Identification of pro-inflammatory cytokines associated with muscle invasive bladder cancer; the roles of IL-5, IL-20, and IL-28A.

We used gene expression profiling to identify inflammatory cytokines that correlate with bladder cancer development. Gene expression profiles of the tissue samples were investigated using cDNA microarrays that contained 103 non-muscle invasive bladder cancers (NMIBC), 62 muscle invasive bladder cancers (MIBC), 58 samples of histologically normal-looking surrounding tissues, and 10 normal, healthy subjects who served as the control cohort for comparison. We grouped the data-sets according to biological characterizations and focused on immune response genes with at least 2-fold differential expression in MIBC vs. controls. The experimental data-set identified 36 immune-related genes that were significantly altered in MIBC samples. In addition, 10 genes were up-regulated and 26 genes were down-regulated in MIBC samples compared with the normal tissues. Among the 10 up-regulated molecules examined, the capacity for both wound-healing migration and invasion was enhanced in response to IL-5, IL-20, and IL-28A in bladder cancer cell lines (253J and EJ cells), compared with untreated cells. The expression levels of IL-5, IL-20, and IL-28A were increased in patients with MIBC. All 3 cytokines and their receptors were produced in bladder cancer cell lines, as determined by real-time PCR, immunoblot analysis and confocal immunofluorescence. Up-regulation of MMP-2 and MMP-9 was found after IL-5, IL-20, and IL-28A stimulation in both cell types. Moreover, an EMSA assay showed that treatment with IL-5, IL-20, and IL-28A induced activation of the transcription factors NF-κB and AP-1 that regulate the MMP-9 promoter. Finally, activation of MAPK and Jak-Stat signaling was observed after the addition of IL-5, IL-20, and IL-28A to bladder cancer cells. This study suggests the presence of specific inflammatory cytokine (IL-5, IL-20, and IL-28A)-mediated association in bladder cancer development. All 3 cytokines may be important new molecular targets for the modulation of migration and invasion in bladder cancer.