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Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis

  • Maddalena Frau
    Affiliations
    Department of Clinical and Experimental Medicine, Laboratory of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy
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  • Francesco Feo
    Affiliations
    Department of Clinical and Experimental Medicine, Laboratory of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy
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  • Rosa M. Pascale
    Correspondence
    Corresponding author. Address: Dipartimento di Medicina Clinica e Sperimentale e Oncologia, Sezione di Patologia Sperimentale e Oncologia, Università di Sassari, via P. Manzella 4, 07100 Sassari, Italy. Tel.: +39 079228105; fax: +39 079228485.
    Affiliations
    Department of Clinical and Experimental Medicine, Laboratory of Experimental Pathology and Oncology, University of Sassari, Sassari, Italy
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Open AccessPublished:May 09, 2013DOI:https://doi.org/10.1016/j.jhep.2013.04.031

      Summary

      Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients’ survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC.

      Abbreviations:

      HCC (hepatocellular carcinoma), ASH (alcoholic steatohepatitis), MDD (methyl deficient diet), SAM (S-adenosylmethionine), MAT (methionine adenosyltransferase), SAH (S-adenosylhomocysteine), SAHH (SAH hydroxylase), GSH (reduced glutathione), BHMT (betaine-homocysteine methyltransferase), MTHF-HMT (5-methyltetrahydrofolate homocysteine methyltransferase), 5′-MTA (5′-methylthioadenosine), CBS (cystathionine β-synthase), PHB1 (prohibitin 1), VLDL (very low density lipoproteins), LDL (low density lipoproteins), PH (partial hepatectomy), DN (dysplastic nodule), GNMT (glycine N-methyltransferase), JAK (Janus kinase), STAT1 (signal transducer and activator of transcription), LKB1 (serine/threonine protein kinase 11), ERK (extracellular signal-regulated kinase), p90RSK (ribosomal protein S6 kinase polypeptide 2), RASGRP3 (RAS guanyl releasing protein 3), HGF (hepatocyte growth factor), MAPK (mitogen-activated protein kinase), PI3K (phosphatadylinositol 3-kinase), AKT (V-AKT murine thymoma viral oncogene homolog), SP1 (specificity protein 1), c-Mybl2 (V-MYB avian myeloblastosis viral oncogene homolog-like 2), NF-kB (nuclear factor kB), AP-1 (activator protein-1), TNFα (tumor necrosis factor α), RBP (mRNA-binding proteins), AUF1 (AUrich RNA binding factor 1), HuR (Hu antigen R), GI (genomic instability), ODC (ornithine decarboxylase), BAX (BCL2-associated x protein), FAS (tumor necrosis factor receptor superfamily, member 6), AP (apurinic/apyrimidinic), APEX1 (endonuclease redox effector APE1/REF-1/APEX1), EGR-1 (early growth response protein-1), ROS (reactive oxygen species), CDC2 (cell division cycle 2), NOS (nitric oxide synthase), AMPK (AMP activated protein kinase), PFK-2 (phosphofructokinase 2), mTORC2 (mammalian target of rapamycin complex), TSC1 (hamartin), TSC2 (tuberin), IKK (inhibitor of kappa light chain gene enhancer in B cells, kinase of), BAK (BCL2 antagonist killer), BCL2 (B-cell cell/lymphoma 2), XIAP (inhibitor of apoptosis, x-linked), USP7 (Ubiquitin-specific-processing protease 7), MDM2 (mouse double minute 2 homolog), NASH (non-alcoholic steatohepatitis), PP2A (protein phosphatase 2A), Spp1 (secreted phosphoprotein 1), DUSP1 (dual-specificity phosphatase 1), SKP2 (S-phase kinase-associated protein 2), CSK1 (CDC28 protein kinase b1), FOXM1 (forkhead box M1B), HIF-1α (hypoxia-inducible factor 1, alpha subunit), MAFK (V-MAF avian musculoaponeurotic fibrosarcoma oncogene family, protein K), PRMT5 (protein arginine methyltransferase 5), JUN (V-JUN avian sarcoma virus 17 oncogene homolog), PIAS1 (protein inhibitor of activated STAT1), Mtap (5’-Methylthioadenosine phosphorylase), HCCB (HCC with better prognosis), HCCP (HCC with poorer prognosis), SL (surrounding liver), ASO (antisense oligonucleotide), Sdc (SAM dacarboxylase), Smr (spermine synthase), Sms (spermidine synthase), PCNA (proliferating cell nuclear antigen)

      Keywords

      Introduction

      Hepatocellular carcinoma (HCC) is a frequent and fatal human cancer, with 0.25–1 million newly diagnosed cases each year [
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      Complex relationships between genetic, etiologic, and environmental risk factors create genotypic and phenotypic heterogeneity within human HCC [
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      ]. Consequently, evaluation of pathogenetic mechanisms and identification of prognostic subtypes of HCC are difficult. A valuable contribution to explore HCC pathogenesis is provided by rodent models in which premalignant and malignant lesions exhibit low heterogeneity, without disturbing environmental influences. [
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      Previous observations that ethionine, an antagonist of methionine, causes cancer [
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      ] cause steatohepatitis, followed by HCC development even in absence of carcinogens administration, encouraged studies on mechanisms regulating availability of the major methyl donor S-adenosylmethionine (SAM) and its role in liver injury, including hepatocarcinogenesis. This review provides an interpretive analysis of recent advances on deregulation of SAM metabolism in liver injury predisposing to liver cancer and determining HCC prognosis. We explore the molecular mechanisms involved in SAM antitumor effect and their contribution to identify new putative prognostic markers and opportunities for targeted therapies.

      Metabolism of S-adenosylmethionine

      Liver is the main source of SAM, synthesized from methionine and ATP in a reaction catalyzed by methionine adenosyltransferases (MATs) [
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      ] (Fig. 1). SAM may be decarboxylated and then channeled into polyamine synthesis, or converted to S-adenosylhomocysteine (SAH) during transmethylation reactions. A reversible reaction catalyzed by SAHH converts SAH to homocysteine and adenosine. Homocysteine may be channeled into the trans-sulfuration pathway leading to cystathionine and GSH synthesis. Alternatively, BHMT catalyzes methionine and dimethylglicine synthesis from homocysteine plus betaine. Homocysteine plus 5-methyltetrahydrofolate leads to methionine and tetrahydrofolate synthesis in a reaction catalyzed by MTHF-HMT. SAH and 5′-MTA, a product of polyamine biosynthesis, may inhibit transmethylation reactions. Interestingly, low SAM levels favor homocysteine re-methylation, whereas high SAM levels activate CBS, whose Km for SAM is 1.2–2 mM, much higher than that of MTHF-HMT (60 μM).
      Figure thumbnail gr1
      Fig. 1Methionine metabolism. SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine; THF, tetrahydrofolate; MTHF, methyl-THF; DMTHF, dimethyl-THF; GN, glycine; DMGN, dimethyl GN; GNMT, glycine N-methyltransferase; SN, sarcosine; Dec-SAM, decarboxylated SAM; SPR, spermine; SPD, spermidine; 5′-MTA, 5′-methylthioedenosine; MAT, methionine adenosyltransferase; MT, methyltransferase; SAHH, SAH hydrolase; CBS, cystathionine beta-synthase; BHMT, betaine-homocysteine methyltransferase; MTHF-HMT, 5-methyltetrahydrofolate homocysteine methyltransferase; MTHFR, methyltetrahydrofolate reductase.; SDC, SAM decarboxylase; SRS, spermine synthase; SDS, spermidine synthase; ODC, ornithine decarboxylase.
      Liver-specific MAT1A encodes for the isozymes MATI and MATIII, tetramer and dimer of the subunit α1, respectively [
      • Ramani K.
      • Mato J.M.
      • Lu S.C.
      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ] (Fig. 1). MAT2A encodes for a α2-subunit, the widely distributed enzyme MATII isoform. MAT2A expression prevails in fetal liver and is substituted by MAT1A in adult liver [
      • Ramani K.
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      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ,
      • LeGros L.
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      Regulation of the human MAT2B gene encoding the regulatory b subunit of methionine adenosyltransferase, MAT II.
      ]. MATI and MATIII isozymes have intermediate (23 μM–1 mM) and high (215 μM–7 mM) Km for methionine, respectively. Thus, physiological liver SAM level (∼60 μM) has low inhibitory effect on MATI and stimulates MATIII activity [
      • Ramani K.
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      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
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      Regulation of the human MAT2B gene encoding the regulatory b subunit of methionine adenosyltransferase, MAT II.
      ]. MATII has the lowest Km (∼4–10 μM) and may be inhibited by the reaction product [
      • Ramani K.
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      • Lu S.C.
      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ]. A third gene, MAT2B, encodes for a β-subunit without catalytic action, which regulates MATII by lowering its Km for methionine and Ki for SAM [
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      Regulation of the human MAT2B gene encoding the regulatory b subunit of methionine adenosyltransferase, MAT II.
      ]. Therefore, β association with the α-subunit renders MATII more susceptible to inhibition by SAM [
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      Regulation of the human MAT2B gene encoding the regulatory b subunit of methionine adenosyltransferase, MAT II.
      ].
      Noticeably, the recent discovery of correlations between GNMT, the main enzyme involved in hepatic SAM catabolism (Fig. 1), and MAT1A, and GNMT and BHMT hepatic proteins, supports a coordinate regulation of methionine cycle enzymes as SAM level determinants [
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      ].

      Effects of variations of SAM:SAH ratio

      Treatment of rats with MDD causes pronounced liver SAM decrease and reduced SAM:SAH ratio [
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      Tissue levels of S-adenosylmethionine and S-adensylhocysteine in rats fed choline-deficient, aminoacid-defined diets for one to five weeks.
      ], lipid peroxidation [
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      Induction of liver cancer by a diet deficient of choline and methionine without added carcinogens.
      ], and fall in phosphatidycholine synthesis because of choline lack and decrease in phosphatidylethanolamine transmethylation. Consequent reduction of lipoprotein assembly and synthesis of membranes involved in lipoprotein secretion leads to steatohepatitis [
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      ]. Lipid peroxidation and SAM decrease also contribute to steatohepatitis by affecting mitochondrial function necessary for fatty acids oxidation. SAM contributes to the stability of PHB1 [
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      ], crucial for maintenance of normal mitochondrial function. SAM pathogenetic role in steatohepatitis is confirmed by the observation that SAM treatment of hepatocytes isolated from MDD-fed rats induces phosphatidycholine synthesis, VLDL and LDL secretion, and decrease in cytoplasmic triacylglycerol [
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      Role of phosphatidylethanolamine methylation in the synthesis of phosphatidylcholine by hepatocytes isolated from choline-deficient rats.
      ].
      Changes in SAM levels are also involved in ASH pathogenesis. In the pre-fibrotic stage of alcoholic rat liver injury, increase of MATII activity, without change of MATI/III activity, is associated with low SAM/SAH ratio, global DNA hypomethylation, c-Myc upregulation, and DNA strand break [
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      Changes in methionine adenosyltransferase and S-adenosylmethionine homeostasis in alcoholic rat liver.
      ]. SAM administration protects from ASH [
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      Effect of the variations of S-adenosyl-L-methionine liver content on fat accumulation and ethanol metabolism in ethanol-intoxicated rats.
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      ,
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      Effects of S-adenosylmethionine on liver methionine metabolism and steatosis with ethanol-induced liver injury in rats.
      ]. Studies on human ASH were inconclusive [
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      ], but long-term treatment with SAM improves survival or delays liver transplantation in patients with alcoholic liver cirrhosis, especially in those with less advanced disease [
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      ]. As concerns chronic hepatitis C, SAM addition with/without betaine to standard therapy with PegIFNa and ribavirin enhances treatment efficacy [
      • Filipowicz M.
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      ]. Further, a biological basis for HCC prevention by SAM in hepatitis B is proposed by the observation that HBx upregulates MAT2A and MAT2β, and reduces MAT1A expression and SAM production in hepatoma cells in vitro [
      • Liu Q.
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      • Wang D.
      • Ma L.
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      The X protein of hepatitis B virus inhibits apoptosis in hepatoma cells through enhancing the methionine adenosyltransferase 2A gene expression and reducing S-adenosylmethionine production.
      ]. Moreover, parenterally administered SAM protects rodents against D-galactosamine [
      • Stramentinoli G.
      • Gualano M.
      • Ideo G.
      Protective role of S-adenosyl-lmethionine on liver injury induced by D-galactosamine in rats.
      ], acetaminophen [
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      Protective role of S-adenosyl-l-methionine against acetaminophen induced mortality and hepatotoxicity in mice.
      ], and CCl4 [
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      • Caballeria J.
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      S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.
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      ] toxicity.
      Different researches showed low SAM/SAH ratio, global DNA hypomethylation, and c-Myc overexpression 0.5 h after partial hepatectomy (PH) in rats fed adequate diet [
      • Garcea R.
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      • Pascale R.M.
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      • Puddu M.
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      Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine: S-adenosylhomocysteine ratio.
      ,
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      • Cai J.
      • Lu S.C.
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      ]. These changes reached a peak at 5–12 h and then progressively returned to pre-PH levels. Maximum c-Ha-Ras and c-Ki-Ras expression occurred 24–30 h after PH, roughly coincident with DNA synthesis peak [
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Simile M.M.
      • Puddu M.
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      • et al.
      Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine: S-adenosylhomocysteine ratio.
      ]. Notably, significant decrease in SAM level and SAM/SAH ratio also occurs in the liver of rats fed adequate diet, during hepatocarcinogenesis induced by different carcinogens and experimental models [
      • Feo F.
      • Garcea R.
      • Pascale R.M.
      • Pirisi L.
      • Daino L.
      • Donaera A.
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      ,
      • Garcea R.
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      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
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      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ,
      • Pascale R.M.
      • Simile M.M.
      • Satta G.
      • Seddaiu M.A.
      • Daino L.
      • Pinna G.
      • et al.
      Comparative effects of l-methionine, S-adenosyl-l-methionine and 5′-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.
      ,
      • Simile M.M.
      • Saviozzi M.
      • De Miglio M.R.
      • Muroni M.R.
      • Nufris A.
      • Pascale R.M.
      • et al.
      Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats.
      ], and persists in dysplastic nodules (DN) and HCC several weeks after arresting carcinogen administration [
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Simile M.M.
      • Puddu M.
      • Ruggiu M.E.
      • et al.
      Protooncogene methylation and expression in regenerating liver and preneoplastic liver nodules induced in the rat by diethylnitrosamine: effect of variations of S-adenosylmethionine: S-adenosylhomocysteine ratio.
      ,
      • Simile M.M.
      • Saviozzi M.
      • De Miglio M.R.
      • Muroni M.R.
      • Nufris A.
      • Pascale R.M.
      • et al.
      Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats.
      ,
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. Furthermore, SAM decrease, with no change in SAH, occurs in human HCC and at a lower extent in the cirrhotic liver surrounding tumor [
      • Calvisi D.F.
      • Simile M.M.
      • Ladu S.
      • Pellegrino R.
      • De Murta V.
      • Pinna F.
      • et al.
      Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis.
      ].
      Normal SAM level and SAM/SAH ratio may be reconstituted by the administration of highly purified SAM during hepatocarcinogenesis [
      • Pascale R.M.
      • Simile M.M.
      • Satta G.
      • Seddaiu M.A.
      • Daino L.
      • Pinna G.
      • et al.
      Comparative effects of l-methionine, S-adenosyl-l-methionine and 5′-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.
      ,
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Simile M.M.
      • Puddu M.
      • Frassetto S.
      • et al.
      Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.
      ,
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. This treatment results in sharp decrease of preneoplastic liver lesions and prevention of DN and HCC development, associated with decrease in labeling index and increase in apoptosis of preneoplastic cells [
      • Pascale R.M.
      • Simile M.M.
      • Satta G.
      • Seddaiu M.A.
      • Daino L.
      • Pinna G.
      • et al.
      Comparative effects of l-methionine, S-adenosyl-l-methionine and 5′-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.
      ,
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Simile M.M.
      • Puddu M.
      • Frassetto S.
      • et al.
      Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.
      ,
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ,
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Feo F.
      Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine.
      ]. In diethylnitrosamine-initiated rats, SAM persistently prevents development of preneoplastic lesions promoted by thiobenzamide [
      • Simile M.M.
      • Saviozzi M.
      • De Miglio M.R.
      • Muroni M.R.
      • Nufris A.
      • Pascale R.M.
      • et al.
      Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats.
      ]. Moreover, human HCC cell lines transfected with MAT1A or cultured in the presence of SAM undergo strong proliferation restraint [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ,
      • Frau M.
      • Tomasi M.L.
      • Simile M.M.
      • Demartis M.I.
      • Salis F.
      • Latte G.
      • et al.
      Role of transcriptional and posttranscriptional regulation of methionine adenosyltransferases in liver cancer progression.
      ]. These observations were recently confirmed by Lu et al. [
      • Lu S.C.
      • Ramani K.
      • Ou X.
      • Lin M.
      • Yu V.
      • Ko K.
      • et al.
      S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model.
      ], which induced orthotropic HCC development by injecting human HCC cell line, H4IIE, in the rat liver parenchyma. Continuous SAM intravenous infusion after tumor cell injection inhibited HCC formation. However, SAM infusion for 24 days did not affect the size of already established tumors. This was explained by a compensatory induction of hepatic GNMT that prevents SAM accumulation. HuH7 cell transfectants, stably overexpressing MAT1A, exhibited higher SAM levels and lower DNA synthesis than control cells [
      • Li J.
      • Ramani K.
      • Sun Z.
      • Zee C.
      • Grant E.G.
      • Yang H.
      • et al.
      Forced expression of methionine adenosyltransferase 1A in human hepatoma cells suppresses in vivo tumorigenicity in mice.
      ]. Lower HCC growth rates, microvessel density, and CD31 and Ki-67 staining, and higher apoptosis occurred in MAT1A-transfected than in control tumors [
      • Li J.
      • Ramani K.
      • Sun Z.
      • Zee C.
      • Grant E.G.
      • Yang H.
      • et al.
      Forced expression of methionine adenosyltransferase 1A in human hepatoma cells suppresses in vivo tumorigenicity in mice.
      ].
      Fall in MAT1A expression associated with MAT2A upregulation occurs in liver cirrhosis and rodent and human HCC, leading to decrease in MAT1A:MAT2A ratio (called MAT1A/MAT2A switch) [
      • Calvisi D.F.
      • Simile M.M.
      • Ladu S.
      • Pellegrino R.
      • De Murta V.
      • Pinna F.
      • et al.
      Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis.
      ,
      • Mato J.M.
      • Corrales F.J.
      • Lu S.C.
      • Avila M.A.
      S-Adenosylmethionine: a control switch that regulates liver function.
      ,
      • Lu S.C.
      • Mato J.M.
      Role of methionine adenosyltransferase and S-adenosylmethionine in alcohol-associated liver cancer.
      ]. MATI/III downregulation, secondary to oxidation of cysteine residue in ATP binding site, and GSH fall occur in the cirrhotic liver [
      • Mato J.M.
      • Alvarez L.
      • Ortiz P.
      • Mingorance J.
      • Durán C.
      • Pajares M.A.
      S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis.
      ,
      • Mato J.M.
      • Corrales F.
      • Martin-Duce A.
      • Ortiz P.
      • Pajares M.A.
      • Cabrero C.
      Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: part I. Biochemical implications.
      ]. SAM administration reconstitutes the GSH pool, protects MATI/III [
      • Mato J.M.
      • Alvarez L.
      • Ortiz P.
      • Mingorance J.
      • Durán C.
      • Pajares M.A.
      S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis.
      ,
      • Mato J.M.
      • Corrales F.
      • Martin-Duce A.
      • Ortiz P.
      • Pajares M.A.
      • Cabrero C.
      Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: part I. Biochemical implications.
      ], and has beneficial effects against liver fibrosis both in rats and humans [
      • Simile M.M.
      • Banni S.
      • Angioni E.
      • Carta G.
      • De Miglio M.R.
      • Muroni M.R.
      • et al.
      5-Methylthioadenosine administration prevents lipid peroxidation and fibrogenesis induced in rat liver by carbon-tetrachloride intoxication.
      ,
      • Mato J.M.
      • Alvarez L.
      • Ortiz P.
      • Mingorance J.
      • Durán C.
      • Pajares M.A.
      S-adenosyl-L-methionine synthetase and methionine metabolism deficiencies in cirrhosis.
      ,
      • Mato J.M.
      • Corrales F.
      • Martin-Duce A.
      • Ortiz P.
      • Pajares M.A.
      • Cabrero C.
      Mechanisms and consequences of the impaired trans-sulphuration pathway in liver disease: part I. Biochemical implications.
      ,
      • Corrales F.
      • Giménez A.
      • Alvarez L.
      • Caballeria J.
      • Pajares M.A.
      • Andreu H.
      • et al.
      S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.
      ]. Being inhibited by its reaction product, MATII upregulation cannot compensate for MATI/III decrease [
      • Finkelstein J.D.
      Metabolic regulatory properties of S-adenosylmethionine and S-adenosylhomocysteine.
      ]. Consequently, decrease in MATI/III:MATII activity ratio strongly contributes, together with the increase in SAM decarboxylation for polyamine synthesis, to sharp SAM decrease [
      • Garcea R.
      • Pascale R.M.
      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
      • et al.
      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ]. Overall, these important findings suggest that MAT1A/MAT2A switch and fall in SAM level are strongly involved in hepatocarcinogenesis. Accordingly, the MAT1A-KO mouse model, characterized by chronic SAM deficiency, even in the presence of MAT2A induction, exhibits hepatomegaly without histologic abnormalities at 3 months of age, and macrovesicular steatosis involving 25–50% of hepatocytes and mononuclear cell infiltration in periportal areas, at 8 months [
      • Lu S.C.
      • Alvarez L.
      • Huang Z.Z.
      • Chen L.
      • An W.
      • Corrales F.J.
      • et al.
      Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation.
      ]. HCC develop in many of these mice at 18 months of age [
      • Lu S.C.
      • Alvarez L.
      • Huang Z.Z.
      • Chen L.
      • An W.
      • Corrales F.J.
      • et al.
      Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation.
      ].
      Remarkably, recent findings showed oxidative stress, steatosis, and fibrosis, followed by HCC development [
      • Martinez-Chantar M.
      • Vázquez-Chantada M.
      • Ariz U.
      • Martínez N.
      • Varela M.
      • Luka Z.
      • et al.
      Loss of the GNMT gene leads to steatosis and hepatocellular carcinoma in mice.
      ], and increased susceptibility to aflatoxin B1-related HCC [
      • Liu S.P.
      • Li Y.S.
      • Lee C.M.
      • Yen C.H.
      • Liao Y.J.
      • Huang S.F.
      • et al.
      Higher susceptibility to aflatoxin B(1)-related hepatocellular carcinoma in glycine N-methyltransferase knockout mice.
      ] in GNMT-KO mice, characterized by elevated SAM liver levels. Global DNA hypomethylation, aberrant expression of DNA methyltransferases 1 and 3b [
      • Liao Y.J.
      • Liu S.P.
      • Lee C.M.
      • Yen C.H.
      • Chuang P.C.
      • Chen C.Y.
      • et al.
      Characterization of a glycine N-methyltransferase gene knockout mouse model for hepatocellular carcinoma: Implications of the gender disparity in liver cancer susceptibility.
      ], aberrant hypermethylation of inhibitors of Ras and JAK/STAT pathways [
      • Lu S.C.
      • Alvarez L.
      • Huang Z.Z.
      • Chen L.
      • An W.
      • Corrales F.J.
      • et al.
      Methionine adenosyltransferase 1A knockout mice are predisposed to liver injury and exhibit increased expression of genes involved in proliferation.
      ], and upregulation of Beta-catenin, cyclin D1, and c-Myc [
      • Martínez-López N.
      • García-Rodríguez J.L.
      • Varela-Rey M.
      • Gutiérrez V.
      • Fernández-Ramos D.
      • Beraza N.
      • et al.
      Hepatoma cells from mice deficient in glycine N-methyltransferase have increased RAS signaling and activation of liver kinase B1.
      ] occur in these mice during HCC development. Furthermore, Ras-mediated LKB1 overactivation, associated with Erk, p90Rsk, and RasGpr3 expression, promotes the proliferation of GNMT-deficient hepatoma cells [
      • Liao Y.J.
      • Liu S.P.
      • Lee C.M.
      • Yen C.H.
      • Chuang P.C.
      • Chen C.Y.
      • et al.
      Characterization of a glycine N-methyltransferase gene knockout mouse model for hepatocellular carcinoma: Implications of the gender disparity in liver cancer susceptibility.
      ]. Interestingly, impairment of liver regeneration in GNMT-KO mice stimulates dormant stem/progenitor cells to replicate, a situation that could favor HCC formation [
      • Martinez-Chantar M.L.
      • Lu S.C.
      • Mato J.M.
      • Luka Z.
      • Wagner C.
      • French B.A.
      • et al.
      The role of stem cells/progenitor cells in liver carcinogenesis in glycine N-methyltransferase deficient mice.
      ]. High liver transaminases, liver injury, fibrosis, and HCC development have been documented in children with GNMT mutation [
      • Lu S.C.
      • Mato J.M.
      S-adenosylmethionine in liver health, injury, and cancer.
      ].
      MAT2A upregulation may also contribute to HCC cell proliferation. In H35 hepatocellular carcinoma cells, MAPK and PI3K/AKT pathways are necessary for HGF-induced cell proliferation and MAT2A upregulation [
      • Pañeda C.
      • Gorospe I.
      • Herrera B.
      • Nakamura T.
      • Fabregat I.
      • Varela-Nieto I.
      Liver cell proliferation requires methionine adenosyltransferase 2A mRNA up-regulation.
      ]. Inhibition of these pathways in H35 cells and fetal liver hepatocytes leads to proliferation restraint, MAT2A under-regulation and MAT1A overexpression [
      • Pañeda C.
      • Gorospe I.
      • Herrera B.
      • Nakamura T.
      • Fabregat I.
      • Varela-Nieto I.
      Liver cell proliferation requires methionine adenosyltransferase 2A mRNA up-regulation.
      ]. Moreover, transfection of MAT2B in HuH7 cells that do not express this subunit results in β-subunit interaction with α2-subunit, DNA synthesis increase, and SAM production decrease, whereas β-subunit downregulation in HepG2 cells, overexpressingMAT2B, diminishes DNA synthesis [
      • Martínez-Chantar M.L.
      • García-Trevijano E.R.
      • Latasa M.U.
      • Martín-Duce A.
      • Fortes P.
      • Caballería J.
      • et al.
      Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.
      ].

      Molecular mechanisms underlying the deregulation of MAT genes

      The presence of numerous CpGs MAT1A and MAT2A promoters motivated the evaluation of the epigenetic regulation of their expression in HCC [
      • Martínez-Chantar M.L.
      • García-Trevijano E.R.
      • Latasa M.U.
      • Martín-Duce A.
      • Fortes P.
      • Caballería J.
      • et al.
      Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma.
      ,
      • Torres L.
      • Avila M.A.
      • Carretero M.V.
      • Latasa M.U.
      • Caballería J.
      • López-Rodas G.
      • et al.
      Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.
      ,
      • Tomasi M.L.
      • Li T.W.
      • Li M.
      • Mato J.M.
      • Lu S.C.
      Inhibition of human methionine adsenosyltransferase 1A transcription by coding region methylation.
      ]. MAT1A downregulation in the cirrhotic liver of CCl4-treated rats and human HepG2 cell line is associated with CCGG sequences methylation in MAT1A promoter [
      • Torres L.
      • Avila M.A.
      • Carretero M.V.
      • Latasa M.U.
      • Caballería J.
      • López-Rodas G.
      • et al.
      Liver-specific methionine adenosyltransferase MAT1A gene expression is associated with a specific pattern of promoter methylation and histone acetylation: implications for MAT1A silencing during transformation.
      ]. In HuH7 cells, MAT1A downregulation was attributed to CCGG methylation at +10 and +80 of the coding region [
      • Tomasi M.L.
      • Li T.W.
      • Li M.
      • Mato J.M.
      • Lu S.C.
      Inhibition of human methionine adsenosyltransferase 1A transcription by coding region methylation.
      ]. MAT2A upregulation in human HCC was associated with CCGG hypomethylation of the gene promoter [
      • Yang H.
      • Huang Z.Z.
      • Zeng Z.
      • Chen C.
      • Selby R.R.
      • Lu S.C.
      Role of promoter methylation in increased methionine adenosyltransferase 2A expression in human liver cancer.
      ]. Recent work [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ] in which the methylation status of all CpGs of MAT1A and MAT2A promoters was examined in rat and human HCC, confirmed these results and showed Mat1A/Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation in Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast growing F344 rats HCC. In slowly growing BN rat HCC, very low changes in Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ]. Furthermore, highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ].
      Various trans-activating factors such as Sp1, c-Mybl2, NF-kB, and AP-1 participate in MAT2A transcriptional upregulation in HCC [
      • Ramani K.
      • Mato J.M.
      • Lu S.C.
      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ]. The mechanisms regulating MAT2B expression are poorly known. Sp1 activates MAT2B promoter [
      • LeGros L.
      • Halim A.B.
      • Chamberlin M.E.
      • Geller A.
      • Kotb M.
      Regulation of the human MAT2B gene encoding the regulatory b subunit of methionine adenosyltransferase, MAT II.
      ]. MAT2B has two dominant splicing variants, variant 1 (V1) and variant 2 (V2), upregulated in HCC. TNFα induces the transcription of only MAT2B V1 by mechanisms involving AP-1 and NF-kB [
      • Ramani K.
      • Mato J.M.
      • Lu S.C.
      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ]. MAT2B V1 promoter expression is stimulated by leptin and inhibited by SAM by mechanisms involving ERK and AKT signaling [
      • Ramani K.
      • Mato J.M.
      • Lu S.C.
      Role of methionine adenosyltransferase genes in hepatocarcinogenesis.
      ].
      Accumulating evidence indicates that a class of mRNA-binding proteins (RBPs) plays a pivotal role in post-transcriptional deregulation of gene expression in cancer cells. Among RBPs, AUF1 enhances mRNA decay, whereas HuR selectively binds to AUrich elements promoting mRNA stabilization [
      • Lal A.
      • Mazan-Mamczarz K.
      • Kawai T.
      • Yang X.
      • Martindale J.L.
      • Gorospe M.
      Concurrent versus individual binding of HuR and AUF1 to common labile target mRNAs.
      ,
      • Kim M.E.
      • Hur J.
      • Jeo S.
      Emerging roles of RNA and RNA-binding protein network in cancer cells.
      ,
      • Vázquez-Chantada M.
      • Fernández-Ramos D.
      • Embade N.
      • Martínez-Lopez N.
      • Varela-Rey M.
      • Woodhoo A.
      • et al.
      HuR/-methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.
      ]. Remarkably, a recent work [
      • Vázquez-Chantada M.
      • Fernández-Ramos D.
      • Embade N.
      • Martínez-Lopez N.
      • Varela-Rey M.
      • Woodhoo A.
      • et al.
      HuR/-methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.
      ] showed Mat1A mRNA decrease in the fetal rat liver, associated with an increase in its interaction with AUF1 and an increase in Mat2A mRNA and its interaction with HuR [
      • Vázquez-Chantada M.
      • Fernández-Ramos D.
      • Embade N.
      • Martínez-Lopez N.
      • Varela-Rey M.
      • Woodhoo A.
      • et al.
      HuR/-methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.
      ] (Supplementary Fig. 1). Immunofluorescence analysis revealed increased HuR and AUF1 protein levels in human livers with HCC suggesting post-transcriptional regulation of MAT proteins in HCC levels of AUF1 [
      • Vázquez-Chantada M.
      • Fernández-Ramos D.
      • Embade N.
      • Martínez-Lopez N.
      • Varela-Rey M.
      • Woodhoo A.
      • et al.
      HuR/-methyl-HuR and AUF1 regulate the MAT expressed during liver proliferation, differentiation, and carcinogenesis.
      ]. Based on these findings, we recently demonstrated a sharp increase of AUF1 and HuR in F344 and human HCC associated with a consistent increase in MAT1A-AUF1 and MAT2A-HuR ribonucleoproteins in both HCC types [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ]. Interestingly, these changes were very low or absent in slowly progressing HCC of BN rats.
      Recent observations attribute reduced MAT1A expression to miRNAs upregulation in HCC [
      • Yang H.
      • Cho M.E.
      • Li T.W.
      • Peng H.
      • Ko K.S.
      • Mato J.M.
      • et al.
      MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma.
      ]. Knockdown of miR-664, miR-485-3p, and miR-495 individually in Hep3B and HepG2 cells, induces MAT1A expression. Hep3B cells tumorigenesis in nude mice is decreased by stable overexpression and increased by knockdown of miRNAs-664/485-3p/495 [
      • Yang H.
      • Cho M.E.
      • Li T.W.
      • Peng H.
      • Ko K.S.
      • Mato J.M.
      • et al.
      MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma.
      ], suggesting that upregulation of these miRNAs contributes to hepatocarcinogenesis by lowering MAT1A expression.
      These observations indicate that both transcriptional and post-transcriptional mechanisms contribute to MAT1A/MAT2A switch and SAM decrease during hepatocarcinogenesis. Moreover, they suggest that MAT1A/MAT2A switch and SAM reduction may have a prognostic value for hepatocarcinogenesis.

      Mechanisms of SAM anti-tumor effect

      It is widely accepted [
      • Ha H.L.
      • Shin H.J.
      • Feitelson M.A.
      • Yu D.Y.
      Oxidative stress and antioxidants in hepatic pathogenesis.
      ,
      • Parola M.
      • Leonarduzzi G.
      • Robino G.
      • Albano E.
      • Poli G.
      • Dianzani M.U.
      On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis.
      ,
      • Hernandez-Munoz R.
      • Diaz-Munoz M.
      • Lopez V.
      • Lopez-Barrera F.
      • Yanez L.
      • Vidrio S.
      • et al.
      Balance between oxidative damage and proliferative potential in an experimental rat model of CCl4-induced cirrhosis: protective role of adenosine administration.
      ] that interaction of DNA with carcinogens and reactive oxygen and nitrogen species, generated during carcinogen metabolism and/or inflammation accompanying early stages of hepatocarcinogenesis, results in genomic instability (GI), leading to somatic point mutations, copy number alterations of individual genes, and gain/loss of chromosomal arms. Several lines of evidence indicate that progressive accumulation of genomic alterations, leading to signaling pathways deregulation, allows initiated cells to evolve to DN and HCC [
      • Ha H.L.
      • Shin H.J.
      • Feitelson M.A.
      • Yu D.Y.
      Oxidative stress and antioxidants in hepatic pathogenesis.
      ,
      • Parola M.
      • Leonarduzzi G.
      • Robino G.
      • Albano E.
      • Poli G.
      • Dianzani M.U.
      On the role of lipid peroxidation in the pathogenesis of liver damage induced by long-standing cholestasis.
      ,
      • Hernandez-Munoz R.
      • Diaz-Munoz M.
      • Lopez V.
      • Lopez-Barrera F.
      • Yanez L.
      • Vidrio S.
      • et al.
      Balance between oxidative damage and proliferative potential in an experimental rat model of CCl4-induced cirrhosis: protective role of adenosine administration.
      ]. The observation that SAM treatment maintains a high GSH pool, in CCl4-intoxicated rats [
      • Corrales F.
      • Giménez A.
      • Alvarez L.
      • Caballeria J.
      • Pajares M.A.
      • Andreu H.
      • et al.
      S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.
      ], suggests a possible chemopreventive role of the SAM antioxidative action. DNA protection from oxidative damage by antioxidants prevents tumor development in various organs, including the liver [
      • De Flora S.
      • Izzotti A.
      • D’Agostin F.
      • Balansky R.M.
      Mechanisms of N-acetylcysteine in the prevention of DNA damage and cancer, with special reference to smoking-related end-points.
      ].
      A sharp increase in polyamine synthesis may also favor fast proliferation of preneoplastic and neoplastic liver cells. Progressive upregulation of the ODC gene and rise in ODC activity and polyamine synthesis occur during rat hepatocarcinogenesis [
      • Feo F.
      • Garcea R.
      • Pascale R.M.
      • Pirisi L.
      • Daino L.
      • Donaera A.
      The variations of S-adenosyl-l-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.
      ,
      • Pascale R.M.
      • Simile M.M.
      • Gaspa L.
      • Daino L.
      • Seddaiu M.A.
      • Pinna G.
      • et al.
      Alterations of ornithine decarboxylase gene during the progression of rat liver carcinogenesis.
      ,
      • Feo F.
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Pirisi L.
      • Frassetto S.
      • et al.
      Early stimulation of polyamine biosynthesis during promotion by phenobarbital of diethylnitrosamine-induced rat liver carcinogenesis. The effects of variations of the S-adenosyl-L-methionine cellular pool.
      ]. Upregulation of polyamine synthesis-related genes also occurs in human HCC [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. SAM may interfere with polyamine synthesis by inhibiting ODC activity [
      • Feo F.
      • Garcea R.
      • Pascale R.M.
      • Pirisi L.
      • Daino L.
      • Donaera A.
      The variations of S-adenosyl-l-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.
      ].
      It should be noted that the effects of SAM on oxidative stress and polyamine synthesis could at least in part depend on accumulation of 5′-MTA [
      • Heby O.
      Role of polyamines in the control of cell proliferation and differentiation.
      ] (Fig. 1), which can also arise from spontaneous splitting of SAM at physiologic temperature and pH [
      • Wu S.E.
      • Huskey W.P.
      • Borchardt R.T.
      • Schowen R.E.L.
      Chiral instability at sulfur of S-adenosylmethione.
      ]. 5′-MTA could undergo oxidation by microsomal mono-oxygenases or auto-oxidation, with formation of sulfoxide and sulfone derivatives, thus exerting a direct antioxidant effect [
      • Brunmark A.
      • Cadenas E.
      Redox and addition chemistry of quinoid compounds and its biological implications.
      ]. 5′-MTA also inhibits CCl4-induced liver fibrosis [
      • Corrales F.
      • Giménez A.
      • Alvarez L.
      • Caballeria J.
      • Pajares M.A.
      • Andreu H.
      • et al.
      S-adenosylmethionine treatment prevents carbon tetrachloride-induced S-adenosylmethionine synthetase inactivation and attenuates liver injury.
      ]. The possibility that ODC inhibition by SAM at least partially depends on its transformation into 5′-MTA is suggested by the observation that SAM preincubation in a cell-free system, in conditions leading to its partial transformation into 5′-MTA, is necessary for strong ODC inhibition to occur in preneoplastic hepatocytes in vitro [
      • Feo F.
      • Garcea R.
      • Pascale R.M.
      • Pirisi L.
      • Daino L.
      • Donaera A.
      The variations of S-adenosyl-l-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.
      ]. 5′-MTA is inhibitory even in the absence of preincubation, and its effect is enhanced when its catabolism is blocked by adenine [
      • Feo F.
      • Garcea R.
      • Pascale R.M.
      • Pirisi L.
      • Daino L.
      • Donaera A.
      The variations of S-adenosyl-l-methionine content modulate hepatocyte growth during phenobarbital promotion of diethylnitrosamine-induced rat liver carcinogenesis.
      ].
      The possible attribution to 5′-MTA of SAM effects is intriguing, and has been the object of accurate analyses. Indeed, SAM was found to be a stronger inhibitor of DNA synthesis and rat hepatocarcinogenesis than 5′-MTA [
      • Garcea R.
      • Pascale R.M.
      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
      • et al.
      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ]. The observation that stable transfectants of HuH7 cells overexpressing MAT1A exhibit higher SAM levels and no change in 5′-MTA content, and are less tumorigenic in vivo than control cells [
      • Lu S.C.
      • Ramani K.
      • Ou X.
      • Lin M.
      • Yu V.
      • Ko K.
      • et al.
      S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model.
      ], strongly supports an anti-tumorigenic effect of SAM independent of 5′-MTA. Furthermore, SAM deficiency during hepatocarcinogenesis is associated with global DNA hypomethylation [
      • Simile M.M.
      • Banni S.
      • Angioni E.
      • Carta G.
      • De Miglio M.R.
      • Muroni M.R.
      • et al.
      5-Methylthioadenosine administration prevents lipid peroxidation and fibrogenesis induced in rat liver by carbon-tetrachloride intoxication.
      ] that is not reversed by 5′-MTA, whereas SAM-induced inhibition of the development of preneoplastic foci in rat liver carcinogenesis is associated with complete recovery of DNA hypomethylation [
      • Garcea R.
      • Pascale R.M.
      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
      • et al.
      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ], and is prevented by the hypomethylating agent 5-azacytidine [
      • Pascale R.M.
      • Simile M.M.
      • Ruggiu M.E.
      • Seddaiu M.A.
      • Satta G.
      • Sequenza M.J.
      • et al.
      Reversal by 5-azacytidine of the S-adenosyl-L-methionine-induced inhibition of the development of putative preneoplastic foci in rat liver carcinogenesis.
      ].
      Global DNA hypomethylation induces GI during hepatocarcinogenesis [
      • Mann C.D.
      • Neal C.P.
      • Garcea G.
      • Manson M.M.
      • Dennison A.R.
      • Berry D.P.
      Prognostic molecular markers in hepatocellular carcinoma: a systematic review.
      ]. AP sites represent the most frequent DNA lesions in cells [
      • Boiteux S.
      • Guillet M.
      AP sites in DNA: repair and biological consequences in Saccharomyces cerevisiae.
      ]. Together with other DNA repair proteins, APEX1 participates in base excision repair [
      • Izumi T.
      • Brown D.B.
      • Naidu C.V.
      • Bhakat K.K.
      • Macinnes M.A.
      • Saito H.
      • et al.
      Two essential but distinct functions of the mammalian AP endonuclease.
      ]. Moreover, APEX1 is also involved in the regulation of gene expression as a redox co-activator of different transcription factors, such as EGR-1, p53, and AP-1 [
      • Tell G.
      • Damante G.
      • Caldwell D.
      • Kelley M.R.
      The intracellular localization of APE1/Ref-1: more than a passive phenomenon?.
      ]. The induction of APEX1 gene by ROS at the transcriptional level [
      • Ramana C.V.
      • Boldogh I.
      • Izumi T.
      • Mitra S.
      Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals.
      ] is part of the defense mechanism against GI [
      • Grösch S.
      • Fritz G.
      • Kaina B.
      Apurinic endonuclease (Ref-1) is induced in mammalian cells by oxidative stress and involved in clastogenic adaptation.
      ].
      A recent work [
      • Tomasi M.L.
      • Iglesias-Ara A.
      • Yang H.
      • Ramani K.
      • Feo F.
      • Pascale M.R.
      • et al.
      S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.
      ] showed increased GI in livers of 1-month-old MAT1A-KO mice, compared to wild type mice, whereas Apex1 mRNA and protein levels were reduced by 20% and 50%, respectively, in these mice of all ages. These changes were correlated with increase in AP sites and reduced expression of APEX1 targets Bax, Fas, and p21 [
      • Tomasi M.L.
      • Iglesias-Ara A.
      • Yang H.
      • Ramani K.
      • Feo F.
      • Pascale M.R.
      • et al.
      S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.
      ]. In cultured human and mouse hepatocytes, MAT1A mRNA decreased whereas APEX1 and c-MYC mRNAs increased. However, APEX1 protein level decreased to 60% of baseline [
      • Tomasi M.L.
      • Iglesias-Ara A.
      • Yang H.
      • Ramani K.
      • Feo F.
      • Pascale M.R.
      • et al.
      S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.
      ] (Supplementary Fig. 2). SAM prevented these changes in cultured hepatocytes, indicating that although SAM inhibits APEX1 transcription, it stabilizes APEX1 protein [
      • Tomasi M.L.
      • Iglesias-Ara A.
      • Yang H.
      • Ramani K.
      • Feo F.
      • Pascale M.R.
      • et al.
      S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.
      ] (Supplementary Fig. 2). This SAM effect on APEX1 regulation might contribute to SAM chemopreventive action and in part explains why chronic SAM deficiency predisposes to HCC.
      The mechanism of APEX1 stabilization by SAM is not known. Recent reports envisage proteasome inhibition by SAM. Simultaneous overexpression of ubiquitin-9 and APEX1 in HeLa cells dramatically lowers APEX1 protein, suggesting ubiquitin-9 is involved in APEX1 protein degradation [
      • Yan M.D.
      • Xu W.J.
      • Lu L.R.
      • Sun L.Y.
      • Liu X.Y.
      • Zheng Z.C.
      Ubiquitin conjugating enzyme Ubc9 is involved in protein degradation of redox factor-1 (Ref-1).
      ]. SAM inhibits chymotrypsin-like and caspase-like activities in 26S proteasome and causes degradation of some of the 26S proteasomal subunits, which is blocked by the proteasome inhibitor MG132 [
      • Tomasi M.L.
      • Tomasi I.
      • Ramani K.
      • Pascale R.M.
      • Xu J.
      • Giordano P.
      • et al.
      S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers.
      ]. Furthermore, SAM and 5′-MTA lower CDC2 expression, upregulated in several cancers, resulting in decreased ubiquitin-9 phosphorylation and expression [
      • Tomasi M.L.
      • Tomasi I.
      • Ramani K.
      • Pascale R.M.
      • Xu J.
      • Giordano P.
      • et al.
      S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers.
      ].
      Nitric oxide (NO) is a product of L-arginine to L-citrulline conversion by NOS. Calcium-independent, inducible iNOS is present in hepatocytes, Kupffer and stellate cells, and cholangiocytes, whereas calcium-dependent eNOS is present in endothelial cells [
      • Pascale R.M.
      • Frau M.
      • Feo F.
      Prognostic significance of iNOS in hepatocellular carcinoma.
      ]. NO may favor HCC development by inducing DNA mutations, in hepatocytes surviving to oxidative stress, and vasodilatation providing premalignant and malignant cells with sufficient metabolites and oxygen. Overproduction of inflammatory cytokines and growth factors during early stages of hepatocarcinogenesis deregulates iNOS [
      • Pascale R.M.
      • Frau M.
      • Feo F.
      Prognostic significance of iNOS in hepatocellular carcinoma.
      ]. Reactive nitrogen species produced via iNOS during chronic hepatitis may play a key role in carcinogenesis by causing DNA damage. iNOS suppression by aminoguanidine results in decreased HCC cell growth, NF-kB and RAS/ERK downregulation, and increased apoptosis in vivo and in vitro [
      • Calvisi D.F.
      • Pinna F.
      • Ladu S.
      • Pellegrino R.
      • Muroni M.R.
      • Simile M.M.
      • et al.
      Aberrant iNOS signalling is under genetic control in rodent liver cancer and potentially prognostic for human disease.
      ]. eNOS activation by AMPK during hepatocarcinogenesis may also contribute to NO production, which is in turn an endogenous AMPK activator [
      • Zhang J.
      • Xi Z.
      • Dong Y.
      • Wang S.
      • Liu C.
      • Zou M.H.
      Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells.
      ], and lowers SAM level by inactivating MATI/III [
      • Vázquez-Chantada M.
      • Ariz U.
      • Varela-Rey M.
      • Embade N.
      • Martínez-Lopez N.
      • Fernández-Ramos D.
      • et al.
      Evidence for an LKB1/AMPK/eNOS cascade regulated by HGF, S-adenosylmethionine and NO in hepatocyte proliferation.
      ] (Fig. 2). On the other hands, survival of SAM-deficient cells in MAT1A-KO mice requires LKB1/AMPK activation. HGF is mitogenic for hepatocytes through LKB1/AMPK activation, which is blocked by SAM [
      • Vázquez-Chantada M.
      • Ariz U.
      • Varela-Rey M.
      • Embade N.
      • Martínez-Lopez N.
      • Fernández-Ramos D.
      • et al.
      Evidence for an LKB1/AMPK/eNOS cascade regulated by HGF, S-adenosylmethionine and NO in hepatocyte proliferation.
      ] (Fig. 2).
      Figure thumbnail gr2
      Fig. 2Effect of SAM on HGF/LKB1/AMPK axis. The HGF/LKB1/AMPK axis enhances NO production via eNOS activation, upregulates the glycolytic key enzyme PFK-2, and induces the nuclear to cytoplasmic HuR translocation, resulting in stabilization of cyclins, p53, and USP7 mRNAs. Hyperactive LKB1 induces p53 hyperphosphorylation. The interaction of phosphorylated p53 with USP7 blocks the negative regulation of p53 by MDM2. SAM inhibits LKB1. This effect is controlled by MATI/III inhibition operated by NO.
      Recent observations indicate that LKB1/AMPK axis activation may contribute to hepatocarcinogenesis through other mechanisms. Consequent to AMPK activation in hepatocytes is nuclear to cytoplasmic HuR translocation, resulting in cyclin mRNAs stabilization. Increased basal LKB1/AMPK axis leads to a rise in cytoplasmic HuR levels, cyclin D1 expression, and cell proliferation [
      • Martínez-Chantar M.L.
      • Vázquez-Chantada M.
      • Garnacho M.
      • Varela-Rey M.
      • Dotor J.
      • Santamaría M.
      • et al.
      S-adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase.
      ] (Fig. 2). Furthermore, AMPK upregulation can contribute to the glycolytic metabolism of cancer cells [
      • Almeida A.
      • Moncada S.
      • Bolaños J.P.
      Nitric oxide switches on glycolysis through the AMP protein kinase and 6-phosphofructo-2-kinase pathway.
      ] through activation of PFK-2, a key enzyme for glycolysis [
      • Calvisi D.F.
      • Frau M.
      • Tomasi M.L.
      • Feo F.
      • Pascale R.M.
      Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison.
      ].
      LKB1 may also regulate AKT-mediated cell survival independently of PI3K, AMPK, and mTORC2 [
      • Martínez-López N.
      • Varela-Rey M.
      • Fernández-Ramos D.
      • Woodhoo A.
      • Vázquez-Chantada M.
      • Embade N.
      • et al.
      Activation of LKB1-Akt pathway independent of PI3 Kinase plays a critical role in the proliferation of hepatocellular carcinoma from NASH.
      ]. A critical role is played by the deubiquitinating enzyme USP7. USP7 contributes to the stability of MDM2, a negative p53 regulator, impairing its self-ubiquitination and degradation. In SAM-deficient hepatocytes, p53 is mostly cytosolic and hyperphosphorylated by several kinases, including hyperactive LKB1 [
      • Cairns R.A.
      • Harris I.S.
      • Mak T.W.
      Regulation of cancer cell metabolism.
      ]. p53 hyperphosphorylation and its interaction with USP7 block the negative regulation by MDM2. Furthermore, active LKB1-induced HuR cytosolic translocation, stabilizes p53 and USP7 mRNAs [
      • Martínez-Chantar M.L.
      • Vázquez-Chantada M.
      • Garnacho M.
      • Varela-Rey M.
      • Dotor J.
      • Santamaría M.
      • et al.
      S-adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase.
      ] (Fig. 2). Thus, LKB1 controls apoptotic response through phosphorylation and cytoplasmic retention of p53, regulation of the de-ubiquitination enzyme USP7, and HuR nucleo-cytoplasmic shuttling. Notably, cytoplasmic staining of p53 and p-LKB1 (Ser428) occurs in a NASH-HCC animal model (from MAT1A-KO mice) and in liver biopsies obtained from human HCC derived from both ASH and NASH [
      • Martínez-López N.
      • Varela-Rey M.
      • Fernández-Ramos D.
      • Woodhoo A.
      • Vázquez-Chantada M.
      • Embade N.
      • et al.
      Activation of LKB1-Akt pathway independent of PI3 Kinase plays a critical role in the proliferation of hepatocellular carcinoma from NASH.
      ]. These findings, however, contrast with the report of a loss of LKB1, identified as an oncosuppressor gene, in cancer cells, including HCC [
      • Cairns R.A.
      • Harris I.S.
      • Mak T.W.
      Regulation of cancer cell metabolism.
      ]. AMPK, activated by LKB1, inhibits AKT signaling turning off mTOR by activating the tumor oncosuppressor complex TSC2/TSC1 [
      • Hezel A.F.
      • Bardeesy N.
      LKB1; linking cell structure and tumor suppression.
      ]. Moreover, AMPK α2 catalytic subunit downregulation is statistically associated with undifferentiated HCC and poor patient prognosis, and AMPK inactivation promotes hepatocarcinogenesis by destabilizing p53 in a p53 deacetylase (SIRTUIN 1)-dependent manner [
      • Lee C.W.
      • Wong L.L.
      • Tse E.Y.
      • Liu H.F.
      • Leong V.Y.
      • Lee J.M.
      • et al.
      AMPK promotes p53 acetylation via phosphorylation and inactivation of SIRT1 in liver cancer cells.
      ]. In complex, the effects of LKB1/AMPK signaling on HCC development are contradictory and probably a comparison between different experimental models and human HCC subtypes may contribute to their complete understanding.
      Other mechanisms of SAM antitumor effects have been envisaged. Forced expression of MAT1A in HCC cell lines results in downregulation of cyclin D1, E2F1, IKK, NF-kB, and antiapoptotic BCL2 and XIAP genes, and upregulation of proapoptotic BAK and BAX genes [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ]. SAM counteracts NF-kB activation in rat preneoplastic foci [
      • García-Román R.
      • Salazar-González D.
      • Rosas S.
      • Arellanes-Robledo J.
      • Beltrán-Ramírez O.
      • Fattel-Fazenda S.
      • et al.
      The differential NF-kB modulation by S-adenosyl-L-methionine, acetylcysteine and quercetin on the promotion stage of chemical hepatocarcinogenesis.
      ] and upregulates the oncosuppressor PP2A that dephosphorylates and inactivates AMPK, pAKT, and pERK [
      • Millward T.A.
      • Zolnierowicz S.
      • Hemmings B.A.
      Regulation of protein kinase cascades by protein phosphatase 2A.
      ,
      • Eichhorn P.J.
      • Creyghton M.P.
      • Bernards R.
      Protein phosphatase 2A regulatory subunits and cancer.
      ]. The lowest SAM levels and PP2A expression occur in both rat and human HCC exhibiting the highest pAKT and pERK expression and proliferation rates [[
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ,
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ,
      • Frau M.
      • Simile M.M.
      • Tomasi M.L.
      • Demartis M.I.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis.
      ] and Frau et al., unpublished results]. ERK and PI3K pathways may be also activated by binding of SPP1 (osteoponin) to integrin receptors in cancer [
      • Zhao J.
      • Dong L.
      • Lu B.
      • Wu G.
      • Xu D.
      • Chen J.
      • et al.
      Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis.
      ]. Reduction of SPP1 expression in MAT1A transfected tumors [
      • Lu S.C.
      • Ramani K.
      • Ou X.
      • Lin M.
      • Yu V.
      • Ko K.
      • et al.
      S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model.
      ] may contribute to ERK and PI3K downregulation.
      SAM level can influence ERK1/2 activity by interfering with DUSP1, a specific ERK inhibitor (Fig. 3). DUSP1 downregulation and ERK1/2 upregulation occur in fast progressing DN and HCC of F344 rats and human HCC [
      • Calvisi D.F.
      • Pinna F.
      • Ladu S.
      • Pellegrino R.
      • Sanna V.
      • Sini M.
      • et al.
      Ras-driven proliferation and apoptosis signalling during rat liver carcinogenesis is under genetic control.
      ,
      • Calvisi D.F.
      • Pinna F.
      • Meloni F.
      • Ladu S.
      • Pellegrino R.
      • Sini L.
      • et al.
      Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated-kinase-mediated control of growth in human hepatocellular carcinoma.
      ]. Conversely, active ERK1/2 phosphorylates the Ser296 residue of DUSP1, thus contributing to its ubiquitination by the SKP2-CKS1 ubiquitin ligase, followed by proteasomal degradation [
      • Calvisi D.F.
      • Pinna F.
      • Ladu S.
      • Pellegrino R.
      • Sanna V.
      • Sini M.
      • et al.
      Ras-driven proliferation and apoptosis signalling during rat liver carcinogenesis is under genetic control.
      ,
      • Calvisi D.F.
      • Pinna F.
      • Meloni F.
      • Ladu S.
      • Pellegrino R.
      • Sini L.
      • et al.
      Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated-kinase-mediated control of growth in human hepatocellular carcinoma.
      ]. On the other hand, ERK1/2 sustains SKP2-CKS1 activity through its target FOXM1 [
      • Calvisi D.F.
      • Pinna F.
      • Ladu S.
      • Pellegrino R.
      • Simile M.M.
      • Frau M.
      • et al.
      Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC.
      ] (Fig. 3). Notably, DUSP1 mRNA and protein levels are markedly reduced in livers of MAT1A-KO mice and in cultured mouse and human hepatocytes, with protein decreasing to lower levels than mRNA [
      • Tomasi M.L.
      • Ramani K.
      • Lopitz-Otsoa F.
      • Rodríguez M.S.
      • Li T.W.
      • Ko K.
      • et al.
      S-adenosylmethionine regulates dual-specificity mitogen-activated protein kinase phosphatase expression in mouse and human hepatocytes.
      ]. SAM treatment protects against the fall in DUSP1 mRNA and protein in cultured mouse and human hepatocytes, and SAM administration to MAT1A-KO mice results in increase in SAM and Dusp1 mRNA and protein levels, and decrease in Erk activity [
      • Tomasi M.L.
      • Ramani K.
      • Lopitz-Otsoa F.
      • Rodríguez M.S.
      • Li T.W.
      • Ko K.
      • et al.
      S-adenosylmethionine regulates dual-specificity mitogen-activated protein kinase phosphatase expression in mouse and human hepatocytes.
      ]. These observations show a control of MAPK by SAM. SAM treatment increases DUSP1 mRNA at transcriptional level, and contributes to increase in DUSP1 protein at post-translational levels, probably through inhibition of its proteasomal degradation [
      • Tomasi M.L.
      • Ramani K.
      • Lopitz-Otsoa F.
      • Rodríguez M.S.
      • Li T.W.
      • Ko K.
      • et al.
      S-adenosylmethionine regulates dual-specificity mitogen-activated protein kinase phosphatase expression in mouse and human hepatocytes.
      ] (Fig. 3). Interestingly, TNF-α/HIF-1α axis sustains the expression of FOXM1 [
      • Xia L.
      • Mo P.
      • Huang W.
      • Zhang L.
      • Wang Y.
      • Zhu H.
      • et al.
      The TNF-α/ROS/HIF-1-induced upregulation of FoxMI expression promotes HCC proliferation and resistance to apoptosis.
      ], which mediates ERK1/2 effects on cell cycle, cell survival, and angiogenesis [
      • Calvisi D.F.
      • Pinna F.
      • Ladu S.
      • Pellegrino R.
      • Simile M.M.
      • Frau M.
      • et al.
      Forkhead box M1B is a determinant of rat susceptibility to hepatocarcinogenesis and sustains ERK activity in human HCC.
      ]. Hypoxia could contribute to ERK1/2 upregulation by reducing SAM level of HCC cells through HIF-1α binding to MAT2A promoter [
      • Liu Q.
      • Liu L.
      • Zhao Y.
      • Zhang J.
      • Wang D.
      • Chen J.
      • et al.
      Hypoxia induces genomic DNA demethylation through the activation of HIF-1α and transcriptional upregulation of MAT2A in hepatoma cells.
      ]. These findings support a suppressive effect of SAM on malignant transformation through ERK1/2 inhibition.
      Figure thumbnail gr3
      Fig. 3Interference of SAM with ERK1/2 inhibition by DUSP1. The inhibition of ERK1/2 activity by DUSP1 is controlled by DUSP1 phosphorylation of Ser296 residue, followed by its ubiquitination by the SKP2–CKS1 ubiquitin ligase and proteasomal degradation, as well as by SKP2–CKS1 activation operated by FOXM1, a major target of ERK1/2 and HIF-1α. SAM enhances DUSP1 inhibitory effect by increasing DUSP1 mRNA at transcriptional level, and by contributing to the increase in DUSP1 protein at post-translational levels, probably through inhibition of its proteasomal degradation.
      Changes of MATs expression may affect cancer cell growth by interfering with protein methylation. A recent study [
      • Reytor E.
      • Pérez-Miguelsanz J.
      • Alvarez L.
      • Pérez-Sala D.
      • Pajares M.A.
      Conformational signals in the C-terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution.
      ] identified two partially overlapping areas at the C-terminal end of the protein involved in cytoplasmic retention and nuclear localization of MATI/III in most rat tissues. Nuclear accumulation of the active enzyme was correlated with histone H3K27 trimethylation, an epigenetic modification associated with DNA methylation, therefore pointing to the need of MATI/III to guarantee SAM supply for specific methylations and, eventually, additional roles. Interestingly, MATIIα also provides SAM locally on chromatin by interacting with chromatin-related proteins involved in histone modification, chromatin remodeling, transcription regulation, and nucleo-cytoplasmic transport [
      • Katoh Y.
      • Ikura T.
      • Hoshikawa Y.
      • Tashiro S.
      • Ito T.
      • Ohta M.
      • et al.
      Methionine adenosyltransferase II serves as a transcriptional corepressor of Maf oncoprotein.
      ,
      • Igarashi K.
      • Katoh Y.
      Metabolic aspects of epigenome: coupling of S-adenosylmethionine synthesis and gene regulation on chromatin by SAMIT module.
      ], This mechanism can regulate MAFK, a member of MAF oncoproteins, which interacts with both MATIIα and MATIIβ [
      • Reytor E.
      • Pérez-Miguelsanz J.
      • Alvarez L.
      • Pérez-Sala D.
      • Pajares M.A.
      Conformational signals in the C-terminal domain of methionine adenosyltransferase I/III determine its nucleocytoplasmic distribution.
      ,
      • Katoh Y.
      • Ikura T.
      • Hoshikawa Y.
      • Tashiro S.
      • Ito T.
      • Ohta M.
      • et al.
      Methionine adenosyltransferase II serves as a transcriptional corepressor of Maf oncoprotein.
      ]. MAFK functions as transcription activator and repressor by forming diverse heterodimers to bind to MAF recognition elements of DNA [
      • Katoh Y.
      • Ikura T.
      • Hoshikawa Y.
      • Tashiro S.
      • Ito T.
      • Ohta M.
      • et al.
      Methionine adenosyltransferase II serves as a transcriptional corepressor of Maf oncoprotein.
      ,
      • Andreu-Pérez P.
      • Esteve-Puig R.
      • de Torre-Minguela C.
      • López-Fauqued M.
      • Bech-Serra J.J.
      • Tenbaum S.
      • et al.
      Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.
      ]. However, the oncogenic role of MAFK and its targets in HCC is unknown. Moreover, ERK1/2 activation, elicited by particular growth factors in different cell lines including HCC cells, may be limited by arginine methylation of RAF proteins by PRMT5 [
      • Andreu-Pérez P.
      • Esteve-Puig R.
      • de Torre-Minguela C.
      • López-Fauqued M.
      • Bech-Serra J.J.
      • Tenbaum S.
      • et al.
      Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.
      ]. Expression of RAF mutants that cannot be methylated affects the amplitude and duration of ERK activation by growth factors [
      • Andreu-Pérez P.
      • Esteve-Puig R.
      • de Torre-Minguela C.
      • López-Fauqued M.
      • Bech-Serra J.J.
      • Tenbaum S.
      • et al.
      Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.
      ]. However, PRMT5 accelerates cell cycle progression through the G1 phase, activates PI3K/AKT and suppresses JNK/c-Jun signaling in lung cancer [
      • Wei T.Y.
      • Juan C.C.
      • Hisa J.Y.
      • Su L.J.
      • Lee Y.C.
      • Chou H.Y.
      • et al.
      Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.
      ]. Apparent discrepancies could depend on PRMT5 localization. PRMT5 and p44/MED50/WD45/WDR77 cytoplasmic co-localization is required for prostate cancer cell growth. In contrast, nuclear PRMT5, present in benign prostate epithelium, inhibits cell growth in a methyltransferase activity-independent manner [
      • Gu Z.
      • Li Y.
      • Lee P.
      • Liu T.
      • Wan C.
      • Wang Z.
      Protein arginine methyltransferase 5 functions in opposite ways in the cytoplasm and nucleus of prostate cancer cells.
      ].
      Finally, HCV protein impairs JAK-STAT signaling by inhibiting STAT1 methylation, which favors STAT1 binding by its inhibitor PIAS1 [
      • Duong F.H.
      • Christen V.
      • Filipowicz M.
      • Heim M.H.
      S-Adenosylmethionine and betaine correct hepatitis C virus induced inhibition of interferon signaling in vitro.
      ]. Remarkably, SAM and betaine restore STAT1 methylation and improve IFNalpha antiviral effect in cell culture [
      • Duong F.H.
      • Christen V.
      • Filipowicz M.
      • Heim M.H.
      S-Adenosylmethionine and betaine correct hepatitis C virus induced inhibition of interferon signaling in vitro.
      ].

      Changes in methionine metabolism and HCC prognosis

      Increasing evidence indicates that the deregulation of various signaling pathways progressively increases with HCC progression and has a prognostic value [
      • Calvisi D.F.
      • Frau M.
      • Tomasi M.L.
      • Feo F.
      • Pascale R.M.
      Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison.
      ]. The comparative analysis of c-Myc and c-Myc/TGFα transgenic mice and of genetically susceptible F344 and genetically resistant BN rats recapitulates the main pathogenetic mechanisms of human HCC, with c-Myc and BN tumors approaching human HCC characterized by better prognosis, and c-Myc/TGFα and F344 HCCs resembling those with shorter survival [
      • Calvisi D.F.
      • Frau M.
      • Tomasi M.L.
      • Feo F.
      • Pascale R.M.
      Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison.
      ,
      • Frau M.
      • Simile M.M.
      • Tomasi M.L.
      • Demartis M.I.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis.
      ,
      • Lee J.S.
      • Chu I.S.
      • Mikaelyan A.
      • Calvisi D.F.
      • Heo J.
      • Reddy J.K.
      • et al.
      Application of comparative functional genomics to identify best-fit mouse models to study human cancer.
      ,
      • Andersen J.B.
      • Loi R.
      • Perra A.
      • Factor V.M.
      • Ledda-Columbano G.M.
      • Columbano A.
      • et al.
      Progenitor-derived hepatocellular carcinoma model in the rat.
      ].
      Decrease in MatI/III:MatII activity ratio occurs in c-Myc and c-Myc/TGFα transgenics, with the lowest values in c-Myc transgenics [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. Sahh gene expression increases in HCC of c-Myc transgenics and in DN and HCC of the double transgenics, suggesting a relatively high production of homocysteine, presumably not associated with rise in GSH because of decreased Cbs levels in tissues of both transgenic models [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ] (Fig. 1). Bhmt expression decreases in dysplastic and neoplastic liver of both transgenic lines, whereas Mthf-hmt expression does not change in c-Myc lesions, showing a sharp increase in HCC of double transgenics [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. As concerns polyamine synthesis, progressive increase in Sdc, Odc, Smr, and Sms mRNAs occurs in dysplastic and neoplastic lesions of both transgenic models, with the highest levels in the lesions of c-Myc/Tgf-a transgenics (Fig. 1). Finally, the expression of Mtap1, encoding a key enzyme for methionine re-synthesis through the salvage pathway [
      • Albers E.
      Metabolic characteristics and importance of the universal methionine salvage pathway recycling methionine from 5’-methylthioadenosine.
      ], increases only in the lesions of double transgenics [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ].
      Similar results were found in HCC with better prognosis (survival >3 years after partial liver resection; HCCB) and poorer prognosis (survival <3 years; HCCP) and their corresponding surrounding liver (SL) [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. In these lesions, MATI:MATII ratio progressively decreases from SL to HCC with the lowest values in HCCP, reflecting the changes in MAT1A:MAT2A expression ratio. A slight increase or no change in SAHH expression and a marked decrease of CBS mRNA occur in human HCCs with better and poorer prognosis and their corresponding SL, with respect to the control liver. As concerns the genes encoding key enzymes of methionine synthesis, BHMT expression decreases in liver lesions of all subgroups, MTHF-HMT does not change and MTAP1 decreases in HCCP. Finally, levels of SDC, ODC, SMR, and SMS progressively increase from SL to HCC, with highest values in HCCP. These observations indicate the association of MAT1A/MAT2A switch, decrease in methionine resynthesis, and increase in SAM utilization for polyamine synthesis with HCC progression. Accordingly, cell proliferation rate of transgenic mice and human HCC are positively correlated with global DNA hypomethylation and GI [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ]. These observations suggest that deregulated methionine metabolism and MATI/III:MATII ratio are implicated in HCC progression and prognosis.
      Figure thumbnail fx1

      Conclusions and future perspectives

      Pleiotropic effects on signal transduction (Fig. 4), associated with decrease in MAT1A expression and SAM levels, favoring hepatocarcinogenesis, include: (a) global DNA hypomethylation, production of oxygen reactive and nitroactive species, and activation of LKB1/AMPK axis, which may induce GI; (b) cell cycle activation following upregulation of c-MYC and genes involved in polyamine synthesis; (c) RAS/ERK, IKK/NF-kB, PI3K/AKT, and NF-kB signaling upregulation, leading to increase in cell proliferation, cell survival, and microvascularization.
      Figure thumbnail gr4
      Fig. 4Pleiotropic effects of SAM treatment during hepatocarcinogenesis. SAM capacity to methylate DNA and stabilize the DNA repair enzyme APEX1, and SAM antioxidant activity reduce genomic instability (GI). The inhibition by SAM of LKB1/AMPK axis increases cytoplasmic concentration of HuR, which stabilizes p53 and USP7 mRNAs. Activation of LKB1 leads to p53 hyperphosphorylation and its interaction with USP7 with consequent block of the negative regulation of p53 by MDM2. Thus, LKB1 controls apoptotic response through phosphorylation and retention of p53 in the cytoplasm, regulation of the de-ubiquitination enzyme USP7, and HuR nucleo-cytoplasmic shuttling. SAM also controls cell growth and survival by inducing PPA2 expression that phosphorylates and inactivates AKT and its targets. Moreover, PPA2 activation and DUSP1 stabilization inhibit RAS/ERK pathway. Finally, SAM affects cell cycle by inhibiting c-MYC expression and polyamine synthesis.
      Most of these changes were discovered in rodent models [
      • Simile M.M.
      • Banni S.
      • Angioni E.
      • Carta G.
      • De Miglio M.R.
      • Muroni M.R.
      • et al.
      5-Methylthioadenosine administration prevents lipid peroxidation and fibrogenesis induced in rat liver by carbon-tetrachloride intoxication.
      ,
      • Garcea R.
      • Pascale R.M.
      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
      • et al.
      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ,
      • Feo F.
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Pirisi L.
      • Frassetto S.
      • et al.
      Early stimulation of polyamine biosynthesis during promotion by phenobarbital of diethylnitrosamine-induced rat liver carcinogenesis. The effects of variations of the S-adenosyl-L-methionine cellular pool.
      ,
      • Tomasi M.L.
      • Iglesias-Ara A.
      • Yang H.
      • Ramani K.
      • Feo F.
      • Pascale M.R.
      • et al.
      S-adenosylmethionine regulates apurinic/apyrimidinic endonuclease 1 stability: implication in hepatocarcinogenesis.
      ,
      • Martínez-López N.
      • Varela-Rey M.
      • Fernández-Ramos D.
      • Woodhoo A.
      • Vázquez-Chantada M.
      • Embade N.
      • et al.
      Activation of LKB1-Akt pathway independent of PI3 Kinase plays a critical role in the proliferation of hepatocellular carcinoma from NASH.
      ,
      • Eichhorn P.J.
      • Creyghton M.P.
      • Bernards R.
      Protein phosphatase 2A regulatory subunits and cancer.
      ]. Previous research on comparative functional genomics to evaluate rodent models for human liver cancer [
      • Frau M.
      • Simile M.M.
      • Tomasi M.L.
      • Demartis M.I.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      An expression signature of phenotypic resistance to hepatocellular carcinoma identified by cross-species gene expression analysis.
      ,
      • Lee J.S.
      • Chu I.S.
      • Mikaelyan A.
      • Calvisi D.F.
      • Heo J.
      • Reddy J.K.
      • et al.
      Application of comparative functional genomics to identify best-fit mouse models to study human cancer.
      ,
      • Andersen J.B.
      • Loi R.
      • Perra A.
      • Factor V.M.
      • Ledda-Columbano G.M.
      • Columbano A.
      • et al.
      Progenitor-derived hepatocellular carcinoma model in the rat.
      ] and other cancers [
      • Ellwood-Yen K.
      • Graeber T.G.
      • Wongvipat J.
      • Iruela-Arispe M.L.
      • Zhang J.
      • Matusik R.
      • et al.
      Myc-driven murine prostate cancer shares molecular features with human prostate tumors.
      ,
      • Sweet-Cordero A.
      • Mukherjee S.
      • Subramanian A.
      • You H.
      • Roix J.J.
      • Ladd-Acosta C.C.
      • et al.
      An oncogenic KRAS2 expression signature identified by cross-species gene-expression analysis.
      ] indicated that molecular pathways associated with specific cancer phenotypes are evolutionarily conserved [
      • Lee J.S.
      • Thorgeirsson S.S.
      Comparative and integrative functional genomics of HCC.
      ]. Accordingly, the progression of both human and rodent HCC prognostic subtypes is correlated with upregulation of MAPK, IKK/NF-kB, JAK/STAT, WNT/FZD, and PI3K/AKT pathways, and cell cycle key genes, downregulation of cell cycle inhibitors [
      • Calvisi D.F.
      • Frau M.
      • Tomasi M.L.
      • Feo F.
      • Pascale R.M.
      Deregulation of signalling pathways in prognostic subtypes of hepatocellular carcinoma: novel insights from interspecies comparison.
      ], and decrease in MAT1A expression and SAM levels [
      • Pascale R.M.
      • Simile M.M.
      • De Miglio M.R.
      • Nufris A.
      • Daino L.
      • Seddaiu M.A.
      • et al.
      Chemoprevention by S-adenosyl-L-methionine of rat liver carcinogenesis initiated by 1,2-dimethylhydrazine and promoted by orotic acid.
      ,
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ]. Some of these changes, including MAT1A/MAT2A switch, are putative prognostic markers of HCC [
      • Cai J.
      • Mao Z.
      • Hwang J.J.
      • Lu S.C.
      Differential expression of methionine adenosyltransferase genes influences the rate of growth of human hepatocellular carcinoma cells.
      ].
      The interference of changes in MAT1A expression and SAM level with several pathways, during hepatocarcinogenesis, opens new therapeutic perspectives. SAM chemopreventive effect for experimental hepatocarcinogenesis has been well documented [
      • Garcea R.
      • Pascale R.M.
      • Daino L.
      • Frassetto S.
      • Cozzolino P.
      • Ruggio M.E.
      • et al.
      Variations in ornithine decarboxylase activity and S-adenosyl-l-methionine and S-methylthioadenosine contents during the development of diethylnitrosamine-induced liver hyperplastic nodules and hepatocellular carcinomas.
      ,
      • Pascale R.M.
      • Simile M.M.
      • Satta G.
      • Seddaiu M.A.
      • Daino L.
      • Pinna G.
      • et al.
      Comparative effects of l-methionine, S-adenosyl-l-methionine and 5′-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.
      ,
      • Simile M.M.
      • Saviozzi M.
      • De Miglio M.R.
      • Muroni M.R.
      • Nufris A.
      • Pascale R.M.
      • et al.
      Persistent chemopreventive effect of S-adenosyl-L-methionine on the development of liver putative preneoplastic lesions induced by thiobenzamide in diethylnitrosamine-initiated rats.
      ,
      • Garcea R.
      • Daino L.
      • Pascale R.M.
      • Simile M.M.
      • Puddu M.
      • Frassetto S.
      • et al.
      Inhibition of promotion and persistent nodule growth by S-adenosyl-L-methionine in rat liver carcinogenesis: role of remodeling and apoptosis.
      ,
      • Frau M.
      • Tomasi M.L.
      • Simile M.M.
      • Demartis M.I.
      • Salis F.
      • Latte G.
      • et al.
      Role of transcriptional and posttranscriptional regulation of methionine adenosyltransferases in liver cancer progression.
      ,
      • Anstee Q.M.
      • Day C.P.
      S-adenosylmethionine (SAMe) therapy in liver disease: a review of current evidence and clinical utility.
      ]. In humans, according to recent observations [
      • Halsted C.H.
      • Medici V.
      Aberrant hepatic methionine metabolism and gene methylation in the pathogenesis and treatment of alcoholic steatohepatitis.
      ,
      • Anstee Q.M.
      • Day C.P.
      S-adenosylmethionine (SAMe) therapy in liver disease: a review of current evidence and clinical utility.
      ,
      • Cholongitas E.
      • Papatheoroditis G.V.
      Review aryicle: novel therapeuic options for chronic hepatitis C.
      ,
      • Morgan T.R.
      Chemoprevention of hepatocellular carcinoma in chronic hepatitis C.
      ], HCC could be prevented by a SAM curative effect on ASH and hepatitis C. An ongoing phase II clinical trial is evaluating SAM as a potential chemopreventive agent in hepatitis C and cirrhosis [
      • Morgan T.R.
      Chemoprevention of hepatocellular carcinoma in chronic hepatitis C.
      ].
      No evidence of SAM therapeutic effect against HCC is available [
      • Frau M.
      • Tomasi M.L.
      • Simile M.M.
      • Demartis M.I.
      • Salis F.
      • Latte G.
      • et al.
      Role of transcriptional and posttranscriptional regulation of methionine adenosyltransferases in liver cancer progression.
      ]. Preliminary approaches should test the effects of stable MAT1A overexpression or MAT2A/MAT2B inhibition in experimental HCC in vivo. Silencing of MAT2A or MAT2B in HepG2 cells inhibits proliferative response to leptin [
      • Ramani K.
      • Yang H.P.
      • Xia M.
      • Iglesias Ara A.
      • Mato J.M.
      • Lu S.C.
      Leptin’s mitogenic effect in human liver cancer cells requires induction of both methionine adenosyltransferase 2A and 2β.
      ]. However, intracellular transduction of viral vectors in vivo still presents numerous limitations [
      • Calvisi D.F.
      • Pascale R.M.
      • Feo F.
      Dissection of signal transduction pathways as a tool for the development of targeted therapies of hepatocellular carcinoma.
      ]. In this context, the therapeutic effect for HCC of a family of fluorinated N,N-dialkylaminostilbene agents, which inhibits colorectal cancer cells proliferation in vitro and in vivo [
      • Zhang W.
      • Sviripa V.
      • Chen X.
      • Shi J.
      • Yu T.
      • Hamza A.
      • et al.
      Fluorinated N, N-Dialkylaminostilbenes repress colon cancer by targeting methionine S-adenosyltransferase 2A.
      ], should be texted. These molecules bind to MATIIα catalytic subunit and inhibit SAM synthesis. They also inhibit WNT/β-catenin signaling [
      • Zhang W.
      • Sviripa V.
      • Chen X.
      • Shi J.
      • Yu T.
      • Hamza A.
      • et al.
      Fluorinated N, N-Dialkylaminostilbenes repress colon cancer by targeting methionine S-adenosyltransferase 2A.
      ], therefore, they could be particularly effective against β-catenin mutated HCC. Furthermore, a recent observation of MAT1A downregulation by miRNAs-664/485-3p/495 overexpression in HCC [
      • Yang H.
      • Cho M.E.
      • Li T.W.
      • Peng H.
      • Ko K.S.
      • Mato J.M.
      • et al.
      MicroRNAs regulate methionine adenosyltransferase 1A expression in hepatocellular carcinoma.
      ], suggests a therapeutic effect of specific anti-miRNAs oligonucleotides. Liver vessels allow entering molecules up to 200 nm in diameter, including antisense oligonucleotides (ASOs) inhibiting or decoying miRNAs. ASOs delivery using pegylated liposomes, biodegradable polymers, lipid nanoparticles is an alternative. However, novel modification, conjugation or formulation strategies may improve effective and safe delivery of anti-miRNAs oligonucleotides. Nonetheless, different miRNAs inhibitors are in preclinical studies, 15 nt ASOs are in clinical trials and 8 nt versions show promise in non-human primates [
      • Broderick J.A.
      • Zamore P.D.
      MicroRNA therapeutics.
      ,
      • Guo J.
      • Friedman S.L.
      The expression patterns and clinical significance of microRNAs in liver diseases and hepatocellular carcinoma.
      ].

      Financial support

      Supported by grants from Associazione Italiana Ricerche sul Cancro ( IG8952 ), Ministero Università e Ricerca ( PRIN 2009 ), Regione Autonoma della Sardegna , Fondazione Banco di Sardegna .

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Supplementary data. data

      • Supplementary Fig. 1

        Schematic representation of AUF1 and HuR interaction with their target mRNAs. In the cytoplasm, MAT2A mRNA is stabilized by its preferential association with HuR, whereas MAT1A is degraded after its association with AUF1.

      • Supplementary Fig. 1

        Effect of SAM on APEX1 expression in hepatocellular carcinoma. The Figure shows the association of decrease in MAT1A expression with increase in c-MYC and APEX1 mRNA levels, the hypothetical link between MAT1A decrease and APEX1 protein ubiquitination and proteasomal degradation, and the possible SAM targets.

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