Both cyclin D1 and estrogens have an essential role in regulating proliferation of breast epithelial cells. We show here a novel role for cyclin D1 in growth regulation of estrogen-responsive tissues by potentiating transcription of estrogen receptor-regulated genes. Cyclin D1 mediates this activation independent of complex formation to a CDK partner. Cyclin D1 activates estrogen receptor-mediated transcription in the absence of estrogen and enhances transcription in its presence. The activation of estrogen receptor by cyclin D1 is not inhibited by anti-estrogens. A direct physical binding of cyclin D1 to the hormone binding domain of the estrogen receptor results in an increased binding of the receptor to estrogen response element sequences, and upregulates estrogen receptor-mediated transcription. These results highlight a novel role for cyclin D1 as a CDK-independent activator of the estrogen receptor.

Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERalpha. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIalpha, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIalpha converts tamoxifen from an ERalpha inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).

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Estrogen receptor alpha (ERα) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERα, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERα dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERα dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERα/14-3-3 interaction. Cocrystallization of the trimeric ERα/ 14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERα-T 594 ) for high-affinity interaction. We confirm that T 594 is a distinct ERα phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T 594 -specific antibody and by mass spectrometry. In line with its ERα/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERα dimerization, inhibits ERα/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERα are provided, together with a small molecule that selectively targets this ERα/14-3-3 interface.T he estrogen receptor alpha (ERα) is a ligand-dependent transcription factor and the driving force of cell proliferation in 75% of all breast cancers. Current therapeutic strategies to treat these tumors rely on selective ER modulators (SERMs), like tamoxifen (TAM) (1) or aromatase inhibitors (AIs) that block estradiol synthesis (2). Although the benefits of treating hormone-sensitive breast cancers with SERMs and AIs are evident, resistance to treatment is commonly observed (3, 4). To overcome resistance, selective ERα down-regulators (SERDs) can for instance be applied that inhibit ERα signaling through receptor degradation (5, 6). Approaches that target the ERα/ DNA or ERα/cofactor interactions are explored as well (5, 7), but other essential steps in the ERα activation cascade are currently unexploited in drug design, also due to a lack of molecular understanding of the processes at hand.One such step that is crucial for many aspects of ERα functioning is ligand-driven receptor dimerization (8, 9). 17β-Estradiol (E2) association with the ERα ligand binding domain (LBD) drives large conformational changes (10) resulting in ERα dissociation from chaperones (11, 12), unmasking of domains for receptor dimerization, and DNA binding (13,14). Whereas the LBD contains the main dimerization domain (15), the extreme C-terminal domain of the receptor (F domain) imposes a restraint on dimerization (15, 16), although the regulation of this remain...