Methylation and silencing of the retinoic acid receptor-beta2 gene in breast cancer.
BACKGROUND
A growing body of evidence supports the hypotheses that the retinoic acid receptor beta2 (RAR-beta2) gene is a tumor suppressor gene and that the chemopreventive effects of retinoids are due to induction of RAR-beta2. RAR-beta2 expression is reduced in many malignant tumors, and we examined whether methylation of RAR-beta2 could be responsible for this silencing.


METHODS
RAR-beta2 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-beta2 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-beta2, and, in 13 of the specimens, RT-PCR analysis was used to detect RAR-beta2 expression.


RESULTS
Cell lines SK-BR-3, T-47D, ZR-75-1, and MCF7 exhibited expression of RAR-beta2 only after demethylation and treatment with ATRA. The first exon expressed in the RAR-beta2 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-beta2 was found in any grade III lesion. An inverse association between methylation and gene expression was found in all grade II lesions. The RAR-beta2 gene from non-neoplastic breast tissue was unmethylated and expressed.


CONCLUSIONS
Methylation of the RAR-beta2 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.
Widschwendter
M
M
Department of Obstetrics and Gynecology, University of Innsbruck, Austria. Martin.Widschwendter@uklibk.ac.at
Berger
J
J
Hermann
M
M
Müller
H M
HM
Amberger
A
A
Zeschnigk
M
M
Widschwendter
A
A
Abendstein
B
B
Zeimet
A G
AG
Daxenbichler
G
G
Marth
C
C
D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics*; D018270:Carcinoma, Ductal, Breast / Q000235:genetics*; D019175:DNA Methylation; D015972:Gene Expression Regulation, Neoplastic; D016153:Genes, Suppressor; D006801:Humans; D016133:Polymerase Chain Reaction; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018168:Receptors, Retinoic Acid