title: Protein kinase A exhibits selective modulation of estradiol-dependent transcription in breast cancer cells that is associated with decreased ligand binding, altered estrogen receptor alpha promoter interaction, and changes in receptor phosphorylation.
abstract: 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 ERalpha, 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 ERalpha phosphorylation. F/I decreased estradiol binding and increased Hsp90 association with ERalpha. Chromatin immunoprecipitation revealed that F/I recruited ERalpha to both pS2 and cMyc promoters at earlier times than estradiol, and F/I shifted estradiol recruitment of ERalpha to earlier time points. F/I induced a unique ERalpha 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 ERalpha phosphorylation.
authors.affiliation: Department of Biochemistry and Cancer Biology, Health Science Campus, University of Toledo, Ohio 43614, USA.
authors.forename: Mariam H
authors.initials: MH
authors.lastname: Al-Dhaheri
authors.forename: Brian G
authors.initials: BG
authors.lastname: Rowan
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D019938:Cyclin D1 / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008024:Ligands; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013629:Tamoxifen / Q000494:pharmacology; D014158:Transcription, Genetic
chemical_list: C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D047628:Estrogen Receptor alpha; D018841:HSP90 Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; C015482:protein kinase modulator; D013629:Tamoxifen; D019938:Cyclin D1; D005576:Colforsin; D004958:Estradiol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015056:1-Methyl-3-isobutylxanthine