Abstract
Cancer stem cells (CSCs) are hypothesized to provide a repository of cells in tumor cell populations that are refractory to chemotherapeutic agents developed for the treatment of differentiated tumor cells. A glioblastoma cancer stem cell line (NSC-11, MDACC) differentiates from a pluripotent state to a progenitor cell-like state when stimulated with a STAT3 phosphorylation inhibitor (WP1193). Recently, a novel autocrine loop (IL-6/STAT3/HIF1alpha) was discovered in pancreatic cancer cells and the object of this study was to probe how perturbations of this loop induce signaling changes in the CSCs. IL-6 induces the JAK pathway, and STAT3 is a downstream target. HIF1\#945; (induced by hypoxia) is a downstream target of STAT3. Six different treatments were studied, including vehicle control, treatment with WP1193 and WP1193 plus Il-6 stimulation, and cell culture conditions with normoxia or hypoxia. Following cell lysis, proteins were enriched in a phosphoprotein enrichment column, reduced, alkylated, and enzymatically digested. Peptides were labeled with isobaric Tandem Mass Tags (TMT6) at amino termini and lysine residues. These six labeled peptide samples were combined into one sample, and phosphopeptides were enriched by hydrophilic interaction (HILIC) chromatography and TiO2-enrichment prior to analysis with an LTQ-Orbitrap mass spectrometer. Protein identification was performed with Proteome Discoverer software and the IPI protein database, and relative peptide quantification was performed simultaneously by comparison of the abundance ratios of the reporter ions from the TMT tags. More than 200 proteins were quantified in this initial study. The largest number of significant protein changes was observed during hypoxic conditions compared to normoxic conditions. In addition to quantification of protein expression levels, differential phosphorylation at specific sites of the same proteins was observed. A novel rapid Western blotting technique (< 1 hour) was used to confirm alterations in protein expression and phosphorylation levels observed in the mass spectrometric experiments. About 15% of the phosphoproteins quantified were linked to the IL-6 pathway and 70% of remaining proteins could be assigned to other interlinked networks. By multiple comparisons between the sample conditions, we observed known and gained novel insights into the contribution of each factor to the IL6/STAT3/HIF1\#945; autocrine loop and CSC differentiation.
Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4893.
100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO
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