Article pubs.acs.org/jpr
SAHA Regulates Histone Acetylation, Butyrylation, and Protein Expression in Neuroblastoma Guofeng Xu,† Jun Wang,† Zhixiang Wu,‡ Lili Qian,‡ Lunzhi Dai,§ Xuelian Wan,‡ Minjia Tan,‡ Yingming Zhao,*,§ and Yeming Wu*,† †
Pediatric Surgery Department, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1650 Kongjiang Road, Shanghai 200092, P. R. China ‡ The Chemical Proteomics Center and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China § Ben May Department for Cancer Research, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, United States S Supporting Information *
ABSTRACT: Emerging evidence suggests that suberoylanilide hydroxamic acid (SAHA), a clinically approved HDAC inhibitor for cutaneous T-cell lymphoma, shows promising clinical benefits in neuroblastoma, the most common extra cranial solid neoplasm with limited choice of therapeutic intervention. However, the molecular mechanism under which the compound exerts its antitumor effect remains elusive. Here we report a quantitative proteomics study that determines changes of protein expression, histone lysine acetylation, and butyrylation in response to SAHA treatment. We detected and quantified 28 histone lysine acetylation and 18 histone lysine butyrylation marks, most of which are dramatically induced by SAHA. Importantly, we identified 11 histone Kbu sites as novel histone marks in human cells. Furthermore, quantitative proteomic analysis identified 5426 proteins, among which 510 proteins were up-regulated and 508 proteins were down-regulated (significant p value 97%) was confirmed by MS prior to protein lysis and mass spectrometric experiments (Supplemental Table S1 in the SI).
Clinical trials are currently undertaken in treating other types of cancers, including neuroblastoma. SAHA regulates gene transcription by targeting class I, class II, and class IV HDACs.11 SAHA has shown strong antitumorigenic effects in neuroblastoma cells12 and has been used by children’s oncology group (COG) in recurrent or resistant/relapsed neuroblastoma trial.13,14 In addition, SAHA has recently been explored for the treatment of neurodegenerative15 and metabolic diseases.16 Despite the great potential of its clinical application, the underlying mechanism by which SAHA regulates histone PTMs, and proteome networks remain largely unknown, particularly in the NB tumor. To fill this knowledge gap, we carried out a comprehensive quantitative proteomic study using mass spectrometry (MS) and stable isotope labeling for cell culture (SILAC) to characterize histone marks and proteome networks that are regulated by SAHA. Our study identified and quantified 28 histone lysine acetylation (Kac) and 18 histone lysine butyrylation (Kbu) sites. In addition, we identified and quantified about 1000 proteins whose expressions are regulated by SAHA. Pathway analysis showed that proteins involved in cellular metabolism are abundant among down-regulated proteins, while DNA-dependent processes such as DNA replication and DNA repair are associated with up-regulated proteins. Together, our study reveals key insights into the molecular mechanism by which SAHA exerts its biological functions.
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Histone Extraction and in-Solution Tryptic Digestion
The core histones were extracted by an acid-extraction method.18 The extracted proteins were digested by trypsin as previously described.19 Briefly, after the proteins were suspended in 100 mM NH4HCO3 (pH 8.0), trypsin was added at an enzyme−substrate ratio of 1:50 (w/w) and then incubated at 37 °C for 16 h. The histone samples were further digested with trypsin (1:100, w/w) at 37 °C for 3 h to ensure the complete digestion. Immunoblotting Analysis
Two micrograms of the histone protein extractions were separated with SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST (0.1% TWEEN 20 in tris-buffered saline (TBS) solution) with 3% BSA (bovine serum albumin), then incubated with pan anti-acetyl-lysine (Kac) antibodies (PTM Biolab, Chicago, IL) at a dilution of 1:3000 (V/V), pan antibutyryl-lysine (Kbu) antibodies (PTM Biolab, Chicago, IL) at a dilution of 1:1000 (V/V), and antiacetyllysine site specific antibodies (PTM Biolab, Chicago, IL) at a dilution of 1:2000 (V/V) at 4 °C overnight, respectively. After washing three times with TBST, the membranes were incubated with secondary antibody coupled to horseradish peroxidase for 1 h. Immunoreactivity was visualized using enhanced chemiluminescence reagents (Life Technologies, USA).
EXPERIMENTAL SECTION
Cell Culture and Reagents
The SH-SY5Y cells were grown in DMEM/F12K medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% of FBS. SAHA (SigmaAldrich, St. Louis, MO) was dissolved in DMSO and stored in −20 °C. Protein A-conjugated agarose beads were from GE Healthcare, and C18 Zip Tips were from Millipore Corporation (Billerica, MA). Other chemicals such as trichloroacetic acid (TCA), formic acid, nicotinamide, acetonitrile (CH3CN), and acetone were from Sigma-Aldrich. DMEM/F12 medium was from Life Technologies (San Diego, CA). Trypsin was from Promega (Fitchburg, MI).
Preparation of Protein Whole-Cell Lysate and In-Solution Tryptic Digestion
Cell Viability Assays
(1 to 2) × 104 cells were seeded in 96-well plates with 100 μL of medium for 24 h and then subjected to the treatment of SAHA for 24, 48, and 72 h, respectively. Cell viability was measured using a cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Japan) according to the manufacturer’s protocol. Briefly, the reagent was added to SH-SY5Y cells growing in 96-well plates, which were then incubated at 37 °C for 2 h in a humidified incubator with 5% CO2. Metabolically active cells converted the water-soluble tetrazolium salt to a yellow-colored formazan dye product. The quantity of formazan product, measured by absorbance at 450 nm, was directly proportional to the number of living cells in culture. Percent viability of the cells treated with SAHA was calculated based on the indicated concentration and time compared with DMSO control.
One volume cell pellets was resuspended in 2 volumes NETN buffer (100 mM NaCl, 20 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 0.5% (v/v), Nonidet P-40 (NP-40) and lysed for 30 min on ice. The lysate was centrifuged at 16 000g for 10 min at 4 °C. The supernatant was transferred to another tube, and the protein concentration was determined by BCA (bicinchoninic acid) assay. For the full proteome analysis, 300 μg total proteins was precipitated with TCA (20%, v/v) at 4 °C. Samples were centrifuged as before, and the pellet was washed with precold acetone twice. Proteins were dissolved in 100 mM NH4HCO3 (pH 8.0) and performed the in-solution digestion as described as previously.20 Briefly, trypsin was added at an enzyme− substrate ratio of 1:50 (w/w) then incubated at 37 °C for 16 h. The samples were reduced with 5 mM dithothreitol at 55 °C for 30 min, and Cys residues were alkylated with 15 mM iodoacetamide at room temperature for 30 min in dark. The alkylation reaction was quenched with 30 mM cysteine for 30 min at room temperature. The samples were further digested with trypsin (1:100, w/w) at 37 °C for 3 h to ensure the complete digestion. The tryptic peptides were then presepa-
SILAC-Based Cell Culture
SH-SY5Y cells were cultured in SILAC medium and checked for SILAC labeling efficiency as previously described.17 Cells were split into 60 mm plates labeled as “heavy” and “light” and grown for at least 6 doubling until the confluence reached 90%. 4212
dx.doi.org/10.1021/pr500497e | J. Proteome Res. 2014, 13, 4211−4219
Journal of Proteome Research
Article
Identification and Quantification of Histone Modification Sites
rated into 20 fractions with a reverse-phase C18 Xbridge column (Waters, Milford, MA) by a preparative HPLC system (Shimadzu, Japan) under high pH condition, as previously reported.21 Each fraction was lyophilized before being subjected to the HPLC−MS/MS analysis.
The modified histone peptides identified from Maxquant were further verified by stringent manual inspection according to criteria as previously described.26 All peptides identified with Cterminal lysine modified acetylation, butyrylation, and di- and trimethylation were removed. The annotated spectra of the identified acetylation and butyrylation sites were provided in Supplemental Table S2 and Supplemental Figure S1 in the SI. Because of the dynamics complexity of histone modifications, trypsin digestion will produce multiple peptide forms for a given modification sites. To achieve the relative quantification information on a histone modification site, we used the sum intensities from all forms of the peptide identified with the modification site. The ratio between the sums of the intensities from all heavy peptides over the sum of light peptides was used to represent the changes of the modification site upon SAHA treatment.
Affinity Enrichment and HPLC−MS/MS Analysis
The immunoaffinity enrichment of the Kac and Kbu peptides from the digested histone peptides was performed as previously described.17,22 Briefly, 1 mg histone tryptic peptides was suspended in 200 μL of NETN buffer, incubated with 50 μL of pan anti-Kac antibody (PTM Biolabs, Chicago, IL) and pan anti-Kbu antibody (PTM Biolabs, Chicago, IL), which were immobilized to protein A agarose beads at 4 °C for 4 h with gentle shaking. The beads were washed with NETN buffer three times, with ENT buffer (100 mM NaCl, 20 mM Tris-Cl pH 8.0, 0.5 mM EDTA, 0.5% (v/v)) twice, and then with water once. The bound peptides were eluted with 0.1% TFA and dried in SpeedVac. The peptide samples were desalted with C18 ZipTips and dissolved in buffer A (0.1% formic acid in water, v/v). Peptide samples were analyzed by an EASY-nLC 1000 system coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA) as described previously.23 The peptides were eluted from a house-made reverse-phase C18 column (10 cm length with 75 μm ID, packed with C18 resin, 3 μm particle size, 90 Å pore size, Dikma Technologies, Lake Forest, CA) with a 2 h gradient of 5 to 90% HPLC buffer B (0.1% formic acid in acetonitrile, v/v) at a flow rate of 300 nL/ min. The AGC targets were one million for full scan with the maximum injection time of 200 ms and five thousand for MS/ MS scan with the maximum injection time of 50 ms, respectively. Survey full-scan MS spectra (from m/z 350 to 1800) were acquired in the Orbitrap with resolution R = 120 000 at m/z 400, followed by MS/MS fragmentation of the 13 most intense ions in the linear ion trap with collisional activated dissociation (CID) energy of 35%.
Statistical and Bioinformatics Analysis
The up-regulation data set and down-regulation data set were classified with a significance t-test p-value cutoff
