Modulation by phenylacetate of early estrogen-mediated events in MCF-7 breast cancer cells

Purpose: Phenylacetate (PA) and its derivatives constitute a group of small aromatic fatty acids that have been of considerable interest due to their anticancer properties in a number of experimental systems. We previously showed that PA can inhibit the growth of estrogen receptor (ER)+ breast cancer cells and that this activity is, at least in part, mediated by the ability of the compound to inhibit transcriptional activation driven by estrogen response elements (EREs). We now shed additional light on the antiestrogenic action of PA by determining its effects on early events in the estrogen-signaling pathway. Methods: MCF-7 breast cancer cells were used in this study. ER-ERE binding activity, and subsequent effects on ER and progesterone receptors (PR), c-myc, and the cyclin-dependent kinase inhibitor p21(ASF1/CP1/MDA-6) (p21) were evaluated using electrophoretic mobility shift assays, real-time RT-PCR, and western blotting methodologies. Effects of PA on p21 promoter activity were assessed in transient transfection experiments utilizing p21 promoter-reporter gene constructs. Results: We demonstrate that PA treatment can block ER-ERE binding activity and that this effect is accompanied by downregulation of PR and c-myc, two genes which are transcriptionally regulated by estrogen through novel-ER- binding sites. Suppression of c-myc by PA is followed by increased mRNA levels of p21, an effect that is mediated by PA activation of the p21 promoter. Forced overexpression of c-myc through co-transfection of MCF-7 cells with a c-myc expression plasmid prevented PA upregulation of p21 promoter activity. Conclusions: These findings confirm the potent antiestrogenic properties of PA, indicate that its effects are mediated by inhibiting ER-ERE interactions, and suggest that downregulation of c-myc is an early event leading to increased p21 expression and cell growth inhibition.