Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers
Ruiying Zhao, … , Guillermina Lozano, Mong-Hong Lee
Ruiying Zhao, … , Guillermina Lozano, Mong-Hong Lee
Published February 7, 2011
Citation Information: J Clin Invest. 2011;121(3):851-865. https://doi.org/10.1172/JCI44111.
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Research Article Oncology

Subunit 6 of the COP9 signalosome promotes tumorigenesis in mice through stabilization of MDM2 and is upregulated in human cancers

Abstract

The mammalian constitutive photomorphogenesis 9 (COP9) signalosome (CSN), a protein complex involved in embryonic development, is implicated in cell cycle regulation and the DNA damage response. Its role in tumor development, however, remains unclear. Here, we have shown that the COP9 subunit 6 (CSN6) gene is amplified in human breast cancer specimens, and the CSN6 protein is upregulated in human breast and thyroid tumors. CSN6 expression positively correlated with expression of murine double minute 2 (MDM2), a potent negative regulator of the p53 tumor suppressor. Expression of CSN6 appeared to prevent MDM2 autoubiquitination at lysine 364, resulting in stabilization of MDM2 and degradation of p53. Mice in which Csn6 was deleted died early in embryogenesis (E7.5). Embryos lacking both Csn6 and p53 survived to later in embryonic development (E10.5), which suggests that loss of p53 could partially rescue the effect of loss of Csn6. Mice heterozygous for Csn6 were sensitized to γ-irradiation–induced, p53-dependent apoptosis in both the thymus and the developing CNS. These mice were also less susceptible than wild-type mice to γ-irradiation–induced tumorigenesis. These results suggest that loss of CSN6 enhances p53-mediated tumor suppression in vivo and that CSN6 plays an important role in regulating DNA damage–associated apoptosis and tumorigenesis through control of the MDM2-p53 signaling pathway.

Authors

Ruiying Zhao, Sai-Ching J. Yeung, Jian Chen, Tomoo Iwakuma, Chun-Hui Su, Bo Chen, Changju Qu, Fanmao Zhang, You-Tzung Chen, Yu-Li Lin, Dung-Fang Lee, Feng Jin, Rui Zhu, Tattym Shaikenov, Dos Sarbassov, Aysegul Sahin, Huamin Wang, Hua Wang, Chien-Chen Lai, Fuu-Jen Tsai, Guillermina Lozano, Mong-Hong Lee

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Figure 4

CSN6 increases the stability of MDM2.

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CSN6 increases the stability of MDM2. (A) CSN6 interacted with MDM2. Lys...
(A) CSN6 interacted with MDM2. Lysates of A549 cells were immunoprecipitated and analyzed with the indicated antibodies. TCE, total cellular extracts. (B) Mapping of the CSN6 binding region within MDM2. Purified GST-MDM2 domains and Flag-CSN6 were subjected to GST-pulldown assay. Specific interactions of MDM2 deletions with CSN6 are indicated. (C) Ectopic expression of CSN6 increased the steady-state protein level of MDM2. EGFP served as a transfection efficiency control as well as a loading control. (D) Enforced expression of CSN6 increased the protein level of MDM2. Myc-CSN6–overexpressing A549, HCT116, and U2OS stable transfectants and vector controls were checked for endogenous MDM2 expression. (E) CSN6 diminished the ubiquitination level of MDM2 in vivo. HA-ubiquitinated MDM2 was immunoprecipitated with anti-HA, then probed with anti-MDM2. Equal amounts of TCE were immunoblotted with the indicated antibodies. (F) CSN6 reduced the ubiquitination level of MDM2 in vitro. GST-MDM2 was incubated with purified Flag-CSN6 or Flag-CSN5 or CSN complex in the presence of E1, E2, and His-Ubiquitin as indicated. (G) CSN6 reduced MDM2 turnover. Cells were treated with 100 μg/ml CHX for the indicated durations. Integrated OD values of MDM2 at each time point were measured. Remaining MDM2 relative to time 0 (set at 100%) is indicated. (H) K364R mutation of MDM2 attenuated the autoubiquitination of MDM2 in vitro. Wild-type and K364R mutant GST-MDM2 or GST-MDM2 were incubated with E1, E2, and His-Ubiquitin as indicated. (I) K364R MDM2 mutant had an extended half-life compared with wild-type MDM2. Remaining MDM2 is indicated.