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. 2019 Oct 30;38(1):438.
doi: 10.1186/s13046-019-1459-6.

GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway

Jingjing Li  1   2 , Qiujie Wang  1 , Yi Yang  1 , Chong Lei  1 , Fan Yang  1 , Li Liang  1 , Chang Chen  3 , Jie Xia  1 , Kai Wang  4 , Ni Tang  5
Affiliations

GSTZ1 deficiency promotes hepatocellular carcinoma proliferation via activation of the KEAP1/NRF2 pathway

Jingjing Li et al. J Exp Clin Cancer Res. .

Abstract

Background: Glutathione S-transferase zeta 1 (GSTZ1) is the penultimate enzyme in phenylalanine/tyrosine catabolism. GSTZ1 is dysregulated in cancers; however, its role in tumorigenesis and progression of hepatocellular carcinoma (HCC) is largely unknown. We aimed to assess the role of GSTZ1 in HCC and to reveal the underlying mechanisms, which may contribute to finding a potential therapeutic strategy against HCC.

Methods: We first analyzed GSTZ1 expression levels in paired human HCC and adjacent normal tissue specimens and the prognostic effect of GSTZ1 on HCC patients. Thereafter, we evaluated the role of GSTZ1 in aerobic glycolysis in HCC cells on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Furthermore, we assessed the effect of GSTZ1 on HCC proliferation, glutathione (GSH) concentration, levels of reactive oxygen species (ROS), and nuclear factor erythroid 2-related factor 2 (NRF2) signaling via gain- and loss- of GSTZ1 function in vitro. Moreover, we investigated the effect of GSTZ1 on diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) induced hepatocarcinogenesis in a mouse model of HCC.

Results: GSTZ1 was downregulated in HCC, thus indicating a poor prognosis. GSTZ1 deficiency significantly promoted hepatoma cell proliferation and aerobic glycolysis in HCC cells. Moreover, loss of GSTZ1 function depleted GSH, increased ROS levels, and enhanced lipid peroxidation, thus activating the NRF2-mediated antioxidant pathway. Furthermore, Gstz1 knockout in mice promoted DEN/CCl4-induced hepatocarcinogenesis via activation of the NRF2 signaling pathway. Furthermore, the antioxidant agent N-acetylcysteine and NRF2 inhibitor brusatol effectively suppressed the growth of Gstz1-knockout HepG2 cells and HCC progression in Gstz1-/- mice.

Conclusions: GSTZ1 serves as a tumor suppressor in HCC. GSH depletion caused by GSTZ1 deficiency elevates oxidative stress, thus constitutively activating the NRF2 antioxidant response pathway and accelerating HCC progression. Targeting the NRF2 signaling pathway may be a promising therapeutic approach for this subset of HCC.

Keywords: Glutathione; Glutathione S-transferase zeta 1; Hepatocellular carcinoma; KEAP1/NRF2 pathway; Oxidative stress.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Fig. 1
Fig. 1
GSTZ1 is downregulated in human HCC tissues and correlates with poor survival of HCC patients. a Flow diagram of phenylalanine and tyrosine catabolism. b GSTZ1 differential plot. Levels of mRNA Gstz1 expression in tumor tissues are shown with normal tissues for comparison. The colored bars represent tumor (red) and normal (blue) tissues. The data are derived from Firebrowse (http://firebrowse.org/). c Kaplan-Meier overall survival curve based on GSTZ1 expression in TCGA LIHC datasets. Median values of overall survival were compared using the log-rank test. d Representative IHC images of GSTZ1 in HCC tissues and tumor-adjacent normal tissues. Magnifications: 200× and 400×. e GSTZ1 expression in 16 cases of HCC and paired non-tumor tissues. For Western blotting, 50 μg protein was loaded per well. Values represent the mean ± standard deviation (SD) (n = 3, performed in triplicate). PAH, 4-phenylalanine monooxygenase; TAT, tyrosine aminotransferase; 4HPP, 4-hydroxyphenylpyruvate; 4HPPD, 4-hydroxyphenylpyruvate dioxygenase; HGA, homogentisate; HGD, 1,2-homogentisate dioxygenase; MAA, maleylacetoacetate; FAA, fumarylacetoacetate; FAH, fumarylacetoacetate hydrolase; FUMA, fumarate; ACA, acetoacetate; SAA, succinylacetoacetate; NT, non-tumor; T, tumor; RSEM, RNA-seq by expectation maximization; IHC, immunohistochemistry
Fig. 2
Fig. 2
GSTZ1 inhibits cell proliferation in HCC cell lines. a Western blotting showing endogenous GSTZ1 protein expression in HCC cell lines. b GSTZ1 overexpression in Huh7 and MHCC-97H cells, and knockout (KO) of GSTZ1 in HepG2 cells were confirmed through immunoblotting. The overexpression model was established by infecting hepatoma cells with adenoviruses expressing GSTZ1 (AdGSTZ1), and adenoviruses expressing GFP (AdGFP) was used as a control. The knockout model was established with the CRISPR-Cas9 system. c-e The proliferation potential of GSTZ1-overexpressing Huh7, MHCC-97H cells, and GSTZ1-KO HepG2 cells. c Representative images and enumeration of EdU-positive cells. d Cell growth curve. e Representative images and determination of the colony formation capacity. For Western blotting, 50 μg protein was loaded per well. Values represent the mean ± SD (n = 3, performed in triplicate), *p < 0.05, **p < 0.01, Student’s t-test (two groups) or one-way ANOVA followed by Tukey tests (three groups). HCC, hepatocellular carcinoma
Fig. 3
Fig. 3
GSTZ1 suppresses the Warburg effect and ROS accumulation in HCC cell lines. a-b The ECAR and OCR in GSTZ1-overexpressing Huh7 cells (left) and GSTZ1-KO HepG2 cells (right) were determined using the Seahorse XF Cell Energy Phenotype test. GSTZ1-overexpressing Huh7 cells were less glycolytic than AdGFP control cells. In contrast, GSTZ1-KO HepG2 cells were more glycolytic than parental cells. a OCR versus ECAR. b ECAR values. c Representative fluorescence images (left) and quantification (right) of ROS levels in GSTZ1-overexpressing SK-Hep1 (top) and GSTZ1-KO HepG2 cells (bottom) stained with the CellROX Orange fluorescence probe. Immunofluorescence intensity was quantified using ImageJ. Values represent mean ± SD values. d Western blotting revealed the levels of 4-HNE modification in proteins in GSTZ1-overexpressing Huh7 cells (top) and GSTZ1-KO HepG2 cells (bottom). e GSSG/GSH ratio for GSTZ1-overexpressing Huh7 cells (top) and GSTZ1-KO HepG2 cells (bottom) determined with Grx1-roGFP2. f Liver GSH levels in wildtype and Gstz1−/− mice, detected via mass spectrometry. For Western blotting, 50 μg protein was loaded per well. Values represent mean ± SD values (n = 3, performed in triplicate), *p < 0.05, **p < 0.01, Student’s t-test (two groups) or one-way ANOVA followed by Tukey tests (three groups). ECAR, extracellular acidification rate; OCR, oxygen consumption rate; HCC, hepatocellular carcinoma; ROS; reactive oxygen species; GSH, glutathione; GSSG: GSH disulfide
Fig. 4
Fig. 4
GSTZ1 negatively regulates the KEAP1/NRF2 pathway in human HCC and in liver tissue of mice. a–c ARE promoter activities in GSTZ1-overexpressing Huh7 cells (left) and GSTZ1-KO HepG2 cells (right). a ARE promoter activities. The NRF2 activator tBHQ (100 μM for 6 h) and the antioxidant NAC (4 mM for 24 h) were used as positive and negative controls, respectively. b Relative mRNA expression of NRF2 target genes in GSTZ1-OE and GSTZ1-KO HCC cells, determined via qRT-PCR. c Western blotting to assess expression levels of NQO1, the NRF2 target, in total cell extracts, and of NRF2 in cytoplasmic and nuclear cell extracts, indicating the effect of GSTZ1 on NRF2 intracellular localization and transcription. β-actin, β-tubulin, and Lamin B1 served as reference proteins for total, cytoplasmic, and nuclear extracts, respectively. d-f Association between GSTZ1 and NQO1 expression in HCC tissues. d Linear regression analysis showing the negative correlation between GSTZ1 and NQO1 mRNA expression in 40 cases of HCC (p = 0.0197; r = − 0.37, Pearson correlation coefficient). e Representative immunohistochemistry images for GSTZ1 and corresponding NQO1 in HCC tissues and tumor-adjacent normal tissues. f Western blotting for GSTZ1 and NQO1 in 9 pairs of HCC and tumor-adjacent normal tissues. For Western blotting, 50 μg protein was loaded per well. Values represent the mean ± SD (n = 3, performed in triplicate), *p < 0.05, **p < 0.01, Student’s t-test. ARE, antioxidant response element; HCC, hepatocellular carcinoma; qRT-PCR, quantitative reverse transcription polymerase chain reaction
Fig. 5
Fig. 5
GSTZ1 deficiency promotes HCC cell growth via activation of the KEAP1/NRF2 pathway. a ROS levels in Gstz1-KO1 cells treated with antioxidant NAC for 24 h or NRF2 inhibitor brusatol for 12 h. b-d Effect of NAC or Bru on the proliferation potential of Gstz1-KO1 cells. b Representative images and colony formation potential. c Cell growth curve. d Representative images (left) and enumeration (right) of EdU positive cells. The final concentrations of NAC and brusatol were 4 mM and 40 nM, respectively. Values represent means ± SD (n = 3, performed in triplicate), **p < 0.01, one-way ANOVA test. HCC, hepatocellular carcinoma; ROS, reactive oxygen species
Fig. 6
Fig. 6
Knockout of Gstz1 promotes DEN/CCl4-induced hepatocarcinogenesis in vivo via activation of the KEAP1/NRF2 pathway. a Schematic representations of the experimental design for WT and Gstz1−/− mice. b Gross appearances of murine livers. Red arrows indicate tumors. c Liver/body weight ratio (left) and number of tumors (right) in each group. d Representative H&E staining and immunohistochemistry staining of Ki67 in liver tumors. e-f Oxidative stress levels in liver tumors. e Representative fluorescence staining of ROS with CellROX Orange probe in hepatic tumors of WT and Gstz1/− mice (left). Intracellular ROS quantification (right). f 4-HNE modification is all proteins in tumor tissues, analyzed via Western blotting. g NRF2 transcriptional activities in liver tumors. Relative Nqo1 mRNA expression, determined via qRT-PCR (Left). NQO1 expression in total cell extracts, and NRF2 expression in cytoplasmic and nuclear extracts, analyzed via Western blotting (Right). h Representative immunohistochemistry images of GSTZ1 and NQO1 in hepatic tumors. For Western blotting, 30 μg protein was loaded per well. Values represent the mean ± SD (n = 3, performed in triplicate), *p < 0.05, **p < 0.01, Student’s t-test (two groups) or one-way ANOVA followed by Tukey tests (four groups). H&E, hematoxylin and eosin; DEN, diethylnitrosamine; HCC, hepatocellular carcinoma; ROS, reactive oxygen species; 4-HNE, 4-hydroxy-2-nonenal; qRT-PCR, quantitative reverse transcription polymerase chain reaction
Fig. 7
Fig. 7
Proposed model for the activation of the KEAP1/NRF2 pathway by ROS. GSTZ1 deficiency activates the GSH-dependent non-enzymatic bypass to catalyze the conversion of MAA to FAA. The GSH-consuming reaction reduces the antioxidant capacity of cells, thus elevating ROS levels. Increased ROS levels impair the ability of KEAP1 to negatively regulate NRF2, leading to NRF2 nuclear translocation and subsequent transactivation, thus promoting HCC proliferation. ROS, reactive oxygen species; MAA, maleylacetoacetate; FAA, fumarylacetoacetate; GSH, glutathione. Bru, brusatol; NAC, N-acetylcysteine
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