Proteomics Analysis of Conditioned Media from Three Breast Cancer Cell Lines
2007; Elsevier BV; Volume: 6; Issue: 11 Linguagem: Inglês
10.1074/mcp.m600465-mcp200
ISSN1535-9484
AutoresVathany Kulasingam, Eleftherios P. Diamandis,
Tópico(s)S100 Proteins and Annexins
ResumoA "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 μg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers. A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 μg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers. Breast cancer is a leading cause of death among women with solid tumors in North America (1van Diest P.J. van der W.E. Baak J.P. Prognostic value of proliferation in invasive breast cancer: a review.J. Clin. Pathol. 2004; 57: 675-681Crossref PubMed Scopus (290) Google Scholar). It is a disease of the middle and late ages of life as 75% of breast cancer is diagnosed in women over the age of 50 (2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J. Clin. 2004; 54: 8-29Crossref PubMed Scopus (3914) Google Scholar). Although breast cancer is less common at a young age, younger women tend to have a more aggressive form of the disease than older women. The 5-year survival rate is close to 97% when the cancer is confined to the breast (2Jemal A. Tiwari R.C. Murray T. Ghafoor A. Samuels A. Ward E. Feuer E.J. Thun M.J. Cancer statistics, 2004.CA Cancer J. Clin. 2004; 54: 8-29Crossref PubMed Scopus (3914) Google Scholar). 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Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.Cancer Res. 1990; 50: 6075-6086PubMed Google Scholar). BT474, a luminal subtype obtained from a stage II localized solid tumor, is positive for ER and progesterone receptor (50–60% of all breast cancer cases) (32Lasfargues E.Y. Coutinho W.G. Redfield E.S. Isolation of two human tumor epithelial cell lines from solid breast carcinomas.J. Natl. Cancer Inst. 1978; 61: 967-978PubMed Google Scholar). This cell line also displays amplification of Her-2/neu or ERBB2 (30% of all breast cancer cases) (33Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene.Science. 1987; 235: 177-182Crossref PubMed Scopus (10014) Google Scholar). Her-2/neu is a cell membrane surface-bound tyrosine kinase involved in signal transduction, leading to cell growth and differentiation. 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These cell lines were cultured in serum-free media (SFM) to ensure that the collected conditioned media (CM) contain no other extraneous proteins except for the secreted or shed proteins from the cancer cells. By collecting and concentrating large volumes of CM produced from cell lines representing seminormal (MCF-10A), non-invasive (BT474), and metastatic origins (MDA-MB-468), the secreted and shed proteins would accumulate in the CM, thereby facilitating their identification through MS. Our comparative proteomics analysis of the CM of MCF-10A, BT474, and MDA-MB-468 identified over 600, 500, and 700 proteins, respectively. A large portion of the proteins was present in all three cell lines; however, a significant portion contained proteins that were unique to each of the lines. Among these were our internal control proteins, human kallikreins 5, 6, and 10, that were identified by MS and ELISA in MDA-MB-468 cells at a concentration ranging from 2 to 50 μg/liter. Members of the human kallikrein family (KLKs) have been implicated in the process of carcinogenesis, and the application of kallikreins as biomarkers for diagnosis and prognosis is currently being investigated. Kallikreins are secreted enzymes that encode for trypsin-like or chymotrypsin-like serine proteases (38Borgono C.A. Diamandis E.P. The emerging roles of human tissue kallikreins in cancer.Nat. Rev. Cancer. 2004; 4: 876-890Crossref PubMed Scopus (557) Google Scholar). Prostate-specific antigen (KLK3), belonging to the family of human tissue kallikreins, and human kallikrein 2 (KLK2) currently have important clinical applications as prostate cancer biomarkers (39Rittenhouse H.G. Finlay J.A. Mikolajczyk S.D. Partin A.W. Human Kallikrein 2 (hK2) and prostate-specific antigen (PSA): two closely related, but distinct, kallikreins in the prostate.Crit. Rev. Clin. Lab. Sci. 1998; 35: 275-368Crossref PubMed Scopus (284) Google Scholar). In addition to the control proteins, various proteases, receptors, protease inhibitors, cytokines, and growth factors were identified. Cellular localization, biological function, and Unigene analyses were performed for the shortened list of candidates consisting of extracellular, membrane, and unclassified proteins. A significant degree of overlap was observed among the proteins identified in this study using a cell culture model and other studies using relevant biological fluids such as NAF and tumor interstitial fluid (TIF). The expression of four candidate molecules was examined in biological fluids, tissues, serum, and breast cytosols. Finally spectral counting analysis revealed promising molecules to investigate further for both understanding the disease and as potential biomarkers for breast cancer. The breast epithelial cell line MCF-10A and the breast cancer cell lines BT-474 and MDA-MB-468 were purchased from the American Type Culture Collection (ATCC), Manassas, VA. MCF-10A was maintained in Dulbecco's modified Eagle's medium and F-12 medium (DMEM/F-12) supplemented with 8% fetal bovine serum, epidermal growth factor (20 ng/ml), hydrocortisone (0.5 μg/ml), cholera toxin (100 ng/ml), and insulin (10 μg/ml). BT-474 and MDA-MB-468 were maintained in phenol red-free RPMI 1640 culture medium (Invitrogen) supplemented with 8% fetal bovine serum. All cells were cultured in a humidified incubator at 37 °C and 5% CO2 in tissue culture T 75-cm2 flasks. Approximately 30 × 106 cells were seeded individually into six 175-cm2 tissue culture flasks per cell line. After 2 days, the RPMI 1640 or DMEM/F-12 media were discarded, and the cells were rinsed twice with 1× PBS. Following this, 30 ml of chemically defined Chinese hamster ovary serum-free medium (Invitrogen) supplemented with glutamine (8 mm) (Invitrogen) were added, and the flasks were incubated for an additional 24 h. The CM were collected and spun down to remove cellular debris. CM were then frozen at −80 °C until further use. A 1-ml aliquot was taken at the time of harvest to measure for total protein (Bradford assay), lactate dehydrogenase (LDH), KLK5, KLK6, and KLK10 via ELISA. The adhered cells were trypsinized and counted using a hemocytometer. This procedure was repeated several times for reproducibility. In addition, 30 ml of the culture media (RPMI 1640 and DMEM/F-12) were subjected to the same conditions as above with no cells added and used for comparison. For the MDA-MB-468 cell lysate experiment, at the end of 24 h in SFM, the adhered cells were lysed using a French press (Thermo Electron) in which the cells are sheared by forcing them through a narrow space. Total protein was measured, and 400 μg of protein from the lysate were added to 60 ml of chemically defined Chinese hamster ovary medium and processed in the same manner as the CM. The cell lysate experiment was performed in duplicate. Two 30-ml CM aliquots were combined (60 ml) for each cell line, creating three biological replicates per cell line, and dialyzed using a 3.5-kDa molecular mass cutoff membrane. The CM were dialyzed in 5 liters of 1 mm ammonium bicarbonate solution overnight at 4 °C with two buffer changes. The dialyzed CM were poured equally into two 50-ml conical tubes. The CM were frozen and lyophilized to dryness. The lyophilized sample was denatured using 8 m urea and reduced with DTT (final concentration, 13 mm; Sigma). Following reduction, the sample was alkylated with 500 mm iodoacetamide (Sigma) and desalted using a NAP5 column (GE Healthcare). The sample was lyophilized and trypsin (Promega)-digested (1:50, trypsin:protein concentration) overnight in a 37 °C waterbath. Following this, the peptides were lyophilized to dryness. The trypsin-digested dry sample was resuspended in 120 μl of mobile phase A (0.26 m formic acid in 10% acetonitrile). The sample was directly loaded onto a PolySULFOETHYL A™ column (The Nest Group, Inc.) containing a hydrophilic, anionic polymer (poly-2-sulfoethyl aspartamide). A 200-Å pore size column with a diameter of 5 μm was used. A 1-h fractionation procedure was performed using an HPLC system (Agilent 1100). A linear gradient of 0.26 m formic acid in 10% acetonitrile as the running buffer and 1 m ammonium formate added as the elution buffer was used. The eluent was monitored at a wavelength of 280 nm. Forty fractions, 200 μl each, were collected every minute after the start of the elution gradient. These 40 fractions were pooled into eight combined fractions (each pool consisting of five fractions) and lyophilized to ∼200 μl. The eight pooled fractions per replicate per cell line were loaded into a ZipTipC18 pipette tip (Millipore; catalogue number ZTC18S096) and eluted in 4 μl of 68% ACN made up of Buffer A (95% water, 0.1% formic acid, 5% ACN, 0.02% TFA) and Buffer B (90% ACN, 0.1% formic acid, 10% water, 0.02% TFA). 80 μl of Buffer A were added, and 40 μl were injected onto a 2-cm C18 trap column (inner diameter, 200 μm). The peptides were eluted from the trap column onto a resolving 5-cm analytical C18 column (inner diameter, 75 μm) with an 8-μm tip (New Objective). The LC setup was coupled on line to a 2-D linear ion trap (LTQ, Thermo Inc.) mass spectrometer using a nano-ESI source in data-dependent mode. Each pooled fraction was run on a 120-min gradient. The eluted peptides were subjected to MS/MS. DTAs were created using the Mascot Daemon (version 2.16) and extract_msn. The parameters for DTA creation were: minimum mass, 300 Da; maximum mass, 4000 Da; automatic precursor charge selection; minimum peaks, 10 per MS/MS scan for acquisition; and minimum scans per group, 1. The resulting raw mass spectra from each pooled fraction were analyzed using Mascot (Matrix Science, London, UK; version 2.1.03) and X!Tandem (Global Proteome Machine Manager, version 2.0.0.4) search engines on the non-redundant International Protein Index (IPI) human database version 3.16 (>62,000 entries). Up to one missed cleavage was allowed, and searches were performed with fixed carbamidomethylation of cysteines and variable oxidation of methionine residues. A fragment tolerance of 0.4 Da and a parent tolerance of 3.0 Da were used for both search engines with trypsin as the digestion enzyme. This operation resulted in eight DAT files (Mascot) and eight XML files (X!Tandem) for each replicate sample per cell line. Scaffold (version Scaffold-01_05_19, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the PeptideProphet algorithm (40Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3912) Google Scholar). Protein identifications were accepted if they could be established at greater than 80.0% probability and contained at least one identified peptide. Protein probabilities were assigned by the ProteinProphet algorithm (41Nesvizhskii A.I. Keller A. Kolker E. Aebersold R. A statistical model for identifying proteins by tandem mass spectrometry.Anal. Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3655) Google Scholar). Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. The DAT and XML files for each cell line plus their respective negative control files (RPMI 1640 or DMEM culture media only) were inputted into Scaffold to cross-validate Mascot and X!Tandem data files. Each replicate sample was designated as one biological sample containing both DAT and XML files in Scaffold and searched with MudPIT (multidimensional protein identification technology) option clicked. The results obtained from Scaffold were processed using an in-house-developed program that generated the protein overlaps between samples. Each protein identification was assigned a cellular localization based on information available from Swiss-Prot, Genome Ontology (GO), Human Protein Reference Database, and other publicly available databases. To calculate the false positive error rate, the individual fractions were analyzed using the "sequence-reversed" decoy IPI human version 3.16 d
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