Toll-like Receptor4Promotes Human Breast Cancer Cells Invasiveness Via LPS Stimulation And Expresses Increasingly Related To Tumor Lymph Node Metastasis From N0to N3in Patients

Objective: Toll-like receptor4(TLR4)-mediated signaling has been implicated in thetumor cell invasion, survival and metastasis in many kinds of cancers. This studyinvestigated the expression and biological role of TLR4in human breast cancerproliferation and metastasis, and proposed its role as a potential target for the therapy ofbreast cancer.Methods: MCF-7and MDA-MB-231are the low and high metastatic human breastcancer cell lines respectively. In this study, LPS was to stimulate human breast cancer cellMCF-7and MDA-MB-231in vitro and the expression of TLR4on the mRNA and proteinlevels was analyzed by RT-PCR, Real-time PCR, FCS and western blotting. After TLR4was activated by LPS, the expression of several genes associated with the cancermetastasis such as MMP-2, MMP-9and VEGF-C, was measured by RT-PCR andReal-time PCR on the mRNA levels; Mean while, their expression on the protein level inthe supernatants of both cells was detected by ELISA. MyD88, which is a very importantprotein in the downstream of TLR4signaling pathway, was analyzed by western blotting;The cytokines in the supernatants of both cells were measured by CBA. In vivo, the murinemetastatic breast tumor model was established by injecting MCF-7and MDA-MB-231celllines into the female BALB/c; The tumor size and weight were observed and weighedrespectively; The expression of TLR4in the tumor tissue was detected byimmunohistochemistry; In addition, the long distance metastasis of transplant tumor wereobserved by Anther indicators. The tumor samples and tumor adjacent tissues of breastcancer patients were obtained, and the expression of TLR4in the above samples wasmeasured by RT-PCR, Real-time PCR and immunohistochemistry.Results: In vitro, using LPS to stimulate human breast cancer cell MCF-7andMDA-MB-231, the expression of TLR4on the mRNA and protein levels increased obvioulsy. However, the proliferation of the both breast cancer cell lines had no significantchanges compared to the controls. In addition, the activation of TLR4notablely promotedthe expression of MMP-2, MMP-9and VEGF-C on the mRNA levels and the secretion insupernatants of both cells. LPS promoted the invasion of MDA-MB-231and MCF-7cellsby transwell assay and by wound healing assay respectively. Moreover, the stimulationwith LPS significantly increased the expression of MyD88, a protein in the downstreamsignaling pathway of TLR4, and caused the secrection of IL-6and IL-10in the humanbreast cancer cells. In vivo, the activation of TLR4with LPS promoted tumorigenesis andformed metastatic lesions in the live in nude mice. Tumor biomarkers CA15-3and CA19-9in mice blood of the experimental group was significantly higher than control group.Combined with the analysis of clinicopathological parameters, the expression of TLR4inhuman breast cancer tissues was higher than normal breast tissues and this is correlatedwith lymph node metastasis from N0to N3in patients.Conclusions: This study indicated that TLR4may participate in the progression andmetastasis of human breast cancer and provide a new therapeutic target against metastasisof breast cancer.