Quantitative Secretome Analysis Reveals that COL6A1 is a Metastasis

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Quantitative Secretome Analysis Reveals that COL6A1 is a MetastasisAssociated Protein Using Stacking Gel-Aided Purification Combined with iTRAQ Labeling Kuo-Hsun Chiu,†,‡ Ying-Hwa Chang,‡,§ Yu-Shun Wu,† Shu-Hui Lee,† and Pao-Chi Liao*,†,||,^ †

Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan Institute of Biopharmaceutical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan Sustainable Environment Research Center, National Cheng Kung University, Tainan, Taiwan ^ Center for Micro/Nano Science and Technology (CMNST), National Cheng Kung University, Tainan, Taiwan

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§

bS Supporting Information ABSTRACT:

In cancer metastasis, secreted proteins play an important role in promoting cancer cell migration and invasion and thus also in the increase of cancer metastasis in the extracellular microenvironment. In this study, we developed a strategy that combined a simple gel-aided protein purification with iTRAQ labeling to quantify and discover the metastasis-associated proteins in the lung cancer cell secretome. Secreted proteins associated with lung cancer metastasis were produced using CL1-0 and CL1-5 cells with different metastatic abilities. Quantitative secretomics analysis identified a total of 353 proteins, 7 of which were considered to be metastasisassociated proteins. These included TIMP1, COL6A1, uPA, and AAT, all of which were higher in CL1-5, and AL1A1, PRDX1, and NID1, which were higher in CL1-0. Six of these metastasis-associated proteins were validated with Western blot analysis. In addition, pathway analysis was performed in building the interaction network between the identified metastasis-associated proteins. Further functional analysis of COL6A1 on the metastatic abilities of CL1 cells was also carried out. An RNA interference-based knock-down of COL6A1 suppressed the metastatic ability of CL1-5 cells; in contrast, a plasmid-transfected overexpression of COL6A1 increased the metastatic ability of CL1-0 cells. This study describes a simple and high throughput sample purification method that can be used for the quantitative secretomics analysis of metastasis-associated proteins. KEYWORDS: isobaric tags for relative and absolute quantitation (iTRAQ), lung cancer, metastasis, secretome, quantitative proteomics

’ INTRODUCTION Metastasis is a complex, multistep process that includes the detachment of tumor cells from a primary lesion, their invasion into vascular or lymphatic vessels, their homing and adherence to destination organs, and the organogenesis of the metastasized cells in their new environment.1,2 During metastatic processes, secreted proteins abound in the extracellular microenvironment and increase tumorigenic metastasis by promoting cancer cell r 2010 American Chemical Society

migration and invasion.3 Secreted proteins enhance the invasive capability of cancer cells by forming fewer stress fibers and by increasing cell motility.4 Several reports have indicated that secreted proteins are associated with cancer metastasis.5-7 Thus, a secretomic analysis would help us to acquire a better compreReceived: August 26, 2010 Published: December 28, 2010 1110

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Journal of Proteome Research hensive understanding of the roles of secreted proteins in cancer metastasis. A proteomics approach is a powerful tool for large-scale studies and has been widely applied in the analysis of several cancer cell secretomes, including those of breast cancer,8-12 lung cancer,13-16 and nasopharyngeal cancer.17,18 Recently, many massspectrometry-based approaches have been described for conducting quantitative proteomics that have included label-free or labeling strategies to enable relative sample quantitation. The advantages of label-free proteomics approaches include the facts that analyses are faster and sample preparation is simpler than that of label-based approaches. However, the major challenges of label-free approaches include the normalization factor selected to reduce inter-run variations in mass spectrometry and computational and statistical analysis of the results.19 In the label-based approaches, the three most widespread methods for protein/ peptide labeling are chemical derivatization (e.g., Isotope Coded Affinity Tags [ICAT] and Isobaric Tags for Relative and Absolute Quantitation [iTRAQ]),20,21 metabolic labeling (e.g., Stable Isotope Labeling of Amino Acids in Culture [SILAC])22,23 and enzymatic labeling during protein digestion using deuterated water.24,25 The iTRAQ-labeling approach incorporates stable isotopes into an NHS-ester derivative amine tagging reagent that is ideally suited for comparative proteomic applications because it provides both quantitation and multiplexing in a single reagent and because the relative intensity of signature reporter ions is used to quantify protein levels and to compare the proteome.26-29 Additional advantages of this approach include the fact that iTRAQ labels each peptide fragment to increase labeling efficiency. What is more, isobarically summing iTRAQlabeled sample sets enhances the intensity of both the precursor and the product ions, thus increasing the number of peptides identified and quantified.30 For all of these reasons, iTRAQ was chosen for use in this study for the quantitative comparison of secretome samples. Most of the recent secretome analyses use two dimensions of separation to deal with the complexity of the secretome.10,11,17,18 Because of issues of compatibility, the contents of secretome samples or of the conditioned medium (CM) must be considered in downstream LC-MS. For instance, high salt contents and complex medium chemicals in a conditioned medium may affect the LC system or the resulting mass spectrometry. There have been several studies investigating cell secretomes using a gel-fractionation method for sample preparation.12,31-34 There is, however, little information in the literature regarding the purification method that can be combined with iTRAQ labeling for quantitative proteomics in the mammalian cell secretome. The secretome contains many exosomes, glycoproteins, and membrane proteins. These types of proteins are not easily extracted or digested, and chemicals contained in the extraction or digestion buffer (such as detergents) may not be compatible with the LC system. Several methods have been developed for the sample preparation of membrane proteins.30,35,36 Lawlor et al. (2009)11 developed an analytical platform that used a “stacking gel” method combined with a label-free approach for highthroughput profiling of the cancer cell secretome. Inspired by the method of Lawlor et al. (2009), we implemented a modified version and developed a novel strategy that combined a sample purification method combined with iTRAQ labeling to quantify secreted protein differences in CL1-0 and CL1-5 cells with different metastatic abilities. We used these two cell lines to discover metastasis-associated proteins through observation of

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differences in metastatic abilities. This method is simpler than that of other gel-fractionation approaches. It allows the secretome to be concentrated in a single gel band containing a sufficient number of proteins for iTRAQ labeling in a typical SDS-PAGE prior to an LC-MS/MS analysis. In our implementation, this strategy provided acceptable throughput in analysis and discovered a novel secreted protein (COL6A1) that is associated with lung cell metastasis.

’ MATERIALS AND METHODS Collection of CL1 Cell Secretome

Lung cancer cell lines (CL1-0 and CL1-5 cells) with different invasive and metastatic capabilities were kindly provided by Dr. P. C. Yang (Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan, Republic of China)37 and were maintained in an RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37 C under 5% CO2. Harvest of Conditioned Media from Cancer Cell Lines

CL1 cells were grown to confluence in tissue culture dishes, washed with serum-free media three times to avoid serum contamination, and incubated in serum-free media for 24 h. The supernatants of the conditioned media (CM) were then harvested and centrifuged to eliminate the intact cells. The supernatants were then concentrated and desalted by centrifugation in Amicon Ultra-15 tubes (molecular weight cutoff 3000 Da; Millipore, Billerica, MA). The protein concentrations of CL1 CM samples were determined using the Bradford protein assay reagent (Biorad). Stacking Gel-Aided Purification Method

A simple protein purification was performed using a gel-aided sample preparation modified from Lawlor et al. (2009).11 The secretome from concentrated CM samples were run on a selfpoured stacking SDS-PAGE gel. The resolving gel portion (0.6 mL of H2O, 2.22 mL of 1.5 M Tris-HCl [pH 8.8], 90 μL of 10% SDS, 6 mL of Bis/Acrylamide, 90 μL of 10% ammonium persulfate, and 5 μL of TEMED) was poured and set to polymerize for one hour. The stacking gel portion (2.9 mL of H2O, 0.5 mL of 1 M Tris-HCl [pH 6.8], 40 μL of 10% SDS, 520 μL of Bis/ Acrylamide, 40 μL of 10% ammonium persulfate, and 4 μL of TEMED) was poured next. A homemade comb (1 mm thickness) was inserted and set to polymerize for 30 min. A total of 100 μg of CM was mixed with 13 μL of H2O, 5 μL of 4X SDS sample buffer, and 2 μL of 0.5 M DTT and then boiled for 10 min. The gel was run at 55 V. The electrophoresis was stopped after the sample had barely passed into the resolving gel, and the gels were then stained using Coomassie Brilliant Blue (CBB) R-250. In-Gel Digestion

The gel pieces corresponding to the CL1 secretomes were diced into ∼1 mm3 pieces. Gel slices were washed and dehydrated three times in 25 mM ammonium bicarbonate (ABC) (pH 7.9) and 50 mM ABC/50% acetonitrile. A protein reduction was subsequently performed by incubating 0.5 M DTT for 1 h at 56 C and then alkylating with 50 μL saturated IAA for 45 min at room temperature in the dark. After two subsequent wash/ dehydration cycles, each gel sample was digested with 20 μg of sequencing-grade modified trypsin (Promega), adding a sufficient volume of 25 mM ammonium bicarbonate to completely saturate the gel. The sample was incubated at 37 C for an overnight digestion. Following the digestion, peptides were 1111

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Journal of Proteome Research extracted twice in 100 μL of 50% ACN in 5% formic acid. The extracted peptides were enriched using OMIX C18 pipet tips (Varian) to remove the effects of any remaining reagents on the iTRAQ labeling. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Labeling

The enriched peptides were labeled with the iTRAQ reagent (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. Briefly, one unit of label (defined as the amount of reagent required to label 100 μg of protein) was thawed and reconstituted in ethanol (70 μL). The peptide mixtures were reconstituted with 20 μL of iTRAQ dissolution buffer. The aliquots of iTRAQ 114, 115, 116, and 117 were combined with the peptide mixtures from the CL1-0 and CL1-5 cell secretomes. The extracted peptide mixtures were then pooled and dried by vacuum centrifugation. The dried peptide mixture was reconstituted and acidified with 10 μL of buffer (5 mM K2HPO4 and 25% ACN [pH 3]) for fractionation by SCX chromatography using an AKTA FPLC system (GE Healthcare). A total of 28 fractionations were generated and were desalted using OMIX C18 pipet tips (Varian) according to the user instructions. NanoLC-ESI-MS/MS

The LC-MS/MS conditions were modified from those of the previous study.30 iTRAQ-labeled samples were reconstituted in eluent buffer A (0.1% (v/v) FA in H2O) and analyzed by LCMS/MS. The buffer B (0.1% (v/v) FA in ACN) gradient started from 0% to 5% at 2 min and then progressed to 37% in 140 min, eluting peptides at 200-300 nL/min. Peptide fragmentation by collision-induced dissociation was performed automatically using the information-dependent acquisition in Analyst QS v1.1 (Applied Biosystems). The method applied a 1-s TOF MS scan and automatically switched to three 2-s product ion scans (MS/ MS) when a target ion reached an intensity of greater than 20 counts. TOF MS scanning was undertaken over the range 4002000 m/z. Product ion scans were undertaken over the range 100-2000 m/z at low resolution. Database Search

For protein identification, data files from the LC-ESI-MS/ MS were batch-searched against the Swiss-Prot human sequence database (version 20090616; 468851 sequences) using the MASCOT algorithm (v2.1.0, Matrix Science, London, U.K.). The peak list in the MS/MS spectra generated under ESI-Q-TOF was extracted with AnalystQS 1.1 (Applied Biosystems) with the default charge state set to 2þ, 3þ, and 4þ. The MS and MS/ MS centroid parameters were set to 10% height percentage and to a merge distance of 0.1 amu. For the MS/MS grouping, the averaging parameters consisted of rejection of spectra with less than five peaks or precursor ions with less than 10 counts/s. Search parameters for peptide and for MS/MS mass tolerance were 1 and 0.5 Da, respectively, with allowance for two missed cleavages made in the trypsin digest and for variable modifications of deamidation (Asn, Gln), oxidation (Met), iTRAQ (Nterminal), iTRAQ (Lys), and carboxyamidomethylation (Cys). Peptides were considered to have been identified if their MASCOT individual ion score was higher than the MASCOT score 20. Protein Quantification

For protein quantitation, data analysis for the iTRAQ experiments was performed with the software Multi-Q.38 The raw data files from QSTAR Pulsar I were converted into files of mzXML format by the program mzFAST, and the search results in MASCOT

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were exported in comma-separated-values (CSV) data format. After the data conversions, Multi-Q selected unique, iTRAQlabeled peptides with confident MS/MS identifications (MASCOT score 20), detected signature ions (m/z 114, 115, 116, and 117), and performed an automated quantitation of peptide abundance. For the detector dynamic range filter, signature peaks with ion counts higher than 1000 or lower than the low quartile were filtered out by Multi-Q. To calculate the average protein ratios, the ratios of quantified, unique iTRAQ peptides were weighted according to their peak intensities to minimize standard deviation. The final protein quantitation results were exported to an output file in CSV data format. Bioinformatics Analysis

The identified proteins were analyzed using the SignalP,39,40 SecretomeP,41,42 and TMpred43 programs to predict the possibility of protein secretion through classic or through nonclassic secretion pathways and the presence of transmembrane domains in the protein sequence. The molecular functions of the identified proteins were determined based on a search against the Human Protein Reference Database (HPRD) (http://www. hprd.org/). For pathway analysis, the accession numbers of the metastasisassociated proteins that were identified as significantly different in CL1-0 and CL1-5 were uploaded and analyzed for evidence of an interaction network using MetaCore from GeneGo Inc., an integrated, manually curated knowledge database of gene/protein lists. Small Interfering RNA

COL6A1 gene silencing was achieved though transfection with COL6A1 siRNA using the siRNA transfection reagent according to the manufacturer’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA). CL1-5 cells (2 105) were seeded in a six-well culture plate in 2 mL of antibiotic-free RPMI 1640 medium supplemented with 10% FBS. Cells were incubated at 37 C in a CO2 incubator until 80% confluent. For each transfection, 0.5 μg of COL6A1 siRNA or control siRNA with 4 μL of siRNA transfection reagent was added to 100 μL of siRNA transfection media. The solution was mixed gently and overlaid onto the cells for 24 h. The media was then aspirated and the cells were maintained in RPMI 1640 medium supplemented with 10% FBS for further study. Over-Expression

Twenty-four hours prior to the transfection experiments, 2 105 CL1-0 cells were grown in RPMI 1640 containing 10% fetal bovine serum (FBS) on six-well culture dishes reaching 70% confluence. CL1-0 cells were then grown in RPMI 1640 media and transfected with plasmids containing COL6A1 and the empty vectors. For each overexpression transfection experiment, 200 μL of RPMI 1640 serum-free medium containing 4 μL of transfection reagent (TurboFect, Fermentas) mixed with 2 μg of plasmid was added to the CL1-0 cells for 24-h incubation. Transfection efficiency was monitored by a Western blotting analysis.

Western Blotting Analysis

Western blotting analysis was used to examine the expression levels of selected proteins. These proteins included PRDX1, NID, AL1A1, COL6A1, uPA, TIM1, AAT. Five to eight micrograms of secreted proteins from the CL1 cell CMs were separated on a 12% SDS-PAGE and transferred to PVDF membranes (Millipore). The membranes were blocked in a 5% nonfat milk solution for 1 h at room temperature and then probed with 1112


Journal of Proteome Research various antibodies against the selected proteins (Santa Cruz Biotechnology) and against anti-R-tubulin (Calbiochem). The membranes were then washed thrice with TBST and incubated with secondary antibody in TBST/2% skim milk. Bound antibody was detected using the Enhanced Chemiluminescence System. Chemiluminescent signals were captured using the Fujifilm LAS 3000 system (Fujifilm). All experiments were performed at least thrice in duplicate. Wound Healing Assay

CL1 cells were seeded in precision-molded inserts (Ibidi, Martinsried, Germany) that created a defined wound gap to monitor cell migration and grown in the RPMI 1640 medium containing 10% FBS according to manufacturer’s protocol. Cells were allowed to close the wound for 24 h. Images were taken at 100 magnification, and photographs were taken at 0 and 24 h at the same position in the wound. The areas of the cell-free zone into which cells migrated (based on the zero line of the linear “wound”) were quantitated under the microscope using ImagePro Plus software (Version 6.0). Migration Assay

A transwell membrane (8-μm pore size, BD Biosciences) was used for a transwell migration assay, The CL1 cells were trypsinized, washed, and kept suspended in their medium without FBS. To the lower wells of the chambers, a migrationinducing medium (with 10% FBS) was added. The upper wells were filled with a serum-free medium with cells (10 000 cells per well), and the lower chambers were filled with an RPMI 1640 medium supplemented with 10% FBS to induce cell migration. After 8 h, the assays were stopped by the removal of the medium from the upper wells and the careful removal of the filters. The filters were fixed with methanol and then stained with 20% Giemsa solution (Sigma). The evaluation of the completed transmigration was performed under the microscope, and the cell number on each filter was counted in five randomly selected fields under a microscope (200). Matrigel Invasion Assay

Cell invasion was examined in a membrane invasion culture system.44 A transwell membrane (8-μm pore size, BD Biosciences) coated with Matrigel basement membrane matrix (2.5 mg/mL; BD Biosciences Discovery Labware) was used for the invasion assay. Cells (1 105) were seeded into the upper wells in an RPMI 1640 medium, and the lower chambers were filled with an RPMI 1640 medium supplemented with 10% FBS. After incubating at 37 C for 24 h, cells on the upper side of the filter membrane were gently removed with cotton swabs. The number of cells migrating through the membrane to the lower side was determined by fixing the membranes with methanol and staining the cells with propidium iodide. The cell number on each filter was counted in five randomly selected fields under a microscope (200). Statistical Analysis

All data were expressed as means with (standard deviations). The statistical significance of the differences between treatments was determined by a Mann-Whitney U test. p < 0.05 was considered to be statistically significant.

’ RESULTS AND DISCUSSION Stacking Gel-Aided Sample Purification Applied in CL1 Cell Secretome Profiling

To prove the feasibility and reproducibility of the stacking gelaided purification method that we combined with the iTRAQ

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labeling used in this study, we first applied the method to a control sample (i.e., bovine serum albumin [BSA]) with a known concentration. Two sets of BSA with same (1:1) and different concentrations (2:1) were subjected to stacking gel-aided purification and in-gel digestion, labeled with two isobaric reagents (i.e., iTRAQ115 and iTRAQ116 for 1:1; iTRAQ 114 and iTRAQ117 for 2:1), were pooled, and were then analyzed by LC-MS/MS. Peptide and protein quantitation was performed on a software Multi-Q38 to automatically process the iTRAQ signature peaks, including peak detection, background subtraction, isotope correction, and peptide normalization to remove systematic errors. In the representative MS/MS spectra of the BSA with the same concentration (1:1), two peptides, (KVPQVSTPTLVEVSR [m/z 643.4] and QTALVELLK [m/z 435.9]), from the BSA were identified (Supplemental Figure 1A, Supporting Information), and the peak of their reporter ions from the three spectra in the m/z 115 and 116 that displayed the average ratio of signature iTRAQ fragments was 1.02. The mean coefficient of variation (CV) was between 14.4% and 18.5%. In the second set of BSA with a concentration of 2:1, two peptides were identified (KVPQVSTPTLVEVSR [m/z 643.4] and QTALVELLK [m/z 435.9]) (Supplemental Figure 1B, Supporting Information), and the m/z 114 and 117 showed that the average ratio of these four peptides was 1.97:1 with a CV of between 15.4 and 17.8%. This can be compared with the results obtained from quantitative proteomics approaches using iTRAQ labeling in which (in iTRAQ experiments) the mean CV was around 20%.21,45,46 These results serve to demonstrate that this sample purification method combined with iTRAQ labeling shows acceptable reproducibility and accuracy in quantitative proteomics analysis. This study was designed to develop a strategy that would combine a simple sample purification and iTRAQ labeling approach. On the basis of the results obtained in our laboratory, secretome samples usually cannot be effectively analyzed using only ultrafiltration combined with in-solution digestion and iTRAQ labeling for LC-MS/MS. A greater amount of proteins (100 μg for each analytical run) than that of the label-free approach was required for iTRAQ labeling in labeling-based quantitative proteomics. CL1 cell secretomes were prepared using a stacking gel-aided purification method that introduced proteins into stacking gels to be concentrated into tight protein bands. A single band concentrating the proteins was cut from the gel and subjected to a standard in-gel tryptic digestion protocol (Figure 1). Digested peptides from the CL1-0 and CL1-5 cell secretomes were labeled with iTRAQ labeling. In the two-run analyses of CL1 cell secretome, we identified a total of 353 proteins, and the associated information of these identified proteins was listed in Supplemental Table 1 (Supporting Information). Large-scale analysis of secretome samples was usually carried out using directly gel-free in-solution digestion or gel-based separation followed by LC-MS/MS or MALDI-TOF MS. In general, more proteins from secretome samples were identified using LC-MS/MS than MALDI-TOF MS. For instance, Matsumoto et al. (2009)13 directly used MALDI-TOF MS to analyze the secretome of a pulmonary large cell neuroendocrine carcinoma cell line (LCN1), and only two peptide fragments of 40 and 19 amino acid residues were identified. Mbeunkui et al. (2005)12 used a 2D LC-MS/MS system to identify secreted proteins, and they obtained an average of 88 proteins in each cancer cell line. Along with the advances in technology for 1113


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Figure 1. Experimental workflow of the secretome sample preparation and of the data analysis. The flowchart shows the various steps of the secretome analysis approach described in this report.

protein separation and identification, more proteins have been identified in secretome samples. Jacobs et al. (2008)10 analyzed human mammary epithelial cell (HMEC) secretome using insolution digestion followed by LC-MS/MS, and obtained 151 proteins that were enriched in the extracellular compartment. This should prove to be very important in sample preparation prior to LC-MS/MS analysis. Piersma et al. (2009)32 compared tC2 reverse-phase cartridge, SCX chromatography, and 1D SDSPAGE gel for preparing H460 secretome samples prior to LCMS/MS, and they identified the most proteins using 1D SDSPAGE gel electrophoresis. Wu et al. (2010)33 also used 1D SDSPAGE gel electrophoresis followed by in-gel digestion and LCMS/MS to investigate 11 cancer types and identified more than 1000 proteins in each cancer type. Therefore, the gel-enhanced fractionation of proteins has proven to be a useful tool in secretome analyses for sample preparation and, more extensively, to obtain a fractionated product of at least a few hundred proteins.32 Lawlor et al. (2009)11 have demonstrated that the number of fractionated gel slices did not reduce the number of identified proteins. A stacking gel-aided purification method using a minimal fractionation strategy can limit the proteins to be concentrated in the tight gel band of a stacking gel; it can thus increase the efficiency of throughput for secretome analysis. In addition, proteins of a broad-range molecular weight (from 7 to 600 kDa) were identified with this method, and it can be assumed to be capable of analyzing most of the range of the protein mixture and of being applied in the analysis of other complex secretome samples. Roelofsen et al. (2009)34 identifed secreted proteins by comparing isotope-labeled amino acid incorporation rates (CILAIR), and used SDS-PAGE for the fractionation of adipose cell

secretome, providing about 340 proteins. Thus the gel-assisted fractionation was also useful as a labeling method. In the present study, we minimized the fractionation number to a single gel band, and identified 353 proteins. We believe that this stacking gel-aided purification method can be further applied in quantitative proteomics approaches with labeling methods. Because the potentials of this method were compatible with those of iTRAQ labeling in its discovery of the metastasis-associated proteins in a secretome, we predicted the possible secretion pathways of these identified proteins using the SignalP, SecretomeP, and TMpred programs to predict the secreted proteins. According to the predictions, 22.1, 31.2, and 83.0% of the identified proteins could be assumed to be secreted proteins depending on their secretion pathways (classic or nonclassic) or the presence of transmembrane domains (Supplemental Table 1 and Figure 2, Supporting Information). In addition, the cellular components and biological processes of these identified proteins were categorized based on the HPRD (Figure 2B and C). The largest proportion of the identified proteins from the CL1 cell secretome functioned in the metabolism and energy pathway. During cancer metastasis, the proteins associated with catalytic activity possess specific binding sites for substrates and lead to catalysis of a biochemical reaction.17 For instance, members of the cathepsin family, including serine, cysteine, and aspartate protease, play important roles in cancer progression and metastasis.47 In addition, some reports have shown that energy metabolism is associated with cancer progression.48-50 Thus, for the proteins associated with energy metabolism (especially those that were potentially secreted into CM), the secretion pathway may be regulated through exosome secretion. For instance, alpha-enolase is an intracellular metabolic 1114



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Figure 2. Classification of the identified proteins of the CL1 secretome. Estimation of contamination of intracellular proteins in conditioned medium (CM). (A) Antitubulin and antiactin antibodies were used for the detection of the intracellular protein due to cell lysis within CM and cell lysate (CE) samples. (B) Identified proteins were predicted as possible secreted proteins according to the SignalP 3.0, SecretomeP 2.0, and TMpred programs. Proteins involved in exosomes were according to the exosome library ExoCarta. The (C) cellular components and (D) biological processes of identified proteins were based on the Human Protein Reference Database (HPRD).

enzyme and is identified in nasopharyngeal carcinoma CM.17 The number of proteins belonging to the classical or nonclassical secretion pathways in this case was less than that belonging to the presenting transmembrane domains. These differences may be the result of the release of CL1-secreted proteins via exosome secretion. In the identified proteins, there were 190 (53.8%) that were involved in exosome secretion (according to the exosome library ExoCarta51). Confirmation of Metastasis-Associated Proteins by Western Blot Analysis

CL1-0 and its cell sublines (CL1-1 to CL1-5) have been widely used to investigate the mechanism of lung cancer metastasis. Previous studies have generally used CL1-0 and CL1-5 to investigate the lung cancer metastasis.52-57 In addition, the advantage of using the parental cell and its subline was to avoid the variations in heterogeneity between cancer cell types. Because CL1-5 is the CL1-0 subline with the most metastatic ability, we compared CL1-0 and its subline, CL1-5 secretome samples, to discover metastasis-associated proteins. We selected a fifth percentile portion of each run of those identified proteins that were supposed to be highly correlated to lung cancer metastasis. We excluded those proteins that were identified in only one-run experiments. Those proteins at a higher

level in CL1-0 or CL1-5 were listed in Table 1. We then selected the potentially secreted proteins with the secretion characteristics using the SignalP 3.0, SecretomeP 2.0, and TMpred programs. Finally, three proteins at higher levels in CL1-0, and four at higher levels in CL1-5 were selected. The selected proteins higher in CL1-0 included retinal dehydrogenase 1 (AL1A1), nidogen-1 (NID-1), and peroxiredoxin-1 (PRDX1). In contrast, the proteins higher in CL1-5 were collagen alpha-1 (VI) chain (COL6A1), metalloprotease inhibitor 1 (TIMP1), urokinase-type plasminogen activator (uPA), and alpha-1-antitrypsin (AAT). Of these seven secreted proteins, six were selected for confirmation using Western blot analysis (Figure 3). These proteins included two proteins that were higher in CL1-0, NID1(Figure 3A), and four that were higher in CL1-5, COL6A1, TIMP1, uPA, and AAT (Figure 3). The Western blot results showed a consistent trend in protein abundance in the quantitative secretomic results. Lung Cancer Metastasis-Associated Secretome Pathway Analysis

The proteins expressed higher in CL1-0 cells that were confirmed by Western blot analysis included PRDX1 and NID1. PRDX1 is a member of the group of small nonseleno peroxidases and is involved in cellular redox regulation and antioxidant 1115


1116

a

Peroxiredoxin-1

Retinal dehydrogenase 1

Urokinase-type plasminogen activator Metalloproteinase inhibitor 1

Alpha-1-antitrypsin

Collagen alpha-1(VI) chain

Q06830

P00352

P00749

P01009

P12109

183

123

93

77

165

179

188

15%

10%

10%

23%

19%

26%

7%

protein protein sequence score coverage

Extracellular matrix structural constituent

Serine-type peptidase activity Extracellular matrix structural constituent Protease inhibitor activity

Aldehyde dehydrogenase activity

Extracellular matrix structural constituent Peroxidase activity

molecuar functiona

subcellular locationa

Cell growth and/or maintenance

Cell growth and/or maintenance Protein metabolism

Up-regulation in CL1-5 Protein metabolism

Metabolism ; Energy pathways Aldehyde metabolism

Extracellular,Endoplasmic reticulum,Lysosome, Cytoplasm Extracellular

Plasma membrane, Cytoplasm,Extracellular Extracellular,Cell surface

Cytoplasm,Nucleus, Nucleolus,Mitochondrion Cytoplasm

Up-regulation in CL1-0 Cell growth and/or maintenance Extracellular

biology functiona

þ þ

-

þ þ

þ

þ

þ

þ

þ

-

þ

þ

þ

þ

þ

þ

þ

þ

þ

þ

10

3

4

7

7

8

7

no. of matehed SignalP SecretomeP TMpred unique peptides

Subcellular locations, molecular function and biological function of the identified proteins were referred to the Human Protein Reference Database (http://www.hprd.org/).

P01033

Nidogen-1

protein name

P14543

Swiss-Prot accession no.

Table 1. Metastasis-Associated Proteins Identified From CL1 Cell Secretome S.D.

3.34 0.69

11.13 5.03

4.52 1.41

9.60 5.66

0.37 0.06

0.55 0.06

0.55 0.04

ratio

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Figure 3. Validation of identified metastasis-associated proteins. Western blot analysis was performed for the metastasis-associated proteins identified from the CL1 cell secretome. Twenty-microgram proteins from three CL1-0 and CL1-5 conditioned media (CM) and cell lysate (CE) and were separated in 12% SDS gel, transferred onto a PVDF membrane, and then probed with NID1 and PRDX1, uPA, TIMP1, AAT and COL6A1 antibodies against the target proteins.

protection in addition to the catalysis of peroxide reduction and to the balancing of cellular H2O2 levels essential for signaling and metabolism.58 Thus, PRDX1 can play a role in inhibiting tumor growth59-61 and is associated with cancer metastasis.62,63 The NIDs (NID-1 and NID-2) bind and form a ternary complex with laminin-1 and collagen type IV and interact with cell receptor molecules. In addition, NIDs have also been reported to regulate cancer cell migration and invasion.64-66 The establishment of lung-cancer metastasis CL1 cell sublines with different metastatic abilities were based on an in vitro invasion chamber,37 and then different sublines (subpopulations) with different metastatic abilities were selected. In other words, the original CL1 population contains cells with different metastatic abilities. The development of cancer cells includes a multistep process that generates a transformation of normal cells into malignant cells,67 and some cells still in the state that does not transforme to a malignant subline with higher metastatic ability, CL1-0 belongs to the subpopulation with the low metastatic ability, the role of proteins, NID-1 and PRDX-1, with higher expression in the CL1-0 cells should be involved in maintenance of cell proliferation or growth, In contrast, the proteins expressed higher in the CL1-5 cells included TIMP1, uPA, AAT, and COL6A1. TIMP1, an inhibitor of matrix metalloproteinases, is correlated with a poor prognosis in cancer. The elevations of TIMP-1 concentrations have been thought to reflect a general up-regulation of MMPs; TIMP-1 may promote cancer progression by enhancing the proliferation of endothelium and angiogenesis, and its function has been reported to be associated with cancer growth and metastasis.68-74 The serine protease uPA, which binds to the specific cell surface receptor uPAR, facilitates the conversion of plasminogen into the serine protease plasmin. Most

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components of the uPA system of plasminogen activation have been linked to cell adhesion and migration through both proteolytic and nonproteolytic mechanisms. In addition, the uPA system of plasminogen activation has been correlated with cancer invasion and metastasis.75-80 AAT is a secretory glycoprotein and is the most abundant serine protease inhibitor in human plasma. Proteolytic enzymes play a significant role in the expression of the malignant phenotype, including the loss of growth regulation, invasiveness and the formation of metastases.71-85 Although it is an inhibitor of the serine protease that contributes to cell invasiveness and metastasis, AAT in elevated serum levels has been observed in the course of a large number of malignant diseases and different cancers.86,87 Collagen VI is a ubiquitous extracellular matrix protein which forms a microfibrillar network in close association with the basement membrane around muscle cells and which interacts with several other matrix constituents.88-92 Because COL6A1 is a major component of the extracellular matrix and is involved in the organization of fibronectin, binding cells, and other types of collagen, including types I and IV.93,94 COL6A1 has been implicated in cell migration and differentiation, and thus changes in COL6A1 gene expression have implications for cancer cell growth. COL6A1 gene expression has been related to cell growth and cancer development and progression.95 However, the mechanism of COL6A1 in cancer metastasis is still unclear, and there have been few studies that have reported the relationship between COL6A1 and cancers. On the basis of the functions of these metastasis-associated proteins, the locations of their functions are mainly in the ECM. To further prove and elucidate the relationship of the metastasis-associated proteins identified by the iTRAQ analysis and their significance in the process of lung cancer metastasis, these selected metastasis-associated proteins were analyzed by applying the MetaCore analytical tool. Biological networks were built as shown in Figure 4. Directly or indirectly regulated protein-protein interaction networks were built by MetaCore analysis based on our identified proteins. Results showed that COL6A1, TIMP1, AAT, and uPA function in the extracellular space and that they interact with NID-1 located in the membrane. NID-1 can bind laminin-1 and collagen type IV to form a protein complex, and the previous study showed that u-PA binds to laminin-NID by interactions that take place in the u-PA amino-terminal region.96 It was proposed that this may facilitate the loading of u-PA between adherent cells and their matrix.96 We assumed that the driving force for uPA secretion should also be mediated by the interaction with NID1. According to the pathway analysis, the interaction of secreted proteins was involved in ECM remodeling. TIMP1 can reduce excessive proteolytic ECM degradation by MMPs, and this balanceof MMPs and TIMP controls the extent of ECM remodeling.97,98 In addition, uPA and plasminogen activators play an important role in the extracellular protease system for ECM remodeling.99 Plasmin directly degrades ECM proteins and also activates a number of MMPs that degrade the ECM proteins and the components of the basal membrane, for example, collagens.100-102 Thus, during metastasis, the role of COL6A1 was mediated via protein-protein interaction in ECM remodeling. Other extracellular proteins that were expressed and identified in the network, such as matrilysin (Matrix Metalloproteinase 7; MMP-7), MMPs, and macrophage migration inhibitory factor 1117



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Figure 4. Pathway analysis of the metastasis-associated secreted proteins. A protein-protein interaction network built using the MetaCore program showed the locations of and interactions among the metastasis-associated proteins. The circles represent the proteins that were identified in this study, and the symbols and links shown on the network illustration are described in the key.

(MIF), have been associated with lung cancer.103-110 The MMP family is recognized as a group of collagenase-related connectivetissue-degrading metalloproteinases capable of degrading multiple components of the extracellular matrix, or stroma, and leading to increased metastasis.111 Because the degradation of the ECM is mediated by MMPs, it is a major step in the process of cancer invasion and metastasis. On the basis of the results obtained from the pathway analysis, the metastasis-associated proteins identified in this study that were determined to be contributing to cancer metastasis interacted with or were regulated by the MMPs. On the basis of the results obtained from the pathway analysis, COL6A1 was directly regulated by proteolysis of matrilysin (MMP-7) and MMP-2 in the ECM (Figure 4). However, the regulation of COL6A1 with MMPs in metastasis has not been reported. In addition, COL6A1 was predicted as the downstream target of macrophage migration inhibitory factor (MIF) in the interaction network. MIF is a protein that is involved in cell-mediated immunity, immunoregulation, and inflammation.108 It has also been implicated in the control of cancer angiogenesis.109,112,113 MIF was also identified in our study (Supplemental Table 1, Supporting Information). Some studies have shown that MIF upregulates the expression of MMPs.115-117 However, the role of COL6A1 in connection to MMPs and MIF is still unclear.

In addition, diseases that were regulated through the interaction network included neoplastic processes (50.98%; p = 6.15 10-15), nonsmall cell carcinoma (47.06%; p = 3.65 10-13), and neoplasm metastasis (47.06%; p = 2.02 10-13). Thus, the results obtained from pathway analysis indicated that the metastasis-associated proteins discovered in this study were involved in carcinoma or metastasis. These results demonstrate that the methodology applied in this study to determine the metastasis-associated proteins was reliable and workable. COL6A1 Significantly Altered the Metastasis Levels in CL1 Cells

The secreted proteins selected in our study provided insight into the molecular interactions of the extracellular matrix and provided valuable information for further lung cancer metastasis studies. Furthermore, to provide a direct proof-of-concept in the methodology for discovering metastasis-associated proteins, we chose COL6A1 for the further functional assay in metastasis because there is little understanding of its biological function in cancer metastasis. Despite its relationship to biological and pathological processes in prostate cancers, the relationship between COL6A1 and metastasis or lung cancer has not yet been determined. Thus, the scientific merit of this study is based in its application for discovering novel metastasis-related proteins in lung cancer. 1118



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Figure 5. Suppression of the migration and invasion abilities of CL1-5 cells by the knock-down of COL6A1. (A) Comparisons are the product of Western blot analysis of the CM and the cell lysate of the COL6A1-siRNA-treated CL1-5 cells. Also indicated are (B) the wound healing assay, (C) the transwell migration assay, and (D) the Matrigel invasion assay of the COL6A1-siRNA-treated CL1-5 cells after 24 h.

We examined the role of COL6A1 in migration and invasion (two important characteristics of cancer metastasis) in CL1 cells. Before we tested the effects of COL6A1 on metastatic ability, Western blot analyses were used for evaluating the efficiency of COL6A1 transfection (Figures 5A and 6A). The knock-down of COL6A1 using COLA1 siRNA in CL1-5 cells completely suppressed COL6A1 expression in CL1-5 cell lysate and the CM at 24 h (Figure 5A). This indicated that the knock-down and transfection efficiency of COL6A1 siRNA were effective. Cell migration was examined by a wound-healing assay. Normal and control CL1-5 cells exhibited a higher migrating capability than CL1-0 cells. COLA1 siRNA decreased the migrating capability in the COL6A1-siRNA-transfected CL1-5 cells at 24 h (Figure 5B). We also performed a transwell migration assay to further confirm these observations. A decrease in migration was observed in the COL6A1-siRNA-transfected CL1-5 cells in comparison with the normal and control CL1-5 cells (Figure 5C). To further investigate whether COL6A1 has an effect on the invasive capability of CL1-5 cells, a Matrigel invasion assay was

performed. CL1-5 cells exhibited a higher invasive capability than CL1-0 cells. COL6A1 siRNA led to a dramatic decrease of invasion in the COL6A1-siRNA-transfected CL1-5 cells at 24 h (Figure 5D). These results demonstrate that the knock-down of COL6A1 expression impairs migration and invasion in CL1-5 cells and that COL6A1 is critical for migration and invasion in CL1-5 cells. We have demonstrated that the knock-down of COL6A1 expression using RNAi technology impaired migration and invasion in CL1-5 cells. The results indicated that COL6A1 was a critical regulator of migration and invasion in lung cancer. In addition to the examination of the role of COL6A1 in CL1-5 cells, we also tested the role of COL6A1 in those CL1-0 cells that had lower metastatic abilities. CL1-0 cells were transfected by COL6A1 to increase COL6A1 expression in the CL1-0 cells and to evaluate the effects of COL6A1 expression on migration and invasion abilities. The overexpression of COL6A1 increased the COL6A1 contents in CL1-0 cell extracts and in the CM (Figure 6A). Compared with the normal and control CL1-0 cells, the overexpression of COL6A1 1119



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Figure 6. Increase of the migration and invasion abilities of CL1-0 cells by the overexpression of COL6A1. (A) Comparisons were performed by Western blot analysis of the CM and the cell lysate of COL6A1 overexpressed CL1-0 cells. Also indicated (B) are the wound healing assay, (C) the transwell migration assay, and (D) the Matrigel invasion assay of the COL6A1 overexpressed CL1-0 cells after 24 h.

increased CL1-0 cells’ migration in the wound healing and transwell experiments at 24 h (Figure 6B and C). In addition, COL6A1 also increased the invasion abilities of CL1-0 cells (Figure 6D). However, the level of metastatic abilities by COL6A1 in CL1-0 cells still cannot reach as the CL1-5 cells. Metastasis is a multistep process, involving a series of sequential, interrelated events that are influenced by multiple interactions of metastatic cells within the host microenvironment.118-121 There are many proteins that may interact with COL6A1 in the ECM in cancer metastasis. Collagen plays an important role in cell adhesion,122-125 and thus we assume that the role of COL6A1 may be that of other collagen chains involved in the adhesion of cells to the extracellular matrix. We thus assumed that an alternative method of COL6A1 regulation might be involved in cell adhesion (proposed in Figure 7) because adhesion is mediated by a series of matrix-associated and cell-surface molecules that interact with each other in a spatially and temporally regulated manner. These molecules, which interact with COL6A1, may contribute to adhesion ability during metastasis.

Because COL6A1 was positively correlated with the CL1 cell metastasis, we suggest that the regulation of MMPs to COL6A1 does not operate through proteolysis to degrade the COL6A1 levels that lead to metastasis in the interaction network (Figure 4). We propose a mechanism for the regulation of COL6A1 in lung metastasis (Figure 7). On the basis of the proposed mechanism, COL6A1 promotes cancer cell migration and invasion by the induction of MIF, which upregulates MMP-2 and MMP-9 expression. A change in the proteolytic degradation of adjacent tissue is required during tumor invasion. MMP-2 and MMP-9 are major contributors to the degradation of gelatin and the collagen extracellular matrix, thereby facilitating cancer cell migration and invasion across tissue boundaries.126,127 MMP-2 has been recognized as a key regulator of tumor differentiation, proliferation, survival, and angiogenesis.128 In this study, the interaction network indicated that COL6A1 should regulate the accumulation of the extracellular matrix by up-regulating MMP-2 and MMP-9 expression, thus 1120



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’ ACKNOWLEDGMENT We thank the National Cheng-Kung University Proteomics Research Core Laboratory for assistance in mass spectrometry analysis for protein identification. This study was supported Grant DOH98-TD-G-111-008 from the Department of Health, Executive Yuan; Grant NSC97-2113-M-006-005-MY3 from the National Science Council; and the NCKU Project of Promoting Academic Excellence & Developing World Class Research Centers from the Ministry of Education of Taiwan. Pathway analyses and data mining were performed using the system provided by the Bioinformatics Core for Genomic Medicine and Biotechnology Development at the National Cheng-Kung University and were supported by a National Science Council grant (NSC 97-3112-B-006 -011 -). We also thank Mr. Corbett Hart Moy from Graduate Institute of Teaching Chinese as a Second Language (at the National Taiwan Normal University) for help in English editing. Figure 7. Proposed mechanism of the association of COL6A1 with lung cancer metastasis. COL6A1 may promote metastasis by interaction with MMP-2, MMP-7, and MIF.

allowing cancer cells to cross tissue boundaries. In the future, the role of COL6A1 among MIF, MMP-2, and MMP-7 expressions will need to be determined for clarification of its relationship to metastasis. Concluding Remarks

In this study, we developed a simple and high-throughput protein purification method for iTRAQ-labeling quantitative secretome analysis. A pathway analysis that constructed protein-protein interactions among selected metastasis-associated proteins showed that the localization of these proteins occurs mainly in the extracellular compartment. In addition, the metastatic abilities of the identified secreted protein COL6A1, a protein associated with lung cancer metastasis, were proven. The methodology of our study was robust and accurate in unearthing proteins for further metastasis studies in the human lung cancer secretome.

’ ASSOCIATED CONTENT

bS

Supporting Information Supplemental Figure 1. The proof-of-concept experiments that show the feasibility and reproducibility of the quantification strategy that combined stacking gel-aided purification with iTRAQ labeling. Two MS/MS spectra for the peptides KVPQVSTPTLVEVSR (m/z 643.42), and QTALVELLK (m/ z 434.98) were derived from BSA prepared in (A) 1:1 and (B) 2:1 ratios. The intensities of iTRAQ reporter ions of these identified peptides (m/z 115 and 116) and (m/z 114 and 117) are depicted. The quantified iTRAQ intensities were (A) 1.11:1 (SD = 0.17) (m/z 116/115) and (B) 1.97:1 (SD = 0.35). Supplemental Table 1. Associated Information of Proteins Identified in CL1 secretomes. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Dr. Pao-Chi Liao Department of Environmental Occupational Health National Cheng Kung University, College of Medicine 138 Sheng-Li Road, Tainan 70428, Taiwan. E-mail: liaopc@mail. ncku.edu.tw. Fax: 886-6-2743748. Author Contributions ‡

These authors contributed to this work equally.

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