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Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts

Abstract

The purpose of this phospho-proteomics study was to demonstrate the broad analysis of cellular protein phosphorylation in cells and tissue as a means to monitor changes in cellular states. As a cancer model, human tumor-derived A431 cells known to express the epidermal growth factor receptor (EGFR) were grown as cell cultures or xenograft tumors in mice. The cells and tumor-bearing animals were subjected to treatments including the EGFR-directed protein kinase inhibitor PK166 and/or EGF stimulation. Whole cell/tissue protein extracts were converted to peptides by using trypsin, and phosphorylated peptides were purified by an affinity capture method. Peptides and phosphorylation sites were characterized and quantified by using a combination of tandem mass spectroscopy (MS) and Fourier transform MS instrumentation (FTMS). By analyzing roughly 106 cell equivalents, 780 unique phosphopeptides from approx 450 different proteins were characterized. Only a small number of these phosphorylation sites have been described previously in literature. Although a targeted analysis of the EGFR pathway was not a specific aim of this study, 22 proteins known to be associated with EGFR signaling were identified. Fifty phosphopeptides were found changed in abundance as a function of growth factor or drug treatment including novel sites of phosphorylation on the EGFR itself. These findings demonstrate the feasibility of using phospho-proteomics to determine drug and disease mechanisms, and as a measure of drug target modulation in tissue.

References

  1. 1

    Ficarro SB, McCleland ML, Stukenberg PT, et al. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisae. Nat Biotechnol 2002;19:01–305.

  2. 2

    Oda Y, Nagasu T, Chait BT. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome. Nat Biotechnol 2001;19:379–382.

  3. 3

    Zhou H, Watts JD, Aebersold R. A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol 2001;19:375–378.

  4. 4

    Yarden Y. The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. Eur J Cancer 2001;37:S3-S8.

  5. 5

    Mendelsohn J, Baselga J. The EGF receptor family as targets for cancer therapy Oncogene 2000;19:6550–6565.

  6. 6

    Voldborg BR, Damstrup L, Spang-Thomsen, M. & Poulsen H. S. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997;12:1197–1206.

  7. 7

    Moscatello DK, Holgado-Madruga M, Godwin, AK, et al. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res 1995;55:5536–5539.

  8. 8

    Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19:183–232.

  9. 9

    Scambia G, Benedetti-Panici P, Ferrandina G, et al. Epidermal growth factor, oestrogen and progesterone receptor expression in primary ovarian cancer: correlation with clinical outcome and response to chemotherapy. Br J Cancer 1995;72:361–366.

  10. 10

    Simpson BJ, Phillips HA., Lessels AM, Langdon SP, Miller WR. c-erbB growth-factorreceptor proteins in ovarian tumours. Int J Cancer 1995;64:202–206.

  11. 11

    Slamon D. J., et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1987;235:177–182.

  12. 12

    Herbst RS. ZD1839: targeting the epidermal growth factor receptor in cancer therapy. Expert Opin. Invest Drugs 2002;11:837–849.

  13. 13

    Moyer JD, Barbacci EG, Iwata KK, et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res 1997;57:4838–4848.

  14. 14

    Traxler P, Bold G, Buchdunger E, et al. Tyrosine kinase inhibitors: from rational design to clinical trials. Med Res Rev 2001;21,499–512.

  15. 15

    Caravatti G, Bruggen J, Buchdunger E, et al. Pyrrolo[2,3-d]Pyrimidine and Pyrazolo[3,4-d] Pyrimidine Derivatives as Selective Inhibitors of the EGF Receptor Tyrosine Kinase. ACS Symposium Series, ed. 796: Anticancer Agents. Oxford University Press, USA:2001;231–244.

  16. 16

    Bruns CJ, Solorzano CC, Harbison MT, et al. Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 2000;60,2926–2935.

  17. 17

    Brandt R, Wong AML, Hynes NE. Mammary glands reconstituted with Neu/ErbB2 transformed HC11 cells provide a novel orthotopic tumor model for testing anti-cancer agents. Oncogene 2001;20:5459–5465.

  18. 18

    Baker CH, Solorzano CC, Fidler IJ. Blockade of vascular endothelial growth factor receptor and epidermal growth factor receptor signaling for therapy of metastatic human pancreatic cancer. Cancer Res 2002;62:1996–2003.

  19. 19

    Ferrari S, Bandi HR, Hofsteenge J, Bussian BM, Thomas G. Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites. J Biol Chem 1991;266(33):22770–22775.

  20. 20

    Ferrari S, Pearson RB, Siegmann M, Kozma SC, Thomas G. The immunosuppressant rapamycin induces inactivation of p70s6k through dephosphorylation of a novel set of sites. J Biol Chem 1993;268(6):4530–4533.

  21. 21

    Sunnarborg SW, Hinkle CL, Stevenson M, et al. Tumor necrosis factor-α converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J Biol Chem 2002; 277(15):12838–12845 (2002).

  22. 22

    Ko TK, Kelly E, Pines J. CrkRS: a novel conserved Cdc2-related protein kinase that colocalises with SC35 speckles. J Cell Sci 2001; 114:2591–2603 (2001).

  23. 23

    Marques F, Moreau JL, Peaucellier G, et al. A new subfamily of high molecular mass CDC2-related kinases with PITAI/VRE motifs. Biochem Biophys Res Commun 2000;279(3): 832–837 (2000).

  24. 24

    Lapidot-Lifson Y, Patinkin D, Prody CA, et al. Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis. Proc Natl Acad Sci USA 1992;89(2):579–583.

  25. 25

    Hauck CR, Sieg DJ, Hsia DA, Loftus JC, Gaarde WA, Monia BP, Schlaepfer DD. Inhibition of focal adhesion kinase expression or activity disrupts epidermal growth factor-stimulated signaling promoting the migration of invasive human carcinoma cells. Cancer Res 2001; 61(19):7079–7090.

  26. 26

    Lu L, Han AP, Chen JJ. Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol Cell Biol 2001; 12:4016–4031.

  27. 27

    Weed SA, Parsons JD. Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene 2001;20:6418–6434.

  28. 28

    Campbell DH, Sutherland RL, Daly RJ. Signaling pathways and structural domains required for phosphorylation of EMSq/cortactin. Cancer Res 1999;59:5376–5385.

  29. 29

    Mariner DJ. Identification of Src phosphorylation sites in the catenin p120ctn. J Biol Chem 2001;276:28006–28013.

  30. 30

    Lu Q. δ-catenin, an adhesive junction-associated protein which promotes cell scattering. J Cell Biol 1999;144:519–532.

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Correspondence to David R. Stover.

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Key Words

  • Phosphoproteomics
  • proteomics
  • Fourier transform mass spectrometry (FTMS)
  • Epidermal Growth Factor Receptor (EGFR)
  • PKI166