h-89 and afimoxifene

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients, and Her-2/ErbB2 expression is associated with decreased sensitivity to TAM. We previously reported that cAMP-dependent protein kinase (PKA)-mediated activator protein-2 (AP-2) activation was responsible for the expression of Her-2/ErbB2 in p53-inactivated mammary epithelial cells (Yang et al., 2006). In the present study, we tested the hypothesis that PKA plays a role in the expression of ErbB2 in tamoxifen-resistant breast cancer cells. Treatment with H-89, a specific PKA inhibitor, suppressed 4-hydroxytamoxifen-induced ErbB2 expression in control MCF-7 cells. In contrast, PKA inhibition by H-89 or cAMP-dependent protein kinase inhibitor l gamma overexpression increased the expression levels of ErbB2 in TAM-resistant MCF-7 (TAMR-MCF-7) cells. Transcriptional regulation of the erbB2 gene depends on two transcription factors, AP-2 and polyomavirus enhancer activator3 (PEA3). H-89 decreased nuclear or total levels of PEA3 in TAMR-MCF-7 cells. Chromatin immunoprecipitation assay results revealed that H-89 treatment reduced PEA3 binding to the proximal Ets binding site of the erbB2 gene promoter. Reporter gene analyses using human erbB2 gene promoter supported the critical role of PEA3 in the overexpression of ErbB2 in TAMR-MCF-7 cells treated with H-89. This deregulated PKA signaling cascades required for the ErbB2 expression may be important for the differential response of TAM-resistant breast cancer cells to EGF/ErbB2 stimuli.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Cell Line, Tumor; Cyclic AMP-Dependent Protein Kinases; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Isoquinolines; Promoter Regions, Genetic; Protein Kinase Inhibitors; Receptor, ErbB-2; Signal Transduction; Sulfonamides; Tamoxifen; Transcription Factor AP-2; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection

Estrogen receptors (ERs) are members of the superfamily of ligand-activated transcription factors. In addition to the classical, hormone-mediated activation, ERs may alternatively be activated in a ligand-independent manner by a variety of agents including growth factors, neurotransmitters and cAMP. It has been demonstrated that the phosphatidylinositol 3 (PI3)-dependent kinase/Akt pathway may activate the ER alpha by increasing the activity of both estrogen independent activation function-1 and estrogen-dependent activation function-2 domains. The Akt phosphorylation site in the ER is Ser167. Phosphorylation of this residue is inhibited by LY294002, which blocks the PI3-kinase/Akt pathway. In the course of studies examining the effects of LY294002 on ligand-independent activation of ERs in L cells, we found that LY294002 exhibits antiestrogenic effects in a dose-dependent manner. By competition binding assays, we found that LY294002 specifically displaced radiolabelled estradiol from ERs with an IC(50) of 11+/-0.06 nM, being an estradiol competitor as effective as the antiestrogens ICI182,780 (IC(50), 21+/-0.13) and 4-OH-tamoxifen (IC(50), 15+/-0.09). Further, LY294002 irreversibly blocked estrogen-induced transactivation of an estradiol-sensitive reporter gene. These findings are of particular importance in the interpretation of studies demonstrating ERs inactivation by the PI3-kinase inhibitor. Our studies show that an apparent block of ER activation cannot be dissociated from inhibition of ligand-mediated events. Thus, this effect can be the result of the ability of LY294002 to bind the ERs and inhibit transactivation of estrogen-regulated genes.

Topics: Animals; Binding Sites; Binding, Competitive; Cells, Cultured; Chromones; Enzyme Inhibitors; Estradiol; Female; Gene Expression Regulation; Genes, Reporter; Genetic Vectors; HeLa Cells; Humans; Imidazoles; Isoquinolines; L Cells; Mice; Morpholines; Phosphoinositide-3 Kinase Inhibitors; Plasmids; Pyridines; Receptors, Estrogen; Sulfonamides; Tamoxifen; Transcription, Genetic