Clathrin-Mediated Endocytosis of Neutrophil Elastase and Cathepsin G by Lung Tumor Epithelial Cells

Abstract 3269

Neutrophil elastase (NE) is an abundantly expressed enzyme with broad substrate specificity and the ability to regulate many components of the tumor microenvironment. Our laboratory has previously demonstrated that NE can gain entry into A549 lung tumor epithelial cells and increase their proliferation via degradation of insulin receptor substrate-1 (IRS-1). We have employed a variety of biochemical and in vitro assays to better define the mechanisms governing the internalization and trafficking of NE within tumor cells. Using radiolabeled ¹²⁵I-NE binding assays, we have determined that NE gains entry into three unique lung tumor epithelial cell lines via a low-affinity/high abundance receptor binding interaction with a dissociation constant (Kd) of 284 nM. NE enters cells via an endocytic pathway that is both clathrin-and dynamin-dependent and caveolin-1- and flotillin-1-independent. The interaction of NE with this putative receptor is also dependent upon an intact tertiary structure, as heat-denatured as well as chemically-denatured NE is not internalized into lung tumor epithelial cells. Uptake is competitively inhibited by the highly homologous proteinase cathepsin G (CG) which, like NE, is internalized into lung tumor epithelial cells via a clathrin- and dynamin-dependent endocytic pathway. Affinity purification studies are currently underway to identify the receptor which mediates uptake of NE into tumor cells. Identification of this receptor may unveil an attractive therapeutic target which would inhibit the uptake of NE into tumor cells while leaving the anti-microbial activities of this proteinase intact.

As NE exhibits broad substrate specificity, we performed an unbiased proteomic screen to uncover additional intracellular targets of NE. Because NE enters the cell via EEA1+ early endosomes, we concentrated on this subcellular compartment to restrict the number of potential targets to those that NE is known to access within the cell. Early endosomal protein was purified from total cell lysates using sucrose gradient fractionation and then subjected to an unbiased mass spectrometry-based proteomic analysis. Early endosomal lysates from NE-treated A549 cells exhibited marked reductions in 8 ubiquitin pathway-associated proteins, 3 prominent splicing factors, and 3 notable proteoglycans. Loss of these targets upon NE treatment was validated by western blot. Additional western blot analyses of total cell lysates from A549 cells (+/−) NE demonstrated changes in gene expression associated with an epithelial-to-mesenchymal transistion (EMT). Specifically, NE-treated cells upregulated alpha-smooth muscle actin, N-cadherin, and fibronectin-EDA, and downregulated E-cadherin in a dose-dependent fashion. These data expand the role of NE in regulating many aspects of solid tumor development, and characterization of the effect of NE on these pathways is currently under investigation.