KiSS-1 is a metastasis suppressor gene reported to be involved in the progression of several solid neoplasias. The loss of KiSS-1 gene expression has been shown to be inversely correlated with increasing tumour stage, distant metastases, 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 proteomic analysis of bladder cancer cells (EJ138) transiently transfected with a vector encompassing the full length KiSS-1 gene using an iTRAQ (isobaric tags for relative and absolute quantitation) approach. Protein extracts collected after 24h and 48h transfection were fractionated, cleaved with trypsin and the resulting peptides labelled with iTRAQ reagents. The labelled peptides were separated by strong cation exchange (SCX) and reversed phase liquid chromatography (LC), and analysed by MALDI TOF/TOF mass spectrometry (MS). Three software packages were utilized for data analysis: ProteinPilot for identification and quantification of differentially expressed proteins, Protein Center for gene ontology (GO) analysis and Ingenuity Pathways to provide insight into biological networks. Comparative analysis among transfected, mock and empty vector exposed cells identified 1529 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 verification analyses by Western Blot using independent transfected replicates. As part of complementary clinical validation strategies, immunohistochemical analyses of proteins regulated by KiSS-1, such as Filamin A, were performed on bladder tumours spotted onto tissue microarrays (n=280). In summary, our study not only served to uncover molecular mechanisms associated with the metastasis suppressor role of KiSS-1 in bladder cancer, but also to reveal the biomarker role of Filamin A in bladder cancer progression and clinical outcome.

Free Article; PMID: 20139371 [PubMed - as supplied by publisher] Source: National Library of Medicine.