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.