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Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes *

Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 46, pp. 38879 –38887, November 18, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes Received for publication, May 25, 2005, and in revised form, August 10, 2005 Published, JBC Papers in Press, September 26, 2005, DOI 10.1074/jbc.M505733200 Chang-Shi Chen, Shu-Chuan Weng, Ping-Hui Tseng, Ho-Pi Lin, and Ching-Shih Chen From the Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210 Despite advances in understanding the role of histone deacety- inhibitors remain elusive. Although they have been shown to activate lases (HDACs) in tumorigenesis, the mechanism by which HDAC the transcription of a defined set of genes through chromatin remodel- inhibitors mediate antineoplastic effects remains elusive. Modifica- ing (4), increasing evidence suggests that modifications of the epigenetic tions of the histone code alone are not sufficient to account for the histone code may not represent the primary cause for HDAC inhibitor- antitumor effect of HDAC inhibitors. The present study demon- mediated growth inhibition and apoptosis in cancer cells (5, 6). To date, strates a novel histone acetylation-independent mechanism by at least two distinct histone acetylation-independent mechanisms have which HDAC inhibitors cause Akt dephosphorylation in U87MG been described for the action of HDAC inhibitors on cellular targets. glioblastoma and PC-3 prostate cancer cells by disrupting HDAC- First, because certain HDAC members can mediate the deacetylation of protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors non-histone proteins, their inhibition interferes with signaling pro- examined, trichostatin A (TSA) and HDAC42 exhibit the highest cesses in which these proteins are involved independently of the activity activity in down-regulating phospho-Akt, followed by suberoylani- of HDAC inhibitors in transcriptional activation. For example, HDAC3 lide hydroxamic acid, whereas MS-275 shows only a marginal effect regulates NFB signaling in the nucleus by deacetylation the Rel-A sub- at 5 M. This differential potency parallels the respective activities unit (7), and HDAC6 modulates microtubule acetylation and chemo- in inducing tubulin acetylation, a non-histone substrate for tactic cell motility by acting as a tubulin deacetylase (8–12). Moreover, HDAC6. Evidence indicates that this Akt dephosphorylation is not Class I HDACs function as a STAT3 deacetylase (13), and the mamma- mediated through deactivation of upstream kinases or activation of lian Class III HDACs SirT1 and SirT2 can deacetylate p53 and tubulin, downstream phosphatases. However, the effect of TSA on phospho- respectively (14, 15). Second, HDACs 1 and 6 have been shown to form Akt can be rescued by PP1 inhibition but not that of protein phos- complexes with protein phosphatase 1 (PP1) (16, 17), of which the com- phatase 2A. Immunochemical analyses reveal that TSA blocks spe- bined deacetylase/phosphatase activities underlie the ability of HDAC1 cific interactions of PP1 with HDACs 1 and 6, resulting in increased to modulate transcriptional activity of cAMP-responsive element-bind- PP1-Akt association. Moreover, we used isozyme-specific small ing protein (16) and that of HDAC6 to regulate microtubule dynamics interfering RNAs to confirm the role of HDACs 1 and 6 as key medi- (17). Also noteworthy is that the binding of HDACs to PP1 was highly ators in facilitating Akt dephosphorylation. The selective action of specific because no association between HDACs and protein phospha- HDAC inhibitors on HDAC-PP1 complexes represents the first tase 2A (PP2A) was noted (17). example of modulating specific PP1 interactions by small molecule Moreover, data from this and other laboratories (18–20) show that agents. From a clinical perspective, identification of this PP1-facil- HDAC inhibitors could facilitate the dephosphorylation of Akt and itated dephosphorylation mechanism underscores the potential use other signaling kinases, although the causative mechanism remains of HDAC inhibitors in lowering the apoptosis threshold for other undefined. In this report, we used TSA to demonstrate that HDAC therapeutic agents through Akt down-regulation. inhibitors facilitate dephosphorylation of Akt by altering the dynamics of HDAC-PP1 complexes. We provide evidence that HDAC inhibitors selectively target HDACs 1 and 6 to disrupt the respective HDAC-PP1 Histone deacetylase (HDAC) is recognized as one of the promising complexes, resulting in increased association of PP1 with Akt. This targets for cancer treatment because many HDAC inhibitors have PP1-facilitated kinase dephosphorylation underscores the diverse func- entered clinical trials in both solid and liquid tumors (1–3). Neverthe- tions of HDAC inhibitors in mediating antineoplastic activities at dif- less, the mechanisms underlying the antiproliferative effects of HDAC ferent cellular levels. * This work was supported by National Institutes of Health Grants CA-94829 and EXPERIMENTAL PROCEDURES CA-112250. The costs of publication of this article were defrayed in part by the pay- Cell Culture—U87MG human glioblastoma cells were kindly pro- ment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vided by Dr. Ing-Ming Chiu (The Ohio State University, Columbus, To whom correspondence should be addressed: College of Pharmacy, 336 Parks Hall, OH), and PC-3 prostate cancer cells were purchased from the American The Ohio State University, 500 12th Ave., Columbus, OH 43210. Tel.: 614-688-4008; Fax: 614-688-8556; E-mail: [email protected]. Type Culture Collection (Manassas, VA). Because both U87MG and The abbreviations used are: HDAC, histone deacetylase; PP1, protein phosphatase 1; PC-3 cells are PTEN-null, they exhibit constitutively active Akt. These PP2A, protein phosphatase 2A; TSA, trichostatin A; SAHA, suberoylanilide hydroxamic cancer cells were cultured in 10% fetal bovine serum (FBS)-supple- acid; HDAC42, N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide; PI3K, phosphatidylinositol 3-kinase; PIP , L- -phosphatidyl-D-myo-inositol 3,4,5-trisphos- mented RPMI 1640 medium containing 100 units/ml penicillin and 100 phate; PDK-1, phosphoinositide-dependent kinase-1; ERK, extracellular signal-re- g/ml streptomycin (Invitrogen). lated kinase; siRNA, small interfering RNA; FBS, fetal bovine serum; p-, phospho-; JNK, c-Jun N-terminal kinase; TBS, Tris-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)- Reagents—Trichostatin A (TSA), calyculin A, and okadaic acid were 2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline; MOPS, purchased from Sigma-Aldrich, and tautomycin was obtained from Cal- 4-morpholinepropanesulfonic acid; MEK, mitogen-activated protein kinase/ERK kinase; Hsp, heat shock protein; CTMP, C-terminal modulator protein. biochem (La Jolla, CA). The HDAC inhibitors suberoylanilide hydrox- NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38879 This is an Open Access article under the CC BY license. Cellular Effect of Histone Deacetylase Inhibitors on Akt amic acid (SAHA) and N-hydroxy-4-(3-methyl-2-phenyl-butyrylami- Flow Cytometry/Apoptosis Assays—After drug treatment as described no)benzamide (HDAC42) were synthesized in our laboratory. HDAC42 above, floating cells were collected, and adherent cells were harvested by belongs to a novel class of hydroxamate-tethered phenylbutyrate deriv- scraping. The combined cells were fixed in ice-cold 70% ethanol and stor- age at20 °C. The cells were centrifuged, washed with PBS, and incubated atives with nanomolar potency in HDAC inhibition (21, 22) and is cur- in 0.2 M phosphate citrate buffer, pH 7.8 at 37 °C to extract low molecular rently undergoing preclinical testing under the Rapid Access to Inter- weight DNA. The samples were stained with propidium iodide solution (10 vention Development Program at the National Cancer Institute. Mouse mg/ml) in the presence of RNase A (100 mg/ml) for 30 min at room tem- antibodies against -tubulin and acetylated -tubulin were from Sigma- 473 308 perature. Cell cycle phase distributions were determined on a FACScort Aldrich. Rabbit antibodies against Akt, Ser -Akt, Thr -Akt, PDK-1, flow cytometer and analyzed by the ModFitLT V3.0 program. p-ERK1/2, p-p38, p-JNK, and various HDAC isozymes were purchased Affinity Purification of HDAC-PP1 Complexes—U87MG cells were from Cell Signaling Technology Inc. (Beverly, MA). Rabbit antibodies treated with TSA at the indicated concentrations in 10% FBS-supple- against PP1, PP2A, and nucleolin, and mouse antibodies against p21 mented RPMI 1640 medium for 48 h. Control cells received the vehicle were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit anti- treatment of 0.1% (v/v) dimethyl sulfoxide. The cells were washed by bodies against p85, p110, histone H3, and acetyl-histone H3 were from phosphate-buffered saline and lysed by the aforementioned Nonidet Upstate Biotechnology Inc. (Lake Placid, NY). Mouse monoclonal anti- P-40 isotonic lysis buffer. Cell extract was incubated with microcystin- actin was from ICN Biomedicals Inc. (Costa Mesa, CA). Rabbit anti- LR-Sepharose (Upstate Biotechnology, Inc.) at 4 °C overnight on a rota- human C-terminal modulator protein (CTMP) antibodies and rabbit tor. After a brief centrifugation, Sepharose beads were collected and anti-TRB3 antibodies were from Alpha Diagnostic International (San washed with the lysis buffer four times. The bound proteins were eluted Antonio, TX) and Oncogene Research Products (San Diego, CA), by 50 l of SDS sample buffer and subjected to SDS-PAGE followed by respectively. Goat anti-rabbit IgG-horseradish peroxidase conjugates immunoblotting with appropriate antibodies. and rabbit anti-mouse IgG horseradish peroxidase conjugates were Co-immunoprecipitation of PP1-Akt Complexes—U87MG cells were from Jackson ImmunoResearch Laboratories (West Grove, PA). The treated with various concentrations of TSA for 48 h and lysed by the siRNA transfection reagent, siRNA transfection medium, and siRNAs aforementioned Nonidet P-40 isotonic lysis buffer with a mixture of against HDAC1 and HDAC6 (catalog numbers sc-29343 and sc-35544, protease inhibitors. After centrifugation at 13000  g for 15 min, the respectively) and a control, scrambled siRNA (catalog number supernatants were collected and incubated with protein A-Sepharose sc-37007) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). beads (Sigma) for 15 min to eliminate nonspecific binding. The mixture Immunoblotting—U87MG and PC-3 cells treated with various con- was centrifuged at 1000  g for 5 min, and the supernatants were centrations of HDAC inhibitors in 10% FBS supplemented RPMI 1640 exposed to Akt or PP1 antibodies in the presence of protein A-Sepha- medium for 48 h were collected and lysed by Nonidet P-40 isotonic lysis rose beads at 4 °C for 2 h. After a brief centrifugation, protein A-Sepha- buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% (v/v) Nonidet P-40, 1 rose beads were collected, washed with the aforementioned lysis buffer mM EDTA, 50 mM NaF, 40 mM -glycerophosphate, and 1 g/ml each four times, suspended in 2 SDS sample buffer, and subjected to West- of aprotinin, pepstatin, and leupeptin). Protein concentrations of the ern blot analysis with antibodies against PP1 or Akt. lysates were determined by using a Bradford protein assay kit (Bio-Rad). Subcellular Fractionation—The nuclear and cytosolic fractions of Equivalent amounts of proteins from each lysate were resolved by SDS- U87MG cells were prepared by using a nuclear/cytosol fraction kit PAGE and then transferred onto Immobilon-nitrocellulose membranes (MBL Co., Watertown, MA) according to the manufacturer’s instruc- (Millipore, Bellerica, MA) in a semidry transfer cell. The transblotted tions. In brief, the cells were cultured to 50% confluency in T-75 flasks, membrane was washed twice with Tris-buffered saline (TBS) contain- treated with TSA for 48 h, and collected by centrifugation. The pelleted ing 0.1% Tween 20 (TBST). After blocking with TBST containing 5% cells were resuspended in 0.2 ml of cytosol extraction buffer A mix nonfat milk for 40 min, the membrane was incubated with the appro- containing dithiothreitol and a mixture of protease inhibitors and were priate primary antibody in TBST containing 1% nonfat milk at 4 °C mixed vigorously on a vortex mixer. The suspension was then incubated overnight. All of the primary antibodies were diluted 1: 2000 in 1% on ice for 10 min, mixed with 11 l of cytosol extraction buffer B, and nonfat milk-containing TBST. After treatment with the primary anti- incubated on ice for 1 min. The lysates were centrifuged at 16,000  g body, the membrane was washed two times with TBST for a total of 20 for 5 min. The supernatant, representing the cytoplasmic fraction, was min, followed by goat anti-rabbit or anti-mouse IgG-horseradish per- transferred to a prechilled tube, and the pellet was resuspended in 100l oxidase conjugates (diluted 1:3000) for 1 h at room temperature and of nuclear extraction buffer mix. The suspension was mixed vigorously washed three times with TBST for a total of 1 h. The immunoblots were on a vortex mixer for 15 s, incubated on ice for 40 min, and centrifuged visualized by ECL chemiluminescence (Amersham Biosciences). at 16,000 g for 10 min to collect the nuclear extract in the supernatant Cell Viability Assay—The effect of the HDAC inhibitors on cell via- fraction. bility was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- Ser/Thr Phosphatase Activity—The Ser/Thr phosphatase activity 2H-tetrazolium bromide (MTT) assay in 6–12 replicates. U87MG and was determined by using a nonradioactive, malachite green-based Ser/ PC-3 cells were seeded and incubated in 96-well, flat-bottomed plates in Thr phosphatase assay kit (Upstate Biotechnology, Inc.) according to 10% FBS-supplemented RPMI 1640 medium 24 h before drug treat- the manufacturer’s instructions. In brief, 5 g of U87MG cell extracts in ment. The cells were exposed to individual HDAC inhibitors at the the aforementioned lysis buffer were incubated with 175 M of the indicated concentrations in 10% FBS-supplemented RPMI 1640 phosphopeptide substrate (K-R-pT-I-R-R) in the phosphatase buffer medium at 37 °C in 5% CO for 48 h. The medium was removed and (20 mM MOPS, pH7.5, 60 mM 2-mercaptoethanol, 0.1 M NaCl, and 0.1 replaced by 200 l of 0.5 mg/ml of MTT in RPMI 1640 medium, and the mg/ml serum albumin) with a total volume of 25 l. The reaction was cells were incubated in the CO incubator at 37 °C for 2 h. Supernatants incubated in a 96-well plate at room temperature for 10 min, terminated were removed from the wells, and the reduced MTT dye was solubilized by adding the malachite green solution, and allowed to stand for 15 min with 200 l/well Me SO. Absorbance was determined on a plate reader to permit color development. Absorption at 650 nm was measured in a at 570 nm. microplate reader, and the serine/threonine phosphatase activity was 38880 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt FIGURE 1. HDAC inhibitor-mediated Akt phos- phorylation in U87MG and PC-3 cells. A, dose- dependent effects of four different HDAC inhibi- tors, including HDAC42, SAHA, MS-275, and TSA on histone H3 acetylation, p21 expression, tubulin acetylation, and phosphorylation state of Akt at 308 473 Thr and Ser . All four inhibitors mediated his- WAF/CIP1 tone hyperacetylation and p21 up-regula- tion but exhibited differential effects on tubulin acetylation and Akt dephosphorylation. U87MG and PC-3 cells were treated with the indicated doses of individual inhibitors for 48 h, and the cell lysates were immunoblotted. B, time-dependent effects of 0.25 and 0.5 M TSA on p-Ser -Akt lev- els vis a` vis p21 expression and histone H3 acetyla- tion in U87 MG cells. The cells were treated with 0.25 or 0.5 M TSA for the indicated time intervals, and the cell lysates were immunoblotted. DMSO, dimethyl sulfoxide. calculated using a standard curve based on free phosphate generated by well followed by incubation for1hinthe dark and three more washes a standard solution. All of the experiments were performed in triplicate. with 300 l of TBS with Tween 20 (0.05% v/v). 100 l of tetramethyl Immunoprecipitated Phosphatidylinositol 3-Kinase (PI3K) Kinase benzidine solution was added to each well and incubated for 20–30 Assay—PI3K activity was determined by the use of a PI3K enzyme- min to allow color development. The reaction was stopped by the addi- linked immunosorbent assay kit from Echelon Biosciences Inc. (Salt tion of 100 l of stop solution (0.5 M H SO ) to each well. Absorbance at 2 4 Lake City, UT) according to the manufacturer’s instruction. U87MG 450 nm was read, and the PI3K activity in each sample was calculated cells were cultured to 50% confluency in 10-cm culture dishes, and the using a standard curve generated by using a PIP standard solution. All medium was removed after TSA treatment. The drug-treated cells were of the experiments were performed in triplicate. washed with 10 ml/dish of ice-cold buffer A (137 mM NaCl, 10 mM Autoradiographic Determination of Phosphoinositide Formation— Tris-HCl, pH 7.4, 1 mM CaCl ,1mM MgCl , and 0.1 mM sodium U87MG cells, cultured in T25 flasks, were labeled with 1 mCi/ml of 2 2 orthovanadate) three times, incubated with 1 ml of ice-cold lysis buffer [ P]orthophosphate (HCl-free; PerkinElmer Life Sciences) in Dulbec- (buffer A plus 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluo- co’s modified Eagle’s phosphate-free and serum-free medium (Invitro- ride) on ice for 20 min, scraped from the dish, transferred to microcen- gen) for 4 h, washed three times with buffer A (30 mM Hepes, pH 7.2, 110 trifuge tubes, and centrifuged. The supernatant was collected, and the mM NaCl, 10 mM KCl, 1 mM MgCl , and 10 mM glucose), and treated protein concentrations were determined by the Bradford assay (Bio- with various concentrations of TSA in 10% FBS-supplemented RMPI Rad). Equal amounts of proteins were treated with 5 l of anti-PI3K 1640 medium for 48 h. The cells were extracted with 3 ml of chloro- antibodies (Upstate Biotechnology, Inc.) at 4 °C for 1 h, to which was form/methanol (1:2, v/v), followed by 4 ml of chloroform, 2.4 M HCl added 60 l of a 50% slurry of protein A-agarose beads in PBS, followed (1:1, v/v), and 1 ml of chloroform four times. The combined organic by incubation at 4 °C for 1 h with mixing. The immunocomplex was phase was dried under a stream of nitrogen and resuspended in 90 lof collected by brief centrifugation, washed with lysis buffer three times, chloroform for TLC analysis by using a 20  20-cm silica gel 60 TLC and incubated for2hat room temperature with 10 lof10 ML-- plate (EM Science) impregnated with 1% potassium oxalate in 50% eth- phosphatidyl-D-myo-inositol 4,5-bisphosphate, 5 lof10 reaction anol. The TLC plate was developed in chloroform/acetone/methanol/ buffer (40 mM MgCl , 200 mM Tris, pH 7.4, 100 mM NaCl, 250 M ATP), acetic acid/water (80:30:26:24:14, v/v/v/v/v) (23). Radioactive spots and 35 l of distilled water. The reaction was terminated by the addition were detected by autoradiography using Kodak X-Omat film, and total of 2.5 l of 100 mM EDTA. After brief centrifugation, 50 l of each PIP was quantified by both densitometry and an AMBIS B scanning reaction mixture was transferred to a 96-well plate, and 50 l of diluted system (San Diego, CA), both of which showed comparable results. L--phosphatidyl-D-myo-inositol 3,4,5-trisphosphate (PIP ) detector Kinase Assay for Phosphoinositide-dependent Kinase-1 (PDK-1)— was added to each well and incubated at room temperature for 1 h. 100 The PDK-1 kinase activity was performed using a PDK-1 kinase assay kit l of reaction mixture from each well was transferred to the corre- (Upstate Biotechnology, Inc.) according to a described procedure (24). sponding well in the detection plate and incubated at room temperature This cell-free assay is based on the ability of recombinant PDK-1 to for1hinthe dark. After three washes with 300 l of TBS with Tween 20 activate its downstream kinase serum- and glucocorticoid-regulated (0.05% v/v), 100 l of the secondary detection reagent was added to each kinase, which in turn phosphorylates the Akt/serum- and glucocorti- NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38881 Cellular Effect of Histone Deacetylase Inhibitors on Akt coid-regulated kinase-specific peptide substrate RPRAATF with 32 32 [- P]ATP. The P-phosphorylated peptide was then separated from residual [- P]ATP by P81 phosphocellulose paper and quantitated by a scintillation counter after three washes with 0.75% phosphoric acid. Isozyme-specific Knockdown of HDACs with siRNA—Isozyme-spe- cific siRNAs were used to attenuate the expression of HDACs 1 and 6 in U87MG cells using reagents obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Transfection of U87MG cells with these siRNA was carried out according to the vendor’s instructions. In brief, the cells were cultured to 40–50% confluency in 10% FBS-supplemented antibi- otic-free RPMI 1640 medium. The HDAC1 and HDAC6 siRNAs were transfected to the cells by the siRNA transfection reagent (Santa Cruz, CA) to the final concentration of 200 nM. A scramble siRNA was used in parallel experiments as a control. The transfected cells were incubated at 37 °C, and the extents of siRNA-mediated down-regulation of HDAC1 and HDAC6 expression were monitored by immunoblotting analysis at different time intervals. It was found that 30-h exposure to siRNA caused more than 80% repression of HDAC expression, and thus it was used through this study. Immunocytochemical Analysis of TSA-mediated Co-localization of PP1 and Akt—U87MG cells were treated with 0.5 M TSA in 10% FBS- supplemented RPMI 1640 medium for different time intervals, washed with Dulbecco’s PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed with PBS. For double staining of phospho-Akt and PP1, the cells were permeabilized with 0.1% Triton X-100 in 1% FBS-containing PBS, treated with rabbit anti-phospho- Ser -Akt (1:500 dilution) at 4 °C for 24 h, and washed with PBS. Sub- sequently, the cells were treated with mouse anti-PP1 (1:100 dilution) for8hat room temperature and washed with PBS. For fluorescent microscopy, Alexa Fluor 488 goat anti-mouse and anti-rabbit IgG (Molecular Probes, Inc.) were used for conjugating PP1 and phospho- Akt, respectively. The nuclear counterstaining was performed using a 4,6-diamino-2-phenylindole-containing mounting medium (Vector, CA) prior to examination. Images of immunocytochemically labeled samples were observed using a Zeiss confocal microscope (LSM510) with an argon laser and a helium-neon laser and appropriate filters (excitation wavelength, 488 nm for PP1, 633 nm for p-Akt, and 543 nm for 4,6-diamino-2-phenylindole). RESULTS FIGURE 2. A, dose-dependent effects of TSA and SAHA on the phosphorylation state of 308 473 Thr - and Ser -Akt vis a` vis ERK1/2, p38, and JNK MAP kinases in U87MG cells. The cells Differential Effects of HDAC Inhibitors on Akt Dephosphorylation— were treated with the indicated concentrations of TSA or SAHA for 48 h, and the cell To shed light onto the causative relationship between HDAC inhibition lysates were immunoblotted. B, dose dependence of the growth inhibitory effects of HDAC42, SAHA, MS-275, and TSA in U87MG and PC-3 cells. The cells were treated with and Akt deactivation, we assessed the effects of four different HDAC individual HDAC inhibitors at the indicated concentrations in 10% FBS-supplemented inhibitors, including HDAC42, SAHA, MS-275, and TSA, on various medium for 48 h, and the cell viability was determined by the MTT assay. Points, means; HDAC-related biomarkers (histone H3 acetylation, p21 expression, and bars, S.D. (n  6 –12). tubulin acetylation) vis a` vis Akt phosphorylation state in U87MG glio- blastoma and PC-3 prostate cancer cells. Of the four HDAC inhibitors 308 473 examination of Akt phosphorylation at both Thr and Ser indicates examined, HDAC42 belongs to a novel class of phenylbutyrate-based that HDAC42 and TSA at submicromolar concentrations were able to HDAC inhibitors (21) and has an IC of 30 nM in inhibiting HDAC substantially reduce phospho-Akt levels. A similar effect on phospho- activity in nuclear extracts. The reported IC values for TSA, SAHA, Akt was not achieved with SAHA until the concentration reached 5 M. and MS-275 are 5–15 nM, 120 nM, and 4.8 M, respectively (2). In contrast, the repressing effect of MS-275 on Akt phosphorylation, Western blot analysis shows that exposure of U87MG and PC-3 cells even at 5 M, was marginal. It is noteworthy that there existed an inverse to these inhibitors led to substantial increases in histone H3 acetylation WAF/CIP1 relationship between the levels of acetylated -tubulin and phospho- and p21 expression (Fig. 1A). However, these four inhibitors Akt, providing a potential link between HDAC6 inhibition and Akt behaved differently with regard to -tubulin acetylation, indicating dif- dephosphorylation. This premise was further supported by the differ- ferences in the respective activities in inhibiting the -tubulin deacety- ential effect of selective siRNA-mediated knockdown of individual lase HDAC6 (8). Although HDAC42 and TSA produced robust hyper- acetylation of -tubulin at submicromolar concentrations, SAHA was HDAC isozymes on phospho-Akt levels (see below). Moreover, the time effective at low micromolar concentrations, and MS-275 was totally course of TSA-mediated Akt dephosphorylation lagged behind that of ineffective, even at 5 M, in inhibiting tubulin deacetylation. Moreover, p21 overexpression and H3 hyperacetylation by at least 12 h (Fig. 1B), 38882 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt TABLE ONE Cell cycle phase distribution of U87MG and PC-3 cells treated with individual HDAC inhibitors at the indicated concentrations in 10% FBS- supplemented medium for 48 h Control cells received Me SO vehicle. Each tabulated percentage represents the average of two independent experiments. Cells in cell cycle phases U87MG PC-3 Sub-G /G G /G SG /M Sub-G /G G /G SG /M 0 1 0 1 2 0 1 0 1 2 Me SO 1.3 69.2 14.5 14.9 0 51.2 22.4 26.4 HDAC-42 0.5 M 0.2 64.8 5.7 29.3 0.2 43.1 22.8 33.9 2.5 M 0.5 71.2 2.5 25.8 0.3 39.6 2.0 58.1 5 M 0.2 69.5 2.5 28.3 4.4 31.2 1.2 62.8 SAHA 0.5 M 0.9 65.9 15.1 17.1 0 56.0 14.1 29.9 2.5 M 0.6 55.3 13.6 30.5 0 44.4 19.4 36.0 5 M 0.7 58.5 10.0 30.8 0 47.7 23.2 29.1 MS-275 0.5 M 0.8 73.3 11.3 14.6 0.1 61.8 10.4 27.7 2.5 M 0.4 81.3 4.2 14.1 0 69.4 2.0 28.6 5 M 0 80.4 4.7 14.9 0 64.2 1.3 34.5 TSA 0.1 M 1.0 64.1 10.7 23.1 0 45.2 7.7 47.1 0.25 M 2.2 70.8 1.8 25.2 0 37.5 2.2 60.3 0.5 M 0.7 64.1 3.2 32.0 6.6 36.6 0 58.2 suggesting that this Akt effect might not be related to these two decreases in the G /G and, in particular, S fractions (TABLE ONE). 0 1 biomarkers. The sub-G /G fraction, an indication of apoptosis, was not observed 0 1 Selective Dephosphorylation of Signaling Kinases—Pursuant to the until the concentrations of HDAC42 and TSA reached 5 and 0.5 M, above finding, a question emerged with regard to the specificity of respectively. Meanwhile, the cell cycle-arresting behavior of SAHA in HDAC inhibitor-mediated kinase dephosphorylation. Accordingly, we these two cell lines differed. It caused dose-dependent increases in the examined the impact of two representative HDAC inhibitors, TSA and G /M population in U87MG cells, but the effect on cell cycle distribu- SAHA, on the phosphorylation status of ERKs, p38, and JNK MAP tion in PC-3 cells changed from G arrest to G /M arrest as the concen- 1 2 kinases versus Akt in U87MG cells (Fig. 2A). As noted, both TSA and tration increased. In contrast, MS-275 induced dose-dependent G cell SAHA caused a modest decrease in phospho-ERK1/2 levels despite cycle arrest in both cell lines. their respective activities in Akt dephosphorylation, whereas those of TSA-mediated Akt Dephosphorylation Is Not Caused by Changes in phospho-p38 and phospho-JNK remained unaffected. These findings the Expression Level of Proteins Involved in Phospho-Akt Regulation— indicate that there existed a certain degree of specificity in HDAC inhib- Mechanistically, this Akt dephosphorylation might be mediated itor-mediated kinase dephosphorylation, suggesting the involvement of through the deactivation of upstream kinases or the activation of down- a unique signaling mechanism. stream phosphatase. To discern the role of transcriptional activation in Differential Effects of HDAC Inhibitors on Cell Proliferation and Cell this drug action, we assessed the expression levels of a series of signaling Cycle—Together, these findings suggest that these four HDAC inhibi- proteins related to the regulation of Akt signaling pathways in TSA- tors exhibited distinct profiles regarding pharmacological targets, treated U87MG cells, which included the p85 regulatory and p110 cat- which might underlie differences in their antitumor activities. For alytic subunits of PI3K, PDK-1, Akt, the negative Akt modulators example, although these agents were able to up-regulate p21 expression CTMP (25) and TRB3 (26), PP1, and PP2A. As shown in Fig. 3A, TSA and histone acetylation, two hallmark features in association with intra- exposure did not alter the expression level of any of these signaling cellular HDAC inhibition, at submicromolar concentrations, their abil- proteins, excluding the involvement of transcriptional activation in ity to suppress cell proliferation varied by almost an order of magnitude altering the status of Akt phosphorylation. (Fig. 2B). Although TSA and HDAC42 were effective in suppressing Furthermore, three lines of evidence argued against the possibility proliferation in U87MG and PC-3 cells at submicromolar concentra- that TSA-mediated Akt deactivation was caused by a decrease in PI3K tions, it would require at least 2.5–5 M for SAHA and MS-275 to attain or PDK-1 kinase activity (Fig. 3B). First, immunoprecipitated PI3K a similar antiproliferative effect. This differential potency paralleled the kinase activity in U87MG cells treated with different doses of TSA respective activities in causing Akt dephosphorylation, suggesting a remained the same as that of the Me SO control (Fig. 3B, left panel, open putative link between Akt down-regulation and the antitumor activities 2 bars). Second, TSA at different doses exhibited no appreciable inhibi- of HDAC42, TSA, and, to a lesser extent, SAHA. Moreover, flow cyto- tory effects on the kinase activity of recombinant PDK-1 (Fig. 3B, left metric analyses indicate that the effect of these HDAC inhibitors on panel, gray bars). Third, autoradiographic analysis of P-labeled phos- growth inhibition was largely attributable to cell cycle arrest in lieu of pholipids demonstrated that the levels of PIP , a PI3K lipid product, and apoptosis. Analyses of cell cycle distribution in drug-treated U87MG and PC-3 cells shows that HDAC42 and TSA induced dose-dependent other inositol lipids in U87MG cells were unaffected by 0.5 M TSA accumulation of cells in the G /M phase, accompanied by comparative treatment (Fig. 3B, right panel). NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38883 Cellular Effect of Histone Deacetylase Inhibitors on Akt Inhibition of PP1 Prevents TSA-mediated Akt Dephosphorylation—In at low doses (100 nM) is selective for PP2A (33, 34), and calyculin A the literature, a number of small molecules such as C -ceramide, 4-hy- lacks selectivity between PP1 and PP2A. Fig. 4 demonstrates that droxynonenal, FTY720, and N-ethylmaleimide have been reported to although none of these inhibitors affected the ability of TSA to inhibit facilitate Akt dephosphorylation by activating PP2A or PP2A-like phos- histone deacetylation, their differential effects on the TSA-mediated phatase (27–31). Consequently, we investigated a potential link between dephosphorylation of Akt was noteworthy. As shown, calyculin A (50 TSA-mediated Akt down-regulation and protein phosphatase activa- nM) and tautomycin (5 M) could completely abrogate the effect of TSA tion by examining the effect of tautomycin, calyculin A, and okadaic on phospho-Akt, whereas okadaic acid lacked appreciable protective acid on phospho-Akt levels in TSA-treated U87MG cells. These three activity against the dephosphorylation. Because PP1 represents a com- compounds exhibit distinct specificity in protein phosphatase inhibi- mon target for calyculin A and tautomycin, this finding suggests the tion, i.e. tautomycin is a highly specific PP1 inhibitor (32), okadaic acid involvement of PP1 in TSA-facilitated kinase dephosphorylation. TSA Disrupts HDAC-PP1 Complexes, Resulting in Increased PP1-Akt Associations—The link between HDAC inhibition and PP1-mediated kinase dephosphorylation could be attributed to two plausible mecha- nisms. First, TSA treatment might lead to increased PP1 activity. Sec- ond, in light of the reported HDAC-PP1 complex formation (16, 17), HDAC inhibitors might cause disruption of these complexes, which would free PP1 to interact with Akt. Of these two mechanisms, the former was refuted by lack of appreciable increases in either the PP1 expression level (Fig. 3A) or the overall Ser/Thr protein phosphatase activity after TSA exposure (data not shown). To evaluate the second possibility, we investigated the expression profile of HDAC isozymes in U87MG cells. Among the seven different isozymes examined, U87MG cells expressed HDACs 1, 2, 3, 5, and 6, but the expression of HDACs 4 and 7 was undetectable. In addition, TSA treatment did not alter the expression level of any of these HDACs (data not shown). To assess the impact of TSA on the dynamics of HDAC-PP1 complexes, we exposed the lysates of drug-treated U87MG cells to microcystin affinity beads to purify PP1-associated complexes (16). Western blot analysis of the affin- ity-purified PP1 complexes indicates that HDACs 1, 3, and 6 could be pulled-down by the affinity beads. Moreover, the levels of affinity bead- bound HDACs 1 and 6 decreased in response to TSA in a dose-depend- ent manner (Fig. 5A). The level of PP1-associated HDAC3, however, remained unaltered by the TSA treatment. Based on the report that the PP1-binding domain of HDAC6 encom- passed the catalytic motif (17), binding of the HDAC inhibitor to the catalytic domain might hinder the binding of PP1 to HDAC6. Presum- ably, HDAC inhibitors could also sequester HDAC1, but not HDAC3, from PP1 association through the same mode of mechanism. In con- FIGURE 3. A, TSA-mediated Akt dephosphorylation is not due to alterations in the expres- trast, the PP1 inhibitor tautomycin did not cause disturbance of the sion level of proteins that are involved in the regulation of Akt phosphorylation. U87MG cells were treated with the indicated doses of TSA for 48 h, and the cell lysates were HDAC-PP1 complex (data not shown), suggesting no overlap between immunoblotted. B, left panel, TSA does not affect the kinase activity of immunoprecipi- the HDAC-binding domain and the catalytic site of PP1. Also notewor- tated Akt in U87MG cells treated with the indicated dose of TSA, nor does it affect the activity of recombinant PDK-1. Right panel, TSA (0.5 M) does not affect PIP level in U87 thy is that TSA-mediated dissociation of HDAC-PP1 complexes did not MG cells. [ P]Orthophosphate-labeled U87MG cells were treated with TSA (500 nM)or significantly change the relative distribution of PP1 or PP2A in the Me SO vehicle for 48 h. The extracted phospholipids were analyzed by TLC separation, followed by autoradiographic analysis as described under “Experimental Procedures.” nucleus or cytoplasm (Fig. 5B). FIGURE 4. The protein phosphatase inhibitors calyculin A (CA), tautomycin (TM), and okadaic acid (OA) exhibit differential effects on TSA- mediated dephosphorylation of Akt and ERKs. U87MG cells were exposed to TSA and individual protein phosphatase inhibitors at the indicated concentrations. After 48 h of incubation, the cell lysates were immunoblotted. DMSO, dimethyl sulfoxide. 38884 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt FIGURE 5. A, TSA selectively disrupts the associa- tion of PP1 with HDACs 1 and 6. U87MG cells were treated with TSA at the indicated concentrations for 48 h. The cell lysates were exposed to microcys- tin affinity beads to purify PP1-associated com- plexes. The affinity-purified complexes were probed with individual HDAC isozyme-specific antibodies by Western blot analysis (WB). B, TSA does not change the relative abundance of PP1 or PP2A in the nucleus or cytoplasm in drug-treated U87MG cells. -Tubulin and nucleolin are used as internal references for the cytoplasmic and nuclear fractions, respectively. In addition, two lines of evidence indicate that the TSA-mediated disruption of HDAC-PP1 complexes was accompanied by a dose-de- pendent increase in PP1-Akt associations. First, immunoprecipitation of PP1 in the lysates of TSA-treated cells followed by Western blotting of Akt, or vice versa, demonstrates a positive correlation between TSA doses and amounts of Akt co-immunoprecipitated with PP1 (Fig. 6A). Second, immunocytochemical examinations show that exposure of U87MG cells to TSA for 4 h led to co-localization of PP1 and phospho- Akt, which, however, was not noted with cells receiving Me SO vehicle (Fig. 6B). The physical interaction between PP1 and Akt led to partial and complete dephosphorylation at 8 and 24 h post-treatment, respectively. Validation of the Involvement of HDACs 1 and 6 in Akt Dephos- phorylation—Together, the above findings suggest that TSA caused PP1-mediated Akt dephosphorylation by sequestering HDACs 1 and 6. To validate the role of these two isozymes in Akt deactivation, we used isozyme-specific siRNAs to selectively knock down the expression of HDACs 1 and 6 and that of a scrambled siRNA as a negative control (Fig. 7A). As shown, repressed expression of HDACs 1 and 6 both led to decreased Akt phosphorylation, whereas control siRNA transfection had no appreciable effect on phospho-Akt levels (Fig. 7B). DISCUSSION The present study demonstrates a novel histone acetylation-indepen- dent mechanism by which HDAC inhibitors mediate the dephospho- rylation of Akt through the disruption of HDAC-PP1 complexes. Together with the previous findings of the functional roles of HDAC- PP1 pairs in the modulation of cAMP-responsive element-binding pro- tein phosphorylation (16) and tubulin acetylation (17), this histone- independent mechanism provides a potential basis to account for the FIGURE 6. TSA treatment leads to increased PP1-Akt associations. A, immunoprecipi- antineoplastic activities of these agents in growth inhibition and apo- tation (IP) of PP1 in lysates of TSA-treated cells, followed by immunoblotting (IB)ofAkt ptosis induction. (left panel) or vice versa (right panel). U87MG cells were treated with TSA at the indicated Of the four inhibitors examined, HDAC42 and TSA were most effec- concentrations, and the cell lysates were exposed to anti-Akt or anti-PP1 in the presence of protein A-Sepharose beads. The immunocomplex was immunoblotted with anti-PP1 tive in facilitating Akt dephosphorylation, followed by SAHA, whereas or anti-Akt. B, immunocytochemical examinations of PP1 and phospho-Akt in U87MG MS-275 exhibited a marginal effect at therapeutically attainable con- cells treated with Me SO vehicle or 500 nM TSA for the indicated times. centrations (5 M). In light of the pivotal role of Akt in cell prolifera- tion, this differential activity in Akt down-regulation might be attrib- ing suggests a role for HDAC6 inhibition in Akt dephosphorylation. uted to differences in the antitumor activities among these four HDAC Consequently, MS-275 was ineffective in causing Akt deactivation as a inhibitors. TSA and HDAC42 was potent in suppressing the prolifera- result of its inability to inhibit HDAC6. tion of U87MG and PC-3 cells, in part, because of their ability to down- The present study further demonstrated a mechanistic link between regulate Akt signaling. It is noteworthy that the activity of these agents PP1 and TSA-mediated Akt dephosphorylation. Our immunochemical to suppress Akt phosphorylation paralleled the respective potency in study showed that TSA blocks specific associations of PP1 with HDACs inducing -tubulin acetylation, a biomarker for HDAC6 (8). This find- 1 and 6, suggesting the involvement of both isozymes in regulating the NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38885 Cellular Effect of Histone Deacetylase Inhibitors on Akt PP1-protein interactions by small molecule agents. From a clinical per- spective, identification of this PP1-facilitated dephosphorylation mech- anism underlies the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through the down- regulation of Akt and ERK signaling. This therapeutic strategy is illus- trated by the ability of HDAC inhibitors to sensitize cancer cells to the apoptotic effects of the Bcl-Abl kinase inhibitor imatinib (42), the Her-2 antibody trastuzumab (19), the receptor tyrosine kinase FLT-3 inhibitor PKC412 (20), the purine analogue fludaribine (43), and the Hsp90 antagonist 17-allylamino-demethoxy geldanamycin (44, 45). In conclusion, the ability of HDAC inhibitors to deactivate Akt through the reorganization of PP1 complexes underlines the complexity of the pharmacological functions of these agents. In light of the clinical application of HDAC inhibitors, a better understanding of this novel histone-independent mechanism will allow the design of more effective strategies to optimize the use of the agents in cancer treatment and/or FIGURE 7. Validation of the involvement of HDACs 1 or 6 in PP1-mediated Akt dephosphorylation. A, selective knockdown of the expression of HDACs 1 and 6 by prevention. isozyme-specific siRNA. 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(2004) Blood 105, 1768–1776 NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38887 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes *

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American Society for Biochemistry and Molecular Biology
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0021-9258
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10.1074/jbc.m505733200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 46, pp. 38879 –38887, November 18, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Histone Acetylation-independent Effect of Histone Deacetylase Inhibitors on Akt through the Reshuffling of Protein Phosphatase 1 Complexes Received for publication, May 25, 2005, and in revised form, August 10, 2005 Published, JBC Papers in Press, September 26, 2005, DOI 10.1074/jbc.M505733200 Chang-Shi Chen, Shu-Chuan Weng, Ping-Hui Tseng, Ho-Pi Lin, and Ching-Shih Chen From the Division of Medicinal Chemistry, College of Pharmacy, The Ohio State University, Columbus, Ohio 43210 Despite advances in understanding the role of histone deacety- inhibitors remain elusive. Although they have been shown to activate lases (HDACs) in tumorigenesis, the mechanism by which HDAC the transcription of a defined set of genes through chromatin remodel- inhibitors mediate antineoplastic effects remains elusive. Modifica- ing (4), increasing evidence suggests that modifications of the epigenetic tions of the histone code alone are not sufficient to account for the histone code may not represent the primary cause for HDAC inhibitor- antitumor effect of HDAC inhibitors. The present study demon- mediated growth inhibition and apoptosis in cancer cells (5, 6). To date, strates a novel histone acetylation-independent mechanism by at least two distinct histone acetylation-independent mechanisms have which HDAC inhibitors cause Akt dephosphorylation in U87MG been described for the action of HDAC inhibitors on cellular targets. glioblastoma and PC-3 prostate cancer cells by disrupting HDAC- First, because certain HDAC members can mediate the deacetylation of protein phosphatase 1 (PP1) complexes. Of four HDAC inhibitors non-histone proteins, their inhibition interferes with signaling pro- examined, trichostatin A (TSA) and HDAC42 exhibit the highest cesses in which these proteins are involved independently of the activity activity in down-regulating phospho-Akt, followed by suberoylani- of HDAC inhibitors in transcriptional activation. For example, HDAC3 lide hydroxamic acid, whereas MS-275 shows only a marginal effect regulates NFB signaling in the nucleus by deacetylation the Rel-A sub- at 5 M. This differential potency parallels the respective activities unit (7), and HDAC6 modulates microtubule acetylation and chemo- in inducing tubulin acetylation, a non-histone substrate for tactic cell motility by acting as a tubulin deacetylase (8–12). Moreover, HDAC6. Evidence indicates that this Akt dephosphorylation is not Class I HDACs function as a STAT3 deacetylase (13), and the mamma- mediated through deactivation of upstream kinases or activation of lian Class III HDACs SirT1 and SirT2 can deacetylate p53 and tubulin, downstream phosphatases. However, the effect of TSA on phospho- respectively (14, 15). Second, HDACs 1 and 6 have been shown to form Akt can be rescued by PP1 inhibition but not that of protein phos- complexes with protein phosphatase 1 (PP1) (16, 17), of which the com- phatase 2A. Immunochemical analyses reveal that TSA blocks spe- bined deacetylase/phosphatase activities underlie the ability of HDAC1 cific interactions of PP1 with HDACs 1 and 6, resulting in increased to modulate transcriptional activity of cAMP-responsive element-bind- PP1-Akt association. Moreover, we used isozyme-specific small ing protein (16) and that of HDAC6 to regulate microtubule dynamics interfering RNAs to confirm the role of HDACs 1 and 6 as key medi- (17). Also noteworthy is that the binding of HDACs to PP1 was highly ators in facilitating Akt dephosphorylation. The selective action of specific because no association between HDACs and protein phospha- HDAC inhibitors on HDAC-PP1 complexes represents the first tase 2A (PP2A) was noted (17). example of modulating specific PP1 interactions by small molecule Moreover, data from this and other laboratories (18–20) show that agents. From a clinical perspective, identification of this PP1-facil- HDAC inhibitors could facilitate the dephosphorylation of Akt and itated dephosphorylation mechanism underscores the potential use other signaling kinases, although the causative mechanism remains of HDAC inhibitors in lowering the apoptosis threshold for other undefined. In this report, we used TSA to demonstrate that HDAC therapeutic agents through Akt down-regulation. inhibitors facilitate dephosphorylation of Akt by altering the dynamics of HDAC-PP1 complexes. We provide evidence that HDAC inhibitors selectively target HDACs 1 and 6 to disrupt the respective HDAC-PP1 Histone deacetylase (HDAC) is recognized as one of the promising complexes, resulting in increased association of PP1 with Akt. This targets for cancer treatment because many HDAC inhibitors have PP1-facilitated kinase dephosphorylation underscores the diverse func- entered clinical trials in both solid and liquid tumors (1–3). Neverthe- tions of HDAC inhibitors in mediating antineoplastic activities at dif- less, the mechanisms underlying the antiproliferative effects of HDAC ferent cellular levels. * This work was supported by National Institutes of Health Grants CA-94829 and EXPERIMENTAL PROCEDURES CA-112250. The costs of publication of this article were defrayed in part by the pay- Cell Culture—U87MG human glioblastoma cells were kindly pro- ment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. vided by Dr. Ing-Ming Chiu (The Ohio State University, Columbus, To whom correspondence should be addressed: College of Pharmacy, 336 Parks Hall, OH), and PC-3 prostate cancer cells were purchased from the American The Ohio State University, 500 12th Ave., Columbus, OH 43210. Tel.: 614-688-4008; Fax: 614-688-8556; E-mail: [email protected]. Type Culture Collection (Manassas, VA). Because both U87MG and The abbreviations used are: HDAC, histone deacetylase; PP1, protein phosphatase 1; PC-3 cells are PTEN-null, they exhibit constitutively active Akt. These PP2A, protein phosphatase 2A; TSA, trichostatin A; SAHA, suberoylanilide hydroxamic cancer cells were cultured in 10% fetal bovine serum (FBS)-supple- acid; HDAC42, N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide; PI3K, phosphatidylinositol 3-kinase; PIP , L- -phosphatidyl-D-myo-inositol 3,4,5-trisphos- mented RPMI 1640 medium containing 100 units/ml penicillin and 100 phate; PDK-1, phosphoinositide-dependent kinase-1; ERK, extracellular signal-re- g/ml streptomycin (Invitrogen). lated kinase; siRNA, small interfering RNA; FBS, fetal bovine serum; p-, phospho-; JNK, c-Jun N-terminal kinase; TBS, Tris-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)- Reagents—Trichostatin A (TSA), calyculin A, and okadaic acid were 2,5-diphenyl-2H-tetrazolium bromide; PBS, phosphate-buffered saline; MOPS, purchased from Sigma-Aldrich, and tautomycin was obtained from Cal- 4-morpholinepropanesulfonic acid; MEK, mitogen-activated protein kinase/ERK kinase; Hsp, heat shock protein; CTMP, C-terminal modulator protein. biochem (La Jolla, CA). The HDAC inhibitors suberoylanilide hydrox- NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38879 This is an Open Access article under the CC BY license. Cellular Effect of Histone Deacetylase Inhibitors on Akt amic acid (SAHA) and N-hydroxy-4-(3-methyl-2-phenyl-butyrylami- Flow Cytometry/Apoptosis Assays—After drug treatment as described no)benzamide (HDAC42) were synthesized in our laboratory. HDAC42 above, floating cells were collected, and adherent cells were harvested by belongs to a novel class of hydroxamate-tethered phenylbutyrate deriv- scraping. The combined cells were fixed in ice-cold 70% ethanol and stor- age at20 °C. The cells were centrifuged, washed with PBS, and incubated atives with nanomolar potency in HDAC inhibition (21, 22) and is cur- in 0.2 M phosphate citrate buffer, pH 7.8 at 37 °C to extract low molecular rently undergoing preclinical testing under the Rapid Access to Inter- weight DNA. The samples were stained with propidium iodide solution (10 vention Development Program at the National Cancer Institute. Mouse mg/ml) in the presence of RNase A (100 mg/ml) for 30 min at room tem- antibodies against -tubulin and acetylated -tubulin were from Sigma- 473 308 perature. Cell cycle phase distributions were determined on a FACScort Aldrich. Rabbit antibodies against Akt, Ser -Akt, Thr -Akt, PDK-1, flow cytometer and analyzed by the ModFitLT V3.0 program. p-ERK1/2, p-p38, p-JNK, and various HDAC isozymes were purchased Affinity Purification of HDAC-PP1 Complexes—U87MG cells were from Cell Signaling Technology Inc. (Beverly, MA). Rabbit antibodies treated with TSA at the indicated concentrations in 10% FBS-supple- against PP1, PP2A, and nucleolin, and mouse antibodies against p21 mented RPMI 1640 medium for 48 h. Control cells received the vehicle were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit anti- treatment of 0.1% (v/v) dimethyl sulfoxide. The cells were washed by bodies against p85, p110, histone H3, and acetyl-histone H3 were from phosphate-buffered saline and lysed by the aforementioned Nonidet Upstate Biotechnology Inc. (Lake Placid, NY). Mouse monoclonal anti- P-40 isotonic lysis buffer. Cell extract was incubated with microcystin- actin was from ICN Biomedicals Inc. (Costa Mesa, CA). Rabbit anti- LR-Sepharose (Upstate Biotechnology, Inc.) at 4 °C overnight on a rota- human C-terminal modulator protein (CTMP) antibodies and rabbit tor. After a brief centrifugation, Sepharose beads were collected and anti-TRB3 antibodies were from Alpha Diagnostic International (San washed with the lysis buffer four times. The bound proteins were eluted Antonio, TX) and Oncogene Research Products (San Diego, CA), by 50 l of SDS sample buffer and subjected to SDS-PAGE followed by respectively. Goat anti-rabbit IgG-horseradish peroxidase conjugates immunoblotting with appropriate antibodies. and rabbit anti-mouse IgG horseradish peroxidase conjugates were Co-immunoprecipitation of PP1-Akt Complexes—U87MG cells were from Jackson ImmunoResearch Laboratories (West Grove, PA). The treated with various concentrations of TSA for 48 h and lysed by the siRNA transfection reagent, siRNA transfection medium, and siRNAs aforementioned Nonidet P-40 isotonic lysis buffer with a mixture of against HDAC1 and HDAC6 (catalog numbers sc-29343 and sc-35544, protease inhibitors. After centrifugation at 13000  g for 15 min, the respectively) and a control, scrambled siRNA (catalog number supernatants were collected and incubated with protein A-Sepharose sc-37007) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). beads (Sigma) for 15 min to eliminate nonspecific binding. The mixture Immunoblotting—U87MG and PC-3 cells treated with various con- was centrifuged at 1000  g for 5 min, and the supernatants were centrations of HDAC inhibitors in 10% FBS supplemented RPMI 1640 exposed to Akt or PP1 antibodies in the presence of protein A-Sepha- medium for 48 h were collected and lysed by Nonidet P-40 isotonic lysis rose beads at 4 °C for 2 h. After a brief centrifugation, protein A-Sepha- buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1% (v/v) Nonidet P-40, 1 rose beads were collected, washed with the aforementioned lysis buffer mM EDTA, 50 mM NaF, 40 mM -glycerophosphate, and 1 g/ml each four times, suspended in 2 SDS sample buffer, and subjected to West- of aprotinin, pepstatin, and leupeptin). Protein concentrations of the ern blot analysis with antibodies against PP1 or Akt. lysates were determined by using a Bradford protein assay kit (Bio-Rad). Subcellular Fractionation—The nuclear and cytosolic fractions of Equivalent amounts of proteins from each lysate were resolved by SDS- U87MG cells were prepared by using a nuclear/cytosol fraction kit PAGE and then transferred onto Immobilon-nitrocellulose membranes (MBL Co., Watertown, MA) according to the manufacturer’s instruc- (Millipore, Bellerica, MA) in a semidry transfer cell. The transblotted tions. In brief, the cells were cultured to 50% confluency in T-75 flasks, membrane was washed twice with Tris-buffered saline (TBS) contain- treated with TSA for 48 h, and collected by centrifugation. The pelleted ing 0.1% Tween 20 (TBST). After blocking with TBST containing 5% cells were resuspended in 0.2 ml of cytosol extraction buffer A mix nonfat milk for 40 min, the membrane was incubated with the appro- containing dithiothreitol and a mixture of protease inhibitors and were priate primary antibody in TBST containing 1% nonfat milk at 4 °C mixed vigorously on a vortex mixer. The suspension was then incubated overnight. All of the primary antibodies were diluted 1: 2000 in 1% on ice for 10 min, mixed with 11 l of cytosol extraction buffer B, and nonfat milk-containing TBST. After treatment with the primary anti- incubated on ice for 1 min. The lysates were centrifuged at 16,000  g body, the membrane was washed two times with TBST for a total of 20 for 5 min. The supernatant, representing the cytoplasmic fraction, was min, followed by goat anti-rabbit or anti-mouse IgG-horseradish per- transferred to a prechilled tube, and the pellet was resuspended in 100l oxidase conjugates (diluted 1:3000) for 1 h at room temperature and of nuclear extraction buffer mix. The suspension was mixed vigorously washed three times with TBST for a total of 1 h. The immunoblots were on a vortex mixer for 15 s, incubated on ice for 40 min, and centrifuged visualized by ECL chemiluminescence (Amersham Biosciences). at 16,000 g for 10 min to collect the nuclear extract in the supernatant Cell Viability Assay—The effect of the HDAC inhibitors on cell via- fraction. bility was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- Ser/Thr Phosphatase Activity—The Ser/Thr phosphatase activity 2H-tetrazolium bromide (MTT) assay in 6–12 replicates. U87MG and was determined by using a nonradioactive, malachite green-based Ser/ PC-3 cells were seeded and incubated in 96-well, flat-bottomed plates in Thr phosphatase assay kit (Upstate Biotechnology, Inc.) according to 10% FBS-supplemented RPMI 1640 medium 24 h before drug treat- the manufacturer’s instructions. In brief, 5 g of U87MG cell extracts in ment. The cells were exposed to individual HDAC inhibitors at the the aforementioned lysis buffer were incubated with 175 M of the indicated concentrations in 10% FBS-supplemented RPMI 1640 phosphopeptide substrate (K-R-pT-I-R-R) in the phosphatase buffer medium at 37 °C in 5% CO for 48 h. The medium was removed and (20 mM MOPS, pH7.5, 60 mM 2-mercaptoethanol, 0.1 M NaCl, and 0.1 replaced by 200 l of 0.5 mg/ml of MTT in RPMI 1640 medium, and the mg/ml serum albumin) with a total volume of 25 l. The reaction was cells were incubated in the CO incubator at 37 °C for 2 h. Supernatants incubated in a 96-well plate at room temperature for 10 min, terminated were removed from the wells, and the reduced MTT dye was solubilized by adding the malachite green solution, and allowed to stand for 15 min with 200 l/well Me SO. Absorbance was determined on a plate reader to permit color development. Absorption at 650 nm was measured in a at 570 nm. microplate reader, and the serine/threonine phosphatase activity was 38880 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt FIGURE 1. HDAC inhibitor-mediated Akt phos- phorylation in U87MG and PC-3 cells. A, dose- dependent effects of four different HDAC inhibi- tors, including HDAC42, SAHA, MS-275, and TSA on histone H3 acetylation, p21 expression, tubulin acetylation, and phosphorylation state of Akt at 308 473 Thr and Ser . All four inhibitors mediated his- WAF/CIP1 tone hyperacetylation and p21 up-regula- tion but exhibited differential effects on tubulin acetylation and Akt dephosphorylation. U87MG and PC-3 cells were treated with the indicated doses of individual inhibitors for 48 h, and the cell lysates were immunoblotted. B, time-dependent effects of 0.25 and 0.5 M TSA on p-Ser -Akt lev- els vis a` vis p21 expression and histone H3 acetyla- tion in U87 MG cells. The cells were treated with 0.25 or 0.5 M TSA for the indicated time intervals, and the cell lysates were immunoblotted. DMSO, dimethyl sulfoxide. calculated using a standard curve based on free phosphate generated by well followed by incubation for1hinthe dark and three more washes a standard solution. All of the experiments were performed in triplicate. with 300 l of TBS with Tween 20 (0.05% v/v). 100 l of tetramethyl Immunoprecipitated Phosphatidylinositol 3-Kinase (PI3K) Kinase benzidine solution was added to each well and incubated for 20–30 Assay—PI3K activity was determined by the use of a PI3K enzyme- min to allow color development. The reaction was stopped by the addi- linked immunosorbent assay kit from Echelon Biosciences Inc. (Salt tion of 100 l of stop solution (0.5 M H SO ) to each well. Absorbance at 2 4 Lake City, UT) according to the manufacturer’s instruction. U87MG 450 nm was read, and the PI3K activity in each sample was calculated cells were cultured to 50% confluency in 10-cm culture dishes, and the using a standard curve generated by using a PIP standard solution. All medium was removed after TSA treatment. The drug-treated cells were of the experiments were performed in triplicate. washed with 10 ml/dish of ice-cold buffer A (137 mM NaCl, 10 mM Autoradiographic Determination of Phosphoinositide Formation— Tris-HCl, pH 7.4, 1 mM CaCl ,1mM MgCl , and 0.1 mM sodium U87MG cells, cultured in T25 flasks, were labeled with 1 mCi/ml of 2 2 orthovanadate) three times, incubated with 1 ml of ice-cold lysis buffer [ P]orthophosphate (HCl-free; PerkinElmer Life Sciences) in Dulbec- (buffer A plus 1% Nonidet P-40 and 1 mM phenylmethylsulfonyl fluo- co’s modified Eagle’s phosphate-free and serum-free medium (Invitro- ride) on ice for 20 min, scraped from the dish, transferred to microcen- gen) for 4 h, washed three times with buffer A (30 mM Hepes, pH 7.2, 110 trifuge tubes, and centrifuged. The supernatant was collected, and the mM NaCl, 10 mM KCl, 1 mM MgCl , and 10 mM glucose), and treated protein concentrations were determined by the Bradford assay (Bio- with various concentrations of TSA in 10% FBS-supplemented RMPI Rad). Equal amounts of proteins were treated with 5 l of anti-PI3K 1640 medium for 48 h. The cells were extracted with 3 ml of chloro- antibodies (Upstate Biotechnology, Inc.) at 4 °C for 1 h, to which was form/methanol (1:2, v/v), followed by 4 ml of chloroform, 2.4 M HCl added 60 l of a 50% slurry of protein A-agarose beads in PBS, followed (1:1, v/v), and 1 ml of chloroform four times. The combined organic by incubation at 4 °C for 1 h with mixing. The immunocomplex was phase was dried under a stream of nitrogen and resuspended in 90 lof collected by brief centrifugation, washed with lysis buffer three times, chloroform for TLC analysis by using a 20  20-cm silica gel 60 TLC and incubated for2hat room temperature with 10 lof10 ML-- plate (EM Science) impregnated with 1% potassium oxalate in 50% eth- phosphatidyl-D-myo-inositol 4,5-bisphosphate, 5 lof10 reaction anol. The TLC plate was developed in chloroform/acetone/methanol/ buffer (40 mM MgCl , 200 mM Tris, pH 7.4, 100 mM NaCl, 250 M ATP), acetic acid/water (80:30:26:24:14, v/v/v/v/v) (23). Radioactive spots and 35 l of distilled water. The reaction was terminated by the addition were detected by autoradiography using Kodak X-Omat film, and total of 2.5 l of 100 mM EDTA. After brief centrifugation, 50 l of each PIP was quantified by both densitometry and an AMBIS B scanning reaction mixture was transferred to a 96-well plate, and 50 l of diluted system (San Diego, CA), both of which showed comparable results. L--phosphatidyl-D-myo-inositol 3,4,5-trisphosphate (PIP ) detector Kinase Assay for Phosphoinositide-dependent Kinase-1 (PDK-1)— was added to each well and incubated at room temperature for 1 h. 100 The PDK-1 kinase activity was performed using a PDK-1 kinase assay kit l of reaction mixture from each well was transferred to the corre- (Upstate Biotechnology, Inc.) according to a described procedure (24). sponding well in the detection plate and incubated at room temperature This cell-free assay is based on the ability of recombinant PDK-1 to for1hinthe dark. After three washes with 300 l of TBS with Tween 20 activate its downstream kinase serum- and glucocorticoid-regulated (0.05% v/v), 100 l of the secondary detection reagent was added to each kinase, which in turn phosphorylates the Akt/serum- and glucocorti- NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38881 Cellular Effect of Histone Deacetylase Inhibitors on Akt coid-regulated kinase-specific peptide substrate RPRAATF with 32 32 [- P]ATP. The P-phosphorylated peptide was then separated from residual [- P]ATP by P81 phosphocellulose paper and quantitated by a scintillation counter after three washes with 0.75% phosphoric acid. Isozyme-specific Knockdown of HDACs with siRNA—Isozyme-spe- cific siRNAs were used to attenuate the expression of HDACs 1 and 6 in U87MG cells using reagents obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Transfection of U87MG cells with these siRNA was carried out according to the vendor’s instructions. In brief, the cells were cultured to 40–50% confluency in 10% FBS-supplemented antibi- otic-free RPMI 1640 medium. The HDAC1 and HDAC6 siRNAs were transfected to the cells by the siRNA transfection reagent (Santa Cruz, CA) to the final concentration of 200 nM. A scramble siRNA was used in parallel experiments as a control. The transfected cells were incubated at 37 °C, and the extents of siRNA-mediated down-regulation of HDAC1 and HDAC6 expression were monitored by immunoblotting analysis at different time intervals. It was found that 30-h exposure to siRNA caused more than 80% repression of HDAC expression, and thus it was used through this study. Immunocytochemical Analysis of TSA-mediated Co-localization of PP1 and Akt—U87MG cells were treated with 0.5 M TSA in 10% FBS- supplemented RPMI 1640 medium for different time intervals, washed with Dulbecco’s PBS, fixed with 4% paraformaldehyde for 30 min at room temperature, and then washed with PBS. For double staining of phospho-Akt and PP1, the cells were permeabilized with 0.1% Triton X-100 in 1% FBS-containing PBS, treated with rabbit anti-phospho- Ser -Akt (1:500 dilution) at 4 °C for 24 h, and washed with PBS. Sub- sequently, the cells were treated with mouse anti-PP1 (1:100 dilution) for8hat room temperature and washed with PBS. For fluorescent microscopy, Alexa Fluor 488 goat anti-mouse and anti-rabbit IgG (Molecular Probes, Inc.) were used for conjugating PP1 and phospho- Akt, respectively. The nuclear counterstaining was performed using a 4,6-diamino-2-phenylindole-containing mounting medium (Vector, CA) prior to examination. Images of immunocytochemically labeled samples were observed using a Zeiss confocal microscope (LSM510) with an argon laser and a helium-neon laser and appropriate filters (excitation wavelength, 488 nm for PP1, 633 nm for p-Akt, and 543 nm for 4,6-diamino-2-phenylindole). RESULTS FIGURE 2. A, dose-dependent effects of TSA and SAHA on the phosphorylation state of 308 473 Thr - and Ser -Akt vis a` vis ERK1/2, p38, and JNK MAP kinases in U87MG cells. The cells Differential Effects of HDAC Inhibitors on Akt Dephosphorylation— were treated with the indicated concentrations of TSA or SAHA for 48 h, and the cell To shed light onto the causative relationship between HDAC inhibition lysates were immunoblotted. B, dose dependence of the growth inhibitory effects of HDAC42, SAHA, MS-275, and TSA in U87MG and PC-3 cells. The cells were treated with and Akt deactivation, we assessed the effects of four different HDAC individual HDAC inhibitors at the indicated concentrations in 10% FBS-supplemented inhibitors, including HDAC42, SAHA, MS-275, and TSA, on various medium for 48 h, and the cell viability was determined by the MTT assay. Points, means; HDAC-related biomarkers (histone H3 acetylation, p21 expression, and bars, S.D. (n  6 –12). tubulin acetylation) vis a` vis Akt phosphorylation state in U87MG glio- blastoma and PC-3 prostate cancer cells. Of the four HDAC inhibitors 308 473 examination of Akt phosphorylation at both Thr and Ser indicates examined, HDAC42 belongs to a novel class of phenylbutyrate-based that HDAC42 and TSA at submicromolar concentrations were able to HDAC inhibitors (21) and has an IC of 30 nM in inhibiting HDAC substantially reduce phospho-Akt levels. A similar effect on phospho- activity in nuclear extracts. The reported IC values for TSA, SAHA, Akt was not achieved with SAHA until the concentration reached 5 M. and MS-275 are 5–15 nM, 120 nM, and 4.8 M, respectively (2). In contrast, the repressing effect of MS-275 on Akt phosphorylation, Western blot analysis shows that exposure of U87MG and PC-3 cells even at 5 M, was marginal. It is noteworthy that there existed an inverse to these inhibitors led to substantial increases in histone H3 acetylation WAF/CIP1 relationship between the levels of acetylated -tubulin and phospho- and p21 expression (Fig. 1A). However, these four inhibitors Akt, providing a potential link between HDAC6 inhibition and Akt behaved differently with regard to -tubulin acetylation, indicating dif- dephosphorylation. This premise was further supported by the differ- ferences in the respective activities in inhibiting the -tubulin deacety- ential effect of selective siRNA-mediated knockdown of individual lase HDAC6 (8). Although HDAC42 and TSA produced robust hyper- acetylation of -tubulin at submicromolar concentrations, SAHA was HDAC isozymes on phospho-Akt levels (see below). Moreover, the time effective at low micromolar concentrations, and MS-275 was totally course of TSA-mediated Akt dephosphorylation lagged behind that of ineffective, even at 5 M, in inhibiting tubulin deacetylation. Moreover, p21 overexpression and H3 hyperacetylation by at least 12 h (Fig. 1B), 38882 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt TABLE ONE Cell cycle phase distribution of U87MG and PC-3 cells treated with individual HDAC inhibitors at the indicated concentrations in 10% FBS- supplemented medium for 48 h Control cells received Me SO vehicle. Each tabulated percentage represents the average of two independent experiments. Cells in cell cycle phases U87MG PC-3 Sub-G /G G /G SG /M Sub-G /G G /G SG /M 0 1 0 1 2 0 1 0 1 2 Me SO 1.3 69.2 14.5 14.9 0 51.2 22.4 26.4 HDAC-42 0.5 M 0.2 64.8 5.7 29.3 0.2 43.1 22.8 33.9 2.5 M 0.5 71.2 2.5 25.8 0.3 39.6 2.0 58.1 5 M 0.2 69.5 2.5 28.3 4.4 31.2 1.2 62.8 SAHA 0.5 M 0.9 65.9 15.1 17.1 0 56.0 14.1 29.9 2.5 M 0.6 55.3 13.6 30.5 0 44.4 19.4 36.0 5 M 0.7 58.5 10.0 30.8 0 47.7 23.2 29.1 MS-275 0.5 M 0.8 73.3 11.3 14.6 0.1 61.8 10.4 27.7 2.5 M 0.4 81.3 4.2 14.1 0 69.4 2.0 28.6 5 M 0 80.4 4.7 14.9 0 64.2 1.3 34.5 TSA 0.1 M 1.0 64.1 10.7 23.1 0 45.2 7.7 47.1 0.25 M 2.2 70.8 1.8 25.2 0 37.5 2.2 60.3 0.5 M 0.7 64.1 3.2 32.0 6.6 36.6 0 58.2 suggesting that this Akt effect might not be related to these two decreases in the G /G and, in particular, S fractions (TABLE ONE). 0 1 biomarkers. The sub-G /G fraction, an indication of apoptosis, was not observed 0 1 Selective Dephosphorylation of Signaling Kinases—Pursuant to the until the concentrations of HDAC42 and TSA reached 5 and 0.5 M, above finding, a question emerged with regard to the specificity of respectively. Meanwhile, the cell cycle-arresting behavior of SAHA in HDAC inhibitor-mediated kinase dephosphorylation. Accordingly, we these two cell lines differed. It caused dose-dependent increases in the examined the impact of two representative HDAC inhibitors, TSA and G /M population in U87MG cells, but the effect on cell cycle distribu- SAHA, on the phosphorylation status of ERKs, p38, and JNK MAP tion in PC-3 cells changed from G arrest to G /M arrest as the concen- 1 2 kinases versus Akt in U87MG cells (Fig. 2A). As noted, both TSA and tration increased. In contrast, MS-275 induced dose-dependent G cell SAHA caused a modest decrease in phospho-ERK1/2 levels despite cycle arrest in both cell lines. their respective activities in Akt dephosphorylation, whereas those of TSA-mediated Akt Dephosphorylation Is Not Caused by Changes in phospho-p38 and phospho-JNK remained unaffected. These findings the Expression Level of Proteins Involved in Phospho-Akt Regulation— indicate that there existed a certain degree of specificity in HDAC inhib- Mechanistically, this Akt dephosphorylation might be mediated itor-mediated kinase dephosphorylation, suggesting the involvement of through the deactivation of upstream kinases or the activation of down- a unique signaling mechanism. stream phosphatase. To discern the role of transcriptional activation in Differential Effects of HDAC Inhibitors on Cell Proliferation and Cell this drug action, we assessed the expression levels of a series of signaling Cycle—Together, these findings suggest that these four HDAC inhibi- proteins related to the regulation of Akt signaling pathways in TSA- tors exhibited distinct profiles regarding pharmacological targets, treated U87MG cells, which included the p85 regulatory and p110 cat- which might underlie differences in their antitumor activities. For alytic subunits of PI3K, PDK-1, Akt, the negative Akt modulators example, although these agents were able to up-regulate p21 expression CTMP (25) and TRB3 (26), PP1, and PP2A. As shown in Fig. 3A, TSA and histone acetylation, two hallmark features in association with intra- exposure did not alter the expression level of any of these signaling cellular HDAC inhibition, at submicromolar concentrations, their abil- proteins, excluding the involvement of transcriptional activation in ity to suppress cell proliferation varied by almost an order of magnitude altering the status of Akt phosphorylation. (Fig. 2B). Although TSA and HDAC42 were effective in suppressing Furthermore, three lines of evidence argued against the possibility proliferation in U87MG and PC-3 cells at submicromolar concentra- that TSA-mediated Akt deactivation was caused by a decrease in PI3K tions, it would require at least 2.5–5 M for SAHA and MS-275 to attain or PDK-1 kinase activity (Fig. 3B). First, immunoprecipitated PI3K a similar antiproliferative effect. This differential potency paralleled the kinase activity in U87MG cells treated with different doses of TSA respective activities in causing Akt dephosphorylation, suggesting a remained the same as that of the Me SO control (Fig. 3B, left panel, open putative link between Akt down-regulation and the antitumor activities 2 bars). Second, TSA at different doses exhibited no appreciable inhibi- of HDAC42, TSA, and, to a lesser extent, SAHA. Moreover, flow cyto- tory effects on the kinase activity of recombinant PDK-1 (Fig. 3B, left metric analyses indicate that the effect of these HDAC inhibitors on panel, gray bars). Third, autoradiographic analysis of P-labeled phos- growth inhibition was largely attributable to cell cycle arrest in lieu of pholipids demonstrated that the levels of PIP , a PI3K lipid product, and apoptosis. Analyses of cell cycle distribution in drug-treated U87MG and PC-3 cells shows that HDAC42 and TSA induced dose-dependent other inositol lipids in U87MG cells were unaffected by 0.5 M TSA accumulation of cells in the G /M phase, accompanied by comparative treatment (Fig. 3B, right panel). NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38883 Cellular Effect of Histone Deacetylase Inhibitors on Akt Inhibition of PP1 Prevents TSA-mediated Akt Dephosphorylation—In at low doses (100 nM) is selective for PP2A (33, 34), and calyculin A the literature, a number of small molecules such as C -ceramide, 4-hy- lacks selectivity between PP1 and PP2A. Fig. 4 demonstrates that droxynonenal, FTY720, and N-ethylmaleimide have been reported to although none of these inhibitors affected the ability of TSA to inhibit facilitate Akt dephosphorylation by activating PP2A or PP2A-like phos- histone deacetylation, their differential effects on the TSA-mediated phatase (27–31). Consequently, we investigated a potential link between dephosphorylation of Akt was noteworthy. As shown, calyculin A (50 TSA-mediated Akt down-regulation and protein phosphatase activa- nM) and tautomycin (5 M) could completely abrogate the effect of TSA tion by examining the effect of tautomycin, calyculin A, and okadaic on phospho-Akt, whereas okadaic acid lacked appreciable protective acid on phospho-Akt levels in TSA-treated U87MG cells. These three activity against the dephosphorylation. Because PP1 represents a com- compounds exhibit distinct specificity in protein phosphatase inhibi- mon target for calyculin A and tautomycin, this finding suggests the tion, i.e. tautomycin is a highly specific PP1 inhibitor (32), okadaic acid involvement of PP1 in TSA-facilitated kinase dephosphorylation. TSA Disrupts HDAC-PP1 Complexes, Resulting in Increased PP1-Akt Associations—The link between HDAC inhibition and PP1-mediated kinase dephosphorylation could be attributed to two plausible mecha- nisms. First, TSA treatment might lead to increased PP1 activity. Sec- ond, in light of the reported HDAC-PP1 complex formation (16, 17), HDAC inhibitors might cause disruption of these complexes, which would free PP1 to interact with Akt. Of these two mechanisms, the former was refuted by lack of appreciable increases in either the PP1 expression level (Fig. 3A) or the overall Ser/Thr protein phosphatase activity after TSA exposure (data not shown). To evaluate the second possibility, we investigated the expression profile of HDAC isozymes in U87MG cells. Among the seven different isozymes examined, U87MG cells expressed HDACs 1, 2, 3, 5, and 6, but the expression of HDACs 4 and 7 was undetectable. In addition, TSA treatment did not alter the expression level of any of these HDACs (data not shown). To assess the impact of TSA on the dynamics of HDAC-PP1 complexes, we exposed the lysates of drug-treated U87MG cells to microcystin affinity beads to purify PP1-associated complexes (16). Western blot analysis of the affin- ity-purified PP1 complexes indicates that HDACs 1, 3, and 6 could be pulled-down by the affinity beads. Moreover, the levels of affinity bead- bound HDACs 1 and 6 decreased in response to TSA in a dose-depend- ent manner (Fig. 5A). The level of PP1-associated HDAC3, however, remained unaltered by the TSA treatment. Based on the report that the PP1-binding domain of HDAC6 encom- passed the catalytic motif (17), binding of the HDAC inhibitor to the catalytic domain might hinder the binding of PP1 to HDAC6. Presum- ably, HDAC inhibitors could also sequester HDAC1, but not HDAC3, from PP1 association through the same mode of mechanism. In con- FIGURE 3. A, TSA-mediated Akt dephosphorylation is not due to alterations in the expres- trast, the PP1 inhibitor tautomycin did not cause disturbance of the sion level of proteins that are involved in the regulation of Akt phosphorylation. U87MG cells were treated with the indicated doses of TSA for 48 h, and the cell lysates were HDAC-PP1 complex (data not shown), suggesting no overlap between immunoblotted. B, left panel, TSA does not affect the kinase activity of immunoprecipi- the HDAC-binding domain and the catalytic site of PP1. Also notewor- tated Akt in U87MG cells treated with the indicated dose of TSA, nor does it affect the activity of recombinant PDK-1. Right panel, TSA (0.5 M) does not affect PIP level in U87 thy is that TSA-mediated dissociation of HDAC-PP1 complexes did not MG cells. [ P]Orthophosphate-labeled U87MG cells were treated with TSA (500 nM)or significantly change the relative distribution of PP1 or PP2A in the Me SO vehicle for 48 h. The extracted phospholipids were analyzed by TLC separation, followed by autoradiographic analysis as described under “Experimental Procedures.” nucleus or cytoplasm (Fig. 5B). FIGURE 4. The protein phosphatase inhibitors calyculin A (CA), tautomycin (TM), and okadaic acid (OA) exhibit differential effects on TSA- mediated dephosphorylation of Akt and ERKs. U87MG cells were exposed to TSA and individual protein phosphatase inhibitors at the indicated concentrations. After 48 h of incubation, the cell lysates were immunoblotted. DMSO, dimethyl sulfoxide. 38884 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 46 •NOVEMBER 18, 2005 Cellular Effect of Histone Deacetylase Inhibitors on Akt FIGURE 5. A, TSA selectively disrupts the associa- tion of PP1 with HDACs 1 and 6. U87MG cells were treated with TSA at the indicated concentrations for 48 h. The cell lysates were exposed to microcys- tin affinity beads to purify PP1-associated com- plexes. The affinity-purified complexes were probed with individual HDAC isozyme-specific antibodies by Western blot analysis (WB). B, TSA does not change the relative abundance of PP1 or PP2A in the nucleus or cytoplasm in drug-treated U87MG cells. -Tubulin and nucleolin are used as internal references for the cytoplasmic and nuclear fractions, respectively. In addition, two lines of evidence indicate that the TSA-mediated disruption of HDAC-PP1 complexes was accompanied by a dose-de- pendent increase in PP1-Akt associations. First, immunoprecipitation of PP1 in the lysates of TSA-treated cells followed by Western blotting of Akt, or vice versa, demonstrates a positive correlation between TSA doses and amounts of Akt co-immunoprecipitated with PP1 (Fig. 6A). Second, immunocytochemical examinations show that exposure of U87MG cells to TSA for 4 h led to co-localization of PP1 and phospho- Akt, which, however, was not noted with cells receiving Me SO vehicle (Fig. 6B). The physical interaction between PP1 and Akt led to partial and complete dephosphorylation at 8 and 24 h post-treatment, respectively. Validation of the Involvement of HDACs 1 and 6 in Akt Dephos- phorylation—Together, the above findings suggest that TSA caused PP1-mediated Akt dephosphorylation by sequestering HDACs 1 and 6. To validate the role of these two isozymes in Akt deactivation, we used isozyme-specific siRNAs to selectively knock down the expression of HDACs 1 and 6 and that of a scrambled siRNA as a negative control (Fig. 7A). As shown, repressed expression of HDACs 1 and 6 both led to decreased Akt phosphorylation, whereas control siRNA transfection had no appreciable effect on phospho-Akt levels (Fig. 7B). DISCUSSION The present study demonstrates a novel histone acetylation-indepen- dent mechanism by which HDAC inhibitors mediate the dephospho- rylation of Akt through the disruption of HDAC-PP1 complexes. Together with the previous findings of the functional roles of HDAC- PP1 pairs in the modulation of cAMP-responsive element-binding pro- tein phosphorylation (16) and tubulin acetylation (17), this histone- independent mechanism provides a potential basis to account for the FIGURE 6. TSA treatment leads to increased PP1-Akt associations. A, immunoprecipi- antineoplastic activities of these agents in growth inhibition and apo- tation (IP) of PP1 in lysates of TSA-treated cells, followed by immunoblotting (IB)ofAkt ptosis induction. (left panel) or vice versa (right panel). U87MG cells were treated with TSA at the indicated Of the four inhibitors examined, HDAC42 and TSA were most effec- concentrations, and the cell lysates were exposed to anti-Akt or anti-PP1 in the presence of protein A-Sepharose beads. The immunocomplex was immunoblotted with anti-PP1 tive in facilitating Akt dephosphorylation, followed by SAHA, whereas or anti-Akt. B, immunocytochemical examinations of PP1 and phospho-Akt in U87MG MS-275 exhibited a marginal effect at therapeutically attainable con- cells treated with Me SO vehicle or 500 nM TSA for the indicated times. centrations (5 M). In light of the pivotal role of Akt in cell prolifera- tion, this differential activity in Akt down-regulation might be attrib- ing suggests a role for HDAC6 inhibition in Akt dephosphorylation. uted to differences in the antitumor activities among these four HDAC Consequently, MS-275 was ineffective in causing Akt deactivation as a inhibitors. TSA and HDAC42 was potent in suppressing the prolifera- result of its inability to inhibit HDAC6. tion of U87MG and PC-3 cells, in part, because of their ability to down- The present study further demonstrated a mechanistic link between regulate Akt signaling. It is noteworthy that the activity of these agents PP1 and TSA-mediated Akt dephosphorylation. Our immunochemical to suppress Akt phosphorylation paralleled the respective potency in study showed that TSA blocks specific associations of PP1 with HDACs inducing -tubulin acetylation, a biomarker for HDAC6 (8). This find- 1 and 6, suggesting the involvement of both isozymes in regulating the NOVEMBER 18, 2005• VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38885 Cellular Effect of Histone Deacetylase Inhibitors on Akt PP1-protein interactions by small molecule agents. From a clinical per- spective, identification of this PP1-facilitated dephosphorylation mech- anism underlies the potential use of HDAC inhibitors in lowering the apoptosis threshold for other therapeutic agents through the down- regulation of Akt and ERK signaling. This therapeutic strategy is illus- trated by the ability of HDAC inhibitors to sensitize cancer cells to the apoptotic effects of the Bcl-Abl kinase inhibitor imatinib (42), the Her-2 antibody trastuzumab (19), the receptor tyrosine kinase FLT-3 inhibitor PKC412 (20), the purine analogue fludaribine (43), and the Hsp90 antagonist 17-allylamino-demethoxy geldanamycin (44, 45). In conclusion, the ability of HDAC inhibitors to deactivate Akt through the reorganization of PP1 complexes underlines the complexity of the pharmacological functions of these agents. In light of the clinical application of HDAC inhibitors, a better understanding of this novel histone-independent mechanism will allow the design of more effective strategies to optimize the use of the agents in cancer treatment and/or FIGURE 7. Validation of the involvement of HDACs 1 or 6 in PP1-mediated Akt dephosphorylation. A, selective knockdown of the expression of HDACs 1 and 6 by prevention. isozyme-specific siRNA. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Nov 18, 2005

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