Molecular Mechanisms And Effects Of Epithelial Cellular Adhesion Molecule (EpCAM) On Gastric Cancer Metastasis

Gastric cancer remains the world's second commonest malignancy, having only been overtaken by lung cancer in the late 1980's, and the leading cause of death from cancer in China. Despite the marked improvements in diagnosis and treatment with the development of endoscope, less than 20% of patients can survive to 5 years after operation. Metastasis is one of the major causes of mortality for gastric cancer patients. In order to explore new effective therapeutic strategy for this disease, it is of important significance to profoundly understand the molecular mechanism of gastric cancer metastasis.The epithelial cellular adhesion molecule (EpCAM) is one of the first tumor-associated-antigens identified by monoclonal antibodies. EpCAM is a member of cell adhesion molecules (CAM) family and often presents as polymers at the cell surface of epithelial cells which mediates Ca2+-independent homotypic cell-cell adhesion. Recent studies have shown that EpCAM, as well as other CAM members, is involved in cell-matrix interactions, cell migration, cell differentiation, morphogenesis, cell cycle, and signaling as well as metabolism. The overexpression of EpCAM in cancers of various origins indicates its relationship with cancer. Previous studies on breast cancer and colon cancer have demonstrated that EpCAM overexpression is closely related with cell proliferation, cell cycle and metastasis. Currently EpCAM has applied in clinic as tumor marker for diagnosis and prognosis. In 2002, a kind of EpCAM mAb, IGN101, was in licensed clinical use in Germany to be adjuvant therapy of breast cancer and rectal cancer postoperation with an increased 7-year survival of 21% and a reduced recurrence rate of 30%. The opening of the first international symposium on EpCAM function and clinical application on 2006 in the Netherlands suggested that this molecule has raised increasing interests of cancer researchers.There are only two reports available on the involvement of EpCAM in gastric cancer, which revealed high-level expression of EpCAM in gastric cancer. The role of EpCAM in gastric cancer and in particular, its molecular mechanisms remain unclear. To explore whether or not EpCAM expression is related to malignant phenotype of gastric cancer, we investigate the expression and role of EpCAM in human gastric cancer. Some possible mechanisms of EpCAM involved are also our interests.【Objectives】(1) To investigate the expression of EpCAM in gastric cancers and the relationship of expression intensity with the clinicopathologic characteristics of gastric cancer, especially the relationship with metastasis. (2) To examine the effects of EpCAM-siRNA on the malignant phenotypes, especially invasion and metastasis of gastric cancer cells by expressional and functional studies. (3) To explore the possible mechanisms of metastatic inhibition in gastric cancer induced by EpCAM-siRNA. 【Methods】(1) The subcellular location and expression of EpCAM in gastric cancer, including metastatic and non-metastatic cases were determined by immunohistochemistry assay.(2) Semiquantitative RT-PCR and Western blot were applied to detect the EpCAM mRNA and protein level in gastric cancer cell lines.(3)Two pairs of siRNA vector of EpCAM were constructed by DNA recombinant technology. (4) EpCAM-siRNAs were transfected into SGC7901 and AGS cells and stable transfected clones were selected by G418 screening. (5) Western blot was performed on different clones to identify the transfection. (6) MTT assay was used to depict the growth curve of the transfectants and the control cells. (7) Flow CytoMeter (FCM) was used to identify the cell cycle distribution of transfectants and control cells. (8)AV/PI staining was performed to detect the apoptosis of these cells. (9)Cloning formation assay on plate and soft agar was applied to test the tumorigenesis in vitro. (10) Adhesion assay, invasion assay and migratory assay were used to determine the abilities of invasion and migration of these cells in vitro. (11) Tail vein metastatic assay were used to observe the abilities of invasion and migration of these cells in vivo. (12) The activities of MMP2 and MMP9 in the conditioned medium were visualized by performing gelatin zymography (13) The expression of MMP2, MMP9, TIMP-1 and TIMP-2 were determined by Western blot.(14) The expression levels of molecules relevant to NFκB singnal pathway were determined by Western blot.【Results】1.EpCAM was over-expressed in gastric cancer tissues and cell lines, especially in metastatic gastric cancer.Immunohistochemistry carried out on 100 paraffin-embedded gastric cancer sections showed that EpCAM was predominantly expressed in the membrane of epithelial cells and occasionally diffused into cytoplasm. In gastric cancer tissues, 74% (74 of 100 cases) EpCAM was overexpressed, significantly higher than that in nonneoplastic tissues. Further analysis of relationship between EpCAM expression and clinicopathological features of the patients revealed a negative association of its expression with degree of tumor differentiation. In addition, EpCAM expression in patients with lymph node metastasis was significantly higher than that in patients without metastasis(90%,45 /50 vs 58%,29/ 50).In metastastic cancer, the staining index was higher than that in non-metastastic cancer(9.91±1.98 versus 5.43±2.32, P<0.05), suggesting a relationship between EpCAM overexpression and gastric cancer metastasis. However, there was no significance of EpCAM expression in primary site and metastastic lymph node of metastastic gastric cancer. Semiquantitative RT-PCR and western blot analysis on gastric cancer cell lines, SGC7901, AGS, GC9811-P, MKN45 and MKN28 revealed that compared with GC9811-P, MKN45 and MKN28 cell lines, EpCAM was highly-expressed more prominently in both of mRNA and protein levels in SGC7901and AGS cell lines.2.EpCAM downregulation inhibited in vitro adhesive, invasive, migratory and in vivo metastatic abilities of gastric cancer cells.To downregulate the expression of EpCAM in gastric cancer cells, two EpCAM-specific siRNA vectors, named EpCAM-si1 and EpCAM-si2, were designed and constructed. These two vectors were transfected into SGC7901 and AGS cells and stable clones were selected by G418 screening and identified by western blot. The two siRNAs had similar effects on silencing EpCAM expression. MTT assay showed that compared with controls, growth of the transfectants slowed down. FCM analysis showed cell cycle arrest in transfectants, that is, cells of tranfectants in G1phrase and S phrase reduced and proliferating index of these cells decreased, while apoptosis of cells remained unchanged. The clone formation assay revealed that clone formation rate of siRNA transfectants was lower than that of control cells. To evaluate the effect of EpCAM on cell adhesion, the ability of SGC7901 (and AGS)-EpCAM siRNAs to adhere to matrigel was investigated by adhesive assay. It was found that EpCAM siRNA could decrease the abilities to adhere to matrigel of SGC7901 and AGS cells which reached the peak at 4h. The influence of EpCAM on the invasive and migratory abilities of gastric cancer cells in vitro were evaluated by invasion assay and migration assay. It was found that EpCAM downregulation by EpCAM siRNA produced a marked inhibition of invasive and migratory abilities of SGC7901 and AGS through matrigel on Boyden chamber assay.Also, Compared with that of parental cells and control cells transfected with empty vector, EpCAM siRNA induced a significantly less visible tumors in liver surface of nude mice inoculated with cancer cells through tail vein.Both in vitro assay and in vivo nude mice assay suggested that EpCAM siRNA had the potential to inhibit many malignant phenotypes of gastric cancer including metastasis.3.MMPs, their inhibitors and NFκB signailing were involved in the inhibition induced by EpCAM siRNA.matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs) are important factors in the process of metastasis of cancer. Therefore, we examined the effect of EpCAM siRNA on the expressions of MMP2, MMP9, TIMP1 and TIMP2 in gastric cancer cells after transfection in both active and inactive form. Our data confirmed that the expression of MMP9 in cytoplasm (inactive form, 92kD) and supernatant (active form, 86kD) of SGC7901 could both be downregulated by EpCAM siRNA, similar results were found about MMP-2. However, the result of TIMP1 was on the contrary. The expression levels of TIMP2 remained unchanged by EpCAM siRNA. We further confirmed our results by antibody treatment assay. SGC7901 and its transfected cells were treated with MMP-2 or MMP9 antibody (0.1μg/ml and 1μg/ml) before performing invasion assay. The results demonstrated that treatment with MMP-2 or MMP9 antibody could inhibit the invasive ability of all cell lines. Inhibiting rates caused by MMP-2 and MMP9 antibody at the concentration of 1μg/ml in SGC7901 parental and empry-transfecting cells were significantly higher than that in EpCAM siRNA transfected cells. The NFκB signalling pathway has been recognized as"cell survival and anti-apoptosis signaling"through upregulation of several genes involved in cell proliferation and cell transformation and regulate the expression of MMPs in transcriptional level. By Western blot, we found EpCAM siRNA could reduce the protein levels of IKKα, IKKβ, NFκB (p65/RelA subunit) and p-IκBα, whereas could increase IκBαlevel. All this results indicated that EpCAM siRNA could suppress the NFκB signaling. Thus, the inhibiton of EpCAM siRNA on gastric cancer cell metastasis might partly correlated with regulation of the expression of MMP-2,9 and TIMP-1 through NFκB signaling pathway.【Conclusion】In conclusion, we provide evidence that EpCAM is highly expressed in metastatic gastric cancer tissues. Knocking down EpCAM expression by specific siRNA can reverse the malignant potential of gastric cancer cells to some degree including the abilities of adhesion, invasion and metastasis. the inhibiton of EpCAM siRNA on gastric cancer cell metastasis might partly correlated with regulation of the expression of MMP-2,9 and TIMP-1 through NFκB signaling pathway.The present work gives the convincing proof of EpCAM as the oncogene in gastric cancer and paves the way for EpCAM as a novel candidate of diagnosis and treatment of gastric cancer metastasis,...