The presence of estrogen receptors (ER) in breast tumors has important implications for both the choice of therapy and for prognosis. Tamoxifen, the most widely used chemotherapeutic agent for breast carcinoma, induces remission in only 50 percent of patients and resistance to the drug develops in most of those who initially respond. The ER antagonist tamoxifen modulates intracellular free Ca2+ concentrations ([Ca2+]i) and is a calmodulin antagonist. Calmodulin, a Ca2+-dependent modulatory protein, is an important component of cell growth and DNA synthesis, and is implicated in neoplastic transformation. Importantly, calmodulin binds ER and may participate in estrogen signal transduction. Therefore, the hypothesis to be evaluated in this proposal is that the Ca2+ mediated binding of calmodulin to ER has an important role in estrogen function and that altered coupling between Ca2+ calmodulin and ER signalling pathways contributes to tamoxifen resistance.
The Specific Aims are: (1) To determine whether the interaction between calmodulin and ER is an important component of receptor signalling, we shall examine ER stability, function and subcellular location. The effects of altering intracellular calmodulin concentration, function and interaction with ER on estrogen-stimulated transcription and subcellular localization of ER will be evaluated. (2) To establish whether Ca2+ and calmodulin regulate tamoxifen function and resistance, [Ca2+]i will be manipulated and measured, subcellular location of proteins will be determined and the binding between calmodulin and ER will be studied; analysis will be performed in both tamoxifen-sensitive and tamoxifen-resistant cell lines. In addition, intracellular Ca2+ and calmodulin concentrations will be altered to determine whether tamoxifen resistance can be modulated. (3) To identify ER-binding proteins that may contribute to tamoxifen resistance, affinity chromatography will be performed; proteins that bind ER in a tamoxifen- or calmodulin-regulated manner will be unambiguously identified by nanoelectrospray tandem mass spectrometry. These studies should provide insight into the function of the interaction between calmodulin and ER, as well as the molecular mechanism of tamoxifen resistance. This knowledge could potentially lead to the ultimate goal of this program, namely the development of novel and specifically targeted pharmacologic agents for the treatment of breast carcinoma.
