Modulation of apoptosis signalling by proteasome inhibition : a single cell analysis

The proteasome inhibitor bortezomib has been successfully used in cancer therapy. Proteasome inhibition modulates various signalling pathways and causes cell death in cancer cells while sparing normal cells. In this thesis we characterise the apoptotic signalling kinetics and sequential events in response to proteasome inhibition in single cells. We identified a novel non-canonical pathway to apoptosis induction in cells with an inhibited intrinsic apoptotic pathway. Proteasome inhibition promoted the apical activation of caspase-8 in these cells. For the first time we demonstrated that autophagy induction in response to proteasome inhibition is critical for caspase-8 activation. Caspase-8 activation resulted in limited apoptosis, which can be further increased by antagonism of the endogenous caspase inhibitor XIAP. Therefore our findings provide an alternative treatment strategy to restore apoptosis susceptibility in highly resistant cancer cells.

Proteasome inhibitors have also been used to re-sensitise resistant cancer cells to TRAIL treatment. Since no kinetic data from single cells are available yet, it is not known whether, when and where the intracellular signalling kinetics of TRAIL-induced apoptosis are affected by proteasome inhibition. We therefore quantified the signalling kinetics of TRAIL-induced apoptosis in response to proteasome inhibition in single cells. Depending on the TRAIL concentration we found two modulation sites, which interfered with TRAIL signalling kinetics upon proteasome inhibition: caspase-8 activation at low doses and the threshold for mitochondria1 permeabilisation at high doses. Our results suggest that upregulation of cFLIP and Mcl-1 by impaired protein degradation were responsible for delayed caspase-8 activation and prolonged caspase-8 activation before MOMP, respectively. Our findings therefore indicate that the synergy between TRAIL and proteasome inhibition is probably based on the stabilisation of active caspases rather than enhanced DISC formation, as is often proposed in the literature.