epithelium (x200), inset (G; x400); C and D, DYS (x200); E and F, ESCC (x200). A, C, and E were stained with H&E. B, D, and F show results of in situ hybridization.

 

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  		Table 2 RARß expression in esophageal samples with different lesions

 
Correlation between Hypermethylation and Silencing of RARß Gene in Esophageal Carcinogenesis.
To elucidate the relationship between DNA hypermethylation and loss of expression of RARß, we compared the results from 13 normal epithelium samples, 6 BCH samples, 7 DYS samples, and 18 ESCC samples (Table 3)Citation . The five samples having both methylation- and unmethylation-specific bands with MU status (Table 3)Citation were the most ambiguous ones, with two samples showing -, two showing -/+, and one sample showing + status in in situ hybridization. The appearance of the methylation-specific band was associated with low or no expression of mRNA. It is not clear whether MU status represents heterozygous DNA hypermethylation. With the other 39 samples (Table 3)Citation , we observed good correlation between hypermethylation and loss of RARß mRNA expression in 37 samples. The two exceptions included one "normal" sample (sample 21), which had no hypermethylation but lacked RARß mRNA expression, and one ESCC sample (sample 22) with a strong methylation band but expressing RARß mRNA, albeit at lower levels. Fisher’s exact test showed that DNA hypermethylation was significantly correlated with transcriptional inactivation of the RARß gene in 13 normal (P = 0.0385), 13 precancerous (BCH and DYS; P = 0.008), and 18 ESCC (P = 0.0025) samples (Table 4)Citation .


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  		Table 4 Correlation between RARß gene methylation status and the loss of mRNA expression by Fisher’s exact test

 
Hypermethylation and loss of expression of RARß appeared to become more frequent as the lesion progressed. In the same resected esophageal samples, if hypermethylation was detected in earlier lesions, it was usually also observed in later lesions. For example, in case 13, hypermethylation started to occur at the BCH stage and became more prominent in the DYS and ESCC stages. In case 22, strong hypermethylation was observed in precancerous lesions (BCH and DYS) and in ESCC (Fig. 1C)Citation . Nevertheless, there were exceptions; for example, although hypermethylation occurred in DYS (sample 19) and normal (sample 30) samples, the ESCC samples had unmethylation status and expressed RARß mRNA. The reason for this discrepancy is not known and remains to be investigated.

Restoration of RARß Expression after DAC Treatment.
Two of the three ESCC cell lines examined showed RARß hypermethylation. Cell line KYSE 510 had only a methylation-specific band, suggesting that it harbored homozygous DNA hypermethylation (Fig. 3)Citation , and KYSE 150 showed both methylation- and unmethylation-specific bands, suggesting that it harbored partial hypermethylation or heterozygosity (Fig. 3)Citation at the CpG islands of the RARß gene. Reverse transcription-PCR analysis showed that KYSE 510 cells did not have detectable RARß mRNA expression (Fig. 3)Citation , whereas KYSE 150 showed weaker mRNA expression (Fig. 3)Citation . The results suggest that heterozygous hypermethylation did not inactivate but lowered the expression of RARß. To determine whether hypermethylation at the CpG islands of the RARß promoter region is responsible for silencing the RARß gene, we treated the cells with DAC, a DNA methyltransferase inhibitor that previously has been shown to reverse the hypermethylation of genes. After DAC treatment for 6 days, the unmethylation-specific band appeared (Fig. 3)Citation , as did the expression of RARß mRNA (Fig. 3)Citation . The restoration of RARß mRNA expression by DAC treatment is consistent with the concept that DNA hypermethylation leads to silencing of the RARß gene. In the DAC-treated cells, the methylation-specific band still existed (Fig. 3)Citation . This partial reversion could be attributable to the inhibition of only the newly synthesized DNA strands (44) . Alternatively, there could be only a selected population of cells that were affected by the DNA methyltransferase inhibitor, whereas other cells were resistant to the treatment (45) .



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  		Fig. 3. RARß methylation status and mRNA expression in ESCC cell lines KYSE150, 450, and 510, and alteration of methylation status and mRNA expression in KYSE 510 by DAC treatment. ESCC cell line KYSE 510 was treated with 8.7 µM DAC for 6 days. MS, methylation-specific PCR band; US, unmethylation-specific PCR band.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Expression of RARß is critical for retinoic acid-induced cell growth and differentiation. In many cancers, including ESCC, RARß expression is frequently lost (15) . In esophageal carcinogenesis, it is not clear when and how expression of RARß is altered. In this study, we hypothesize that CpG island hypermethylation of the RARß gene is a key mechanism for the silencing of this gene, and this process was investigated systematically in human esophageal tissues with different grades of lesions as well as in ESCC cell lines. Hypermethylation of the RARß promoter region was observed in some of the histologically normal esophageal epithelial samples. In some of these samples, both methylation- and unmethylation-specific bands were observed. An interesting possibility is that hypermethylation had already occurred in some apparently normal cells. The percentage of samples with hypermethylation increased in BCH (43%) and further increased in DYS (58%), which was similar to that in ESCC (70%) samples. Some of the ESCC samples also had both methylation- and unmethylation-specific bands. If we assume that the tumor developed from the same clone of epithelial cells, the minor unmethylation-specific bands (case 13 in Table 3Citation ) are likely attributable to contamination by stromal cells. Another possibility is that in some ESCC samples, only one allele of the RARß gene is hypermethylated, as we observed in the KYSE 150 cell line (Fig. 3)Citation . In this case, the methylation- and unmethylation-specific bands are expected to be of equal intensity. This was observed in cases 20, 26, 32, and 40 (Table 3)Citation .

We used in situ hybridization to determine RARß mRNA expression in human ESCC samples because PCR-based analysis always involves the risk of possible contamination by other cells in the sample. RNA analysis using laser-capture-microdissected samples is technically very difficult and was not successful in the preliminary experiment. Loss of expression of RARß was observed in samples with histologically normal epithelium and BCH (19 and 24%, respectively) and at increased frequencies in DYS and ESCC samples (43 and 58%, respectively). Our results for samples with lesions were similar to those reported by Qiu et al. (15) . The loss of RARß expression in normal esophageal epithelium has not been reported previously.

The loss of RARß mRNA expression correlated well with DNA hypermethylation in Fisher’s exact test, showing statistical significance for samples in each category of normal, precancerous lesions, or ESCC. A more rigorous correlation analysis is planned to determine whether hypermethylation is correlated with the loss of RARß expression in each individual sample. The results from the five samples bearing MU status suggest that methylation of the RARß promoter decreases the level of mRNA expression. Of the 39 samples with clear methylated or unmethylated status, 37 showed a good concordance with the mRNA level. In two samples that did not show a concordance, one normal sample had no mRNA expression but showed no hypermethylation. This could be attributable to other mechanisms, such as gene deletion or mutation. The other, an ESCC sample with strong methylation bands but also with RARß mRNA expression, is more intriguing. One possibility is that the samples were taken from different parts of a heterogeneous carcinoma.

All of the above results suggest that hypermethylation of the RARß promoter occurs rather early in some individuals and that the frequency of this event increases during esophageal carcinogenesis. The mechanism for this hypermethylation reaction is not known. Esophagitis is rather common in this high-risk population in Linzhou (46 , 47) , and chronic inflammation could be a driving force for the hypermethylation. It has been suggested that increased cell turnover induced by chronic inflammation could make some CpG islands progressively lose protection against methylation (48) .

The involvement of CpG island hypermethylation in the silencing of the RARß gene is also supported by the observations that treatment of KYSE 510 cells (with hypermethylation of the RARß promoter and no mRNA expression) with DAC caused partial reversion of RARß promoter hypermethylation and reexpression of mRNA. Silencing of the RARß gene would affect the normal differentiation of esophageal epithelial cells and promote carcinogenesis. If hypermethylation-associated silencing of the RARß gene is a significant event in esophageal carcinogenesis, we may expect that once such an event occurred in an individual’s esophageal epithelium, it should also be observed in samples with more severe lesions. This was indeed observed in most of the esophageal samples (Table 3)Citation , but there were some exceptions. In two cases (19 and 30), hypermethylation was observed in normal and DYS samples, but not in the corresponding ESCC samples. It is possible that the group of cells with hypermethylation in normal or DYS tissues, in the absence of other key molecular alterations, did not progress or had not progressed to cancer and that the clinically observed carcinomas originated from different clones of epithelial cells. The reversion of hypermethylation may also occur in the normal sample, but this is less likely in the DYS sample.

Our results suggest that DNA hypermethylation of RARß promoter CpG islands, which leads to gene silencing, is a rather early event in esophageal squamous carcinogenesis. Dietary or pharmaceutical agents that can inhibit this process may prevent or delay the development of ESCC. In cancer patients, activation of RARß expression has been proposed as a therapeutic approach (49 , 50) . Our results provide support for the use of a selected DNA methyltransferase inhibitor for the prevention and treatment of esophageal cancer.


   ACKNOWLEDGMENTS
 
We thank Dr. Yutaka Shimada (Kyoto University, Kyoto, Japan) for providing the KYSE cancer cell lines for this study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by NIH Grant CA65871 (to C. S. Y.).

1 Yimin Wang and Ming Zhu Fang contributed equally in this study. Back

2 To whom requests for reprints should be addressed, at Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020. Phone: (732) 445-5360; Fax: (732) 445-0687; E-mail: csyang{at}rci.rutgers.edu Back

3 The abbreviations used are: RAR, retinoic acid receptor; ESCC, esophageal squamous cell carcinoma; DAC, 2'-deoxy-5-azacytidine; BCH, basal cell hyperplasia; DYS, dysplasia; MSP, methylation-specific PCR. Back

Received 4/21/03; revised 7/14/03; accepted 7/29/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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