Differential Expression of Novel Tyrosine Kinase Substrates during Breast Cancer Development

• DOI: 10.1074/mcp.m700395-mcp200
• Publication Date: 2007-09-14T00:30:01Z
• Authors: Yunhao Chen; Lee-Yee Choong; Qingsong Lin; Robin Philp; Chee-Hong Wong; Boon-Keong Ang; Yee-Ling Tan; Marie-Chiew-Shia Loh; Choy-Leong Hew; Nilesh Shah; Brian J. Druker; Poh-Kuan Chong; Yoon-Pin Lim
• Keywords: 0301 basic medicine; 570; Spectrometry, Mass, Electrospray Ionization; 610; Breast Neoplasms; Protein-Tyrosine Kinases; Immunohistochemistry; Substrate Specificity; 3. Good health; 03 medical and health sciences; Tandem Mass Spectrometry; Cell Line, Tumor; Humans; Phosphorylation; Chromatography, Liquid

To identify novel tyrosine kinase substrates that have never been implicated in cancer, we studied the phosphoproteomic changes MCF10AT model of breast cancer progression using a combination phosphotyrosyl affinity enrichment, iTRAQ™ technology, and LC-MS/MS. Using complementary MALDI- ESI-based mass spectrometry, 57 unique proteins comprising kinases, phosphatases, other signaling were detected to undergo differential phosphorylation during disease progression. Seven these (SPAG9, Toll-interacting protein (TOLLIP), WBP2, NSFL1C, SLC4A7, CYFIP1, RPS2) validated be substrates. SPAG9, TOLLIP, NSFL1C further proven authentic targets epidermal growth factor Iressa (gefitinib). A closer examination revealed expression bicarbonate transporter, was down-regulated 64% 25 matched normal tumor clinical samples. The TOLLIP cancers heterogeneous with 25% showing higher compared tissues 35% reverse trend. Preliminary studies on hand, did not show between diseased states. This is first time SLC4A7 discovered as are also associated human development. Future molecular functional will provide insights into roles etiology cancer. Understanding critical its management. could achieved by creating database genetic aberrations initiation, maintenance, However, practically impossible study an individual because physicians' intervention. Although use samples from patients different stages one option, differences patients' environmental backgrounds complicate analysis. Consequently experimental models valuable successfully used events development (1Clark E.A. Golub T.R. Lander E.S. Hynes R.O. Genomic analysis metastasis reveals essential role for RhoC.Nature. 2000; 406: 532-535Crossref PubMed Scopus (1308) Google Scholar, 2Kang Y. Siegel P.M. Shu W. Drobnjak M. Kakonen S.M. Cordon-Cardo C. Guise T.A. Massague J. multigenic program mediating bone.Cancer Cell. 2003; 3: 537-549Abstract Full Text PDF (2043) 3Yang Mani S.A. Donaher J.L. Ramaswamy S. Itzykson R.A. Come Savagner P. Gitelman I. Richardson A. Weinberg Twist, master regulator morphogenesis, plays metastasis.Cell. 2004; 117: 927-939Abstract (3084) Scholar). comprises series isogenic, xenograft-derived cell lines mimic (4Dawson P.J. Wolman S.R. Tait L. Heppner G.H. Miller F.R. MCF10AT: evolution proliferative disease.Am. Pathol. 1996; 148: 313-319PubMed 5Miller Xenograft premalignant disease.J. Mammary Gland Biol. Neoplasia. 5: 379-391Crossref (89) Scholar) has characterized terms cytogenetics, apoptosis, transforming factor-β signaling, proteomics (6Chong B.E. Hamler R.L. Lubman D.M. Ethier S.P. Rosenspire A.J. Differential screening mapping nonporous reversed-phase HPLC coupled spectrometric analysis.Anal. Chem. 2001; 73: 1219-1227Crossref (71) 7Chong Rapid profiles non-porous high performance liquid chromatography separation matrix-assisted laser desorption/ionization time-of-flight spectral analysis.Rapid Commun. Mass Spectrom. 1999; 13: 1808-1812Crossref (34) 8Chong F. Protein identification isolated spectrometry.Rapid 1998; 12: 1986-1993Crossref 9Starcevic S.L. Diotte N.M. Zukowski K.L. Cameron M.J. Novak R.F. Oxidative DNA damage repair lineage (PBD).Toxicol. Sci. 75: 74-81Crossref (35) 10Starcevic Elferink Progressive resistance apoptosis Natl. Cancer Inst. 93: 776-782Crossref (25) 11Tang B. Vu Booker T. Santner S.J. Anver M.R. Wakefield L.M. TGF-β switches suppressor prometastatic progression.J. Clin. Investig. 112: 1116-1124Crossref (342) 12Worsham Pals G. Schouten J.P. Tiwari N. van Spaendonk R. High-resolution immortalization, transformation, MCF10 model.Breast Res. Treat. 2006; 96: 177-186Crossref (65) major deficiency most tools their inability detect low abundance proteins. Phosphoproteomics hand offers several advantages. (i) It form subproteomics allows detection would otherwise masked whole proteome approach. (ii) Unlike microarray, it open system discovery phosphoproteins studied. In turn, contribute our understanding biology. (iii) Because phosphoproteomics focuses kinases and/or substrates, may implication target-directed therapeutics either or epidemiology drug targets. For reasons, interested exploit studying done before. We previously developed two-dimensional PAGE cleavable ICAT-based dissect pathways map phosphoprotein contents tumors, respectively (13Chen Low T.Y. Choong L.Y. Ray R.S. Tan Y.L. Toy Lin Q. Ang B.K. Wong C.H. Lim Li Hew C.L. Sze N.S. Druker B.J. Y.P. identified Endofin, DCBLD2, KIAA0582 EGF cells.Proteomics. 2007; 7: 2384-2397Crossref (47) 14Lim Diong L.S. Qi Epstein R.J. Phosphoproteomic fingerprinting anticancer action cells.Mol. Ther. 2: 1369-1377PubMed 15Lim C.Y. Ooi L.L. Selective hyperphosphorylation cytoskeletal stress primary cancers: implications adjuvant kinase-inhibitory drugs.Clin. 10: 3980-3987Crossref (36) this study, adapted relatively newer isobaric tags relative absolute quantification (iTRAQ™) 1The abbreviations are: iTRAQ, quantitation; PV, pervanadate; EGF, factor; EGFR, receptor; FAK, focal adhesion kinase; IPI, International Index; IHC, immunohistochemistry; EF, error HEK, embryonic kidney; EPHA2, Ephrin A2; ER, estrogen BIN, biological interaction network; cytoplasmic FMR1 interacting 1 isoform 1; protein; VCP, transitional endoplasmic reticulum ATPase/89kDa protein. technology profile level aims displayed levels model. These candidates subsequently investigated involvement setting. PY20H mouse monoclonal anti-phosphotyrosine antibody, SHP2, Cortactin, Paxillin, plakoglobin, SHC1, β-catenin, EPS8 antibodies BD Biosciences. P130CAS antibody Neomarkers (Fremont, CA); A2 receptor Zymed Laboratories Inc. (Carlsbad, E-cadherin Cell Signaling Technology (Beverly, MA); Chemicon (Temecula, CYFIP Upstate Biotechnology (Lake Placid, NY); PSMC4 Atlas Antibodies AB (Stockholm, Sweden); receptor-type tyrosine-protein phosphatase kappa precursor Abcam (Cambridge, UK); RPS2 Abnova Corp. (Taipei, Taiwan). cDNAs WBP2 Origene (Rockville, MD). Anti-V5 tag pcDNA™3.1 Directional TOPO® vector Invitrogen. ECL kit purchased GE Healthcare, prestained weight markers Bio-Rad, protease inhibitor mixture Roche Applied Science, polyvinylidene difluoride membranes Millipore (Bedford, MA). Sodium orthovanadate 3% (0.9 m) hydrogen peroxide Sigma-Aldrich. Xenograft-derived (MCF10A1, MCF10AT1KCl.2, MCF10CA1h, MCF10CA1aCl.1) obtained Dr. Fred at Barbara Ann Karmanos Institute (Detroit, MI). Cells cultured described elsewhere (11Tang incubated 37 °C humidified atmosphere containing 5% CO2 routinely serum-starved 16 h before stimulation mm pervanadate 15 min °C. Fresh 0.45 m stock generated mixing equal volume 0.9 sodium H2O2. During stimulation, final concentration 1:454 dilution solution. extraction, cells grown 150-mm tissue culture dishes rinsed ice-cold PBS lysis ml non-denaturing buffer (50 Tris-HCl (pH 7.5), 0.5% Triton X-100, Igepal, 150 NaCl, EDTA, 50 NaF, plus inhibitors mixture). lysates clarified centrifugation 4 microcentrifuge 14,000 rpm 10 min. Estimation performed bicinchoninic acid assay (Pierce). Immunoblotting immunoprecipitation according previous reports (14Lim 16Lim B.C. Guy G.R. Association atypical C isotypes docker FRS2 fibroblast signaling.J. 274: 19025-19034Abstract (30) protocol xenograft reviewed approved Institutional Animal Care Use Committee National University Singapore compliance international guidelines care animals scientific purpose. A1 (107), 1k (5 × 106), 1h (106), 1a (3 105) Matrigel (BD Biosciences) (1:1) injected subcutaneously flanks severe combined immunodeficiency mice observed weekly. Histological assessment conducted certified pathologist. Serum-starved treated lysed. From large scale preparations lysates, portion analysis, retained subsequent validation. former purpose, 60 mg total each line purification via 4G10 antibody-based immunoaffinity purification. reported except enriched eluted PBS-based rather than Tris-based latter incompatible iTRAQ reagents After four determined, they then denatured, cysteines blocked (Applied Biosystems, Foster City, CA). Each sample digested trypsin provided reagent overnight (16 h) labeled follows: A1, 114 tag; 1k, 115 1h, 116 1a, 117 tag. As (PV)-induced phosphoproteome had amounts ranging μg, two vials same isotopic ensure complete labeling. pooled cleaned up cation exchange cartridge kit. desalted, lyophilized, analyzed ESI-LC/MS/MS MALDI-TOF-TOF. On-line QSTAR-XL hybrid quadrupole tandem spectrometer Biosystems) LC Packings (Dionex, Sunnyvale, CA) FAMOS autoinjector unit, Switchos 10-port valve UltiMatePLUS nanoflow pumping unit. Sample trapping C18 peptide (300 μm 5 mm, Packings) flow 0.1% formic μl/min. Following wash step diverted back through 100 nl/min Switchos. Peptides application gradient 0 90% acetonitrile over 40 separated passing column packed house consisted 75-μm 10-cm 5-μm packing (Column Engineering, Ontario, Canada). eluting sprayed directly orifice spectrometer, which run information-dependent acquisition mode selecting all 2+ 4+ charged ions signal intensity greater 8 counts/s range 300–2000 amu. CID nitrogen gas setting 4, collision energy set automatic, allowing increased increasing ion mass. fraction aliquot searched against subset Internation Index (IPI) (European Bioinformatics Institute, Hinxton, UK) Mascot (Matrix London, search engine. results exclusion list based masses matching peptides maximum 2000 masses. excluded MS/MS fragmentation when remaining rerun. quantification, data files (*.wiff) together ProteinPilot™ software version 2.0 UniProt (release 11, downloaded May 2007 267,354 entries 16,602 entries; European Institute) Paragon™ algorithm. parameters allowed cysteine modification methyl methanethiosulfonate, emphasis, modifications programmed threshold (unused protscore (conf)) 1.3 achieve 95% confidence. bias correction option executed concerned chemically induced can take place extents various cells. MALDI UltiMate™ (Dionex-LC equipped Probot™ spotting device. captured 0.3 1-mm trap (3-μm PepMap™, Å) washed 0.05% TFA followed 0.2 50-mm (Monolithic PS-DVB) Packings). mobile phase B 98% H2O, 2% ACN 80% 20% ACN, 0.04% TFA, respectively. elution 0–60% 20 rate 2.7 fractions mixed matrix solution (7 mg/ml α-cyano-4-hydroxycinnamic 130 μg/ml ammonium citrate 75% ACN) 5.4 μl/min 25-nl tee (Upchurch Scientific) onto 192-well stainless steel target plates Probot microfraction collector speed s/well. ABI 4700 Proteomics Analyzer MALDI-TOF/TOF operating result-independent mode. Typically 1000 shots accumulated well sample. analyses kV pressure ∼1 10−6 torr. noise ratio 100, 6000 spectrum. precursors 10,000 accumulated. GPS Explorer™ 3.5 create engine (version 2.1; Matrix Science) identifications. IPI 3.14, released January 2006, 57,366 sequences) restricted tryptic peptides. Cysteine methanethiolation; N-terminal labeling; lysine; serine, threonine, phosphorylation; methionine oxidation selected variable modifications. One missing cleavage allowed. Precursor tolerance ppm, fragment Da. Maximum rank 2, minimum score confidence interval (peptide) 95%. GSP Explorer 3.5. normalization 10-cm2 dish until 80–90% confluence (∼1 107 exponentially dividing cells). Total RNA extracted Purescript isolation (Gentra Qiagen, West Sussex, manufacturer's protocol. strand cDNA synthesis, 2 μg transcribed following conditions: 1× avian myeloblastosis virus transcriptase reaction (Promega), 30 units RNasin® ribonuclease dNTP (Invitrogen), oligo(dT) (Invitrogen). RT-PCR triplicates pooled. integrity assessed PCR amplification control gene, glyceraldehyde-3-phosphate dehydrogenase. Negative (without template) included check contaminating genomic DNA. Evaluation gene TaqMan® Gene Expression Assay gene-specific primers NSF1LC (Hs00739840_mH), (Hs00600857_m1), dehydrogenase (HS99999905_m1) Biosystems. Sample-specific loaded quadruplicates microplate cDNA/well 7900HT system. Quantification “normal” comparative CT method manual. Frozen Hospital General freshly prepared IHC fixing 10% neutral buffered formalin (Sigma) °C, subjecting ThermoShandon processor, embedding paraffin. Sections warmed oven, dewaxed three xylene, passaged graded ethanol (100, 95, 70%) double distilled H2O. (Abgent), (Abcam), SPAG9 antigen retrieval cooking 121 Tris-EDTA buffer, pH 9.0. (Chemicon Inc.), Target Retrieval Solution (DakoCytomation, Glostrup, Denmark) 95 quenching endogenous peroxidase activity H2O2 blocking BSA min, sections 1:50 dilution, 1:100 dilution. Detection Envision+/horseradish (DakoCytomation). All slides counterstained Gill's hematoxylin dehydrated, mounted light microscopic evaluation. Interpretation eosin analysis/scoring pathologist maintain consistency. design differentially phosphorylated shown Fig. 1A. MCF10A1, modeled after epithelium, MCF10AT1K.cl2, MCF10CA1a.cl1, epithelium grade lesions, They abbreviated study. tumors subcutaneous injection nude (note tumorigenic) grew rates grades validating indeed reflective (Fig. 1B Supplemental 1). prepare potent inhibitor, enhance presentation untreated stimulated PV probed reveal overall cellular 1C, top panel). intensities few (marked arrows) stronger stimulation. amount remarkably low, suggesting deficient more kinases. affinity-captured antibodies, trypsin-digested, (17Ross P.L. Huang Y.N. Marchese J.N. Williamson Parker K. Hattan Khainovski Pillai Dey Daniels Purkayastha Juhasz Martin Bartlet-Jones He Jacobson Pappin D.J. Multiplexed quantitation Saccharomyces cerevisiae amine-reactive tagging reagents.Mol. Proteomics. 1154-1169Abstract (3695) Scholar), spectrometry determine increase coverage identifications generated, separate made, 114, 115, 116, 117, ratios 115:114, 116:114, 117:114 indicate potentially respect A1. Table I shows (gene symbol, 2; name, 3) quantity (columns 6–8) antibody-enriched lines. S.D. (EF) columns 9–11 indicator ProteinPilot MALDI-based dataset. Additional information such sequence, m/z value, sites single assignment 1. (including group reporting) LC-MS/MS derived (A B) 3, Not platform (Supplemental 2B) Only those hits minimal best/total ESI/ProteinPilot-based method, 3 where Hence Tables proteins/isoforms (when sequence(s) sufficient discriminatory power). case, member, usually highest rank/score search, listed tables representative group.Table IRelative PV-stimulated Open table new tab cutoff values classify expressed, calculated median EF data. worked out 0.27 1.2 upper (1.27 (for S.D.) EF)) lower (0.73 0.83 applied Proteins below considered underexpressed, whereas above overexpressed. Taking point 30% variation acceptable main technical variation, minimized pooling effect. Another least needed taken consideration variations approach (18Gan C.S. Chong P.K. Pham T.K. Wright P.C. Technical, (iTRAQ).J Proteome 6: 821-827Cr