MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

Abstract

KRAS mutations are primary genetic lesions leading to pancreatic cancer. The promoter of human KRAS contains a nuclease-hypersensitive element (NHE) that can fold in G4-DNA structures binding to nuclear proteins, including MAZ (myc-associated zinc-finger). Here, we report that MAZ activates KRAS transcription. To knockdown oncogenic KRAS in pancreatic cancer cells, we designed oligonucleotides that mimic one of the G-quadruplexes formed by NHE (G4-decoys). To increase their nuclease resistance, two locked nucleic acid (LNA) modifications were introduced at the 3′-end, whereas to enhance the folding and stability, two polycyclic aromatic hydrocarbon units (TINA or AMANY) were inserted internally, to cap the quadruplex. The most active G4-decoy (2998), which had two para-TINAs, strongly suppressed KRAS expression in Panc-1 cells. It also repressed their metabolic activity (IC50 = 520 nM), and it inhibited cell growth and colony formation by activating apoptosis. We finally injected 2998 and control oligonucleotides 5153, 5154 (2 nmol/mouse) intratumorally in SCID mice bearing a Panc-1 xenograft. After three treatments, 2998 reduced tumor xenograft growth by 64% compared with control and increased the Kaplan–Meier median survival time by 70%. Together, our data show that MAZ-specific G4-decoys mimicking a KRAS quadruplex are promising for pancreatic cancer therapy.

The MAZ transcription factor is a downstream target of the oncoprotein Cyr61/CCN1 and promotes pancreatic cancer cell invasion via CRAF–ERK signaling

“…Moreover, studies found that sequestering the MAZ protein by a stable KRas promoter analog prevents MAZ from activating K-Ras transcription and delays tumor growth in mice (7). Given these studies, we hypothesized that the regulation of phenotypic shift and aggressive behavior of PDAC cells by MAZ could be mediated through regulation of K-Ras transcription.…”

“…It plays critical role in PDAC development and progression (30,50). Previous studies have suggested MAZ activates K-Ras transcription via binding into the G4-DNA of the K-Ras promoter in pancreatic cancer cells (7). However, analysis of Human Pancreatic Adenocarcinoma Array found that MAZ ablation in Panc-1 cells minimally, but nonsignificantly decreased the K-Ras mRNA expression while the mRNA expression of various cancer-promoting genes like MMP1, MMP-7, VEGF, IL6, Cyp2E1, and CDC42 were markedly reduced ( Figures 5A and 5B).…”

“…These include human heart, brain, placenta, lung, liver, skeletal muscle, and pancreas (3,4). MAZ activates the expression of a variety of genes such as c-Myc, insulin, VEGF, RAS gene family and serotonin-1A receptor (5)(6)(7)(8)(9), and can simultaneously terminate transcription of eNOS and wild-type p53 (10,11). Studies demonstrate that MAZ plays a critical role in the progression of various cancers including breast, prostate, liver, and pancreas (2,6,(12)(13)(14).…”

“…Additional genetic, epigenetic, dosage or environmental factors may be required to raise the threshold of the activity of K-Ras signal for tumorigenesis (20,30,31), yet these factors have not been fully elucidated. A study has shown MAZ upregulates K-Ras transcription via binding into the G4-DNA of the K-Ras promoter in pancreatic cancer cells (7,32). However, a mechanistic link between the oncogenic role of MAZ and KRas activation has not yet been elucidated.…”

G‐Quadruplex Supramolecular Assemblies in Photochemical Upconversion

“…[22][23][24][25][26][27][28][29][30] Covalent attachment of the TINA molecule in guanine-rich sequences led to formation of inter-and intramolecular G4-DNAs with enhanced kinetic and thermodynamic parameters. [31][32][33][34][35] In comparison to pyrene, the phenylethynylpyrene-1-yl moiety in the TINA molecule has higher fluorescence quantum yield and is less sensitive to oxygen quenching even in aqueous solutions, [36][37][38] which is beneficial for PUC. Recently, we examined TINA-modified G4-DNAs forming parallel tetramolecular assemblies in which TINA molecule is present at the 5'-end, between T and dG at the 5'-end of the dTG 4 T sequence and in the middle of the G-tract of dTG 6 T sequence (Figure 1 D).…”