The Effect Of Caldesmon And Its Phosphorylation On Metastatic Breast Cancer Cell Mobility

Breast cancer is the most frequently diagnosed cancer in women around the world. Uncontrolled growth and excessive migratory activities are the hallmarks of breast cancer cells. Both processes involve the actin cytoskeleton, which maintains and changes the cell shape in concert with actions of the contractile apparatus as a result of cell signaling. It is well established that polymerization and depolymerization of actin filaments plays a central role in controlling a broad spectrum of cellular activities, including cell division and migration, in addition to exo- and endocytosis, apoptosis and inflammation and gene expression. A large number of actin-binding proteins (ABP) participate in the organization and remodeling of the actin cytoskeleton. One such ABP found in nearly all vertebrate cells is caldesmon (CaD).There are two alternatively spliced isoforms of CaD derived from a single gene: the heavy caldesmon (h-CaD), found exclusively in differentiated SMCs, and the light isoform (l-CaD), present in nearly all types of vertebrate cells. Large number of evidences have supported that l-CaD can regulate the mobility of normal cells, such as epithelial, endothelial and fibroblast cells, but very little research has addressed the role of l-CaD in breast cancer cells mobility especially the highly metastatic breast cancer cells. Curiously, in many non-metastatic tumor and transformed cells l-CaD is down-regulated, but in high aggressive metastatic tumor cells, the expression of l-CaD is significant up-regulated, so we think l-CaD may play a critical role in maintaining the high mobility of metastatic breast cancer cells. In order to explore the mechanism by which l-CaD regulates breast cancer cell mobility and provide new insights into nature of metastasis of breast cancer, in this thesis, we used human epithelial metastatic breast cancer cells MDA MB-231 as experimental model, First, we used in vitro biophysical studies to clarify the effect of l-CaD on actin polymerization; Second, by siRNA and exogenous gene force-expression, the effect of l-CaD on cell mobility was evaluated; Third, the unphosphorylated l-CaD mimics peptide was synthetised and introduced into MDA MB-231 cells to detect the effect of l-CaD on cell mobility and tumor formation. Our studies mainly include:①In vitro biophysical study, we used pyren labeled G-actin in F-actin buffer to detect the progress of actin filament formation. By adding different mount of unphosphorylated or phosphorylated l-CaD respectively in different F-actin stage, we checked the actin filament formation by pyren fluorescence. The results showed that the unphosphorylated l-CaD can bind to the F-actin rapidly and has dual effect on the polymerization of G-actin to form F-actin; The phosphorylated l-CaD had some binding effect on F-actin but there was little promotion in actin polymerization.②In cellar level, western-blotting was used to detect the expression and phosphorylation level of l-CaD in metastatic breast cancer cell MDA MB-231, non-metastatic cell MCF-7 and human mammary epithelial cell HMEC. The results showed that MCF-7, like many transformed and tumor cells, contained very little l-CaD. On the contrary, the expression level of l-CaD in the metastatic MDA-MB231 cells was rather high. However, although the total content of l-CaD in MDA-MB231 cells was comparable to that in the normal HMEC cells, the phosphorylation level of endogenous l-CaD in MDA-MB231 was much higher than in HMEC cells, indicating that the l-CaD is under much more dynamic modulation in MDA-MB231 cells than in HMEC cells. Such modulation may be related to the metastatic properties of these aggressive breast tumor cells.③The expression of l-CaD was knocked down by siRNA packaged in lentivirus. First, western-blotting was used to detect the knock-down efficiency; Second, the F-actin was stained by rhodamine phalloidin to check the effect of l-CaD on cytoskeleton; Third, transell assay was used to measure the mobility of cells and then the traction force measurement and cell adhesion assay were performed to detect the sub-step of cell migration process. The results showed that the loss of l-CaD expression would break down the cytoskeleton of MDA MB-231 cells and the migration including its sub-steps would be inhibited by the l-CaD knocked down.④The MDA MB-231 and MCF-7 cells were transfected with EGFP wild type l-CaD plasmid and its phosphorylation mutant A1234 which mimics unphosphorylatable l-CaD and D1234 which mimics always phosphorylated l-CaD on ERK and PAK sites, the EGFP empty vehicle was used as control. The western-blotting was used to detect the expression efficiency of exogenous plasmid; The F-actin was stained by rhodamine phalloidin to check the effect of l-CaD on cytoskeleton; The transwell assay, traction force measurement and cell adhesion assay were performed to measure the effect of l-CaD on cell mobility. The results showed that wild type l-CaD and A1234 transfected cells exhibited thicker and more stable cytoskeleton, the traction force and cell adhesion were increased but the whole cell migration was blocked in both MDA MB231 and MCF-7 cells; The D1234 mutant had lithe effect on cytoskeleton in MDA MB-231 and MCF-7 cells; Both cells exhibited similar traction force and cell adhesion to the control cells. But, D1234 mutant decrease the migration activities in MDA MB-231 cells but not in MCF-7 cells.⑤The peptide which mimics the unphosphorylated mutant of l-CaD in ERK and PAK sites was generated in vitro. By TAT sequence, we introduced the peptide into MDA MB-231 cells. First, the cytoskeleton structure was detected by by rhodamine phalloidin staining; Second, cell mobility activities were measured by transwell assay, traction force measurement and cell adhesion assay; Third, cell proliferation was checked by alamar blue stain; Forth, the MDA MB-231 cells incubated with peptide were injected in nude mice to detect the effect of peptide in tumor growth. The results showed that the peptide has significant passive effect on tumor cell proliferation and the cell mobility, further more the peptide decreased the tumor formation of MDA MB-231 cells.