The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells
Uta Kossatz, … , Jeffrey D. Singer, Nisar P. Malek
Uta Kossatz, … , Jeffrey D. Singer, Nisar P. Malek
Published October 11, 2010
Citation Information: J Clin Invest. 2010;120(11):3820-3833. https://doi.org/10.1172/JCI41959.
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Research Article Oncology

The cyclin E regulator cullin 3 prevents mouse hepatic progenitor cells from becoming tumor-initiating cells

Abstract

Cyclin E is often overexpressed in cancer tissue, leading to genetic instability and aneuploidy. Cullin 3 (Cul3) is a component of the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase that is involved in the turnover of cyclin E. Here we show that liver-specific ablation of Cul3 in mice results in the persistence and massive expansion of hepatic progenitor cells. Upon induction of differentiation, Cul3-deficient progenitor cells underwent substantial DNA damage in vivo and in vitro, thereby triggering the activation of a cellular senescence response that selectively blocked the expansion of the differentiated offspring. Positive selection of undifferentiated progenitor cells required the expression of the tumor suppressor protein p53. Simultaneous loss of Cul3 and p53 in hepatic progenitors turned these cells into highly malignant tumor-initiating cells that formed largely undifferentiated tumors in nude mice. In addition, loss of Cul3 and p53 led to the formation of primary hepatocellular carcinomas. Importantly, loss of Cul3 expression was also detected in a large series of human liver cancers and correlated directly with tumor de-differentiation. The expression of Cul3 during hepatic differentiation therefore safeguards against the formation of progenitor cells that carry a great potential for transformation into tumor-initiating cells.

Authors

Uta Kossatz, Kai Breuhahn, Benita Wolf, Matthias Hardtke-Wolenski, Ludwig Wilkens, Doris Steinemann, Stephan Singer, Felicitas Brass, Stefan Kubicka, Brigitte Schlegelberger, Peter Schirmacher, Michael P. Manns, Jeffrey D. Singer, Nisar P. Malek

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Figure 2

Differentiation of Cul3-knockout hepatic progenitor cells leads to the induction of DNA damage and cellular senescence.

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Differentiation of Cul3-knockout hepatic progenitor cells leads to the i...
(A) Western blot analysis of known Cul3 substrates cyclin E, Aurora B, and p21 at 4 and 8 weeks after birth. Arrow indicates the cyclin E band. Actin was used as a loading control and was performed as noted in the methods section. Noncontiguous lanes from the same blot were spliced. Numbers indicate the different mice analyzed. (B) Immunofluorescence staining of γ-H2AX in Cul3-knockout and wild-type mice positive and negative for Cre recombinase. Arrows in Cul3loxP/loxP AlfpCre (Cul3 KO) livers indicate cells that accumulated DNA damage. In wild-type Cre+ and Cre– livers, no γ-H2AX (left panels) staining can be detected (compare Supplemental Figure 2C). DAPI staining is shown in the right panels. (C) Quantification of DNA damage in CD34-, CD133-, and CK14-positive cells (at least 100 cells from 3–4 mice were analyzed). The graph shows quantification of double-positive cells. (D) Representative pictures of round- and oval-shaped CK14-positive cells (left panels). DAPI is shown in the right panels. CK14-positive cells cannot be found in wild-type livers. (E) Western blot analysis for senescence-associated markers p15 and p16 in whole liver lysates in Cul3loxP/loxP AlfpCre livers at 4 and 8 weeks after birth in comparison with wild-type liver lysates, in which no p15 or p16 could be detected. Numbers indicate the different mice analyzed. Noncontiguous lanes from the same blot were spliced. (F) Quantification of the expression of p16 in different liver cells at 4 and 8 weeks (200 CD34- or CK14-positive cells in 3 independent experiments). Hepatocytes were identified by typical morphology (Supplemental Figure 1E). Scale bars: 10 μm. *P < 0.05, **P < 0.005.