Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases Adaikkalam Vellaichamy1,2, Arun Sreekumar1,2,6¤*, John R. Strahler5, Theckelnaycke Rajendiran1,2, Jindan Yu1,2,7, Sooryanarayana Varambally1,2,6, Yong Li1,2, Gilbert S. Omenn3,4,6, Arul M. Chinnaiyan1,2,6., Alexey I. Nesvizhskii2,3.* 1 Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan, United States of America, 2 Department of Pathology, University of Michigan, Ann Arbor, Michigan, United States of America, 3 Center for Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, Michigan, United States of America, 4 Internal Medicine and Human Genetics, University of Michigan, Ann Arbor, Michigan, United States of America, 5 Michigan Proteome Consortium, University of Michigan, Ann Arbor, Michigan, United States of America, 6 Comprehensive Cancer Center, University of Michigan, Ann Arbor, Michigan, United States of America, 7 Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America Abstract Prostate cancer remains the most common malignancy among men in United States, and there is no remedy currently available for the advanced stage hormone-refractory cancer. This is partly due to the incomplete understanding of androgen-regulated proteins and their encoded functions. Whole-cell proteomes of androgen-starved and androgen- treated LNCaP cells were analyzed by semi-quantitative MudPIT ESI- ion trap MS/MS and quantitative iTRAQ MALDI- TOF MS/MS platforms, with identification of more than 1300 high-confidence proteins. An enrichment-based pathway mapping of the androgen-regulated proteomic data sets revealed a significant dysregulation of aminoacyl tRNA synthetases, indicating an increase in protein biosynthesis- a hallmark during prostate cancer progression. This observation is supported by immunoblot and transcript data from LNCaP cells, and prostate cancer tissue. Thus, data derived from multiple proteomics platforms and transcript data coupled with informatics analysis provides a deeper insight into the functional consequences of androgen action in prostate cancer. Citation: Vellaichamy A, Sreekumar A, Strahler JR, Rajendiran T, Yu J, et al. (2009) Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases. PLoS ONE 4(9): e7075. doi:10.1371/journal.pone.0007075 Editor: Lennart Randau, Yale University, United States of America Received July 3, 2009; Accepted July 29, 2009; Published September 18, 2009 Copyright: 2009 Vellaichamy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded in part by the Michigan Technology Tri-Corridor, Michigan Economic Development Corporation grant 687 for the Michigan Proteomics Alliance for Cancer Research (GSO, AMC, AS, AV), the National Institutes of Health grants U54DA021519 for the National Center for Integrative Biomedical Informatics (GSO, AMC), R01CA126239 (AN), RO1CA133458 (AS, AMC) U01 CA111275 (AMC), 1K99CA129565-01A1 (JY), and P50 CA69568 (AMC, AS), Department of Defense PC080665 (JY), and the Fund for Discovery of the University of Michigan Comprehensive Cancer Center (AS). AMC is supported by a Clinical Translational Science Award from the Burroughs Welcome Foundation and is an Investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nesvi@med.umich.edu (AN); asreekumar@mcg.edu (AS) ¤ Current address: Medical College of Georgia Cancer Center, Medical College of Georgia, Augusta, Georgia, United States of America . These authors contributed equally to this work. Introduction Cancer of the prostate (PCa) is the most commonly diagnosed cancer among men in the United States, with an estimated 200,000 new cases and 29,000 deaths for 2008 [1]. Many reports suggest that androgens which are required for normal develop- ment of the prostate also support the growth and progression of PCa. Androgens exert their cellular effects via binding to the androgen receptor (AR), a member of the nuclear hormone receptor (NR) super family. AR, in turn, binds to androgen response elements (AREs) in the promoter and enhancer regions of target genes, acting as a transcriptional regulator by transducing the cognate hormone signal in the nucleus [2,3]. Prostate cancer therapy through androgen ablation initially achieves regression of tumors; however, a more aggressive, hormone-refractory form of the tumor (HR-PCa) may evolve, with subsequent mortality. Although multiple groups have interrogated androgen-regulated changes at the transcriptome and proteome levels using gene expression arrays and mass spectrometry, respectively [4,5,6,7,8,9,10], there is still a lack of considerable understanding on the functional consequence of the hormone action during prostate cancer development. Partly this deficiency is a reflection of the sparseness in the proteomic data; due to the limitations of proteomic technologies, the total number of proteins identified and quantified in a single study is modest. In addition, there are technical challenges from such under-representation of proteins and in assessing the statistical significance of differences in protein expression. To overcome these challenges, and to obtain a deeper insight into androgen-regulated proteomic alterations in prostate cancer, we applied a two-pronged strategy. First, we enhanced the breath of the androgen-regulated proteome profiled in this study by employing two complementary mass-spectrometry (MS) platforms: one that involves isotope labeling of peptides with an iTRAQ PLoS ONE | www.plosone.org 1 September 2009 | Volume 4 | Issue 9 | e7075