Breast Cancer Research and Treatment

, Volume 155, Issue 2, pp 261–271 | Cite as

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

  • Johanna M. Tuomela
  • Jouko A. Sandholm
  • Mika Kaakinen
  • Katherine L. Hayden
  • Kirsi-Maria Haapasaari
  • Arja Jukkola-Vuorinen
  • Joonas H. Kauppila
  • Petri P. Lehenkari
  • Kevin W. Harris
  • David E. Graves
  • Katri S. Selander
Preclinical study

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.

Keywords

Toll-like receptor 9 Invasion LL-37 Telomeric G-quadruplex DNA 9-mer hairpin DNA 

Notes

Acknowledgments

The authors wish to thank Ms. Christine Pressey for skillful assistance with the qPCR assays.

Compliance with ethical standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Financial support

This work was funded by grants from the Department of Defense (W81XWH—10—1—0308, K.S.S., D.G.), Finnish Cancer Organizations (J.S.), Finnish Cultural Foundation (J.T.), Maud Kuistila Memorial Foundation (J.T.), Orion-Farmos Research Foundation (J.T.), K. Albin Johansson Foundation (J.T.), and Cancer Foundation of South-Western Finland (J.T).

Supplementary material

10549_2016_3683_MOESM1_ESM.pdf (283 kb)
Supplementary material 1 (PDF 283 kb)

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Johanna M. Tuomela
    • 1
    • 2
    • 3
  • Jouko A. Sandholm
    • 1
    • 2
    • 4
  • Mika Kaakinen
    • 5
    • 6
  • Katherine L. Hayden
    • 7
  • Kirsi-Maria Haapasaari
    • 8
  • Arja Jukkola-Vuorinen
    • 9
  • Joonas H. Kauppila
    • 5
    • 8
    • 10
  • Petri P. Lehenkari
    • 5
    • 8
    • 10
  • Kevin W. Harris
    • 1
    • 2
    • 11
  • David E. Graves
    • 2
    • 7
  • Katri S. Selander
    • 1
    • 2
    • 12
    • 13
  1. 1.Division of Hematology-Oncology, Department of MedicineUniversity of Alabama at BirminghamBirminghamUSA
  2. 2.Comprehensive Cancer CenterUniversity of Alabama at BirminghamBirminghamUSA
  3. 3.Department of Cell Biology and Anatomy, Institute of BiomedicineUniversity of TurkuTurkuFinland
  4. 4.Turku Centre for BiotechnologyUniversity of Turku and Åbo Akademi UniversityTurkuFinland
  5. 5.Department of Anatomy and Cell BiologyUniversity of OuluOuluFinland
  6. 6.Oulu Center for Cell–Matrix Research, Faculty of Biochemistry and Molecular Medicine, Biocenter OuluUniversity of OuluOuluFinland
  7. 7.Department of ChemistryUniversity of Alabama at BirminghamBirminghamUSA
  8. 8.Department of PathologyUniversity of OuluOuluFinland
  9. 9.Department of OncologyUniversity Hospital of OuluOuluFinland
  10. 10.Department of SurgeryOulu University HospitalOuluFinland
  11. 11.Birmingham Veterans Affairs Medical CenterBirminghamALUSA
  12. 12.Department of PathologyLapland Central HospitalRovaniemiFinland
  13. 13.Department of ChemistryUABBirminghamUSA

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