Bax degradation by the ubiquitin/proteasome-dependent pathway: involvementin tumor survival and progression

Previously we reported that proteasome inhibitors were able to overcome Bcl -2-mediated protection from apoptosis. Here we show that inhibition of the proteasome activity in Bcl-2-overexpressing cells accumulates the proapopto tic Bar protein to mitochondria/cytoplasm, where it interacts to Bcl-2 prot ein. This event was followed by release of mitochondrial cytochrome c into the cytosol and activation of caspase-mediated apoptosis. In contrast, prot easome inhibition did not induce any apparent changes in Bcl-2 protein leve ls. In addition, treatment with a proteasome inhibitor increased levels of ubiquitinated forms of Bar protein, without any effects on Bar mRNA express ion. We also established a cell-free Bar degradation assay in which an in v itro-translated, S-35-labeled Bar protein can be degraded by a tumor cell p rotein extract, inhibitable by addition of a proteasome inhibitor or deplet ion of the proteasome or ATP, The Bar degradation activity can be reconstit uted in the proteasome-depleted supernatant by addition of a purified 20S p roteasome or proteasome-enriched fraction. Finally, by using tissue samples of human prostate adenocarcinoma, we demonstrated that increased levels of Bar degradation correlated well with decreased levels of Bar protein and i ncreased Gleason scores of prostate cancer. Our studies strongly suggest th at ubiquitin/proteasome-mediated Bar degradation is a novel survival mechan ism in human cancer cells and that selective targeting of this pathway shou ld provide a unique approach for treatment of human cancers, especially tho se overexpressing Bcl-2.