Regulation of autophagic-lysosomal protein degradation in transformed cells

Abstract

In transformed cells the processing and translocation of lysosomal proteases are altered and protease inhibitors are deregulated. These alterations increase the capacity of neoplastic cells for degradation of extracellular matrix constituents and are thought to contribute to tumor invasion. I have tested the hypothesis that deregulation of intracellular protein degradation by lysosomes may also contribute to tumorigenesis by conferring a growth advantage to neoplastic cells, particularly in conditions of nutritional stress. In these conditions normal cells up-regulate the degradation of their cytoplasmic proteins to maintain amino acid and glucose homeostasis. To this end, I have studied protein turnover and the inducibility of lysosomal protein degradation in one mesenchymal (F9 12-1a) and three bronchial epithelial (BEAS-2B, a SV40 transformed clone of normal human bronchial cells (NHBE); BZR, a ras transfected tumorigenic clone of BEAS-2B; Calu-1, an epidermoid carcinoma cell line) transformed cells in culture. Protein degradation and synthesis were measured as the release of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine from cells and incorporation of {dollar}\rm\sp{lcub}14{rcub}C{dollar}-valine into proteins. Autophagic degradation of cytoplasm was quantified by ultrastructural morphometry. I found that in F9 12-1a, BEAS-2B, and BZR cells basal proteolysis was down-regulated. In the transformed bronchial cells basal protein degradation was unaffected by cell density and did not appear to be regulated in parallel with rates of protein synthesis and cell proliferation. Prolonged nutritional deprivation up-regulated basal proteolysis in Calu-1 cells, this change was associated with the development of a more differentiated cellular phenotype. The induction of malignant phenotype did not affect basal proteolysis. Amino acid deprivation stimulated autophagic protein degradation equally well (2-fold) in transformed and normal cells. Serum growth factors did not regulate proteolysis in either normal or transformed bronchial cells. Induction of differentiated phenotype by {dollar}\rm Ca\sp{lcub}2+{rcub}{dollar} or prolonged nutritional deprivation increased the inducibility of autophagic protein degradation in normal and transformed epithelial cells and upregulated proteolysis in response to deprivation of growth factors in Calu-1 cells. I conclude that transformation decreases the basal degradation of cytoplasmic proteins in some neoplastic cells. Down-regulation of proteolysis occurs independently of protein synthesis. Lysosomal proteolysis of transformed cells is not down-regulated. In normal and transformed cells proteolysis can be strikingly augmented by autophagy during acute or chronic nutritional stresses and can be further enhanced by inducing cell differentiation.