Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro

• Published in: Breast cancer research and treatment Vol. 155; no. 2; pp. 261 - 271
• Main Authors: Tuomela, Johanna M., Sandholm, Jouko A., Kaakinen, Mika, Hayden, Katherine L., Haapasaari, Kirsi-Maria, Jukkola-Vuorinen, Arja, Kauppila, Joonas H., Lehenkari, Petri P., Harris, Kevin W., Graves, David E., Selander, Katri S.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.01.2016; Springer; Springer Nature B.V
• Subjects: Antimicrobial Cationic Peptides - genetics; Breast cancer; Breast Neoplasms - genetics; Breast Neoplasms - pathology; Cancer cells; Cancer genetics; Cancer research; Cancer therapies; Cell adhesion & migration; Cell Line, Tumor; Chemotherapy; Deoxyribonucleic acid; DNA; DNA Fragmentation; DNA, Neoplasm - genetics; Female; G-Quadruplexes; Genetic research; Humans; Ligands; Medicine; Medicine & Public Health; Neoplasm Invasiveness - genetics; Neoplasm Invasiveness - pathology; Oncology; Preclinical Study; Proteases; Protein expression; RNA; RNA, Messenger - genetics; Telomere - genetics; Toll-Like Receptor 9 - genetics; 9-mer hairpin DNA; LL-37; Toll-like receptor 9; Invasion; Telomeric G-quadruplex DNA
• ISSN: 0167-6806; 1573-7217
• DOI: 10.1007/s10549-016-3683-5

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor widely expressed in cancers. We previously showed that synthetic and self-derived DNA fragments induce TLR9-mediated breast cancer cell invasion in vitro. We investigated here the invasive effects of two nuclease-resistant DNA fragments, a 9-mer hairpin, and a G-quadruplex DNA based on the human telomere sequence, both having native phosphodiester backbone. Cellular uptake of DNAs was investigated with immunofluorescence, invasion was studied with Matrigel-assays, and mRNA and protein expression were studied with qPCR and Western blotting and protease activity with zymograms. TLR9 expression was suppressed through siRNA. Although both DNAs induced TLR9-mediated changes in pro-invasive mRNA expression, only the telomeric G-quadruplex DNA significantly increased cellular invasion. This was inhibited with GM6001 and aprotinin, suggesting MMP- and serine protease mediation. Furthermore, complexing with LL-37, a cathelicidin-peptide present in breast cancers, increased 9-mer hairpin and G-quadruplex DNA uptake into the cancer cells. However, DNA/LL-37 complexes decreased invasion, as compared with DNA-treatment alone. Invasion studies were conducted also with DNA fragments isolated from neoadjuvant chemotherapy-treated breast tumors. Also such DNA induced breast cancer cell invasion in vitro. As with the synthetic DNAs, this invasive effect was reduced by complexing the neoadjuvant tumor-derived DNAs with LL-37. We conclude that 9-mer hairpin and G-quadruplex DNA fragments are nuclease-resistant DNA structures that can act as invasion-inducing TLR9 ligands. Their cellular uptake and the invasive effects are regulated via LL-37. Although such structures may be present in chemotherapy-treated tumors, the clinical significance of this finding requires further studying.