Abstract 1617: Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets

Abstract

Abstract INTRODUCTION: LC-MS/MS proteomics is an essential technology to help unravel the complex molecular events that lead to and propagate cancer. We have developed an analytical workflow to quantify the expression and phosphorylation status of thousands of proteins simultaneously in frozen resected human pancreatic tumor (T), relative to matched non-tumor (NT) tissue. These measurements enabled us to determine activity of anti-neoplastic drug targets and other signaling proteins. METHODS: Peptides resulting from tryptic digestion of proteins extracted from frozen tissue of pancreatic ductal adenocarcinoma and background pancreas (n=12), were labelled with tandem mass tags (TMT 8-plex), separated by strong cation exchange chromatography, then were analysed by LC-MS/MS directly or first enriched for phosphopeptides using Fe3+ and TiO2, prior to analysis. In-house, commercial and freeware bio-informatics platforms were used to identify relevant biological events from the complex dataset. RESULTS: Of 2,101 proteins identified, 152 demonstrated significant difference in expression between tumor and non-tumor tissue. They included proteins that are known to be up-regulated in pancreatic cancer (e.g. Mucin-1), but the majority were new candidate markers such as homeodomain interacting protein kinase 1 & Myosin light chain kinase. Of the 6,543 unique phosphopeptides identified (6,284 unique phosphorylation sites), 635 showed significant regulation, particularly those from proteins involved in cell migration (Rho GTPase signaling proteins such as Rho guanine nucleotide exchange factors & Serine/threonine-protein kinase MRCKα) as well as proteins involved in disassembly of cell-cell junctions (tight junction, adherens junction) and formation of cell-extracellular matrix (ECM) junctions (focal adhesions). We quantified activator & inhibitory phosphorylation sites on FYN, AKT1, HDAC1&2, GSK3α&β, RAF kinases, MAPKs (p38, ERK1&2), PKCs, Casein Kinases and >20 others, as well as their downstream substrates some of which were often found to be highly modulated (≥ 2 fold) in T versus NT in different cases. CONCLUSION: Application of our LC-MS/MS proteomic workflow to frozen resected human pancreatic T versus NT tissue elucidated molecular events likely contributing to pancreatic cancer in each case, particularly those contributing to cell migration, and in future may help clinicians predict the best targeted anti-cancer therapy bespoke for an individual patient. Citation Format: David Britton, Yoh Zen, Stefan Selzer, Vikram Mitra, Alberto Quaglia, Debashis Sarker, Leandro Castellano, Justin Stebbing, Julia Gee, Rob Nicholson, Nigel Heaton, Ian Pike. Quantification of pancreatic cancer proteome & phosphorylome: Indicates molecular events likely contributing to cancer & activation status of drug targets. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2014-1617