Methylation and silencing of the retinoic acid receptor-β2 gene in breast cancer

Abstract

Background: A growing body of evidence supports the hypotheses that the retinoic acid receptor β2 (RAR-β2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-β2. RAR-β2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-β2 could be responsible for this silencing. Methods: RAR-β2 expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) analysis in eight breast cancer cell lines that were either treated with the demethylating agent 5-aza-2'-deoxycytidine and subsequently with all-trans-retinoic acid (ATRA) or left untreated. Sodium bisulfite genomic sequencing was used to determine the locations of 5- methylcytosines in the RAR-β2 genes of three of these cell lines. In 16 breast cancer biopsy specimens and non-neoplastic breast tissue, methylation- specific PCR was used to determine the methylation status of RAR-β2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-β2 expression. Results: Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-β2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-β2 transcript was methylated in cell lines ZR-75-1 and SK-BR-3. Six breast cancer specimens showed methylation in the same region of the gene. No expression of RAR-β2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-β2 gene from non-neoplastic breast tissue was unmethylated and expressed. Conclusions: Methylation of the RAR-β2 gene may be an initial step in breast carcinogenesis; treatment of cancer patients with demethylating agents followed by retinoic acid may offer a new therapeutic modality.