Retinoic acid receptor α2 is a growth suppressor epigenetically silenced in MCF-7 human breast cancer cells

Retinoic acid (RA) receptor (RAR) β2 has been shown to be underexpressed in human breast cancer cells, including MCF-7 cells, and recent reports have suggested that hypermethylation of the RARβ2 promoter and 5′-UTR is the underlying cause. Here we show that RARα2 is also underexpressed in MCF-7 breast cancer cells, at both the message and the protein level, relative to normal or nontumorigenic breast epithelial cells. Bisulfite sequencing of the CpG island in the RARα2 promoter revealed highly penetrant and uniform cytosine methylation in MCF-7 cells. Pretreatment with the DNA methyltransferase inhibitor, azacytidine, followed by treatment with RA and a histone deacetylase inhibitor, trichostatin A, resulted in partial promoter demethylation and RARα2 induction, which strongly suggested that promoter hypermethylation is responsible for RARα2 underexpression. We compared the outcome of ectopic expression in MCF-7 cells of matched levels of RARα2 and RARβ2. On the basis of a clonogenic assay, RARα2 displayed ligand-dependent growth-suppressive activity similar to that of RARβ2; thus, 10 and 20 nM RA inhibited clonogenic growth by 52 and 80%, respectively, in RARα2-transfected cells compared with 75 and 77%, respectively, in RARβ2-transfected cells. We conclude that the silencing of the RARα2 promoter by hypermethylation may play a contributory role in the dysregulation of RA signaling in mammary tumorigenesis.