The deubiquitinase inhibitor b-AP15 induces strong proteotoxic stress and mitochondrial damage.

Human cancers are characterized by intrinsic or acquired resistance to apoptosis and evasion of apoptosis has been proposed to contribute to treatment resistance. Bis-benzylidine piperidone compounds, containing α,β-unsaturated carbonyl functionalities, have been extensively documented as being effective in killing apoptosis-resistant cells and to display promising antineoplastic activities in a number of tumor models. We here explored the phenotypic response of colon cancer cells to b-AP15, a bis-benzylidine piperidone previously shown to inhibit the proteasome deubiquitinases (DUBs) USP14 and UCHL5. Whereas similar overall mRNA and protein expression profiles were induced by b-AP15 and the clinically available proteasome inhibitor bortezomib, b-AP15 induced stronger increases of chaperone expression. b-AP15 also induced a stronger accumulation of polyubiquitinated proteins in exposed cells. These proteins were found to partially colocalize with organelle structures, including mitochondria. Mitochondrial oxidative phosphorylation decreased in cells exposed to b-AP15, a phenomenon enhanced under conditions of severe proteotoxic stress caused by inhibition of the VCP/p97 ATPase and inhibition of protein translocation over the ER. We propose that mitochondrial damage caused by the association of misfolded proteins with mitochondrial membranes may contribute to the atypical cell death mode induced by b-AP15 and related compounds. The robust mode of cell death induction by this class of drugs holds promise for treatment of tumor cells characterized by apoptosis resistance.