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 beta-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.
Gopalan
B
B
Department of Thoracic and Cardiovascular Surgery, The University of Texas, MD Anderson Cancer Center, Houston, TX 77030, USA.
Shanker
M
M
Scott
A
A
Branch
C D
CD
Chada
S
S
Ramesh
R
R
D000256:Adenoviridae; D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D020134:Catalytic Domain / Q000235:genetics; D045744:Cell Line, Tumor; D005260:Female; D015316:Genetic Therapy; D006801:Humans; D007378:Interleukins / Q000096:biosynthesis* / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D025801:Ubiquitin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects / Q000235:genetics; D000099180:Interleukin-24