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

Toll-like receptor 9 (TLR9) is a cellular DNAreceptor, 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 ERa 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 qRTPCR, 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 ERa 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 MDAMB- 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 ERa, which also inhibited NF-jB activation by TLR9-ligands. In conclusion, expression of TLR9 isoforms A and B can be detected in clinical breast cancer specimens. The ERa 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.