Differential protein expression profiling by iTRAQ-2DLC-MS/MS of human bladder cancer EJ138 cells transfected with the metastasis suppressor KiSS-1 gene

Abstract

e16043 Background: The use of isobaric tags for relative and absolute quantization (iTRAQ) followed by multidimensional liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis is emerging as a powerful methodology for biomarker and drug target discovery. KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 expression has been shown to be inversely correlated with increasing tumor stage and poor overall survival in bladder tumors. In order to identify the molecular pathways associated with the metastasis suppressor role of KiSS-1 in bladder cancer, we carried out a proteome discovery analysis using an iTRAQ approach. Methods: Bladder cancer cells (EJ138) were transiently transfected with a vector encompassing the full length KiSS-1 gene. Protein extracts collected after 24h and 48h transfection were fractionated, digested with trypsin and treated with iTRAQ reagents. The labelled peptides were separated through Strong Cation Exchange (SCX) and Reversed Phase LC and analysed by MALDI TOF/TOF MS. Several software packages were utilized for data analysis: ProteinPilot, Protein Center for gene ontology (GO) analysis and Ingenuity Pathway. Results: Comparative analysis among transfected, mock and empty vector exposed cells have identified more than 800 proteins with high confidence (>99%), showing high correlation rates among replicates (>70%). The involvement of the identified proteins in biological networks has served to characterize molecular pathways associated with KiSS-1 expression and to select critical candidates for validation analyses by Western Blot using independent transfected replicates. As part of complementary clinical validation strategies, inmunohistochemical analyses performed in metastatic bladder tumours spotted onto tissue microarrays (n = 175) have revealed the role of KiSS-1, ezrin and filamin in bladder cancer progression. Conclusions: Our proteomic study not only has served to reveal molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer, but also to identify novel potential metastatic biomarkers for patients affected with bladder tumors. No significant financial relationships to disclose.