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Abstract

Many pathologies, most notably cancer, are accompanied by aberrant proteolytic
activity which can stem from altered localization of commonly intracellular proteases.
Thereby, we focused our research on atypical proteolysis in the tumour microenvironment
mediated by apoptotic caspases and cysteine cathepsins.
Caspases are the main proteases that drive apoptosis, a form of programmed cell death,
which is commonly dysregulated in various pathologies. Apoptosis is predominately an
immunologically silent form of cell death that can become immunogenic during progression
to secondary necrosis, thus providing an opportunity for extracellular presence of active
apoptotic caspases. By triggering apoptosis in Jurkat E6.1 cells we demonstrated that
apoptotic caspases-3 and -7 can be found in the extracellular millieu of secondary necrotic
cells. Furthermore, we showed that they can then cleave extracellular domains of
membrane-bound proteins from MDA-MB-231 breast cancer cells, a function generally
attributed to proteases from metalloprotease and cathepsin families.
We were also focused on the extracellular roles of cysteine cathepsins K, L and S. For
cathepsins L and S, it has already been shown that they can act as sheddases, with at least
one of the substrates pointing towards their involvement in the activation or regulation of
immune response. Therefore, we aimed at elucidating whether cathepsin-mediated
extracellular proteolysis could play a role in regulation of immune response. Low level of
activation of the complement system is a known factor contributing to the
immunosuppressive nature of the tumour microenvironment. By treating MDA-MB-231
breast cancer cells with cathepsins we have shown that they blunt the deposition of the
components of the complement system, thereby blunting the immune response.
In the case of cathepsin K, using melanoma cell line HT-144, we have identified the
extracellular domain of MMP-14 as one of its extracellular substrates. Consequently, we
tried to ascertain whether this could lead to further activation of extracellular
metalloproteases. We have detected increased gelatinolytic activity in the sheddomes of
treated cells, but were unable to identify the exact enzymes responsible for the activation.
Furthermore, we observed that cathepsin K treatment results in detachment of cancer
cells, but we could not elucidate whether solely cathepsin K-mediated cleavages are
responsible for the detachment or whether the process is accelerated by downstream
activation of extracellular metalloproteases.
Our research provides the first evidence for the potential involvement of apoptotic
caspases-3 and -7 in the extracellular proteolytic network and expands our knowledge about
the extracellular roles of cysteine cathepsins by implicating cathepsins L and S in regulation
of immune response and identifying MMP-14 as an extracellular substrate of cathepsin K.