Key Laboratory for Experimental Teratology of the Ministry of Education and Biomedical Isotope Research Center, School of Basic Medical Sciences, Shandong University, Jinan, Shandong, China.
Cell Death Dis. 2019 Dec 18;10(12):954. doi: 10.1038/s41419-019-2187-8.
A novel, highly selective biomarker is urgently needed to predict and monitor triple-negative breast cancer (TNBC) because targeting molecules are not currently available. Although associated with various malignant tumors, the role of toll-like receptor 5 (TLR5) in TNBC remains uncertain. We aimed to define the effects of TLR5 in TNBC to determine whether it could serve as a prognostic and monitoring indicator for TNBC. We established TNBC cell line 4T1 with low TLR5 expression (GFP tag; TLR5 4T1) and with normal TLR5 expression (GFP tag; TLR5 4T1) using lentivirus-shRNA-TLR5 knockdown transfection and negative lentivirus transfection, respectively. Detected by western blot and qPCR, we found knockdown of TLR5 resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist1, which were related to EMT (epithelial-mesenchymal transition). In addition, downregulation of TLR5 increased the invasion and migration of 4T1 cells in vitro, which were investigated by CCK-8 and wound healing, as well as transwell assay and colony formation. Furthermore, the metastatic ability of TLR5 4T1 cells to the lungs was also increased compared to TLR5 4T1 cells in vivo. To verify the effect of TLR5 as a monitor indicator, mice bearing TLR5 and TLR5 4T1 tumors injected with I-anti-TLR5 mAb or isotype I-IgG were assessed by whole body phosphor-autoradiography and fluorescence imaging in vivo. Phosphor-autoradiography of model mice revealed early tumors at 6 days after inoculation with TLR5 4T1, but not TLR5 4T1 cells. Intratumoral accumulation of radioactivity positively correlated with TLR5 expression, and fluorescence imaging in vivo revealed both TLR5 and TLR5 4T1 tumors. Our results suggested that downregulation of TLR5 in TNBC increased tumor invasiveness and EMT expression via TRAF6 and SOX2 pathway and TLR5 could serve as a prognostic and monitoring indicator for TLR5-positive tumors.
一种新型的、高度选择性的生物标志物迫切需要被开发出来，以用于预测和监测三阴性乳腺癌（TNBC），因为目前还没有针对该疾病的靶向分子。尽管 Toll 样受体 5（TLR5）与多种恶性肿瘤相关，但它在 TNBC 中的作用仍不确定。我们旨在明确 TLR5 在 TNBC 中的作用，以确定其是否可以作为 TNBC 的预后和监测指标。我们通过慢病毒-shRNA-TLR5 敲低转染和阴性慢病毒转染，分别建立了 TLR5 低表达（GFP 标签；TLR5 4T1）和 TLR5 正常表达（GFP 标签；TLR5 4T1）的 TNBC 细胞系 4T1。通过 Western blot 和 qPCR 检测，我们发现 TLR5 敲低导致 TLR5 和 E-钙黏蛋白表达降低，而 N-钙黏蛋白、波形蛋白、纤连蛋白、TRAF6、SOX2 和 Twist1 的表达增加，这些蛋白与 EMT（上皮-间充质转化）有关。此外，下调 TLR5 增加了 4T1 细胞在体外的侵袭和迁移能力，这通过 CCK-8 和划痕愈合实验、Transwell 实验和集落形成实验进行了研究。此外，与 TLR5 4T1 细胞相比，TLR5 4T1 细胞在体内向肺部转移的能力也增加了。为了验证 TLR5 作为监测指标的作用，我们将携带 TLR5 和 TLR5 4T1 肿瘤的小鼠分别注射 I-anti-TLR5 mAb 或同型 IgG，并通过全身磷自动放射性和荧光成像进行评估。模型小鼠的磷自动放射性显示，在接种 TLR5 4T1 后 6 天即可检测到早期肿瘤，但接种 TLR5 4T1 细胞后则不能。肿瘤内放射性物质的积累与 TLR5 的表达呈正相关，而体内荧光成像则显示 TLR5 和 TLR5 4T1 肿瘤均有表达。我们的研究结果表明，下调 TNBC 中的 TLR5 通过 TRAF6 和 SOX2 通路增加了肿瘤的侵袭性和 EMT 表达，TLR5 可作为 TLR5 阳性肿瘤的预后和监测指标。
