Abstract LB-136: Phenotypic discrimination of mutant adenomatous polyposis coli (APC) expressing cells and APC corrected cells by shotgun proteomics
Authors: Patrick J. Halvey, Bing Zhang, Robbert J. Slebos, Robert J. Coffey, Daniel C. Liebler
Journal: Cancer Research (2010)

Abstract Much research has focused on the disruptive effect of genetic mutations on key cell signaling pathways, e.g. KRAS, BRAF, APC, and p53. However, the system-wide impact of cancer-related mutations in cells remains poorly understood. Recently, shotgun proteomic approaches, which incorporate improved peptide separation techniques and enhanced sensitivity, have been used to investigate the molecular events associated with subtle phenotypic variation. Adenomatous polyposis coli (APC) mutations occur in 60-70% of colorectal cancer cases and play a central role in early adenoma formation. Here we use shotgun proteomics on a standardized liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform to assess proteomic differences between mutant APC-expressing SW480 cells (nonsense mutation at codon 1338) and APC-corrected SW480 cells. The use of an isogenic cell model provides a control for variation in background protein expression. Tryptic peptides from whole cell lysates were separated by isoelectric focusing (IEF) and analyzed by LC-MS/MS on a LTQ mass spectrometer. Database searches with the resulting MS/MS spectra identified 4930 proteins across all groups (6 replicates per cell type, 4.2% protein FDR). Of the identified proteins, 93% were observed in both mutant and corrected cell lines, indicating that a relatively small fraction of the proteome is affected by correction of the APC mutation. We performed hierarchical clustering on differentially expressed proteins, calculating pairwise similarity based on the Pearson's correlation coefficient and using the average linkage. We found strong proteomic clustering of mutant APC replicates and APC corrected replicates, thus demonstrating the ability of shotgun proteomics to differentiate between subtle biological phenotypes. We also carried out parallel measurements of transcript levels using microarray analyses. Combining proteomic and microarray data in a single clustering analysis showed high concordance of the results obtained by the two orthogonal methods. As expected, several members of the canonical Wnt signaling pathway were found to be differentially expressed between mutant corrected cells; these include ß-catenin, which is activated in Wnt signaling in the absence of APC, and Dickkopf-related protein 4 (DKK4), a known antagonist of the signaling pathway. In addition to known APC-related proteins, we identified a set of differentially expressed proteins that had not previously been linked to the Wnt pathway. These proteins are involved in Ca2+ homeostasis, guanine nucleotide exchange apoptosis, and other functions. Shotgun proteomics can detect proteomic differences between subtly different phenotypes that differ at a single genetic locus. In this example, the approach provides new insights into the mechanism of mutant APC-associated tumorigenesis. (Supported by NIH grant U54CA126479.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-136.