Molecular Mechanisms of Protein Kinase A Signaling Pathway on Estrogen Receptor Action in Breast Cancer
Estrogen receptor á (ERá) is transcriptional factor that promotes breast cancer growth by inducing a set of genes that regulate cell cycle and proliferation. ERá is activated by estrogen binding and/or by crosstalk with different signaling pathways. Here, PKA regulation of ERá action was investigated in tamoxifen-sensitive MCF7 and T47D, and tamoxifen-resistant MCF7-LCC2 breast cancer cells. Forskolin/IBMX (F/I), which elevates cAMP, induced ligand-independent activation of ERá but repressed estradiol activation of ERá in PKA-dependent manner as assessed by estrogen response element (ERE)-reporter gene assays. However, F/I exhibited gene specific effects on endogenous ER-regulated genes. F/I enhanced estradiol induction of pS2 and cMyc that was correlated with rapid and cyclic ERá recruitment to the respective promoters as assessed by chromatin immunoprecipitation assays. In contrast, F/I repressed estradiol induction of cyclin D1 mRNA and protein that was correlated with inhibition of estradiol-dependent breast cancer growth. To identify potential mechanisms by which F/I regulates ERá action, estradiol binding, Hsp90 interaction with ERá and ERá phosphorylation were examined. F/I blocked estradiol binding to ERá in all cell lines that was correlated with an increase in Hsp90 association with ERá. Importantly, F/I induced a unique ERá phosphorylation profile (inhibition of serine 118 phosphorylation and increase serine 305 phosphorylation) that was distinct from phosphorylation profiles regulated by estradiol and estradiol + F/I. F/I inhibition of serine 118 phosphorylation was correlated with a decrease in ERK1/ERK2 phosphorylation. Serine 305 204 phosphorylation was required for ligand-independent activation of ERá by F/I as well as the F/I suppression of ERá activation by estradiol. Development of tamoxifen resistance in breast cancer and the paucity of biomarkers that predict tamoxifen sensitivity illustrate an urgent need to identify novel proteins that regulate tamoxifen sensitivity and/or serve as surrogate markers of tamoxifen response. Two dimensional protein analysis and mass spectrometry were applied to identify proteins induced by tamoxifen in tamoxifen-sensitive T47D cells. Six novel tamoxifen-regulated proteins were identified (calreticulin, synapse-associated protein, CD2 antigen-binding protein-2, D-3-phosphoglycerate dehydrogenase, and pyridoxine 5’-phosphate oxidase). Tamoxifen regulation or proteins occurred at the mRNA level. These proteins could serve as markers for tamoxifen sensitivity in breast cancer cells.
School:University of Toledo Health Science Campus
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:molecular mechanisms protein kinase a estrogen receptor breast cancer and tamoxifen gene expression
Date of Publication:01/01/2006