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Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network : an initial study in GSC11 glioblastoma stem cells

Nilsson, Carol L LU ; Dillon, Roslyn; Devakumar, Arugadoss; Shi, Stone D-H; Greig, Michael J; Rogers, John C; Krastins, Bryan; Rosenblatt, Michael; Kilmer, Gregory and Major, Michael, et al. (2010) In Journal of Proteome Research 9(1). p.430-443
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/HIF1alpha) 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... (More)

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/HIF1alpha) 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/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.

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@article{09c599f6-7245-4297-881a-74328c8cedb7,
  abstract     = {<p>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/HIF1alpha) 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 (&lt;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/HIF1alpha autocrine loop and the CSC response to perturbations by hypoxia, inhibition of STAT3 phosphorylation, and IL-6 stimulation.</p>},
  author       = {Nilsson, Carol L and Dillon, Roslyn and Devakumar, Arugadoss and Shi, Stone D-H and Greig, Michael J and Rogers, John C and Krastins, Bryan and Rosenblatt, Michael and Kilmer, Gregory and Major, Michael and Kaboord, Barbara J and Sarracino, David and Rezai, Taha and Prakash, Amol and Lopez, Mary and Ji, Yongjie and Priebe, Waldemar and Lang, Frederick and Colman, Howard and Conrad, Charles A.},
  issn         = {1535-3893},
  keyword      = {Blotting, Western,Chemokines,Chromatography, Liquid,Glioblastoma,Humans,Hypoxia,Hypoxia-Inducible Factor 1, alpha Subunit,Interleukin-6,Models, Biological,Neoplastic Stem Cells,Nitric Oxide Synthase Type II,Phosphopeptides,Phosphoproteins,Phosphorylation,Proteome,STAT3 Transcription Factor,Signal Transduction,Tandem Mass Spectrometry,Tryptophan,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {1},
  pages        = {430--443},
  publisher    = {The American Chemical Society},
  series       = {Journal of Proteome Research},
  title        = {Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network : an initial study in GSC11 glioblastoma stem cells},
  url          = {http://dx.doi.org/10.1021/pr9007927},
  volume       = {9},
  year         = {2010},
}