Estrogen receptor (ER) modulators produce distinct tissue-specific biological effects, but within the confines of the established models of ER action it is difficult to understand why. Previous studies have suggested that there might be a relationship between ER structure and activity. Different ER modulators may induce conformational changes in the receptor that result in a specific biological activity. To investigate the possibility of modulator-specific conformational changes, we have applied affinity selection of peptides to identify binding surfaces that are exposed on the apo-ERs ␣ and ␤ and on each receptor complexed with estradiol or 4-OH tamoxifen. These peptides are sensitive probes of receptor conformation. We show here that ER ligands, known to produce distinct biological effects, induce distinct conformational changes in the receptors, providing a strong correlation between ER conformation and biological activity. Furthermore, the ability of some of the peptides to discriminate between different ER ␣ and ER ␤ ligand complexes suggests that the biological effects of ER agonists and antagonists acting through these receptors are likely to be different.

Estrogen receptor alpha transcriptional activity is regulated by distinct conformational states that are the result of ligand binding. Phage display was used to identify peptides that interact specifically with either estradiol- or tamoxifen-activated estrogen receptor alpha. When these peptides were coexpressed with estrogen receptor alpha in cells, they functioned as ligand-specific antagonists, indicating that estradiol-agonist and tamoxifen-partial agonist activities do not occur by the same mechanism. The ability to regulate estrogen receptor alpha transcriptional activity by targeting sites outside of the ligand-binding pocket has implications for the development of estrogen receptor alpha antagonists for the treatment of tamoxifen-refractory breast cancers.

The transcription factor c-MYC is stabilized and activated by phosphorylation at serine 62 (S62) in breast cancer. Protein phosphatase 2A (PP2A) is a critical negative regulator of c-MYC through its ability to dephosphorylate S62. By inactivating c-MYC and other key signaling pathways, PP2A plays an important tumor suppressor function. Two endogenous inhibitors of PP2A, I2PP2A, Inhibitor-2 of PP2A (SET oncoprotein) and cancerous inhibitor of PP2A (CIP2A), inactivate PP2A and are overexpressed in several tumor types. Here we show that SET is overexpressed in about 50-60% and CIP2A in about 90% of breast cancers. Knockdown of SET or CIP2A reduces the tumorigenic potential of breast cancer cell lines both in vitro and in vivo. Treatment of breast cancer cells in vitro or in vivo with OP449, a novel SET antagonist, also decreases the tumorigenic potential of breast cancer cells and induces apoptosis. We show that this is, at least in part, due to decreased S62 phosphorylation of c-MYC and reduced c-MYC activity and target gene expression. Because of the ubiquitous expression and tumor suppressor activity of PP2A in cells, as well as the critical role of c-MYC in human cancer, we propose that activation of PP2A (here accomplished through antagonizing endogenous inhibitors) could be a novel antitumor strategy to posttranslationally target c-MYC in breast cancer.breast cancer therapy | phosphatase activator T he c-MYC (MYC) oncoprotein is overexpressed in human breast cancer and this is associated with poor clinical outcome (1, 2). Expression of MYC is regulated at multiple levels, including protein stability, which is increased in several cancer types (1,3,4). MYC stability is regulated in part by sequential and interdependent phosphorylation at two conserved residues, threonine 58 (T58) and serine 62 (S62) (5). MYC is phosphorylated at S62 (pS62) through the mitogen-activated protein kinase (MAPK) pathway or cyclin-dependent kinase (CDK) activation in response to growth signals and this modification increases its stability and oncogenic activity (5-8). When growth signals cease, GSK3, in a manner dependent upon prior phosphorylation at S62, phosphorylates T58 (pT58) (5, 6). T58 phosphorylation facilitates protein phosphatase 2A (PP2A)-mediated dephosphorylation of pS62 and recruitment of the E3 ubiquitin ligase SCF Fbw7 to initiate proteasomal destruction of MYC (9, 10). This process is facilitated by AXIN1, which helps nucleate a destruction complex for MYC at target gene promoters (11, 12). Our previous work has shown that MYC stability is increased in breast cancers and that this correlates with high pS62-and low pT58-MYC (4).PP2A is a ubiquitously expressed, heterotrimeric serinethreonine (S/T) phosphatase that mediates 30-50% of cellular S/T phosphatase activity (13). Target specificity of PP2A is directed by a variable regulatory (B) subunit, and we have shown that B56α is the isoform that directs PP2A to MYC (9, 13). Human cell transformation requires inhibition of PP2A activity and, in an siRNA screen, B56α, ...