Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)-positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110A and p110B, in ER + breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110A RNAi inhibited growth and promoted apoptosis in all tested ER + breast cancer cells under estrogen deprived-conditions, whereas p110B RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110A/p110B inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER + breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110A and p110B catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER + breast cancer. [Cancer Res 2009;69(9):3955-62]

AR is necessary for AR nuclear export and is dominant over the NLS in the DNAbinding domain and hinge region in the absence of hormone. Our findings suggest that androgen can regulate NES AR and, subsequently, the NLS of the AR, providing a mechanism by which androgen regulates AR nuclear/ cytoplasmic shuttling. Estrogen receptor ␣ and mineralocorticoid receptor also contain functional NES, suggesting that this ligand-regulated NES is conserved among steroid receptors. Androgen receptor (AR)1 is a member of the steroid receptor superfamily that regulates gene expression in a ligand-dependent manner (1, 2). AR is necessary for male accessory organ development and is involved in prostate cancer progression. The human AR contains 918 amino acids and consists of the N-terminal transactivation domain (1-555), DNA-binding domain (DBD) (556 -623), hinge region (624 -665), and C-terminal ligand-binding domain (LBD) (666 -918) (3). The DBD, hinge region, and LBD of AR share a high degree of homology with other steroid receptors. Intracellular localization of AR and other steroid receptors such as glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) is ligand-dependent. The limit for passive diffusion across the nuclear pore complex has been suggested to fall within the range of 20 -40 kDa (4). Larger proteins such as AR, which is ϳ100 kDa in size, must be actively transported through the nuclear pore complex to enter or leave the nucleus. AR is localized to the cytoplasm in the absence of ligand and translocates into the nucleus in the presence of ligand (5-7). Likewise, nuclear AR can be exported to the cytoplasm upon ligand withdrawal (8). These observations indicate that AR contains both a nuclear localization signal (NLS) and nuclear export signal (NES) and that their activities are regulated by androgen either directly or indirectly. Because nuclear localization is necessary for steroid receptors to transactivate their target genes, the ligand-dependent nuclear import/export represents a critical mechanism by which steroids regulate the activity of their cognate receptors.The signal involved in the transport of steroid receptors from the cytoplasm to the nucleus has been studied extensively. Mutagenesis studies of the human AR have defined a bipartite NLS in the DBD and hinge region at amino acids 617-633 (9). This NLS is composed of two clusters of basic amino acids (underlined) separated by 10 amino acid residues: RKCY-EAGMTLGARKLKK. Similar NLS were found in other steroid receptors. In addition to the NLS in the DBD and hinge region, Picard and Yamamoto (10) demonstrated the existence of a ligand-dependent NLS present in the LBD of GR. When fused to ␤-galactosidase or to N-terminal fragments of the GR lacking the NLS, the LBD of GR conferred ligand-dependent nuclear localization to the fusion proteins. Similarly, a ligand-dependent NLS also exists in the LBD of AR, which is capable of inducing nuclear import in the absence of the NLS in the DBD and hinge region (11,12).Studies on sequences responsible...

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BACKGROUND-Androgen receptor (AR) is the key molecule in androgen-refractory prostate cancer. Despite androgen ablative conditions, AR remains active and is necessary for the growth of androgen-refractory prostate cancer cells. Nuclear localization of AR is a prerequisite for its transcriptional activation. We examined AR localization in androgen-dependent and androgenrefractory prostate cancer cells.

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