The anti-apoptotic protein clusterin is induced by cancer chemotherapy, stabilized by the proteasome, and degraded by intracellular proteases

B20

Background: Cancer is generally treated with a combination of chemotherapy, radiation, and surgery. Highly malignant, p53-negative cancer cells are partially resistant to chemotherapy, and an important candidate mechanism is the induction of clusterin, which is strongly induced by chemotherapy and promotes drug resistance. Clusterin is capable of inhibiting apoptosis by binding to Bax or promoting apoptosis by binding to the DNA repair protein Ku70 in the nucleus. We have analyzed the regulatory pathways that control clusterin expression after chemotherapy.Experimental procedures: MDA-MB-231 human breast cancer cells were treated with doxorubicin in combination with pharmacological inhibitors the proteasome, and clusterin expression was analyzed by western blot. Clusterin expression was then suppressed by RNAi, and its effect on apoptosis was analyzed by cleavage of PARP and pro-caspase 3.Results: Clusterin expression was strongly induced by doxorubicin, as previously published. However, clusterin was degraded following treatment with doxorubicin in combination with the proteasome inhibitor epoxomicin in a dose-dependent manner, suggesting that clusterin is stabilized by proteasome activity. Thus, clusterin is similar to other anti-apoptotic proteins, such as the transcription factor NF-κB, which are inactivated by proteasome inhibitors. In contrast, clusterin levels were increased following treatment with the proteasome inhibitor MG132, either alone or in combination with doxorubicin. Using RNAi directed to the third exon of clusterin, we determined that clusterin suppressed MG132-induced apoptosis. MG132 differs from epoxomicin in that it inhibits several non-proteasome proteases. Our findings suggest a model in which MG132-sensitive proteases degrade clusterin following chemotherapy, suppressing apoptosis.Conclusions: Proteasome inhibitors are a promising class of anti-neoplastic drugs that are currently in clinical trials. Clusterin is an anti-apoptotic target protein for highly specific proteasome inhibitors, suggesting that clusterin may be a useful marker for proteasome inhibitor activity in tumors. In addition, our results are consistent with a model in which cellular proteases inhibit clusterin and promote apoptosis following chemotherapy. This protealytic activity towards clusterin is suppressed by the proteasome and by histone deacetylases (see accompanying abstract). As a result, clusterin expression may be an important parameter for individualized treatment with proteasome inhibitors.