title: G4-DNA formation in the HRAS promoter and rational design of decoy oligonucleotides for cancer therapy.
abstract: HRAS is a proto-oncogene involved in the tumorigenesis of urinary bladder cancer. In the HRAS promoter we identified two G-rich elements, hras-1 and hras-2, that fold, respectively, into an antiparallel and a parallel quadruplex (qhras-1, qhras-2). When we introduced in sequence hras-1 or hras-2 two point mutations that block quadruplex formation, transcription increased 5-fold, but when we stabilized the G-quadruplexes by guanidinium phthalocyanines, transcription decreased to 20% of control. By ChIP we found that sequence hras-1 is bound only by MAZ, while hras-2 is bound by MAZ and Sp1: two transcription factors recognizing guanine boxes. We also discovered by EMSA that recombinant MAZ-GST binds to both HRAS quadruplexes, while Sp1-GST only binds to qhras-1. The over-expression of MAZ and Sp1 synergistically activates HRAS transcription, while silencing each gene by RNAi results in a strong down-regulation of transcription. All these data indicate that the HRAS G-quadruplexes behave as transcription repressors. Finally, we designed decoy oligonucleotides mimicking the HRAS quadruplexes, bearing (R)-1-O-[4-(1-Pyrenylethynyl) phenylmethyl] glycerol and LNA modifications to increase their stability and nuclease resistance (G4-decoys). The G4-decoys repressed HRAS transcription and caused a strong antiproliferative effect, mediated by apoptosis, in T24 bladder cancer cells where HRAS is mutated.
authors.affiliation: Department of Medical and Biological Science, School of Medicine, Udine, Italy.
authors.forename: Alexandro
authors.initials: A
authors.lastname: Membrino
authors.forename: Susanna
authors.initials: S
authors.lastname: Cogoi
authors.forename: Erik B
authors.initials: EB
authors.lastname: Pedersen
authors.forename: Luigi E
authors.initials: LE
authors.lastname: Xodo
mesh_terms: D001483:Base Sequence; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000378:metabolism; D015195:Drug Design; D054856:G-Quadruplexes; D006367:HeLa Cells; D006801:Humans; D007211:Indoles / Q000378:metabolism; D054833:Isoindoles; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000627:therapeutic use*; D017354:Point Mutation / Q000235:genetics; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D000090063:Proto-Oncogene Mas; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D015854:Up-Regulation / Q000235:genetics; D001749:Urinary Bladder Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology
chemical_list: D004268:DNA-Binding Proteins; D007211:Indoles; D054833:Isoindoles; C000715470:MAS1 protein, human; D009841:Oligonucleotides; D000090063:Proto-Oncogene Mas; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C076035:c-MYC-associated zinc finger protein; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); C013647:phthalocyanine