Interactions of IGF-II and Cathepsin D in MCF-7 Breast Cancer Cells

Abstract

A primary role of the IGF-II/M6P receptor is to target lysosomal enzymes from the golgi to the lysosomes. This receptor has distinct binding sites for IGF-II and M6P, however, reciprocal interactions between these ligands have been observed (Kiess et al. 1989, 1990). Since IGF-II modulates the routing of cathepsin D in MCF-7 cells by blocking the intracellular binding of cathepsin D to the IGF-II/M6P receptor (De León et al. 1996), we hypothesized that expressing a mutant form of IGF-II that does not bind the IGF-II/M6P receptor will not interfere with lysosomal enzyme trafficking. In our present study, we report the effect in cathepsin D trafficking when Arg54 Arg55 IGF-II is expressed in MCF-7 breast cancer cells. Arg54 Arg55 IGF-II has no affinity for the IGF-II/M6P receptor but maintains a high affinity for the IGF-I and insulin receptors (Sakano et al. 1991). Western and radioimmunoassay analyses demonstrated that transfected MCF-7 cells secrete high levels of mutant IGF-II. Receptor binding assays using CM from Arg54 Arg55 IGF-II secreting cells also confirmed that indeed, the mutant IGF-II has a significant decreased binding to the IGF-II/M6P receptor. Utilizing this model and testing our hypothesis by Western blotting and metabolic labeling analyses, we demonstrated that Arg54 Arg55 IGF-II expression does not affect cathepsin D routing. This data, thus, confirms our hypothesis that IGF-II interference with cathepsin D routing is receptor mediated. Additional studies with this model also showed that morphological changes, indicative of a more aggressive and invasive phenotype, were associated with IGF-II expression. Specifically, changes in the expression of extracellular matrix (ECM) proteins such as vitronectin, Cadherin-5, and E-Cadherin were observed in cells expressing IGF-II. In particular, free-floating Arg54 Arg55 IGF-II cells displayed a significant reduction in an unknown 140 kD complex observed in adhering Arg54 Arg55 IGF-II cells. Thus, changes in IGF-II alter extracellular matrix proteins and facilitate the expression of a phenotype characteristic of more aggressive cancer cells. In summary, our studies demonstrate that lysosomal enzyme trafficking of cathepsin D is not affected when Arg54 Arg55 IGF-II is overexpressed, thus, providing direct evidence that the IGF-II effect is receptor mediated. The morphological changes observed in the transfected cells provide further evidence of the importance of IGF-II interactions with the IGF-II/M6P receptor in breast cancer