Elimination of C/EBPα through the ubiquitin-proteasome system promotes the development of liver cancer in mice
Guo-Li Wang, … , Milton Finegold, Nikolai A. Timchenko
Guo-Li Wang, … , Milton Finegold, Nikolai A. Timchenko
Published June 1, 2010
Citation Information: J Clin Invest. 2010;120(7):2549-2562. https://doi.org/10.1172/JCI41933.
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Research Article Oncology

Elimination of C/EBPα through the ubiquitin-proteasome system promotes the development of liver cancer in mice

Abstract

Despite significant advancements in our understanding of cancer development, the molecular mechanisms that underlie the formation of liver cancer remain largely unknown. C/EBPα is a transcription factor that regulates liver quiescence. Phosphorylation of C/EBPα at serine 193 (S193-ph) is upregulated in older mice and is thought to contribute to age-associated liver dysfunction. Because development of liver tumors is associated with increasing age, we investigated the role of S193-ph in the development of liver cancer using knockin mice expressing a phospho-mimetic aspartic acid residue in place of serine at position 193 (S193D) of C/EBPα. The S193D isoform of C/EBPα was able to completely inhibit liver proliferation in vivo after partial hepatectomy. However, treatment of these mice with diethylnitrosamine/phenobarbital (DEN/PB), which induces formation of liver cancer, actually resulted in earlier development of liver tumors. DEN/PB treatment was associated with specific degradation of both the S193-ph and S193D isoforms of C/EBPα through activation of the ubiquitin-proteasome system (UPS). The mechanism of UPS-mediated elimination of C/EBPα during carcinogenesis involved elevated levels of gankyrin, a protein that was found to interact with the S193-ph isoform of C/EBPα and target it for UPS-mediated degradation. This study identifies a molecular mechanism that supports the development of liver cancer in older mice and potential therapeutic targets for the prevention of liver cancer.

Authors

Guo-Li Wang, Xiurong Shi, Simon Haefliger, Jingling Jin, Angela Major, Polina Iakova, Milton Finegold, Nikolai A. Timchenko

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Figure 4

Cdc2 phosphorylates C/EBPα at S193 in Hep3B2 cells and restores growth inhibitory activity of C/EBPα.

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Cdc2 phosphorylates C/EBPα at S193 in Hep3B2 cells and restores growth i...
(A) Ectopic expression of cdc2 leads to phosphorylation of C/EPBα at S193. WT and S193A-C/EBPα were cotransfected with cdc2 into Hep3B2 cells. Expression of proteins was examined by Western blotting with antibodies to total C/EBPα, ph-S193-C/EBPα, cdc2, and β-actin. Un, untransfected cells. (B) Proteins were separated by 2D electrophoresis, transferred to the membrane, and probed with Abs to C/EBPα. Positions of ph-S193 isoforms (a and b) are shown by red arrows. (C) Cdc2 inhibits proliferation of Hep3B2 cells via restoration of growth inhibitory activity of C/EBPα. Hep3B2 cells were transfected with pAdTrack-cdc2, with pAdTrack-cdc2 plus siRNA to C/EBPα, and with pAdTrack-cdc2 plus scramble siRNA (control). Percentage of single cells (growth arrest) and proliferating cells was calculated. Upper image shows expression of C/EBPα and cdc2 in transfected cells. Data shown are mean ± SD. (D) C/EBPα is phosphorylated at S193 at 48 hours after PH. Nuclear extracts from livers at 48 hours after PH were examined by 2D gel Western blot approach. The bottom image shows 2D separation of the samples treated with alkaline phosphatase (CIP). (E) Expression of C/EBPα, cdk4, and cdc2 after PH. Western blotting was performed with the nuclear extracts isolated at different time points after PH. (F) Switch of interactions of C/EBPα with cdk4 to the interactions with cdc2 during liver regeneration. Upper image shows examination of C/EBPα in extracts used for the IP. Bottom image shows Western blotting of C/EBPα IPs with Abs to ph-S193 isoform of C/EBPα, cdk4, and cdc2. Heavy and light chains of IgGs are shown.