title: Pyrrole-imidazole polyamide-mediated silencing of KCNQ1OT1 expression induces cell death in Wilms' tumor cells.
abstract: KvDMR (an intronic CpG island within the KCNQ1 gene) is one of the imprinting control regions on human chromosome 11p15.5. Since KvDMR exists within the promoter region of KCNQ1OT1 (antisense transcript of KCNQ1), 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 KCNQ1OT1. Indeed, de-methylation of KvDMR accompanied by uncontrolled overexpression of KCNQ1OT1 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 KCNQ1OT1 expression might suppress its oncogenic potential. In the present study, we designed two pyrrole-imidazole (PI) polyamides, termed PI-a and PI-b, which might have the ability to bind to CCAAT boxes of the KCNQ1OT1 promoter region, and investigated their possible antitumor effect on Wilms' tumor-derived G401 cells. Gel retardation assay demonstrated that PI-a and PI-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 KCNQ1OT1 was significantly decreased when treated with PI-a and PI-b simultaneously but not with either PI-a or PI-b single treatment. Consistent with these results, the combination of PI-a and PI-b resulted in a significant reduction in viability of G401 cells in a dose-dependent manner. Furthermore, FACS analysis demonstrated that combinatory treatment with PI-a and PI-b induces cell death as compared with control cells. Taken together, our present observations strongly suggest that the combinatory treatment with PI polyamides targeting KCNQ1OT1 might be a novel therapeutic strategy to cure patients with tumors over-expressing KCNQ1OT1.
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Shinsuke
authors.initials: S
authors.lastname: Yoshizawa
authors.affiliation: Division of General Medicine, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Kiminobu
authors.initials: K
authors.lastname: Sugito
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Shota
authors.initials: S
authors.lastname: Uekusa
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kawashima
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Reina
authors.initials: R
authors.lastname: Hoshi
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Watanabe
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Hirano
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Furuya
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Masuko
authors.affiliation: Innovative Therapy Research Group, Nihon University Research Institute of Medical Science, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Ueno
authors.affiliation: Innovative Therapy Research Group, Nihon University Research Institute of Medical Science, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Noboru
authors.initials: N
authors.lastname: Fukuda
authors.affiliation: Division of General Medicine, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Soma
authors.affiliation: Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba, Japan.
authors.forename: Toshinori
authors.initials: T
authors.lastname: Ozaki
authors.affiliation: Department of Pediatric Surgery, Nihon University School of Medicine, Tokyo, Japan.
authors.forename: Tsugumichi
authors.initials: T
authors.lastname: Koshinaga
authors.affiliation: Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute, Chiba, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nagase
mesh_terms: D001562:Benzimidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D020868:Gene Silencing; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000494:pharmacology*; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000235:genetics*; D009757:Nylons / Q000138:chemical synthesis / Q000494:pharmacology*; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011758:Pyrroles / Q000138:chemical synthesis / Q000494:pharmacology*; D009396:Wilms Tumor / Q000188:drug therapy / Q000235:genetics*
chemical_list: D001562:Benzimidazoles; D007093:Imidazoles; C485621:KCNQ1OT1 long non-coding RNA, human; D009757:Nylons; D024642:Potassium Channels, Voltage-Gated; D011758:Pyrroles