TLR5: A prognostic and monitoring indicator for triple-negative breast cancer.

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.