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, 22 (6), 1025-34

Autophagy Inhibits Oxidative Stress and Tumor Suppressors to Exert Its Dual Effect on Hepatocarcinogenesis

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Autophagy Inhibits Oxidative Stress and Tumor Suppressors to Exert Its Dual Effect on Hepatocarcinogenesis

Y Tian et al. Cell Death Differ.

Abstract

The role of autophagy in carcinogenesis is controversial and apparently complex. By using mice with hepatocyte-specific knockout of Atg5, a gene essential for autophagy, we longitudinally studied the role of autophagy in hepatocarcinogenesis. We found that impairing autophagy in hepatocytes would induce oxidative stress and DNA damage, followed by the initiation of hepatocarcinogenesis, which could be suppressed by the antioxidant N-acetylcysteine. Interestingly, these mice developed only benign tumors with no hepatocellular carcinoma (HCC), even after the treatment with diethylnitrosamine, which induced HCC in wild-type mice. The inability of mice to develop HCC when autophagy was impaired was associated with the induction of multiple tumor suppressors including p53. Further analysis indicated that the induction of p53 was associated with the DNA-damage response. Tumorigenesis studies using an established liver tumor cell line confirmed a positive role of autophagy in tumorigenesis and a negative role of p53 in this process when autophagy was impaired. Our studies thus demonstrate that autophagy is required to maintain healthy mitochondria and to reduce oxidative stress and DNA damage to prevent the initiation of hepatocarcinogenesis. However, once hepatocarcinogenesis has been initiated, its presence is also required to suppress the expression of tumor suppressors to promote the development of HCC.

Figures

Figure 1
Figure 1
Development of hepatic tumors in L-Atg5-KO mice. (a) Immunoblot analysis of Atg5, LC3 and p62 in the liver of 4-month old Atg5-WT and L-Atg5-KO mice and in the liver tumors of 10-month old L-Atg5-KO mice. Actin served as the loading control. Two or more tissue samples were analyzed to ensure the reproducibility of the results. (b) Liver tumor incidence in Atg5-WT and L-Atg5-KO mice; ‘n' indicates the number of mice analyzed. The increase of tumor incidence starting from 6 months of age was statistically significant (P<0.01). (c) Left panel, a typical liver of L-Atg5-KO mice at 10 months of age; right panel, H&E staining of a representative hepatic adenoma isolated from a 10-month-old mouse; and middle panel, H&E staining of the liver tissue of an age-matched control mouse. LC3-I, non-lipidated LC3; LC3-II, lipidated LC3; NT, non-tumor tissue; T, tumor tissue
Figure 2
Figure 2
Induction of oxidative DNA damage in the liver of L-Atg5-KO mice. (a) Electron microscopy of mitochondria in the hepatocytes of 4-month-old wild-type (WT) and L-Atg5-KO mice. Scale bar, 1 μm. (b) FACS analysis of mitochondrial membrane potential. The horizontal axis measured the green JC1 fluorescence and the vertical axis measured the red JC1 fluorescence. Boxed areas indicated mitochondria with high membrane potentials. See Materials and methods for details. (c) FACS analysis of ROS in mouse hepatocytes after staining with DCFDA, a fluorogenic dye that measures ROS in cells. (d) Analysis of 4-HNE in the liver of WT and L-Atg5-KO mice and the spleen of L-Atg5-KO mice at different age. (e) Analysis of 8-OXO-dG levels. In (d) and (e), five mice were analyzed for every time point and the results represented the mean. **P<0.01 when the liver of L-Atg5-KO mice were compared with their spleen or with the liver of the WT mice. (f) Immunostaining of γ-H2AX (brown-color staining) in the normal liver tissue sections of 4-month-old mice. The liver tumor tissue section of a 6-month-old L-Atg5-KO mouse was also analyzed. NT, non-tumor tissue; T, tumor tissue
Figure 3
Figure 3
Reduction of liver tumor incidence of L-Atg5-KO mice by NAC. Comparison of the liver tumor incidence (a), tumor number (b) and tumor size (c) in L-Atg5-KO mice without and with treatment of NAC; ‘n' indicates the number of mice analyzed. *P<0.05; **P<0.01. In (a), the data were analyzed by χ2, and in (b) and (c), they were analyzed by the Student's t-test
Figure 4
Figure 4
Effect of Atg5 KO on hepatocarcinogenesis in mice treated with DEN. Wild-type and L-Atg5-KO mice were injected with DEN (5 mg/kg body weight) at 16, 23, 30 and 37 days of age. (a) Liver tumor incidence at 4, 6 and 8 months of age. **P<0.01. (b) H&E staining of a typical adenoma from 6-month-old L-Atg5-KO mice (left panel) and HCC from wild-type mice (right panel). The adenomas were well-circumscribed and consisted of sheets of hepatocytes with a bubbly vacuolated cytoplasm. The HCC of wild-type mice displayed a macrotrabecular pattern with clear-cell morphology and nuclear irregularity. (c) Immunoblot analysis of glypican-3. (Upper two panels) Eight liver tumor nodules were randomly selected from four wild-type mice for immunoblot analysis of glypican-3 and actin. Non-tumor tissues from two different wild-type mice were used as the controls. (Lower two panels) One liver tumor tissue was randomly selected from each of six L-Atg5-KO mice for immunoblot analysis. The non-tumor tissues of two wild-type mice and two L-Atg5-KO mice were also analyzed to serve as the controls
Figure 5
Figure 5
Immunoblot analysis of tumor suppressors in the liver of wild-type (WT) and L-Atg5-KO mice. Immunoblot was conducted to analyze the expression of various tumor suppressors in non-tumor (NT) and tumor (T) liver tissues of WT (a) and L-Atg5-KO mice (b) that had been treated with DEN. In (b), the non-tumor liver tissues of WT mice were used as the control. Two mice were used for all of the studies to ensure the reproducibility of the results. The asterisk denotes the hyperphosphorylated Rb
Figure 6
Figure 6
Analysis of the mechanisms of p53 inductions by Atg5 KO. Immunoblot analyses of: (a) ATM and Chk2 and their phosphorylated forms; and (b) MDM2 in the liver of wild-type (WT) and L-Atg5-KO mice
Figure 7
Figure 7
Effects of Atg5 and p53 on tumorigenesis of HepG2 cells in nude mice. (a) Immunoblot analysis of Atg5, p53, LC3, p62 and actin in stable HepG2 cells expressing various shRNAs. (b) Nude mice were subcutaneously injected with HepG2 cells that expressed various shRNAs. Tumor volumes were measured at different time points after injection. The difference of tumor sizes between control HepG2 cells and HepG2 cells with Atg5 knockdown was statistically significant starting from 30 days (P<0.05 on day 30 and P<0.01 after day 30). The difference between HepG2 cells with Atg5 or p53 knockdown and HepG2 cells with Atg5 and p53 double knockdown was statistically significant starting from 35 days (P<0.05 on day 35 and P<0.01 after day 35). The tumor growth of control HepG2 cells was terminated 50 days after the injection owing to the large tumor size. (c) Representative mice with tumors of HepG2 cells
Figure 8
Figure 8
Induction of cell deaths in HepG2 tumor tissues in nude mice. (a) HepG2 tumor tissue sections were H&E-stained (top panels) or immunostained for cleaved caspase-3 (bottom panels). For H&E staining, areas boxed were enlarged and shown in the upper right corner. (b) Percentage of cells with activated caspase-3 in HepG2 tumor tissues. The results represent the mean of 20 different viewing fields. *P<0.05; **P<0.01. (c) Immunoblot analysis of caspase-3 and PARP in HepG2 tumor tissues

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