Abstract
PurposeActivation of Toll like receptors (TLRs) signaling has been implicated in promoting malignant cell invasion and metastatic potential. Previously we demonstrated that increased TLR-9 expression predicted poor survival in oral cancer patients. The objective of this study is to further investigate the roles and potential molecular mechanisms of TLR-9 signaling in human oral cancer cell invasion.MethodsCell migration, invasion and protein expression were detected by wound healing assay, Transwell chambers model and western blot. The secretion and activity levels of metalloproteinases-2/9 were quantified by ELISA and Gelatin zymography. EMSA and ChIP assays were employed to detect the activity of AP-1signal pathway. TLR-9 siRNA transfection was used to regulate the expression and activity of TLR-9 in oral cancer cell line HB cells.ResultThe results of both wound healing assay and in vitro Transwell assay revealed that activation of TLR-9 induced dose- and time- dependent migration and invasion of HB cells. An increased expression, secretion and activity of MMP-2 were observed upon the treatment of CpG-ODN. The TLR-9 signaling-mediated MMP-2 expression appeared to be a consequence of AP-1 activation, because that their DNA binding activity was enhanced by CpG-ODN treatment. All these influences were efficiently repressed by the knockdown of TLR-9 through siRNA or pretreatment of an AP-1 inhibitor.ConclusionActivation of TLR-9 signaling could promote human oral cancer HB cells invasion with the induction of MMP-2 presentation by attenuating AP-1 binding activity, suggesting a novel anti-metastatic application for TLR-9 targeted therapy in oral cancer in the future.
Highlights
Oral Squamous Cell Carcinoma (OSCC) is the most common malignancy in the head and neck region, presenting approximately 389,000 new cases yearly [1,2]
The purpose of this study is to investigate the influence of TLR9 signaling on in-vitro invasiveness of HB cells, a human oral cancer cell line; with special emphasis on synthesis and secretion of matrix metalloproteinases (MMPs)-2 and MMP-9
Scratch wound migration assay shown 0.8 mM CpG-ODN effectively promoted the migration of HB cells into the wounded area in 24 h, which was determined as movement of cells into the wound (Fig. 1A and C)
Summary
Oral Squamous Cell Carcinoma (OSCC) is the most common malignancy in the head and neck region, presenting approximately 389,000 new cases yearly [1,2]. TLR-2 and TLR-9 signaling were found to promote tumor cell migration in breast cancer[9,10], TLR-4 and TLR-9 signaling were demonstrated to accelerate cell invasion in prostate cancer[11,12], and activation of TLR-2 and TLR-4 signaling were shown to stimulate tumor cell movement in colon cancer[13,14]. These findings suggest that investigation of the relationship between TLRs signaling and tumor invasion may shed new light for effective prevention of cancer metastatic spread
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