MDA-7 IL-24, a novel tumor suppressor cytokine is ubiquitinated and regulated by the ubiquitin-proteasome system, and inhibition of MDA-7 IL-24 degradation enhances the antitumor activity

Steady-state protein levels are determined by the balance between protein synthesis and degradation. Protein half-lives are determined primarily by degradation, and the major degradation pathways involve either lysosomal destruction or an ATP-dependent process involving ubiquitination to target proteins to the proteosome. Studies have shown that multiple tumor-suppressor proteins are ubiquitinated and degraded by the 26S proteasome. In the present study, we investigated whether the tumor suppressor/cytokine melanoma differentiation-associated gene-7/interleukin-24 gene (MDA-7/IL-24) protein is ubiquitinated and its degradation controlled by the proteasome. Treatment of ovarian (2008) and lung (H1299) tumor cells with adenoviral delivery of mda-7 (Ad-mda7) or Ad-mda7 plus the proteosome inhibitor MG132 showed that MDA-7 protein expression was dependent upon proteosome activity. Western blot and immunoprecipitation analyses verified that the MDA-7 protein was ubiquitinated and that ubiquitinated-MDA-7 levels were increased in MG132-treated cells. These results were confirmed using small interfering RNA (siRNA)-mediated knockdown of ubiquitin. Furthermore, ubiquitinated MDA-7 protein was degraded by the 26S proteasome, as MDA-7 accumulation was observed only when cells were treated with MG132 but not with lysosome or protease inhibitors. Inhibition of the catalytic β-5 subunit of the 20S proteasome using siRNA resulted in MDA-7 protein accumulation. Finally, treatment of tumor cells with Ad-mda7 plus the proteasome inhibitor bortezomib resulted in increased tumor cell killing. Our results show that MDA-7/IL-24 is ubiquitinated and degraded by the 26S proteasome. Furthermore, inhibition of MDA-7 degradation results in enhanced tumor killing, identifying a novel anticancer strategy.