Monitoring Phosphoproteomic Response to Targeted Kinase Inhibitors Using Reverse-Phase Protein Microarrays

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 795)

Abstract

Phosphoproteomic networks mediated by protein kinases are the key drivers of proliferative and survival signals underlying human cancers, and as such a number of kinases have been the subject of intensive drug discovery efforts. A key question that must be answered during clinical development is whether a kinase inhibitor is effectively inhibiting its appropriate target kinase and pathway in the tumor. Reverse-phase protein arrays (RPMAs) offer the ability to analyze behavior of entire signaling networks in response to drug treatment and thus have promise as a technology for monitoring cellular response to kinase inhibitors. We have shown that it is possible to use RPMAs to detect phosphorylation changes in key multiple signaling pathway proteins in response to targeted inhibitors of EGFR, MEK, and PI3 kinase.

Key words

MEK inhibitor PI3 Kinase inhibitor Reverse-phase protein array Pharmacodynamic biomarker 

References

  1. 1.
    Grant, S. K. (2009) Therapeutic Protein Kinase Inhibitors, Cell. Mol. Life Sci, 66(7), 1163–1177.Google Scholar
  2. 2.
    Zhang, J., Yang, P. L., and Gray, N. S. (2009) Targeting cancer with small molecule kinase inhibitors. Nat. Rev. Cancer 9, 28–39.PubMedCrossRefGoogle Scholar
  3. 3.
    Kohno, M., and Pouyssegur, J. (2006) Targeting the ERK signaling pathway in cancer therapy. Ann. Med. 38, 200–211.PubMedCrossRefGoogle Scholar
  4. 4.
    Ihle, N. T., and Powis, G. (2009) Take your PIK: phosphatidylinositol 3-kinase inhibitors race through the clinic and toward cancer therapy. Mol. Cancer Ther. 8, 1–9.PubMedCrossRefGoogle Scholar
  5. 5.
    Carnero, A., Blanco-Aparicio, C., Renner, O., Link, W., and Leal, J. F. (2008) The PTEN/PI3K/AKT signalling pathway in cancer, therapeutic implications. Curr. Cancer Drug Tar. 8, 187–198.CrossRefGoogle Scholar
  6. 6.
    Bareschino, M. A., Schettino, C., Troiani, T., Martinelli, E., Morgillo, F., and Ciardiello, F. (2007) Erlotinib in cancer treatment., Ann. Oncol. 18 Suppl 6, vi35–41.Google Scholar
  7. 7.
    Brown, A. P., Carlson, T. C., Loi, C. M., and Graziano, M. J. (2007) Pharmacodynamic and toxicokinetic evaluation of the novel MEK inhibitor, PD0325901, in the rat following oral and intravenous administration. Cancer Chemoth. Pharm. 59, 671–679.CrossRefGoogle Scholar
  8. 8.
    Folkes, A. J., Baker, S. J., Chuckowree, I. S., Eccles, S. A., Hayes, A., Hancox, T. C., Latif, M. A., Olivero, A. G., Patel, S., and Shuttleworth, S. J. (2008) The discovery of GDC-0941: A potent, selective, orally bioavailable inhibitor of class I PI3 kinase for the treatment of cancer. in AACR Annual Meeting, San Diego.Google Scholar
  9. 9.
    Espina, V., Wulfkuhle, J., Calvert, V. S., Liotta, L. A., and Petricoin, E. F., 3rd. (2008) Reverse phase protein microarrays for theranostics and patient-tailored therapy. In Methods in molecular biology (Clifton, N.J) 441, 113–128.Google Scholar
  10. 10.
    Wulfkuhle, J. D., Edmiston, K. H., Liotta, L. A., and Petricoin, E. F., 3 rd. (2006) Technology insight: pharmacoproteomics for cancer – promises of patient-tailored medicine using protein microarrays. Nat. Clin. Pract. Oncol. 3, 256–268.PubMedCrossRefGoogle Scholar
  11. 11.
    Espina, V., Wulfkuhle, J. D., Calvert, V. S., Petricoin, E. F., 3rd, and Liotta, L. A. (2007) Reverse phase protein microarrays for monitoring biological responses. In Methods in molecular biology (Clifton, N.J) 383, 321–336.Google Scholar
  12. 12.
    Boyd, Z. S., Wu, Q. J., O’Brien, C., Spoerke, J., Savage, H., Fielder, P. J., Amler, L., Yan, Y., and Lackner, M. R. (2008) Proteomic analysis of breast cancer molecular subtypes and biomarkers of response to targeted kinase inhibitors using reverse-phase protein microarrays. Mol. Cancer Ther. 7, 3695–3706.PubMedCrossRefGoogle Scholar
  13. 13.
    Secrist, J. P., Burns, L. A., Karnitz, L., Koretzky, G. A., and Abraham, R. T. (1993) Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. J. Biol. Chem. 1993 Mar 15;268(8), 5886–5893.PubMedCrossRefGoogle Scholar
  14. 14.
    Steinberg, T. H., Jones, L. J., Haugland, R. P., and Singer, V. L. (1996) SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein, Anal Biochem. 239(2), 223–237.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Theranostics HealthRockvilleUSA
  2. 2.Department of Development Oncology DiagnosticsGenentech Inc.South San FranciscoUSA

Personalised recommendations