Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.
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.
Nilsson
Carol L
CL
Pfizer Global Research and Development, 10770 Science Center Drive, San Diego, California 92121, USA. Carol.Nilsson@pfizer.com
Dillon
Roslyn
R
Devakumar
Arugadoss
A
Shi
Stone D-H
SD
Greig
Michael
M
Rogers
John C
JC
Krastins
Bryan
B
Rosenblatt
Michael
M
Kilmer
Gregory
G
Major
Michael
M
Kaboord
Barbara J
BJ
Sarracino
David
D
Rezai
Taha
T
Prakash
Amol
A
Lopez
Mary
M
Ji
Yongjie
Y
Priebe
Waldemar
W
Lang
Frederick F
FF
Colman
Howard
H
Conrad
Charles A
CA
D015153:Blotting, Western; D018925:Chemokines / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D005909:Glioblastoma / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D015850:Interleukin-6 / Q000378:metabolism*; D008954:Models, Biological; D014411:Neoplastic Stem Cells / Q000737:chemistry* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry / Q000379:methods; D014364:Tryptophan / Q000378:metabolism
D018925:Chemokines; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C508600:IL6 protein, human; D015850:Interleukin-6; D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D014364:Tryptophan; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II