iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer Ishtiaq Rehman 1 * . , Caroline A. Evans1,2., Adam Glen1, Simon S. Cross3, Colby L. Eaton1, Jenny Down 1 , Giancarlo Pesce1, Joshua T. Phillips4, Ow Saw Yen 2 , George N. Thalmann5, Phillip C. Wright2, Freddie C. Hamdy6 1 Department of Human Metabolism, The Medical School, The Mellanby Centre for Bone Research, University of Sheffield, Sheffield, United Kingdom, 2 Biological and Environmental Systems Group, Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Sheffield, United Kingdom, 3 Academic Unit of Pathology, Department of Neuroscience, Faculty of Medicine, Dentistry and Health, University of Sheffield, Sheffield, United Kingdom, 4 Department of Urology, City Hospital, Birmingham, United Kingdom, 5 Department of Urology, Anna-Seiler-Haus, University of Bern, InselspitalInselspital, Bern, Switzerland, 6 Nuffield Department of Surgical Sciences, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom Abstract A major challenge in the management of patients with prostate cancer is identifying those individuals at risk of developing metastatic disease, as in most cases the disease will remain indolent. We analyzed pooled serum samples from 4 groups of patients (n = 5 samples/group), collected prospectively and actively monitored for a minimum of 5 yrs. Patients groups were (i) histological diagnosis of benign prostatic hyperplasia with no evidence of cancer ‘BPH’, (ii) localised cancer with no evidence of progression, ‘non-progressing’ (iii) localised cancer with evidence of biochemical progression, ‘progressing’, and (iv) bone metastasis at presentation ‘metastatic’. Pooled samples were immuno-depleted of the 14 most highly abundant proteins and analysed using a 4-plex iTRAQ approach. Overall 122 proteins were identified and relatively quantified. Comparisons of progressing versus non-progressing groups identified the significant differential expression of 25 proteins (p,0.001). Comparisons of metastatic versus progressing groups identified the significant differential expression of 23 proteins. Mapping the differentially expressed proteins onto the prostate cancer progression pathway revealed the dysregulated expression of individual proteins, pairs of proteins and ‘panels’ of proteins to be associated with particular stages of disease development and progression. The median immunostaining intensity of eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), one of the candidates identified, was significantly higher in osteoblasts in close proximity to metastatic tumour cells compared with osteoblasts in control bone (p = 0.0353, Mann Whitney U). Our proteomic approach has identified leads for potentially useful serum biomarkers associated with the metastatic progression of prostate cancer. The panels identified, including eEF1A1 warrant further investigation and validation. Citation: Rehman I, Evans CA, Glen A, Cross SS, Eaton CL, et al. (2012) iTRAQ Identification of Candidate Serum Biomarkers Associated with Metastatic Progression of Human Prostate Cancer. PLoS ONE 7(2): e30885. doi:10.1371/journal.pone.0030885 Editor: Karl X. Chai, University of Central Florida, United States of America Received August 26, 2011; Accepted December 27, 2011; Published February 15, 2012 Copyright: 2012 Rehman 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 supported by grants from PROMET (project no. FP6-LSH-5-2004-018858), and P-MARK (contract no. LSHC-CT-2004-503011), under the sixth EU Framework programme and the NCRI ProMPT (Prostate cancer Mechanisms of progression and Treatment) collaborative grant to Dr. Hamdy, and grants from Engineering and Physical Sciences Research (EPSRC) to Dr. Wright: EP/E036252/1 and GR/S84347/01. We are grateful to Dr. Nicholas Hoyle at Roche Diagnostics, Penzberg, Germany for his support towards consumables and reagents for the project. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. Competing Interests: We are grateful to Dr. Nicholas Hoyle at Roche Diagnostics, Penzberg, Germany for his support towards consumables and reagents for the project. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: i.rehman@sheffield.ac.uk . These authors contributed equally to this work. Introduction In Europe and the US, prostate cancer is the second most common cancer diagnosis and the third most common cause of cancer-related deaths in men [1,2]. Moreover, the incidence has increased since the widespread introduction of prostate specific antigen (PSA) testing [3]. Most patients with prostate cancer are diagnosed at an early stage, but even with screening over 7% of cases develop metastatic disease [4]. In men with distant metastasis the prognosis is poor, with an average survival of 24 to 48 months. Bone is the most common site for prostate cancer metastasis and is associated with bone pain, spinal cord compression and marrow failure [4]. Currently, bone metastatic lesions are determined by imaging such as isotope bone scanning, however, the identification of a serum based biomarker(s) for predicting the susceptibility of patients to develop bone metastasis could enable a more accurate clinical assessment of the disease and help guide therapy. The diagnosis of prostate cancer is most commonly made by a triad of serum prostate specific antigen (PSA) measurements, digital rectal examination (DRE), and histological assessment of transrectal ultrasound (TRUS) guided biopsy material [5]. Although PSA is a FDA approved biomarker for prostate cancer detection, its usefulness is controversial as it has been shown to be unreliable for disease diagnosis, and in particular for distinguishing PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30885