The antitumor activity of tamoxifen in patients with breast cancer may be determined, in part, by tumor levels of AIB1 and HER-2. Thus, AIB1 may be an important diagnostic and therapeutic target.

Regulation of nuclear receptor gene expression involves dynamic and coordinated interactions with histone acetyl transferase (HAT) and deacetylase complexes. The estrogen receptor (ER␣) contains two transactivation domains regulating ligand-independent and -dependent gene transcription (AF-1 and AF-2 (activation functions 1 and 2)). ER␣-regulated gene expression involves interactions with cointegrators (e.g. p300/ CBP, P/CAF) that have the capacity to modify core histone acetyl groups. Here we show that the ER␣ is acetylated in vivo. p300, but not P/CAF, selectively and directly acetylated the ER␣ at lysine residues within the ER␣ hinge/ligand binding domain. Substitution of these residues with charged or polar residues dramatically enhanced ER␣ hormone sensitivity without affecting induction by MAPK signaling, suggesting that direct ER␣ acetylation normally suppresses ligand sensitivity. These ER␣ lysine residues also regulated transcriptional activation by histone deacetylase inhibitors and p300. The conservation of the ER␣ acetylation motif in a phylogenetic subset of nuclear receptors suggests that direct acetylation of nuclear receptors may contribute to additional signaling pathways involved in metabolism and development.

The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. Steroid receptors, including the androgen receptor (AR), are members of the nuclear receptor (NR) superfamily which generally function as ligand-dependent transcriptional regulators (9, 31, 84). The AR is expressed in a variety of cell types and plays an important role in development, male sexual differentiation, and prostate cellular proliferation. The functional domains of the AR (termed A to F) are conserved with other members of the classical receptor subclass. The C-terminal region of the AR, including the hinge region and ligand-binding domain (LBD), is responsible for ligand binding and dimerization. The well-conserved DNA binding domain consists of 68 amino acids with two zinc finger structures. The N-terminal region contributes to transcriptional activation through its activation function 1 (AF-1) (5). In contrast to several other hormone-regulated NRs, the AR lacks an intrinsic AF-2 function in the LBD. The LBD, which consists of 12 ␣ helices projecting away from the hormone-binding pocket in the absence of ligand, undergoes substantial conformational changes in the presence of ligand. The folding of the most carboxyl-terminal helix 12 over the ligand-binding pocket in turn creates new structural surfaces that bind coactivators required for efficient transactivation.Several AR coactivators have been identified, including the p160 proteins, the p300/CREB-binding protein (CBP) family, Ubc9, ARA70, ARA55, and TIP60 (1,5,15,68,92). The efficient recruitment of coactivators to the AR involves an association between both the AR amino terminus and the LBD (5, 34). The coactivator proteins regulate gene expression through several distinct mechanisms. CBP and the related functional homologue p300 (CBP/p300) convey a bridging function between the DNA-bound transcription factor and the basal apparatus and provide a scaffold to assemble high-molecular-weight enhanceosomes (reviewed in reference 26). In addition, the cointegrator proteins p300/CBP share the capacity to acetylate histones, which correlates, under certain circumstances, with their transcriptional coactivator function (54,87). Acetylation facilitates binding of transcription factors to * Corresponding author. Mailing address: The Albert Einstein Can-

Results: Expression of the two isoforms correlated with each other, as well as with ER. Additional analyses revealed that patients with PR-positive tumors but high PR-A:PR-B ratios, which were often caused by high PR-A levels, were 2.76 times more likely to relapse than patients with lower ratios, indicating resistance to tamoxifen.Conclusions: This study suggests that knowledge of the PR-A:PR-B ratio may identify a subgroup of ER-positive/ PR-positive patients with node-positive breast cancer that benefit poorly from endocrine therapy.

Purpose: Breast cancer is a hormone-dependent cancer, and the presence of estrogen receptor ␣ (ER-␣) in tumors is used clinically to predict the likelihood of response to hormonal therapies. The clinical value of the second recently identified ER isoform, called ER-␤, is less clear, and there is currently conflicting data concerning its potential role as a prognostic or predictive factor.Experimental Design: To assess whether ER-␤ expression is associated with clinical outcome, protein levels were measured by immunoblot analysis of a retrospective bank of tumor cell lysates from 305 axillary node-positive patients. A total of 119 received no adjuvant therapy, and 186 were treated with tamoxifen only. The median follow-up time was 65 months. Univariate and multivariate Cox regression modeling was done to assess the prognostic and predictive significance of ER-␤ expression.Results: Expression of ER-␤ protein did not correlate significantly with any other clinical variables, including ER and progesterone levels (as measured ligand binding assay), tumor size, age, or axillary nodal status. In the untreated population, those patients whose tumors who expressed both receptor isoforms exhibited the most favorable outcome as compared with those patients who had lost ER-␣ expression. However, there was no association between ER-␤ levels alone and either disease-free or overall survival in the untreated patient population. In contrast, in both univariate and multivariate analyses, high levels of ER-␤ predicted an improved disease-free and overall survival in patients treated with adjuvant tamoxifen therapy.Conclusions: These findings provide evidence that ER-␤ may be an independent predictor of response to tamoxifen in breast cancer. Furthermore, these results suggest that ER-␤ may influence tumor progression in ways different from those mediated by the ER-␣ isoform.

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