Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide affecting over one-third of the population in the U.S. It has been associated with obesity, type 2 diabetes, hyperlipidemia, and insulin resistance and is initiated by the accumulation of triglycerides in hepatocytes. Isolated hepatic steatosis (IHS) remains a benign process, while a subset develops superimposed inflammatory activity and progression to nonalcoholic steatohepatitis (NASH) with or without fibrosis. However, the molecular mechanisms underlying NAFLD progression are not completely understood. Liver biopsy is still required to differentiate IHS from NASH as easily accessible noninvasive biomarkers are lacking. In terms of treatments for NASH, pioglitazone, vitamin E, and obeticholic acid have shown some benefit. All of these agents have potential complications associated with long-term use. Nowadays, a complex hypothesis suggests that multiple parallel hits are involved in NASH development. However, the 'key switch' between IHS and NASH remains to be discovered. We have recently shown that knocking out enzymes involved in S-adenosylmethionine (SAMe) metabolism, the main biological methyl donor in humans that is abundant in the liver, will lead to NASH development in mice. This could be due to the fact that a normal SAMe level is required to establish the proper ratio of phosphatidylethanolamine to phosphatidylcholine that has been found to be important in NAFLD progression. New data from humans have also suggested that these enzymes play a role in the pathogenesis of NAFLD and that some of SAMe cycle metabolites may serve as noninvasive biomarkers of NASH. In this review, we discuss the evidence of the role of SAMe in animal models and humans with NAFLD and how studying this area may lead to the discovery of new noninvasive biomarkers and possibly personalized treatment for NASH.

Nonalcoholic fatty liver disease (NAFLD) is a heterogeneous and complex disease that is imprecisely diagnosed by liver biopsy. NAFLD covers a spectrum that ranges from simple steatosis, nonalcoholic steatohepatitis (NASH) with varying degrees of fibrosis, to cirrhosis, which is a major risk factor for hepatocellular carcinoma. Lifestyle and eating habit changes during the last century have made NAFLD the most common liver disease linked to obesity, type 2 diabetes mellitus and dyslipidemia, with a global prevalence of 25%. NAFLD arises when the uptake of fatty acids (FA) and triglycerides (TG) from circulation and de novo lipogenesis saturate the rate of FA β-oxidation and very-low density lipoprotein (VLDL)-TG export. Deranged lipid metabolism is also associated with NAFLD progression from steatosis to NASH, and therefore, alterations in liver and serum lipidomic signatures are good indicators of the disease's development and progression. This review focuses on the importance of the classification of NAFLD patients into different subtypes, corresponding to the main alteration(s) in the major pathways that regulate FA homeostasis leading, in each case, to the initiation and progression of NASH. This concept also supports the targeted intervention as a key approach to maximize therapeutic efficacy and opens the door to the development of precise NASH treatments.

Methionine adenosyltransferase genes encode enzymes responsible for the biosynthesis of S-adenosylmethionine, the principal biological methyl donor and precursor of polyamines and glutathione. Mammalian cells express three genes - MAT1A, MAT2A, and MAT2B - with distinct expression and functions. MAT1A is mainly expressed in the liver and maintains the differentiated states of both hepatocytes and bile duct epithelial cells. Conversely, MAT2A and MAT2B are widely distributed in non-parenchymal cells of the liver and extrahepatic tissues. Increasing evidence suggests that methionine adenosyltransferases play significant roles in the development of cancers. Liver cancers, namely hepatocellular carcinoma and cholangiocarcinoma, involve dysregulation of all three methionine adenosyltransferase genes. MAT1A reduction is associated with increased oxidative stress, progenitor cell expansion, genomic instability, and other mechanisms implicated in tumorigenesis. MAT2A/MAT2B induction confers growth and survival advantage to cancerous cells, enhancing tumor migration. Highlighted examples from colon, gastric, breast, pancreas and prostate cancer studies further underscore methionine adenosyltransferase genes' role beyond the liver in cancer development. In this subset of extra-hepatic cancers, MAT2A and MAT2B are induced via different regulatory mechanisms. Understanding the role of methionine adenosyltransferase genes in tumorigenesis helps identify attributes of these genes that may serve as valuable targets for therapy. While S-adenosylmethionine, and its metabolite, methylthioadenosine, have been largely explored as therapeutic interventions, targets aimed at regulation of MAT gene expression and methionine adenosyltransferase protein-protein interactions are now surfacing as potential effective strategies for treatment and chemoprevention of cancers. Impact statement This review examines the role of methionine adenosyltransferases (MATs) in human cancer development, with a particular focus on liver cancers in which all three MAT genes are implicated in tumorigenesis. An overview of MAT genes, isoenzymes and their regulation provide context for understanding consequences of dysregulation. Highlighting examples from liver, colon, gastric, breast, pancreas and prostate cancers underscore the importance of understanding MAT's tumorigenic role in identifying future targets for cancer therapy.

Methionine adenosyltransferases (MATs) are essential enzymes for life as they produce S-adenosylmethionine (SAMe), the biological methyl donor required for a plethora of reactions within the cell. Mammalian systems express two genes, MAT1A and MAT2A, which encode for MATα1 and MATα2, the catalytic subunits of the MAT isoenzymes, respectively. A third gene MAT2B, encodes a regulatory subunit known as MATβ which controls the activity of MATα2. MAT1A, which is mainly expressed in hepatocytes, maintains the differentiated state of these cells, whilst MAT2A and MAT2B are expressed in extrahepatic tissues as well as non-parenchymal cells of the liver (e.g., hepatic stellate and Kupffer cells). The biosynthesis of SAMe is impaired in patients with chronic liver disease and liver cancer due to decreased expression and inactivation of MATα1. A switch from MAT1A to MAT2A/MAT2B occurs in multiple liver diseases and during liver growth and dedifferentiation, but this change in the expression pattern of MATs results in reduced hepatic SAMe level. Decades of study have utilized the Mat1a-knockout (KO) mouse that spontaneously develops non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) to elucidate a variety of mechanisms by which MAT proteins dysregulation contributes to liver carcinogenesis. An increasing volume of work indicates that MATs have SAMe-independent functions, distinct interactomes and multiple subcellular localizations. Here we aim to provide an overview of MAT biology including genes, isoenzymes and their regulation to provide the context for understanding consequences of their dysregulation. We will highlight recent breakthroughs in the field and underscore the importance of MAT's in liver tumorigenesis as well as their potential as targets for cancer therapy.

The discovery of the microRNA (miRNA) family of small RNAs as fundamental regulators of post-transcriptional gene expression has fostered research on their importance in every area of biology and clinical medicine. In the particular area of liver metabolism and disease, miRNAs are gaining increasing importance. By focusing on two fundamental hepatic biosynthetic pathways, glutathione and methionine, we review recent advances on the comprehension of the role of miRNAs in liver pathophysiology and more specifically of models of hepatic cholestasis/fibrosis and hepatocellular carcinoma.

Methionine adenosyltransferase 2B (MAT2B) encodes for variant proteins V1 and V2 that interact with GIT1 to increase ERK activity and growth in human liver and colon cancer cells. MAT2B or GIT1 overexpression activates MEK. This study explores the mechanism for MEK activation. We examined protein-protein interactions by co-immunoprecipitation and verified by confocal microscopy and pull-down assay using recombinant or in vitro translated proteins. Results were confirmed in an orthotopic liver cancer model. We found that MAT2B and GIT1-mediated MEK1/2 activation was not mediated by PAK1 or Src in HepG2 or RKO cells. Instead, MAT2B and GIT1 interact with B-Raf and c-Raf and enhance recruitment of Raf proteins to MEK1/2. MAT2B-GIT1 activates c-Raf, which is the key mediator for MEK/12 activation, because this still occurred in RKO cells that express constitutively active B-Raf mutant. The mechanism lies with the ability of MAT2B-GIT1 to activate Ras and promote B-Raf/c-Raf heterodimerization. Interestingly, MAT2B but not GIT1 can directly interact with Ras, which increases protein stability. Finally, increased Ras-Raf-MEK signaling occurred in phenotypically more aggressive liver cancers overexpressing MAT2B variants and GIT1. In conclusion, interaction between MAT2B and GIT1 serves as a scaffold and facilitates signaling in multiple steps of the Ras/Raf/MEK/ERK pathway, further emphasizing the importance of MAT2B/GIT1 interaction in cancer growth.

Prohibitin 1 (PHB1) is a mitochondrial chaperone that regulates cell growth. Phb1 knock-out mice exhibit liver injury and hepatocellular carcinoma (HCC). Phb1 knock-out livers show induction of tumor growth-associated genes, H19 and insulin-like growth factor 2 (Igf2). These genes are controlled by the imprinting control region (ICR) containing CCCTC-binding transcription factor (CTCF)-binding sites. Because Phb1 knock-out mice exhibited induction of H19 and Igf2, we hypothesized that PHB1-mediated regulation of the H19-Igf2 axis might control cell proliferation in normal hepatocytes. H19 and Igf2 were induced (8-20-fold) in 3-week-old Phb1 knock-out livers, in Phb1 siRNA-treated AML12 hepatocytes (2-fold), and HCC cell lines when compared with control. Phb1 knockdown lowered CTCF protein in AML12 by ∼30% when compared with control. CTCF overexpression lowered basal H19 and Igf2 expression by 30% and suppressed Phb1 knockdown-mediated induction of these genes. CTCF and PHB1 co-immunoprecipitated and co-localized on the ICR element, and Phb1 knockdown lowered CTCF ICR binding activity. The results suggest that PHB1 and CTCF cooperation may control the H19-Igf2 axis. Human HCC tissues with high levels of H19 and IGF2 exhibited a 40-50% reduction in PHB1 and CTCF expression and their ICR binding activity. Silencing Phb1 or overexpressing H19 in the mouse HCC cell line, SAMe-D, induced cell growth. Blocking H19 induction prevented Phb1 knockdown-mediated growth, whereas H19 overexpression had the reverse effect. Interestingly H19 silencing induced PHB1 expression. Taken together, our results demonstrate that the H19-Igf2 axis is negatively regulated by CTCF-PHB1 cooperation and that H19 is involved in modulating the growth-suppressive effect of PHB1 in the liver.

Prohibitin1 (PHB1) is a mitochondrial chaperone with diverse functions that include cell proliferation, apoptosis, and mitochondrial homoeostasis. Liver-specific Phb1 knockout (KO) mice develop spontaneous injury and hepatocellular carcinoma (HCC). Our previous work demonstrated that PHB1 negatively regulates the H19-insulin-like growth factor 2 (IGF2)-H19-IGF2 axis signaling pathway and E-box activity in hepatocytes and HCC cells. Phb1 KO livers exhibited increased expression of multiple wingless/integrated (WNT) target genes compared to control littermates. Therefore, we hypothesized that PHB1 is a negative regulator of WNT-beta-catenin signaling in the liver. Analysis of livers from Phb1 KO mice demonstrated an activation of the WNT-beta-catenin pathway as determined by phosphorylation of glycogen synthase kinase 3 (GSK3)betaserine [Ser]9 and protein kinase B (AKT)Ser473. Phb1 KO livers showed increased messenger RNA (mRNA) levels of multiple WNT ligands, with Wnt7a (79-fold), Wnt10a (12-fold), and Wnt16 (48-fold) being most highly overexpressed compared to control littermates. Subcellular fractionation of liver cells from Phb1 KO mice indicated that hepatocytes are the main source of WNT ligands. Immunostaining and cellular colocalization analysis of Phb1 KO livers demonstrated expression of WNT7a, WNT10a, and WNT16 in hepatocytes. Chromatin immunoprecipitation revealed increased binding of transcription factor E2F1 (E2F1) to the Wnt10a promoter in Phb1 KO livers and WNT9A in HepG2 cells. PHB1 silencing in HepG2 cells activated WNT signaling, whereas its overexpression caused inactivation of this pathway. PHB1 silencing in HepG2 cells induced the expression of multiple WNT ligands of which WNT9A induction was partly regulated through E2F1. Conclusion: PHB1 acts as a negative regulator of WNT signaling, and its down-regulation causes the induction of multiple WNT ligands and downstream activation of canonical WNT-beta-catenin signaling in murine liver and human HCC cells, in part through E2F1.

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and inhibits apoptosis via Bcl-2 by unknown mechanism. Methionine adenosyltransferase 2A (MAT2A) encodes for MATα2, the catalytic subunit of the MATII isoenzyme that synthesizes S-adenosylmethionine (SAMe). Ubc9, Bcl-2 and MAT2A expression are up-regulated in several malignancies. Exogenous SAMe decreases Ubc9 and MAT2A expression and is pro-apoptotic in liver and colon cancer cells. Here we investigated whether there is interplay between Ubc9, MAT2A and Bcl-2. We used human colon and liver cancer cell lines RKO and HepG2, respectively, and confirmed key finding in colon cancer specimens. We found MATα2 can regulate Bcl-2 expression at multiple levels. MATα2 binds to Bcl-2 promoter to activate its transcription. This effect is independent of SAMe as MATα2 catalytic mutant was also effective. MATα2 also directly interacts with Bcl-2 to enhance its protein stability. MATα2's effect on Bcl-2 requires Ubc9 as MATα2's stability is influenced by sumoylation at K340, K372 and K394. Overexpressing wild type (but not less stable MATα2 sumoylation mutants) protected from 5-fluorouracil-induced apoptosis in both colon and liver cancer cells. Colon cancer have higher levels of sumoylated MATα2, total MATα2, Ubc9 and Bcl-2 and higher MATα2 binding to the Bcl-2 P2 promoter. Taken together, Ubc9's protective effect on apoptosis may be mediated at least in part by sumoylating and stabilizing MATα2 protein, which in turn positively maintains Bcl-2 expression. These interactions feed forward to further enhance growth and survival of the cancer cell.

The principal methyl donor of the cell, S-adenosylmethionine (SAMe), is produced by the highly conserved family of methionine adenosyltranferases (MATs) via an ATP-driven process. These enzymes play an important role in the preservation of life, and their dysregulation has been tightly linked to liver and colon cancers. We present crystal structures of human MATα2 containing various bound ligands, providing a "structural movie" of the catalytic steps. High- to atomic-resolution structures reveal the structural elements of the enzyme involved in utilization of the substrates methionine and adenosine and in formation of the product SAMe. MAT enzymes are also able to produce S-adenosylethionine (SAE) from substrate ethionine. Ethionine, an S-ethyl analog of the amino acid methionine, is known to induce steatosis and pancreatitis. We show that SAE occupies the active site in a manner similar to SAMe, confirming that ethionine also uses the same catalytic site to form the product SAE.