G4-DNA formation in the HRAS promoter and rational design of decoy oligonucleotides for cancer therapy

PLoS One. 2011;6(9):e24421. doi: 10.1371/journal.pone.0024421. Epub 2011 Sep 8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Proliferation
  • DNA-Binding Proteins / metabolism
  • Drug Design*
  • G-Quadruplexes*
  • HeLa Cells
  • Humans
  • Indoles / metabolism
  • Isoindoles
  • Molecular Sequence Data
  • Oligonucleotides / therapeutic use*
  • Point Mutation / genetics
  • Polymorphism, Genetic
  • Promoter Regions, Genetic*
  • Protein Binding
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins p21(ras) / genetics*
  • Sp1 Transcription Factor / metabolism
  • Transcription Factors / metabolism
  • Transcription Initiation Site
  • Transcription, Genetic
  • Up-Regulation / genetics
  • Urinary Bladder Neoplasms / drug therapy*
  • Urinary Bladder Neoplasms / genetics
  • Urinary Bladder Neoplasms / pathology

Substances

  • DNA-Binding Proteins
  • Indoles
  • Isoindoles
  • MAS1 protein, human
  • Oligonucleotides
  • Proto-Oncogene Mas
  • Sp1 Transcription Factor
  • Transcription Factors
  • c-MYC-associated zinc finger protein
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)
  • phthalocyanine