Abstract
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 125I-anti-TLR5 mAb or isotype 125I-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.
Highlights
Membrane-bound toll-like receptors (TLRs) play key roles in innate and adaptive immunity
Detected by western blot and quantitative PCR (qPCR), we found knockdown of toll-like receptor 5 (TLR5) resulted in decreased expression of TLR5 and E-cadherin and increased expression of N-cadherin, vimentin, fibronectin, TRAF6, SOX2, and Twist[1], which were related to epithelial–mesenchymal transition (EMT)
We quantified the protein and mRNA expression of TLR5, E-cadherin, N-cadherin, vimentin, fibronectin, TRAF6, SOX2 and the transcription factor, Twist[1], by western blot and qPCR to verify the effects of TLR5 on EMT (Fig. 1a and b, respectively)
Summary
Membrane-bound toll-like receptors (TLRs) play key roles in innate and adaptive immunity. They are primarily expressed in some types of immunocytes and recognize conserved pathogen-associated molecular patterns (PAMPs)[1,2,3,4]. Breast cancer is the most common cancer among women, and it is associated with high mortality rates[15,16,17]. It can be classified into TNBC (triple-negative breast cancer) and non-TNBC subtypes. The TNBC type accounts for 12–24% of all breast cancers and is more aggressive and prone to metastasis than the non-TNBC type.
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