Protein kinase A exhibits selective modulation of estradiol-dependent transcription in breast cancer cells that is associated with decreased ligand binding, altered estrogen receptor α promoter interaction, and changes in receptor phosphorylation

Inhibition of protein kinase A (PKA) promotes estrogen-dependent growth of MCF7 breast cancer cells, although the mechanisms by which PKA regulates estrogen receptor ( ER) function remain unclear. In this study elevation of cAMP by forskolin/ 3-isobutyl-1-methylxanthine (F/I) suppressed estradiol-dependent MCF7 and T47D breast cancer cell growth but not tamoxifen-resistant MCF7-LCC2 cells. Although F/I induced ligand independent activation of ER alpha, F/I also decreased estradiol-dependent reporter gene transcription. Overexpression of PKA or PKA inhibitor (PKI) demonstrated that F/I effects on repression of estradiol action occurred through the PKA pathway. 8CPT-2Me-cAMP, a selective inducer of non-PKA signaling, did not alter ER-dependent transcription. In contrast to F/I effects on reporter genes, F/I exhibited gene-specific effects on endogenous, ER-regulated genes. F/I enhanced estradiol induction of pS2 and cMyc but repressed estradiol induction of cyclin D1 mRNA and protein in MCF7 cells. To explore likely mechanisms by which F/I regulated ER, experiments examined estradiol binding, Hsp90 interaction, promoter recruitment, and ER alpha phosphorylation. F/I decreased estradiol binding and increased Hsp90 association with ER alpha. Chromatin immunoprecipitation revealed that F/I recruited ER alpha to both pS2 and cMyc promoters at earlier times than estradiol, and F/I shifted estradiol recruitment of ER alpha to earlier time points. F/I induced a unique ER alpha phosphorylation profile (increase in serine 305 and decrease in serine 118 phosphorylation) that was distinct from estradiol and estradiol + F/I. Taken together, F/I signaling through PKA selectively regulates estradiol-dependent genes in breast cancer, which is associated with reduced ligand binding and changes in promoter interaction and ER alpha phosphorylation.