Studies On Invasion And Metastasis Associated Genes In Esophageal Carcinoma

Esophageal cancer is one of the ten most common malignancy, although we have developed and made significant progress in the treatment and diagnosis of tumor, it still threatened the health of human. Esophageal cancer is characterized by rapid clinical progression and poor prognosis due to adjacent tissue invasion and distant organs metastasis at a very early stage. Many researches have suggested that tumor microenvironment and interstitial cells play an important role in mediating invasion and metastasis. These malignant tumors always consume a large quantity of oxygen, contain acidosis,growth factor,proteases or proteolytic enzymes and the inflammatory cells. The special microinviroment not only influence the prolifation,invasion,migration,adhesion and angiogenesis of cancer cells but also have profound influence on interstitial cells related with tumor. Recent studies show that metastasis of tumor were determined by the coordination and cooperation between cancer cells and interstitial cells. Interstitial cells participate the whole process of metastasis.Our research aimed at realizing the relationship between the metastasis and microvascular endothelial cells of esophageal cancer. Microvascular endothelial cells(ECEC) were separated by MACS system from fresh samples. We found that ECEC can promote the prolifation and have anti-apoptotic ability similar with cancer cells. Analysis of gene expression pattern in ECEC by microarray, there are a lot of genes specially high expressed in ECEC such as chemotatic factor superfamily and some genes related with tumor metastasis. The invasion and migration ability of esophageal cancer cells was significantly improved when these cells co-cultured with ECEC cells (P<0.01) And this improvement depends on the direct contact of tumor cells with ECEC. ECEC can promote the growth,invasion and lung metastasis of xenografts when ECEC and cancer cells were mixed injected into nude mice (P<0.01). Analysis of gene expression pattern in cancer cells when co-cultured with ECEC by microarray, we found that there are so many up-regulated genes related with tumor invasion and metastasis. After function verification of these genes such as AREG,EREG,MMP1,IL6,MMP3,IL1B,ADM and Cyr61, we found that transfection of AREG and EREG can significantly promote the invasion and migration of tumor cells (P<0.01). AREG and EREG gene transfection can also promote the growth of human esophageal cancer cells transplanted subcutaneously into nude mice in vivo (P<0.01). Compared with control, mice of AREG and EREG group showed shorten survival time (P<0.01). In short, ECEC can ptomote the invasion and metastasis of tumor cells in vitro and in vivo. Additionally, ECEC play a critical role in invasion promotion by regulating gene expression of the co-cultured tumor cells. Meanwhile, we put forwards an incisive analysis of the gene EREG. First, we found there was high relationship whit expression of EREG and invasion ability of esophageal cancer cell lines. Immunofluorescence revealed that EREG was over-expressed in esophageal cancer cell lines with high-invasive potential. Immunohistochemical analysis showed EREG was over-expressed in esophageal cancer (P<0.01). The expression of EREG was closely related with local invasion (T) and lymph node metastasis (N). The patients with EREG high expression always show shorten survival time compared with EREG low or negative patients. The over-expressed EREG is a valuable prognostic factor independent of lymph node metastasis or local invasion. The further analysis on the function and molecular mechanisms of EREG indicated that EREG significantly induced changes in cell morphology. EREG high-expressed cells often with a spindle shape and growing loosely. Neutralizing antibody of EREG further confirmed its invasion promotion ability. By immunofluorescence analysis of F-actin and total actin, there was more filopodia and lamellipodia in the cells transfected with EREG. And we also observed that there are more nuclear import of c-Jun protein in EREG transfected cells. Molecular mechanism analysis indicated that treatment of cells with EREG resulted in invasion and redistribution of cytoskeletal proteins of tumor cells and led to a biphasic activation of EGFR and Src/FAK pathway. Based on these programs we can make a conclusion:ECEC can promote the invasion and metastasis of tumor cell in vitro and in vivo, ECEC specially high expressed genes of chemotatic factor superfamily, ECEC cells specially up-regulate a group of metastasis genes when co-cultured with tumor cells, one of the genes EREG which up-regulated by ECEC plays very important roly in the invasion and metastasis of esophageal carcinoma. All of these results have not been reported as yet.A highly invasive sub-cell line EC9706-P4 was established, the expression profile of the EC9706-P4 and the parental cell line EC9706 was analyzed by gene microarray analysis. TFPI-2 (Tissue factor pathway inhibitor 2) was identified as a candidate invasion and metastasis inhibitor of esophageal carcinoma cells. TFPI-2 has been implicated as a metastasis-associated gene in many types of tumors. Here we describe the potential involvement of TFPI-2 in the development of esophageal carcinoma. Western blotting revealed that TFPI-2 was down-regulated in 75% of esophageal carcinomas and in most esophageal carcinoma cell (ESCC) lines. Immunohistochemistry revealed that TFPI-2 was significantly down-regulated in tumor tissues and lymph node metastases. Experimental overexpression of TFPI-2 in KYSE450, KYSE510, YES2, and EC9706 cells significantly inhibited their invasive ability. Overexpression of TFPI-2 in EC9706 cells inhibited xenograft tumor growth and invasion into surrounding tissues, as well as reducing lung metastasis. Further studies demonstrated that recombinant TFPI-2 protein significantly inhibited the activity of matrix metalloproteinases and tumor-related angiogenesis. Parenteral treatment with recombinant TFPI-2 protein significantly suppressed xenograft tumor growth and metastasis. Together, these data indicated that TFPI-2 inhibits tumor invasion and angiogenesis both in vitro and in vivo, and suggest a potentially important therapeutic role for recombinant TFPI-2 in the treatment of malignant esophageal carcinomas.Accumulating evidence suggests a role for microRNAs in human carcinogenesis as novel types of tumor suppressors or oncogenes. In this study, we aimed to identify microRNAs species involved in the regulation of tumor invasion and growth. Using quantitative RT-PCR analysis, we demonstrated that mir-663 was down-regulated in human gastric cancer cell lines in contrasted to normal cells. Transient introduction of mir-663 lentiviral vector into human gastric cancer cell lines, BGC823 and SNU5, induced the modality changes and proliferation suppression of these cells. In addition, mir-663 disturbed the DNA content and induced mitotic catastrophe phenotype in tumor cells. More over, mir-663 also suppressed in vivo growth of BGC823 and SNU5 cells.Western-blot analyses revealed up-regulation of cyclin B by mir-663 introduction. Our results provided evidence that down-regulation of mir-663 in tumor cells may contribute to aberrant cell hyperplasia, leading to gastric cancer development. Mir-663 might function as a potent suppressor for tumor gene therapy.