title: Quantitative phosphoproteomic analysis of the STAT3/IL-6/HIF1alpha signaling network: an initial study in GSC11 glioblastoma stem cells.
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 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.
authors.affiliation: Pfizer Global Research and Development, 10770 Science Center Drive, San Diego, California 92121, USA. Carol.Nilsson@pfizer.com
authors.forename: Carol L
authors.initials: CL
authors.lastname: Nilsson
authors.forename: Roslyn
authors.initials: R
authors.lastname: Dillon
authors.forename: Arugadoss
authors.initials: A
authors.lastname: Devakumar
authors.forename: Stone D-H
authors.initials: SD
authors.lastname: Shi
authors.forename: Michael
authors.initials: M
authors.lastname: Greig
authors.forename: John C
authors.initials: JC
authors.lastname: Rogers
authors.forename: Bryan
authors.initials: B
authors.lastname: Krastins
authors.forename: Michael
authors.initials: M
authors.lastname: Rosenblatt
authors.forename: Gregory
authors.initials: G
authors.lastname: Kilmer
authors.forename: Michael
authors.initials: M
authors.lastname: Major
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Kaboord
authors.forename: David
authors.initials: D
authors.lastname: Sarracino
authors.forename: Taha
authors.initials: T
authors.lastname: Rezai
authors.forename: Amol
authors.initials: A
authors.lastname: Prakash
authors.forename: Mary
authors.initials: M
authors.lastname: Lopez
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Ji
authors.forename: Waldemar
authors.initials: W
authors.lastname: Priebe
authors.forename: Frederick F
authors.initials: FF
authors.lastname: Lang
authors.forename: Howard
authors.initials: H
authors.lastname: Colman
authors.forename: Charles A
authors.initials: CA
authors.lastname: Conrad
mesh_terms: 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
chemical_list: 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