CCL21/CCR7 Axis Inducing EMT To Promote Invasion And Directed Migration Of Human Breast Cancer Cells

Background Breast carcinoma is one of the most common malignant tumors in women, and cancer metastasis is one of the important factors resulting in tumor recurrence after treatment.In recent years, the chemokine receptorCCR7 has been focused on for several years in the field of tumor metastasis research. For example, it has been certified that CCR7 was highly expressed in breast carcinoma, gastric carcinoma, esophageal carcinoma, thyroid papillary carcinoma and so on, and was closely associated with lymph node metastasis. The ligands for CCR7 werecalled secondary lymphoid tissue chemokine 19/21 (CCL19/CCL21), which were expressed specifically in secondary lymphoid tissue including lymph nodes,peyer’s patches,spleen and lymphatic endothelium.Thus, the CCR7 and its ligand CCL19/CCL21 were recognized as specificity forleading tumor cells to migrate to lymph nodes.However, little is known about molecular mechanisms of CCR7 mediating tumor cell migration to lymph nodes.Epithelial-mesenchymal transition(EMT) of cancer cells has been also the research focus for recent years.During EMT process,cancercells were gain the ability to move freely in cell matrix, which were characterized by loss of epithelial phenotype, gain of mesenchymal phenotype and changes in cell morphology and function.Thereby,EMT plays an important role in cancer metastasis. Whether CCR7/CCL21 or CCL19axis could activate EMT during initiating invasion and directed migration of human breast cancer cells has not been reported before thisresearch.Objective Breast cancer was choosn as the research target, to explore whether CCL21/CCR7 axis could induce EMT process and thereby promote invasion and directed migration of human breast cancer cells.Materials and methods1. Immunohistochemical stainings were performed to detect the expressions of the CCR7 and the EMT associated markers including N-cadherin and slug in paraffin sections from 60 cases of female primary breast carcinoma tissues.2. The breast carcinoma cell lines 1428, MCF-7 and MDA-MB-231 were induced by CCL21 for 48h respectively. Then the expression of CCR7 and EMT associated markers including E-cadherin, N-cadherin, slug and Vimentin were detected by Western-blotting and Real-time PCR respectively; Wound healing assay and Matrigel invasion assay were also employed to investigate the role of CCL21/CCR7 signal in breast cancer cell invading and migrating.3. To interfere CCR7 gene expression, the breast cancer cells(MCF-7^ MDA-MB-231) were transfected with CCR7 shRNA (17428,17432)or control shRNA plasmid (NC-shRNA) using Lipofectamine 2000. Then, each cell line wasstimulated by CCL21, followed by Western-blotting to detect expressions of CCR7 and EMT associated markers,by wound healing assay and matrigel invasion assayto investigate the migration ability of breast cancer cell.4. Statistical analysis was carried out on above experimental results using software SPSS17.0.Results1. CCR7, N-cadherin and Slug were highly expressed in 60%(36/60), 76.67%(46/60) and65%(40/60) respectively in 60 cases of female primary breast carcinoma tissue, and significantly associated with lymph node metastases as well as with clinical pathological stage. Furthermore, the expression of CCR7 was significantly correlated to that of N-cadherin and Slug.2. In vitro, afterstimulating the cell lines with CCL21,the levels of both proteins and mRNAs for the E-cadherin was down-regulated,whilethe CCR7, N-cadherin, slug and Vimentin were up-regulated in each cell line.Also, the number of both tumor cell migration and invasionwereincreased after treatedwith CCL21.3. After knocking down the CCR7 gene of cell lines, followed by stimulating the cell lines with CCL21,the levels of both protein and mRNA expressions of E-cadherin, N-cadherin, Slug, Vimentinwere not changed in the presence of CCL21. Also, the cancer cell migration and invasion induced by CCL21 were significantly abolished in the condition of CCR7 knockdown.Conclusion CCR7, N-cadherin and Slug were abundantly expressed in breast carcinoma, and were closely related to the lymph node metastasis; CCL21/CCR7 axis could activate EMT process to promote invasion anddirected migration of breast carcinoma cells.