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 3

Development of tumors is accelerated in C/EBPα-S193D knockin mice.

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Development of tumors is accelerated in C/EBPα-S193D knockin mice. (A) P...
(A) Pictures of livers at different time points after DEN injection. Arrows show large tumor nodules observed in livers of mice treated with DEN for 25 weeks. (B) DNA synthesis is increased in livers of S193D mice. Bar graphs show percentage of BrdU-positive hepatocytes in tumor sections of livers of WT and S193D mice. Summary of results with 3 animals of each genotype is shown. Data shown are mean ± SD. (C) Expression of C/EBPα and cell-cycle proteins in WT and S193D livers at 25 weeks after DEN injection. Nuclear extracts were examined by Western blotting with antibodies shown on the right. (D) The mutant S193D isoform of C/EBPα is specifically degraded during liver tumor development. Western blotting was performed with WT and S193D livers at 6, 20, 25, and 30 weeks after injection of DEN. 2 isoforms of C/EBPα are shown by arrows. (E) cdc2 is elevated during DEN-mediated carcinogenesis and phosphorylates C/EBPα at S193 before degradation of C/EBPα. Western blotting was performed with nuclear extracts of WT mice. Bottom: C/EBPα was IP from nuclear extracts, and the IPs were probed with antibodies to cdc2 and ph-S193 C/EBPα. Bar graphs show a ratio of S193-ph isoform of C/EBPα to total C/EBPα protein. (F) Location of the predicted cdc2 sites within C/EBPα and generation of mutant constructs. (G) Cdc2 phosphorylates C/EBPα at S193 and S21. Cdc2 was IP from nuclear extracts isolated from the liver at 30 weeks after DEN treatments. Kinase assay was performed with WT C/EBPα and with C/EBPα mutants S21A, S193A, and S21A–S193A (Dm).