| Esophageal cancer is the eighth most common cancer.Esophageal Squmous cell carcinoma(ESCC) accounts for＞90%of the esophageal carcinoma in Asian countries. The 5-year survival rate for ESCC patients is as low as about 10%-30%for many years.Invasion and metastasis have still been the obstacle to successful ESCC treatment.The mechanisms underlying invasion and metastasis remain poorly understood due to the lack of adequate metastatic models for more comprehensive biological investigation.In the present study,we developed an in vivo spontaneous metastasis model of ESCC.By initially injecting nude mice subcutaneously(s.c) with human ESCC EC9706 cells,isolating the human tumor cells from the resultant mouse lung tumors and then growing explants of these tumors in culture,two derivative human sublines with different metastatic phenotypes,designated MLuB1 and MLuC1,were sequentially obtained.The incidence of mice with lung metastasis produced by MLuC1 was higher than that of MLuB1(87%for MLuC1 and 22%for MLuB1). Further selection of MLuC1 cells yielded cell lines(MLuC21,MLuC22,MLuC23 and MLuC24) with a short latency period.The third-round selection sublines MLuC333 and MLuC314(from lymph node metastases) were then obtained.The gene expression profiles of MLuB1 and MLuC1 sublines were compared with cDNA arrays containing 5,000 known genes.Forty-serven genes were differentially expressed≥2.0 - fold,in which 19 genes were overexpressed and 28 underexpressed in MLuC1 cells.Changes in RNA expression were confirmed by semi-quantitative RT-PCR analysis.Some of these genes were related to tumor progression,such as high mobility group AT-hook 2(HMGA2),carbonic anhydraseⅫ(CA12),cathepsin S(Cath) and Laminin-5(Ln-5)γ2,etc.The findings of these differentially expressed genes will contribute to further studies on the mechanisms underlying the invasion and metastasis of ESCC.Ln-5γ2 was one of the up-regulated genes in MLuC1 cells.An increasing number of reports have demonstrated preferential distribution ofγ2 chain at the invasive front of tumors and associated with clinical behavior of cancers including ESCC.This may have important pathophysiological consequences,as proteolytically processed forms ofγ2 thereof are known to promote migration of a multitude of epithelial cells in vitro. The pro-motility function ofγ2 by proteolytic processing indicates it may contribute to the elevated cell migration under tumor cell invasion.MT1-MMP was the main enzyme responsible for Ln-5γ2 cleavage.The relationship between Ln-5γ2 and MT1-MMP in ESCC is unclear.Western-blotting analysis reavealed that degradated fragments of Ln-5γ2 and active form of MT1-MMP in MLuC1 was significantly higher than those in MLuB1.Immunohistochemistry was performed on 121 formalin-fixed,paraffin-embedded specimens of ESCC.Expression of Ln-5γ2 and MT1-MMP was observed in 61.9%(75/121) and 71.9%(87/121) tumors,respectively. Sixty eases(49.5%) presented co-expression of Ln-5γ2 and MT1-MMP. Co-expresson of these proteins was significantly associated with the depth of invasion. Moreover,proteolytic co-activation of Ln-5γ2 and MT1-MMP were frequently found in tumor tissues,whereas in the correponding normal esophageal tissues,there are only intact forms ofγ2 and MT1-MMP.siRNA-mediated silencing of MT1-MMP significantly reduced production ofγ2' andγ2x in MLuC1 cells and in turn inhibited cell migration.The results indicate that MT1-MMP is probably an enzyme responsible for Ln-5γ2 cleavage in ESCC and that interaction between MT1-MMP and Ln-5γ2 may play a critical role in promoting invasion and metastasis of human ESCC. |
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