Abnormal nitrosothiol metabolism in hepatocellular carcinoma
Authors: Juliette Martin, Jean-François Dufour
Journal: Journal of Hepatology (2010)

COMMENTARY ON: S-nitrosylation from GSNOR deficiency impairs DNA repair and promotes hepatocarcinogenesis. Wei W, Li B, Hanes MA, Kakar S, Chen X, Liu L. Sci Transl Med 2010;2(19):19ra13. Copyright (2010). Abstract reprinted with permission from AAAS. http://www.ncbi.nlm.nih.gov/pubmed/20371487 Abstract: Human hepatocellular carcinoma (HCC) is associated with elevated expressions 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, was 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. Hepatocellular carcinoma is comprised of a heterogeneous group of tumors [[1]Dufour J.F. Johnson P. Liver cancer: from molecular pathogenesis to new therapies: summary of the EASL single topic conference.J Hepatol. 2010; 52: 296-304Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Complex transcriptomic analyses have led to the molecular classification of hepatocellular tumors into several subtypes [2Boyault S. Rickman D.S. de Reynies A. Balabaud C. Rebouissou S. Jeannot E. et al.Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets.Hepatology. 2007; 45: 42-52Crossref PubMed Scopus (882) Google Scholar, 3Lee J.S. Thorgeirsson S.S. Genome-scale profiling of gene expression in hepatocellular carcinoma: classification, survival prediction, and identification of therapeutic targets.Gastroenterology. 2004; 127: S51-55Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 4Chiang D.Y. Villanueva A. Hoshida Y. Peix J. Newell P. Minguez B. et al.Focal gains of VEGFA and molecular classification of hepatocellular carcinoma.Cancer Res. 2008; 68: 6779-6788Crossref PubMed Scopus (497) Google Scholar]. The multiplicity of tumor subtypes renders the development of treatment strategies especially challenging because the molecules that can be targeted by drugs will differ according to the subtype. In fact, there are few target molecules active in the pathogenesis of HCC that are shared among tumor subtypes. mTOR is one targetable molecule that is shared by many HCC subtypes; it is inhibited by sirolimus and everolimus and is active in almost half of all HCCs studied [[5]Sieghart W. Fuereder T. Schmid K. Cejka D. Werzowa J. Wrba F. et al.Mammalian target of rapamycin pathway activity in hepatocellular carcinomas of patients undergoing liver transplantation.Transplantation. 2007; 83: 425-432Crossref PubMed Scopus (133) Google Scholar]. Another interesting target molecule is Ras, which phosphorylates Raf, the target of sorafenib, and is active in the majority of HCCs [[6]Calvisi D.F. Ladu S. Gorden A. Farina M. Conner E.A. Lee J.S. et al.Ubiquitous activation of Ras and Jak/Stat pathways in human HCC.Gastroenterology. 2006; 130: 1117-1128Abstract Full Text Full Text PDF PubMed Scopus (595) Google Scholar]. As a possible addition to this short list, Limin Liu and co-workers have uncovered a new carcinogenic pathway with therapeutic potential. S-nitroso-glutathione (GSNO) is a metabolite, which is formed by the nitrosylation of GSH by nitric oxide (NO) in the presence of oxygen and metabolized by GSNO reductase (GSNOR). GSNO acts as a NO donor, a reservoir of nitrosyl residues capable of forming proteins adducts. Nitrosylation of Cys residues in proteins serves a post-translational regulatory function in many processes, including the inactivation of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase (AGT). O6-alkylguanine can pair with a thymidine rather than a cytosine and yields G:C to A:T transitions. Liu and colleagues probed the relationship between the tumorigenesis of HCC, the levels of NO, and the S-nitrosylation of AGT. Their experiments were designed to ask whether GSNOR was an influential player in liver tumorigenesis. Liu et al. previously generated mice that harbor a targeted deletion of GSNOR. GNSOR−/− mice accumulate S-nitrosothiols and have increased levels of protein S-nitrosylation [[7]Liu L. Yan Y. Zeng M. Zhang J. Hanes M.A. Ahearn G. et al.Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock.Cell. 2004; 116: 617-628Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar]. Liu now reports that GNSOR−/− mice have a 10-fold higher incidence of spontaneous liver tumors than controls. In an effort to elucidate the mechanisms involved, they generated mice deficient in both GNSOR−/− and inducible nitric oxide synthase (iNOS−/−) and demonstrated that the accumulation of nitroso-compounds, which are mutagenic by virtue of the increased production of O6-alkylguanines, is a critical step. GNSOR−/−/iNOS−/− mice produced much less nitroso-compounds but were no more susceptible to the development of spontaneous or chemically-induced (diethylnitrosamine) liver tumors than wild-type mice. In diethylnitrosamine treated GNSOR−/− mice, the activity of AGT was massively decreased whereas in GNSOR−/−/iNOS−/− mice, AGT activity did not change. Decreased AGT was associated with diminished DNA repair, which was assessed by measuring O6-ethyl dG six days after challenge with diethylnitrosamine. Furthermore, after exposure to LPS to induce iNOS, the level of S-nitrosylated AGT, which represents inactivated AGT, was equally low in GSNOR−/− and controls, absent in GSNOR−/−/iNOS−/− but was rescued by a proteasome inhibitor in GSNOR−/− only. Finally, Liu examined tissues of 24 patients with HCC and showed the activity of GSNOR to be 80–90% lower than in the adjacent non tumoral tissue in 10 patients. This lower GSNOR activity correlated well with a decreased expression of the protein. In these same tissues the expression of AGT was also diminished (Fig. 1). These data open up perspectives for therapeutic applications. In HCC, inducible NOS is frequently activated and nitrite/nitrate levels elevated. This raises the question of the suitability of iNOS as a target molecule. Notwithstanding the genetic approach chosen by Liu et al. the treatment of GSNOR−/− mice with an iNOS inhibitor would have been equally as informative. Further to their observation that the level of nitrosylated AGT is degraded by the proteasome, a remaining question is whether proteasome inhibitors could not only rescue the protein level but also the enzymatic DNA repair activity and protect against the carcinogenesis process in GNSOR−/− mice. Proteasome inhibitors have already proven beneficial in oncology (bortezomide, Velcade®). An even more intriguing question is whether a selective defect of DNA repair in hepatic tumors could be advantageous. Are these tumors amenable to treatment with alkylating agents? Such a strategy has had some success in treating glioblastomas. The expression of AGT is frequently silenced in glioblastomas due to promoter methylation, with the result that some glioblastomas are particularly susceptible to the alkylating drug, temozolomide [[8]Hegi M.E. Diserens A.C. Gorlia T. Hamou M.F. de Tribolet N. Weller M. et al.MGMT gene silencing and benefit from temozolomide in glioblastoma.N Engl J Med. 2005; 352: 997-1003Crossref PubMed Scopus (5432) Google Scholar]. Notwithstanding the important new information delivered in this paper, a few weaknesses stand out. The small number of human HCC samples (24) precludes a firm conclusion regarding the prevalence of altered GSNOR expression in the HCC patient population. The concurrent determination of iNOS in these samples should have been presented. Other important targets of S-nitrosylation, in addition to AGT should have been mentioned. The mechanism by which GSNOR is down-regulated is not addressed in humans. The authors only speculate about a gene deletion in region 4q23, a region of frequent chromosomal deletions, or genetic/epigenetic defects. The control experiment confirming the absence of iNOS in the double knockout model is missing as is immunohistochemistry evidence of GNSOR and AGT deficiency in mouse and human livers. Finally, there is no demonstration of O6-ethyl dG in human samples. Despite these omissions, this work sheds new light on a liver carcinogenic pathway that may be relevant to a majority of patients. The next step should therefore be to focus on identifying those patients whose tumors depend on abnormal S-nitrosothiol metabolism by measuring circulating nitrite/nitrate levels and offer them specific pharmacological intervention accordingly. The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.