Abstract
Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.
Highlights
Cancer is the second greatest cause of death in the developed world
Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X
Matrigel and fibronectin were from Becton Dickinson; all secondary antibodies, conjugated with Alexa Fluor were from Invitrogen; control siRNA, goat anti b2-integrin, goat anti aenolase and goat anti c-enolase antibodies were from Santa Cruz Biotechnology; anti-profilin 1 (C-terminal) antibody and mouse anti b-actin antibody were from Sigma; goat polyclonal anticathepsin X antibody, recognizing pro- and mature forms, was from R&D Systems; mouse monoclonal (X22) anti-clathrin antibody was from Abcam; anti-rabbit HRP and anti-mouse HRP antibodies were from Millipore
Summary
Cancer is the second greatest cause of death in the developed world. To improve prevention, diagnosis and treatment, it is necessary to understand the molecular mechanisms of tumor development and progression in order that targets for the development of effective drugs and diagnostic tools can be identified. A number of molecules have been suggested to promote malignant processes, among them being cysteine cathepsins, such as cathepsin X [1,2]. Cathepsin X is up-regulated in prostatic intraepithelial neoplasia and prostate cancer [2,3] and suggested to be involved in the early stages of tumor development [2]. Cathepsin X is upregulated in gastric cancer [4] and hepatocellular carcinoma [5]. In the latter it may induce an epithelial to mesenchymal transition, an important process promoting tumor metastasis and malignancy by increasing cell motility and decreasing cell-cell adhesion [5]
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