Proteomic Analysis of Microvesicles Derived from Human Colorectal

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Proteomic Analysis of Microvesicles Derived from Human Colorectal Cancer Cells Dong-Sic Choi,†,§ Jae-Min Lee,†,§ Gun Wook Park,‡ Hyeon-Woo Lim,† Joo Young Bang,⊥ Yoon-Keun Kim,† Kyung-Hoon Kwon,‡ Ho Jeong Kwon,| Kwang Pyo Kim,⊥ and Yong Song Gho*,† Department of Life Science and Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea, Mass Spectrometer Development Team, Korea Basic Science Institute, Daejeon 305-333, Republic of Korea, Department of Biotechnology, College of Engineering, Yonsei University, Seoul 120-749, Republic of Korea, and Institute of Biomedical Science and Technology, Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea Received April 04, 2007

Microvesicles (MV) are membrane vesicles secreted from the plasma and endosomal membrane compartment by various cell types such as hematopoietic, epithelial, and tumor cells. Actively growing tumor cells shed MV, and the rate of shedding increases in malignant tumors. Although recent progress in this area has revealed that tumor-derived MV play multiple roles in tumor growth and metastasis via immune escape, tumor invasion, and angiogenesis, the mechanism of vesicle formation and the biological roles of tumor-derived MV are not understood. Here, we report the first global proteomic analysis of highly purified MV from human colorectal cancer cells. Using 1D SDS gel electrophoresis and nano-LC–MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. We identified 49 proteins involved in the biogenesis of MV, including annexins, ADP-ribosylation factors, and Rab proteins. We also identified 28 proteins that may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Taken together with previously reported results, our observations suggest that tumor-derived MV may act as communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. This information will help elucidate the biogenesis and functions of tumor-derived MV, and aid in the development of effective vaccines for various cancers, including colorectal cancer. Keywords: microvesicles • exosomes • microparticles • communicasomes • colorectal cancer • tolerosomes • proteomics • HT29

Introduction Cell-to-cell communication is an essential process in living organisms. Intercellular communication can occur via soluble mediators or direct membrane contact between the signaling and target cells.1 Another mechanism has been described recently in both prokaryotic and eukaryotic cells whereby the release and uptake of secreted membrane vesicles transfers small packages of information to target cells.2 Membrane vesicles derived from eukaryotic cells are called microvesicles (MV), whereas those from bacteria are called outer membrane vesicles, suggesting that this process has been conserved throughout evolution.2 * To whom correspondence should be addressed. Phone: 82-54-279-2345. Fax: 82-54-279-8609. E-mail: [email protected]. † Pohang University of Science and Technology. § These authors contributed equally to this work. ‡ Korea Basic Science Institute. ⊥ Konkuk University. | Yonsei University.

4646 Journal of Proteome Research 2007, 6, 4646–4655 Published on Web 10/24/2007

MV are spherical, bilayered proteolipids with an average diameter of 0.03-1 µm. They are enriched in various bioactive materials, including proteins, lipids, and mRNA. MV are produced by various hematopoietic and nonhematopoietic cells, including reticulocytes, mast cells, T cells, B cells, platelets, dendritic cells, epithelial cells, and tumor cells.3,4 Recent biological and proteomic studies of MV derived from various origins have provided insight into the mechanisms of vesicle formation and the biological functions of MV.5–16 Although the mechanisms of MV formation are not clear, two independent mechanisms have been proposed: (1) cells release relatively large MV (0.1–1 µm) from the plasma membrane in a calcium-dependent manner during membrane blebbing, or (2) exosomes, which are smaller, more homogeneous-sized MV (30–100 nm), are secreted from the endosomal membrane compartment after the fusion of secretory granules with the plasma membrane.3,4 MV contain numerous proteins and lipids similar to those in the cellular membranes from which they originate. Moreover, they engulf some cytoplasmic proteins and 10.1021/pr070192y CCC: $37.00

 2007 American Chemical Society

Proteomics of Colorectal Cancer Cell-Derived Microvesicles mRNA during their formation, and hijack various infectious particles such as human immunodeficiency virus and prions. Increasing evidence suggests that MV are communicasomes, that is, extracellular organelles that play diverse roles in intercellular communication. Communicasomes function via the direct stimulation of target cells as signaling complexes; the transfer of membrane receptors, proteins, and mRNA between cells; and the delivery of infectious agents.3,4 In vivo and in vitro experiments have shown that MV shedding is elevated and continuous in actively growing tumor cells.1 Tumor-derived MV are equipped with the tumor surface antigens, immune-suppressing cytokines, integrins, proteases, and angiogenic molecules required for immune escape, tumor invasion, and neovascularization. Thus, MV play multiple roles in tumor growth and metastasis.1,3,4 Furthermore, the anticancer drug doxorubicin accumulates in MV shed by tumor cells, suggesting that drug-resistant tumor cells may use vesicle shedding as a drug efflux mechanism.17,18 These observations suggest that the inhibition of vesicle shedding and the modulation of MV function may be worthwhile approaches for cancer therapy. In cancer patients, serum levels of MV are both significantly elevated and positively correlated with malignancy,19,20 suggesting that tumor-derived MV could serve as an important diagnostic indicator for cancer. In addition, tumor-derived MV harbor tumor surface antigens, which could be used to stimulate immunological responses against tumor cells. These characteristics of tumor-derived MV have been used to develop cell-free cancer vaccines that are now being tested in clinical trials.21 Colorectal cancer (CRC) is the second most common type of malignancy in the Western world.22 CRC cells release MV known as tolerosomes, which induce T-cell apoptosis via Fas ligand and TNF-related apoptosis inducing ligand.23,24 Although proteomic studies of CRC cell-derived MV have been performed, these studies were limited and identified only a small number of proteins: only 27 and 40 proteins were identified in MV derived from human and mouse CRC cells, respectively.5,6 Therefore, a global proteomic study of CRC cell-derived MV is needed to determine how CRC cells release MV, as well as the pathological functions of MV. Therefore, we analyzed the global proteome of highly purified MV from HT29, a human CRC cell line. Using 1D SDS gel electrophoresis and nano-LC–MS/MS analyses, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, comprising 235 previously known microvesicular proteins, as well as 181 proteins that have not been identified in MV derived from various origins.5–16 We identified several proteins involved in vesicle formation and the diverse biological functions of tumorderived MV. Our results provide a number of indicators that will not only increase the understanding of the biogenesis and pathological functions of tumor-derived MV, but also stimulate the development of effective vaccines against cancers, including CRC.

Experimental Procedures Cell Cultures. Human CRC cells (HT29) were obtained from the American type Culture Collection and were cultured in RPMI-1640 medium (Invitrogen Corporation, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen Corporation), 100 units/mL penicillin, and 100 mg/ mL streptomycin. The cultures were incubated in humidified air in 5% CO2 at 37 °C.

research articles Apoptosis Assay. HT29 cells were cultured in serum-free RPMI-1640 or RPMI-1640 supplemented with 10% fetal bovine serum. After 24 h of incubation, cells were trypsinized and stained with Annexin V-FITC (Becton Dickinson, San Diego, CA) according to the manufacturer’s specifications. FACS analysis was performed on a FACSCalibur (Becton Dickinson) immediately after addition of propidium iodide. Experiments were carried out in triplicate, and all data were analyzed using the Cell Quest software package (Becton Dickinson). MV Isolation. Confluent HT29 cells were washed twice with phosphate-buffered saline (PBS) and then grown in serum-free RPMI-1640. After 24 h of incubation, the conditioned medium was centrifuged once at 500g for 10 min, and then twice at 800g for 15 min The supernatant was concentrated by ultrafiltration using the QuixStand Benchtop System (GE Healthcare, Bucks, U.K.) with a 100-kDa hollow fiber membrane (GE Healthcare). To enrich the MV, the concentrated supernatant was added onto 0.8 and 2.7 M sucrose cushions in 20 mM HEPES/150 mM NaCl buffer (pH 7.2) and then ultracentrifuged at 100 000g for 70 min. After centrifugation, the interface between the 0.8 and 2.7 M sucrose cushions was collected and diluted 5-fold in PBS. We repeated the sucrose cushion ultracentrifugation three times. For further purification of MV, sucrose density gradient ultracentrifugation was performed. Briefly, sucrose cushionenriched MV were mixed with 2.8 M sucrose and pipetted into the bottom of a 4-mL tube. We then added 3 mL each of 1.6 and 0.6 M sucrose in 20 mM HEPES/150 mM NaCl buffer (pH 7.2), and the tubes were ultracentrifuged at 175 000g for 16 h. Ten fractions of equal volume were collected from the bottom of the tubes, diluted with 9 mL of PBS, and then ultracentrifuged at 150 000g for 2 h. Finally, the purified MV were resuspended in PBS, and the protein concentration of each fraction was determined using refractometry and Bradford dye assays (Bio-Rad Laboratories, Hercules, CA). All fractions were stored at -80 °C until use. Electron Microscopy of MV. The purified MV were applied to glow-discharged carbon-coated copper grids (EMS, Matfield, PA). After allowing the MV to absorb for 3 min, the grids were rinsed with droplets of deionized water and positive-stained with a mixture of 2% methylcellulose and 4% uranylacetate (Ted Pella, Redding, CA). Electron micrographs were recorded using a JEM 1011 microscope (Jeol, Japan) at an acceleration voltage of 100 kV. Western Blotting. Whole-cell lysate (WCL, 10–20 µg) and MV (0.5–2 µg) were separated by SDS-PAGE and then transferred to a polyvinylidene fluoride membrane. The blocked membrane was then incubated with the indicated antibodies. The immunoreactive bands were visualized using a chemiluminescent substrate. 1D SDS-PAGE and In-Gel Digestion. The purified MV (60 µg) were electrophoresed on a 4-20% gradient Novex Trisglycine gel (Invitrogen Corporation). After electrophoresis, the gel was stained with GelCode Blue Stain Reagent (Pierce, Rockford, IL) and cut into 10 slices of equal size. Each gel slice was destained with 200 µL of 50% acetonitrile (ACN) in 50 mM NH4HCO3, dehydrated with 50 µL of ACN, and dried. In-gel tryptic digestion was carried out overnight at 37 °C using a 12.5 ng/mL solution of sequencing-grade modified trypsin (Promega, Madison, WI) in 50 mM NH4HCO3. Tryptic peptides resulting from the digestion were extracted with 25 µL of 50% ACN in 5% formic acid for 20 min. The extracted peptides were dried and reconstituted with 0.1% formic acid. Journal of Proteome Research • Vol. 6, No. 12, 2007 4647

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Figure 1. Purification and characterization of HT29-derived MV. (A) Fractions of sucrose density gradients were analyzed by Western blotting. CD63 and CD81, marker proteins of MV, were strongly detected in fraction 3. Note that the density of fraction 3 was ∼1.16 g/mL, which is consistent with the typical microvesicular density of 1.13–1.19 g/mL.3 Molecular weight standards are indicated on the left (kDa). (B) Electron microscopy revealed that purified MV varied in size from 40 to 150 nm. The samples were not contaminated by cellular debris or protein aggregates. Scale bar ) 100 nm. (C) Western blotting showed that cytochrome c, a mitochondrial protein found in apoptotic bodies, and GM130, a protein found in the cis-Golgi apparatus, were not detected in the purified MV, while they were present in whole-cell lysate (WCL).

Nano-LC-ESI–MS/MS. The reconstituted tryptic peptides were loaded onto a homemade microcapillary C18 column (75 µm × 10 cm). Buffer A consisted of 0.1% formic acid in H2O, and buffer B consisted of 0.1% formic acid in ACN. Separation by LC was conducted using linear gradients of buffer B (3–30% over 50 min; 30–50% over 15 min; 50–90% over 5 min) at a flow rate of 250 nL/min. The separated peptides were then analyzed on an LTQ linear ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA). The electrospray voltage was set at 2.0 kV, and the threshold for switching from MS to MS/MS was 500 counts. The normalized collision energy for MS/MS was 35% of the main RF amplitude, and the duration of activation was 30 ms. All spectra were acquired in data-dependent mode. Each full MS scan was followed by nine MS/MS scans corresponding to the nine most intense peaks from the full MS scan. The peak repeat count for dynamic exclusion was 1, and the repeat duration was 30 s. The dynamic exclusion duration was set at 180 s, the exclusion mass width was (1.5 Da, and the list size of dynamic exclusion was 50. Database Searches. Only MS/MS spectra for doubly charged ions with m/z 700–3500 were searched within the normal human IPI protein database (IPI-HUMAN.v.3.17). To assess the false-assignment distribution, a reversed sequence database was created from the IPI human protein database.25 The search parameters allowed for one missed cleavage for trypsin. The TurboSEQUEST protein search algorithm (Thermo Electron) was used with precursor and fragment ion mass tolerances of 1.5 and 1 Da, respectively. Oxidation on Met (+16 Da) was selected as a variable modification. After using SEQUEST to search the combined databases (IPI and reversed database), the peptide sequences with the highest cross-correlation (Xcorr) values were collected by in-house software to produce the protein list. False-positive rates were estimated using a previously described algorithm,26 and the PeptideProphet F score was computed using the SEQUEST scores for Xcorr, ∆Cn, and SpRank.27 To increase the confidence level, the protein hit score (PHS), which counts the number of peptide hits, was included to add significance to the unique peptides and to devalue the peptides common to several proteins.26 Because the PHS deducts the effect of shared peptides, selecting a PHS value 4648

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higher than 1 is a more rigorous condition than “multiple peptides hit.” Gene Ontology Annotation. To obtain the subcellular localization and biological processes of the proteins involved, we searched the HPRD (http://www.hprd.org/) and PANTHER (http://www.pantherdb.org/) consortium databases.

Results and Discussion Purification and Characterization of HT29-Derived MV. To isolate MV with high purity, MV secreted by HT29 cells were purified in two sequential steps. In the first step, MV were isolated from the serum-free culture supernatant using a combination of differential centrifugation to remove cells and cell debris, ultrafiltration using a 100-kDa hollow fiber membrane to concentrate MV, and ultracentrifugation onto sucrose cushions.28,29 In the second purification step, MV were further purified using sucrose density gradients to remove nonmembranous proteins, protein aggregates, and denatured MV. To determine where MV were localized in the sucrose fractions, each fraction was subjected to Western blotting and probed for the presence of the MV marker proteins CD63 and CD81 (Figure 1A). MV settled at a density of ∼1.16 g/mL, consistent with the typical microvesicular density of 1.13–1.19 g/mL.3 Electron microscopy of purified MV revealed that almost all were small, closed vesicles ranging from 40 to 150 nm in size; apoptotic bodies, cellular debris, and protein aggregates were not detected (Figure 1B). These purified MV were similar in shape and size to MV isolated from human CRC cells.5 Furthermore, cytochrome c, a mitochondrial protein found in apoptotic bodies, and GM130, a protein found in the cis-Golgi apparatus, were not detected in MV, while they were present in WCL (Figure 1C). Serum contains MV of various origins, and some proteomic studies have been performed on MV derived from cells cultured in the presence of MV-depleted serum.7–9 Although we can speculate that MV shed in serum-free medium may be different from those in the presence of serum, MV were isolated from culture supernatant of HT29 cells incubated in serum-free medium to avoid potential contamination from serum-derived vesicles and proteins. FACS analysis showed that the overall viability of HT29 cells cultured in the presence of serum was

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Table 1. Summary of Data Analysis and the Number of Protein Assignments According to PHS Values

first trial second trial third trial

no. of MS/MS spectrab

no. of filtered peptides (error rate < 1%)

proteins assigned by single peptide

proteins assigned by multiple peptides

proteins of PHS < 1

proteins of PHS ) 1

proteins of PHS > 1

20977 23304 22170

2332 2328 2160

1073 972 985

863 880 783

1308 1245 1173

190 181 182

438 426 413

a Note that proteins assigned by PHS > 1 are more reliable than those assigned by multiple peptides. 3500.

97.00 ( 0.09%; the percentage of apoptotic (annexin V-FITClabeled cells), necrotic (propidium iodide-labeled cells), and secondary necrotic cells (propidium iodide and annexin VFITC-labeled cells) were 0.166 ( 0.07%, 2.63 ( 0.17%, and 0.22 ( 0.11%, respectively. When HT29 cells were cultured in serumfree medium, 94.56 ( 0.43% cells were viable; 0.31 ( 0.12%, 4.73 ( 0.41%, and 0.39 ( 0.12% of cells entered an early apoptosis, necrosis, and secondary necrosis, respectively. These results indicated that apoptosis and necrosis were not induced by incubation of HT29 cells in serum-free medium. The fact that the density of apoptotic bodies is 1.24 –1.28 g/ml30 suggests that apoptotic bodies coming from the few cells undergoing spontaneous apoptosis in the serum-free culture should be removed during the sucrose density gradient centrifugation (Figure 1A). When purified MV were examined under electron microscopy and by Western blotting, apoptotic bodies and apoptotic marker protein, cytochrome c, were not detected (Figure 1B,C). Although we could not completely exclude the possibility of contamination with scarce amounts of apoptotic bodies, all these results suggest that purified MV derived from serum-free cultured HT29 cells may not contain apoptotic bodies from the few cells undergoing spontaneous apoptosis in the serum-free culture. Proteomic Analysis of HT29-Derived MV. To determine the protein composition of MV isolated from HT29 cells, microvesicular proteins were separated by 1D SDS-PAGE on 4–20% gel. The gel was then sliced into 10 bands of equal size and subjected to trypsin digestion, and the extracted peptides were analyzed by nano-LC–MS/MS. We obtained 20 977, 23 304, and 22 170 MS/MS spectra from three independent nano-LC–MS/ MS analyses (Table 1). Using only MS/MS spectra derived from doubly charged ion-peaks of 700–3500 m/z,31 we searched the IPI human forward and reversed sequence databases using SEQUEST. From high-confidence peptide sequences with an error rate < 1%, we identified 1936 (F score > 2.071), 1852 (F score > 2.191), and 1768 (F score > 2.280) proteins in the first, second, and third trials, respectively. Because peptides that are shared by multiple proteins are less informative than unique peptides, we used PHS to strengthen the protein identification. Our analysis showed that proteins with PHS > 1 were identified by multiple peptides that are unique and shared among only a few proteins. We identified a total of 547 microvesicular proteins from three independent experiments with high confidence (PHS > 1); 438, 426, and 413 microvesicular proteins in the first, second, and third trials, respectively. The proteins identified and the detailed peptide data are provided in Supporting Tables 1 and 2 in Supporting Information, respectively. Notably, the three independent proteomic analyses of HT29-derived MV showed high reproducibility (Figure 2). Four hundred and sixteen of 547 microvesicular proteins were identified at least in two trials with high confidence (PHS > 1); 314 and 102 proteins were identified in three and two trials, respectively (Supporting Table 3 in Supporting Information). Furthermore, 92 of 102 proteins identified only in two trials

b

Charge of +2 only; m/z of the precursor is 700–

Figure 2. Venn diagrams depicting overlap in proteins identified in three replicate runs. A total of 547 proteins were identified from the three trials of HT29-derived MV, and 416 microvesicular proteins were identified at least in two trials with high confidence (PHS > 1).

Figure 3. Validation of HT29-derived microvesicular proteins. To validate microvesicular proteins identified by MS/MS in this study, Western blotting was performed for both whole-cell lysate (WCL) and MV using the indicated antibodies. Beta-Actin, ezrin, HSP90, LAMP 1, beta-catenin, NG2, galectin-4, and JUP were detected in both WCL and purified MV. Beta-catenin, NG2, galectin-4, and JUP have never been identified in MV derived from various origins, whereas beta-Actin, ezrin, HSP90, and LAMP 1 are markers of MV derived from various origins.

were present in the other trial with PHS e 1. This high rate of identification of the same proteins in separate trials suggests that the procedures used for protein identification are highly reproducible and that the identifications are reliable. In summary, we identified 416 microvesicular proteins, which were observed at least in two trials including 181 as yet unreported proteins (Supporting Table 3 in Supporting Information). We further analyzed these proteins. Western blottings showed that HT29-derived MV contained beta-Actin, ezrin, HSP90, LAMP 1, beta-catenin, chondroitin sulfate proteoglycan 4 (NG2), galectin-4, and junction plakoglobin (JUP) that were identified by MS/MS analysis in this study (Figure 3). Beta-catenin, NG2, galectin-4, and JUP have never been identified in MV derived from various origins, whereas beta-Actin, ezrin, HSP90, and LAMP 1 are markers of MV derived from various origins.3,4 Journal of Proteome Research • Vol. 6, No. 12, 2007 4649

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Figure 4. Comparison of the number of proteins identified in MV of human and mouse CRC cells from two earlier studies,5,6 and reported here from work on an HT29-derived MV preparation. Proteins common to two or more of the samples are shown in areas where the circles overlap.

Previous proteome studies of human and mouse CRC cellderived MV identified 27 and 40 proteins, respectively.5,6 We can also compare the number of MV-derived proteins identified in our study to those of two earlier studies (Figure 4 and Supporting Table 3 in Supporting Information). Note that 389 of the 416 microvesicular proteins reported here have not been identified in previous studies; 12 and 20 proteins of HT29derived MV overlapped with those of human and mouse CRC cell-derived MV, respectively. Actin, alpha-enolase, HSP90, pyruvate kinase M1, and pyruvate kinase M2 were found in all three CRC cell-derived MV studies. These proteins have also been identified in MV derived from various origins including platelet, urine, seminal fluid, malignant lymphocytes, and mesothelioma cells.9–13 Furthermore, the expression of alphaenolase, HSP90, pyruvate kinase M1, and pyruvate kinase M2 is significantly up-regulated in cancers including human CRC, suggesting that these microvesicular proteins may be involved in tumor progression.32–34 We further compared the microvesicular proteins reported here to those of human CRC cells, T84-DRB1*0401/CIITA-derived MV.5 Of the 27 proteins identified in the previous study, 12 proteins including Actin, alphaenolase, glyceraldehyde-3-phosphate dehydrogenase, HSP90, keratin 1, keratin 9, keratin 10, lactate dehydrogenase A, pyruvate kinase M1, pyruvate kinase M2, tubulin beta-2 chain, and tumor-associated calcium signal transducer 1 were identified in all three trials of our study (Supporting Table 3 in Supporting Information). Furthermore, dipeptidyl peptidase IV, creatine kinase chain B, and phosphoglycerate kinase 1 were identified in one trial of our study. HLA class I A24, HLA-DR R class II, cell-surface A33 antigen, EPS8, major vault protein, microsomal dipeptidase, syntaxin 3, syntaxin-binding protein 2, and 4 unnamed proteins were not identified in HT29-derived MV. Although we could not completely exclude the other possibilities, the discrepancy of findings between the studies may have been due to differences in mass analyses and cells. In the previous study, MALDI-TOF–MS analysis was used to identify proteins present in MV derived from T84-DRB1*0401/ CIITA cells that were doubly transfected with the HLADRB1*0401 molecule and the transactivator CIITA, resulting in constitutive expression of MHC class I and class II molecules. It is well-known that ESI-MS/MS and MALDI-TOF–MS analyses of the same sample usually identify different sets of proteins.35 Furthermore, the previous proteomic studies of MV derived from various origins revealed the presence of common, as well as cell type-specific, proteins.5–16 4650

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Choi et al. Annotation of Identified Proteins. We classified the identified microvesicular proteins by subcellular localization and biological process. We used the HPRD and PANTHER database to annotate 262 and 278 of the 416 identified proteins, respectively (Supporting Table 3 in Supporting Information and Figure 5). Figure 5A shows the subcellular localization of the identified proteins. The majority of the proteins came from the cytoplasm (47.3%), plasma membrane (19.9%), and extracellular space (8.4%). In terms of major biological processes, we identified proteins involved in cell structure and motility (14.6%), signal transduction (14.3%), intracellular protein traffic (12.9%), and protein metabolism and modification (12.1%) (Figure 5B). A number of proteins that are involved in metabolism and energy pathways (9.0%), immunity and defense (7.8%), transport (6.8%), developmental process (4.9%), nucleic acid metabolism (3.6%), cell adhesion (2.7%), protein targeting and localization (1.7%), oncogenesis (1.2%), apoptosis (0.7%), and other processes were also identified. It is noteworthy that the subcellular distribution and biological functions of the identified proteins were consistent with those of previous studies.10,11 The previous proteomic studies of MV derived from various origins revealed the presence of common, as well as cell typespecific, proteins.5–16 Previous studies have reported a number of the same proteins found in HT29-derived MV.3,4 These common proteins include annexins, ADP-ribosylation factors, clathrin, Rab proteins, transferrin receptor, heat shock proteins, tetraspanins, integrins, cell adhesion molecules, proteins involved in signal transduction, cytoskeletal proteins, metabolic enzymes, translational elongation factors, and nuclear proteins (Supporting Table 3 in Supporing Information). When we compared the microvesicular proteins reported here to those of urine-derived MV,11 94 of the 416 proteins present in HT29derived MV overlapped with those of urine-derived MV (Supporting Table 3 in Supporting Information). Most of these proteins have been known as microvesicular marker proteins: CD9, cytoskeletal proteins (Actin, cofilin, moesin, and tubulin), heat shock proteins (HSP70 and HSP90), metabolic enzymes (alpha-enolase, pyruvate kinase, and thioredoxine peroxidase), proteins involved in membrane transport and fusion (annexin II, annexin IV, annexin V, clathrin, Rab-1B, Rab-7, and ESCRT proteins including Alix and charged multivesicular body protein 2a), and proteins involved in signal transduction (14-3-3 protein and Gi2R).3,4 The microvesicular marker proteins including CD82 and Rab GDP dissociation inhibitor were identified only in this study, while annexin VI, CD13, and Tsg 101 were observed only in urine-derived MV. The other microvesicular marker proteins such as dipeptidyl peptidase IV, flotillin, and Rab-2A identified in urine-derived MV were seen only in one trial of our study. We also reported 181 proteins that have never been identified in MV derived from various origins. Some of these proteins are related to cell adhesion and cell structure, for example, tetraspanin-8, galectin-4, claudin-3, villin-1, alpha-catenin, and epithelial-cadherin. Some are related to protein trafficking and signal transduction including charged multivesicular body protein 2a, synaptosomal-associated protein 23, myristoylated alanine-rich C-kinase substrate, ephrin-B1, beta-catenin, and lung cancer oncogene 7 (RACK1). Therefore, our proteomic analysis is concordant with the current knowledge of the subcellular localization and functions of microvesicular proteins.3,4 In addition, the newly identified microvesicular proteins may further contribute to the understanding of the biogenesis and function of MV (see discussion below).

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Figure 5. Classification of identified proteins by subcellular localization and biological process. (A) The subcellular localization of 262 proteins as determined by HPRD database. (B) Biological processes of 278 identified proteins as determined by PANTHER.

Proteins Involved in MV Biogenesis. Although the mechanism of MV formation is not clear, vesicles are released from surface membranes in a calcium-dependent manner during membrane blebbing. They are also secreted from the endosomal membrane compartment after the fusion of secretory granules with the plasma membrane.1 In HT29-derived MV, we identified 49 proteins involved in biogenesis, including annexins, small GTPases of the ADP-ribosylation factors and Rab proteins, ERM (ezrin/radixin/moesin) proteins, components of the ESCRT complex, clathrin, dynein, and Rab GDP dissociation inhibitor, among others (Table 2). Small GTPases of the Rab superfamily regulate docking, fusion, and transport of vesicles in endocytosis and exocytosis.36 Numerous studies have established that different Rab proteins are localized to distinct intracellular compartments where they play central roles in the proper targeting to and fusion of vesicles with the correct destination organelles. It has been proposed that exosomes, nonplasma membrane-derived MV, are released into the extracellular milieu via multiple processes: vesicles from the clathrin-dependent endocytic pathway or trans-Golgi network are delivered to early/sorting endosomes, and then move to late endosomes characterized by their internal vesicles as multivesicular bodies (MVBs); exosomes are released by cells after MVBs fuse with the plasma membrane.3,4 We identified Rab-1B, Rab-5B, Rab-5C, Rab-6A, Rab-7, Rab-8A, Rab-10, Rab-11A, Rab-11B, Rab-33B, and Rab35 (Table 2). Rab-1B, Rab-6A, Rab-10, Rab-11A, Rab-11B, and Rab-33B were involved in vesicular transport between the endoplasmic reticulum–Golgi-plasma membrane, while Rab5B and Rab-5C regulated clathrin-coated vesicle transport from the plasma membrane to the early/sorting endosomes. Rab-7 was essential for vesicle transport from the early endosomes to MVBs, since a dominant negative Rab-7 mutant strongly inhibited this pathway. Rab-8A regulated polarized membrane transport to the basolateral membrane in epithelial cells,37 and in intestinal epithelial cells, different types of MV were released from the apical and basolateral sides,5 suggesting that Rab-8A may play an important role in basolateral MV biogenesis. A recent study showed that Rab-35 regulates an endocytic recycling pathway, while its function in membrane traffic is still not clearly defined.38 Taken together with previously reported results, the presence of so many types of Rab indicates that HT29-derived MV originate from various intracellular compartments. It will be of interest to determine the exact roles of microvesicular Rab proteins in MV biogenesis. All Rab proteins identified in this study have previously been identified as microvesicular proteins that are present in MV

derived from various origins including urine.9–12 Although most Rab proteins are constitutively expressed in all mammalian cells, several Rab proteins including Rab-1B, Rab-7, and Rab10 have been shown to be up-regulated in human tumors.39,40 Therefore, we speculate that dysregulation of Rab gene expression may cause elevated MV shedding in actively growing tumor cells.19,20 However, the relationship between the expression levels of Rab proteins and overall MV shedding rate in normal and tumor cells must first be addressed. Proteins Involved in the Pathological Functions of HT29Derived MV. Tumor growth and metastasis involves a war against hosts well equipped with defense systems, and tumor invasion, immune escape, and angiogenesis are critical for tumor growth and metastasis to occur.41,42 Although the physiological and pathological functions of tumor-derived MV are not clear, growing evidence suggests that MV shedding is elevated in actively growing tumor cells, and that tumor-derived MV play multiple roles in tumor growth and metastasis by promoting tumor invasion, immune escape, and angiogenesis.1,3,4 As shown in Table 3, many of the microvesicular proteins that we identified may function in tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. Angiogenesis, the formation of new blood vessels from preexisting vessels, occurs under various normal and pathological conditions.43 In physiological processes, angiogenesis is finely tuned by a balance of stimulatory and inhibitory factors. However, persistent and unregulated growth of new capillaries is crucial for sustained tumor growth, because it allows oxygenation and nutrient perfusion to the tumor as well as removal of waste. Neovascularization is also responsible for the increased entry of tumor cells into the circulation and metastasis. It is believed that tumor and stromal cells secrete angiogenic factors such as vascular endothelial growth factor and basic fibroblast growth factor (bFGF) and that tumorassociated angiogenesis occurs by the action of these factors.43 Furthermore, growing evidence suggests that MV derived from tumor cells and platelets also possess angiogenic activities; vesicular components such as sphingomyelin, CD147, tetraspanin-8, vascular endothelial growth factor, and bFGF are likely involved in MV-mediated neovascularization.44–47 In this study, we identified several microvesicular proteins that are believed to be involved in tumor-associated angiogenesis: ADAM 10, CD44, NG2, ephrin-B1, macrophage migration inhibitory factor (MIF), RACK1, and tetraspanin-8. To the best of our knowledge, this is the first study to report that NG2, ephrin-B1, and RACK1 are microvesicular proteins. Although further studies should Journal of Proteome Research • Vol. 6, No. 12, 2007 4651

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Table 2. Identified Proteins Involved in MV Biogenesis

family

Annexin

ARF

ERM

ESCRT

Rab

Other

IPI accession number

protein

IPI00218918 IPI00549413 IPI00455315 IPI00418169 IPI00221225 IPI00329801 IPI00414320 IPI00215914 IPI00215917 IPI00215918 IPI00215919 IPI00003527 IPI00479359 IPI00219365 IPI00017367 IPI00382452 IPI00004416 IPI00246058 IPI00008964 IPI00017344 IPI00016339 IPI00023526 IPI00016342 IPI00028481 IPI00016513 IPI00429190 IPI00020436 IPI00021475 IPI00300096 IPI00020984 IPI00024067 IPI00456969 IPI00005578 IPI00643041 IPI00004503 IPI00010418 IPI00419585 IPI00646304 IPI00031461 IPI00019345 IPI00015148 IPI00329719 IPI00010438 IPI00216231 IPI00478874 IPI00479018 IPI00299086 IPI00549343 IPI00030911

Annexin A1 Annexin A1 Annexin A2 Annexin A2 isoform 1 Annexin A4 Annexin A5 Annexin A11 ADP-ribosylation factor 1 ADP-ribosylation factor 3 ADP-ribosylation factor 4 ADP-ribosylation factor 5 Ezrin-radixin-moesin-binding phosphoprotein 50 Ezrin Moesin Radixin Splice Isoform 1 of Charged multivesicular body protein 1a Charged multivesicular body protein 2a Alix Ras-related protein Rab-1B Ras-related protein Rab-5B Ras-related protein Rab-5C Ras-related protein Rab-6A Ras-related protein Rab-7 Ras-related protein Rab-8A Ras-related protein Rab-10 Ras-related protein Rab-11A Ras-related protein Rab-11B Ras-related protein Rab-33B Ras-related protein Rab-35 Calnexin precursor Clathrin heavy chain 1 Dynein heavy chain, cytosolic EH-domain-containing protein 4 GTP-binding nuclear protein Ran LAMP1 protein Myosin Ic Peptidyl-prolyl cis–trans isomerase A Peptidylprolyl isomerase B precursor Rab GDP dissociation inhibitor beta Ras-related protein Rap-1A precursor Ras-related protein Rap-1b precursor Splice Isoform 1 of Myosin Id Splice Isoform SNAP-23a of Synaptosomal-associated protein 23 Splice Isoform SNAP-23b of Synaptosomal-associated protein 23 Syntenin isoform 2 Syntenin isoform 3 Syntenin-1 Vesicle-associated membrane protein 3 Vesicle-associated membrane protein 8

be carried out, HT29-derived MV equipped with several kinds of angiogenic molecules may promote tumor growth and metastasis by increasing tumor neovascularization. Several recent studies have shown that human cancer cells release MV containing ADAM 10, a disintegrin and metalloproteinase, and L1, a type I membrane glycoprotein that is overexpressed in many types of human carcinoma, including CRC.48,49 ADAM 10 cleaves L1, which then stimulates in vitro and in vivo angiogenesis via the activation of endothelial Rvβ3 and promotes the migration, invasion, and growth of tumor cells.48–50 Furthermore, ADAM 10 itself has angiogenic activity, suggesting that MV containing ADAM 10 should be involved in tumor-induced angiogenesis.51 CD44, a multistructural and 4652

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previous description in other cell-derived MV proteomics

Yes6,7,9,12,13 Yes6,7,9,12,13 Yes6–9,11–13 Yes6,9,11,13 Yes6,8–13 Yes9,11,12,15 Yes9,11 Yes10,11 Yes9,11 Yes9,11 Yes9 Yes8–11,13 Yes6,8–11,13,14 Yes8,9,11

Yes8,9,11 Yes9,10 Yes9,11 Yes9,11 Yes11,12 Yes6,9–11 Yes9,11,12 Yes9–11 Yes9,11 Yes9–11 Yes11,12 Yes9,11 Yes7,9,10 Yes6,7,9,10,14 Yes7 Yes10,11 Yes9 Yes11 Yes9,10,12 Yes7,9,10 Yes8–10 Yes10,11 Yes10,11,15 Yes10

Yes8,9,11 Yes11 Yes10

multifunctional cell surface molecule, regulates tumor progressionandmetastasis,aswellastumor-associatedangiogenesis.52–55 Overexpression of CD44 occurs in human cancer including CRC, and CD44 promotes resistance to apoptosis and increased invasion in human CRC cells.52,53 Evidence shows the enhanced expression of CD44 on tumor-associated vessels, suggesting that the molecule is involved in tumor angiogenesis.54,55 NG2 is a transmembrane chondroitin sulfate proteoglycan, and its expression is associated with neovascularization during tumor development and wound healing.56,57 It has been shown that NG2 positively regulates angiogenesis via its direct interaction with angiogenic growth factors such as bFGF and plateletderived growth factor.58 Furthermore, cells continuously release

research articles

Proteomics of Colorectal Cancer Cell-Derived Microvesicles Table 3. Identified Proteins Involved in the Pathological Functions of HT29-Derived MV

protein

IPI accession number

14-3-3 proteins 14-3-3 protein beta/alpha 14-3-3 protein epsilon 14-3-3 protein eta 14-3-3 protein gamma 14-3-3 protein theta 14-3-3 protein zeta/delta Acid phosphatase 1 isoform c ADAM 10

IPI00216318 IPI00000816 IPI00216319 IPI00220642 IPI00018146 IPI00021263 IPI00219861 IPI00013897

Alix Beta-catenin CD44 CD82 CD9 Chondroitin sulfate proteoglycan 4 Claudin-3 Ephrin-B1 Epithelial-cadherin Galectin-4 Junction plakoglobin Lung cancer oncogene 7 Macrophage migration inhibitory factor

IPI00246058 IPI00017292 IPI00305064 IPI00020446 IPI00215997 IPI00019157 IPI00007364 IPI00024307 IPI00025861 IPI00009750 IPI00554711 IPI00641950 IPI00293276

Peroxiredoxin-1 Phosphatidylethanolamine-binding protein Plexin B2 Ras-related protein R-Ras Ras-related protein R-Ras2 Syntenin-1 Tetraspanin-8

IPI00000874 IPI00219446 IPI00398435 IPI00020418 IPI00012512 IPI00299086 IPI00015872

pathological functions of HT29-derived MV

previous description in other cell-derived MV proteomics

Tumor growth

the extracellular domain of NG2 produced by cell surface proteolysis, and this soluble NG2 has a potent angiogenic activity.57 All these observations suggest that NG2 associated with MV should be angiogenic and promote tumor growth and metastasis by increasing tumor-associated neovascularization. Recent studies have demonstrated that ephrin-B1 is involved in tumor progression by promoting neovascularization and tumor invasion in several human cancers.59,60 Furthermore, overexpression of ephrins has been associated with increased tumor growth and tumorigenicity, as well as poor prognosis.61 MIF functions as a pluripotent cytokine, and it is involved in a broad spectrum of pathophysiological events associated with inflammation and the immune response. Furthermore, several reports have linked MIF to fundamental processes controlling cell proliferation, cell survival, angiogenesis, and tumor progression.62–64 An antibody for MIF suppresses tumor growth via blockage of the tumor-induced angiogenesis, while tumor growth and the associated angiogenesis are significantly enhanced in MIF transgenic mice compared to control mice.64 Furthermore, several studies have reported that MIF is highly expressed in several tumors, and that the expression of MIF is associated with enhanced angiogenesis.65 In human CRC and nonsmall cell lung cancer, the expression of RACK1 is upregulated, and RACK1 might contribute to tumor growth via the induction of neovascularization as well as the protection of tumor cells from apoptosis.66 Tetraspanin-8/CO-029 (human homologue of rat D6.1A) promotes tumor growth via the induction of systemic angiogenesis, and its expression is associated with poor prognoses in patients with gastrointestinal cancer.47 Furthermore, tetraspanin-8-contaning MV derived

Tumor growth Angiogenesis, tumor growth, migration, invasion Tumor growth Tumor growth Angiogenesis, tumor growth, invasion Immune modulation, invasion Immune modulation, invasion Angiogenesis Tumor growth Angiogenesis, invasion Tumor growth Tumor growth Tumor growth Angiogenesis, tumor growth Angiogenesis, immune modulation, tumor growth, invasion Tumor growth Tumor growth Angiogenesis, invasion Invasion Invasion Invasion Angiogenesis

Yes9–12 Yes8–12 Yes9 Yes9–12 Yes9–11 Yes9–12 Yes10 Yes10 Yes8,9,11 Yes9 Yes6 Yes10,11

Yes9,11,12 Yes9–12,15 Yes9–12

Yes9 Yes8,9,11

from rat pancreatic adenocarcinoma cells strongly induced angiogenesis in vivo; it also induced endothelial cell branching in vitro.47 In CRC and pancreatic cancer, the expression of tetraspanin 8 is up-regulated in tumor cells,67 suggesting that the presence of tetraspanin-8 on MV could serve as diagnostic markers for these cancers.

Conclusion We used 1D SDS-PAGE and LC–MS/MS analyses to identify proteins in MV derived from human CRC cells. On the basis of a stringent filter allowing only MS/MS spectra of doubly charged ions of 700–3500 m/z and peptides with an error rate of less than 1% after forward and reverse sequence database searches, we identified a total of 547 microvesicular proteins from three independent experiments with high confidence; 416 proteins were identified at least in two trials, including 181 as yet unreported proteins. In combination with previously reported results, many of the microvesicular proteins that we identified may function in MV biogenesis and tumorigenesis via promotion of migration, invasion, and growth of tumor cells, immune modulation, metastasis, and angiogenesis. This work has taken us one step nearer to an integrated view of tumor-derived MV with regard to their mechanisms of biogenesis and function. Furthermore, our results will stimulate the development of effective vaccines against cancer, including CRC. Sporadic CRC develops through randomly acquired somatic mutations; most (85%) are microsatellite stable, while the other subset comprises DNA microsatellite unstable CRC (15%).68 Since the HT29 cells used in this study are microsatJournal of Proteome Research • Vol. 6, No. 12, 2007 4653

research articles ellite stable, comparative proteomic studies on MV derived from CRC cells that are not microsatellite stable would provide valuable data.

Acknowledgment. This work was supported by grants from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea (0320380-2), POSTECH Research Fund, and the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MOST) (R15-2004-033-05001-0). Dong-Sic Choi and Jae-Min Lee were recipients of Brain Korea 21 fellowship. Supporting Information Available: Supporting Table 1, proteins identified in HT29-derived MV; Supporting Table 2, peptides identified in HT29-derived MV; Supporting Table 3, summary of identified proteins and their functional classification. This material is available free of charge via the Internet at http:// pubs.acs.org. References (1) Ratajczak, J.; Wysoczynski, M.; Hayek, F.; Janowska-Wieczorek, A.; Ratajczak, M. Z. Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication. Leukemia 2006, 20, 1487–1495. (2) Mashburn-Warren, L. M.; Whiteley, M. Special delivery: vesicle trafficking in prokaryotes. Mol. Microbiol. 2006, 61, 839–846. (3) Thery, C.; Zitvogel, L.; Amigorena, S. Exosomes: composition, biogenesis and function. Nat Rev. Immunol. 2002, 2, 569–579. (4) Fevrier, B.; Raposo, G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 2004, 16, 415–421. (5) Van Niel, G.; Raposo, G.; Candalh, C.; Boussac, M.; Hershberg, R.; Cerf-Bensussan, N.; Heyman, M. Intestinal epithelial cells secrete exosome-like vesicles. Gastroenterology 2001, 121, 337–349. (6) Van Niel, G.; Mallegol, J.; Bevilacqua, C.; Candalh, C.; Brugiere, S.; Tomaskovic-Crook, E.; Heath, J. K.; Cerf-Bensussan, N.; Heyman, M. Intestinal epithelial exosomes carry MHC class II/peptides able to inform the immune system in mice. Gut 2003, 52, 1690– 1697. (7) Banfi, C.; Brioschi, M.; Wait, R.; Begum, S.; Gianazza, E.; Pirillo, A.; Mussoni, L.; Tremoli, E. Proteome of endothelial cell-derived procoagulant microparticles. Proteomics 2005, 5, 4443–4455. (8) Mears, R.; Craven, R. A.; Hanrahan, S.; Totty, N.; Upton, C.; Young, S. L.; Patel, P.; Selby, P. J.; Banks, R. E. Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Proteomics 2004, 4, 4019–4031. (9) Miguet, L.; Pacaud, K.; Felden, C.; Hugel, B.; Martinez, M. C.; Freyssinet, J. M.; Herbrecht, R.; Potier, N.; van Dorsselaer, A.; Mauvieux, L. Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization. Proteomics 2006, 6, 153–171. (10) Garcia, B. A.; Smalley, D. M.; Cho, H.; Shabanowitz, J.; Ley, K.; Hunt, D. F. The platelet microparticle proteome. J. Proteome Res. 2005, 4, 1516–1521. (11) Pisitkun, T.; Shen, R. F.; Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 13368–13373. (12) Utleg, A. G.; Yi, E. C.; Xie, T.; Shannon, P.; White, J. T.; Goodlett, D. R.; Hood, L.; Lin, B. Proteomic analysis of human prostasomes. Prostate 2003, 56, 150–161. (13) Hegmans, J. P.; Bard, M. P.; Hemmes, A.; Luider, T. M.; Kleijmeer, M. J.; Prins, J. B.; Zitvogel, L.; Burgers, S. A.; Hoogsteden, H. C.; Lambrecht, B. N. Proteomic analysis of exosomes secreted by human mesothelioma cells. Am. J. Pathol. 2004, 164, 1807–1815. (14) Wubbolts, R.; Leckie, R. S.; Veenhuizen, P. T.; Schwarzmann, G.; Mobius, W.; Hoernschemeyer, J.; Slot, J. W.; Geuze, H. J.; Stoorvogel, W. Proteomic and biochemical analyses of human B cellderived exosomes. Potential implications for their function and multivesicular body formation. J. Biol. Chem. 2003, 278, 10963– 10972. (15) Jin, M.; Drwal, G.; Bourgeois, T.; Saltz, J.; Wu, H. M. Distinct proteome features of plasma microparticles. Proteomics 2005, 5, 1940–1952.

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