Cathepsin X inhibitors impair the resistence of tumor cells to antiprotease therapy

Abstract

The progression of malignant diseases is associated with excessive activity of the proteolytic enzymes involved in tumor growth, angiogenesis, migration, invasion and metastasis. Among them is a group of cysteine cathepsins that are present in normal cells in endosomes and lysosomes and are responsible for intracellular protein catabolism. In tumor cells these enzymes undergo various changes regarding their expression, activity and localization. Cathepsin B has appeared as the most important tumor promoter. In lysosomes it acts predominantly as an exopeptidase (carboxypeptidase) involved in protein catabolism and autophagy, whereas in tumors, with increased endopeptidase activity, it is responsible for degrading proteins of the extracellular matrix (ECM). Cathepsin B is overexpressed in practically all tumor types and inhibition of its endopeptidase activity has frequently been reported to reduce tumor growth and metastasis in animal cancer models. In recent years, several specific inhibitors of cathepsin B with favorable pharmacological properties have been developed and become candidates for clinical testing. They provide very potent antitumor effects although,  after a time, their effectiveness is reduced. We and others have recently found that the main factor of this resistance is delayed over-expression of a similar proteolytic enzyme, cathepsin X. Like cathepsin B, cathepsin X also acts as a carboxypeptidase. However, in tumors it does not switch to endopeptidase activity to degrade the ECM but enhances tumor cell migration and invasion by alternative mechanisms, predominantly through the regulation of integrin receptors and tumor suppressor protein profilin 1. Its higher expression and activity is therefore associated with the renewed ability of tumor cells to migrate and invade, although in this case without degradation of the ECM. 

Our hypothesis is that inhibitors of cathepsin X may decrease the resistance of tumor cells to anti-peptidase therapy, in particular to therapy with inhibitors of cathepsin B.  In the proposed research project we will obtain  inhibitors of cathepsin X that will be: i) small molecules that bind reversibly and tightly to cathepsin X, ii) specific for  cathepsin X and iii) water-soluble and non-toxic for mammalian cells. The signaling pathways leading to induced overexpression of Cat X as a resistance factor in anti-protease therapy will also be determined, in this way identifying additional targets whose regulation could impair the resistance. 

New inhibitors will be evaluated in in vitro and in vivo models of tumor migration, invasion, angiogenesis and metastasis, in combination with known inhibitors of cathepsin B and other peptidase inhibitors. The new compounds will be prepared using advanced methods of structure based drug design and synthesis, virtual screening of compound libraries and enzyme kinetics. The structures with the best binding score will be synthesized, purified, and characterized. Formulations that will enable further in vitro and in vivo testing will be developed. Selected compounds will be evaluated in in vitro models of tumor adhesion, migration, invasion and angiogenesis that resemble pathological processes of tumor progression. Moreover, new inhibitors of cathepsin X will be tested in vivo in the mouse models of tumor growth and metastasis that we developed earlier for evaluation of the efficacy of the inhibitors of cathepsin B and other cysteine proteases. They include C57Bl/6 mice and LPB-1 tumor cells in the model of mouse fibrosarcoma, and FVB/N mice tumor cells and their transgenic cell line FVB/PyMT in the model of breast cancer. Our work will constitute an innovative approach to the prevention of drug resistance, enable faster translation of existing anti-peptidase tumor therapy into clinical testing, and promise more effective treatment of cancer patients.