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(American Journal of Pathology. 2001;158:299-303.)
© 2001 American Society for Investigative Pathology

Regular Articles

Biallelic Inactivation of Retinoic Acid Receptor ß2 Gene by Epigenetic Change in Breast Cancer

Qifeng Yang^*, Ichiro Mori^*, Liang Shan^*, Misa Nakamura^*, Yasushi Nakamura^*, Hirotoshi Utsunomiya^*, Goro Yoshimura, Takaomi Suzuma, Takeshi Tamaki, Teiji Umemura, Takeo Sakurai and Kennichi Kakudo^*

From the Second Department of Pathology^*
and Department of Surgery,
Wakayama Medical College, Wakayama, Japan; and the Laboratory of Experimental Carcinogenesis,
National Cancer Institute, Bethesda, Maryland

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
A growing body of evidence supports the hypotheses that retinoic acid receptor ß2 (RAR ß2) is a tumor suppressor gene. Although the loss of RAR ß2 expression has been reported in many malignant tumors, including breast cancer, the molecular mechanism is still poorly understood. We hypothesized that loss of RAR ß2 activity could result from multiple factors, including epigenetic modification and loss of heterozygosity (LOH). Using methylation-specific polymerase chain reaction and LOH analysis, we found that biallelic inactivation via epigenetic changes of both maternal and paternal alleles, or epigenetic modification of one allele combined with genetic loss of the remaining allele, could completely suppress RAR ß2 expression in breast cancer. Thus, it is possible that substantial numbers of human cancers arise through suppressor gene silencing via epigenetic mechanisms that inactivate both alleles. Because of this, chromatin-remodeling drugs may provide a novel strategy for cancer prevention and treatment.

	Introduction

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The regulation of cell growth and differentiation by retinoids in normal, premalignant, and malignant cells is thought to result from both direct and indirect effects on retinoid gene expression mediated by nuclear receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs). These receptors are ligand-activated transcription factors and members of the steroid hormone receptor superfamily, and both consist of three subtypes: , ß, and . Retinoid receptors activate transcription in a ligand-dependent manner by binding as RAR/RXR heterodimers or RXR homodimers to retinoic acid response elements (RAREs) located in the promoter regions of target genes.

One of the target genes of retinoid receptors is RAR ß, which has a central role in the growth regulation of mammary epithelial cells. RAR ß maps to chromosome 3p24, a region that exhibits a high frequency (45%) of loss of heterozygosity (LOH) in primary breast tumors.⁶ Retroviral transduction of breast tumor cell lines with RAR ß2 results in inhibition of tumor cell proliferation.⁷ RAR ß2 levels were found to be decreased or suppressed in a number of malignant tumors, including lung carcinoma, squamous cell carcinoma of the head and neck, and breast cancer.^8-10 These findings suggest that RAR ß2 plays an important role in limiting the growth of many cell types, and that the loss of this regulatory activity is associated with tumorigenesis. To understand why RAR ß activity is down-regulated or lost in malignant tumors, intense efforts have been directed at identifying possible alterations that affect either the RAR ß2 promoter or its regulatory factors.^7,10,11 Intriguingly, methylation of the RAR ß2 promoter region has recently been described in breast cancer.^12-14 The aim of this study was to further elucidate the mechanism of complete RAR ß2 inactivation in breast cancer.

	Materials and Methods

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Cell Lines and Tissue Samples

Human female breast cancer cell lines T-47 D and MRK-nu-1 were obtained from Japan Cancer Research Resources Bank (Tokyo, Japan). A total of 45 paired tumor and peripheral blood samples were collected from the Affiliated Kihoku Hospital of Wakayama Medical College, Japan. Tumor samples were snap-frozen at -70°C immediately after resection. All specimens underwent histological examination by two pathologists to confirm diagnosis of adenocarcinoma through evaluation of more than 90% of sample tumor cells.

DNA and RNA Extraction

DNA and RNA extractions were performed using the QIAamp Tissue Kit and the Rneasy Kit (both Qiagen GmbH, Hilden, Germany), respectively, according to the manufacturer’s protocols.

Microsatellite Analysis of LOH

Analysis of polymerase chain reaction (PCR)-based LOH was performed by using three microsatellite markers flanking chromosome 3p24: D3S 1283, D3S 1293, and D3S 1286. All primer sequences and their locations were obtained from human genetic linkage maps. PCR was carried out in reaction volumes of 50 µl containing 100 ng genomic DNA as template, 1x PCR buffer, 200 µmol/L dNTP mix, 300 nmol/L forward primer, 300 nmol/L reverse primer, and 2.5 units Taq DNA polymerase. Each microsatellite marker was amplified from paired normal and tumor DNA samples by PCR under the following reaction conditions: 94°C for 2 minutes for one cycle; followed by 35 cycles of 94°C for 1 minute, 52–60°C for 30 seconds, and 72°C for 45 seconds, with a final incubation step at 72°C for 5 minutes. Ten-microliter aliquots of the PCR products were then loaded onto 6% denaturing polyacrylamide gels, and separated by electrophoresis at 350 volts for 3 to 6 hours. Gels were stained using the PlusOne DNA Silver Staining Kit in a GeneStain Automated Gel Stainer (Pharmacia Biotech AB, Uppsala, Sweden). Two observers analyzed the staining results visually and recorded allele imbalance when there was clear reduction in the intensity of one allele amplified from tumor DNA samples.

Mutation Analysis

To amplify the region containing the ßRARE, we used the following primer pairs: sense primer 5'-GGA GTT GGT GAT GTCAGA CTA G-3', position 737–758; and antisense primer 5'-GAT CCC AAG TTC TCC TTC CAA G-3', position 1062–1040 of RAR ß2 promoter region (GenBank accession no. X56849). After an initial denaturation step at 94°C for 1 minute, DNA was amplified through 35 cycles of 30 seconds denaturing at 94°C, 30 seconds annealing at 60°C, and 45 seconds extension at 72°C in a DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, CT). Expected fragment length was 324 bp. PCR fragments were purified using the QIAquick Purification kit (Qiagen, Chatsworth, CA), and 1-µl aliquots used in sequencing reactions using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). After purification using Centri-Sep Spin Columns, samples were resuspended in 20 µl of template suppression reagent for sequence analysis in an Applied Biosystems Model 310 Sequencer (Applied Biosystems). Sequencing was performed on both DNA strands.

Evaluation of Methylation Status of the RAR ß2 Promoter Region

Approximately 1.0 µg of each DNA sample was bisulfite modified using a commercial kit (CpGenome DNA modification kit, Oncor Inc., Gaithersburg, MD) according to the manufacturer’s instructions. Bisulfite-modified DNA was PCR amplified by using the primer pairs RARß-M and RARß-U, as previously described.¹⁵ Samples positive for RAR ß2 promoter region methylation were confirmed using the PCR-based HpaII restriction enzyme assay according to the procedure detailed by Kane et al.¹⁶ Briefly, genomic DNA samples were digested with HpaII or MspI, or incubated without restriction enzymes. The 550-bp region upstream of the RARß2 gene, which contains 5 HpaII sites, was then amplified using the primer pairs: forward primer, 5'-TGC TCA ACG TGA GCC AGG A-3', position 554–572; and reverse primer, 5'-AGG CTT GCT CGG CCA ATC CA-3', position 1105–1086 of RAR ß2 promoter region (GenBank accession no. X56849).

Cell Culture

RPMI 1640 growth medium was supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cultures were grown at 37°C in a 5% CO₂ atmosphere. Cells were seeded at 4 x 10⁴ cells per T 75 flask on day 0 and treated for 24 hours on days 2, 3, and 4 with 5-aza-2'-deoxycytidine (5-Aza-CdR). On day 6, cells were harvested for analysis of RAR ß2 promoter region methylation status and for RAR ß2 protein production.

Reverse Transcription (RT)-PCR for RAR ß2

One-microgram aliquots of RNA were reverse-transcribed in 20-µl reaction volumes at 42°C using the SuperScript Preamplification System (Life Technologies, Gaithersburg, MD), and 2-µl aliquots of cDNA then subjected to RT-PCR. Primers for exon 5 (sense primer 5'-ATC GAT GCC AAT ACT GTC GA-3', position 503–522) and exon 6 (antisense primer 5'-GAC TCG ATG GTC AGC ACT G-3', position 744–426) were designed according to published RAR ß2 sequence¹⁷ (GenBank accession no. AF157483). RT-PCR with actin primers (sense primer 5'-GCT CGT CTG CGA CAA CGG CTC-3' and antisense primer 5' -CAA ACA TGA TCT GGG TCA TCT TCT C-3') was used as an internal RNA control. PCR products were analyzed on 2% agarose gels.

Western Blot Analysis

Total protein extracts were prepared in lysis buffer containing 40 mmol/L HEPES (pH 7.4), 1% triton-X 100, 10% glycerol, 0.1% sodium dodecyl sulfate, and 1 mmol/L phenylmethylsulfonylfluoride. Protein concentrations were determined using the BCA protein assay kit (Pierce, Rockford, IL). Ten-microgram aliquots of total protein from each sample were analyzed in 10 to 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Membranes were blocked for 1 hour at room temperature using 5% skim milk (Becton Dickinson, Sparks, MD) and then incubated overnight at 4°C with RAR ß primary mouse monoclonal antibody raised against a C-terminal RARß epitope (SA-179; Biomol Research Laboratories, Plymouth Meeting, PA) at a dilution of 1:1000. After three 8-minute washes with phosphate-buffered saline containing 0.04% Tween 20, membranes were incubated for 1 hour with horseradish peroxidase-coupled anti-IgG secondary antibody (1:1000). An actin immunoblot (1:200 dilution; Sigma, St. Louis, MO) was used to confirm the presence of protein in the cell extracts. Blots were visualized using the ECL Western blotting analysis system (Amersham, Buckinghamshire, UK) according to the manufacturer’s protocols.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Loss of Heterozygosity (LOH) on Chromosome 3p24

We performed LOH analysis on chromosome 3p24 at three loci: D3S 1283, D3S 1293, and D3S 1286. All patients were informative for at least one locus, and the overall LOH frequency at 3p24 involving at least one marker was 24% (11/45). When individual markers were analyzed, LOH was found in 24% of breast cancers at D3S 1283, 32% at D3S 1293, and 24% at D3S 1286 (Figure 1) . Retention of both maternal and paternal alleles was detected in both T-47D and MRK-nu-1 cell lines (data not shown).

View larger version (39K): [in this window] [in a new window]   Figure 1. LOH at chromosome 3p24 in breast cancer. Three microsatellite markers were analyzed for LOH in surgical specimens obtained from breast cancer patients and compared with matched peripheral blood leukocyte samples. Representative examples of heterozygotes with LOH are shown. N, normal DNA (leukocyte DNA); T, tumor DNA.

We analyzed RAR ß2 expression in the two tumor cell lines and in 11 breast cancers that exhibited LOH using RT-PCR and Western blot analysis. RAR ß2 mRNA expression was observed in T-47D cells, and in 7 of 11 tumors (64%; Figure 2 ). This result suggests that LOH alone cannot completely suppress RAR ß2 expression.

View larger version (33K): [in this window] [in a new window]   Figure 2. RT-PCR analysis of RAR ß2 expression in breast cancer cell lines and tumors. T-47D and MRK-nu-1 cell lines were untreated by 5-Aza-CdR. All patient samples used exhibited LOH. RAR ß2 mRNA expression was not observed in MRK-1 cells, or in cases k36, k41, k18, and k118.

To examine the molecular mechanisms of RAR ß2-loss in breast cancer, we sequenced the RAR ß2 gene promoter for evidence of mutations. No mutations were found in RAR ß2 promoter region at positions 737 to 1062 (GenBank accession no. X56849) in any of the breast cancer tumors or cell lines. We then examined the methylation status of the RAR ß2 promoter region for evidence of epigenetic change. Using methylation-specific PCR, both methylated and unmethylated alleles were found in the T-47D cell line, but only methylated alleles were demonstrated in the MRK-nu-1 cell line (Figure 4A) .

View larger version (71K): [in this window] [in a new window]   Figure 4. Treatment with 5-Aza-CdR induces demethylation and RAR ß2 protein expression in breast cancer cell lines. T-47D and MRK-nu-1 cells were left untreated or incubated for 3 days with 5-Aza-CdR. A: Methylation-specific PCR analysis of cell line DNA before (lanes 1, 2, 5, and 6) and after (lanes 3, 4, 7, and 8) 5.0 µmol/L 5-Aza-CdR treatment. Extracted DNA was treated with bisulfite as described in the text, and amplified by PCR using primers specific for methylated (M lanes) and unmethylated (U lanes) DNA. The presence of a 146-bp PCR product in lanes marked U indicates that the corresponding RAR ß2 gene is unmethylated; product in lanes marked M indicates that RAR ß2 is methylated. B: Western blot analysis of RAR ß2 protein expression in cell lines before and after treatment for 3 days with 1.0 µmol/L (lanes 2 and 5) and 5.0 µmol/L (lanes 3 and 6) 5-Aza-CdR, in comparison with untreated cells (lanes 1 and 4).

View larger version (27K): [in this window] [in a new window]   Figure 3. Analysis of RAR ß2 promoter methylation status of breast tumor samples. PCR amplification products resulting from the RAR ß2 promoter region before or after digestion are shown. Case k36, methylated; k58, unmethylated.

To determine whether RAR ß2 promoter methylation could be further linked to loss of RAR ß2 expression, the cancer cell lines were treated with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR). Treatment of cells with 1.0 and 5.0 µmol/L 5-Aza-CdR for 3 days led to complete demethylation of both the T-47D and MRK-nu-1 cell lines (Figure 4A) . Moreover, as shown in Figure 4B , 5-Aza-CdR successfully restored RAR ß2 protein expression in MRK-nu-1 cells that harbored methylated promoter regions. Up-regulation of RAR ß2 protein expression was observed in T-47D cells that previously contained both methylated and unmethylated promoters. These data indicate that RAR ß2 promoter methylation leads to the down-regulation or loss of RAR ß2 transcription in breast cancer cell lines.

Biallelic Inactivation of RAR ß2 in Breast Cancer

By Western blot analysis, small amounts of wild-type RAR ß2 protein were detectable in T-47D but not MRK-nu-1 cells (Figure 4B) , demonstrating that both wild-type RAR ß2 alleles were inactivated in MRK-nu-1. In many cancers, biallelic inactivation of suppressor genes is the result of mutation of one allele, followed by deletion of the remaining allele.¹⁸ This two-step process can be observed as LOH involving polymorphic markers linked to suppressor gene loci.¹⁸ When LOH at the RAR ß2 locus was analyzed in the breast cancer cell lines, retention of both maternal and paternal alleles was detected in both T-47D and MRK-nu-1 cells. After 5-Aza-CdR treatment, RAR ß2 induction was detected in the MRK-nu-1 cell line, and RAR ß2 up-regulation observed in the T-47D cell line. Thus, monoallelic inactivation in T-47D and biallelic inactivation in MRK-nu-1 appears to be caused by epigenetic modification. Furthermore, of the 11 tumor samples that exhibited LOH, the four cases with methylation showed complete loss of RAR ß2 transcripts. Therefore, biallelic inactivation of suppressor genes may also result from epigenetic modification of one allele followed by gene deletion of the remaining allele.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reduced RAR ß2 mRNA expression has been observed in several solid tumors, including lung carcinoma, squamous cell carcinoma, and breast cancer.^8-10 A growing body of evidence supports the hypotheses that the RAR ß2 gene is a tumor suppressor gene^19-21 and that the chemopreventive effects of retinoids are due to RAR ß2 induction.²² However, the mechanism of RAR ß2 suppression remains largely unknown.

The RAR ß2 gene promoter includes a RARE motif that can be activated by RAR/retinoid X receptor (RXR) heterodimers. However, in breast cancer cell lines, retinoic acid does not induce RAR ß2 gene expression, despite tumor cells being able to trans-activate an exogenous ßRARE.²³ These facts support the concept that the endogenous receptors contain point mutations or polymorphisms that disrupt function. However, in our present study, we detected no mutations or polymorphisms within the ßRARE promoter. Likewise, no mutations were found in the promoter region of RAR ß2 in HeLa cervical carcinoma cells that also lack RAR ß2.²⁴ Thus, it is likely that the RAR ß2 promoter region is not a target for mutation in many cancers, and other mechanisms for RAR ß2 suppression should be considered. Cote et al¹⁵ first suggested that methylation may be a mechanism responsible for the lack of RAR ß2 gene expression in a colon carcinoma cell line. Data presented in this report demonstrate that epigenetic silencing of the RAR ß2 gene promoter may be an important event in breast cancer, and that an epigenetic silencing mechanism was closely associated with RAR ß2 gene promoter methylation.

RAR ß2 is located at chromosome 3p24, and LOH of chromosome 3p24 is a common event in breast cancer.⁶ Therefore, we also performed LOH analysis on the cell line and tumor samples using microsatellite markers. It is thought that LOH alone cannot completely suppress RAR ß2 expression, as many genes can be expressed monoallelically.^25,26 Indeed, the lack of correlation between retinoic acid receptor ß2 expression and LOH at chromosome 3p24 has been demonstrated in esophageal cancer.²⁷ In our present study, seven breast cancer samples with LOH at 3p24 showed RAR ß2 expression, which suggested that RAR ß2 can be expressed in a monoallelic fashion.

Biallelic inactivation has been demonstrated for many suppressor genes, such as p53 and APC, and is usually caused by mutational inactivation of one allele accompanied by deletion or loss of the cognate wild-type allele, via mechanisms that result in LOH.¹⁸ However, we found that biallelic inactivation of the RAR ß2 gene could result either from epigenetic inactivation of both parental alleles, or from epigenetic modification of one allele and deletion of the remaining allele. These findings suggest that epigenetic gene inactivation is a highly efficient mechanism of silencing RAR ß2 gene expression. Transcriptional repression by DNA methylation can be mediated through a sequence-independent process that involves changes in chromatin structure and histone acetylation levels. Binding of the transcriptional repressor MeCP2 to methylated DNA is followed by recruitment of a complex containing a transcriptional corepressor and a histone deacetylase. Deacetylation of histones is associated with reduced transcription levels, perhaps through tighter nucleosomal packing.²⁸ On RXR-RAR heterodimer binding to RARE, chromatin structure undergoes dynamic, reversible changes in and around the RAR ß2 promoter.²⁹ Therefore, elucidation of mechanisms underlying epigenetic inactivation of RAR ß2 may be relevant to the understanding of many different types of human cancer.

This study established that RAR ß2 promoter region methylation was strongly associated with epigenetic gene silencing, and that the silencing mechanism was sufficient to accomplish biallelic inactivation of the RAR ß2 gene that could be reversed by exposure to demethylating agents. Knowledge of epigenetic changes at RAR ß2 may have implications for both cancer therapy and prevention. It is tempting to speculate that demethylating agents might have a role in cancer prevention for individuals who are at risk for cancer or for individuals in whom RAR ß2 promoter methylation is detected as an early neoplastic change. Moreover, knowledge of RAR ß2 methylation state in primary breast cancers may be useful to identify tumors that are more likely to respond to RA-therapy.

	Acknowledgements

 
We thank Professor R. L. Momparler (Departement de Pharmacologie, Universite de Montreal and Centre de Recherche Pediatrique, Hopital Ste-Justine, Quebec, Canada) for suggestions and encouragement.

	Footnotes

 
Address reprint requests to Qifeng Yang, Second Department of Pathology, Wakayama Medical College, 811–1 Kimiidera, Wakayama City, 641-0012 Japan. E-mail: yang-qf{at}mail.wakayama-med.ac.jp

Accepted for publication September 25, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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