Abstract LB-8: Liquid chromatography - mass spectrometry proteomics to monitor the activity of multiple phosphorylation signaling pathways in cancer
Authors: David Britton, Justin Stebbing, Laura Roca Alonso, Claire Russell, Christopher Loessner, Stephan Jung, Malcolm Ward, Ian Pike, Leandro Castellano
Journal: Cancer Research (2012)

Abstract Introduction: The translation of genomic pre-disposition into a disease phenotype is driven primarily by protein expression and a complex network of post-translational modifications (PTM). One such PTM, phosphorylation, affords exquisite regulation of protein function by modulating protein folding, substrate affinity, stability, and activity and is proven to modulate the activity of thousands of onco- and tumor suppressor proteins. Phosphorylation-induced dysregulation of key checkpoints in signaling pathways that drive cell division, proliferation, differentiation, and apoptosis contributes to the cancer phenotype. A priori knowledge of which phosphorylation events are driving a particular tumor will assist in drug target validation, compound selection and ultimately treatment optimization for individual patients. We have developed a liquid chromatography mass spectrometry proteomics approach to monitor the activity of these signaling pathways in cancer cells. Experimental procedure: Peptides resulting from tryptic digestion of proteins from a human breast cancer cell line (BT474 +/− lapatinib, heregulin, lapatinib+heregulin), were labeled with tandem mass tags (TMT®) and passed through phospho-peptide enrichment columns (IMAC or TiO2). The enriched phospho-peptides were then analyzed by LC-MS/MS to identify and quantify the level of protein phosphorylation in each sample relative to a spiked internal reference sample. Data summary: In total we identified 4046 phosphorylation sites on 1054 phospho-proteins in BT474 cells under the various treatment conditions stated above. Lapatinib and heregulin were shown to significantly modulate phosphorylation on key proteins involved in 32 KEGG signaling pathways, including: the pancreatic cancer pathway (11 phospho-proteins), cell cycle pathway (18 phospho-proteins) and the ERBB signaling pathway, where we observed modulation of phosphorylation sites on 11 phospho-proteins including ERBB2 (pT701, pS703, pY877, pS1050/1051, pS1054, pS1083, pS1107, pS1166, pY1248 and 4 additional novel sites), AKT1 (pS124, pS126, pS129, pS137), and GSK3B (pS9, pY216 and an additional novel site). Conclusion: The global analysis of site-specific phosphorylation on key signaling pathways relevant to a specific cancer phenotype has been developed. Our initial data provide further insight into the on- and off-target effects of the ERBB1/ERBB2 tyrosine kinase inhibitor lapatinib. These data may help define optimal combination treatments in ERBB1/ERBB2 positive tumors. The application of this method to other tumor types and therapeutic agents is widely recommended. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-8. doi:1538-7445.AM2012-LB-8