Identification of the Amyloid β-Protein Precursor and Cystatin C as Novel Epidermal Growth Factor Receptor Regulated Secretory Proteins in Nasopharyngeal Carcinoma by Proteomics Can-E Tang,#,† Yong-Jun Guan,#,‡ Bin Yi,#,† Xin-Hui Li,† Ke Liang,† Hai-Yan Zou,† Hong Yi,† Mao-Yu Li,† Peng-Fei Zhang,† Cui Li,† Fang Peng,† Zhu-Chu Chen,†,‡ Kai-Tai Yao,‡ and Zhi-Qiang Xiao*,† Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China, and Cancer Research Institute, Xiangya School of Medicine, Central South University, Changsha 410078, China Received December 6, 2009
The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-Rstimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-R for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid β-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-R stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-R stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC. Keywords: Nasopharyngeal carcinoma • epidermal growth factor receptor • secreted proteins • amyloid β-protein precursor • cystatin C
Introduction Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China, where the incidence rate is as high as 15-50 per 100 000 people per year.1 Overexpression of the epidermal growth factor receptor (EGFR) is common in NPC,2-6 and about 85% of the Chinese patients with NPC have moderate to strong expression of EGFR.7 The increased expression of the receptor is often associated with the increased amount of its ligands’ EGF and TGF-R production in the NPC cells, which form an autocrine stimulatory pathway in NPC.8 Moreover, overexpression of EGFR in primary tumors was associated with the tumor metastasis, recurrence, and poor * To whom correspondence should be addressed. Tel.: (86)731-4327239. Fax: (86)731-4327321. E-mail:
[email protected]. # These authors contributed equally to this work. † Xiangya Hospital. ‡ Xiangya School of Medicine. 10.1021/pr100663p
2010 American Chemical Society
survival in patients with NPC.7,9,10 Recent data have proposed EGFR as a new target for anti-NPC therapy.11,12 These studies suggest that EGFR plays a crucial role in the development and progression of NPC. EGFR is a transmembrane glycoprotein consisting of an extracellular ligand binding domain, a transmembrane region, and an intracellular protein tyrosine kinase domain.13 The natural ligands EGF and TGF-R bind to the extracellular domain of EGFR and activate the receptor and its downstream signal transduction pathways such as PI3K and MAPK pathways, ultimately causing activation or modulation of various cellular processes including a protein secretion.14 During cancer development and progression, tumor cells continually secrete proteins that are involved in intercellular communication, cell growth, adhesion, motility, and invasion.15 Therefore, identification of secreted proteins in NPC cells after EGFR activation will yield insight into the alterations in the profile of secreted Journal of Proteome Research 2010, 9, 6101–6111 6101 Published on Web 09/30/2010
research articles proteins associated with the EGFR activation and provide important information on the role and mechanism of EGFR in the NPC. Secretome is referred to as the proteins secreted or shed by the cells through the classical and nonclassical secretory pathway. In the classical secretory pathway, proteins containing a signal peptide are translocated from the cytoplasm of the cell into the lumen of the endoplasmic reticulum (ER). From the ER they are transported to the Golgi apparatus and finally to the plasma membrane where they are released into the extracellular compartment. However, in the nonclassical secretory pathway, proteins are synthesized without a signal peptide and maintain a predominantly cytosolic distribution until they are exported from the cell via an ER/Golgi-independent pathway.16-18 Since secreted proteins are known to regulate a variety of biological functions and are involved in a multitude of pathological processes, investigation of secreted proteins is helpful to elucidate carcinogenesis. Furthermore, investigation of secreted proteins may represent an attractive strategy for isolating and identifying potential tumor biomarker candidates, as secreted proteins are more likely to be detected in body fluids such as serum or plasma.19-22 Recently, secretomics have been used to investigate the role and mechanism of oncogenes and tumor suppressor genes, discovering their regulating secretory proteins associated with biological functions.23,24 Although previous studies have used a proteomic approach to investigate the EGFR signaling pathway, identifying some novel EGFR-regulated intracellular proteins,25-28 little is known concerning EGFR-regulated secreted proteins. Investigation of EGFR-regulated secreteome can identify the target secretory proteins of the EGFR signaling pathway, which may be an alternative way to decipher the role and mechanism of EGFR in NPC. In this study, we aimed to investigate the target secretory proteins of EGFR in NPC by comparative proteomic analysis of the conditioned media from TGF-R-stimulated and unstimulated NPC cell line CNE-2 cells. As a result, 16 nonredundant differential secretory proteins were identified by 2-DE and mass spectrometry. We further validated that two differential secretory proteins APP and cystatin C are EGFRregulated proteins in both NPC cells and clinical NPC tissues, and upregulation of APP by activated EGFR could increase the growth and motility of NPC cells. The data will provide useful information for further study of the role and mechanism of EGFR in NPC.
Methods and Materials Materials. Immobiline pH-gradient (IPG) DryStrips (pH 3-10, length 24 cm), IPG buffer (pH 3-10), DryStrip cover fluids, thiourea, urea, CHAPS, DTT, Pharmalyte (pH 3-10), bromophenol blue, Bis, TEMED, commassie brilliant blue G-250, molecular weight marker, Tris-base, SDS, glycine, horseradish peroxidase-conjugated goat antirabbit and antimouse IgG, and the enhanced chemiluminescence (ECL) system were purchased from Amersham Biosciences (Stockholm, Sweden). Sequencing-grade modified trypsin was obtained from Promega (Madison, WI, USA). PVDF membrane was obtained from Millipore (Boston, MA, USA). Polyclonal rabbit antihuman EGFR antibody was from Santa Cruz Biotechnology (Santa Cruz, CA, USA), monoclonal mouse antihuman APP N-terminus/ectodomain antibody (22C11) from Chemicon (Temekula, CA, USA), polyclonal rabbit antihuman 6102
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Tang et al. cystatin C antibody from Abcam, Inc. (Cambridge, MA, USA), and monoclonal mouse antihuman β-actin antibody from Sigma-Aldrich (St. Louis, MO, USA). Mercaptoethanol, iodoacetamide, R-cyano-4-hydroxycinnamic acid (CCA), and HCl were from Sigma-Aldrich. All buffers were prepared with Milli-Q water. Cell Line and Tissue Specimens. The human poorly differentiated NPC cell line CNE-2 with high expression of EGFR has been described previously.29 29 cases of formalin-fixed and paraffin-embedded archival NPC tissues were obtained from the Department of Pathology, the Second Xiangya Hospital of Central South University, China, according to institutional regulations and used for immunohistochemistry. On the basis of the 1978 WHO classification,30 all NPCs were histopathologically diagnosed as poorly differentiated squamous cell carcinomas. Cell Culture and Treatment. CNE-2 cells were cultured to 60-70% confluency in DMEM medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA) at 37 °C, washed four times with the serum-free medium, and then were serum-starved in the serum-free medium supplemented with 5 µg/mL of transferrin (Sigma-Aldrich) and 5 µg/mL of insulin (Sigma-Aldrich) for 12 h. After being serum-starved, the cells were incubated in the serum-free medium supplemented with 30 ng/mL of TGF-R (Sigma-Aldrich) for 24 h or mocktreated as a control. After TGF-R stimulation, the conditioned medium was carefully removed and filtered using a 0.45 µm syringe filter to remove suspended cells. The filtered media were then desalinated and concentrated to 0.25 mL by ultrafiltration centrifugation in Amicon Ultra-15 tubes (5 kDa molecular weight cutoff; Millipore). The concentration of the proteins was determined using the BCA protein assay kit (Pierce, Rockford, IL, USA) and stored at -70 °C for 2-DE. To examine whether the differential secretory proteins APP and cystatin C are regulated by EGFR, CNE-2 cells were cultured in the serum-free medium supplemented with 5 µg/mL of transferrin and 5 µg/mL of insulin for 12 h, washed four times with the serum-free medium, and then were pretreated with 5 µM EGFR tyrosine kinase inhibitor PD153035 (Calbiochem, Darmstadt, Germany), 100 nM PI3K inhibitor Wortmannin (Calbiochem), or 25 µM MEK inhibitor PD98058 (Calbiochem) for 2 h, followed by incubation with 30 ng/mL of TGF-R for 24 h. Control cells were treated with the vehicle dimethyl sulfoxide (DMSO; Sigma-Aldrich) only. After treatment, both the cells and supernatant were collected for Western blot analysis. Two-Dimensional Gel Electrophoresis. The purified conditioned media from TGF-R-stimulated and unstimulated CNE-2 cells were used for comparative proteomics analysis. Two-dimensional gel electrophoresis (2-DE) was performed to separate proteins from the conditioned media as previously described by us.31 Briefly, 600 µg of protein samples was diluted to 450 µL with a rehydration solution [7 mol/L urea, 2 mol/L thiourea, 0.2% DTT, 0.5% (v/v) pH 3-10 IPG buffer, and trace bromophenol blue] and applied to IPG strips (pH 3-10; L, 24 cm) by 14 h rehydration at 30 V. The proteins were focused successively for 1 h at 500 V, 1 h at 1000 V, and 8.5 h at 8000 V to give a total of 68 kVh on an IPGphor (Amersham Biosciences). Focused IPG strips were equilibrated for 15 min in a solution (6 mol/L urea, 2% SDS, 30% glycerol, 50 mmol/L Tris-HCl, pH 8.8, and 1%, DTT) and then for an additional 15 min in the same solution except that DTT was replaced by 2.5% iodoacetamide. After equilibration, SDS-PAGE was performed
Proteomics and EGFR on the Ettan DALT II system (Amersham Biosciences). After SDS-PAGE, the Blue Silver staining method, a modified Neuhoff’s colloidal coomassie blue G-250 stain, was used to visualize the protein spots in the 2-DE gels. Triplicate gels were made for each conditioned medium. Image Analysis. The stained 2-DE gels were scanned with MagicScan software on Imagescanner (Amersham Biosciences) and analyzed using the PDQuest system (Bio-Rad Laboratories, Hercules, CA, USA) according to the protocols provided by the manufacturer. To minimize the contribution of experimental variations, three separate gels were prepared for each conditioned medium. The gel spot pattern of each gel was summarized in a standard after spot matching. Thus, we obtained one average gel for each conditioned medium. Spot intensities were quantified by calculation of spot volume after normalization of the image using the total spot volume normalization method multiplied by the total area of all the spots. The change index was defined as the ratio between the spot percentage relative volumes in the TGF-R-treated medium and untreated medium. Proteins were classified as being differentially expressed between the two conditioned media when spot intensity showed a difference of g2-fold variation in TGF-R-treated medium in comparison to untreated medium. Significant differences in protein levels were determined by Student’s t-test with a set value of P < 0.05. Mass Spectrometry Analysis. All the differential protein spots were excised from stained gels using punch, and in-gel trypsin digestion was performed as previously described by us.31 The tryptic peptide was mixed with a CCA matrix solution. An amount of 1 µL of the mixture was analyzed with a Voyager System DE-STR 4307 MALDI-TOF Mass Spectrometer (MS) (ABI, Foster City, CA, USA) to get a peptide mass fingerprint (PMF). In PMF map database searching, a Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Peptide matching and protein searches against the Swiss-Prot database were performed using the Mascot search engine (http://www.matrixscience.com/) with a mass tolerance of (50 ppm. Western Blot. Western blot was performed to detect the levels of the differential secretory proteins (APP and cyststin C) in the conditioned media and whole cell extracts of CNE-2 cells as previously described by us.31 Briefly, 40 µg of lysates was separated by 8% SDS-PAGE and transferred to a PVDF membrane. Blots were blocked with 3% nonfat dry milk for 2 h at room temperature and then incubated with 1:1000 dilution of monoclonal mouse anti-APP N-terminus/ectodomain antibody or 1:500 dilution of polyclonal rabbit anticystatin C antibody for 2 h at room temperature, followed by incubation with 1:3000 dilution of horseradish peroxidase-conjugated goat antimouse or antirabbit IgG for 1 h at room temperature. The signal was visualized with an ECL detection reagent and quantitated by densitometry using the ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA). β-Actin was detected simultaneously using 1:5000 dilution of monoclonal mouse anti-β-actin antibody (Sigma-Aldrich) as a loading control. Additionally, Western blot was performed to examine the distribution of β-actin, an abundant cytosolic protein, in the conditioned medium and whole cell extract of CNE-2 after serum starvation. Three independent experiments were done. Cell Growth Analysis. MTT assay was carried out to detect the effect of secretory APP on NPC cell growth. Briefly, CNE-2 cells were seeded in 96-well culture plates (1 × 104/well) in
research articles 150 µL of DMEM medium supplemented with 10% FBS. After 12 h incubation, monoclonal mouse antihuman APP Nterminus/ectodomain antibody (final concentration, 2.5 µg/ mL) was added into the medium to block the biological effects of APP, and the cells were grown for 6 days. Every 24 h, 20 µL of MTT (5 mg/mL, Sigma-Aldrich) was added to wells, and the medium was removed after 4 h of incubation. An amount of 150 µL of DMSO (sigma-Aldrich) was added to each well for 10 min at room temperature. The absorbance of each well was read with a Bio-Tek Instruments EL310 Microplate Autoreader at 490 nm. Control cells were treated with the same concentration of mouse control IgG1 (Cymbus Technology, Chandlers Ford, United Kingdom). Additionally, CNE-2 cells were cultured in DMEM medium with or without 10% fetal bovine serum for 36 h as described above, and MTT assay was performed to detect the effect of serum starvation on the viability of the CNE-2 cells. Three independent experiments were performed in triplicate. In Vitro Cell Migration Assay. In vitro cell migration assay was conducted to determine the effect of secretory APP on NPC cell migration in 24-well transwell chambers (Costar, Cambridge, MA, USA) as previously described by us.31 The upper and lower culture compartments of each well in the transwell chambers are separated by polycarbonate membranes (8 µm pore size). To assess the ability of the cells to penetrate the polycarbonate membrane, 1.25 × 104 cells in 0.5 mL of DMEM medium containing 0.5% FBS and monoclonal mouse antihuman APP N-terminus/ectodomain antibody (final concentration, 2.5 µg/mL) were placed into the upper compartment of the wells, and 0.75 mL of DMEM medium containing 1% FBS was placed into the lower compartment. The transwell chambers were incubated for 24 h at 37 °C in humidified 5% CO2 atm. Migratory cells attached underneath the chamber membrane were stained with a Diff-Quik stain kit (Dade Behring, Newark, DE, USA) and counted in five random fields with an inverted microscope (at ×200 magnification). Migratory ability was defined as the average cell numbers that penetrated the membrane per field. Control cells were treated with the same concentration of mouse control IgG1 (Cymbus Technology). Three independent experiments were performed in triplicate. Immunohistochemistry. Immunohistochemical analysis of EGFR, APP, and cystatin C was performed on formalin-fixed and paraffin-embedded tissue sections using the standard immunohistochemical technique. Tissue sections (4 µm) were deparaffinized in xylene, rehydrated in a graded ethanol series, and treated with an antigen retrieval solution (10 mmol/L sodium citrate buffer, pH 6.0). The sections were incubated with polyclonal rabbit antihuman EGFR antibody (1:200 dilution), monoclonal mouse antihuman APP N-terminus/ ectodomain antibody (1:200 dilution), or polyclonal rabbit anticystatin C antibody (1:400 dilution) overnight at 4 °C and then were incubated with 1:1000 dilution of biotinylated secondary antibody followed by an avidin-biotin peroxidase complex (DAKO, Carpinteria, CA, USA) according to the manufacturer’s instructions. Finally, tissue sections were incubated with 3′,3′-diaminobenzidine (Sigma-Aldrich) until a brown color developed and counterstained with Harris’ modified hematoxylin. In negative controls, primary antibodies were omitted. Evaluation of Staining. Sections were blindly evaluated by two investigators in an effort to provide a consensus on staining patterns by light microscopy. EGFR, APP, and cystatin C protein staining was assessed according to the methods previously Journal of Proteome Research • Vol. 9, No. 12, 2010 6103
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Figure 1. (A) Representative 2-DE maps of the conditioned medium from TGF-R-stimulated CNE-2 and unstimulated CNE-2 cells. Eighteen differential secreted protein spots that have been identified by MS are marked with arrows. (B) A close-up image of the region of 2-D gels shows the upregulation of protein spot 3 (APP) and downregulation of protein spot 10 (cystatin C) in the medium from TGF-Rstimulated CNE-2 cells compared with the control medium. (C) A representative result of Western blot analysis shows the upregulation of APP expression and downregulation of cystatin C in the medium from TGF-R-stimulated CNE-2 cells compared with the control medium. TGF-R-CM, the conditioned medium from TGF-R-stimulated CNE-2 cells; Control CM, the conditioned medium from TGF-Runstimulated CNE-2 cells; Loading control, nontarget protein bands in Coomassie blue staining-1D-gel before transferring to a PVDF membrane.
described by us.31 Each case was rated according to a score that added a scale of intensity of staining to the area of staining. At least 10 high-power fields were chosen randomly, and >1000 cells were counted for each section. The intensity of staining was graded on the following scale: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, intense staining. The area of staining was evaluated as follows: 0, no staining of cells in any microscopic fields; 1+, 60% stained positive. The minimum score when summed (extension + intensity) was therefore 0 and the maximum 6. A combined staining score (extension + intensity) of e2 was considered to be negative staining; a score between 3 and 4 was considered to be moderate staining; that between 5 and 6 was considered to be strong staining. Secretory Protein Analysis. To do secretory protein prediction of the identified proteins, we used program SignalP (http://www.cbs.dtu.dk/services/SignalP) with Hidden Markov models for predicting the presence of the secretory signal peptide,32,33 program SecretomeP (http://www.cbs.dtu.dk/ services/SecretomeP) for divining nonsignal peptide-triggered protein secretion,34 and PubMed database searching (http://www.ncbi.nlm.nih.gov) for comparing with the secretory proteins reported in the literature. Statistical Analysis. The statistical software package SPSS 13.0 was used in this study. Single comparisons were performed using Student’s t test or Mann-Whitney’s U test. The possible correlations in the expression levels of EGFR, APP, and cystatin C in NPC were analyzed using partial correlation analysis. All statistical tests were two-sided. Differences were considered statistically significant for P-values
