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

TY - JOUR

T1 - Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network

T2 - an initial study in GSC11 glioblastoma stem cells

AU - Nilsson, Carol L

AU - Dillon, Roslyn

AU - Devakumar, Arugadoss

AU - Shi, Stone D-H

AU - Greig, Michael J

AU - Rogers, John C

AU - Krastins, Bryan

AU - Rosenblatt, Michael

AU - Kilmer, Gregory

AU - Major, Michael

AU - Kaboord, Barbara J

AU - Sarracino, David

AU - Rezai, Taha

AU - Prakash, Amol

AU - Lopez, Mary

AU - Ji, Yongjie

AU - Priebe, Waldemar

AU - Lang, Frederick

AU - Colman, Howard

AU - Conrad, Charles A.

PY - 2010/1

Y1 - 2010/1

N2 - 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.

AB - 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.

KW - Blotting, Western

KW - Chemokines

KW - Chromatography, Liquid

KW - Glioblastoma

KW - Humans

KW - Hypoxia

KW - Hypoxia-Inducible Factor 1, alpha Subunit

KW - Interleukin-6

KW - Models, Biological

KW - Neoplastic Stem Cells

KW - Nitric Oxide Synthase Type II

KW - Phosphopeptides

KW - Phosphoproteins

KW - Phosphorylation

KW - Proteome

KW - STAT3 Transcription Factor

KW - Signal Transduction

KW - Tandem Mass Spectrometry

KW - Tryptophan

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1021/pr9007927

DO - 10.1021/pr9007927

M3 - Article

VL - 9

SP - 430

EP - 443

JO - Journal of Proteome Research

JF - Journal of Proteome Research

SN - 1535-3893

IS - 1

ER -