Abstract
KRAS is a GTPase involved in the proliferation signaling of several growth factors. The KRAS gene is GC-rich, containing regions with known and putative G-quadruplex (G4) forming regions. Within the middle of the G-rich proximal promoter, stabilization of the physiologically active G4mid structure downregulates transcription of KRAS; the function and formation of other G4s within the gene are unknown. Herein we identify three putative G4-forming sequences (G4FS) within the KRAS gene, explore their G4 formation, and develop oligonucleotides targeting these three regions and the G4mid forming sequence. We tested Polypurine Reverse Hoogsteen hairpins (PPRHs) for their effects on KRAS regulation via enhancing G4 formation or displacing G-rich DNA strands, downregulating KRAS transcription and mediating an anti-proliferative effect. Five PPRH were designed, two against the KRAS promoter G4mid and three others against putative G4FS in the distal promoter, intron 1 and exon 5. PPRH binding was confirmed by gel electrophoresis. The effect on KRAS transcription was examined by luciferase, FRET Melt2, qRT-PCR. Cytotoxicity was evaluated in pancreatic and ovarian cancer cells. PPRHs decreased activity of a luciferase construct driven by the KRAS promoter. PPRH selectively suppressed proliferation in KRAS dependent cancer cells. PPRH demonstrated synergistic activity with a KRAS promoter selective G4-stabilizing compound, NSC 317605, in KRAS-dependent pancreatic cells. PPRHs selectively stabilize G4 formation within the KRAS mid promoter region and represent an innovative approach to both G4-stabilization and to KRAS modulation with potential for development into novel therapeutics.
Highlights
KRAS is a 21-kD GTPase that plays a role in cell survival, proliferation, and differentiation [1,2]
Mutation of the KRAS gene has been identified as a transforming oncogenic event, where it creates an unstable environment allowing for more mutational selection and increasingly aggressive disease [5]
G40 s formed within the proximal KRAS promoter have been previously described [37,51], including the physiologically active KRAS G4mid sequence
Summary
KRAS is a 21-kD GTPase that plays a role in cell survival, proliferation, and differentiation [1,2]. It is constitutively expressed, but active only when GTP-bound. Single point mutations of the KRAS gene abolish inherent GTP hydrolysis; these mutations render the protein constitutively active. In the absence of a mutation, increased KRAS activity in human tumors is the result of gene amplification, overexpression, or increased upstream activation [3]. The genomic amplification of KRAS, in particular, is associated with metastatic disease and poor prognosis in hormone-related cancers such as ovarian cancer [6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]
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