ARTICLE pubs.acs.org/jpr Proteomic Consequences of a Single Gene Mutation in a Colorectal Cancer Model Patrick J. Halvey,†,‡ Bing Zhang,§ Robert J. Coﬀey,||,^ Daniel C. Liebler,†,‡ and Robbert J. C. Slebos*,‡,|| †Department of Biochemistry, ‡Jim Ayers Institute for Precancer Detection and Diagnosis, §Department of Biomedical Informatics, D)epartment of Cancer Biology, and ^Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6350, United States bS Supporting Information ABSTRACT: The proteomic eﬀects of speciﬁc cancer-related muta- tions have not been well characterized. In colorectal cancer (CRC), a relatively small number of mutations in key signaling pathways appear to drive tumorigenesis. Mutations in adenomatous polyposis coli (APC), a negative regulator of Wnt signaling, occur in up to 60% of CRC tumors. Here we examine the proteomic consequences of a single gene mutation by using an isogenic CRC cell culture model in which wildtype APC expression has been ectopically restored. Using LC MS/MS label free shotgun proteomics, over 5000 proteins were identiﬁed in SW480Null (mutant APC) and SW480APC (APC restored). We observed 155 signiﬁcantly diﬀerentially expressed proteins between the two cell lines, with 26 proteins showing opposite expression trends relative to gene expression measurements. Protein changes corresponded to previously characterized features of the APCNull phenotype: loss of cell adhesion proteins, increase in cell cycle regulators, alteration in Wnt signaling related proteins, and redistribution of β-catenin. Increased expression of RNA processing and isoprenoid biosynthetic proteins occurred in SW480Null cells. Therefore, shotgun proteomics reveals proteomic diﬀerences associated with a single gene change, including many novel diﬀerences that fall outside known target pathways. KEYWORDS: shotgun proteomics, label-free quantitation, multiple reaction monitoring (MRM), LC APC MS/MS, colorectal cancer, ’ INTRODUCTION Mass spectrometry-based shotgun proteomics is a powerful tool for the interrogation of biological systems. Recent techno- logical advances allow thousands of proteins to be routinely iden- tiﬁed from submilligram quantities of tissues or cells.1 Increased depth of proteome coverage lends itself to the investigation of molecular signatures, which underlie complex disease processes. For instance, in cancer, thousands of mutations have been identi- ﬁed but the precise relationship between genomic variation and cancer phenotype remains largely unclear.2 Individual mutations may bring about proteomic changes that otherwise would not be predicted based on known gene function. In-depth proteomic analysis is required to deﬁne the broader landscape of protein networks and signaling pathways associated with cancer-related mutations. Determination of the proteomic consequences of individual mutations in tissues presents a major challenge due to the tremendous genetic heterogeneity between individual patient tumors.3 Isogenic cell lines present an attractive model system, wherein the phenotypic eﬀects of a single gene defect can be assessed in the context of uniform background gene/protein ex- pression. Colorectal cancer (CRC) cell models are ideally suited for the examination of proteomic eﬀects of single gene changes. Several mutations in key signaling pathways have been identiﬁed in CRC. These “driver mutations” have been mapped to speciﬁc events during tumor progression from normal epithelia to carcinoma.4 One essential driver mutation occurs in adenoma- tous polyposis coli (APC), and is among the most frequently observed genetic aberrations in colorectal adenomas and cancers.5 APC mutations (or allelic loss) occur in 70 80% of adenomas and carcinomas.6,7 While many other amino acid-changing mutations are associated with CRC, most can be classiﬁed as “passenger” muta- tions, that is, they do not confer a selective growth advantage on the tumor and are propagated randomly by clonal expansion.4,8 Therefore, by focusing on proteomic changes assocziated with a single driver mutation, one may decipher the protein subnet- works that underlie tumor development. APC occurs in complex with Axin (AXIN1) and glycogen synthase kinase-3β (GSK3B) and plays a role in the proteolytic degradation of β-catenin (CTNNB1). Decreases in CTNNB1 levels result in the inhibition of TCF/LEF-mediated transcription, Received: September 8, 2011 Published: November 21, 2011 r 2011 American Chemical Society 1184 dx.doi.org/10.1021/pr2009109 | J. Proteome Res. 2012, 11, 1184–1195