Increased expression, membrane association and secretion of cathepsins B, D and L are observed in transformed fibroblasts and in murine and human tumors, including human breast tumors. We have shown that, during the preneoplastic to neoplastic transition of MCF-10 human breast epithelial cells (induced by transfection with oncogenic ras), the trafficking of cathepsins B and D, but not L, is altered. The altered trafficking results in the localization of cathepsins B and D in separate peripheral vesicles and cell surface regions in the ras-transfected cells, as contrasted with their colocalization in perinuclear vesicles in the parental cells. Receptor and non-receptor mediated mechanisms could lead to altered trafficking, membrane association, and secretion of cathepsins. For cathepsin B, alternative splicing in the 5'-UTR and the presence of multiple transcription initiation sites may regulate its level of expression and its localization. By 5'-RACE in a human glioblastoma, we found that 25% of transcripts would encode a truncated cathepsin B protein lacking part of the propeptide as well as the signal peptide necessary for delivery to the endoplasmic reticulum. Thus, cathepsin B translated from these transcripts should be cytoplasmic. Secretion of procathepsins by tumors may result from their increased expression and swamping of receptors that deliver these enzymes to lysosomes. However, in the ras- transfected MCF-l0 cells, increases in expression at the mRNA level for cathepsins B, D and L were not seen, nor was increased secretion of procathepsins B and L. On the other hand, secretion of cathepsin B (mature and pro forms) could be readily effected from the ras-transfected MCF10 cells by conditions/agents that induce reorganization of the cytoskeleton. Our studies to date have established a link between malignant progression of human breast epithelial cells and altered trafficking of cathepsins B and D.
Our specific aims i n this proposal are to determine, in the immortal and ras-transfected MCF-10 cells,: 1) the subcellular distribution and secretion of cathepsin D with a new dansyl substrate; 2) whether truncated forms of cathepsin B (transcripts and protein) are present; 3) whether pro, mature and/or truncated cathepsins B and D are associated with peripheral vesicles and/or cell surface; 4) whether delivery to and/or association of cathepsins B and D with peripheral vesicles and cell surface is mediated by any of the known receptors for lysosomal enzymes; 5) the identity of vesicles labeling for cathepsins B, D and L and of ras-related proteins associated with these vesicles; and 6) whether inhibition of ras will alter the differential trafficking of cathepsins B, D and L and the invasive phenotype of the ras-transfected MCF-10 cells. The present studies may provide information that is important not only to breast carcinoma, but also to other human tumors in whose malignant progression ras has been implicated.
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