Differential cathepsin responses to inhibitor-induced feedback: E-64 and cystatin C elevate active cathepsin S and suppress active cathepsin L in breast cancer cells

Cathepsins are powerful proteases, once referred to as the lysosomal cysteine proteases, that have been implicated in breast cancer invasion and metastasis, but pharmaceutical inhibitors have suffered failures in clinical trials due to adverse side effects. Scientific advancement from lysosomotropic to cell impermeable cathepsin inhibitors have improved efficacy in treating disease, but off-target effects have still been problematic, motivating a need to better understand cellular feedback and responses to treatment with cathepsin inhibitors. To address this need, we investigated effects of E-64 and cystatin C, two broad spectrum cathepsin inhibitors, on cathepsin levels intra- and extracellularly in MDA-MB-231 breast cancer cells. Cathepsins S and L had opposing responses to both E-64 and cystatin C inhibitor treatments with paradoxically elevated amounts of active cathepsin S, but decreased amounts of active cathepsin L, as determined by multiplex cathepsin zymography. This indicated cellular feedback to selectively sustain the amounts of active cathepsin S even in the presence of inhibitors with subnanomolar inhibitory constant values. These differences were identified in cellular locations of cathepsins L and S, trafficking for secretion, co-localization with endocytosed inhibitors, and longer protein turnover time for cathepsin S compared to cathepsin L. Together, this work demonstrates that previously underappreciated cellular compensation and compartmentalization mechanisms may sustain elevated amounts of some active cathepsins while diminishing others after inhibitor treatment. This can confound predictions based solely on inhibitor kinetics, and must be better understood to effectively deploy therapies and dosing strategies that target cathepsins to prevent cancer progression.