Discovery of Retinoblastoma-Associated Binding Protein 46 as a Novel Prognostic Marker for Distant Metastasis in Nonsmall Cell Lung Cancer by Combined Analysis of Cancer Cell Secretome and Pleural Effusion Proteome Chih-Liang Wang,†,‡,¶ Chun-I Wang,#,¶ Pao-Chi Liao,| Chi-De Chen,# Ying Liang,§ Wen-Yu Chuang,O Ying-Huang Tsai,† Hua-Chien Chen,§ Yu-Sun Chang,§ Jau-Song Yu,#,§,∇ Chih-Ching Wu,§,⊥ and Chia-Jung Yu*,#,§,∇,⊥ Division of Pulmonary Oncology and Interventional Bronchoscopy, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, Taiwan, Graduate Institute of Biomedical Sciences, Biology, Chang Gung University, Tao-Yuan, Taiwan, Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Molecular Medicine Research Center, Chang Gung University, Tao-Yuan, Taiwan, Department of Pathology, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan, and Department of Cell and Molecular Biology, Chang Gung University, Tao-Yuan, Taiwan Received February 18, 2009
Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer, which is one of the most prominent causes of cancer-related mortality worldwide. Discovery of serum tumor markers could facilitate early NSCLC detection and metastatic prognosis. Here, we simultaneously analyzed the NSCLC cell secretome and proteomic profiles of pleural effusion from lung adenocarcinoma patients for NSCLC biomarker discovery. Retinoblastoma-associated binding protein 46 (RbAp46), one of the proteins detected both in NSCLC cell secretome and pleural effusion proteome, was chosen for further evaluation. Both of RbAp46 mRNA and protein levels were upregulated significantly in NSCLC cancer tissues. Serum levels of RbAp46 were markedly higher in NSCLC patients than in healthy controls, and a combination of RbAp46 and CEA could outperform CEA alone in discriminating NSCLC patients from healthy persons. Importantly, elevated serum RbAp46 level was highly correlated with NSCLC distant metastasis. Moreover, knockdown of RbAp46 inhibited the migration ability of lung cancer cells. Our data collectively suggest that RbAp46 serves as a novel biomarker and prognosticator for NSCLC, and is involved in lung cancer cell migration. Keywords: Nonsmall cell lung cancer • Biomarker • Secretome • Pleural effusion • Retinoblastomaassociated binding protein 46
Introduction Lung cancer is one of the most prominent causes of cancer death over the world. The persistent poor survival for lung cancer patients is largely attributable to late stage at diagnosis. Nonsmall cell lung cancer (NSCLC), including squamous cell carcinoma, adenocarcinoma, large-cell carcinoma, and some * To whom should be addressed. Department of Cell and Molecular Biology, Chang Gung University, 259 Wen-Hwa first Road, Kwei-Shan, Tao-Yuan, Taiwan. Tel: 886-3-2118800, ext. 3424. Fax: 886-3-2118042. E-mail:
[email protected];
[email protected]. † Department of Thoracic Medicine, Chang Gung Memorial Hospital. ‡ Graduate Institute of Clinical Medical Sciences, Chang Gung University. ¶ These authors contributed equally to this work. # Graduate Institute of Biomedical Sciences, Biology, Chang Gung University. | Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University. § Molecular Medicine Research Center, Chang Gung University. O Department of Pathology, Chang Gung Memorial Hospital. ∇ Department of Cell and Molecular Biology, Chang Gung University. ⊥ These authors share equal contribution as senior authors.
4428 Journal of Proteome Research 2009, 8, 4428–4440 Published on Web 08/06/2009
rare subtypes, is the most common type of lung cancer, making up about 80% of all cases.1,2 The overall 5-year survival for NSCLC remains at only 15%; however, if the cancer is detected at stage IA, the 5-year survival often exceeds 80%.3 Radiological image studies, including chest X-ray and computer tomogram (CT), provide inappropriate accuracy for cancer diagnosis and disease staging.4,5 Positron emission tomography (PET) provides functional information of tumor activity and higher sensitivity (80-100%) for metastasis detection, but it is limited for routine utility because of its variable specificity (40-100%) and availability.6 Thus, it is urgent to find a good method for early cancer detection and metastatic discovery to improve survival rate. Tumor markers are biological parameters that can be measured in the serum, plasma, urine, saliva, or pleural effusion of suspected cancer patients. Sera proteins, such as CEA, CYFRA 21-1, squamous cell carcinoma antigen, neuron-specific enolase, progastrin-releasing peptide, tumor M2-pyruvate kinase, 10.1021/pr900160h CCC: $40.75
2009 American Chemical Society
research articles
RbAp46 as a Novel Prognostic Marker for Distant Metastasis C-reactive protein, and CA125 are reported as potential markers to indicate cancer presence, facilitate histological analysis as well as predict progression.7,8 Because of the limited sensitivity or specificity of potential markers described above, it is not currently recommended or encouraged in routine clinical practice for lung cancer.9 Proteomic approaches have been widely applied for the analysis of malignant diseases, especially in the field of plasma/ serum tumor marker identification.10,11 Beside the accessible human body fluids, identification of proteins secreted by or shed from cancer cell lines may greatly enhance the discovery of potential cancer plasma/serum biomarkers. Recently, several potential biomarkers discovered from cancer cell secretome analysis have been reported,12-19 such as collapsin response mediator protein-2 as a colon cancer marker;12 Mac-2 binding protein as a oral cancer marker;13 cathepsin D, syntenin, and gp100 as uveal melanoma biomarkers;14 and protein gene product 9.5, translationally controlled tumor protein, tissue inhibitors of metalloproteinases-2, and triosephosphate isomerase as the lung cancer biomarkers.15 These studies indicate that identification of cancer cell secreted proteins seems to be an efficient strategy for discovery of cancer markers, those can be measured in body fluid of suspected cancer patients. Most importantly, secretome data sets would offer a good opportunity to find promising candidates for early diagnosis as well as prognosis of cancer. Pleural effusion, an accumulation of pleural fluid, contains proteins originating from plasma filtrate, released by inflammatory cells or epithelial cell. Pleural effusion usually occurs in patients with congestive cardiac failure, bacterial pneumonia, tuberculosis, pulmonary embolism, cancer, and physical trauma.20 Biochemical examination of pleural fluid is usually performed to identify the cause of a pleural effusion. Several studies have shown that pleural effusions contain proteins of potential diagnostic value. These potential biomarkers include lung surfactant protein A (SP-A),21 carcinoembryonic antigen (CEA),21 cystatin C,22 vascular endothelial growth factor (VEGF),23 and pigment epithelium-derived factor (PEDF).24,25 In addition, in some patients with malignant lung carcinoma, the cancer cells could be identified in pleural effusion by cytological test. It is possible that cancer cells can migrate from primary tumor sites into pleural cavity through vascular invasion and lymphatic obstruction26 and then secrete protein into this fluid. Thus, there is a good opportunity to discover potential biomarkers for lung cancer from pleural effusion. Previously, Tyan et al. performed the proteomic profiling of pleural effusion from lung adenocarcinoma patients using twodimensional nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry (2D nanoHPLC-ESI-MS/MS) system and two-dimensional gel electrophoresis analyses.27-29 Till now, it is the best completed database for the diversity and relative abundance of various proteins found in the pleural exudates. The database contains 140 proteins, and would provide not only information on the nature of protein present in human pleural effusion, but also potential protein diagnostic markers to be examined in further investigations.28 In this study, we propose that combined analysis of NSCLC cell secretome and pleural effusion proteome form lung adenocarcinoma patients could be a feasible strategy to efficiently identify NSCLC potential serum biomarkers. We analyzed the secretome of two NSCLC cell lines, CL1-0 and CL1-5, by the onedimensional gel electrophoresis in conjunction with nano
liquid-chromatography tandem mass spectrometry (GeLC-MS/ MS) approach. This secretome data set was then combined with pleural effusion proteomic data set to search the potential targets for lung cancer serum biomarkers. Retinoblastoma associated binding protein 46 (RbAp46), one of the 22 candidates, was neither reported as plasma/serum protein nor reported as lung cancer marker and was chosen for further validation. We confirmed the overexpression of RbAp46 in lung cancer tissues by quantitative real-time PCR and immunohistochemistry. Importantly, we found serum levels of RbAp46 were significantly higher in lung cancer patients, compared to healthy controls, and positively correlated with distal metastasis of NSCLC. Moreover, knockdown of RbAp46 inhibited the migration ability of lung cancer cells. Our data collectively suggest that RbAp46 serves as a novel biomarker and prognosticator for NSCLC, and is involved in lung cancer cell migration.
Materials and Methods Cell Culture. CL1-0 and CL1-5 cells, established from a 64year-old man with a poorly differentiated lung adenocarcinoma, were kindly provided by Dr. P. C. Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China).30 CL1-0 and CL1-5 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) plus antibiotics at 37 °C in a humidified atmosphere of 95% air/5% CO2. Harvest of Conditioned Media from Cancer Cell Lines. Conditioned media from the various cancer cell lines were collected and processed as previously described.31 Briefly, cancer cells were grown to confluence in tissue culture dishes, washed with serum-free media, and incubated in serum-free media for 24 h. The supernatants were then harvested and centrifuged to eliminate the intact cells. Then, the supernatants were concentrated and desalted by centrifugation in Amicon Ultra-15 tubes (molecular weight cutoff 5000 Da; Millipore, Billerica, MA). The cells left on the dishes were washed twice with phosphate-buffered saline (PBS) and lysed in hypotonic buffer (10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 50 mM NaF, 20 mM Na4P2O7, 1 mM Na3VO4, 1 mM PMSF, 1 mM benzamidine, 0.5 µg/mL leupeptin, and 1% Triton-X100) on ice for 15 min. The cell lysates were collected and then sonicated on ice followed by centrifugation at 10 000g for 25 min at 4 °C. The resulting supernatants were used as the cell extracts. The protein concentrations of the various samples were determined using the BCA protein assay reagent from Pierce (Rockford, IL). Determination of Cell Viability. Cancer cell lines were cultured in complete medium for 24 h and then cells were grown in complete medium or serum-free medium for additional 24 h. Cells in the media and attached on the plates were collected separately, and the numbers of viable and dead cells in each fraction were determined using the trypan blue dye exclusion assay. The percentage of cell viability was expressed as the ratio of total viable cells to the sum of total viable and dead cells. One-Dimensional SDS-PAGE and In-Gel Digest of Proteins. Protein (50 µg) was resolved on 8-14% gradient SDS-PAGE and stained with 0.5% Coomassie Brillant Blue G-250. The whole gel lane was cut into 70 pieces and subjected to in-gel tryptic digestion, essentially as previously described.12 Briefly, the gel pieces were destained in 10% methanol, and then dehydrated in acetonitrile and dried in a SpeedVac. The proteins were Journal of Proteome Research • Vol. 8, No. 10, 2009 4429
research articles reduced with 25 mM NH4HCO3 containing 10 mM dithiothreitol at 60 °C for 30 min and alkylated with 55 mM iodoacetamide at room temperature for 30 min. After reduction and alkylation, proteins were digested using sequencing grade modified porcine trypsin (20 µg/mL) (Promega, Madison, WI) overnight at 37 °C. Peptides were extracted with acetonitrile, and dried in a SpeedVac. Reverse Phase Liquid Chromatography-Tandem Mass Spectrometry. Each peptide mixture was reconstituted in HPLC buffer A (0.1% formic acid, Sigma, St. Louis, MO), loaded into a trap column (Zorbax 300SB-C18, 0.3 × 5 mm, Agilent Technologies, Wilmington, DE) at a flow rate of 0.2 µL/min in HPLC buffer A, and separated on a resolving 10-cm analytical C18 column (inner diameter, 75 µm) with a 15-µm tip (New Objective, Woburn, MA). The peptides were eluted by a linear gradient of 0-10% HPLC buffer B (99.9% ACN containing 0.1% formic acid) for 3 min, 10-30% buffer B for 35 min, 30-35% buffer B for 4 min, 35-50% buffer B for 1 min, 50-95% buffer B for 1 min, and 95% buffer B for 8 min at a flow rate of 0.25 µL/min across the analytical column. The LC setup was coupled on-line with a 2-D linear ion trap mass spectrometer LTQ-Orbitrap (Thermo Fisher, San Jose, CA) operated using the Xcalibur 2.0 software (Thermo Fisher). Intact peptides were detected in the Orbitrap at a resolution of 30 000. Internal calibration was performed using the ion signal of (Si(CH3)2O)6H+ at m/z 445.120025 as a lock mass.32 The datadependent procedure that alternated between one MS scan followed by six MS/MS scans for the six most abundant precursor ions in the MS survey scan was applied. The m/z values selected for MS/MS were dynamically excluded for 180 s. The electrospray voltage applied was 1.8 kV. Both MS and MS/ MS spectra were acquired using the one microscan with a maximum fill-time of 1000 and 100 ms for MS and MS/MS analysis, respectively. Automatic gain control was used to prevent overfilling of the ion trap; 5 × 104 ions were accumulated in the ion trap for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 2000 Da. Database Searching and Bioinformatics. The resulting MS/ MS spectra were searched using SEQUEST algorithm (Thermo Fisher) against a nonredundant International Protein Index (IPI) human sequence database v3.26 (released February 2007; 67 665 sequences; 28 353 548 residues) from the European Bioinformatics Institute (http://www.ebi.ac.uk/IPIhuman.html). Search parameters included differential amino acid mass shifts for oxidized methionine (16 Da) and fixed modification for carbamidomethyl cysteine. All search results were filtered with the Xcorr (1+ g 1.9, 2+ g 2.3, 3+ g 3.75), Rsp (e3), and DeltaCn (g0.1). The random sequence database was used to estimate false positive rates for peptide matches, and the false positive rate for the peptide sequence matches using the criteria was estimated to be
