S-nitrosylation from GSNOR deficiency impairs DNA repair and promotes hepatocarcinogenesis

Abstract

Human hepatocellular carcinoma (HCC) is associated with elevated expression of inducible nitric oxide synthase (iNOS), but the role of nitric oxide in the pathogenesis of HCC remains unknown. We found that the abundance and activity of S-nitrosoglutathione reductase (GSNOR), a protein critical for control of protein S-nitrosylation, were significantly decreased in approximately 50% of patients with HCC. GSNOR-deficient mice were very susceptible to spontaneous and carcinogen-induced HCC. During inflammatory responses, the livers of GSNOR-deficient mice exhibited substantial S-nitrosylation and proteasomal degradation of the key DNA repair protein O(6)-alkylguanine-DNA alkyltransferase. As a result, repair of carcinogenic O(6)-alkylguanines in GSNOR-deficient mice was significantly impaired. Predisposition to HCC, S-nitrosylation and depletion of alkylguanine-DNA alkyltransferase, and accumulation of O(6)-alkylguanines were all abolished in mice deficient in both GSNOR and iNOS. Thus, our data suggest that GSNOR deficiency, through dysregulated S-nitrosylation, may promote HCC, possibly by inactivating a DNA repair system.

Figures

Fig. 1

GSNOR is frequently deficient in human HCC. (A) GSNOR activity in HCC (red) and associated noncancerous liver (green) tissues from 24 patients. P, patient. Data are from 2–4 independent experiments with the mean indicated by the bar. (B) The GSNOR activity in the HCCs is significantly lower than that in the noncancerous liver tissues in the 24 patients (P = 0.01; Wilcoxon rank-sum test). (C) Western blot analysis of GSNOR. NT, non-tumor liver tissue; HCC, cancer tissue.

Fig. 2

GSNOR−/− mice are predisposed to spontaneous hepatocellular tumors. (A) Liver of a GSNOR−/− mouse with two tumors (arrows). (B) Hematoxylin and eosin (H&E)-stained section of HCC from a GSNOR−/− mouse. Scale bar, 100 µm. (C) Hepatocellular (HC) tumor-free survival curves from Kaplan-Meier analysis. Wild-type (WT) (n = 53) versus GSNOR−/− (n = 54), P = 0.007 by Log-rank test. (D, E and F) Incidence of HCC, HCA, and all hepatocellular tumors (All) in all the mice (D), males (E) (26 WT, 29 GSNOR−/−), and females (F) (27 WT, 25 GSNOR−/−). Tumor incidence is shown by a percentage of mice with tumor. Significant differences between wild-type and age-matched GSNOR−/− mice are indicated by one (P < 0.05) or two (P < 0.005) asterisks.

Fig. 3

Spontaneous hepatocellular tumors in the GSNOR−/− background is decreased by deletion of iNOS. GSNOR−/−iNOS−/− (n = 28), GSNOR−/− (n = 29), and wild-type (n = 26) males were analyzed histopathologically. Tumor incidence is shown by a percentage of mice with tumor. Incidence of all hepatocellular tumors (P = 0.007) and of HCC alone (P = 0.013) in GSNOR−/−iNOS−/− mice was significantly lower than in GSNOR−/− mice.

Fig. 4

Diethylnitrosamine-induced tumor development is increased in GSNOR−/− mice. (A and B) Representative livers of male wild-type and GSNOR−/− mice 10 months after DEN (5 µg/g) injection. (C) Number of tumors (≥1 mm) per mouse and (D) maximal tumor diameters (mean ± SE) in DEN-treated wild-type (WT) (n = 13), GSNOR−/− (KO) (n = 10), and GSNOR−/−iNOS−/− (DKO) (n = 14) mice. GSNOR−/− mice develop significantly more tumors than wild-type (P < 0.001) and GSNOR−/−iNOS−/− (P < 0.001) mice; the maximal tumor size in GSNOR−/− is significantly bigger than that in wild-type (P < 0.001) and GSNOR−/−iNOS−/− (P = 0.003).

Fig. 5

O6-alkylguanine-DNA alkyltransferase is depleted in livers of DEN-challenged GSNOR−/− mice. (A) AGT activity in liver lysates of mice 6 days after DEN (37.5 µg/g) injection. Data are mean (± SE) from 4 wild-type (WT), 5 GSNOR−/− (KO), and 3 GSNOR−/−iNOS−/− (DKO) mice. The AGT activity in GSNOR−/− mice is significantly lower than that in wild-type (P < 0.004) and GSNOR−/−iNOS−/− (P < 0.001) mice. (B) Lactate dehydrogenase (LDH) activity in liver lysates of the DEN-challenged mice described in (A). Data are mean (± SD) from 4 wild-type, 3 GSNOR−/−, and 3 GSNOR−/−iNOS−/− mice. (C) Immunoblot of AGT, GSNOR, and β-actin in livers of wild-type, GSNOR−/−, and GSNOR−/−iNOS−/− mice before or 6 days after DEN injection. The amount of liver lysate in the last lane, 7.5 µg, is 30% of that in the other lanes. (D) Immunoblot of AGT and GAPDH in livers of mice 4 days after treatment with DEN or DEN followed by the proteasome inhibitor MG262.

Fig. 6

Repair of O6-ethyldeoxyguanosine in livers of GSNOR−/− mice is impaired. Immuno-slot blot of genomic DNA from livers of wild-type (WT), GSNOR−/−(KO), and GSNOR−/−iNOS−/− (DKO) mice before (−DEN) or 2 and 6 days after DEN injection. The DNA (1 ug per slot) is probed with monoclonal antibodies against O6-ethyldeoxyguanosine (O6-ethyl-dG) and O2-ethyldeoxythymidine (O2-ethyl-dT), respectively.

Fig. 7

S-nitrosylation and inactivation of AGT in livers of GSNOR−/− mice. (A) Immunoblot of AGT and β-actin in livers of wild-type (WT), GSNOR−/− (KO), and GSNOR−/−iNOS−/− (DKO) mice treated with no LPS (−LPS), LPS (+LPS), or LPS followed by the proteasome inhibitor MG262 (+LPS +MG262). (B) Detection of S-nitrosylated AGT (SNO AGT) by the biotin switch assay and AGT immunoblot. Samples were GSNOR−/−iNOS−/− liver lysate treated with no chemical (−), 300 µM cysteine (Cys), 300 µM S-nitroso-cysteine (SNO), 1 mM dithiothreitol (DTT), or 300 µM hydrogen peroxide (H2O2). (Bottom row) Immunoblot of total AGT in each sample. (C) Biotin switch analysis of endogenously S-nitrosylated AGT in livers of LPS-challenged mice. MG262 was injected into some of the mice to inhibit proteasomal degradation of AGT. (Bottom row) Total AGT.