Cyclin E1 and cyclin-dependent kinase 2 are critical for initiation, but not for progression of hepatocellular carcinoma

Abstract

E-type cyclins E1 (CcnE1) and E2 (CcnE2) are regulatory subunits of cyclin-dependent kinase 2 (Cdk2) and thought to control the transition of quiescent cells into the cell cycle. Initial findings indicated that CcnE1 and CcnE2 have largely overlapping functions for cancer development in several tumor entities including hepatocellular carcinoma (HCC). In the present study, we dissected the differential contributions of CcnE1, CcnE2, and Cdk2 for initiation and progression of HCC in mice and patients. To this end, we tested the HCC susceptibility in mice with constitutive deficiency for CcnE1 or CcnE2 as well as in mice lacking Cdk2 in hepatocytes. Genetic inactivation of CcnE1 largely prevented development of liver cancer in mice in two established HCC models, while ablation of CcnE2 had no effect on hepatocarcinogenesis. Importantly, CcnE1-driven HCC initiation was dependent on Cdk2. However, isolated primary hepatoma cells typically acquired independence on CcnE1 and Cdk2 with increasing progression in vitro, which was associated with a gene signature involving secondary induction of CcnE2 and up-regulation of cell cycle and DNA repair pathways. Importantly, a similar expression profile was also found in HCC patients with elevated CcnE2 expression and poor survival. In general, overall survival in HCC patients was synergistically affected by expression of CcnE1 and CcnE2, but not through Cdk2. Our study suggests that HCC initiation specifically depends on CcnE1 and Cdk2, while HCC progression requires expression of any E-cyclin, but no Cdk2.

Keywords: DNA repair; HCC; cell cycle; diethylnitrosamine; liver.

Fig. 1.

CcnE1, but not CcnE2, is critical for DEN-driven hepatic tumor development. Analysis of 40-wk-old WT (n = 22), CcnE2^−/− (n = 19), and CcnE1^−/− (n = 16) mice after DEN treatment. (A) Serum ALT levels. (B) Liver:body weight ratio (%). (C) Tumor incidence. The proportion of mice with ≥1 liver nodule is shown. (D) Macroscopic appearance of livers and HCC. Three representative livers per group are shown. (E) Number of macroscopic HCC nodules and the cumulative liver tumor diameter (tumor size) for each mouse. *P < 0.05; **P < 0.005; ***P < 0.001.

Fig. 2.

CcnE1 is a key factor for c-myc–driven hepatocarcinogenesis. Mice with hepatocyte-specific overexpression of c-myc (myc^tg, n = 16) and myc^tgCcnE1^−/− double-transgenic mice (n = 11) were euthanized at the age of 70 wk and analyzed for the number and size of macroscopic HCC nodules. (A) Tumor incidence. (B) Representative macroscopic appearance of livers from c-myc^tg and myc^tgCcnE1^−/− mice. (C) Number of macroscopic HCC nodules for each mouse in the study. (D) Cumulative tumor diameter (mm) for each animal. (E) Liver:body weight ratio (%). *P < 0.05; **P < 0.005.

Fig. 3.

HCC development is attenuated by hepatocyte-specific inactivation of Cdk2. Cdk2^f/f (n = 9) and Cdk2^Δhepa (n = 8) mice were treated with DEN at the age of 14 d and euthanized at the age of 40 wk. (A) Cdk2 deletion efficiency in the liver (Δhepa) was verified by PCR using hepatic cDNA and by immunoblot (IB). (B) Macroscopic appearance of representative livers from 40-wk-old Cdk2^f/f and Cdk2^Δhepa animals. (C) Scatter plots showing the number of macroscopic HCC nodules per mouse (Left), cumulative tumor diameter (mm, Middle) and liver:body weight ratio (%, Right) for each animal. (D) Gene expression analysis of cyclin E1 (Left), cyclin E2 (Middle), and cyclin A2 (Right) in livers of DEN-treated Cdk2^f/f (n = 9) and Cdk2^Δhepa (n = 8) animals. Expression levels were calculated as fold induction in comparison with untreated WT controls (Cdk2^f/f ctrl, n = 3). *P < 0.05; **P < 0.005.

Fig. 4.

Different requirements for CcnE1 in precancerous hepatoma cells and advanced HCC cells. (A and B) Growth curves. Relative (rel.) values are shown. (A) untreated CcnE1^f/fpreCL and CcnE1^f/fHCC cells. (B–E) CcnE1^f/fpreCL and CcnE1^f/fHCC cells were infected with adv-EGFP or adv-EGFP/cre and analyzed at indicated time points after infection. (B) Growth curves of CcnE1^f/fpreCL and CcnE1^f/fHCC cells after infection. (C) Immunoblot analysis of CcnE1, phospho-Rb, CcnA2, cre-Rekombinase (cre) and activated Caspase-3 (act. Casp3). (D) Infected CcnE1^f/fpreCL and CcnE1^f/fHCC cells were gated for EGFP expression and analyzed for DNA-content by FACS. Sub-G1, Apoptotic cells. (E) Infected CcnE1^f/fpreCL cells (green) were subjected to confocal live cell fluorescence microscopy. A time window of 7 h is shown. White arrow: dividing cell; Red arrows: apoptosis-like cell death. *P < 0.05; **P < 0.005; ***P < 0.001.

Fig. 5.

Cdk2-kinase activity is critical for proliferation of precancerous murine hepatoma cells but dispensable for tumor progression. (A) Basal gene expression of CcnE1, CcnE2 and CcnA2 in Cdk2^f/fpreCL and Cdk2^f/fHCC cells in comparison with total liver RNA (Liver) from untreated donor mice of the same genetic background (Cdk2^f/f). (B–D) Cdk2^f/fpreCL and Cdk2^f/fHCC cells were treated with adv-EGFP (EGFP) or adv-EGFP/cre (cre) or left untreated (untr.) (B) Growth curves of Cdk2^f/fpreCL and Cdk2^f/fHCC cells. (C) Immunoblot analysis of CcnE1, CcnA2, Cdk2 and Rb phosphorylation 3 d after Cdk2 deletion. (D) Cdk2 (IP:Cdk2), CcnE1 (IP:E1) and CcnE2 (IP:E2) kinase complexes were immunoprecipitated and subjected to in vitro kinase assay using recombinant histone H1 as substrate. Input: 10% of the supernatant (Sn) was analyzed for β-Actin expression. ***P < 0.001.

Fig. 6.

Common properties and gene signatures of CcnE1-independent mouse hepatoma cells and HCC patients with high CcnE2 expression. (A) Four independent CcnE1^f/fpreCL cell lines (preCL) and three independent HCC-derived cell lines (HCC) were infected with adv-cre to delete CcnE1 or treated with adv-EGFP control virus. qPCR gene expression analysis for CcnE1, CcnE2, and CcnA2 in seven cell lines with (f/f) or without (^−/−) CcnE1 expression is shown. H, primary hepatocytes from CcnE1^f/f mice (n = 3) were used as reference. (B and C) Bioinformatic analysis of 369 human HCC samples based on data generated by the Cancer Genome Atlas (TCGA) Research Network. (B) Overall survival comparison analysis of patient cohorts with high (i.e., above median) or low (i.e., below median) expression of CcnE1, CcnE2, CcnE1, and CcnE2 or Cdk2. P values are indicated within diagrams. (C) Scatter plots showing high positive Spearman Correlation of Ect2, Msh2, Rad21, E2F1, and Rbbp7 with CcnE2 expression in HCC patients. p, adjusted P value; R, Spearman coefficient. **P < 0.005; ***P < 0.001.