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. 2017 Jan 30;6(1):e292.
doi: 10.1038/oncsis.2016.91.

Egr-1 regulates irradiation-induced autophagy through Atg4B to promote radioresistance in hepatocellular carcinoma cells

W-X Peng  1   2 , Y-Y Wan  1 , A-H Gong  2 , L Ge  3 , J Jin  2 , M Xu  3 , C-Y Wu  1
Affiliations

Egr-1 regulates irradiation-induced autophagy through Atg4B to promote radioresistance in hepatocellular carcinoma cells

W-X Peng et al. Oncogenesis. .

Abstract

Although hepatocellular carcinoma (HCC) is usually response to radiation therapy, radioresistance is still the major obstacle that limits the efficacy of radiotherapy for HCC patients. Therefore, further investigation of underlying mechanisms in radioresistant HCC cells is warranted. In this study, we determined the effect of early growth response factor (Egr-1) on irradiation-induced autophagy and radioresistance in HCC cell lines SMMC-7721 and HepG2. We showed that autophagy-related gene 4B (Atg4B) is induced by Egr-1 upon ionizing radiation (IR) in HCC cells. Luciferase reporter assays and chromatin immunoprecipitation (ChIP) revealed that Egr-1 binds to the Atg4B promoter to upregulate its expression in HCC cells. Suppression of Egr-1 function by dominant-negative Egr-1 dampens IR-induced autophagy, cell migration, and increases cell sensitivity to radiotherapy. Together, these results suggest that Egr-1 contributes to HCC radioresistance through directly upregulating target gene Atg4B, which may serve as a protective mechanism by preferential activation of the autophagy.

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Figures

Figure 1
Figure 1
Egr-1 promotes radioresistance in HCC cells. (a) Egr-1 expression was rapidly induced by radiation treatment. Western blot analysis of Egr-1 expression after different doses of IR treatment. (b) Survival of cells was examined by CCK-8 assay. SMMC-7721 and HepG2 cells were infected with Ad-GFP or Ad-DN-Egr-1 followed by IR (8 Gy) treatment, **P<0.01 vs Ad-GFP group. (c, d) Suppression of Egr-1 transcription activity inhibits colony-formation abilities of HCC cells, ***P<0.001 vs Ad-GFP group. (e) Suppression of Egr-1 transcription activity enhances IR- induced apoptosis.
Figure 2
Figure 2
Egr-1 promotes cell migration upon IR. (a, b) Suppression of Egr-1 attenuates migration capability of HCC cells after IR treatment. (a) Photography and (b) quantitative chart of the transwell assay without matrigel coated was employed to detect the cell migration ability in SMMC-7721 and HepG2 cells infected with Ad-GFP or Ad-DN-Egr-1, ***P<0.001 vs Ad-GFP group. (c) Depletion of Egr-1 by Ad-DN-Egr-1 significantly inhibited the expression of Vimentin, N-Cadherin and Snail in SMMC-7721 and HepG2 cells determined by western blots analysis.
Figure 3
Figure 3
IR-induced autophagy facilitates radioresistance of HCC cells. (a) IR treatment induces autophagy as dose-dependent manner. The expression of autophagic marker Atg4B, LC3 and p62 were detected by western blot. (b) Inhibition of autophagy decreases cell viability after IR treatment. SMMC-7721 and HepG2 cells were pre-treated with 3-MA (2 mm) and CQ (30 μM) for 24 h, then cells were exposure to IR at 8 Gy. Cell viability of cell was measured by CCK-8 72 h after IR treatment. (c) Depletion of autophagy promotes IR-induced cell apoptosis. SMMC-7721 and HepG2 cells were pre-treated with 3-MA (2 mM) and CQ (30 μm) for 24 h, then cells were exposure to IR at 8 Gy. The expression of apoptotic markers cleaved caspase-3, Bax, Bcl-2 and autophagy markers Atg4B, p62 and LC3 were explored by western blot.
Figure 4
Figure 4
Egr-1 directly binds to Atg4B promoter. (a) Schematic description of three potential Egr-1 binding sites and the mutation sites on the human Atg4B promoter. (b) Egr-1 regulates Atg4B expression. Atg4B mRNA and protein level were measured by qPCR and western blot, respectively. (c) Atg4B promoter activity under stimulation by IR exposure. (a) SMMC-7721 were infected with Ad-GFP or Ad-DN-Egr-1. (b) Wild-type or mutant Atg4B promoter constructs were transfected into SMMC-7721 cells along with different combinations of expression vectors and/or their vector. (d) Identification of Egr-1 binding to Atg4B promoter in vivo by ChIP assay. Lysates from SMMC-7721 cells were subjected to ChIP by using ChIP assay kit from CST. Sonicated chromatin was used as input, Histone H3 antibody was used as positive control, and IgG was used as negative control (IgG). The band of ChIP-PCR products amplified by Atg4B-ChIP primers were shown.
Figure 5
Figure 5
Egr-1 promotes HCC Cells resistance to IR by facilitating irradiation-induced autophagy. (a) Repression of Egr-1 attenuates IR-induced autophagy. Cells were infected with Ad-GFP or Ad-DN-Egr-1. Western blot examines the expression of p62 and LC3 after IR (8 Gy) treatment. (b, c) Overexpression Egr-1 restores cell apoptosis caused by inhibition of autophagy by 3-MA, CQ or Atg4B siRNA. Cells were infected by Ad-GFP or Ad-Egr-1, followed 3-MA (2 mM), CQ (30 μm) treatment (b) or siRNA (100 nM) transfection. (c) The expression of apoptotic markers C-caspase-3, Bax, Bcl-2 and autophagy markers Atg4B and LC3 were detected by western blot. (d) Overexpression of Egr-1 increases colony-formation ability of HCC cells. Photography and graph indicates colony numbers of each experimental group.
Figure 6
Figure 6
Atg4B is upregulated in human hepatocellular carcinoma specimens. (a) Atg4B expression in normal liver tissue and hepatocellular carcinoma specimens. Images were taken from the Human Protein Atlas (http://www.proteinatlas.org) online database. (b) Oncomine data showing Atg4B expression in normal vs tumor of liver (n=22). (c) Atg4B mRNA expression in the TCGA liver cancer RNAseq (IlluminaHiSeq; N=371) data set.
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