CCAAT/enhancer binding protein α predicts poorer prognosis and prevents energy starvation-induced cell death in hepatocellular carcinoma

Abstract

CCAAT enhancer binding protein α (C/EBPα) plays an essential role in cellular differentiation, growth, and energy metabolism. Here, we investigate the correlation between C/EBPα and hepatocellular carcinoma (HCC) patient outcomes and how C/EBPα protects cells against energy starvation. Expression of C/EBPα protein was increased in the majority of HCCs examined (191 pairs) compared with adjacent nontumor liver tissues in HCC tissue microarrays. Its upregulation was correlated significantly with poorer overall patient survival in both Kaplan-Meier survival (P=0.017) and multivariate Cox regression (P=0.028) analyses. Stable C/EBPα-silenced cells failed to establish xenograft tumors in nude mice due to extensive necrosis, consistent with increased necrosis in human C/EBPα-deficient HCC nodules. Expression of C/EBPα protected HCC cells in vitro from glucose and glutamine starvation-induced cell death through autophagy-involved lipid catabolism. Firstly, C/EBPα promoted lipid catabolism during starvation, while inhibition of fatty acid beta-oxidation significantly sensitized cell death. Secondly, autophagy was activated in C/EBPα-expressing cells, and the inhibition of autophagy by ATG7 knockdown or chloroquine treatment attenuated lipid catabolism and subsequently sensitized cell death. Finally, we identified TMEM166 as a key player in C/EBPα-mediated autophagy induction and protection against starvation.

Conclusion: The C/EBPα gene is important in that it links HCC carcinogenesis to autophagy-mediated lipid metabolism and resistance to energy starvation; its expression in HCC predicts poorer patient prognosis.

Figure 1

Up-regulation of C/EBPα in human primary HCC tissues predicted poorer patient survival. (A) The C/EBPα protein was determined by immunohistochemical staining in HCC tissue microarrays and is summarized in the right panel. (B) A Kaplan-Meier survival analysis showed that the patients with upregulated C/EBPα had poorer overall survival. (C) The HCC patients were ranked into four different groups according to the difference between C/EBPα expression in tumor (TU) and in nontumor (NT) tissues, with 0 for no difference and 3 for the highest up-regulation. The number of patients analyzed in each group and P values are indicated. (D,E) Kaplan-Meier curves showed the overall survival of HCC patients subgrouped by C/EBPα and serum AFP level (D) or C/EBPα and vascular invasion (E). Disc, discordant risk assessments: high C/EBPα expression and low risk predicted by AFP (<300 ng/mL)/vascular invasion or vice versa. Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus.

Figure 2

We found that C/EBPα was required for generation of xenograft tumor. (A) Five million C/EBPα-expressing cells (Hep3B and stable negative control shNC) and C/EBPα–silenced stable cells (sh4 and sh7) were inoculated into the left or right flank, respectively, in Balb/c nude mice. After 30 days of observation, solid tumor nodules were removed from dead mice. Tumor size was determined by a caliper and calculated using the formula volume = (width × width × length)/2. *P < 0.01. The numbers of solid nodules developed at day 30 are shown above. (B) Solid nodules were collected from dead mice at different days and subjected to hematoxylin and eosin staining. Representative histopathological hematoxylin and eosin–stained images with 4× and 20× magnification are shown. Arrows indicate regions impaired by necrotic cell death. H&E, hematoxylin and eosin.

Figure 3

Hepatocarcinoma cells were protected from energy starvation–induced cell death by C/EBPα. (A) The stable C/EBPα-expressing shNC control cells and C/EBPα–silenced cells (sh4 and sh7) were starved in glucose- and glutamine-free Dulbecco's modified Eagle's medium (Glu+Gln starvation) for 2 days. Cell images are shown in the upper panel, followed by cell cycle profiles with the proportion of sub-G₁ dead cells indicated as mean ± standard deviation. Western blotting in the lower panel shows the expression of C/EBPα. (B) Cells were starved in glucose- and glutamine-free Dulbecco's modified Eagle's medium, fetal bovine serum–free Dulbecco's modified Eagle's medium, or fetal bovine serum– and amino acid–double free Earle's Balanced Salt Solution medium for 2 days. (C,D) The C/EBPα-expressing Hep3B and Huh7 and C/EBPα-deficient HepG2 and HCC-M cells were starved as above. (E) Overexpression of C/EBPα using a metallothionein inducible promoter system (induced by 100 µM zinc chloride) in C/EBPα-deficient HCC-M cells resulted in partial protection against starvation-induced cell death. *P < 0.05 compared with cells cultured in full medium. Abbreviations: FBS, fetal bovine serum; EBSS, Earle's Balanced Salt Solution; MT, metallothionein.

Figure 4

Lipid catabolism was essential for C/EBPα-mediated protection against energy starvation. Cells expressing C/EBPα (Hep3B and shNC) and C/EBPα-silenced stable cells (sh4 and sh7) were starved in glucose- and glutamine-free medium. The intracellular levels of adenosine triphosphate (A) and triglyceride (B) were determined in a time course. (C) Fatty acid beta-oxidation rates in Hep3B and sh4 cells were determined after 2-hour starvation. Intracellular levels of acetyl-coenzyme A were determined as in A. (D) The Hep3B cells were either pretreated with the liposynthesis inhibitor C75 (5 µg/mL), simvastatin (statin, 10 µM), and diethylum-belliferyl phosphate (50 µM) for 2 weeks before starvation or treated with the fatty acid beta-oxidation inhibitor etomoxir and ranolazine in starvation medium for 3 days. Dead cells were determined by sub-G₁ assay as above. *P < 0.05 compared with the corresponding Hep3B and shNC cells of the same time point, #P < 0.05 compared with unstarved cells. (E) Oxygen consumption rates in Hep3B shNC cells were determined by Seahorse mitochondrial stress analysis, under basal conditions and in response to the indicated inhibitors (n = 4). Basal and maximum respiration rates are summarized in the right panel. *P < 0.05 compared with resting cells, #P < 0.05 compared with the corresponding starved cells. Abbreviations: ATP, adenosine triphosphate; CoA, coenzyme A; DEBP, diethylum-belliferyl phosphate; Eto, etomoxir; Rano, ranolazine; DMSO, dimethyl sulfoxide; FCCP, fluorocarbonyl cyanide phenylhydrazone; Rot+AA, rotenone and antimycin A; FU, full medium; OCR, oxygen consumption rate.

Figure 5

Autophagy was essential for C/EBPα-mediated lipid catabolism and protection against starvation. (A) Cells expressing C/EBPα (shNC) and C/EBPα-silenced stable cells (sh4 and sh7) were treated with or without 25 µM of the lysosomal inhibitor CQ in glucose- and glutamine-free medium for 3 hours. Flux of LC3-II was determined by western blotting. (B) Cells expressing C/EBPα (Hep3B) and C/EBPα-deficient HepG2 and HCC-M cells were treated as in A. (C) The Hep3B cells were silenced of ATG7 or C/EBPα by specific siRNAs for 2 days before starvation treatment (siNC for nonspecific siRNA) for 3 days. Dead cells were determined by sub-G₁ assay (left panel) and the proteins by western blotting (right panel). (D,E) The Hep3B cells were starved with or without CQ for 2 days. Dead cells were determined (D), and the intracellular triglyceride level was calculated as in Fig. 3B. (F) The shNC and sh4 cells were starved with or without CQ (100 µM). Fatty acid beta-oxidation rates were determined (left panel), and oxygen consumption rates were separately determined by mitochondrial stress assay (n = 4, right panel). Abbreviations: FU, full medium; NG, glucose- and glutamine-free medium; NS, nonspecific staining; DMSO, dimethyl sulfoxide.

Figure 6

In C/EBPα-expressing Hep3B cells TMEM166 was required for the induction of autophagy. (A) The TMEM166 protein was determined by western blotting in stable cell lines. (B-D) In Hep3B cells TMEM166 was silenced by specific siRNA before starvation. The sub-G₁ cell deaths were determined (B), western blotting is shown (C), and intracellular triglyceride levels were measured (D). (E,F) The TMEM166 protein was overexpressed in the low-expressing HCC-M cells and then starved. Abbreviations: FU, full medium; NG, glucose- and glutamine-free medium.