Abstract
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Purpose: A novel highly selective biomarker is needed for the diagnosis and treatment of triple-negative breast cancer (TNBC), for which no targeting molecules are currently available. Toll-like receptor 5 (TLR5) was reported to be associated with a variety of malignant tumors; however, the role of TLR5 in TNBC remains poorly understood. This study aimed to investigate the effect of TLR5 in TNBC to determine whether TLR5 could be used as a target for early noninvasive TNBC diagnosis. Methods: A 4T1 cell line with low TLR5 expression was established with lentivirus-shRNA-TLR5 knockdown transfection (GFP tag, named TLR5- 4T1); TLR5 and epithelial-mesenchymal transition (EMT)-associated protein and mRNA expression were detected via western blot and qPCR. CCK-8, wound healing, and transwell and colony formation assays were performed to investigate whether lower TLR5 expression affected the invasion and migration of 4T1 cells. A lung metastasis experiment was performed to study the metastatic abilities in vivo. Finally, TLR5+/- 4T1-bearing mice were injected with 125I-anti-TLR5 mAb and isotype 125I-IgG and whole body phosphor-autoradiography and fluorescence imaging were performed in vivo.
Results: TLR5 knockdown resulted in lower expression of TLR5 and E-cadherin and higher expression of N-cadherin, vimentin, fibronectin, and the transcription factor, Twist1. Higher proliferation and stronger invasion were detected in TLR5- 4T1 cells in vitro. Phosphor-autoradiography of model mice showed TLR5+ 4T1 tumors early on day 6 after tumor cell inoculation, whereas no image was obtained for TLR5- 4T1 at the same time. Further, we found that radioactivity in the tumors was positively correlated with TLR5 expression and that in vivo fluorescence imaging successfully showed both TLR5+ and TLR5- 4T1 tumor tissues. Interestingly, we found that TLR5 knockdown resulted in early lung tumor metastasis. Lung tumor was more clearly visible in TLR5- 4T1 mice than in TLR5+ 4T1 mice.Conclusion: Downregulation of TLR5 in TNBC increased tumor invasiveness and EMT. Our data provides a novel target molecule for early TLR5+ tumor diagnosis.
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