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

Clinical Proteomics volume 1, pages 69–80 (2004)Cite this article

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. 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.

    Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    PubMed  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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).

    PubMed  CAS  Google Scholar 

  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).

    Article  PubMed  CAS  Google Scholar 

  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.

    Article  PubMed  CAS  Google Scholar 

  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.

    PubMed  CAS  Google Scholar 

  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.

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. MDS Proteomics Inc., 1670 Discovery Dr., Charlottesville, VA

    David R. Stover, Jennifer Caldwell, Jarrod Marto, Karen Root, Olga Ornatsky, Chris Orsi & Nina Radosevic

  2. 25 I Attwell Drive, M9Q 7H4, Toronto, Canada

    David R. Stover, Jennifer Caldwell, Jarrod Marto, Karen Root, Olga Ornatsky, Chris Orsi & Nina Radosevic

  3. Oncology Research, Novartis Pharma AG, CH-4002, Basel, Switzerland

    Juergan Mestan, Michael Stumm & Doriano Fabbro

  4. Oncology Research, Novartis Institutes for Biomedical Research, 02139, Cambridge, MA

    David R. Stover

  5. Banting and Best Department of Medical Research, University of Toronto, 112 College St., M5G IL6, Toronto, ON, Canada

    Linda Liao & Michael F. Moran

Authors
  1. David R. Stover
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  2. Jennifer Caldwell
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  3. Jarrod Marto
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  4. Karen Root
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  5. Juergan Mestan
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  6. Michael Stumm
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  7. Olga Ornatsky
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  8. Chris Orsi
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  9. Nina Radosevic
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  10. Linda Liao
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  11. Doriano Fabbro
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  12. Michael F. Moran
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Correspondence to David R. Stover.

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Stover, D.R., Caldwell, J., Marto, J. et al. Differential phosphoprofiles of EGF and EGFR kinase inhibitor-treated human tumor cells and mouse xenografts. Clin Proteom 1, 69–80 (2004). https://doi.org/10.1385/CP:1:1:069

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Key Words

Clinical Proteomics

ISSN: 1559-0275