Department of Gynecology and Obstetrics, Medical University Innsbruck, Innsbruck, Austria.
Cancer Sci. 2010 Apr;101(4):1059-66. doi: 10.1111/j.1349-7006.2010.01491.x. Epub 2010 Jan 12.
Toll-like receptor 9 (TLR9) activates the innate immune response when exposed to non-methylated CpG-DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age-matched tumor-free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen-receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone-receptor (HR)-negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG-DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor-kappa B (NF-kappaB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT-20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG-DNA with TLR9, which diminished when the cells were exposed to methylated CpG-DNA (mCpG-DNA) or control GpC-DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines.
Toll 样受体 9（TLR9）在暴露于非甲基化 CpG-DNA 时激活先天免疫反应。最近发现 TLR9 表达于先前表现为全基因组低甲基化的癌细胞中。我们着手研究 TLR9 在乳腺癌和卵巢癌细胞中的表达和分子活性。首先，我们证实了转移性乳腺癌患者（n = 18）血清中低甲基化 DNA 水平高于年龄匹配的无肿瘤女性（n = 18）。在乳腺癌细胞系和组织中，TLR9 mRNA 表达与雌激素受体（ER）状态相关（n = 124，P = 0.005）。表达也与乳腺癌（n = 138，P = 0.04）和卵巢癌标本的肿瘤分级增加相关。福尔马林固定石蜡包埋（FFPE）乳腺癌组织的免疫组织化学分析显示，激素受体（HR）阴性标本中 TLR9 蛋白表达更高（n = 116，P < 0.001）。通过体外划痕试验，我们观察到过表达 TLR9 的细胞系在受到 CpG-DNA 刺激时表现出更高的细胞迁移率。在评估乳腺癌中 TLR9 的分子活性时，我们发现核因子-kappa B（NF-kappaB）活性与 TLR9 mRNA 表达呈强正相关（相关系数 r = 0.7，P < 0.001）。最后，BT-20 和 Hs578T 乳腺癌细胞系的免疫荧光分析显示 CpG-DNA 与 TLR9 部分共定位，当细胞暴露于甲基化 CpG-DNA（mCpG-DNA）或对照 GpC-DNA 时共定位减少。总之，我们证明 TLR9 表达与乳腺癌和卵巢癌标本的分化不良相关，并且 TLR9 过表达和低甲基化 DNA 刺激增强了癌细胞系的迁移能力。
