Targeting CanineKITPromoter by Candidate DNA G-Quadruplex Ligands

Abstract

G-quadruplexes (G4) are nucleic acid secondary structures frequently assumed by G-rich sequences located mostly at telomeres and proto-oncogenes promoters. Recently, we identified, in canine KIT (v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog) promoter, two G-rich sequences able to fold into G4: d_kit1 and d_kit2_A16. In this study, an anthraquinone (AQ1) and an anthracene derivative (AN6), known to stabilize the G4 structures of the corresponding human h_kit1 and h_kit2, were tested on the canine G4 and in two canine mast cell tumor (MCT) cell lines (C2 and NI-1) to verify their capability to down-regulate KIT expression. The cytotoxicity of AQ1 and AN6 was determined using the Alamar Blue test; also the constitutive expression of KIT and other proto-oncogenes containing G4 structures in their promoter (BCL2, VEGFa, VEGFR2, KRAS, and TERT) was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Then the time-and dose-dependent effects of both ligands on target gene expression were assessed by qRT-PCR. All target genes were constitutively expressed up to 96 hours of culture. Both ligands decreased KIT mRNA levels and c-kit protein amount, and AN6 was comparatively fairly more effective. DNA interaction studies and a dualluciferase gene reporter assay performed on a noncancerous canine cell line (Madin-Darby Canine Kidney cells) proved that this down-regulation was the result of the interaction of AN6 with KIT proximal promoter. Interestingly, our results only partially overlap with those previously obtained in human cell lines, where AQ1 was found as the most effective compound. These preliminary data might suggest AN6 as a promising candidate for the selective targeting of canine KIT-dependent tumors.

Whole-Transcriptome Profiling of Canine and Human in Vitro Models Exposed to a G-Quadruplex Binding Small Molecule

“…On a comparative basis, we observed a higher number of DEGs in the human versus the dog cell line. This result supports preliminary analysis on C2 cells where a slight reduction of KIT mRNA and protein levels was observed upon treatment with AQ1 33 . This can be reasonably connected to the different sequence and distribution of the G4 forming domains in the human versus canine promoters although the modulation of distinct cellular pathways cannot be ruled out.…”

“…The post-transcriptional mechanisms of regulation and the emerging evidence that 5′- and 3′-untranslated regions (5′- and 3′-UTRs) as well as open reading frames (ORFs) contain putative RNA G4, might play an important role in tumor biology, e.g., switching on/off some tumor-related pathways 61 . From previous data, the AQ1-mediated downregulation of KIT was confirmed also at the protein level in human and canine in vitro models 31 – 33 . Hence, the protein evaluation of the kit-downstream pathways could substantiate current transcriptional results.…”

“…We previously used a targeted approach to assess the effect of AQ1 both in human and canine KIT -dependent cell lines (from two to almost forty-fold inhibition) 31 – 33 . Results demonstrated a significant inhibition of cell proliferation and downregulation of KIT and BCL2 induced by AQ1 whereas other prototypical oncogenes as hTERT , MYC , and PDGF were not highly modulated by this compound.…”

“…In perspective, it could be of interest to extend the RNA-Seq analysis also to a non-tumor cell line, to distinguish any non-specific effects. From previous information obtained in MDCK cells, these are much more resistant to AQ1 in term of cytotoxicity and survival 33 , but a more in-depth study investigating also non tumor-specific pathways is still missing. Finally, in terms of pathways analysis (DAVID), a limit derived by the actual scarce annotation of dog genome in comparison with the human one, and this should be consistently taken into consideration in the approach to future studies.…”

“…S1 ) 32 . They showed a high degree of homology with the corresponding human sequences, and preliminary studies were conducted to assess their interaction with AQ1 33 . These studies on G4 structures were executed using a targeted-gene approach; nevertheless, it is of interest to extend the study of AQ1 transcriptional effects to a wider scenario, like the whole-transcriptome.…”