Breast Cancer Research and Treatment

, Volume 132, Issue 2, pp 411–419 | Cite as

Estrogen receptor-α and sex steroid hormones regulate Toll-like receptor-9 expression and invasive function in human breast cancer cells

  • Jouko Sandholm
  • Joonas H. Kauppila
  • Christine Pressey
  • Johanna Tuomela
  • Arja Jukkola-Vuorinen
  • Markku Vaarala
  • Martin R. Johnson
  • Kevin W. Harris
  • Katri S. Selander
Preclinical study


Toll-like receptor 9 (TLR9) is a cellular DNA-receptor, which is widely expressed in cancer. Synthetic TLR9-ligands induce cancer cell invasion in vitro, but the role of TLR9 in cancer pathophysiology remains unclear. Increased TLR9 expression has been, however, detected in estrogen receptor negative (ER−) breast cancers. In this study, we investigated the effects of ERα expression and sex steroid hormones on TLR9 expression in human ER+ (MCF-7, T47-D) and ER− (MDA-MB-231) breast cancer cell lines in vitro. We also studied TLR9 mRNA expression in archival breast cancer specimens (n = 12) with qRT-PCR, using primer sets that detect only the TLR9A isoform or the isoforms A and B (TLR9A/B). The TLR9 mRNA expression was detected in 10/12 specimens with both primer sets, and in 1/12 with only the TLR9A or the TLR9A/B primer sets. The basal TLR9 mRNA expression levels were significantly lower in the ER+ cell lines as compared with the ER− MDA-MB-231 cells. The transfection of ERα cDNA into MDA-MB-231 cells also resulted in down-regulation of TLR9 expression. While sex steroids had no effect on TLR9 expression in MCF-7 cells, testosterone (10−8 M) induced TLR9 expression in MDA-MB-231 and T47-D cells. Although bicalutamide blocked this testosterone effect in MDA-MB-231 cells, in T47-D cells bicalutamide increased TLR9 expression and only partially blocked the testosterone effects. Estradiol (10−8 M) induced TLR9 expression in T47-D cells. The invasive effects of synthetic TLR9-ligands were augmented by testosterone in vitro. This effect was lost in TLR9 siRNA MDA-MB-231 cells and also decreased by over-expression of ERα, which also inhibited NF-κB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERα and sex steroid hormones regulate TLR9 expression and invasive effects in the breast cancer cells. Also, the commonly used hormonal cancer therapy bicalutamide affects TLR9 expression.


Toll-like receptor 9 Invasion Estradiol Testosterone 



This study was financially supported by grants from the Northern Finnish Duodecim Foundation (A. J.-V.), Finnish Cultural Foundation, Lapland Funds (A. J.-V. and K. S. S.), Cancer Foundation of Northern Ostrobotnia, Oulu University Scholarship Foundation (J. H. K.), and Elsa U. Pardee Foundation (K. S. S.).


  1. 1.
    Akira S, Hemmi H (2003) Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 85:85–95PubMedCrossRefGoogle Scholar
  2. 2.
    Matsumoto M, Funami K, Tanabe M et al (2003) Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol 171:3154–3162PubMedGoogle Scholar
  3. 3.
    Nishiya T, DeFranco AL (2004) Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors. J Biol Chem 279:19008–19017PubMedCrossRefGoogle Scholar
  4. 4.
    Schmausser B, Andrulis M, Endrich S et al (2004) Expression and subcellular distribution of toll-like receptors TLR4, TLR5 and TLR9 on the gastric epithelium in Helicobacter pylori infection. Clin Exp Immunol 136:521–526PubMedCrossRefGoogle Scholar
  5. 5.
    Schmausser B, Andrulis M, Endrich S, Muller-Hermelink HK, Eck M (2005) Toll-like receptors TLR4, TLR5 and TLR9 on gastric carcinoma cells: an implication for interaction with Helicobacter pylori. Int J Med Microbiol 295:179–185PubMedCrossRefGoogle Scholar
  6. 6.
    Leifer CA, Kennedy MN, Mazzoni A, Lee C, Kruhlak MJ, Segal DM (2004) TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 173:1179–1183PubMedGoogle Scholar
  7. 7.
    Wagner H (2004) The immunobiology of the TLR9 subfamily. Trends Immunol 25:381–386PubMedCrossRefGoogle Scholar
  8. 8.
    Akira S, Takeda K, Kaisho T (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2:675–680PubMedCrossRefGoogle Scholar
  9. 9.
    Merrell MA, Ilvesaro JM, Lehtonen N et al (2006) Toll-like receptor 9 agonists promote cellular invasion by increasing matrix metalloproteinase activity. Mol Cancer Res 4:437–447PubMedCrossRefGoogle Scholar
  10. 10.
    Ilvesaro JM, Merrell MA, Li L et al (2008) Toll-like receptor 9 mediates CpG oligonucleotide-induced cellular invasion. Mol Cancer Res 6:1534–1543PubMedCrossRefGoogle Scholar
  11. 11.
    Ilvesaro JM, Merrell MA, Swain TM et al (2007) Toll like receptor-9 agonists stimulate prostate cancer invasion in vitro. Prostate 67:774–781PubMedCrossRefGoogle Scholar
  12. 12.
    Mempel M, Voelcker V, Kollisch G et al (2003) Toll-like receptor expression in human keratinocytes: nuclear factor kappaB controlled gene activation by Staphylococcus aureus is toll-like receptor 2 but not toll-like receptor 4 or platelet activating factor receptor dependent. J Invest Dermatol 121:1389–1396PubMedCrossRefGoogle Scholar
  13. 13.
    Droemann D, Albrecht D, Gerdes J et al (2005) Human lung cancer cells express functionally active Toll-like receptor 9. Respir Res 6:1PubMedCrossRefGoogle Scholar
  14. 14.
    Gonzalez-Reyes S, Marin L, Gonzalez L et al (2010) Study of TLR3, TLR4 and TLR9 in breast carcinomas and their association with metastasis. BMC Cancer 10:665–674PubMedCrossRefGoogle Scholar
  15. 15.
    Nurmenniemi S, Kuvaja P, Lehtonen S et al (2010) Toll-like receptor 9 ligands enhance mesenchymal stem cell invasion and expression of matrix metalloprotease-13. Exp Cell Res 316:2676–2682PubMedCrossRefGoogle Scholar
  16. 16.
    McKelvey KJ, Highton J, Hessian PA (2011) Cell-specific expression of TLR9 isoforms in inflammation. J Autoimmun 36(1):76–86PubMedCrossRefGoogle Scholar
  17. 17.
    Du X, Poltorak A, Wei Y, Beutler B (2000) Three novel mammalian toll-like receptors: gene structure, expression, and evolution. Eur Cytokine Netw 11:362–371PubMedGoogle Scholar
  18. 18.
    Chuang TH, Ulevitch RJ (2000) Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw 11:372–378PubMedGoogle Scholar
  19. 19.
    Jukkola-Vuorinen A, Rahko E, Vuopala KS et al (2008) Toll-like receptor-9 expression is inversely correlated with estrogen receptor status in breast cancer. J Innate Immun 1:59–68PubMedCrossRefGoogle Scholar
  20. 20.
    Berger R, Fiegl H, Goebel G et al (2010) Toll-like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors. Cancer Sci 4:1059–1066CrossRefGoogle Scholar
  21. 21.
    Baker AE, Brautigam VM, Watters JJ (2004) Estrogen modulates microglial inflammatory mediator production via interactions with estrogen receptor beta. Endocrinology 145:5021–5032PubMedCrossRefGoogle Scholar
  22. 22.
    Calippe B, Douin-Echinard V, Delpy L et al (2010) 17Beta-estradiol promotes TLR4-triggered proinflammatory mediator production through direct estrogen receptor alpha signaling in macrophages in vivo. J Immunol 185:1169–1176Google Scholar
  23. 23.
    Calippe B, Douin-Echinard V, Laffargue M et al (2008) Chronic estradiol administration in vivo promotes the proinflammatory response of macrophages to TLR4 activation: involvement of the phosphatidylinositol 3-kinase pathway. J Immunol 180:7980–7988PubMedGoogle Scholar
  24. 24.
    Paimela T, Ryhanen T, Mannermaa E et al (2007) The effect of 17beta-estradiol on IL-6 secretion and NF-kappaB DNA-binding activity in human retinal pigment epithelial cells. Immunol Lett 110:139–144PubMedCrossRefGoogle Scholar
  25. 25.
    Fahey JV, Wright JA, Shen L et al (2008) Estradiol selectively regulates innate immune function by polarized human uterine epithelial cells in culture. Mucosal Immunol 1:317–325PubMedCrossRefGoogle Scholar
  26. 26.
    Neve RM, Chin K, Fridlyand J et al (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527PubMedCrossRefGoogle Scholar
  27. 27.
    Merrell M, Suarez-Cuervo C, Harris KW, Vaananen HK, Selander KS (2003) Bisphosphonate induced growth inhibition of breast cancer cells is augmented by p38 inhibition. Breast Cancer Res Treat 81:231–241PubMedCrossRefGoogle Scholar
  28. 28.
    Kallio A, Guo T, Lamminen E et al (2008) Estrogen and the selective estrogen receptor modulator (SERM) protection against cell death in estrogen receptor alpha and beta expressing U2OS cells. Mol Cell Endocrinol 289:38–48PubMedCrossRefGoogle Scholar
  29. 29.
    Ren T, Xu L, Jiao S et al (2009) TLR9 signaling promotes tumor progression of human lung cancer cell in vivo. Pathol Oncol Res 15:623–630PubMedCrossRefGoogle Scholar
  30. 30.
    Qiu J, Wang X, Guo X, Zhao C, Wu X, Zhang Y (2009) Toll-like receptor 9 agonist inhibits ERalpha-mediated transactivation by activating NF-kappaB in breast cancer cell lines. Oncol Rep 22:935–941PubMedCrossRefGoogle Scholar
  31. 31.
    Ando S, De Amicis F, Rago V et al (2002) Breast cancer: from estrogen to androgen receptor. Mol Cell Endocrinol 193:121–128PubMedCrossRefGoogle Scholar
  32. 32.
    Peters AA, Buchanan G, Ricciardelli C et al (2009) Androgen receptor inhibits estrogen receptor-alpha activity and is prognostic in breast cancer. Cancer Res 69:6131–6140PubMedCrossRefGoogle Scholar
  33. 33.
    Yao CL, Kong P, Wang ZY et al (2008) Cloning and expression analysis of two alternative splicing toll-like receptor 9 isoforms A and B in large yellow croaker, Pseudosciaena crocea. Fish Shellfish Immunol 25:648–656PubMedCrossRefGoogle Scholar
  34. 34.
    Franch R, Cardazzo B, Antonello J, Castagnaro M, Patarnello T, Bargelloni L (2006) Full-length sequence and expression analysis of Toll-like receptor 9 in the gilthead seabream (Sparus aurata L.). Gene 378:42–51PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Jouko Sandholm
    • 1
  • Joonas H. Kauppila
    • 2
    • 3
    • 5
  • Christine Pressey
    • 1
  • Johanna Tuomela
    • 1
  • Arja Jukkola-Vuorinen
    • 4
  • Markku Vaarala
    • 3
    • 5
  • Martin R. Johnson
    • 6
  • Kevin W. Harris
    • 1
    • 7
  • Katri S. Selander
    • 1
    • 8
  1. 1.Department of MedicineUniversity of Alabama at BirminghamBirminghamUSA
  2. 2.Department of Anatomy and Cell BiologyUniversity of OuluOuluFinland
  3. 3.Department of SurgeryOulu University HospitalOuluFinland
  4. 4.Department of Oncology and RadiotherapyOulu University HospitalOuluFinland
  5. 5.Department of PathologyOulu University HospitalOuluFinland
  6. 6.Department of Pharmacology and ToxicologyUniversity of Alabama at BirminghamBirminghamUSA
  7. 7.Birmingham Veterans Affairs Medical CenterBirminghamUSA
  8. 8.Department of Medicine, Division of Hematology-OncologyUniversity of Alabama at BirminghamBirminghamUSA

Personalised recommendations