Studies On Invasion And Metastasis Associated Genes In Esophageal Carcinoma

Esophageal cancer is the fourth most common malignancy in China and its 5-years survival rate is less than 10%. 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. Therefore, disrupting invasion and metastasis is essential to develop effective treatments and longer the survival time of esophageal cancer. To investigate genes involved in esophageal carcinoma invasion and metastasis, we applied two different strategies, as by microarray comparison high invasive and metastasis potential subcell line with parent cell line or by building functional mAb libray fowolling functional mAb screen and antigen identification.For microarray analyses, an in vitro metastasis model via matrigel invasion clonal selection approach was employed, and a highly invasive sub-line EC9706-P4 was established. Then the expression profile of the sub-line and the parental cell line EC9706 was analyzed by gene microarray analysis. By this screen, TM4SF3 (transmembrane 4 superfamily 3) was identified as a candidate promoter for esophageal carcinoma cell invasion and metastasis. Western blotting revealed that TM4SF3 was overexpressed in 57.1% (8/14) of esophageal carcinomas and esophageal carcinoma cell lines with high-invasive potential. RNAi knockdown and ectopic expression indicated it promoted cell migration and invasion. Upregulating TM4SF3 expression in EC9706 cells promoted xenograft tumor invading into surrounding tissues, enhanced lung metastasis, and shortened the lifespan of mice in a spontaneous metastasis model. Further studies demonstrated that ADAM12m was upregulated by TM4SF3 overexpression in vitro and in vivo. Abrogating up-expression of ADAM12m by siRNA significantly suppressed TM4SF3-mediated invasion. Furthermore, a SNP of 397bp (G→C) in TM4SF3 abrogated ADAM12m upregulated by TM4SF3, and decreased its promoting invasion and metastasis potential.By gene microarray analysis, we also identified ADAM12m, a membrane-anchored form of a disintegrin and metalloprotease, was over expressed in EC9706-P4 cells. Immunohistochemical analysis on 119 cases tissue microarray and 98 cases paired primary cancer tissues and lymph nodes with tumor metastasis showed ADAM12m was overexpressed in tumor, and gradient upregulated in tumor invasion and lymph node metastasis. The overexpressed ADAM12m was a valuable prognostic factor independent of lymph node metastasis or local invasion. RNAi-mediated knockdown and ectopic expression of ADAM12m showed that ADAM12m promoted esophageal carcinoma metastasis in vitro and in vivo. Subsequent mechanistic studies showed that ADAM12m overexpression participated in focal adhesion and conferred metastatic properties to cancer cells by promoting FAK phosphorylation and ILK expression. The ADAM12 mediated FAK phosphorylation is integrinβ3 dependent and due to the enhancement of integrinβ3-FAK interaction. Furthermore, the correlation of ADAM12m overexpression with up-regulated FAK phosphorylation and ILK expression was frequently observed in clinical samples. Thus, ADAM12m overexpression and the related pathway activation, should be frequent events in esophageal carcinoma progression, and contributes to the cancer invasion and metastasis. Therefore, AMAM12 may be a valuable target for the treatment of esophageal carcinoma invasion and metastasis.To screen functional mAbs associated with esophageal carcinoma invasion and metastasis, an esophageal carcinoma functional monoclonal antibodies library containing 1,260 clones, was prepared by immunized Balb/c mouse with nucleated cells separated from metastatic esophageal carcinoma primary tumor. From this library, 545 membrane reactive clones were got by viable cell immunofluorescence assay. Then 485 clones, which did not or weakly reacted with normal esophageal tissue were selected out by immunohistochemitry. By migration, invasion, adhesion and proliferation assays, 70 clones showed the ability of migration inhibition, 7 clones showed the ability of invasion inhibition, 5 clones showed the ability of proliferation inhibition, 32 clones could inhibit the adhesion of esophageal tumor cells with HLEC and 48 clones could inhibit or promote the adhesion of esophageal tumor cells with LYEC. Then 20 of these clones were picked out for further assayed and at last 8 clones were cultured to produce antibody for antibody affinity assay. Three antigens purified by antibody affinity assay were identified by MALDI- TOF and LCQ-TOF, and were homology to hDUS2 (12B8 mAb), 1FAK1 (3D11 mAb) and GRLF1 (4B9 mAb). These functional mAb or antigens recognized by them might have potential application for development of therapies targeting esophageal carcinoma invasion and metastasis. Moreover, these mAb might be valuable for investigate the mechanism of esophageal carcinoma invasion and metastasis.