Study Of The Relationship And Mechanism Of E-cadherin With Ovary Cancer

Ovary carcinoma is one of the most frequent malignant tumors of female reproductive system, and its incidence in the world has recently been increasing. For patient with ovary carcinoma, distant metastases are the major cause of mortality. Metastasis is a multistep process. The initial and the most critical steps of metastasis include detachment of malignant cells from the primary tumor and invasion into surrounding tissue. In normal tissues, cells are tightly adhered with each other, so that they are generally not allowed to migrate freely. However, the malignant cells are more loosely associated, and can freely detach from primary tumor and migrate out.In recent years, many research work have showed that the epithelial cadherin (E-cadherin) is a very important regulator of the tumor cell adhesion. E-cadherin is a 120kD transmembrane glycoprotein coded by the E-cadherin gene located in chromosome 16q22.1. It connects epithelial cells via homotypic calcium-dependent interactions. The reduced expression of E-cadherin has been implicated in several features of tumor pathology: tumor development, cell differentiation, invasion, metastasis, and prognosis in various human cancers, such as breast, esophageal, gastric, oral, hepatocellular, thyroid, bladder, and lung cancer. Low expression of E-cadherin has been regarded as one of the main molecular events involved in dysfunction of the cell-cell adhesion system, triggering cancer invasion and metastasis. therefore, E-cadherin is regarded as the tumor metastasis suppressor.E-cadherin-mediated cell adhesion requires intracellular attachment of this glycoprotein to the actin cytoskeleton via members of the cateninfamily includingα-catenin (102 kD),β-catenin (92 kD), andγ-catenin (80 kD). It is indicated thatβ-catenin links E-cadherin toα-catenin and, consequently to the actin microfilament network of the cytoskeleton. As compared withα-catenin,β-catenin interacts with E-cadherin more directly. Many research results has suggested thatβ-catenin acts as tumor suppressor involved in cell-cell adhesion. The mutation or loss ofβ-catenin gene induced cell disaggregation independently. After being transfected withβ-catenin cDNA, tumor cells recovered aggregation and adhension. In addition to its role on cell-cell adherens junctions,β-catenin is also a transducer/transcriptional factor in signal transduction pathways. Specially,β-catenin is a critical component of the Wnt signaling system.β-catenin is to the nuclear and combines with Tcf/Lef when the cytoplasrnic level ofβ-catenin is elevated due to the activation of Wnt signaling or the mutation of APC gene,β-catenin gene etc. Then theβ-catenin-Tcf/Lef complex act as a transcription molecule which resulting in the trascription of genes such as c-myc, cyclin D1,MMP-7, CD44 etc and finally leading to the malignant transform of the cells.In addition to E-cadherin and catenins as the importment adheres mechanism, caveolae and caveolin-1 are also critical for the cell-cell adhesion. Caveolae, 50–100 nm protein-coated invaginations of the plasma membrane, play an important role in endocytosis and signal transduction. Caveolin-1, a 22 kD integral membrane protein, is a major component of caveolae and is important for the form and function of caveolae. The caveolin-1 may functionally regulate the activation of caveolae-associated signaling molecules, and have close relationship with APC-β-catenin-Tcf/Lef signals. caveolin-1 expression in mRNA and protein levels are both downregulated during cell transformation by oncogene Ha-Ras, v-Abl, Myc, Neu. Moreover, low caveolin-1 protein expression is also observed in a number of human cancers, including human breast, lung, colon, and ovarian carcinomas, suggesting a negatively regulatory role for caveolin-1 in tumor development. Consistent with these observations, the proliferation rate and tumor metastasis ability of caveolin-1-deficient cells are significantly increased. But this view has been controversial because caveolin-1 over-expression is seen in a few number of cancer such as prostate cancer that can promotetumor cells proliferation and metastasis. It has shown that caveolin-1 has bidirection characteristics in tumor biology.Recent years, along with the further research of the tumor metastasis mechanism, some cytokines have been discovered to induce the tumor cell metastasis. The cytokines is combined with its receptor to cause the tumor cell migration by signal transduction system. Cell motility is an important factor for the process of invasion and metastasis of tumors and is affected by extracellular signals such as growth factors. Epidermal growth factor (EGF) is the first discovered multieffect growth factor which can stimulate epithelial cell migration and DNA synthesis. EGF also plays an important role in over proliferation of tumor cells. EGF produces a marked effect only when combining with its receptor. The EGF receptor (EGFR) is known to localize in caveolae and are down regulated through their interaction with the caveolin-1 scaffolding domain. The EGFR is expressed in most mesenchymal and epithelial cells, and its signal transduction induces intrinsic tyrosine kinase activity finally affects cell growth and proliferation. Enhanced expression of the EGFR was related to poor prognosis and aggressive progress in many human tumors. It has been reported that EGF promotes tumor cell motility and migration. However, its downstream target related to cell motility is still unclear. Growth factors have been discovered to induce the disassembly of E-cadherin-mediated cell–cell adhesion, promote proliferation and motility of cancer cells.Hepatocyte growth factor (HGF) or scatter factor(SF)is a multifunctional peptide secreted by interstitial cell. HGF acts as a mitogen, morphogen and motogen for epithelial cells. It signals via c-Met, a tyrosine kinase receptor, which undergoes tyrosine phosphorylation and activation. HGF/c-Met pathway also contributes to survival, angiogenesis, metastasis and growth of tumor. However, it is still unclear that the downstream target of HGF/c-Met signal transduction related to cell proliferation, invasion and migration. Kamei et al reported that E-cadherin internalization in response to HGF was accompanied by the disruption of cell-cell adhesion and scattering of cells.In conclusion, E-cadherin is the important tumor metastasis suppressor. Distant metastases of ovary carcinoma are the major cause of mortality.However, there are few reports describing the molecular mechanism of ovary carcinoma metastasis, especially the interrelationship between E-cadherin and ovary carcinoma metastasis. In this study, we used human ovary serous cystadenocarcinoma cell lines, HO-8910Pm and HO-8910 with high and low metastatic potential respectively (established by Zhejiang Cancer Institute, Hangzhou, China) and nude mice as experimental models to confirm the roles of expression level of E-cadherin on metastasis of ovary carcinoma and signal transduction molecules regulating cell adhesion. This experiment will supply a new target of drug design for anti-metastasis of ovary carcinoma.The main reseach work is followings:1. Experimental study on the different expression of E-cadherin,β-catenin and caveolin-1 between both high metastasatic HO-8910Pm cell line and low metastatic HO-8910 cell line.First, this study explored the role of expression of E-cadherin,β-catenin and caveolin-1 in both high and low metastatic HO-8910Pm and HO-8910 cells on metastasis of tumor cells. MTT assay and adhesion assay were used to detect the cancer cells proliferation ability and adhesion ability to ECM Gel. The cell invasion and migration abilities were assessed by using Transwell chambers. The expression of E-cadherin,β-actin and caveolin-1 in the protein and mRNA level was detected by immunofluorescence staining analyses, Western blotting analyses and RT-PCR. The results showed that the abilities of cells proliferation, adhesion, invasion and migration in HO-8910Pm cells were significantly higher than that of HO-8910 cells. The expressions of E-cadherin,β-catenin and caveolin-1 were seen mainly in HO-8910 cells but were difficult to detect on HO-8910Pm cells either at protein level or at mRNA level (p<0.05). These data indicated that the low expression of E-cadherin,β-actin and caveolin-1 may be correlated with the increased invasion and metastasis abilities of human ovary carcinoma.2. Effects of RNAi-mediated gene silencing of E-cadherin expression on thebiologic behaviors of HO-8910 cellsTo establish whether the loss of E-cadherin was responsible for increased invasive and migratory capabilities of ovary carcinoma cells, HO-8910 cells were transfected with E-cadherin siRNA to silence theexpression of the target gene. Immunofluorescence staining and Western blotting analyses showed that the expression level of E-cadherin was significantly reduced in experimental cells, comparing with control cells. And the cells transfected with control siRNA can not silence the expression of target gene. The above results suggested that the RNAi was effective. The further experiment in vitro showed that the proliferatiive,invasive and migratory capability of HO-8910/RNAi cells were all higher than that of the controls. Furthermore, the effect of E-cadherin down-regulation of HO-8910 cells on metastasis was examined in nude mice. A significant increase was seen in mean tumor volume after E-cadherin silencing (p<0.05). These results suggested that the partial loss of E-cadherin was sufficient to promote the proliferation and metastasis of ovary carcinoma.3. Effects of EGF or HGF on E-cadherin-mediated cell-cell adhesion in HO8910 cells.To determine whether EGF or HGF affects the E-cadherin-mediated cell-cell adhesion, HO-8910 cells were treated with 20ng/ml EGF or 10ng/ml HGF for 72 h. The changes of HO8910 cells biological behaviour after treatment were estimated by morphological assay, cell proliferation assay, in vitro cell invasion assay, migration assay, and scratch wound migration assay. The expression changes of E-cadherin,β-catenin and caveolin-1 with EGF or HGF treatment were investigated by Western blotting, immunofluorescence staining, and RT-PCR analysis. The results indicated that EGF or HGF induced the epithelial-like to fibroblastoid conversion of HO8910 cell line, increased the cell proliferation, and stimulated cell invasion and migration, respectively. The low membrane expression of E-cadherin andβ-catenin was accompanied by a down-regulation of caveolin-1 expression after treatment with EGF or HGF. In addition, these ligands resulted in a lower membrane associated signal of E-cadherin,β-catenin and caveolin-1 in these cancer cells (p<0.05). These results suggest that signalling via EGF/EGFR and HGF/c-Met are likely the most important mediators of E-cadherin-mediated cell-cell adhesion in human ovary carcinoma cells. Caveolin-1 may inhibit tumor metastasis through mediating E-cadherin/β–catenin signal passageway.In conclusion, the above research work shows that E-cadherin ismetastasis suppressor of HO-8910 cells. The reduced expression of E-cadherin is closely related with tumor invasion and metastasis. E-cadherin-mediated cell-cell adhesion may be negatively regulated via EGF/EGFR and HGF/c-Met signal passageways and positively regulated by caveolin-1. These results suggest that above signal transduction molecules may become new effective targets to inhibit tumor metastasis. This will supply a new idea for designing drugs inhibiting tumor metastasis.