Role And Mechanism For Sox-3 In Lymph Node Metastasis Of Esophageal Squamous Cell Carcinoma

Background and ObjectivesEsophageal cancer(EC)is the eighth most common malignancy in the world and ranks sixth among all cancers in mortality.despite the development of multiple disciplinary treatment during the past 40 years,esophageal cancer 5-year overall survival(OS)have improved only slightly and still remain low at 19 percent.The two most common histological types of esophageal carcinoma include esophageal squamous cell carcinoma(ESCC)and esophageal adenocarcinoma(EAC).In China,esophageal cancer is the third most frequently diagnosed cancer and the fourth leading cause of cancer death,with ESCC as the predominant histological subtype and accounts for nearly 95% of all EC.Compared with EAC,ESCC has a even poorer prognosis with 10-year overall survival of about 20% postoperatively.Lymph node status is the single most important prognostic factor in ESCC,with an increasing number of metastatic lymph nodes being associated with a progressively poor prognosis.But the mechanisms that control lymph node metastasis(LNM)are unclear,and to date,efforts to identify effective methods for prediction and treatment of lymph node metastasis in ESCC have proven to be primarily unsuccessful.Sox-3 belongs to the Sox B1 subgroup of transcriptional activators.Sox-3 is highly expressed and has important roles in maintaining the broad developmental potential and identity of neural stem cells in the vertebrate embryo,with expression decreases as development proceeds.Recently Sox-3 has been suggested to be one of the proto-oncogenes and involved in tumorigenesis.During previously clinical study,we found that Sox-3 expression was significantly upregulated in ESCC,and positively correlated with lymph node metastasis and patient survival,suggesting that Sox-3 overexpression in ESCC plays a crucial role in lymph node metastasis.It has been proven that Increased lymphatic vessel density(LVD)in and around tumors is associated with lymph node metastasis formation and poor prognosis in ESCC.The most extensively studied molecular system that signals for tumor lymphangiogenesis and is associated with lymphatic spread from primary cancers is the VEGF-C/VEGF-D/VEGFR-3 signaling axis.High expressions of VEGF-C and/or VEGF-D were strongly associated with disease stage,lymphatic vessel density,lymphatic invasion and regional lymph node metastasis in patients with ESCC.Taken together,Sox-3 expression in ESCC was hypothesized to promote lymph node metastasis and associated with VEGF-C/D-VEGFR3 signaling axis.To investigate the role and mechanism for Sox-3 in lymph node metastasis of ESCC,the human ESCC cell line with different Sox-3 expression were used to explore the role and mechanism for Sox-3 in lymph node metastasis of esophageal squamous cell carcinoma by Western blot,qRT-PCR,ELISA,IHC,Tube formation,Transwell cell invasion,Wound healing assays in vitro and in vivo.This study provided a new direction for the diagnosis and treatment of lymph node metastasis in ESCC.Methods1.Western blot and qRT-PCR were used to determine the significant difference of Sox-3 expression between ESCC cell models that have been built through recombinant lentivirus Sox-3 siRNA interference.The role of Sox-3 in proliferation,apoptosis,invasion,migration and lymphangiogenesis was examined by MTT cell proliferation experiment,flow cytometry,Transwell invasion assays,wound healing assays and human lymphatic endothelial cell tube formation assay on ESCC.2.TE-10 cells and Sox-3 siRNA TE-10 cells were inoculated into the right axillary subcutaneous tissue to built nude mouse transplantation tumor model.Volumes of primary tumor and ipsilateral axillary lymph nodes together with histopathology examination were measured to explore the Influence of high Sox-3 expression on tumorigenesis and lymph node metastasis in ESCC,and the correlation between Sox-3 and VEGF-C/D expression was detected through Western blot and immunohistochemistry.3.The influence of Sox-3 expression on the expression and secretion of VEGF-C/D was examined by Western blot and ELISA in vitro.Tube formation assay was performed with lymphatic endothelial cell which VEGFR-3 was blocked by it’s antibody(SAR131675)to explore the role and mechanism for VEGF-C and VEGF-D in lymph node metastasis of ESCC promoted by Sox-3.Result1.The significant difference of Sox-3 expression between ESCC cell models was identified by Western blot and qRT-PCR with Sox-3 expression on TE-10 cell linessign if icantly inhibited by recombinant lentivirus Sox-3 siRNA interference.Through MTT cell proliferation experiment,flowcytometry,Transwell invasionassays,wound healing assays and human lymphatic endothelial cell tube formation assay,we found that high expression of Sox-3 on ESCC could significantly promote cell proliferation,invasion,migration ability and lymphangiogenesis,and inhibit cell apoptosis.2.Nude mouse transplantation tumor model was built by inoculating the right axillary subcutaneous tissue with TE-10 cells and Sox-3 siRNA TE-10 cells.No significant difference of growth rate and the final tumor volume of inoculated TE-10 cell with different Sox-3 expression.But the volumes of the ipsilateral axillary lymph nodes and the ratios of metastatic to total dissected ipsilateral axillary lymph nodes were statistically increased with higher Sox-3 expression.Through Western blot and immunohistochemistry,the positive correlation between Sox-3 and VEGF-C/D expression was identified with both VEGF-C and VEGF-D expression upregulated in transplantation tumor with higher Sox-3 expression.Suggested that high Sox-3 expression in ESCC may promote lymph node metastasis associated with VEGF-C/VEGF-D/VEGFR-3.3.Compared with higher Sox-3 expression,the expressions as well as the secretion of VEGF-C and VEGF-D were both downregulated on interfered TE-10 cells with lower Sox-3 expression detected by Western blot and ELISA in vitro.Tube formation assay revealed that after the function of VEGFR-3 on lymphatic endothelial cell was blocked by SAR131675,the tube formation ability of both higher and lower Sox-3 expression TE-10 cells were significantly reduced with no significant difference between them.Suggesting that high sox3 expression can up-regulate the expression of VEGF-C and VEGF-D in ESCC cells,and VEGF-C/D was required in Sox3’s promotion to lymphangiogenesis.Conclusions1.High expression of Sox-3 on ESCC could significantly promote cell proliferation,invasion and migration ability,and inhibit cell apoptosis in vitro,but the influence of Sox-3 expression on tumor growth rate and the final tumor volume was not found in vivo.2.Sox-3 overexpression in ESCC could promote lymphangiogenesis in vitro and lymph node metastasis in vivo.3.The mechanisms underlying lymphatic metastasis induced by Sox-3 in ESCC probably was the lymphangiogenesis via upregulation of VEGF-C and VEGF-D.