Qiu Jianming, Wang Xiaojian, Guo Xinhai, Zhao Chenyin, Wu Xuhui, Zhang Yongkui
Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, P.R. China.
Oncol Rep. 2009 Oct;22(4):935-41. doi: 10.3892/or_00000520.
Primarily, Toll-like receptor 9 (TLR9) is a specific receptor for microbial DNA in human immune cells. TLR9 has been found to be a promising target in tumor immunotherapy but the direct effect of its activation on tumor cells remains unknown. In this study, we examined the effect of TLR9 ligation on estrogen receptor alpha (ERalpha)-mediated transactivation of breast cancer. Luciferase report gene assays, RNA interference of TLR9 and Chromatin immunoprecipitation were performed to measure the effect of TLR9 ligation on ERalpha-mediated transactivity of T47D and MCF-7 cells. Bromodeoxyuridine incorporation assay was used to examine the effect of TLR9 ligation on estrogen (E2)-induced proliferation of breast cancer cells. We also investigated the mechanism for the effect of TLR9 ligation on ERalpha-mediated transactivity. We found that ERalpha-mediated transcription via estrogen response element of human breast cancer cells line T47D was significantly suppressed when treated with 17beta-estradiol in combination with TLR9 agonist CpG oligonucleotides and this effect of CpG was dependent on TLR9. Furthermore, nuclear factor kappaB (NF-kappaB) inhibitor BAY 11-7082 could abolish the inhibitory effect of CpG oligonucleotides on ERalpha-mediated transactivation. We also confirmed the effect of CpG oligonucleotides on ERalpha-mediated transactivation in the breast cancer cell line MCF-7 forced to stably overexpress TLR9. Finally, we observed that CpG oligonucleotides were also able to inhibit estrogen-induced proliferation of breast cancer cells as a consequence of decreased ERalpha-mediated transactivation. Taken together, our data suggest that TLR9 signal pathway, by activating NF-kappaB, negatively regulates ERalpha-mediated transactivation of breast cancer. Thus, TLR9 agonist inhibits the proliferation of breast cancer cells in response to estrogen.
主要地，Toll样受体9（TLR9）是人类免疫细胞中微生物DNA的特异性受体。TLR9已被发现是肿瘤免疫治疗中有前景的靶点，但其激活对肿瘤细胞的直接作用仍不清楚。在本研究中，我们检测了TLR9连接对雌激素受体α（ERα）介导的乳腺癌反式激活的影响。进行荧光素酶报告基因检测、TLR9的RNA干扰和染色质免疫沉淀，以测量TLR9连接对T47D和MCF-7细胞中ERα介导的反式活性的影响。使用溴脱氧尿苷掺入试验检测TLR9连接对雌激素（E2）诱导的乳腺癌细胞增殖的影响。我们还研究了TLR9连接对ERα介导的反式活性产生影响的机制。我们发现，当用17β-雌二醇联合TLR9激动剂CpG寡核苷酸处理时，人乳腺癌细胞系T47D中通过雌激素反应元件的ERα介导的转录显著受到抑制，且CpG的这种作用依赖于TLR9。此外，核因子κB（NF-κB）抑制剂BAY 11-7082可消除CpG寡核苷酸对ERα介导的反式激活的抑制作用。我们还证实了CpG寡核苷酸对稳定过表达TLR9的乳腺癌细胞系MCF-7中ERα介导的反式激活的作用。最后，我们观察到由于ERα介导的反式激活减少，CpG寡核苷酸也能够抑制雌激素诱导的乳腺癌细胞增殖。综上所述，我们的数据表明，TLR9信号通路通过激活NF-κB，负向调节ERα介导的乳腺癌反式激活。因此，TLR9激动剂抑制乳腺癌细胞对雌激素的增殖反应。
