Androgen receptor acetylation governs trans activation and MEKK1-induced apoptosis without affecting in vitro sumoylation and trans-repression function

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. The AR acetylation site point mutants showed wild-type trans repression of NF-kappa B, AP-1, and Sp1 activity; wild-type sumoylation in vitro; wild-type ligand binding; and ligand-induced conformational changes. However, acetylation-deficient AR mutants were selectively defective in DHT-induced trans activation of androgen-responsive reporter genes and coactivation by SRC1, Ubc9, TIP60, and p300. The AR acetylation site mutant showed 10-fold increased binding of the N-CoR corepressor compared with the AR wild type in the presence of ligand. Furthermore, histone deacetylase 1 (HDAC1) bound the AR both in vivo and in cultured cells and HDAC1 binding to the AR was disengaged in a DHT-dependent manner. MEKK1 induced AR-dependent apoptosis in prostate cancer cells. The AR acetylation mutant was defective in MEKK1-induced apoptosis, suggesting that the conserved AR acetylation site contributes to a pathway governing prostate cancer cellular survival. As AR lysine residue mutations that abrogate acetylation correlate with enhanced binding of the N-CoR repressor in cultured cells, the conserved AR motif may directly or indirectly regulate ligand-dependent corepressor disengagement and, thereby, ligand-dependent trans activation.     

SIRT1与基因转录

 SIRT1（silent mating type information regulation 2 homolog 1）是一种具有NAD-依赖的蛋白去乙酰化酶活性的多功能转录调节因子.在体内通过对几种控制代谢及内分泌信号的转录因子去乙酰化作用来调节其活性.从而广泛参与调控哺乳动物细胞寿命的不同信号通路及糖代谢,胰岛素分泌等多条代谢通路,预示着SIRT1在医学临床应用和研究中可能极具应用价值.     

The PPARγ ligand ciglitazone regulates androgen receptor activation differently in androgen-dependent versus androgen-independent human prostate cancer cells

The androgen receptor (AR) regulates growth and progression of androgen-dependent as well as androgen-independent prostate cancer cells. Peroxisome proliferator-activated receptor gamma (PPARγ) agonists have been reported to reduce AR activation in androgen-dependent LNCaP prostate cancer cells. To determine whether PPARγ ligands are equally effective at inhibiting AR activity in androgen-independent prostate cancer, we examined the effect of the PPARγ ligands ciglitazone and rosiglitazone on C4-2 cells, an androgen- independent derivative of the LNCaP cell line. Luciferase-based reporter assays and Western blot analysis demonstrated that PPARγ ligand reduced dihydrotestosterone (DHT)-induced increases in AR activity in LNCaP cells. However, in C4-2 cells, these compounds increased DHT-induced AR driven luciferase activity. In addition, ciglitazone did not significantly alter DHT-mediated increases in prostate specific antigen (PSA) protein or mRNA levels within C4-2 cells. siRNA-based experiments demonstrated that the ciglitazone-induced regulation of AR activity observed in C4-2 cells was dependent on the presence of PPARγ. Furthermore, overexpression of the AR corepressor cyclin D1 inhibited the ability of ciglitazone to induce AR luciferase activity in C4-2 cells. Thus, our data suggest that both PPARγ and cyclin D1 levels influence the ability of ciglitazone to differentially regulate AR signaling in androgen-independent C4-2 prostate cancer cells.     

Reciprocal roles of DBC1 and SIRT1 in regulating estrogen receptor α activity and co-activator synergy

Estrogen receptor α (ERα) plays critical roles in development and progression of breast cancer. Because ERα activity is strictly dependent upon the interaction with coregulators, coregulators are also believed to contribute to breast tumorigenesis. Cell Cycle and Apoptosis Regulator 1 (CCAR1) is an important co-activator for estrogen-induced gene expression and estrogen-dependent growth of breast cancer cells. Here, we identified Deleted in Breast Cancer 1 (DBC1) as a CCAR1 binding protein. DBC1 was recently shown to function as a negative regulator of the NAD-dependent protein deacetylase SIRT1. DBC1 associates directly with ERα and cooperates synergistically with CCAR1 to enhance ERα function. DBC1 is required for estrogen-induced expression of a subset of ERα target genes as well as breast cancer cell proliferation and for estrogen-induced recruitment of ERα to the target promoters in a gene-specific manner. The mechanism of DBC1 action involves inhibition of SIRT1 interaction with ERα and of SIRT1-mediated deacetylation of ERα. SIRT1 also represses the co-activator synergy between DBC1 and CCAR1 by binding to DBC1 and disrupting its interaction with CCAR1. Our results indicate that DBC1 and SIRT1 play reciprocal roles as major regulators of ERα activity, by regulating DNA binding by ERα and by regulating co-activator synergy.     

White button mushroom (Agaricus bisporus) disrupts androgen receptor signaling in human prostate cancer cells and patient-derived xenograft

White button mushroom (WBM) (Agaricus bisporus) is a potential prostate cancer (PCa) chemo-preventative and therapeutic agent. Our clinical phase І trial of WBM powder in patients with biochemically recurrent PCa indicated that WBM intake reduced the circulating levels of prostate-specific antigen (PSA). We hypothesized that WBM exerts its effects on PCa through the androgen receptor (AR) signaling axis. Therefore, we conducted a reverse translational study with androgen-dependent PCa cell lines (LNCaP and VCaP) and patient-derived-xenografts (PDX) from a prostate tumor (TM00298). In both LNCaP and VCaP cells, western blots and qRT-PCR assays indicated that WBM extract (6–30 mg/mL) suppressed DHT-induced PSA expression and cell proliferation in a dose-dependent manner. Immunofluorescence analysis of AR revealed that WBM extract interrupted the AR nuclear-cytoplasmic distribution. PSA promotor-luciferase assay suggested that WBM extract inhibited DHT-induced luciferase activity. RNA-Seq on WBM-treated LNCaP cells confirmed that WBM treatment suppressed the androgen response pathways and cell-cycle control pathways. Our PDX showed that oral intake of WBM extract (200 mg/kg/d) suppressed tumor growth and decreased PSA levels in both tumors and serum. In the present study, we also identified a conjugated linoleic acid isomer (CLA-9Z11E) as a strong AR antagonist by performing LanthaScreen TR-FRET AR Coactivator Interaction Assays. The inhibitory effect of CLA-9Z11E (IC50: 350 nM) was nearly two times stronger than the known AR antagonist, cyproterone acetate (IC50: 672 nM). The information gained from this study improves the overall understanding of how WBM may contribute to the prevention and treatment of PCa.     

Prediction and confirmation of a switch-like region within the N-terminal domain of hSIRT1

Many proteins display conformational changes resulting from allosteric regulation. Often only a few residues are crucial in conveying these structural and functional allosteric changes. These regions that undergo a significant change in structure upon receiving an input signal, such as molecular recognition, are defined as switch-like regions. Identifying these key residues within switch-like regions can help elucidate the mechanism of allosteric regulation and provide guidance for synthetic regulation. In this study, we combine a novel computational workflow with biochemical methods to identify a switch-like region in the N-terminal domain of human SIRT1 (hSIRT1), a lysine deacetylase that plays important roles in regulating cellular pathways. Based on primary sequence, computational methods predicted a region between residues 186–193 in hSIRT1 to exhibit switch-like behavior. Mutations were then introduced in this region and the resulting mutants were tested for allosteric reactions to resveratrol, a known hSIRT1 allosteric regulator. After fine-tuning the mutations based on comparison of known secondary structures, we were able to pinpoint M193 as the residue essential for allosteric regulation, likely by communicating the allosteric signal. Mutation of this residue maintained enzyme activity but abolished allosteric regulation by resveratrol. Our findings suggest a method to predict switch-like regions in allosterically regulated enzymes based on the primary sequence. If further validated, this could be an efficient way to identify key residues in enzymes for therapeutic drug targeting and other applications.