Cellular and Molecular Life Sciences

, Volume 68, Issue 11, pp 1983–2002 | Cite as

Global molecular dysfunctions in gastric cancer revealed by an integrated analysis of the phosphoproteome and transcriptome

  • Tiannan Guo
  • Sze Sing Lee
  • Wai Har Ng
  • Yi Zhu
  • Chee Sian Gan
  • Jiang Zhu
  • Haixia Wang
  • Shiang Huang
  • Siu Kwan Sze
  • Oi Lian Kon
Research Article


We integrated LC-MS/MS-based and protein antibody array-based proteomics with genomics approaches to investigate the phosphoproteome and transcriptome of gastric cancer cell lines and endoscopic gastric biopsies from normal subjects and patients with benign gastritis or gastric cancer. More than 3,000 non-redundant phosphorylation sites in over 1,200 proteins were identified in gastric cancer cells. We correlated phosphoproteome data with transcriptome data sets and reported the expression of 41 protein kinases, 5 phosphatases and 65 phosphorylated mitochondrial proteins in gastric cancer cells. Transcriptional expression levels of 190 phosphorylated proteins were >2-fold higher in gastric cancer cells compared to normal stomach tissue. Pathway analysis demonstrated over-presentation of DNA damage response pathway and underscored critical roles of phosphorylated p53 in gastric cancer. This is the first study to comprehensively report the gastric cancer phosphoproteome. Integrative analysis of the phosphoproteome and transcriptome provided an expansive view of molecular signaling pathways in gastric cancer.


Gastric cancer Phosphoproteome Transcriptome Protein antibody array Protein kinase Protein phosphatase Mitochondria DNA damage response 



Receptor tyrosine kinase


Mass spectrometry


High-performance liquid chromatography


False discovery rate


Electrostatic repulsion-hydrophilic interaction chromatography


Strong cation exchange


Immobilized metal ion affinity chromatography


DNA damage response



This work is supported by the National Cancer Centre of Singapore Research Fund. This work is also supported by grants from the Ministry of Education (ARC: T206B3211 to SKS) and the Agency for Science, Technology and Research (BMRC: 07/1/22/19/531 to SKS) of Singapore.

Supplementary material

18_2010_545_MOESM1_ESM.pdf (377 kb)
Supplementary material 1 (PDF 378 kb)
18_2010_545_MOESM2_ESM.xls (2.3 mb)
Supplementary material 2 (XLS 2,341 kb)


  1. 1.
    Peek RM Jr, Blaser MJ (2002) Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat Rev Cancer 2:28–37PubMedCrossRefGoogle Scholar
  2. 2.
    Wagner AD, Moehler M (2009) Development of targeted therapies in advanced gastric cancer: promising exploratory steps in a new era. Curr Opin Oncol 21:381–385PubMedCrossRefGoogle Scholar
  3. 3.
    Sawyers C (2004) Targeted cancer therapy. Nature 432:294–297PubMedCrossRefGoogle Scholar
  4. 4.
    Di Cosimo S, Baselga J (2010) Management of breast cancer with targeted agents: importance of heterogeneity. [corrected]. Nat Rev Clin Oncol 7:139–147PubMedCrossRefGoogle Scholar
  5. 5.
    Esteva FJ, Yu D, Hung MC, Hortobagyi GN (2010) Molecular predictors of response to trastuzumab and lapatinib in breast cancer. Nat Rev Clin Oncol 7:98–107PubMedCrossRefGoogle Scholar
  6. 6.
    Daub H, Specht K, Ullrich A (2004) Strategies to overcome resistance to targeted protein kinase inhibitors. Nat Rev Drug Discov 3:1001–1010PubMedCrossRefGoogle Scholar
  7. 7.
    Xu AM, Huang PH (2010) Receptor tyrosine kinase coactivation networks in cancer. Cancer Res 70:3857–3860PubMedCrossRefGoogle Scholar
  8. 8.
    Engelman JA, Zejnullahu K, Mitsudomi T, Song Y, Hyland C, Park JO, Lindeman N, Gale CM, Zhao X, Christensen J, Kosaka T, Holmes AJ, Rogers AM, Cappuzzo F, Mok T, Lee C, Johnson BE, Cantley LC, Janne PA (2007) MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316:1039–1043PubMedCrossRefGoogle Scholar
  9. 9.
    Nahta R, Yu D, Hung MC, Hortobagyi GN, Esteva FJ (2006) Mechanisms of disease: understanding resistance to HER2-targeted therapy in human breast cancer. Nat Clin Pract Oncol 3:269–280PubMedCrossRefGoogle Scholar
  10. 10.
    Stommel JM, Kimmelman AC, Ying H, Nabioullin R, Ponugoti AH, Wiedemeyer R, Stegh AH, Bradner JE, Ligon KL, Brennan C, Chin L, DePinho RA (2007) Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies. Science 318:287–290PubMedCrossRefGoogle Scholar
  11. 11.
    Cutsem EV, Kang Y, Chung H, Shen L, Sawaki A, Lordick F, Hill J, Lehle M, Feyereislova A, Bang Y (2009) Efficacy results from the ToGA trial: a phase III study of trastuzumab added to standard chemotherapy (CT) in first-line human epidermal growth factor receptor 2 (HER2)-positive advanced gastric cancer (GC). J Clin Oncol 27:18s Suppl. abstr LBA4509CrossRefGoogle Scholar
  12. 12.
    Dragovich T, Campen C (2009) Anti-EGFR-targeted therapy for esophageal and gastric cancers: an evolving concept. J Oncol 2009:804108PubMedGoogle Scholar
  13. 13.
    Iwasaki J, Nihira S (2009) Anti-angiogenic therapy against gastrointestinal tract cancers. Jpn J Clin Oncol 39:543–551PubMedCrossRefGoogle Scholar
  14. 14.
    Comoglio PM, Giordano S, Trusolino L (2008) Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat Rev Drug Discov 7:504–516PubMedCrossRefGoogle Scholar
  15. 15.
    Arkenau HT (2009) Gastric cancer in the era of molecularly targeted agents: current drug development strategies. J Cancer Res Clin Oncol 135:855–866PubMedCrossRefGoogle Scholar
  16. 16.
    Grabsch H, Sivakumar S, Gray S, Gabbert HE, Muller W (2010) HER2 expression in gastric cancer: rare, heterogeneous and of no prognostic value—conclusions from 924 cases of two independent series. Cell Oncol 32:57–65PubMedGoogle Scholar
  17. 17.
    Huang PH, White FM (2008) Phosphoproteomics: unraveling the signaling web. Mol Cell 31:777–781PubMedCrossRefGoogle Scholar
  18. 18.
    Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221PubMedCrossRefGoogle Scholar
  19. 19.
    Diella F, Gould CM, Chica C, Via A, Gibson TJ (2008) Phospho.ELM: a database of phosphorylation sites—update 2008. Nucleic Acids Res 36:D240–D244PubMedCrossRefGoogle Scholar
  20. 20.
    Diella F, Cameron S, Gemund C, Linding R, Via A, Kuster B, Sicheritz-Ponten T, Blom N, Gibson TJ (2004) Phospho.ELM: a database of experimentally verified phosphorylation sites in eukaryotic proteins. BMC Bioinformatics 5:79PubMedCrossRefGoogle Scholar
  21. 21.
  22. 22.
    Bodenmiller B, Campbell D, Gerrits B, Lam H, Jovanovic M, Picotti P, Schlapbach R, Aebersold R (2008) PhosphoPep—a database of protein phosphorylation sites in model organisms. Nat Biotechnol 26:1339–1340PubMedCrossRefGoogle Scholar
  23. 23.
    Gnad F, Ren S, Cox J, Olsen JV, Macek B, Oroshi M, Mann M (2007) PHOSIDA (phosphorylation site database): management, structural and evolutionary investigation, and prediction of phosphosites. Genome Biol 8:R250PubMedCrossRefGoogle Scholar
  24. 24.
    Knight ZA, Lin H, Shokat KM (2010) Targeting the cancer kinome through polypharmacology. Nat Rev Cancer 10:130–137PubMedCrossRefGoogle Scholar
  25. 25.
    Tonks NK (2006) Protein tyrosine phosphatases: from genes, to function, to disease. Nat Rev Mol Cell Biol 7:833–846PubMedCrossRefGoogle Scholar
  26. 26.
    Alonso A, Sasin J, Bottini N, Friedberg I, Osterman A, Godzik A, Hunter T, Dixon J, Mustelin T (2004) Protein tyrosine phosphatases in the human genome. Cell 117:699–711PubMedCrossRefGoogle Scholar
  27. 27.
    Vintonyak VV, Antonchick AP, Rauh D, Waldmann H (2009) The therapeutic potential of phosphatase inhibitors. Curr Opin Chem Biol 13:272–283PubMedCrossRefGoogle Scholar
  28. 28.
    Gan CS, Guo T, Zhang H, Lim SK, Sze SK (2008) A comparative study of electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) versus SCX-IMAC-based methods for phosphopeptide isolation/enrichment. J Proteome Res 7:4869–4877PubMedCrossRefGoogle Scholar
  29. 29.
    Guo T, Gan CS, Zhang H, Zhu Y, Kon OL, Sze SK (2008) Hybridization of pulsed-Q dissociation and collision-activated dissociation in linear ion trap mass spectrometer for iTRAQ quantitation. J Proteome Res 7:4831–4840PubMedCrossRefGoogle Scholar
  30. 30.
    Zhu Y, Guo T, Park JE, Li X, Meng W, Datta A, Bern M, Lim SK, Sze SK (2009) Elucidating in vivo structural dynamics in integral membrane protein by hydroxyl radical footprinting. Mol Cell Proteomics 8:1999–2010PubMedCrossRefGoogle Scholar
  31. 31.
    Fenyo D, Beavis RC (2003) A method for assessing the statistical significance of mass spectrometry-based protein identifications using general scoring schemes. Anal Chem 75:768–774PubMedCrossRefGoogle Scholar
  32. 32.
    Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard DM, Yang X, Shi W, Bryant SH (2004) Open mass spectrometry search algorithm. J Proteome Res 3:958–964PubMedCrossRefGoogle Scholar
  33. 33.
    Obenauer JC, Cantley LC, Yaffe MB (2003) Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs. Nucleic Acids Res 31:3635–3641PubMedCrossRefGoogle Scholar
  34. 34.
    Bodenmiller B, Mueller LN, Mueller M, Domon B, Aebersold R (2007) Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods 4:231–237PubMedCrossRefGoogle Scholar
  35. 35.
    Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80:62–76PubMedCrossRefGoogle Scholar
  36. 36.
    Alves G, Wu WW, Wang G, Shen RF, Yu YK (2008) Enhancing peptide identification confidence by combining search methods. J Proteome Res 7:3102–3113PubMedCrossRefGoogle Scholar
  37. 37.
    Zhang H, Guo T, Li X, Datta A, Park JE, Yang J, Lim SK, Tam JP, Sze SK (2010) Simultaneous characterization of glyco- and phospho-proteomes of mouse brain membrane proteome with electrostatic repulsion hydrophilic interaction chromatography (ERLIC). Mol Cell Proteomics 9:635–647PubMedCrossRefGoogle Scholar
  38. 38.
    Sarg B, Helliger W, Talasz H, Forg B, Lindner HH (2006) Histone H1 phosphorylation occurs site-specifically during interphase and mitosis: identification of a novel phosphorylation site on histone H1. J Biol Chem 281:6573–6580PubMedCrossRefGoogle Scholar
  39. 39.
    Beausoleil SA, Villen J, Gerber SA, Rush J, Gygi SP (2006) A probability-based approach for high-throughput protein phosphorylation analysis and site localization. Nat Biotechnol 24:1285–1292PubMedCrossRefGoogle Scholar
  40. 40.
    Schwartz D, Gygi SP (2005) An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 23:1391–1398PubMedCrossRefGoogle Scholar
  41. 41.
    Amanchy R, Periaswamy B, Mathivanan S, Reddy R, Tattikota SG, Pandey A (2007) A curated compendium of phosphorylation motifs. Nat Biotechnol 25:285–286PubMedCrossRefGoogle Scholar
  42. 42.
    Slaughter DP, Southwick HW, Smejkal W (1953) Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer 6:963–968PubMedCrossRefGoogle Scholar
  43. 43.
    Subramanian H, Roy HK, Pradhan P, Goldberg MJ, Muldoon J, Brand RE, Sturgis C, Hensing T, Ray D, Bogojevic A, Mohammed J, Chang JS, Backman V (2009) Nanoscale cellular changes in field carcinogenesis detected by partial wave spectroscopy. Cancer Res 69:5357–5363PubMedCrossRefGoogle Scholar
  44. 44.
    Kuniyasu H, Yasui W, Yokozaki H, Kitadai Y, Tahara E (1993) Aberrant expression of c-met mRNA in human gastric carcinomas. Int J Cancer 55:72–75PubMedCrossRefGoogle Scholar
  45. 45.
    Heideman DA, Snijders PJ, Bloemena E, Meijer CJ, Offerhaus GJ, Meuwissen SG, Gerritsen WR, Craanen ME (2001) Absence of tpr-met and expression of c-met in human gastric mucosa and carcinoma. J Pathol 194:428–435PubMedCrossRefGoogle Scholar
  46. 46.
    Smolen GA, Sordella R, Muir B, Mohapatra G, Barmettler A, Archibald H, Kim WJ, Okimoto RA, Bell DW, Sgroi DC, Christensen JG, Settleman J, Haber DA (2006) Amplification of MET may identify a subset of cancers with extreme sensitivity to the selective tyrosine kinase inhibitor PHA-665752. Proc Natl Acad Sci USA 103:2316–2321PubMedCrossRefGoogle Scholar
  47. 47.
    Nicholson RI, Gee JM, Harper ME (2001) EGFR and cancer prognosis. Eur J Cancer 37 (Suppl 4):S9–S15PubMedCrossRefGoogle Scholar
  48. 48.
    Kanta SY, Yamane T, Dobashi Y, Mitsui F, Kono K, Ooi A (2006) Topoisomerase II alpha gene amplification in gastric carcinomas: correlation with the HER2 gene. An immunohistochemical, immunoblotting, and multicolor fluorescence in situ hybridization study. Hum Pathol 37:1333–1343PubMedCrossRefGoogle Scholar
  49. 49.
    Tokuyasu N, Shomori K, Nishihara K, Kawaguchi H, Fujioka S, Yamaga K, Ikeguchi M, Ito H (2008) Minichromosome maintenance 2 (MCM2) immunoreactivity in stage III human gastric carcinoma: clinicopathological significance. Gastric Cancer 11:37–46PubMedCrossRefGoogle Scholar
  50. 50.
    Yuan W, Chen Z, Wu S, Ge J, Chang S, Wang X, Chen J (2009) Expression of EphA2 and E-cadherin in gastric cancer: correlated with tumor progression and lymphogenous metastasis. Pathol Oncol Res 15:473–478PubMedCrossRefGoogle Scholar
  51. 51.
    Zhou YN, Xu CP, Han B, Li M, Qiao L, Fang DC, Yang JM (2002) Expression of E-cadherin and beta-catenin in gastric carcinoma and its correlation with the clinicopathological features and patient survival. World J Gastroenterol 8:987–993PubMedGoogle Scholar
  52. 52.
    Yamamoto S, Tomita Y, Hoshida Y, Takiguchi S, Fujiwara Y, Yasuda T, Doki Y, Yoshida K, Aozasa K, Nakamura H, Monden M (2006) Expression of hepatoma-derived growth factor is correlated with lymph node metastasis and prognosis of gastric carcinoma. Clin Cancer Res 12:117–122PubMedCrossRefGoogle Scholar
  53. 53.
    Nikolova M, Guenova M, Taskov H, Dimitrova E, Staneva M (1998) Levels of expression of CAF7 (CD98) have prognostic significance in adult acute leukemia. Leuk Res 22:39–47PubMedCrossRefGoogle Scholar
  54. 54.
    Esseghir S, Reis-Filho JS, Kennedy A, James M, O’Hare MJ, Jeffery R, Poulsom R, Isacke CM (2006) Identification of transmembrane proteins as potential prognostic markers and therapeutic targets in breast cancer by a screen for signal sequence encoding transcripts. J Pathol 210:420–430PubMedCrossRefGoogle Scholar
  55. 55.
    Kaira K, Oriuchi N, Imai H, Shimizu K, Yanagitani N, Sunaga N, Hisada T, Ishizuka T, Kanai Y, Endou H, Nakajima T, Mori M (2009) Prognostic significance of l-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in early stage squamous cell carcinoma of the lung. Cancer Sci 100:249–254CrossRefGoogle Scholar
  56. 56.
    Prager GW, Poettler M, Schmidinger M, Mazal PR, Susani M, Zielinski CC, Haitel A (2009) CD98hc (SLC3A2), a novel marker in renal cell cancer. Eur J Clin Invest 39:304–310PubMedCrossRefGoogle Scholar
  57. 57.
    Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078PubMedCrossRefGoogle Scholar
  58. 58.
    Harper JW, Elledge SJ (2007) The DNA damage response: 10 years after. Mol Cell 28:739–745PubMedCrossRefGoogle Scholar
  59. 59.
    Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S (2002) The protein kinase complement of the human genome. Science 298:1912–1934PubMedCrossRefGoogle Scholar
  60. 60.
    Thomson M (2002) Evidence of undiscovered cell regulatory mechanisms: phosphoproteins and protein kinases in mitochondria. Cell Mol Life Sci 59:213–219PubMedCrossRefGoogle Scholar
  61. 61.
    Horbinski C, Chu CT (2005) Kinase signaling cascades in the mitochondrion: a matter of life or death. Free Radic Biol Med 38:2–11PubMedCrossRefGoogle Scholar
  62. 62.
    Deng N, Zhang J, Zong C, Wang Y, Lu H, Yang P, Wang W, Young GW, Korge P, Lotz C, Doran P, Liem DA, Apweiler R, Weiss JN, Duan H, Ping P (2010) Phosphoproteome analysis reveals regulatory sites in major pathways of cardiac mitochondria. Mol Cell Proteomics (In press) (doi: 10.1074/mcp.M110.000117)
  63. 63.
    Deng WJ, Nie S, Dai J, Wu JR, Zeng R (2010) Proteome, phosphoproteome, and hydroxyproteome of liver mitochondria in diabetic rats at early pathogenic stages. Mol Cell Proteomics 9:100–116PubMedCrossRefGoogle Scholar
  64. 64.
    Cui Z, Hou J, Chen X, Li J, Xie Z, Xue P, Cai T, Wu P, Xu T, Yang F (2010) The profile of mitochondrial proteins and their phosphorylation signaling network in INS-1 beta cells. J Proteome Res 9:2898–2908PubMedCrossRefGoogle Scholar
  65. 65.
    Reinders J, Wagner K, Zahedi RP, Stojanovski D, Eyrich B, van der Laan M, Rehling P, Sickmann A, Pfanner N, Meisinger C (2007) Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase. Mol Cell Proteomics 6:1896–1906PubMedCrossRefGoogle Scholar
  66. 66.
    Ito J, Taylor NL, Castleden I, Weckwerth W, Millar AH, Heazlewood JL (2009) A survey of the Arabidopsis thaliana mitochondrial phosphoproteome. Proteomics 9:4229–4240PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Tiannan Guo
    • 1
    • 2
  • Sze Sing Lee
    • 1
  • Wai Har Ng
    • 1
  • Yi Zhu
    • 2
  • Chee Sian Gan
    • 2
  • Jiang Zhu
    • 3
  • Haixia Wang
    • 3
  • Shiang Huang
    • 3
  • Siu Kwan Sze
    • 2
  • Oi Lian Kon
    • 1
  1. 1.Division of Medical Sciences, Humphrey Oei Institute of Cancer ResearchNational Cancer Centre SingaporeSingaporeSingapore
  2. 2.School of Biological SciencesNanyang Technological UniversitySingaporeSingapore
  3. 3.Center for Stem Cell Research and Application, Union HospitalHuazhong University of Science and TechnologyWuhanPeople’s Republic of China

Personalised recommendations