Molecular Cancer Therapeutics 971
Histone deacetylase inhibitor LAQ824 down-regulates Her-2 and sensitizes human breast cancer cells to trastuzumab, taxotere, gemcitabine, and epothilone B Lianne Fuino,1 Purva Bali,1 Sylvie Wittmann,1 Sreenivasa Donapaty,1 Fei Guo,1 Hirohito Yamaguchi,1 Hong-Gang Wang,1 Peter Atadja,2 and Kapil Bhalla1 1 Department of Interdisciplinary Oncology, Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, FL and 2 Novartis Pharmaceutical Inc., Summit, NJ
active against human breast cancer cells and has the potential to improve the efficacy of trastuzumab, taxotere, gemcitabine, and epothilone B against breast cancer with Her-2/neu amplification. (Mol Cancer Ther. 2003;2:971 – 984)
Introduction Abstract Histone deacetylase inhibitors induce hyperacetylation of the amino-terminal lysine residues of the core nucleosomal histones, which results in chromatin remodeling and altered gene expression. Present studies demonstrate that exposure to a novel hydroxamic acid analogue histone deacetylase inhibitor, LAQ824, induced p21WAF1and p27KIP1 and caused growth arrest and apoptosis of human breast cancer SKBR-3 and BT-474 cells that possess amplification and overexpression of Her-2/neu. Treatment with LAQ824 depleted the mRNA and protein levels of Her-2/neu-encoded Her-2, which was associated with attenuation of pAKT, c-Raf-1, and phosphorylated mitogen-activated protein kinase levels. LAQ824 also induced the acetylation of heat shock protein (hsp) 90, resulting in inhibition of its binding to ATP, which has been shown to impair the chaperone association of hsp 90 with its client proteins, Her-2, AKT, and c-Raf-1. Consistent with this, treatment with LAQ824 shifted the binding of Her-2 from hsp 90 to hsp 70, promoting proteasomal degradation of Her-2. Thus, LAQ824 depletes Her-2 through two mechanisms: attenuation of its mRNA levels and promotion of its degradation by the proteasome. Following LAQ824 treatment, the cell membrane association, autotyrosine phosphorylation, and colocalization of Her2 with HER-3 also declined. Cotreatment with LAQ824 significantly increased trastuzumab-induced apoptosis of BT-474 and SKBR-3 cells. This was associated with greater attenuation of Her-2, c-Raf-1, and pAKT levels. LAQ824 also enhanced taxotere-induced, epothilone Binduced, and gemcitabine-induced apoptosis of BT-474 and SKBR-3 cells. These findings suggest that LAQ824 is
Received 5/6/03; revised 7/9/03; accepted 8/18/03. The costs of publication of this article were defrayed in part by the payment 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. Requests for Reprints: Kapil Bhalla, Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, MRC 3 East, Room 3056, Tampa, FL 33612. Phone: (813) 903-6861; Fax: (813) 903-6817. E-mail:
[email protected]
Despite recent progress in our understanding of its genetic and molecular basis, advanced and metastatic breast cancer remains incurable (1). Encouragingly, a number of agents with diverse cellular targets and mechanisms of actions have been developed that are active against breast cancer cells (2). Approximately 25% of breast cancers have amplification and overexpression of Her-2/neu oncogene (3), which encodes for a member of the family of epidermal growth factor receptor tyrosine kinases (4). Dimerization of Her-2 with HER-3 results in activation and signaling through the phosphatidylinositol 3-kinase (PI3K)/AKT and Ras-Raf-extracellular signal-regulated kinase (Erk) pathways, promoting cell proliferation and survival, and is associated with poor prognosis in breast cancer (4, 5). Her-2-mediated signaling also confers resistance to apoptosis due to antibreast cancer agents, including tubulin-polymerizing agents (TPAs; e.g., taxotere), as well as antiestrogen treatment with tamoxifen (6 – 8). Conversely, repression of Her-2 attenuates its antiapoptotic signaling and suppresses Her-2-induced malignant phenotype, making Her-2 an excellent therapeutic target in breast cancer (9, 10). Indeed, a recombinant, humanized, monoclonal anti-Her-2 antibody, trastuzumab (Herceptin), has been shown to dephosphorylate and down-regulate Her-2 levels and signaling as well as to exhibit clinical efficacy against breast cancer (11). Cotreatment with trastuzumab also improves the antibreast cancer activity of chemotherapeutic agents, especially TPAs (12 – 15). However, resistance to trastuzumab, given alone or in combination with chemotherapeutic agents, is common, involving mechanisms that involve the activation of PI3K/AKT and/or Erk1/2 signaling (16 – 18). This highlights the need to develop new strategies that would target the mechanisms of resistance and enhance sensitivity of breast cancer to trastuzumab and chemotherapeutic agents (19). Geldanamycin (GA) and its less toxic analogue 17allylamino-demethoxygeldanamycin (17-AAG) are inhibitors of the heat shock protein (hsp) 90, which is an intracellular chaperone for a number of client proteins that include protein kinases and nuclear hormone receptors (20 – 22). Among the protein kinases that are chaperoned by hsp 90 are Her-2, AKT, c-Raf-1, and Bcr-Abl (23 – 26).
972 LAQ824 Lowers Her-2 and Induces Apoptosis
Depletion of c-Raf-1 and inhibition of Erk1/2 phosphorylation as well as attenuation of pAKT by GA or 17-AAG leads to disruption of the Raf-mitogen-activated protein kinase (MAPK)/Erk kinase-MAPK (Erk1/2) and PI3K/ AKT signaling, resulting in cytostasis and apoptosis of cancer cells (27). Treatment with GA or 17-AAG has also been demonstrated to down-regulate Her-2 and its downstream signaling (28). This is associated with sensitization of breast cancer cells to chemotherapeutic agents (29). Thus, a hsp 90 inhibitor has the potential to exert dual antibreast cancer effect (i.e., depletion of Her-2 and its signaling as well as attenuation of AKT and c-Raf-1 activity that may be independent of down-regulation of Her-2 signaling). Whether this would translate into superior clinical efficacy of 17-AAG with or without antibreast cancer agents, including Herceptin, TPAs, and gemcitabine, has not been confirmed. Acetylation, phosphorylation, and methylation are among the key post-translational modifications of the amino-terminal tails of core nucleosomal histones (30). The combinatorial nature of the histone modifications represents a specific epigenetic code called the ‘‘histone code’’ (31). Distinct modifications either increase or decrease the interaction affinity of the nonhistone protein transcriptional complexes with DNA, thereby regulating dynamic transitions between transcriptionally active and silent states of the chromatin (32). Histone acetyltransferases and histone deacetylases (HDAC) catalyze the acetylation and deacetylation of the lysine residues in the histone tails, respectively (33, 34). The acetylation of histones H3 and H4 at specific lysine residues by histone acetyltransferases is associated with transcriptional activation; hence, the histones associated with active genes are highly acetylated (30). Conversely, histones associated with repressed heterochromatin are hypoacetylated due to the activity of HDACs (34). Treatment with HDAC inhibitors (HDI) causes hyperacetylation of the aminoterminal lysine residues in the nucleosomal histones, which has been shown to transcriptionally up-regulate p21WAF1 (referred to as p21) expression (35). This is associated with cell cycle arrest and apoptosis of cancer and leukemia cells (36 – 38). The hydroxamic acid analogues (e.g., trichostatin A [TSA] and suberoylanilide hydroxamic acid [SAHA]) have been demonstrated to induce p21 and p27KIP1 (referred to as p27) as well as to exert potent in vitro and in vivo anticancer effects (35 – 39). Depsipeptide (FK228), a natural product HDI, was also shown to cause acetylation of the hsp 90 in lung cancer cells (40). This was shown to destabilize the chaperone complex of hsp 90 with its client proteins and targeted them for degradation by the proteasome (40). Recently, a cinnamic acid hydroxamate, LAQ824, has been demonstrated to act as a potent HDI and exerts anticancer effects at nanomolar concentration (41). In the present studies, we took a more comprehensive look at the effects of treatment with LAQ824 on the mRNA and protein expression of Her-2/neu gene and on the cell cycle status and apoptosis of cultured human breast cancer cells. We determined
whether LAQ824 treatment would also induce the acetylation of hsp 90 and disrupt its chaperone association with Her-2, leading to the proteasomal degradation and depletion of Her-2. Furthermore, whether cotreatment with LAQ824 would enhance the apoptosis induced by antibreast cancer agents, including trastuzumab, taxotere, epothilone B (Epo B), and gemcitabine, was also probed in the present studies.
Materials and Methods Reagents LAQ824 and Epo B were kindly provided by Novartis Corp. (Basel, Switzerland). Taxotere was a gift from Aventis Pharmaceuticals (Bridgewater, NJ). Trastuzumab was a gift from Genentech (South San Francisco, CA). Taxotere and Epo B were made at a concentration of 10 mM in DMSO and stored at 20jC. 17-AAG was kindly provided by the Developmental Therapeutics Branch of Cancer Therapy Evaluation Program/National Cancer Institute/NIH (Bethesda, MD). Proteosome inhibitor, N-acetyl leucyl-leucyl norlucinal (ALLnL), was purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal antipoly(ADP-ribose) polymerase (PARP) was purchased from Cell Signaling (Beverly, MA). Anti-Bax, anti-cIAP2, and anti-Mcl-1 polyclonal antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-hsp 70 and anti-hsp 90 antibodies were purchased from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada). Other antibodies for the immunoblot analyses to detect the levels of proteins, including Bcl-xL, pAKT, AKT, p21, and p27, were obtained, as described previously (42, 43). Cell Culture The human breast cancer cell lines BT-474 and SKBR-3 were obtained from American Type Culture Collection (Manassas, VA) and maintained in the recommended culture medium. Culture mediums were supplemented with 10% fetal bovine serum, 100-units/ml penicillin, and 100-Ag/ml streptomycin (Life Technologies, Inc., Grand Island, NY) at 37jC in a humidified 5% CO2 incubator (44). Flow Cytometric Analysis of Cell Cycle Status The flow cytometric evaluation of the cell cycle status was performed according to a previously described method (42, 44). The percentage of cells in the G1, S, and G2-M phases were calculated using Multicycle software (Phoenix Flow Systems, San Diego, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltertrazolium Bromide Assay Cytotoxic effect of the drugs was determined by the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay, as described previously (45). Cells (1 – 5 103 in 100 Al/well) were grown overnight in 96well plates. Fresh media (100 Al) containing the designated drug was added for specified exposure intervals. At the completion of incubation, media was replaced with fresh
Molecular Cancer Therapeutics 973
complete media (100 Al). Three hours before the end of the incubation period, 20 Al of PBS containing MTT (5 mg/ml) was added to each well. Following this, the plates were centrifuged at 200 g for 5 min and the media was removed. The precipitate was then resuspended in 100 Al of DMSO. The absorbance was measured on a plate reader at 540 nm. Each experiment was performed in triplicate. Apoptosis Assessment by Annexin V Staining After drug treatment, cells were resuspended in 100Al staining solution containing Annexin V fluorescein and propidium iodide in a HEPES buffer (Annexin V-FITC, BD Pharmingen, San Diego, CA). Following incubation at room temperature for 15 min, cells were analyzed by flow cytometry (42, 44). Annexin V binds to those cells that express phosphatidylserine on the outer layer of the cell membrane, and propidium iodide stains the cellular DNA of those cells with a compromised cell membrane. This allows for the discrimination of live cells (unstained with either fluorochrome) from apoptotic cells (stained only with Annexin V) and necrotic cells (stained with both Annexin V and propidium iodide). Morphology of Apoptotic Cells After drug treatment, cells were washed with PBS and resuspended in the same buffer. Cytospin preparation of 50 103 cells were fixed and stained with Wright-Giemsa stain (Biochemical Sciences Inc., Swedesboro, NJ). Cell morphology was determined by light microscopy. In all, five different fields were randomly selected for counting at least 500 cells. The percentage of apoptotic cells was calculated for each experiment, as described previously (42, 44). Western Blot Analyses Western blot analyses of Her-2, c-Raf-1, AKT, pAKT, p21, p27, acetylated histones H3 and H4 proteins, and aˆ-actin were performed using specific antisera or monoclonal antibodies (see above), as described previously (42, 44). Horizontal scanning densitometry was performed on Western blots by using acquisition into Adobe PhotoShop (Adobe Systems Inc., San Jose, CA) and analysis by the NIH Image Program (NIH, Bethesda, MD). The expression of aˆactin was used as a control. Immunoprecipitation Following the designated treatments, cells were lysed in the lysis buffer (1% SDS, 1% Triton X-100, 0.5% deoxycholate, 150-mM NaCl, 2-mM EDTA, 1-mM phenylmethylsulfonyl fluoride, 10-Ag/ml leupeptin) for 1 h, and the nuclear and cellular debris was cleared by centrifugation. Protein G agarose beads were washed twice with radioimmunoprecipitation assay (RIPA)-1 buffer (50-mM Tris [pH 7.5], 150-mM NaCl, 1-mM EDTA, 1% NP40, 0.1% deoxycholate) and incubated with hsp 90-specific monoclonal antibody (1 in 100; Stressgen) at 4jC for 2 h. After washing the protein G and the antibody mix with RIPA-1 buffer, 100 Ag of total cell lysates were added and incubated overnight at 4jC. The immunoprecipitates were washed thrice in RIPA-1 buffer and proteins were eluted with the SDS sample loading buffer prior to the immunoblot analyses (44).
Immunoprecipitation of Conformationally Changed Bax Cells are lysed in 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfate (CHAPS) lysis buffer (150-mM NaCl, 10-mM HEPES [pH 7.4], 1% CHAPS) containing protease inhibitors. Immunoprecipitation is performed in lysis buffer by using 500 Tg of total cell lysate and 2.5 Tg of anti-Bax 6A7 monoclonal antibody (Sigma). The resulting immune complexes as well as the supernatants are subjected to immunoblotting analysis with anti-Bax polyclonal rabbit antiserum, as described previously (46). Immunofluorescent Microscopy for Her-2 and HER-3 Expressions Cells were washed twice in PBS, fixed in 4% paraformaldehyde for 30 min, and permeabilized in 0.5% Triton X-100/ PBS for 15 min. After preblocking with 3% BSA/PBS, cells were incubated with primary antibodies at 37jC for 1 h followed by three washes in PBS and incubation with FITC-conjugated or rhodamine-conjugated secondary antibodies for 1 h at 37jC. Cells were washed in PBS before nuclear staining with 0.1 Ag/ml of 4V,6-diamino-2-phenylinodole (DAPI) and analysis by a fluorescence microscopy, as described previously (47). Controls for immunomicroscopy experiments included use of isotype and subclasses matched monoclonal antibodies when employing monoclonals and preimmune serum when employing polyclonal antibodies. Northern Blot Analysis Northern blot analysis of Her-2/neu mRNA was performed using a previously reported method (43). Total RNA was extracted from treated and untreated cells using RNeasy Mini Kit (Qiagen Inc., Valencia, CA). Total RNA (20 Ag) was separated on 1% agarose gel containing formaldehyde (0.22 M) and transferred overnight to an uncharged nylon membrane by capillary transfer in 10 SSC. Membranes were treated with UV Crosslinker (Stratagene, La Jolla, CA) and subsequently probed with 32 P-a[dCTP]-radiolabeled probe. A 1.6-kb fragment of Her2/neu cDNA was used as the probe. Real-Time Quantitative ReverseTranscription-PCR Total RNA was isolated from cells using RNeasy Mini Kit (Qiagen) and real-time reverse transcription-PCR analysis was performed, as described previously (42). Two microliters of total RNA (0.1 Ag) were added to 23 Al of a reaction mix (Taqman RT-PCR Reagent Kit, Perkin-Elmer, Boston, MA) consisting of 19.7 Al of H2O, 0.15 Al of enzyme mix, 0.5 Al of forward primer, 0.5 Al of reverse primer, 1.0 Al of Her2/neu gene-specific oligonucleotide probe, 0.5 Al of histone H1 forward primer, 0.5 Al of histone H1 reverse primer, and 0.5 Al of histone-specific oligonucleotide probe, combined and placed in a 96-well plate. The plate was transferred to an ABI 7700 thermocycler (Perkin-Elmer), and samples were processed at 48jC for 30 min and then at 95jC for 10 min. These were followed by 35 amplification cycles (95jC, 15 s and 60jC, 1 min) during which fluorescent signals of Her-2/neu and histone H1 probes were measured, as described previously. Serial dilutions of 106 to 101 of Her-2/neu plasmid DNA and histone H1 plasmid DNA
974 LAQ824 Lowers Her-2 and Induces Apoptosis
were used as a reference standard of fluorescent signals for copy number, as described previously (42). All the probes carried a 5V FAM reporter label and a 3V TAMRA quencher group. All primers and probes were synthesized by PE-Applied Biosystems (Perkin-Elmer). Chromatin Immunoprecipitation Assay Chromatin immunoprecipitation (ChIP) analysis was performed by a slight modification of a previously described method (35). Cells were incubated overnight at a density of 0.25106 cells/ml at 37jC with 5% CO2. On the next day, cells were cultured with 0, 50, 100, or 250 nM of LAQ824 for 24 h. Formaldehyde was then added to the cells to a final concentration of 1%, and the cells were gently shaken at room temperature for 10 min. Following this, the cells were pelleted and suspended in 1 ml of ice-cold PBS containing protease inhibitors (Complete, Boehringer Mannheim, Indianapolis, IN). Cells were again pelleted, resuspended in 0.5 ml of SDS lysis buffer (1% SDS/1.0-mM EDTA/50-mM Tris-HCl [pH 8.1]), and incubated on ice for 20 min. Lysates were sonicated with 15-s bursts. Debris was removed from samples by centrifugation for 20 min at 15,000 g at 4jC. An aliquot of the chromatin preparation (100 Al) was set aside and designated as input fraction. The supernatants were diluted 3-fold in the immunoprecipitation buffer (0.01% SDS/1.0% Triton X-100/1.2-mM EDTA/ 16.7-mM Tris-HCl [pH 8.1]/150-mM NaCl), and 80 Al of 50% protein A-Sepharose slurry containing 20-Ag sonicated salmon sperm DNA and 1-mg/ml BSA in the TE buffer (10-mM Tris-HCl [pH 8.0]/1-mM EDTA) were added and incubated by rocking for 2 h at 4jC. Beads were pelleted by centrifugation, and supernatants were placed in fresh tubes with 5 Ag of the antiacetylated histone H3 antibody, antiacetylated histone H4 antibody, or normal rabbit serum and incubated overnight at 4jC. Protein A-Sepharose slurry (60 Al) was added, and the samples were rocked for 1 h at 4jC. Protein A complexes were centrifuged and washed thrice for 5 min each with immunoprecipitation buffer and twice for 5 min each with immunoprecipitation buffer containing 500-mM NaCl. Immune complexes were eluted twice with 250 Al of elution buffer (1% SDS/0.1-M NaHCO3) for 15 min at room temperature. Twenty microliters of 5-M NaCl were added to the combined eluates, and the samples were incubated at 65jC for 24 h. EDTA, TrisHCl (pH 6.5), and proteinase K were then added to the samples at a final concentration of 10 mM, 40 mM, and 0.04 Ag/Al, respectively. The samples were incubated at 37jC for 30 min. Immunoprecipitated DNA (both immunoprecipitation samples and input fraction) was recovered by phenol/chloroform extraction and ethanol precipitation and analyzed by PCR. The Her-2/neu-specific primers were used to perform PCR on DNA isolated from ChIP experiments and input samples. The optimal reaction conditions for PCR were determined for each primer pair. Parameters were denaturation at 95jC for 1 min and annealing at 56jC for 1 min followed by elongation at 72jC for 1 min. PCR products were analyzed by 2.5% agarose/ ethidium bromide gel electrophoresis. The primer pair used for Her-2/neu was forward primer: 5V AAC ACA TCC
CCC TCC TTG ACT ATC 3V and reverse primer: 5V AGC TTC ACT TTC TCC CTC TCT TCG 3V. The size of the amplified product was 330 bp. The ratio of immunoprecipitated DNA to input DNA was calculated for each treatment and primer set. The fold increase after treatment with LAQ824 was calculated from the indicated ratio. Acetylation of hsp 90 and Its Binding to ATPSepharose Untreated or LAQ824-treated cells were lysed in TNESV buffer (50-mM Tris, 2-mM EDTA, 100-nM NaCl, 1-nM sodium vanadate, 1% NP40 at pH 7.5), and total cellular proteins were quantified using the BCA Protein Assay (Pierce Biotechnology, Inc., Rockford, IL). hsp 90 was immunoprecipitated from 200 Ag of total protein using mouse hsp 90 antibody, and immunoprecipitates were immunoblotted with antiacetyl lysine antibody, as described previously (41). Alternatively, cell lysates were prepared after drug treatment and hsp 90 was affinity precipitated from 200 Ag of total cellular protein using ATP-Sepharose beads. The concentration of hsp 90 in the precipitates was assessed by Western blot analysis using a polyclonal anti-hsp 90 antibody (41). Preparation of Detergent-Soluble and DetergentInsoluble Fractions After the designated drug treatments, cells were lysed with TNSEV buffer (50-mM Tris-HCl [pH 7.5], 2-mM EDTA, 100-mM NaCl, 1-mM sodium orthovanadate, 1% NP40 containing 20-Ag/ml aprotonin, 20-Ag/ml leupeptin, 1-mM phenylmethylsulfonyl fluoride, 25-mM NAF, and 5-mM Nethylmaleimide; 40). The insoluble fraction (pellet) was solubilized with SDS buffer (80-mM Tris [pH 6.8], 2% SDS, 100-mM DTT, and 10% glycerol). Fifty micrograms of proteins from the NP40 soluble and insoluble fractions were separated on 7.5% SDS polyacrylamide gel and analyzed by Western blotting (44). Statistical Analyses Data were expressed as means F SEM. Comparisons used Student’s t test or ANOVA, as appropriate. P values of
