Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1α signaling network: An initial study in GSC11 glioblastoma stem cells

Carol L. Nilsson, Roslyn Dillon, Arugadoss Devakumar, Stone D.H. Shi, Michael Greig, John C. Rogers, Bryan Krastins, Michael Rosenblatt, Gregory Kilmer, Michael Major, Barbara J. Kaboord, David Sarracino, Taha Rezai, Amol Prakash, Mary Lopez, Yongjie Ji, Waldemar Priebe, Frederick F. Lang, Howard Colman, Charles A. Conrad

Research output: Contribution to journalArticle

86 Scopus citations

Abstract

Initiation and maintenance of several cancers including glioblastoma (GBM) may be driven by a small subset of cells called cancer stem cells (CSCs). CSCs may provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of tumors. STAT3 is a key transcription factor associated with regulation of multiple stem cell types. Recently, a novel autocrine loop (IL-6/STAT3/HIF1α) has been observed in multiple tumor types (pancreatic, prostate, lung, and colon). The objective of this study was to probe perturbations of this loop in a glioblastoma cancer stem cell line (GSC11) derived from a human tumor by use of a JAK2/STAT3 phosphorylation inhibitor (WP1193), IL-6 stimulation, and hypoxia. A quantitative phosphoproteomic approach that employed phosphoprotein enrichment, chemical tagging with isobaric tags, phosphopeptide enrichment, and tandem mass spectrometry in a high-resolution instrument was applied. A total of 3414 proteins were identified in this study. A rapid Western blotting technique (<1 h) was used to confirm alterations in key protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 10% of the phosphoproteins were linked to the IL-6 pathway, and the majority of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed expected changes and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1α autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

Original languageEnglish (US)
Pages (from-to)430-443
Number of pages14
JournalJournal of Proteome Research
Volume9
Issue number1
DOIs
StatePublished - Jan 4 2010

Keywords

  • Cancer stem cells
  • Glioblastoma multiforme
  • HIF1α
  • IL-6
  • LC-MS/MS
  • LTQ-Orbitrap
  • Phosphopeptide enrichment
  • Quantitative phosphoproteomics
  • STAT3 phosphorylation inhibition
  • Western blot

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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  • Cite this

    Nilsson, C. L., Dillon, R., Devakumar, A., Shi, S. D. H., Greig, M., Rogers, J. C., Krastins, B., Rosenblatt, M., Kilmer, G., Major, M., Kaboord, B. J., Sarracino, D., Rezai, T., Prakash, A., Lopez, M., Ji, Y., Priebe, W., Lang, F. F., Colman, H., & Conrad, C. A. (2010). Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1α signaling network: An initial study in GSC11 glioblastoma stem cells. Journal of Proteome Research, 9(1), 430-443. https://doi.org/10.1021/pr9007927