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. 2022 Apr;6(4):665-678.
doi: 10.1002/hep4.1838. Epub 2021 Oct 23.

Impact of Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

Norifumi Iseda  1 , Shinji Itoh  1 , Tomoharu Yoshizumi  1 , Takahiro Tomiyama  1 , Akinari Morinaga  1 , Kyohei Yugawa  1   2 , Masahiro Shimokawa  1   3 , Tomonari Shimagaki  1 , Huanlin Wang  1 , Takeshi Kurihara  1 , Yoshiyuki Kitamura  4 , Yoshihiro Nagao  1 , Takeo Toshima  1 , Noboru Harada  1 , Kenichi Kohashi  2 , Shingo Baba  4 , Kousei Ishigami  4 , Yoshinao Oda  2 , Masaki Mori  1
Affiliations

Impact of Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status

Norifumi Iseda et al. Hepatol Commun. 2022 Apr.

Abstract

We examined phosphorylated nuclear factor erythroid 2-related factor 2 (P-NRF2) expression in surgically resected primary hepatocellular carcinoma (HCC) and investigated the association of P-NRF2 expression with clinicopathological features and patient outcome. We also evaluated the relationship among NRF2, cancer metabolism, and programmed death ligand 1 (PD-L1) expression. In this retrospective study, immunohistochemical staining of P-NRF2 was performed on the samples of 335 patients who underwent hepatic resection for HCC. Tomography/computed tomography using fluorine-18 fluorodeoxyglucose was performed, and HCC cell lines after NRF2 knockdown were analyzed by array. We also analyzed the expression of PD-L1 after hypoxia inducible factor 1α (HIF1A) knockdown in NRF2-overexpressing HCC cell lines. Samples from 121 patients (36.1%) were positive for P-NRF2. Positive P-NRF2 expression was significantly associated with high alpha-fetoprotein (AFP) expression, a high rate of poor differentiation, and microscopic intrahepatic metastasis. In addition, positive P-NRF2 expression was an independent predictor for recurrence-free survival and overall survival. NRF2 regulated glucose transporter 1, hexokinase 2, pyruvate kinase isoenzymes L/R, and phosphoglycerate kinase 1 expression and was related to the maximum standardized uptake value. PD-L1 protein expression levels were increased through hypoxia-inducible factor 1α after NRF2 overexpression in HCC cells. Conclusions: Our large cohort study revealed that P-NRF2 expression in cancer cells was associated with clinical outcome in HCC. Additionally, we found that NRF2 was located upstream of cancer metabolism and tumor immunity.

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Figures

FIG. 1
FIG. 1
IHC staining of P‐NFR2 in patients with HCC.
FIG. 2
FIG. 2
Relationship between P‐NRF2 and SUVmax. The median SUVmax values with P‐NRF2 negativity and positivity were 3.38 (IQR 2.91‐4.21) and 4.89 (IQR 3.66‐6.73), respectively (P < 0.0001).
FIG. 3
FIG. 3
Kaplan‐Meier curves showing the survival of patients with HCC according to the expression of P‐NRF2. (A) RFS in all patients. (B) OS in all patients.
FIG. 4
FIG. 4
Relationship between NRF2 and metabolism in HCC. (A) Heatmap shows distinct differences in mRNA expression in NRF2‐high cells and NRF2‐low cells. (B,C) GLUT1, HK2, PKLR, and PGK1 expression were assessed using quantitative real‐time PCR in NRF2‐overexpressing cells and control cells. Abbreviations: ACLY, ATP Citrate Lyase; ACO1, Aconitase 1; ACO2, Aconitase 2; ACTB, Actin Beta; AGL, Amylo‐Alpha‐1, 6‐Glucosidase, 4‐Alpha‐Glucanotransferase; ALDOB, Aldolase, Fructose‐Bisphosphate B; ALDOC, Aldolase, Fructose‐Bisphosphate C; B2M, Beta‐2‐Microglobulin; BPGM, Bisphosphoglycerate Mutase; CS, Citrate Synthase; Ctrl, control; DLAT, Dihydrolipoamide S‐Acetyltransferase; DLD, Dihydrolipoamide Dehydrogenase; DLST, Dihydrolipoamide S‐Succinyltransferase; ENO1, Enolase 1; ENO1, Enolase 1; ENO3, Enolase 3; FBP1, Fructose‐Bisphosphatase 1; FBP2, Fructose‐Bisphosphatase 2; FH, Fumarate Hydratase; G6PC, Glucose‐6‐Phosphatase Catalytic Subunit ; G6PC3, Glucose‐6‐Phosphatase Catalytic Subunit 3; G6PD, Glucose‐6‐Phosphate Dehydrogenase; GALM, Galactose Mutarotase; GAPDH, Glyceraldehyde‐3‐Phosphate Dehydrogenase; GBE1, 1,4‐Alpha‐Glucan Branching Enzyme 1; GCK, Glucokinase; GPI, Glucose‐6‐Phosphate Isomerase; GSK3A, Glycogen Synthase Kinase 3 Alpha; GSK3B, Glycogen Synthase Kinase 3 Beta; GYS1, Glycogen Synthase 1; GYS2, Glycogen Synthase 2; H6PD, Hexose‐6‐Phosphate Dehydrogenase/Glucose 1‐Dehydrogenase; HK3, Hexokinase 3; HPRT1, Hypoxanthine Phosphoribosyltransferase 1; IDH1, Isocitrate Dehydrogenase (NADP(+)) 1; IDH2, Isocitrate Dehydrogenase (NADP(+)) 2; IDH3A, Isocitrate Dehydrogenase (NAD(+)) 3 Catalytic Subunit Alpha; IDH3B, Isocitrate Dehydrogenase (NAD(+)) 3 Non‐Catalytic Subunit Beta; IDH3G, Isocitrate Dehydrogenase (NAD(+)) 3 Non‐Catalytic Subunit Gamma; MDH1, Malate Dehydrogenase 1; MDH1B, Malate Dehydrogenase 1B; MDH2, Malate Dehydrogenase 2; PC, Pyruvate Carboxylase; PCK1,Phosphoenolpyruvate Carboxykinase 1; PCK2, Phosphoenolpyruvate Carboxykinase 2; PDHA1, Pyruvate Dehydrogenase E1 Subunit Alpha 1; PDHB, Pyruvate Dehydrogenase E1 Subunit Beta; PDK1, Pyruvate Dehydrogenase Kinase 1; PDK2, Pyruvate Dehydrogenase Kinase 2; PDK3, Pyruvate Dehydrogenase Kinase 3; PDK4, Pyruvate Dehydrogenase Kinase 4; PDP2, Pyruvate Dehyrogenase Phosphatase Catalytic Subunit 2; PDPR, Pyruvate Dehydrogenase Phosphatase Regulatory Subunit; PFKL, Phosphofructokinase, Liver Type; PGAM2, Phosphoglycerate Mutase 2; PGK2, Phosphoglycerate Kinase 2; PGLS, 6‐Phosphogluconolactonase; PGM1, Phosphoglucomutase 1; PGM2, Phosphoglucomutase 2; PGM3, Phosphoglucomutase 3; PHKA1, Phosphorylase Kinase Regulatory Subunit Alpha 1; PHKB, Phosphorylase Kinase Regulatory Subunit Beta; PHKG1, Phosphorylase Kinase Catalytic Subunit Gamma 1; PHKG2, Phosphorylase Kinase Catalytic Subunit Gamma 2; PRPS1, Phosphoribosyl Pyrophosphate Synthetase 1; PRPS1L1, Phosphoribosyl Pyrophosphate Synthetase 1 Like 1; PRPS2, Phosphoribosyl Pyrophosphate Synthetase 2; PYGL, Glycogen Phosphorylase L; PYGM, Glycogen Phosphorylase, Muscle Associated; RBKS, Ribokinase; RPE, Ribulose‐5‐Phosphate‐3‐Epimerase; RPIA, Ribose 5‐Phosphate Isomerase A; RPLP0, Ribosomal Protein Lateral Stalk Subunit P0; SDHA, Succinate Dehydrogenase Complex Flavoprotein Subunit A; SDHB, Succinate Dehydrogenase Complex Iron Sulfur Subunit B; SDHC, Succinate Dehydrogenase Complex Subunit C; SDHD, Succinate Dehydrogenase Complex Subunit D; SUCLA2, Succinate‐CoA Ligase ADP‐Forming Subunit Beta; SUCLG1, Succinate‐CoA Ligase GDP/ADP‐Forming Subunit Alpha; SUCLG1, Succinate‐CoA Ligase GDP/ADP‐Forming Subunit Alpha; TKT, Transketolase; TPI1, Triosephosphate Isomerase 1; UGP2, UDP‐Glucose Pyrophosphorylase 2.
FIG. 5
FIG. 5
Overexpression of NRF2 increases PD‐L1 expression through HIF1α. (A) Control and NRF2‐overexpressing Hep3B and HuH7 cell lines were treated with IFN‐γ. (B) NRF2‐overexpressing cells with control and HIF1A knockdown were treated with IFN‐γ.
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