The Effects Of E-cadherin On The Survival Of Tumor Cells After Introvasation

Metastasis is a multistep process facilitated by the evolution of anoikis-resistant subsets of cancer cells that are capable of surviving in the bloodstream during dissemination after they detach from the primary tumor and its stroma. Anoikis, a Greek word meaning loss of "home" or "homelessness" was originally defined by Frisch a decade ago as a unique phenomenon reflecting apoptotic cell death as a direct consequence of insufficient cell-matrix interaction and was later recognized as a potentially significant factor in tumor angiogenesis and metastasis. Although the underlying mechanisms rendering anoikis resistance in cancer cells are not fully understood, and most researches involving anoikis foucs on the connection between Cell-matrix interactions. Whereas, our study aims to determine the role that homophilic E-cadherin plays in promoting or suppressing survival of tumor cells when they enter the blood circulation and explore the possible mechanisms.The thesis includes two parts:1. Establishment of E-cadh(+) cell strain and conformation of its related porpertiesIn order to study the effects of E-cadherin on the survival of tumor cells after introvasation, firstly we need to establish the properly cell models. High invasive and metastatic breast cancer cell lines and tumor tissues are frequently associated with the down-regulation of E-cadh expression or function perturbed. So we choose MDA-MB-231 human breast carcinoma cell line which is E-cadh negative as our our cellular model. Human mammary carcinoma cell MDA-MB-231 was tansfected with the plasmid E-cadh-pcDNA3. G418 were used to screen cell strains, and E-cadh positive clones, Ecad-231-7 and Ecad-231-9, were confirmed by Western blot. Moreover, induction of E-cadh expression promoted a remarkable change in the morphology of MDA-MB-231 cells, which form epithelium-like clusters, whereas control clones maintained the fusiform appearance. E-cadh as a adhesion molecule, its influence on hemotypic, heterotypic adhesion and cell detachement were assayed by the ability of adhesion of these cells after forced expression. Results shows E-cadh expression could enhance hemotypic self adhesion, heterotypic cell-cell adhesion and cell-ECM adhesion (p<0.01). The effects of Cell-cell adhesion mediated by E-cadh was further transmitted inside the cells by its interactions withβ-catenin. So we detected the interactions of E-cadh andβ-catenin by immunoprecipitation and the changes of protein level of β-catenin and its target gene cyclin D1 by WB, and Results shows that Exogenous E-cadh could combine with endogenousβ-catenin, and expression ofβ-catenin was up regulated whereas Cyclin D1 was down regulated. MTT assay also demonstrated that E-cadh could inhibit cell proliferation (p<0.01)Our conclusion is that E-cadh could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involvingβ-catenin and Cyclin D1.2. The Effects of E-cadherin on the survival of tumor cells after introvasationResults from several studies have suggested that defects in E-cadh play a significant role in the malignant progression of tumors. Breast cancer, which often exhibits defects in E-cadh, is highly aggressive, with most of the patients having metastatic disease at the time of diagnosis. To obtain additional insight into the mechanism of anoikis induced by E-cadh, we use a recombinant plasmid E-cadh pcDNA3 encoding human E-cadh to infecte E-cadh negative breast carcinoma cell line MDA-MB-231, and successfully established two monoclonal tumor cell strains stably expressing E-cadh, which named Ecad-231-7 and Ecad-231-9. Ability to grow under anchorage-independent conditions is one of the major hallmarks of malignant cells. Key to this is the capacity of cells to suppress anoikis, or programmed cell death induced by detachment from the extracellular matrix. To imitate this phenomenon in vitro, we grew tumor cells under suspension conditions by plating them to extra-low adhesion six wells to prevent attachment to underlying plastic.Under normal culture conditions for 48 h, Apoptosis assays and cell cycle assay such as PI staining revealed no significant apoptosis and distinct cell percentages of S phase in E-cadh-expressing cells or E-cadh-negative cells. Interestingly, when tumor cells were detached and incubated in suspension for 48 h, they showed obvious reduction in S phase from37.9±2.7% to9.9±0.8%(p<0.01) for Ecad-231-7, and they underwent more obvious apoptosis (from2.0% to4.2%) (p<0.01). This is in accompanied by increased Cleaved PARP expression, which is a downstream effectors of Caspase 3.To verify that E-cadh could impact tumor cells anoikis, calcium chelator EDTA was added to disturb the hemophilic binding of E-cadh. When E-cadh expressing tumor cells were incubated in suspension with medium containing 0.02%EDTA for 48 h, larger percentage of cells underwent apoptosis (p<0.01), which comply with the up regulation of Cleaved PARP and down regulation of Cyclin D1, and E-cadh maintaining at a higher level. What is more, when tumor cells were cultured in suspension in the serum-free medium, the percentage of apoptosis cells showed no distinguished differences between E-cadh-negative MDA-MB-231 cells and E-cadh-expressing Ecad-231-7 cells, but the latter showed the distinguished G1-S arrest, whereas, Ecad-231-9 showed lower percentage of apoptosis cells because of the reduction of E-cadh protein levels under this culture conditions.As a functional phenomenon, anoikis can suppress expansion of oncogenically transformed cells by preventing proliferation at migrating locations, whereas migrating tumor cells that are resistant to anoikis induction can grow at inappropriate locations. Elevated expression of E-cadh inhibited the proliferation of tumor cells at suspension conditions and then increased their susceptibility to anoikis, and this effect was mediated, at least in part, by the Caspase 3 apoptosis pathway. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a fundational basis for the correlation between defects in E-cadh and anoikis resistance.