title: Telomeric G-quadruplex-forming DNA fragments induce TLR9-mediated and LL-37-regulated invasion in breast cancer cells in vitro.
abstract: 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.
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.|Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.|Department of Cell Biology and Anatomy, Institute of Biomedicine, University of Turku, Turku, Finland.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Tuomela
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.|Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.|Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
authors.forename: Jouko A
authors.initials: JA
authors.lastname: Sandholm
authors.affiliation: Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland.|Oulu Center for Cell-Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Oulu, Finland.
authors.forename: Mika
authors.initials: M
authors.lastname: Kaakinen
authors.affiliation: Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Hayden
authors.affiliation: Department of Pathology, University of Oulu, Oulu, Finland.
authors.forename: Kirsi-Maria
authors.initials: KM
authors.lastname: Haapasaari
authors.affiliation: Department of Oncology, University Hospital of Oulu, Oulu, Finland.
authors.forename: Arja
authors.initials: A
authors.lastname: Jukkola-Vuorinen
authors.affiliation: Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland.|Department of Pathology, University of Oulu, Oulu, Finland.|Department of Surgery, Oulu University Hospital, Oulu, Finland.
authors.forename: Joonas H
authors.initials: JH
authors.lastname: Kauppila
authors.affiliation: Department of Anatomy and Cell Biology, University of Oulu, Oulu, Finland.|Department of Pathology, University of Oulu, Oulu, Finland.|Department of Surgery, Oulu University Hospital, Oulu, Finland.
authors.forename: Petri P
authors.initials: PP
authors.lastname: Lehenkari
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA.|Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.|Birmingham Veterans Affairs Medical Center, Birmingham, AL, USA.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Harris
authors.affiliation: Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA.|Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Graves
authors.affiliation: Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA. Katriselander@uabmc.edu.|Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL, USA. Katriselander@uabmc.edu.|Department of Pathology, Lapland Central Hospital, Rovaniemi, Finland. Katriselander@uabmc.edu.|Department of Chemistry, UAB, CHEM 201 1720 2nd Ave South, Birmingham, AL, 35294-1240, USA. Katriselander@uabmc.edu.
authors.forename: Katri S
authors.initials: KS
authors.lastname: Selander
mesh_terms: D023181:Antimicrobial Cationic Peptides / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology*; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D054856:G-Quadruplexes; D006801:Humans; D008024:Ligands; D009361:Neoplasm Invasiveness / Q000235:genetics* / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D016615:Telomere / Q000235:genetics*; D051217:Toll-Like Receptor 9 / Q000235:genetics*; D054804:Cathelicidins
chemical_list: D023181:Antimicrobial Cationic Peptides; D004273:DNA, Neoplasm; D008024:Ligands; D012333:RNA, Messenger; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D054804:Cathelicidins
keywords: 9-mer hairpin DNA; Invasion; LL-37; Telomeric G-quadruplex DNA; Toll-like receptor 9