Ilvesaro Joanna M, Merrell Melinda A, Li Li, Wakchoure Savita, Graves David, Brooks Sonja, Rahko Eeva, Jukkola-Vuorinen Arja, Vuopala Katri S, Harris Kevin W, Selander Katri S
Division of Hematology-Oncology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294-3300, USA.
Mol Cancer Res. 2008 Oct;6(10):1534-43. doi: 10.1158/1541-7786.MCR-07-2005.
Toll-like receptor 9 (TLR9) belongs to the innate immune system and recognizes microbial and vertebrate DNA. We showed previously that treatment with the TLR9-agonistic ODN M362 (a CpG sequence containing oligonucleotide) induces matrix metalloproteinase-13-mediated invasion in TLR9-expressing human cancer cell lines. Here, we further characterized the role of the TLR9 pathway in this process. We show that CpG oligonucleotides induce invasion in macrophages from wild-type C57/B6 and MyD88 knockout mice and in human MDA-MB-231 breast cancer cells lacking MyD88 expression. This effect was significantly inhibited in macrophages from TLR9 knockout mice and in human MDA-MB-231 breast cancer cells stably expressing TLR9 small interfering RNA or dominant-negative tumor necrosis factor receptor-associated factor 6 (TRAF6). Sequence modifications to the CpG oligonucleotides that targeted the stem loop and other secondary structures were shown to influence the invasion-inducing effect in MDA-MB-231 cells. In contrast, methylation of the cytosine residues of the parent CpG oligonucleotide did not affect the TLR9-mediated invasion compared with the unmethylated parent CpG oligonucleotide. Finally, expression of TLR9 was studied in clinical breast cancer samples and normal breast epithelium with immunohistochemistry. TLR9 staining localized in epithelial cells in both cancer and normal samples. The mean TLR9 staining intensity was significantly increased in the breast cancer cells compared with normal breast epithelial cells. In conclusion, our results suggest that TLR9 expression is increased in breast cancer and CpG oligonucleotide-induced cellular invasion is mediated via TLR9 and TRAF6, independent of MyD88. Further, our findings suggest that the structure and/or stability of DNA may influence the induction of TLR9-mediated invasion in breast cancer.
Toll样受体9（TLR9）属于先天性免疫系统，可识别微生物和脊椎动物的DNA。我们之前的研究表明，用TLR9激动剂ODN M362（一种含CpG序列的寡核苷酸）处理可诱导表达TLR9的人癌细胞系中基质金属蛋白酶-13介导的侵袭。在此，我们进一步阐述了TLR9通路在此过程中的作用。我们发现，CpG寡核苷酸可诱导野生型C57/B6和MyD88基因敲除小鼠的巨噬细胞以及缺乏MyD88表达的人MDA-MB-231乳腺癌细胞发生侵袭。在TLR9基因敲除小鼠的巨噬细胞以及稳定表达TLR9小干扰RNA或显性负性肿瘤坏死因子受体相关因子6（TRAF6）的人MDA-MB-231乳腺癌细胞中，这种效应受到显著抑制。针对茎环和其他二级结构对CpG寡核苷酸进行的序列修饰显示会影响MDA-MB-231细胞中的侵袭诱导效应。相比之下，与未甲基化的亲本CpG寡核苷酸相比，亲本CpG寡核苷酸中胞嘧啶残基的甲基化并不影响TLR9介导的侵袭。最后，我们用免疫组织化学方法研究了临床乳腺癌样本和正常乳腺上皮中TLR9的表达情况。TLR9染色定位于癌症样本和正常样本的上皮细胞中。与正常乳腺上皮细胞相比，乳腺癌细胞中TLR9的平均染色强度显著增加。总之，我们的结果表明，乳腺癌中TLR9表达增加，且CpG寡核苷酸诱导的细胞侵袭是通过TLR9和TRAF6介导的，与MyD88无关。此外，我们的研究结果表明，DNA的结构和/或稳定性可能会影响乳腺癌中TLR9介导的侵袭的诱导。
