Thiazolidinediones Mimic Glucose Starvation in Facilitating Sp1 Degradation through the Up-Regulation of -Transducin Repeat-Containing Protein

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress -catenin and cyclin D1 by up-regulating the E3 ligase SCF , we hypothesized that -transducin repeat-containing protein ( -TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with -TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed -TrCP. Although ectopic expression of -TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous -TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box (DSGAGS) that mediates -TrCP recognition and encompasses a glycogen synthase kinase 3 (GSK3 ) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinasemediated phosphorylation of Thr739 and GSK3 -mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate -TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy. In addition to maintaining the basal transcription of housekeeping genes, increasing evidence indicates that the transcription factor Sp1 also plays an important role in regulating the expression of a host of key effectors of signaling pathways governing cell cycle progression, cell proliferation, angiogenesis, apoptosis, and metastasis (Wierstra, 2008). These target proteins include receptor tyrosine kinases and their growth factor ligands, cyclin-dependent kinase inhibitors, c-Myc, Mdm2, Mcl-1, survivin, XIAP, Fas ligand, PUMA, and death receptor 5 (Wierstra, 2008). Moreover, Sp1 This work was supported by the National Institutes of Health National Cancer Institute [Grant CA112250] and the Department of Defense Prostate Cancer Research Program [Grant W81XWH-09-1-0198]. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.109.055376. ABBREVIATIONS: PPAR , peroxisome proliferator-activated receptor; AR, androgen receptor; -TrCP, -transducin repeat-containing protein; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; GSK3 , glycogen synthase kinase 3 ; GST, glutathione transferase; HA, hemagglutinin; JNK, Jun NH2-terminal kinase; MEK, mitogen-activated protein kinase kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; WT, wild-type; F-TrCP, F-box-deleted -transducin repeat-containing protein; IP, immunoprecipitation; IB, immunoblotting; HA-UB, hemagglutinin-ubiquitin; Hsp90, heat shock protein 90; MAP, mitogen-activated protein; STE buffer, STE buffer, Tris/NaCl/EDTA/ dithiothreitol/phenylmethylsulfonyl fluoride; M-PER, mammalian protein extraction reagent; PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; IKK , I B kinase ; siRNA, small interfering RNA; FBS, fetal bovine serum; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PD98059, 2 -amino-3 -methoxyflavone; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; SB216763, 3-(2,4-dichlorophenyl)-4-(1methyl-1H-indol-3yl)-1H-pyrrole-2,5-dione; SP600125, anthra[1,9-c,d]pyrazol-6(2H)-one; PD169316, 4-(4-fluorophenyl)2-(4-nitrophenyl)-5-(4pyridyl)1H-imidazole; OSU-CG12, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)thiazolidine-2,4-dione. 0026-895X/09/7601-47–57$20.00 MOLECULAR PHARMACOLOGY Vol. 76, No. 1 Copyright © 2009 The American Society for Pharmacology and Experimental Therapeutics 55376/3485364 Mol Pharmacol 76:47–57, 2009 Printed in U.S.A. 47 at A PE T Jornals on Jne 9, 2017 m oharm .aspeurnals.org D ow nladed from interacts with a diversity of transcription factors, oncogenes, and tumor suppressors as part of the mechanism regulating its transcriptional activity, which underscores the complexity of its biological activities and its implications for tumorigenesis. Consequently, Sp1 overexpression has been linked to tumor progression and poor prognosis in many human cancers, including those of stomach, liver, thyroid, and pancreas (Kitadai et al., 1992; Liétard et al., 1997; Shi et al., 2001; Chiefari et al., 2002; Wang et al., 2003; Jiang et al., 2004, 2008; Yao et al., 2004). In addition, siRNA-mediated Sp1 knockdown decreased tumor growth and/or metastasis of gastric and pancreatic cancers in animal model studies (Jiang et al., 2004; Yuan et al., 2007). Together, these findings underscore the translational value of targeting dysregulated Sp1 expression in cancer therapy. However, despite advances in understanding Sp1’s biological functions, the mechanism that controls the turnover of this transcriptional factor remains unclear. Under physiological conditions, metabolic stress, such as glucose starvation, or stimulation with cAMP promotes the degradation of Sp1 through a proteasome-dependent pathway (Han and Kudlow, 1997; Su et al., 1999, 2000). We recently demonstrated that members of the thiazolidinedione family of peroxisome proliferator-activated receptor(PPAR ) agonists were able to promote proteasomal degradation of Sp1 in prostate cancer cells, leading to PPAR independent transcriptional repression of androgen receptor (AR) (Yang et al., 2007). This mechanistic finding provides a molecular basis for the pharmacological exploitation of thiazolidinediones to develop potent Sp1-targeted agents, among which the ciglitazone-derived analog OSU-CG12 represents an optimal agent (Yang et al., 2008). Although devoid of PPAR activity, OSU-CG12 facilitated Sp1 degradation at low micromolar concentrations in a manner qualitatively similar to that of glucose starvation. In light of the therapeutic potential of targeting Sp1 in cancer therapy, we embarked on characterizing the mechanism by which OSU-CG12 and glucose starvation facilitate the proteasomal degradation of Sp1. It is noteworthy that OSU-CG12 and related thiazolidinediones promote the degradation of -catenin, cyclin D1, and other cell-cycle regulatory proteins in LNCaP cells by up-regulating the expression of -transducin repeat-containing protein ( -TrCP), an F-box component of the Skp1-Cul1F-box protein E3 ubiquitin ligase (Wei et al., 2007, 2008). In this study, we obtained several lines of evidence to support the mechanistic link between -TrCP and Sp1 degradation in response to OSU-CG12 and glucose deprivation. Furthermore, mutational and modeling analyses indicate that -TrCP recognizes Sp1 through its DpSGAGpS motif, in which extracellular signal-regulated kinases (ERKs) and GSK3 play an obligatory role. Materials and Methods Cell Line, Culture, and Reagents. LNCaP androgen-responsive and PC-3 androgen-unresponsive prostate cancer cells and MCF-7 estrogen receptor-positive breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in 10% fetal bovine serum (FBS)-supplemented RPMI 1640 or F-12/Dulbecco’s modified Eagle’s medium at 37°C in a humidified incubator containing 5% CO2. In experiments assessing the effects of glucose deprivation, cells were cultured in glucose-free RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% FBS. Ciglitazone and its PPAR -inactive derivative 2CG and OSU-CG12 were synthesized according to a published procedure (Yang et al., 2008). The following pharmacological agents were purchased from various sources: MG132, PD98059, U0126, lithium chloride, SB216763, and SP600125 were from Sigma-Aldrich (St. Louis, MO); and PD169316 and cycloheximide were from Calbiochem (San Diego, CA). Stock solutions of these agents were made in DMSO and added to medium with a final DMSO concentration of 0.1%. Antibodies against various proteins were obtained from the following sources: mouse monoclonal antibodies: -catenin, Wee1, and p53 were from Santa Cruz Biotechnology (Santa Cruz, CA); hemagglutinin (HA) and Myc were from Roche (Indianapolis, IN); -TrCP and Skp2 were from Invitrogen; Flag was from Sigma; -actin was from MP Biomedicals (Irvine, CA); rabbit antibodies: Sp1, AR, estrogen receptor (ER) , p-Ser9GSK3 , GSK3 , p-Ser473-Akt, Akt, p-Thr202/Tyr204-ERK, ERK, p-Thr183/Tyr185-Jun NH2-terminal kinase (JNK), JNK, p-Ser63-cJun, c-Jun, p-Thr180/Tyr182-p38 and p38, p-Thr334-MAPKAPK-2, MAPKAPK-2, c-Raf, I B kinase (IKK ), and Stat3 were from Cell Signaling Technology (Danvers, MA); I B was from Santa Cruz Biotechnology; glutathione transferase (GST) was from Sigma; and Fbx4 was from Rockland (Gilbertsville, PA). The HA-tagged GSK3 K85A plasmid was purchased from Addgene (Cambridge, MA). Dominant-negative (kinase-defective) mitogen-activated protein kinase kinase (MEK) 1 (K97A) and constitutively active MEK1 (S218/222D) subcloned into pCMVHA were prepared as described previously (Slack et al., 1999). Plasmid Construction and Site-Directed Mutagenesis. To achieve the expression of Flag-tagged wild-type (WT) Sp1 protein, the pCMVSp1 plasmid (Yang et al., 2007) encoding full-length Sp1 was subcloned into EcoRI/XbaI sites of the p3XFLAG-CMV26 expression vector. Plasmids encoding various Sp1 mutations were generated from pWT Sp1-Flag by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). Primers used to generate Sp1 mutations were as follows: S728A, 5 -GTGGGCACTTTGCCCCTGGACGCTGGGGCAGGT-3 and 5 -ACCTGCCCCAGCGTCCAGGGGCAAAGTGCCCAC-3 ; S732A, 5 -CTGGACAGTGGGGCAGGTGCAGAAGGCAGTGGCACTGCC-3 and 5 GGCAGTGCCACTGCCTTCTGCACCTGCCCCACTGTCCAG-3 ; and T739A, 5 -AGGCAGTGGCACTGCCGCTCCTTCAGCCCTTATTA-3 and 5 -TAATAAGGGCTGAAGGAGCGGCAGTGCCACTGCCT-3 . To generate the construct for expression of F-box (residues 180– 226)-deleted -TrCP ( F-TrCP), the DNA sequence encoding amino acid residues 1 to 179 of -TrCP was PCR-amplified from the -TrCP-Myc plasmid (Wei et al., 2007) with the primers 5 CCGATATAAGCTTATGGACCCGGCCGAG-3 (forward) and 5 GCGCGCGAAGCTTATCTCTCTGCAACAT-3 (reverse), which were flanked by HindIII restriction sites. The sequence encoding -TrCP amino residues 227 to 605 was obtained by digestion of -TrCP-Myc plasmid with HindIII/XbaI. The resulting fragments were incubated with those corresponding to residues 1 to 179 followed by the ligation into the pMyc4-CMV14 expression vector (Yang et al., 2005) to generate the F-TrCP-Myc plasmid. All constructs were verified by DNA sequencing. RNA Isolation and Semiquantitative PCR Analysis. Total RNA was isolated from drug-treated LNCaP cells using the RNeasy mini kit (Qiagen, Valencia, CA) and then reverse-transcribed to cDNA using the Omniscript RT Kit (Qiagen) according to the manufacturer’s instructions. Primers used for PCR were as follows: Sp1, 5 -GGCGAGAGGCCATTTATGTGT-3 and 5 -AGTGGCATCAACGTCATGCA-3 ; AR, 5 -ACACATTGAAGGCTATGAATGTC-3 and 5 -TCACTGGGTGTGGAAATAGATGGG-3 ; ER , 5 -ACTGCATCAGATCCAAGGGAACG-3 and 5 -GGCAGCTCTCATGTCTCCAGCAGA-3 ; and -actin, 5 -TCTACAATGAGCTGCGTGTG-3 and 5 -GGTCAGGATCTTCATGAGGT-3 . PCR products were separated electrophoretically in 1% agarose gels and visualized by ethidium bromide staining. Transient Transfection, RNA Interference, and Luciferase Assay. Cells were transfected with various plasmids using Nucleo48 Wei et al. at A PE T Jornals on Jne 9, 2017 m oharm .aspeurnals.org D ow nladed from fector kit R of the Amaxa Nucleofector system (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s instructions. Cells were then seeded into six-well plates (5 10 cells/well) and incubated in 10% FBS-containing medium for 24 h before drug treatment. Transfection efficiency was 75% as determined by cotransfection with pmaxGFP plasmids and visualization of green fluorescent protein-positive cells by fluorescence microscopy. For siRNA experiments, cells were nucleofected with scrambled or TrCP siRNA (Santa Cruz Biotechnology) and seeded into six-well plates (5 10 cells/well) for drug treatments and subsequent analyses. For the luciferase assay, LNCaP cells expressing AR promoterluciferase and herpes simplex virus thymidine kinase promoterRenilla reniformis luciferase were prepared as we described previously (Yang et al., 2007) and transfected with WT or mutant forms of Sp1. Cells were cultured in 24-well plates (1 10 cells/well) in 10% FBS-containing RPMI 1640 medium for 24 h and treated with OSU-CG12 for the indicated intervals. Reporter gene assays were then performed as we reported previously (Yang et al., 2007). Immunoprecipitation and Immunoblotting. Ectopically expressed and endogenous proteins were immunoprecipitated from cell lysates after various treatments. These proteins included Flagtagged WT and mutant Sp1, endogenous Sp1, and -TrCP-Myc. Immunoprecipitation with anti-Flag, anti-Myc, and anti-Sp1 antibodies followed by immunoblotting for various target proteins were performed as described previously (Wei et al., 2007). In Vivo Ubiquitination Assay. Cells were transfected with the expression vector for HA-tagged ubiquitin in combination with the plasmid for WT or mutant (S732A) Flag-tagged Sp1, the plasmid for WT or F-TrCP-Myc, or scrambled or -TrCP siRNA. After nucleofection with plasmids or siRNA, cells were cultured in six-well plates for 24 h and then treated with 5 M OSU-CG12 for 12 or 36 h, followed by 12-h cotreatment with the proteasome inhibitor MG132. Cells were harvested into M-PER buffer containing 1% protease inhibitor cocktail and centrifuged at 13,000g for 20 min. The supernatants were collected, preincubated with protein A-agarose for 15 min, and centrifuged at 1000g for 5 min. One-tenth volume of each supernatant was stored at 4°C for use as the input sample for immunoblotting, and the remainder was incubated with anti-Sp1 or anti-Flag affinity gels overnight at 4°C. Immunoprecipitates were centrifuged, collected, washed, suspended in 2 SDS sample buffer, and subjected to Western blot analysis with antibodies against HA, Flag, or Sp1. GST Pull-Down Assay. The expression and purification of GSTfusion proteins and the pull-down of cellular proteins were performed as described previously (Wei et al., 2007). In brief, GST, and the GST-TrCP and GST-Skp2 fusion proteins were expressed in Escherichia coli strain BL21 (DE3) by isopropyl-1-thio-D-galactopyranoside induction for 3 h at 37°C. After centrifugation at 7,000g for 10 min, bacterial pellets were suspended in 10 ml of STE buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride), and lysed by sonication on ice. The lysates were centrifuged for 20 min at 30,000g, and the pellets were dissolved in 10 ml of 1.5% N-laurylsarcosine (sarkosyl)-containing STE buffer at 4°C for 1 h. After centrifugation at 30,000g for 20 min, supernatants were collected and mixed with 2.5 ml of 2% Triton X-100. Bacterial lysates were subjected to Western blot analysis with GST antibody for assessment of fusion protein expression. Recombinant GST and GST-fusion proteins were purified from supernatants by incubation with glutathione-Sepharose beads, which were used for the pull-down of Sp1 variants from