Caveolin-1 mediates the expression and localization of cathepsin B, pro-urokinase plasminogen activator and their cell-surface receptors in human colorectal carcinoma cells

Cathepsin B and pro-urokinase plasminogen activator (pro-uPA) localize to the caveolae of HCT 116 human colorectal carcinoma cells, an association mediated by active K-RAS. In this study, we established a stable HCT 116 cell line with a gene encoding antisense caveolin-1 (AS-cav-1) to examine the effects of caveolin-1, the main structural protein of caveolae, on the expression and localization of cathepsin B and pro-uPA, and their cell-surface receptors p11 and uPA receptor (uPAR), respectively. AS-cav-1 HCT 116 cells secreted less procathepsin B than control (empty vector) cells as measured by immunoblotting and pepsin activation of the proenzyme. Expression and secretion of pro-uPA was also downregulated in AS-cav-1 HCT 116 cells. Localization of cathepsin B and pro-uPA to caveolae was reduced in AS-cav-1 HCT 116 cells, and these cells expressed less total and caveolae-associated p11 and uPAR compared with control cells. Previous studies have shown that uPAR forms a complex with caveolin-1 and beta1-integrin, and we here show that downregulation of caveolin-1 also suppressed the localization of beta1-integrin to caveolae of these cells. Finally, downregulation of caveolin-1 in HCT 116 cells inhibited degradation of the extracellular matrix protein collagen IV and the invasion of these cells through Matrigel. Based on these results, we hypothesize that caveolin-1 affects the expression and localization of cathepsin B and pro-uPA, and their receptors, thereby mediating cell-surface proteolytic events associated with invasion of colon cancer cells

Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.

The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as beta1-integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating beta1-integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of beta1-integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of beta1-integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells

Cathepsin B contributes to Na + hyperabsorption in cystic fibrosis airway epithelial cultures: Cathepsin B and ENaC

In cystic fibrosis (CF) lung disease, the absence of functional CF transmembrane conductance regulator results in Cl−/HCO3− hyposecretion and triggers Na+ hyperabsorption through the epithelial Na+ channel (ENaC), which contribute to reduced airway surface liquid (ASL) pH and volume. Prostasin, a membrane-anchored serine protease with trypsin-like substrate specificity has previously been shown to activate ENaC in CF airways. However, prostasin is typically inactive below pH 7.0, suggesting that it may be less relevant in acidic CF airways. Cathepsin B (CTSB) is present in both normal and CF epithelia and is secreted into ASL, but little is known about its function in the airways. We hypothesized that the acidic ASL seen in CF airways may stimulate CTSB to activate ENaC, contributing to Na+ hyperabsorption and depletion of CF ASL volume. In Xenopus laevis oocytes, CTSB triggered α- and γENaC cleavage and induced an increase in ENaC activity. In bronchial epithelia from both normal and CF donor lungs, CTSB localized to the apical membrane. In normal and CF human bronchial epithelial cultures, CTSB was detected at the apical plasma membrane and in the ASL. CTSB activity was significantly elevated in acidic ASL, which correlated with increased abundance of ENaC in the plasma membrane and a reduction in ASL volume. This acid/CTSB-dependent activation of ENaC was ameliorated with the cell impermeable, CTSB-selective inhibitor CA074, suggesting that CTSB inhibition may have therapeutic relevance. Taken together, our data suggest that CTSB is a pathophysiologically relevant protease that activates ENaC in CF airways

Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ

The breast tumor microenvironment regulates progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). However, it is unclear how interactions between breast epithelial and stromal cells can drive this progression and whether there are reliable microenvironmental biomarkers to predict transition of DCIS to IDC

Imaging Proteolysis by Living Human Breast Cancer Cells

Malignant progression is accompanied by degradation of extracellular matrix proteins. Here we describe a novel confocal assay in which we can observe proteolysis by living human breast cancer cells (BT20 and BT549) through the use of quenched-fluorescent protein substrates. Degradation thus was imaged, by confocal optical sectioning, as an accumulation of fluorescent products. With the BT20 cells fluorescence was localized to pericellular focal areas that coincide with pits in the underlying matrix. In contrast, fluorescence was localized to intracellular vesicles in the BT549 cells, vesicles that also label for lysosomal markers. Neither intracellular nor pericellular fluorescence was observed in the BT549 cells in the presence of cytochalasin B, suggesting that degradation occurred intracellularly and was dependent or endocytic uptake of substrate. In the presence of a cathepsin B-selective cysteine protease inhibitor, intracellular fluorescence was decreased ∼90% and pericellular fluorescence decreased 67% to 96%, depending on the protein substrate. Matrix metallo protease inhibitors reduced pericellular fluorescence ∼50%, i.e., comparably to a serine and a broad spectrum cysteine protease inhibitor. Our results suggest that: 1) a proteolytic cascade participates in pericellular digestion of matrix proteins by living human breast cancer cells, and 2) the cysteine protease cathepsin B participates in both pericellular and intracellular digestion of matrix proteins by living human breast cancer cells

Functional Imaging of Proteolysis: Stromal and Inflammatory Cells Increase Tumor Proteolysis

The underlying basement membrane is degraded during progression of breast and colon carcinoma. Thus, we imaged degradation of a quenched fluorescent derivative of basement membrane type IV collagen (DQ-collagen IV) by living human breast and colon tumor spheroids. Proteolysis of DQ-collagen IV by HCT 116 and HKh-2 human colon tumor spheroids was both intracellular and pericellular. In contrast, proteolysis of DQ-collagen IV by BT20 human breast tumor spheroids was pericellular. As stromal elements can contribute to proteolytic activities associated with tumors, we also examined degradation of DQ-collagen IV by human monocytes/macrophages and colon and breast fibroblasts. Fibroblasts themselves exhibited a modest amount of pericellular degradation. Degradation was increased 4–17-fold in cocultures of fibroblasts and tumor cells as compared to either cell type alone. Inhibitors of matrix metalloproteinases, plasmin, and the cysteine protease, cathepsin B, all reduced degradation in the cocultures. Monocytes did not degrade DQ-collagen IV; however, macrophages degraded DQ-collagen IV intracellularly. In coculture of tumor cells, fibroblasts, and macrophages, degradation of DQ-collagen IV was further increased. Imaging of living tumor and stromal cells has, thus, allowed us to establish that tumor proteolysis occurs pericellularly and intracellularly and that tumor, stromal, and inflammatory cells all contribute to degradative processes

Functional imaging of tumor proteolysis

The roles of proteases in cancer are now known to be much broader than simply degradation of extracellular matrix during tumor invasion and metastasis. Furthermore, proteases from tumor-associated cells (e.g., fibroblasts, inflammatory cells, endothelial cells) as well as tumor cells are recognized to contribute to pathways critical to neoplastic progression. Although elevated expression (transcripts and proteins) of proteases, and in some cases protease inhibitors, has been documented in many tumors, techniques to assess functional roles for proteases require that we measure protease activity and inhibition of that activity rather than levels of proteases, activators, and inhibitors. Novel techniques for functional imaging of protease activity, both in vitro and in vivo, are being developed as are imaging probes that will allow us to determine protease activity and in some cases to discriminate among protease activities. These should be useful clinically as surrogate endpoints for therapies that alter protease activities

Functional Live-Cell Imaging Demonstrates that β-Integrin Promotes Type IV Collagen Degradation by Breast and Prostate Cancer Cells

The ability of tumor cells to adhere to, migrate on, and remodel extracellular matrices is mediated by cell surface receptors such as β 1 -integrins. Here we conducted functional live-cell imaging in real time to investigate the effects of modulating β 1 -integrin expression and function on proteolytic remodeling of the extracellular matrix. Human breast and prostate cancer cells were grown on reconstituted basement membrane containing a quenched fluorescent form of collagen IV. Generation of cleavage products and the resulting increases in fluorescence were imaged and quantified. Decreases in the expression and activity of β 1 -integrin reduced digestion of quenched fluorescent-collagen IV by the breast and prostate cancer cells and correspondingly their invasion through and migration on reconstituted basement membrane. Decreased extracellular matrix degradation also was associated with changes in the constituents of proteolytic pathways: decreases in secretion of the cysteine protease cathepsin B, the matrix metalloproteinase (MMP)-13, and tissue inhibitors of metalloproteinases (TIMP)-1 and 2; a decrease in expression of MMP-14 or membrane type 1 MMP; and an increase in secretion of TIMP-3. This is the first study to demonstrate through functional live-cell imaging that downregulation of β 1 -integrin expression and function reduces proteolysis of collagen IV by breast and prostate cancer cells

Mutant K-ras Regulates Cathepsin B Localization on the Surface of Human Colorectal Carcinoma Cells

Cathepsin B protein and activity are known to localize to the basal plasma membrane of colon carcinoma cells following the appearance of K-ras mutations. Using immunofluorescence and subcellular fractionation techniques and two human colon carcinoma cell lines—one with a mutated K-ras allele (HCT 116) and a daughter line in which the mutated allele has been disrupted (HKh-2)—we demonstrate that the localization of cathepsin B to caveolae on the surface of these carcinoma cells is regulated by mutant K-ras. In HCT 116 cells, a greater percentage of cathepsin B was distributed to the caveolae, and the secretion of cathepsin B and pericellular (membrane-associated and secreted) cathepsin B activity were greater than observed in HKh-2 cells. Previous studies established the light chain of annexin II tetramer, p11, as a binding site for cathepsin B on the surface of tumor cells. The deletion of active K-ras in HKh-2 cells reduced the steady-state levels of p11 and caveolin-1 and the distribution of p11 to caveolae. Based upon these results, we speculate that cathepsin B, a protease implicated in tumor progression, plays a functional role in initiating proteolytic cascades in caveolae as downstream components of this cascade (e.g., urokinase plasminogen activator and urokinase plasminogen activator receptor) are also present in HCT 116 caveolae