Pharmacological Protease Inhibitor Preserves Proteolytic Activity In Breast Cancer Cells: Computational Models To Probe Unexpected Cellular Responses

Abstract

Cysteine cathepsins are powerful elastases and collagenases, produced by breast cancer cells and tumor associated macrophages, capable of remodeling extracellular matrix and driving tumor growth and metastasis. Cathepsin expression and activity are regulated by endogenous protein inhibitors as well as proteolytic interactive networks that are not completely understood. To date, no cathepsin inhibitors have cleared clinical trials due to side effects including increased stroke risk. Surprisingly, cathepsin inhibitor treatment has been associated with increased cathepsin expression and activity. This unexpected increase in cathepsin activity could be contributing to observed side effects, but mechanisms responsible for this increased activity have not been identified. Furthermore, this increase in activity following inhibitor treatment is not consistent across different members of the cathepsin family. We have previously published that MDA‐MB‐231 breast cancer cells treated with the pharmacological cathepsin inhibitor E64 have increased active cathepsin S and decreased active cathepsin L, despite no change in total detectable cathepsin protein or mRNA. Subsequent experiments have revealed similar responses to E64 in other cancer and macrophage cell lines, suggesting this response to inhibitors is a biological phenomenon, which if elucidated would inform cathepsin inhibitor treatment in cancer. The objective of this work is to use mathematical modeling to test multiple mechanisms capable of explaining cathepsin specific changes in activity following inhibitor treatment.A series of mathematical models were developed in R to test potential hypothesized mechanisms responsible for increased active enzyme following inhibitor treatment. The models consist of series of ordinary differential equations based on principles of generalized mass action, capable of simulating the production, activation, inhibition and degradation of cathepsin L and S in breast cancer cells. This model architecture allows us to investigate different potential regulatory nodes in the cathepsin “life‐cycle”.We have previously published that cathepsins can degrade other cathepsins, a phenomenon we termed cathepsin cannibalism. Our first model tested the hypothesis that E64 was inhibiting cathepsin L, preventing it from degrading active cathepsin S. However, this model was not able to recapitulate the experimental results. We next tested the hypothesis that E64 could be stabilizing cathepsin S, increasing its activity over time. We found that the formation of stable cathepsin S E64 complexes could explain the observed increase in cathepsin S activity, while the loss of cathepsin L activity could be explained by disrupting binding with intracellular substrates or inhibitors. The model also predicted the intracellular production and degradation of cathepsin L is significantly greater than for cathepsin S. This hypothesis was supported experimentally using the translational inhibitor cycloheximide, which revealed cathepsin L intracellular turnover proceeds significantly more quickly than cathepsin S. Taken together, these results suggest that protease inhibitors can cause both accumulation of active proteases as well as depletion of active proteases, with no appreciable change in total protein. This is important to the design and successful application of future protease inhibitors to prevent off‐target inhibition and clinical trial ending side effects.Support or Funding InformationThis work was funded by NSF CBET‐0939511.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.