eIF3a mediates HIF1α-dependent glycolytic metabolism in hepatocellular carcinoma cells through translational regulation

Abstract

eIF3a is the largest subunit of eIF3 complex and is a key player in translational control. Recently eIF3a is recognized as a proto-oncogene, which is overexpressed and connected to tumorigenesis of many cancers. However, the mechanistic roles of eIF3a during the tumorigenesis remain largely elusive. Here, we report that depletion of eIF3a significantly reduced HIF1α protein level and cellular glycolysis ability. Mechanistically, we found that eIF3a regulates HIF1α protein synthesis through internal ribosomal entry site (IRES)-dependent translation. Importantly, through analyses of our own sample collection, we found that eIF3a is overexpressed in hepatocellular carcinoma (HCC) tissues, and a high level of eIF3a predicts poor prognosis of HCC patients. TCGA analyses further confirmed that eIF3a is coincident with an elevated activity of HIF1α pathway genes. Collectively, we identify eIF3a as a regulator for glycolysis through HIF1α IRES-dependent translational regulation, which may be a potential therapeutic target for HCC.

Keywords: HCC; HIF1α; eIF3a; glycolysis.

Figure 1

eIF3a depletion led to decreased HIF1α protein level in cancer cells. A. WB analyses of HIF1α, eIF3a and β-Actin (as control) in the indicated Huh-7 cells under CoCl₂ treatment. B. RT-qPCR analyses of HIF1α in the control and eIF3a KD Huh-7 cells under the indicated treatment and data are represented as mean ± SD from three biological replicates. *P < 0.05, T test. C. WB analyses of HIF1α and eIF3a in the control and eIF3a KD Huh-7 cells under the treatment of PHD inhibitor DMOG. β-Actin was used as control. D. WB analyses of HIF1α, eIF3a and β-Actin (as control) in the indicated HepG2 cells under CoCl₂ treatment. E. WB analyses of HIF1α, eIF3a and β-Actin (as control) in the control, eIF3a KD and eIF3a KD Huh-7cells with the indicated rescuing construct under CoCl₂ treatment.

Figure 2

eIF3a depletion led to decreased cellular glycolytic activity in HCC cells. A. RT-qPCR analyses of the indicated genes in the control and eIF3a KD Huh-7 cells and data are represented as mean ± SD from three biological replicates. *P < 0.05; **P < 0.01, T test. B. Glucose uptake analyses using analog 2-NBDG for the indicated Huh-7 cells. 2-NBDG incorporation was determined by flow cytometry. The representative histograms of triplicate samples (left) and geometrical mean fluorescence intensities (right) are shown. **P < 0.01, T test. C, D. Left, kinetic ECAR response of indicated cells to glucose (10 mM), oligomycin (1 μM) and 2-DG (50 mM). Right, quantification of glycolysis, glycolysis capacity and glycolytic reserve of indicated Huh-7 and HepG2 cells, respectively. All data are represented as mean ± SD from three biological replicates. **P < 0.01; ***P < 0.001, T test.

Figure 3

eIF3a regulates HIF1α protein translation through IRES-dependent mechanism. A. WB analyses of HIF1α, eIF3a and β-Actin (as control) in the control and eIF3a KD Huh-7 cells under the indicated treatments. B. Schematic representation of the bicistronic reporter constructs used in the study. C, D. Reporter gene analyses of indicated IRESes activities in the control and eIF3a KD Huh-7 and HepG2 cells, respectively. The ratios of firefly luciferase activities over renilla luciferase activities were calculated and presented as mean ± SD from three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001, T test. E. WB analyses of HIF1α, eIF3a and β-Actin (as control) in the control and eIF3a KD Huh-7 cells stably carrying the vector control or the inducible FLAG-HIF1α (non-IRES) expression construct under indicated treatments. F. Left, kinetic ECAR response of indicated cells to glucose (10 mM), oligomycin (1 μM) and 2-DG (50 mM). Right, quantification of glycolysis, glycolysis capacity and glycolytic reserve of indicated Huh-7 cells. All data are represented as mean ± SD from three biological replicates. *P < 0.05; **P < 0.01; ***P < 0.001, T test.

Figure 4

Elevated eIF3a expression and its correlation with HIF1α activities in the aggressive type of liver cancer. A. IHC analyses of eIF3a levels in 106 HCC tissues. T, tumor, E, cancer embolus and P, paratumor as control. Three representative cases were shown in the upper; the IHC staining intensities of all 106 cases were quantified and shown in the bottom. B. Prognostic analyses of eIF3a expression in 106 HCC patients. C. Pearson correlation analyses of the mRNA levels of eIF3a and indicated HIF1α target genes (Data from TCGA). The correlation coefficient (R) and P value (by two-sided t-test) were indicated. TPM represents transcripts per million.