For a long time, cysteine cathepsins were considered primarily as proteases crucial for nonspecific bulk proteolysis in the endolysosomal system. However, this view has dramatically changed, and cathepsins are now considered key players in many important physiological processes, including in diseases like cancer, rheumatoid arthritis, and various inflammatory diseases. Cathepsins are emerging as important players in the extracellular space, and the paradigm is shifting from the degrading enzymes to the enzymes that can also specifically modify extracellular proteins. In pathological conditions, the activity of cathepsins is often dysregulated, resulting in their overexpression and secretion into the extracellular space. This is typically observed in cancer and inflammation, and cathepsins are therefore considered valuable diagnostic and therapeutic targets. In particular, the investigation of limited proteolysis by cathepsins in the extracellular space is opening numerous possibilities for future break-through discoveries. In this review, we highlight the most important findings that establish cysteine cathepsins as important players in the extracellular space and discuss their roles that reach beyond processing and degradation of extracellular matrix (ECM) components. In addition, we discuss the recent developments in cathepsin research and the new possibilities that are opening in translational medicine.

In recent decades, dysregulation of proteases and atypical proteolysis have become increasingly recognized as important hallmarks of cancer, driving community-wide efforts to explore the proteolytic landscape of oncologic disease. With more than 100 proteases currently associated with different aspects of cancer development and progression, there is a clear impetus to harness their potential in the context of oncology. Advances in the protease field have yielded technologies enabling sensitive protease detection in various settings, paving the way towards diagnostic profiling of disease-related protease activity patterns. Methods including activity-based probes and substrates, antibodies, and various nanosystems that generate reporter signals, i.e., for PET or MRI, after interaction with the target protease have shown potential for clinical translation. Nevertheless, these technologies are costly, not easily multiplexed, and require advanced imaging technologies. While the current clinical applications of protease-responsive technologies in oncologic settings are still limited, emerging technologies and protease sensors are poised to enable comprehensive exploration of the tumor proteolytic landscape as a diagnostic and therapeutic frontier. This review aims to give an overview of the most relevant classes of proteases as indicators for tumor diagnosis, current approaches to detect and monitor their activity in vivo, and associated therapeutic applications.

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.

Highly-selective fluorogenic substrate and activity-based probe for monitoring cathepsin L activity in the breast cancer cell line MDA-MB-231.