Mechanism of Action of G-Quadruplex–Forming Oligonucleotide Homologous to the Telomere Overhang in Melanoma

Gagan Chhabra,Luke Wojdyla,Mark Frakes,Zachary Schrank,Brandon Leviskas,Marko Ivancich,Pooja Vinay,Ramesh Ganapathy,Benjamin E Ramirez,Neelu Puri

doi:10.1016/j.jid.2017.11.021

Abstract

T-oligo, a guanine-rich oligonucleotide homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. Guanine-rich oligonucleotides can form G-quadruplexes (G4), which are stabilized by the hydrogen bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using nondenaturing PAGE, nuclear magnetic resonance, and immunofluorescence. Using an anti-G-quadruplex antibody, we showed that T-oligo can form G4 in the nuclei of melanoma cells. Furthermore, using DNase I in a nuclease degradation assay, G4-T-oligo was found to be more stable than single-stranded T-oligo. G4-T-oligo had decreased antiproliferative effects compared with single-stranded T-oligo. However, G4-T-oligo has similar cellular uptake as single-stranded T-oligo, as shown by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the antiproliferative activity of T-oligo. T-oligo also inhibited mRNA expression of human telomerase reverse transcriptase, a catalytic subunit of telomerase that was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be up-regulated and bound by T-oligo, suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies show that T-oligo can form a G-quadruplex and that the antitumor effects of T-oligo may be mediated through POT1/TRF2 and via human telomerase reverse transcriptase inhibition through JNK activation.

Highlights

Telomeres have become an attractive target for anticancer therapeutics because of near-universal overexpression of telomerase in tumors, which endows malignant cells with immortality (Crees et al, 2014; Ruden and Puri, 2013)
Our earlier studies indicated that SS-T-oligo may possibly recruit the shelterin complex, a group of six proteins regulating telomeric stability and homeostasis, away from the telomere, uncapping the telomere overhang and causing DNA-damage response (DDR) (Pitman et al, 2013)
We show that the anticancer signaling mechanism of T-oligo involves human telomerase reverse transcriptase and JNK, which further suggests that T-oligo induces DDR signals through inhibition of telomerase and telomereassociated proteins

Summary

Introduction

Telomeres have become an attractive target for anticancer therapeutics because of near-universal overexpression of telomerase in tumors, which endows malignant cells with immortality (Crees et al, 2014; Ruden and Puri, 2013). Guanine-rich oligonucleotides (GROs), including single-stranded (SS) telomere homolog oligonucleotides, are known to have potent anticancer effects when administered to several malignant cell types both in vitro and in vivo (Crees et al, 2014). One well-studied 11-base T-oligo (dGTTAGGGTTAG), which is homologous to the 30-telomere overhang sequence, induces potent DNA damage responses (DDRs) in several cancers (Eller et al, 2002; Puri et al, 2004, 2014; Ruden and Puri, 2013; Wojdyla et al, 2014; Yaar et al, 2007) and has minimal or no toxicity on normal cells (Puri et al, 2004, 2014).

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