15-Deoxy-Δ^12,14-prostaglandin J₂ inhibits transcriptional activity of estrogen receptor-α via covalent modification of DNA-binding domain

The cyclopentenone 15-deoxy-Δ^12,14-prostaglandin J ₂ (15d-PGJ₂) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-7 (PPARγ)-dependent and PPARγ-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-α (ERα) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ₂ inhibits both 17β-estradiol (E₂)-dependent and E₂-independent ERα transcriptional activity by PPARγ-independent mechanism. In addition, 15d-PGJ₂ directly modifies ERα protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ ₂ into ERα, both in vitro and in vivo. Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys²²⁷ and Cys²⁴⁰) within the COOH-terminal zinc finger of ERα DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ₂. Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ₂ inhibits DNA binding of ERα and subsequent repression of ERα target gene expression, such as pS2 and c-Myc. Therefore, our results suggest that 15d-PGJ₂ can block ERα function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERα DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERα transcriptional activity.