Invasion of Selectively Permeable Basement Membranes by Breast Cancer Cells
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The selectively permeable basement membranes and the associated extracellular matrix of sea urchin embryos have been utilized as an invasion substrate for metastatic mammary epithelial cells. This study was completed with the goal of understanding the mechanism through which tumor cells invade basement membranes. Peptides containing the minimal sequence arginine-glycine-asparatic acid (RGD), but not the sequence arginine-glycine-glutamic acid (RGE), were capable of decreasing invasion frequencies of sea urchin basement membranes by metastatic breast carcinoma cells (SUM 52 PE) by up to ten-fold. Thus, RGD-binding integrins may function in invasion. Immunostaining shows that SUM 52 PE cells are producing the α5β1 and α3β1 integrins, but not the α2β1 integrin. Blocking anti-α5 and anti-β1 integrin monoclonal antibodies efficiently inhibited invasion by SUM 52 PE on sea urchin basement membranes; however, blocking anti-α3 monoclonal antibodies had no effect. Hence, the α5β1 integrin (the fibronectin receptor) functions in invasion of sea urchin basement membranes by metastatic breast cancer cells. Since SUM 52 PE cells are grown under serum-free conditions, and sea urchin basement membranes are naturally serum-free, the effect of plasma fibronectin on invasion was studied. Metastatic SUM 52 PE cells invaded sea urchin basement membranes in the presence of serum, but failed to invade under serum-free conditions. Adding soluble plasma fibronectin to serum-free invasion assays in concentrations ranging from 4 to 400 µg/ml fully restored the invasive behavior of the cells. Depleting fibronectin from serum by affinity chromatography destroyed the ability of the cells to invade sea urchin basement membranes, but adding fibronectin to the medium made with the depleted serum fully restored the invasive ability of the cells. Therefore, fibronectin elicits invasion. It was also found that the 120kDa fibronectin fragment containing the cell-binding domain is as capable of inducing invasion as native fibronectin, but the 70kDa fragment of fibronectin which contains the N-terminus does not elicit invasion. Thus, the cell binding domain of fibronectin is responsible for the invasive behavior of the cells. Untreated SUM 52 PE cells stained for fibronectin, but when the cells were treated with 50µg/ml cycloheximide, they did not stain for fibronectin. Thus, the cells are producing cellular fibronectin as well as the α5β1 integrin. Plasma fibronectin elicits invasion but cellular fibronectin does not. This leads to the conclusion that invasion is dependent on the presence of plasma fibronectin and the expression of the α5β1 integrin.