Abstract
More than 380,000 new cases of bladder cancer are diagnosed worldwide, accounting for ∼150,200 deaths each year. To discover potential biomarkers of bladder cancer, we employed a strategy combining laser microdissection, isobaric tags for relative and absolute quantitation labeling, and liquid chromatography-tandem MS (LC-MS/MS) analysis to profile proteomic changes in fresh-frozen bladder tumor specimens. Cellular proteins from four pairs of surgically resected primary bladder cancer tumor and adjacent nontumorous tissue were extracted for use in two batches of isobaric tags for relative and absolute quantitation experiments, which identified a total of 3220 proteins. A DAVID (database for annotation, visualization and integrated discovery) analysis of dysregulated proteins revealed that the three top-ranking biological processes were extracellular matrix organization, extracellular structure organization, and oxidation-reduction. Biological processes including response to organic substances, response to metal ions, and response to inorganic substances were highlighted by up-expressed proteins in bladder cancer. Seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. Immunohistochemical analyses showed significantly elevated levels of three proteins-SLC3A2, STMN1, and TAGLN2-in tumor cells compared with noncancerous bladder epithelial cells, and suggested that TAGLN2 could be a useful tumor tissue marker for diagnosis (AUC = 0.999) and evaluating lymph node metastasis in bladder cancer patients. ELISA results revealed significantly increased urinary levels of both STMN1 and TAGLN2 in bladder cancer subgroups compared with control groups. In comparisons with age-matched hernia urine specimens, urinary TAGLN2 in bladder cancer samples showed the largest fold change (7.13-fold), with an area-under-the-curve value of 0.70 (p < 0.001, n = 205). Overall, TAGLN2 showed the most significant overexpression in individual bladder cancer tissues and urine specimens, and thus represents a potential biomarker for noninvasive screening for bladder cancer. Our findings highlight the value of bladder tissue proteome in providing valuable information for future validation studies of potential biomarkers in urothelial carcinoma.
Highlights
From the ‡Department of Urology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; §School of Medicine, College of Medicine, Chang Gung University, Taoyuan, Taiwan; ¶Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan; ʈDepartment of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan; **Department of Pathology, Chang Gung Memorial Hospital, Taoyuan, Taiwan; ‡‡Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan; §§Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
To understand the presence in urine of bladder tissue proteins associated with bladder cancer, we first compared the list of 131 up-regulated bladder tissue proteins with 1939 normal urinary proteins, downloaded from the Sys-Bodyfluid database compiled by Li et al [27]
This comparison revealed that carbonic anhydrase 2 (CA2), phosphoglycerate kinase 1 (PGK1), SFN, and TXN had been identified in the normal urine proteome
Summary
Clinical Specimens—Fresh-frozen specimens of surgically resected primary bladder tumor and adjacent nontumorous tissues were obtained from four patients (two females and two males). Fresh-frozen tumor specimens for iTRAQ analysis were obtained from bladder cancer patient volunteers diagnosed at the Chang Gung Memorial Hospital, Taoyuan, Taiwan. The ratio of each protein was quantified using the Libra program, a module within the TPP software package that performs quantification on MS/MS spectra that have multiplexed labeled peptides. The final score was obtained by multiplying the intensity (I) by the percentage of positive-stained cells (P) Using this system, we classified protein expression level in bladder cancer tissue and adjacent urothelium cells into three groups for each candidate cancer biomarker: low staining, 0 –99 for TAGLN2, and SLC3A2, and 0 –150 for STMN1; moderate staining, 100 –199 for TAGLN2, 100 –159 for SLC3A2, and 151–200 for STMN1; strong staining, Ն200 for TAGLN2, Ն160 for SLC3A2, and Ն201 for STMN1.
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