The Mammary Epithelial Cell Secretome and Its Regulation by Signal Transduction Pathways Jon M. Jacobs,†,§ Katrina M. Waters,†,4 Loel E. Kathmann,‡ David G. Camp, II,†,§ H. Steven Wiley,†,⊥ Richard D. Smith,†,§ and Brian D. Thrall*,†,‡ Systems Biology Program, Cell Biology and Biochemistry, Biological Separations and Mass Spectrometry, Computational Biology Groups, and Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 Received July 15, 2007
Extracellular proteins released by mammary epithelial cells are critical mediators of cell communication, proliferation, and organization, yet the actual spectrum of proteins released by any given cell (the secretome) is poorly characterized. To define the set of proteins secreted by human mammary epithelial cells (HMEC), we combined analytical and computational approaches to define a secretome protein set based upon probable biological significance. Analysis of HMEC-conditioned medium by liquid chromatography–mass spectrometry resulted in identification of 889 unique proteins, of which 151 were found to be specifically enriched in the extracellular compartment when compared with a database of proteins expressed in whole HMEC lysates. Additional high mass accuracy analysis revealed 36 proteins whose extracellular abundance increased after treatment with phorbol ester (PMA), a protein kinase C agonist and general secretagogue. Many of the PMA stimulated proteins have been reported to be aberrantly expressed in human cancers and appear to be coregulated as multigene clusters. By inhibiting PMA-mediated transactivation of the epidermal growth factor receptor (EGFR), a pathway critically required for normal HMEC function, we found that the secretion of specific matrix metalloproteases was also coordinately regulated through EGFR transactivation. This study demonstrates a tiered strategy by which extracellular proteins can be identified and progressively assigned to classes of increasing confidence and regulatory importance. Keywords: proteome of secreted proteins • mammary epithelial cell • phorbol ester • epidermal growth factor receptor • mass spectrometry
Introduction Extracellular proteins released through secretory pathways, or by ectodomain shedding, are major effectors of autocrine signaling and intercellular communication. Because secreted proteins are primary regulators of tissue function and homeostasis, they are particularly promising as candidates for therapeutic manipulation and disease biomarkers. Thus, much effort has been expended on identifying the total set of secreted proteins (the secretome).1–8 Among the challenges in defining the “secretome” are the large numbers of proteins that can potentially be released and the multitude of cellular pathways that are involved in their regulation. Previous computational and genomic analyses have predicted that between 1000 and 2000 unique soluble proteins may be secreted from an average mammalian cell.7,8 However, the diversity of the secretome is * Corresponding author. Brian D. Thrall, Cell Biology and Biochemistry Group, Pacific Northwest National Laboratory, Richland, Box 999, Mail Stop P7-56, WA, 99352. Tel. 509-376-3809. Fax509-376-6767. E-mail: brian.thrall@ pnl.gov. † Systems Biology Program. ‡ Cell Biology and Biochemistry. § Biological Separations and Mass Spectrometry. 4 Computational Biology Groups. ⊥ Environmental Molecular Science Laboratory.
558 Journal of Proteome Research 2008, 7, 558–569 Published on Web 01/01/2008
almost certainly cell-specific, and such proteins can be derived from multiple mechanisms, including regulated translocation of secretory vesicles, constitutive secretion of newly expressed proteins, and metalloprotease-dependent ectodomain cleavage (shedding) of membrane bound proteins. Because the structural basis of many of these regulatory processes is poorly understood, it is currently difficult to predict extracellular proteins on the basis of their sequence alone. In human mammary epithelial cells (HMEC), the central role of extracellular proteins in modulating cell function is particularly evident, as cell proliferation and motility necessary for normal development and mammary gland morphogenesis are dependent on proteins secreted or shed into the extracellular space.9–12 Conversely, the aberrant secretion or shedding of proteins is commonly associated with disease, including cancer,13–17 and the accessibility of these proteins from clinically relevant samples such as plasma,18,19 nipple aspirate fluid,15 and tumor interstitial fluid20 makes them particularly attractive as potential early biomarkers of mammary disease. Previous approaches to globally characterize secreted proteins have predominantly employed analytical methods such as 2D-gel electrophoresis combined with mass spectrometric analysis of conditioned medium proteins.1–3,5 However, these 10.1021/pr0704377 CCC: $40.75
2008 American Chemical Society
Mammary Epithelial Cell Secretome approaches have typically resulted in identification of a restricted number of proteins, in part because of limitations in sample throughput, sensitivity, and dynamic range. Methods that employ high mass accuracy mass spectrometry (MS) coupled with capillary liquid chromatography (LC) separations to quantify peptides previously identified using LC-MS/MS methods can help overcome these challenges.21–23 However, all sensitive proteomic technologies still face the same challenge of discriminating the authenticity of specific cellular compartment members. This is especially true for extracellular proteins, which can arise in the medium by multiple mechanisms, including active secretion and regulated proteolytic release from the cell membrane, low levels of cell death occurring during cell culture, or even through contaminants originally present in the medium. Thus, a systematic approach to assigning identified proteins as specifically targeted to the extracellular space is needed. Among the most potent stimuli identified for cellular protein release is the phorbol ester and tumor promoter, phorbol 12myristate 13-acetate (PMA). PMA stimulates both exocytic pathways as well as pathways involving metalloproteasedependent ectodomain cleavage and shedding of membraneanchored proteins,24–28 two of the major cellular mechanisms of membrane protein turnover.29,30 The rapid induction of exocytic and ectodomain shedding responses by PMA has been attributed to activation of protein kinase C (PKC) along with activation of downstream effectors such as the mitogen-activated protein kinases (MAPK).24,27 However, PMA also transactivates the epidermal growth factor receptor (EGFR), in part by stimulating the shedding of EGFR autocrine ligands.24,31,32 This is particularly important in HMEC, as earlier work has demonstrated that autocrine activation of EGFR is essential for normal HMEC proliferation, motility, and organization into three-dimensional organoid structures.10,12,33,34 Thus, PMA not only is useful as a general secretegogue for investigating the HMEC secretome but also provides a useful stimulus to concurrently evaluate whether specific secretory responses necessary for normal HMEC function are regulated through EGFR transactivation. In this study, we applied a systematic global proteomics approach to (1) determine the overall repertoire of extracellular proteins expressed in HMEC and (2) begin classifying proteins whose shedding and secretion is regulated by key signaling pathways that control HMEC function. By combining mass spectrometry-based analysis with bioinformatics comparisons across databases, we confidently identified 151 proteins that are enriched in the extracellular compartment and 36 proteins whose secretion was specifically stimulated by PMA. Interestingly, many of these regulated proteins have been previously reported to be aberrantly expressed in human cancers, and a subset of matrix metalloproteases and their upstream regulators were found to be coordinately regulated by EGFR transactivation. Our study illustrates an approach for determining which proteins are likely to be important in regulating cell behavior.
Experimental Procedures Cell Culture and Reagents. Human mammary epithelial cells (HMEC strain 184A1 L5)35 were obtained from Martha Stampfer (Lawrence Berkeley National Laboratory). These cells synthesize their own basement membrane matrix and are highly dependent on EGFR autocrine signaling for normal proliferation.12,33,35 HMECs were routinely cultured in DFCI-1 medium supplemented with 12.5 ng/mL of human epidermal growth factor (EGF) (Calbiochem, San Diego, CA) as previously described.10
research articles Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma Chemical (St. Louis, MO), and the highly selective EGFR kinase inhibitor, PD153035,36 and MEK inhibitor, U0126,37 were purchased from Calbiochem and dissolved as a concentrated stock in dimethylsulfoxide. The EGFR neutralizing monoclonal antibody 225 (225 mAb), purified from hybridoma supernatants, was a gift from Dr. Lee Opresko (Pacific Northwest National Laboratory). All other reagents were of cell culture or molecular biology grade. For analysis of protein shedding and secretion, HMECs (passage 14) were grown to approximately 80% confluence in 100 mm2 dishes (10 per group), washed with phosphate-buffered saline to facilitate the removal of the fetal bovine serum, and exposed to treatment chemicals diluted in medium consisting of serum-free DFCI-1 supplemented with 1 µM insulin and 10 µg/mL of hydrocortisone. Although all treatments were conducted in serum-free medium, the same lot of serum used in previous proteomic studies of HMEC23,38 was also used to prepare cultures for this study. The serumfree medium composition was derived from preliminary experiments, where it was determined that insulin and hydrocortisone were supplements that were essential for maintaining cell viability during the treatment period (results not shown). For proteomic analysis by LC-MS/MS or liquid chromatography coupled with Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR), treatments included PMA (200 nM), either alone or in the presence of EGFR kinase inhibitor for 24 h. Control samples received an equal concentration of the dimethylsulfoxide vehicle. In experiments where EGFR signaling was selectively blocked by 225 mAb, the final concentration of neutralizing antibody was 10 µg/mL, based on previous work that showed this was sufficient to block EGFR transactivation.10 A total of 180 mL of medium volume per treatment group was utilized for all MS/MS and MS-based analyses at an initial protein concentration of 10 µg/mL. Sample Preparation. The steps used for processing conditioned media were identical for all cellular conditions described. Because of the extensive sample volumes and fractionation necessary for analysis, it was necessary to pool samples within treatment groups for LC-MS/MS analysis. FTICR-MS analyses (discussed below) were performed with three technical replicates. Following treatment, media was collected, and the volume was reduced 10-fold by lyophilization, followed by removal of low molecular weight species using a PD-10 desalting column per manufacturer’s instructions (Amersham Pharmacia Biotech, Uppsala, Sweden). The protein fractions were combined and lyophilized again to reduce volume by 20-fold. We generally observed ∼80% sample retention, based on the amount of protein initially found in the media volume in comparison to the amount of protein obtained for tryptic digestion. Soluble proteins were then denatured using 8 M urea, reduced with 5 mM TBP (SigmaAldrich, St. Louis, MO) at 37 °C for 60 min, after which the sample was alkylated by incubation with 15 mM iodoacetamide for 1.5 h at room temperature. Samples were then diluted 8-fold for preparation for digestion. Sequencing-grade modified porcine trypsin was prepared as instructed by the manufacturer (Promega, Madison, WI) and added to all protein samples at a 1:50 (w/w) trypsin-to-protein ratio for 5 h at 37 °C. The digestions were stopped by boiling for 5 min followed by cooling on ice. The samples were then stored at -80 °C until time for separation. Peptide Separation. HMEC peptide samples were subjected to strong cation exchange chromatography (SCX) using a Journal of Proteome Research • Vol. 7, No. 2, 2008 559
research articles PolyLC Polysulfethyl A 200- × 4.6-mm column (Columbia, MD) preceded by a 10- × 4.6-mm guard column with a flow rate of 1 mL/min. The separations were performed with a Shimadzu LC-10A system using a Unicam 4225 UV/vis detector (Thermo Electron, Waltham, MA, USA) with mobile phases consisting of solvent A (10 mM ammonium formate, 25% acetonitrile (ACN), pH 3.0) and solvent B (500 mM ammonium formate, 25% ACN, pH 6.8). Once the sample was injected, the run was isocratic for 10 min at 100% solvent A, followed by an initial gradient from 100% solvent A to 50% solvent B for 50 min. A steeper gradient to 100% solvent B lasting 10 min was then performed and held isocratically at 100% solvent B for 15 min. A total of 70 -1 mL fractions were collected using a Shimadzu FRC-10A fraction collector. Fractions at the beginning and end of the separation were combined resulting in a total of 41 fractions for each sample, which were lyophilized to dryness and stored at -80 °C until time for analysis. Reversed-Phase LC Separation and MS/MS Analysis. The reversed phase capillary liquid chromatography system is composed of a 150-µm i.d. × 360-µm o.d. × 65-cm capillary (Polymicro Technologies Inc., Phoenix, AZ) fitted with a 2 µm retaining mesh and packed with 5 µm Jupiter C18 stationary phase (Phenomenex, Torrence, CA). Mobile phase C consisted of 0.05% trifluoroacetic acid (TFA) and 0.2% acetic acid in water, and mobile phase D consisted of 0.1% TFA and 90% ACN in water. The exponential gradient mixing of mobile phase C with mobile phase D (1.8 µL/min) began while maintaining constant pressure (5000 psi) for 20 min following a 10 µL injection of the sample (1.0 µg/µL). The capillary column was interfaced with a Finnigan LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) using an electrospray ionization source manufactured in-house. The initial MS scan utilized an m/z range of 400–2000 after which three of the most abundant ions were selected for MS/MS analysis using a collisional energy setting of 45%. Dynamic exclusion was used to prevent repeated analysis of the same high-abundance ion. The temperature of the heated capillary and the electrospray voltage were 200 °C and 2.2 kV, respectively. LC-MS/MS Data Analysis. Peptide identification used SEQUEST39 to match the MS/MS fragmentation spectra with peptides from the IPI human database (Aug 2006 version, 61 225 entries, no enzyme search, ( 3 Da tolerance for parent MS peak). The criteria selected for filtering followed methods based upon searches against the sequence-reversed IPI human database which provides a target of 95% confidence in peptide identifications.40 Reverse database searching resulted in a specific false discovery rate (FDR) of 5.2% at the peptide level and a FDR of 1.0% at the protein level. Briefly, protein identifications were retained if their identified peptide met the following criteria: (1) SEQUEST DelCN value of g0.10 and (2) SEQUEST correlation score (Xcorr) g1.5 for charge state 1+ and full tryptic peptides; Xcorr g3.1 for partial tryptic peptides; Xcorr g1.9 for charge state 2+ and full tryptic peptides; Xcorr g3.8 for partial tryptic peptides; Xcorr g2.9 for charge state 3+ and full tryptic peptides; Xcorr g4.5 for partial tryptic peptides. Only proteins identified by at least two unique peptides are reported. A mass tag database containing the calculated mass and normalized elution time for each identified peptide was generated for subsequent LC-FTICR analysis. The program ProteinProphet was used to consolidate redundant protein identifications from the results.41 Additional database searching included using the Bovine NCBI database (downloaded in March 2006; 560
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Jacobs et al. 33 543 sequence entries). Comparison of results between the two searches (human IPI and Bovine NCBI) was performed by blasting each list of SEQUEST identified peptides against the opposing database to determine the total number of unique and overlapping peptides for each respective data set. Analysis of HMEC Proteins by LC-FTICR. LC-FTICR analysis of HMEC peptide samples used a 9 T FTICR instrument, designed and constructed in our laboratory, coupled to the same high-resolution reversed-phase capillary LC described in the previous section.42 All LC-FTICR analyses were conducted in triplicate. The acquired FTICR spectra (105 resolution) were processed to obtain peak lists containing the monoisotopic mass, observed charge, and integrated peak area of the major ions in each spectrum.21 The accurate masses were calibrated using the masses of internal calibrant species infused at the beginning and end of each LC-FTICR analysis. For identification, the peak lists for each analysis were matched against a mass tag database constructed from the LC-MS/MS analyses of the HMEC medium samples from this study, combined with LC-MS/MS analyses from our previous proteomic studies of HMEC whole cell lysates.23,38,43 This matching is performed by finding the groups of species in the data from adjacent spectra comprising a peptide peak, computing a corresponding median monoisotopic mass, performing an alignment of both mass and elution time,44 and then comparing the mass and elution time of the group with the mass and normalized elution time of each peptide in the HMEC mass tag database (match tolerance of (5 ppm and (0.02 normalized elution time).45 In instances where multiple database mass tags fell within the match tolerance of a specific detected peak, we applied a probability algorithm to identify the most likely match based upon closeness of the mass accuracy and elution time information, as previously described.46 Immunoblot and ELISA Analysis. The level of EGFR transactivation was determined by immunoprecipitation of total EGFR followed by analysis of phosphotyrosine levels by Western blot. Briefly, cells were lysed in RIPA buffer (50 mM TrisCl, pH 7.2, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate, 4 mM iodoacetate) supplemented with protease inhibitors, and the lysate was centrifuged at 10 000g for 5 min to remove debris. Up to 1 mg of total protein was incubated with 25 µL of anti-EGFR 225Ab cross-linked to protein A/G beads (Ultralink, Pierce Chemical) for 30 min at 4 °C with gentle rocking. The immunoprecipitated complexes were washed several times followed by elution in SDS-PAGE loading buffer for Western blot analysis. Detection of phosphorylated EGFR was performed using either an antibody against phosphotyrosine (Transduction Laboratories) or an antibody that selectively recognizes EGFR phosphorylated at Tyr1173 (Santa Cruz Biotechnology, Santa Cruz, CA). For analysis of ERK protein activation, Western blots were performed using an antiactive ERK antibody that recognizes the dual-phosphorylated forms of ERK1 and ERK2 (Santa Cruz Biotechnology). All Western blot experiments were performed using chemiluminescent detection with a Kodak luminescent imager. For analysis of secreted MMP-1, MMP-9, and MMP-10 proteins, commercial ELISA assays were used per manufacturer’s instructions (R&D Systems, Minneapolis, MN). Total cellular protein levels were measured by the BCA assay (Pierce, Rockford, IL) and used for normalizing the ELISA measurements.
Mammary Epithelial Cell Secretome
Figure 1. Scheme for experimental analysis of the HMEC secretome. Shown is the experimental design for comparative proteomic analysis of the HMEC-conditioned medium for identification and quantification of differentially secreted proteins. The main flow of the study involved the tryptic digestion of the isolated protein samples followed by two independent MS analyses: (1) LC-MS/MS analysis providing the identification of the peptide, creation of the mass and time tag database, and spectra count, and (2) LC-FTICR accurate mass measurement providing a quantitative peak area-based value for complementary comparison.
Results Identification of Proteins from Conditioned Medium. Figure 1 outlines the overall approach taken to characterize the HMEC secretome under constitutive and induced conditions. Briefly, HMEC cultures of equivalent cell densities were treated with medium alone or PMA. For comparison of EGFR-dependent responses (discussed in following sections), additional treatment groups received PMA combined with a selective EGFR kinase inhibitor (PD153035)36 or inhibitor alone. After 24 h, conditioned medium from each of the treatment groups was collected, and the proteins were isolated and tryptically digested for MS analysis (see Materials and Methods for details). A portion of the digested peptides were fractionated using offline strong cation exchange (SCX) chromatography and then subjected to LC-MS/MS analysis for identification of the peptide and its precursor protein. After SEQUEST analysis, FDR calculation, and protein redundancy reduction using ProteinProphet (see Materials and Methods), a combined total of 889 proteins (6802 peptides) were identified across all treatment groups, with at least two peptides per protein (see Supplemental Table 1). Data Filtering for Extracellular Proteins. A major challenge when sampling proteins found in the extracellular space is how to distinguish between those targeted to the extracellular space via specific cellular mechanisms versus those species that arise due to nonspecific events, such as low levels of cell death during culture. To determine which of the identified proteins were enriched in the extracellular space, we compared peptide abundances of proteins identified in conditioned medium from this study with peptide abundances previously identified by analyses with the same methods using total cell lysates (see top of Figure 2). While spectra counts provide a semiquantitative abundance measure, this approach is a useful first step
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Figure 2. Schematic of data filtering used for determination of secretome proteins. The 889 proteins identified in conditioned medium were compared with previous HMEC global cell lysate results providing a subset list of 177 proteins that were classified as enriched in the extracellular compartment. Additional filtering was conducted by comparison of human peptide sequences with the bovine database to remove potential carryover proteins arising from bovine serum used in cell culture, resulting in a final subset of 151 secretome proteins.
for identifying a prospective pool of proteins for further analyses.47 In previous studies using total cell lysates, we have identified 4155 proteins expressed in HMEC.23,38,43 This database includes proteins identified in whole cell lysates derived from the same cell samples that were the source of conditioned medium in this study.23 Based on spectra counts, we determined that 121 of the proteins identified in conditioned medium were found exclusively in the extracellular compartment. An additional 56 proteins were identified with at least twice as many peptide counts in the conditioned medium as compared to the cell lysate studies. Thus, in comparing across our entire HMEC proteome database, 177 of the total 889 proteins identified were found to be localized primarily to the extracellular compartment (see Supporting Information). The cells used in this study were extensively washed to remove protein components arising from the fetal bovine serum (FBS) used in culture, and treatments were conducted in serum-free medium. However, it is possible that measurable amounts of FBS components could carry over to the downstream analysis. To clarify the contribution of potential serum protein contaminants, we filtered the data further by conducting blast searches against the NCBI bovine database (see Materials and Methods for details). This resulted in identifying 9 out of the 177 proteins which contained at least one unique bovine peptide sequence. Proteins with multiple unique bovine peptides included alpha-fetoprotein and hemoglobin-alpha. Other proteins with only a single unique bovine peptide included insulin, a series of laminin subunits, and a series of proprotein convertase subtypes. These nine proteins were removed from our secretome list, since at least some component was bovine based. An additional 17 proteins were found that did not have a unique human peptide sequence (i.e., complete identify between the human and bovine sequence). To be conservative, these proteins were removed from further analysis as well. This resulted in a final set of 151 proteins for further analysis. To estimate whether the data filtering approaches used were effective in enriching for potential secreted and shed proteins, we compared the distribution of Gene Ontology (GO) annotaJournal of Proteome Research • Vol. 7, No. 2, 2008 561
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Figure 3. Comparative summary of annotated cellular localization distributions. Shown is the GO protein distribution of cellular compartments for the 4155 global HMEC protein data set, the 889 proteins identified in conditioned medium in the current study, and the filtered 151 secretome protein subset. Results shown are based upon concurrent reporting of categories (i.e., one protein can fall within multiple GO categories). Across all data sets, 87% of the proteins were annotated within GO.
tions for cellular localization for our global cell lysate data sets (4155 proteins), all proteins identified in conditioned medium (889 proteins), and the 151 proteins in our final secretome subset. The results shown in Figure 3 clearly demonstrate the data filtering strategy significantly enriched for membrane and extracellular proteins and reduced nuclear and mitochondrial proteins. Excluding proteins whose intracellular localization is not annotated (or unknown), 79% of the final 151 proteins selected are annotated as extracellular and/or membrane proteins, and the remaining fraction is predominantly cytoplasmic. Although GO annotations are not absolute indicators, the overall distribution is consistent with a significant enrichment for shed and secreted proteins. Identification of PMA- and EGFR-Regulated Proteins. A stringent criterion for a secreted protein is that its extracellular levels will be modulated by known secretagogues, such as PMA. Indeed, comparison of the total number of peptide spectra between control and PMA-treated samples suggests clear treatment-related differences for many identified proteins. Examples of proteins that appear to be selectively released into the extracellular space by HMEC upon PMA stimulation, based on the total number of peptide identifications per protein, are shown in Table 1. Among the most abundantly represented class of proteins identified are lysosomal cysteine proteinases, including members of the cathepsin family. The high representation of this class of proteins provides confidence that the identified proteomic response involves PMA-mediated stimulation of secretory vesicles in HMEC. Additional classes of proteins include both urokinase- and tissue-type plasminogen activators and members of the matrix metalloproteinase and kallikrein serine protease families. For several proteins that are known to be exported through secretory pathways, such as kallikrein 5, kallikrein 6, and cathepsin D, the overall peptide coverage obtained was sufficient to predict the signal peptide sequence, which is cleaved prior to secretion (see Supporting Information).48,49 This result provides an additional level of 562
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Jacobs et al. confidence that these proteins are regulated through PMAdependent stimulation of exocytosis. In addition to these secreted proteins, transmembrane proteins potentially released from HMEC through ectodomain shedding mechanisms were also identified. Examples include the proteoglycan syndecan-4 and members of the ephrin family of receptor tyrosine kinase ligands, whose regulated shedding in other cell types is associated with directing cell migration and guidance.50,51 It is noteworthy that a previous study in HMEC also identified syndecan-4 as a PMA-stimulated protein,52 which also provides confidence in the reproducibility of our results. PMA was chosen for these studies because it acts as a general stimulus for both ectodomain shedding and protein secretory pathways. Although PMA is a protein kinase C agonist, it has also been shown to transactivate EGFR in other cell types.24,31 The results shown in Figure 4 demonstrate that this mechanism of signaling is conserved in HMEC as well. Based on immunoprecipitation of total EGFR followed by Western blot with a phosphotyrosine-specific antibody, we found that PMA treatment caused a rapid increase in tyrosine phosphorylation of the EGFR, apparent within 15 min (Figure 4A). Although the magnitude of PMA-induced EGFR phosphorylation was less than that induced by direct activation with EGF (1 ng/mL) (Figure 4B), PMA treatment caused a more sustained increase in EGFR phosphorylation than did EGF, in that it remained elevated above untreated controls even after 24 h. The increased phosphorylation of EGFR induced by PMA was also markedly reduced by cotreatment with PD153035, a selective inhibitor of EGFR autophosphorylation,36 indicating that the PMA-sensitive phosphorylation sites involve tyrosine residues required for EGFR kinase activity (Figure 4D). Western blot analysis using an antibody that selectively recognizes dualphosphorylated and active ERK proteins demonstrated that PMA also caused a robust activation of the ERK pathway within 15 min (Figure 4E). Furthermore, inhibition of PMA-induced EGFR transactivation by cotreatment with PD153035 also dramatically reduced ERK activation, as did selective inhibition of the upstream activator of ERK (MEK) using U0126 (Figure 4F). These results demonstrate that the increased EGFR phosphorylation observed in response to PMA treatment is coupled to functional downstream signaling through the canonical MAPK pathway. Spectra counts provide a qualitative measure of abundance and were useful in identifying candidate proteins whose release was regulated by PMA. To provide an independent and more quantitative measure of abundance, we also used LC-FTICR analysis to obtain high mass accuracy measurements. For this approach, the calculated masses and chromatographic elution times of each peptide identified by the LC-MS/MS analyses were used to develop a peptide mass tag database. The mass tags developed from the current study were combined with mass tags from previous proteomic studies using HMEC.23,38,43 The combined database was then used as a reference to identify and quantify peptides based upon integrated peak area values obtained by LC-FTICR. Conditioned medium protein samples derived from control cells (medium alone) and cells treated with PMA, EGFR inhibitor alone, or PMA combined with EGFR inhibitor were analyzed in triplicate by LC-FTICR, and the subsequent high mass accuracy isotope distributions were identified using the reference database. A total of 459 proteins were identified by LC-FTICR (with at least two peptides per protein), and 404 of these proteins (88%) overlapped with the 889 total proteins
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Mammary Epithelial Cell Secretome Table 1. Example Proteins Identified in HMEC Conditioned Medium By LC-MS/MS control protein
Cathepsin L2 Syndecan-4 Precursor Kallikrein 6 Serum Amyloid A2 Inhibin β a-chain LISCH Protein Tissue-type Plasminogen Activator Cathepsin B Interstitial Collagenase (MMP-1) Stromelysin 2 (MMP-10) SPARC-related modular calcium-binding protein Semaphorin 7A Extracellular Matrix Protein 1 Brain-Specific Serine Protease
unique
a
PMA total
b
unique
a
totalb
sequencec
3 4 0 2 0 3 0
3 5 0 2 0 3 0
23 15 16 4 9 9 6
65 49 35 18 28 18 19
K.AVATVGPISVAMDAGHSSFQFYK.S R.ETEVIDPQDLLEGR.Y R.LARPAKLSELIQPLPLER.D K.RGPGGAWAAEVISNAR.E R.LFQQQKHPQGSLDTGEEAEEVGLKGER.S R.VVATKQGNAVTLGDYYQGR.R R.IKGGLFADIASHPWQAAIFAK.H
1 0
1 0
10 11
16 15
K.ILRGQDHCGIESEVVAGIPRTDQYWEK.I K.AFQLWSNVTPLTFTK.V
0 0
0 0
7 4
12 11
R.LIADDFPGVEPK.V R.RFTDYCDLNKDKVISLPELK.G
1 2
1 2
7 9
12 13
K.GHVGQDRVDFGQTEPHTVLFHEPGSSSVWVGGR.G R.VTPNLMGHLCGNQR.V
0
0
4
7
R.AQGGGALRAPSQGSGAAAR.S
a Number of unique peptides identified for the protein in the untreated or PMA sample. b Total number of peptides identified for the protein in the untreated or PMA sample. c The sequence shown is a representative sequence for the protein identified in the analysis.
tions and is displayed as a linked cluster analysis. Overall, the general treatment-related patterns between the two data sets compared well. A total of 36 proteins were found to change in abundance in response to PMA by at least 2-fold compared to controls, based on both spectra count and LC-FTICR ion measurements (Figure 5B). This consensus PMA-regulated subset is based on the conservative criteria that a minimum of 10 peptide identifications were observed by LC-MS/MS across all treatment groups. Figure 4. PMA stimulates EGFR transactivation. HMECs were treated with either PMA (200 nM) (panel A) or 10 ng/mL of EGF (panel B) for the times indicated. Total EGFR was immunoprecipitated and probed using an antiphosphotyrosine antibody by Western blot as a measure of EGFR transactivation. (C) Control samples where total EGFR was immunoprecipitated and then probed during Western blot using an antitotal EGFR antibody. (D) HMECs were treated with PMA in the presence or absence of a selective inhibitor of EGFR kinase activity, PD153035 (100 nM) for 2 h, followed by immunoprecipitation of total EGFR and Western blot analysis using an antibody against phospho-EGFR (Tyr1173). (E) Western blot analysis of phosphorylated ERK after 15 min treatment either with PMA alone or in the presence of PD153035. (F) Western blot analysis of phosphorylated ERK after 15 min of treatment with PMA alone or in the presence of the MEK inhibitor, U0126 (10 µM). The results shown are representative of at least three experiments. The double band in panels E and F are from the antibody recognizing the phosphorylated forms of both ERK1 (p44) and ERK2 (p42).
previously identified by LC-MS/MS analysis of conditioned medium. LC-FTICR peak area values were obtained for 85 of the original 151 proteins designated as our secretome subset. The mean values and coefficient of variation for these analyses are provided in the Supporting Information. A comparison of the treatment related patterns obtained by LC-MS/MS spectra count data and LC-FTICR peak area measurements for the 85 proteins represented by both data sets is shown in Figure 5A. To compare between these different abundance measurements, each data set was normalized as a fractional abundance value across the four treatment condi-
More than half of the PMA-stimulated subset consists of proteins with known proteolytic or extracellular matrix function, reinforcing the notion that the predominant effect of PMAinitiated signaling in HMEC is targeted toward stimulating proteins that remodel the extracellular environment. Another intriguing characteristic of the PMA-stimulated proteins is the high fraction of proteins that represent potential biomarkers of human cancer. Multiple PMA-stimulated proteins are documented to be differentially expressed in a variety of human cancers, and several are currently regarded as prognostic clinical markers (see Table 2 and references in Supporting Information). Examples include several members of the kallikrein, matrix metalloproteinase, and cathepsin families of proteases. Other proteins in this group include inhibin-βA, urokinase plasminogen activator, and an extracellular matrix protein (ECM1) reported to be expressed at higher levels in mammary cancer compared to normal mammary tissue.53–55 In some cases, PMA treatment stimulated the release of different protein isoforms from the same gene. For instance, two variants of serum amyloid protein, a secreted protein differentially expressed in multiple cancer types,56,57 were identified. A second example is dermokine, a protein known to have multiple isoforms and splice variants, some of which have been shown to be highly secreted during epithelial differentiation.58,59 The β and γ isoforms of dermokine were identified by multiple unique peptides specific to the isoform, as well as with peptide sequences common to both isoforms (see Supporting Information). Proteins not previously known to be expressed in mammary cells were also identified. For Journal of Proteome Research • Vol. 7, No. 2, 2008 563
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Jacobs et al.
Figure 5. Treatment-related secretome patterns measured by complementary MS analyses. (A) Heat map representation comparing treatment-related effects based upon LC-MS/MS spectral (peptide) count abundance measurements and LC-FTICR peak intensity results for 85 proteins. For comparison, the results are displayed as the normalized fractional abundance across treatment groups. This value (indicated by the color scale at the top) was calculated by dividing the peptide count or peak area for a treatment group by the sum of the peptide counts or peak areas across all treatment groups. Thus, proteins with equal representation in each of the 4 groups would have a normalized abundance of 0.25, whereas proteins which are represented in only 1 of 4 treatments would have a value of 1.0. (B) Consensus subset of proteins which showed at least 2-fold stimulation or repression by PMA treatment compared to controls as determined by both LC-MS/MS and LC-FTICR measurements. The left side panels highlight the functional category for each protein derived from the GO database annotation, illustrating the predominant effect of PMA on proteins involved in proteolytic and extracellular matrix functions. 564
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Mammary Epithelial Cell Secretome Table 2. PMA-Stimulated Proteins Differentially Expressed in Human Cancer*
a
protein
gene
cancer types
refsa
Kallikrein 5 Kallikrein 6 Kallikrein 10 MMP-1 MMP-9 MMP-10 Cathepsin L Cathepsin L2 Inhibin βA Extracellular Matrix Protein Serum Amyloid Protein Urokinase Plasminogen Activator Elafin/Peptidase Inhibitor3 Cadherin-3
KLK5 KLK6 KLK10 MMP1 MMP9 MMP10 CTSL CTSL2 INHBA ECM1 SAA1 PLAU PI3 CDH3
mammary; ovarian; prostate ovarian; uterine (serous); colorectal ovarian; tamoxifen resistant mammary colorectal; esophageal; melanoma; ovarian; mammary endometrial; mammary; nonsmall cell lung (NSCLC) NSCLC; esophageal; mammary; astrocytoma mammary; pancreatic; lung mammary; colorectal; cervical mammary; ovarian; prostate thyroid; metastatic mammary ovarian; lung; early colorectal mammary; broadly expressed in solid tumors lung; esophageal mammary; prostate
(1–4) (1, 5–7) (1, 8) (9–13) (14–18) (19–22) (23–26) (27, 28) (29–32) (33, 34) (35–37) (38–41) (42, 43) (44–46)
Additional references for Table 2 are provided as Supporting Information.
instance, a protein initially annotated “unknown” in the IPI database was identified through further peptide sequence alignment (BLAST) to be HLAR 698 (human homologue of mouse suprabasin). Our identification of this protein in HMECconditioned medium validates a previous prediction that it is a secreted protein.7 The results shown in Figure 5 also reveal a subset of the PMA-stimulated proteins whose cellular release is inhibited by blocking EGFR autocrine signaling. Interestingly, while EGFR autocrine activity is thought to be a general requirement for HMEC proliferation, motility, and morphogenesis,12,33,60,61 only a select group of related extracellular proteins identified appear to be strongly dependent on EGFR. These include three functionally related members of the matrix metalloproteinase family (MMP-1, MMP-9, MMP-10). PMA treatment strongly induced the secretion of these proteins, and this effect was markedly inhibited when cells were concurrently treated with PD153035. Orthogonal Validation of MS Results. Because the MMPs are thought to be critical to normal mammary epithelial cell behavior and development,11 additional experiments were designed to provide orthogonal validation of the combined MS analysis approach taken in this study. To further assess the dependencies of MMP secretion on EGFR-dependent ERK signaling, ELISA assays were conducted to measure secreted levels of MMP-1, MMP-9, and MMP-10 in conditioned medium from cells stimulated with PMA, in the presence or absence of selective inhibitors of EGFR or downstream ERK activation. We found that blocking EGFR transactivation with either the kinase inhibitor PD153035 or EGFR neutralizing antibody 22562 caused a marked reduction in secretion of each of the MMPs (Figure 6). Although the absolute level of induction by PMA varied among the three MMPs, cotreatment with PD153035 resulted in 74–77% inhibition of PMA-induced secretion of these proteins. Cotreatment with concentrations of 225 mAb (10 µg/ mL) previously shown to block EGFR transactivation10 was slightly more effective in inhibiting MMP secretion (82-85%). Inhibition of ERK activation by cotreatment with U0126 (Figure 4F), a selective inhibitor of the upstream activator of ERK,37 resulted in a complete blockade of PMA-induced secretion of all three MMPs evaluated (Figure 6). Thus, these results validate the MS results and demonstrate that the PMA-induced secretion of these proteins is mediated through EGFR-dependent activation of the ERK pathway.
Figure 6. PMA-induced matrix metalloproteinase secretion is mediated by EGFR-dependent ERK activation. Levels of secreted MMPs as determined by ELISA 24 h after treatment with media only (open bars), PMA (gray bars), PMA combined with PD153035 (single hatched bars), PMA combined with 10 µg/mL of 225mAb (double hatched bars), or PMA combined with 10 µM U0126 (black bars). Note: Values for the different MMPs are normalized to mg cell protein and are displayed on a log scale. The results shown are the mean ( s.d. from triplicate analysis within a single experiment, which was repeated twice with similar results.
Discussion Extracellular proteins play an essential role in governing mammary cell behavior through autocrine and paracrine signaling and by providing a local microenvironment necessary for cell motility and proliferative capacity. However, defining the overall repertoire of proteins that are released from cells in a regulated manner is challenging, due in part to the multiple cellular mechanisms that control these processes, the low abundance of many extracellular proteins, and the potential for confounding factors such as nonspecific release due to cell death in culture. In this study, we demonstrate a tiered strategy for filtering protein identifications obtained by sensitive LCMS methods to permit assignment to the extracellular compartment with additional confidence. With the comprehensive and sensitive LC-MS methods used, we identified 889 proteins in conditioned medium using strict criteria. By comparing our results with results from whole cell proteome analyses, proteins which were exclusively found or highly enriched in the extracellular space could be identified. After further eliminating contaminating serum proteins through cross-species database comparison, a subset of 151 proteins which have a high likelihood of being regulated as extracellular proteins was Journal of Proteome Research • Vol. 7, No. 2, 2008 565
research articles determined. Although to our knowledge this represents the most comprehensive data set of this type for human mammary epithelial cells, it is likely a conservative estimate of the secretome. With the data filtering approaches used, it is possible that some proteins observed in previous studies of whole cell lysates were not included in this subset, yet are bona fide secreted proteins that are induced under different growth conditions (such as in the presence of serum). Nonetheless, comparison of the distribution of GO annotations for cell localization before and after data filtering (Figure 3) demonstrates the approach specifically enriched for membrane and extracellular proteins. With this additional level of confidence, this restricted subset of proteins should therefore be considered a good initial definition of the secretome of mammary epithelial cells. One of the goals of cell proteomics is the identification of cellular response pathways either that are novel or whose importance in a specific cell type is poorly recognized. The use of PMA as a general stimulus allowed parsing of the initial global list of extracellular proteins into a defined subset of proteins whose extracellular release is regulated through secretory or shedding pathways. A major target of PMA regulation was the stimulated release of proteins involved in proteolytic function (Figure 5). In particular, ∼40% of the extracellular proteins stimulated by PMA are known proteases or modulators of protease activity. The proteins regulated by PMA also represent examples of several of the major cellular pathways for protein release, including regulated release through stimulated exocytosis (i.e., lysosomal proteases) and enhanced expression of proteins that are constitutively secreted (i.e., MMPs). A surprising result from this analysis is the high fraction of PMA-stimulated extracellular proteins identified that have been previously shown to be aberrantly expressed in various human cancers (Table 2), despite the fact that the HMEC cell line used is nontumorigenic. The effects of PMA are selective, since only a limited fraction of the total 151 secretome proteins identified were specifically stimulated by PMA. As PMA is a tumor promoter, it may be expected that this result reflects signaling pathways activated by PMA that are constitutively upregulated in some human cancers. Consistent with this interpretation, several of the PMA-stimulated proteins identified in this study were also recently reported to be released from cancer cells at higher levels compared to normal cells. These include not only proteins released by classical secretory pathways but also proteins released by regulated proteolysis from the cell membrane. As an example, extracellular levels of syndecan-4 reported to be elevated in pancreatic cancer63 are thought to be controlled through ectodomain shedding. For the subset of PMA-induced proteins not listed in Table 2, the expression/ secretory patterns in human cancer are not well characterized and represent excellent opportunities for further investigation. It is also apparent from our results that the signaling pathways stimulated by PMA result in a coordinated secretion of functionally related proteins. The coordinated secretion of some classes of proteins may reflect their chromosomal regulation as a multigene cluster. For instance, the kallikrein genes are tandemly arranged on region q13.4 of chromosome 19, representing the largest contiguous cluster of proteases in the human genome.64 Our results indicate that the expression and secretion of KLK5, KLK6, and KLK10 gene products are coregulated in mammary cells. Another example of coordinated regulation of secreted proteins is demonstrated with the 566
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Jacobs et al. identification of the dermokine and suprabasin (HLAR 698) gene products. These proteins have been primarily linked to epidermal cells and to our knowledge have not been shown to be secreted by mammary cells. Interestingly, these genes are closely aligned in the same orientation on human chromosome 19q13.1 in a locus termed the stratified epithelium secreted peptides complex (SSC).58,59 While the precise function of these proteins remains unknown, both are implicated in epidermal differentiation, and a previous study in mice showed suprabasin is induced by PMA treatment.65 As PMA causes the differentiation and growth arrest of HMEC,66 our results suggest a potential autocrine or paracrine role for this gene complex in mammary cell differentiation. Another important outcome of this study is finding that the secretion of several proteins within a matrix metalloproteinase cascade is coordinately regulated through “indirect” mechanisms requiring EGFR transactivation. Two of the EGFRdependent MMPs identified in HMEC (MMP-1, MMP-9) are also present in human mammary tumor interstitial fluid,20 consistent with their proposed roles in regulating tumor invasion and angiogenesis. The significance of EGFR transactivation as a conserved physiological signaling mechanism is suggested by the growing number of growth factor receptor– ligands, cytokines, and physical agents demonstrated to stimulate transactivation in more than 60 cell types.67 In general, these studies have focused on the mitogenic aspects of EGFR signaling cross-talk. However, we found that the secretion of MMP-1, MMP-9, and MMP-10 was also regulated through EGFR-dependent transactivation. The sustained transactivation of EGFR in response to PMA (Figure 4A) is consistent with previous studies suggesting prolonged activation of the ERK is necessary for induction of MMP-9.68,69 Although the mechanisms of transactivation are not clear, our demonstration that MMP secretion is blocked by both an EGFR kinase inhibitor and a neutralizing EGFR antibody suggests that the MMP response is triggered by PMA-induced shedding of EGFR ligands. Indeed, recent experiments using protein microarray assays have demonstrated PMA stimulates the shedding of EGFR ligands in HMEC, including amphiregulin and transforming growth factor R (Richard Zangar, PNNL, personal communication). The coordinated regulation of several MMPs through EGFR transactivation could be a useful cellular strategy because several of these enzymes function cooperatively as an enzymatic cascade during tissue remodeling.70 Although the MMPs are secreted as pro-zymogens, the presence of catalytic amounts of plasmin generated by plasminogen activator activity is sufficient to fully activate MMP-1, MMP-9, and MMP-10.70 It is noteworthy that not only are the MMPs regulated in an EGFR-dependent manner but also the upstream urokinase plasminogen activator also demonstrated EGFR dependence (Figure 5). Once secreted, MMP-1 can also be activated by MMP-10,71 and it has been reported that MMP-10 also activates MMP-9.72 In addition to the known functional overlap between extracellular activation of these enzymes, synergy in the overall matrix remodeling activity may also be gained through their coordinated transcriptional regulation by the EGFR signaling pathway. To our knowledge, this is the first report demonstrating the coordinated secretion of an extracellular proteolytic cascade through a transactivation-dependent mechanism. While PMA was used as a general stimulus in this study, prior work from our laboratory demonstrated that TNF-induced secretion of MMP-9 is also EGFR-dependent.10 A study report-
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Mammary Epithelial Cell Secretome ing that osteopontin induces urokinase-type plasminogen activator in an EGFR-dependent manner73 indicates that transactivation-dependent MMP regulation could be a general response to different physiological stimuli.
Conclusions In summary, we have demonstrated a systematic approach for determining the secretome in nontumorigenic HMEC. In addition to the use of PMA as a general secretegogue, we find that comparison of results from analysis of conditioned medium proteins with whole cell lysate proteomes is a valuable strategy for filtering protein identifications obtained using highly sensitive LC-MS technologies, permitting identification of a prospective pool of extracellular proteins with additional confidence. The approach should be generally applicable for other cell types and for comparing secretome responses between normal and cancer cells. Furthermore, our results reveal a role for transactivation of the EGFR signaling pathway in regulating matrix metalloprotease secretion by mammary cells. These findings may be particularly important for understanding the relationships between amplified EGFR signaling and cell invasiveness during mammary carcinogenesis. Abbreviations: EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; HMEC, human mammary epithelial cell; LC-FTICR-MS, liquid chromatography coupled Fourier transform ion cyclotron resonance mass spectrometry; LC-MS/ MS, liquid chromatography-tandem mass spectrometry; MEK, extracellular signal-regulated kinase; MMP, matrix metalloproteinase; PMA, phorbol 12-myristate 13-acetate.
Acknowledgment. Support for portions of this research was provided by the Laboratory Directed Research Development Program through the Biomolecular Systems Initiative, the NIH National Center for Research Resources (RR018522), and the Environmental Molecular Sciences Laboratory (EMSL) at Pacific Northwest National Laboratory (PNNL). The EMSL is a national scientific user facility sponsored by the U.S. Department of Energy Office of Biological and Environmental Research. PNNL is operated by Battelle for the DOE under Contract DE-ACO6-76RLO 1830. Supporting Information Available: Three supplemental tables, two supplemental figures, and additional references that support the data presented are provided. These include: Supplemental Table 1, total list of unique peptides, masses, and SEQUEST scores for the 889 proteins identified in this study. The protein group numbers derived from Protein Prophet as well as gene identifiers are listed for reference. Raw MS/MS spectra files (DTA files) used in the creation of the mass tag database can be obtained by contacting the corresponding author. Supplemental Table 2, list of 151 proteins which were found to be enriched or exclusively identified in the extracellular compartment when compared with previous HMEC cell lysate proteomic databases. Also included are spectra counts, mean FTICR ion peak areas, and coefficient of variation information for untreated controls, PMA treatment, EGFR inhibitor alone (PD), and combined PMA and EGFR inhibitor (PD + PMA). Supplemental Figure 1, LC-MS/MS peptide coverage for selected secreted proteins. Supplemental Figure 2, LC-MS/MS identification of dermokine splice variants. Supplemental References: Additional references in support of Table
2 of the manuscript. This material is available free of charge via the Internet at http://pubs.acs.org.
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