Pyrrole-imidazole polyamide-mediated silencing of KCNQ1OT1 expression induces cell death in Wilms' tumor cells

KvDMR (an intronic CpG island within the KCNQI gene) is one of the imprinting control regions on human chromosome 11p15.5. Since KvDMR exists within the promoter region of KCNQIOT1 (antisense transcript of KCNQI), it is likely that genomic alterations of this region including deletion, paternal uniparental disomy and de-methylation in maternal allele lead to aberrant overexpression of KCNQIOT1. Indeed, de-methylation of KvDMR accompanied by uncontrolled overexpression of KCNQIOT1 occurs frequently in Beckwith-Wiedemann syndrome (BWS), and around 10% of BWS patients developed embryonal tumors (Wilms' tumor or hepatoblastoma). These observations strongly suggest that silencing of KCNQIOTI expression might suppress its oncogenic potential. In the present study, we designed two pyrrole-imidazole (PI) polyamides, termed P1-a and PI-b, which might have the ability to bind to CCAAT boxes of the KCNQIOT1 promoter region, and investigated their possible antitumor effect on Wilms' tumor-derived G401 cells. Gel retardation assay demonstrated that P1-a and P1-b specifically bind to their target sequences. Microscopic observations showed the efficient nuclear access of these PI polyamides. Quantitative real-time PCR analysis revealed that the expression level of KCNQIOTI was significantly decreased when treated with P1-a and P1-b simultaneously but not with either P1-a or P1-b single treatment. Consistent with these results, the combination of P1-a and P1-b resulted in a significant reduction in viability of G401 cells in a dose-dependent manner. Furthermore, FACS analysis demonstrated that combinatory treatment with P1-a and P1-b induces cell death as compared with control cells. Taken together, our present observations strongly suggest that the combinatory treatment with PI polyamides targeting KCNQIOTI might be a novel therapeutic strategy to cure patients with tumors overexpressing KCNQIOTI.