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.
Highlights
The ras gene family consists of three functional proto-oncogenes (HRAS, NRAS and KRAS) that encode for guanine-binding proteins sharing a high homology (p21RAS) [1]
Our study provides compelling evidence that HRAS transcription is regulated by the interplay between Sp1, myc associated zinc-finger transcription factor (MAZ) and G4-DNA, which acts as a transcription repressor
As previous studies have shown that MAZ binds to G4-DNA from the murine KRAS [24] and c-myb [39] promoters, we explored whether it binds to the HRAS quadruplexes
Summary
The ras gene family consists of three functional proto-oncogenes (HRAS, NRAS and KRAS) that encode for guanine-binding proteins sharing a high homology (p21RAS) [1]. Given the crucial role of HRAS overexpression and mutations in the tumorigenesis of bladder cancer, one attractive therapeutic strategy could be to inhibit HRAS transcription with molecules that are able to impair the activity of the gene promoter. For this aim we asked how HRAS transcription is regulated. For their potent antiproliferative effect in T24 urinary bladder cancer cells, G4-decoys seem to be very promising effector drugs for urinary bladder cancer therapy
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