Sumoylation of methionine adenosyltransferase alpha 1 promotes mitochondrial dysfunction in alcohol-associated liver disease.

BACKGROUND AND AIMS: Methionine adenosyltransferase alpha1 (MATα1) is responsible for the biosynthesis of S-adenosylmethionine in normal liver. Alcohol consumption enhances MATα1 interaction with peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1), which blocks MATα1 mitochondrial targeting, resulting in lower mitochondrial MATα1 content and mitochondrial dysfunction in alcohol-associated liver disease (ALD) in part through upregulation of cytochrome P450 2E1. Conversely, alcohol intake enhances SUMOylation, which enhances cytochrome P450 2E1 expression. MATα1 has potential SUMOylation sites, but whether MATα1 is regulated by SUMOylation in ALD is unknown. Here, we investigated if MATα1 is regulated by SUMOylation and, if so, how it impacts mitochondrial function in ALD. APPROACH AND RESULTS: Proteomics profiling revealed hyper-SUMOylation of MATα1, and prediction software identified lysine 48 (K48) as the potential SUMOylation site in mice (K47 in humans). Experiments with primary hepatocytes, mouse, and human livers revealed that SUMOylation of MAT1α by SUMO2 depleted mitochondrial MATα1. Furthermore, mutation of MATα1 K48 prevented ethanol-induced mitochondrial membrane depolarization, MATα1 depletion, and triglyceride accumulation. Additionally, CRISPR/CRISPR associated protein 9 gene editing of MATα1 at K48 hindered ethanol-induced MATα1-PIN1 interaction, degradation, and phosphorylation of MATα1 in vitro. In vivo, CRISPR/CRISPR associated protein 9 MATα1 K48 gene-edited mice were protected from ethanol-induced fat accumulation, liver injury, MATα1-PIN1 interaction, mitochondrial MATα1 depletion, mitochondrial dysfunction, and low S-adenosylmethionine levels. CONCLUSIONS: Taken together, our findings demonstrate an essential role for SUMOylation of MATα1 K48 for interaction with PIN1 in ALD. Preventing MATα1 K48 SUMOylation may represent a potential treatment strategy for ALD.

Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis.

BACKGROUND & AIMS: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. METHODS: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). RESULTS: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. CONCLUSION: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. IMPACT AND IMPLICATIONS: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

Reciprocal Regulation Between Forkhead Box M1/NF-κB and Methionine Adenosyltransferase 1A Drives Liver Cancer.

BACKGROUND AND AIMS: Forkhead box M1 (FOXM1) and nuclear factor kappa B (NF-ĸB) are oncogenic drivers in liver cancer that positively regulate each other. We showed that methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor in the liver and inhibits NF-ĸB activity. Here, we examined the interplay between FOXM1/NF-κB and MAT1A in liver cancer. APPROACH AND RESULTS: We examined gene and protein expression, effects on promoter activities and binding of proteins to promoter regions, as well as effects of FOXM1 inhibitors T0901317 (T0) and forkhead domain inhibitory-6 (FDI-6) in vitro and in xenograft and syngeneic models of liver cancer. We found, in both hepatocellular carcinoma and cholangiocarcinoma, that an induction in FOXM1 and NF-κB expression is accompanied by a fall in MATα1 (protein encoded by MAT1A). The Cancer Genome Atlas data set confirmed the inverse correlation between FOXM1 and MAT1A. Interestingly, FOXM1 directly interacts with MATα1 and they negatively regulate each other. In contrast, FOXM1 positively regulates p50 and p65 expression through MATα1, given that the effect is lost in its absence. FOXM1, MATα1, and NF-κB all bind to the FOX binding sites in the FOXM1 and MAT1A promoters. However, binding of FOXM1 and NF-κB repressed MAT1A promoter activity, but activated the FOXM1 promoter. In contrast, binding of MATα1 repressed the FOXM1 promoter. MATα1 also binds and represses the NF-κB element in the presence of p65 or p50. Inhibiting FOXM1 with either T0 or FDI-6 inhibited liver cancer cell growth in vitro and in vivo. However, inhibiting FOXM1 had minimal effects in liver cancer cells that do not express MAT1A. CONCLUSIONS: We have found a crosstalk between FOXM1/NF-κB and MAT1A. Up-regulation in FOXM1 lowers MAT1A, but raises NF-κB, expression, and this is a feed-forward loop that enhances tumorigenesis.

Methionine Adenosyltransferase α1 Is Targeted to the Mitochondrial Matrix and Interacts with Cytochrome P450 2E1 to Lower Its Expression.

Methionine adenosyltransferase α1 (MATα1, encoded by MAT1A) is responsible for hepatic biosynthesis of S-adenosyl methionine, the principal methyl donor. MATα1 also act as a transcriptional cofactor by interacting and influencing the activity of several transcription factors. Mat1a knockout (KO) mice have increased levels of cytochrome P450 2E1 (CYP2E1), but the underlying mechanisms are unknown. The aims of the current study were to identify binding partners of MATα1 and elucidate how MATα1 regulates CYP2E1 expression. We identified binding partners of MATα1 by coimmunoprecipitation (co-IP) and mass spectrometry. Interacting proteins were confirmed using co-IP using recombinant proteins, liver lysates, and mitochondria. Alcoholic liver disease (ALD) samples were used to confirm relevance of our findings. We found that MATα1 negatively regulates CYP2E1 at mRNA and protein levels, with the latter being the dominant mechanism. MATα1 interacts with many proteins but with a predominance of mitochondrial proteins including CYP2E1. We found that MATα1 is present in the mitochondrial matrix of hepatocytes using immunogold electron microscopy. Mat1a KO hepatocytes had reduced mitochondrial membrane potential and higher mitochondrial reactive oxygen species, both of which were normalized when MAT1A was overexpressed. In addition, KO hepatocytes were sensitized to ethanol and tumor necrosis factor α-induced mitochondrial dysfunction. Interaction of MATα1 with CYP2E1 was direct, and this facilitated CYP2E1 methylation at R379, leading to its degradation through the proteasomal pathway. Mat1a KO livers have a reduced methylated/total CYP2E1 ratio. MATα1's influence on mitochondrial function is largely mediated by its effect on CYP2E1 expression. Patients with ALD have reduced MATα1 levels and a decrease in methylated/total CYP2E1 ratio. Conclusion: Our findings highlight a critical role of MATα1 in regulating mitochondrial function by suppressing CYP2E1 expression at multiple levels.

Mechanisms of MAFG Dysregulation in Cholestatic Liver Injury and Development of Liver Cancer.

BACKGROUND & AIMS: MAF bZIP transcription factor G (MAFG) is activated by the farnesoid X receptor to repress bile acid synthesis. However, expression of MAFG increases during cholestatic liver injury in mice and in cholangiocarcinomas. MAFG interacts directly with methionine adenosyltransferase α1 (MATα1) and other transcription factors at the E-box element to repress transcription. We studied mechanisms of MAFG up-regulation in cholestatic tissues and the pathways by which S-adenosylmethionine (SAMe) and ursodeoxycholic acid (UDCA) prevent the increase in MAFG expression. We also investigated whether obeticholic acid (OCA), an farnesoid X receptor agonist, affects MAFG expression and how it contributes to tumor growth in mice. METHODS: We obtained 7 human cholangiocarcinoma specimens and adjacent non-tumor tissues from patients that underwent surgical resection in California and 113 hepatocellular carcinoma (HCC) specimens and adjacent non-tumor tissues from China, along with clinical data from patients. Tissues were analyzed by immunohistochemistry. MAT1A, MAT2A, c-MYC, and MAFG were overexpressed or knocked down with small interfering RNAs in MzChA-1, KMCH, Hep3B, and HepG2 cells; some cells were incubated with lithocholic acid (LCA, which causes the same changes in gene expression observed during chronic cholestatic liver injury in mice), SAMe, UDCA (100 µM), or farnesoid X receptor agonists. MAFG expression and promoter activity were measured using real-time polymerase chain reaction, immunoblot, and transient transfection. We performed electrophoretic mobility shift, and chromatin immunoprecipitation assays to study proteins that occupy promoter regions. We studied mice with bile-duct ligation, orthotopic cholangiocarcinomas, cholestasis-induced cholangiocarcinoma, diethylnitrosamine-induced liver tumors, and xenograft tumors. RESULTS: LCA activated expression of MAFG in HepG2 and MzChA-1 cells, which required the activator protein-1, nuclear factor-κB, and E-box sites in the MAFG promoter. LCA reduced expression of MAT1A but increased expression of MAT2A in cells. Overexpression of MAT2A increased activity of the MAFG promoter, whereas knockdown of MAT2A reduced it. MAT1A and MAT2A had opposite effects on the activator protein-1, nuclear factor-κB, and E-box-mediated promoter activity. Expression of MAFG and MAT2A increased, and expression of MAT1A decreased, in diethylnitrosamine-induced liver tumors in mice. SAMe and UDCA had shared and distinct mechanisms of preventing LCA-mediated increased expression of MAFG. OCA increased expression of MAFG, MAT2A, and c-MYC, but reduced expression of MAT1A. Incubation of human liver and biliary cancer cells lines with OCA promoted their proliferation; in nude mice given OCA, xenograft tumors were larger than in mice given vehicle. Levels of MAFG were increased in human HCC and cholangiocarcinoma tissues compared with non-tumor tissues. High levels of MAFG in HCC samples correlated with hepatitis B, vascular invasion, and shorter survival times of patients. CONCLUSIONS: Expression of MAFG increases in cells and tissues with cholestasis, as well as in human cholangiocarcinoma and HCC specimens; high expression levels correlate with tumor progression and reduced survival time. SAMe and UDCA reduce expression of MAFG in response to cholestasis, by shared and distinct mechanisms. OCA induces MAFG expression, cancer cell proliferation, and growth of xenograft tumors in mice.

Prohibitin 1 suppresses liver cancer tumorigenesis in mice and human hepatocellular and cholangiocarcinoma cells.

Prohibitin 1 (PHB1) is best known as a mitochondrial chaperone, and its role in cancer is conflicting. Mice lacking methionine adenosyltransferase α1 (MATα1) have lower PHB1 expression, and we reported that c-MYC interacts directly with both proteins. Furthermore, c-MYC and MATα1 exert opposing effects on liver cancer growth, prompting us to examine the interplay between PHB1, MATα1, and c-MYC and PHB1's role in liver tumorigenesis. We found that PHB1 is highly expressed in normal hepatocytes and bile duct epithelial cells and down-regulated in most human hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). In HCC and CCA cells, PHB1 expression correlates inversely with growth. PHB1 and MAT1A positively regulate each other's expression, whereas PHB1 negatively regulates the expression of c-MYC, MAFG, and c-MAF. Both PHB1 and MATα1 heterodimerize with MAX, bind to the E-box element, and repress E-box promoter activity. PHB1 promoter contains a repressive E-box element and is occupied mainly by MAX, MNT, and MATα1 in nonmalignant cholangiocytes and noncancerous tissues that switched to c-MYC, c-MAF, and MAFG in cancer cells and human HCC/CCA. All 8-month-old liver-specific Phb1 knockout mice developed HCC, and one developed CCA. Five-month-old Phb1 heterozygotes, but not Phb1 flox mice, developed aberrant bile duct proliferation; and one developed CCA 3.5 months after left and median bile duct ligation. Phb1 heterozygotes had a more profound fall in the expression of glutathione synthetic enzymes and higher hepatic oxidative stress following left and median bile duct ligation. CONCLUSION: We have identified that PHB1, down-regulated in most human HCC and CCA, heterodimerizes with MAX to repress the E-box and positively regulates MAT1A while suppressing c-MYC, MAFG, and c-MAF expression; in mice, reduced PHB1 expression predisposes to the development of cholestasis-induced CCA. (Hepatology 2017;65:1249-1266).

Deregulated methionine adenosyltransferase α1, c-Myc, and Maf proteins together promote cholangiocarcinoma growth in mice and humans(‡).

UNLABELLED: c-Myc induction drives cholestatic liver injury and cholangiocarcinoma (CCA) in mice, and induction of Maf proteins (MafG and c-Maf) contributes to cholestatic liver injury, whereas S-adenosylmethionine (SAMe) administration is protective. Here, we determined whether there is interplay between c-Myc, Maf proteins, and methionine adenosyltransferase α1 (MATα1), which is responsible for SAMe biosynthesis in the liver. We used bile duct ligation (BDL) and lithocholic acid (LCA) treatment in mice as chronic cholestasis models, a murine CCA model, human CCA cell lines KMCH and Huh-28, human liver cancer HepG2, and human CCA specimens to study gene and protein expression, protein-protein interactions, molecular mechanisms, and functional outcomes. We found that c-Myc, MATα1 (encoded by MAT1A), MafG, and c-Maf interact with one another directly. MAT1A expression fell in hepatocytes and bile duct epithelial cells during chronic cholestasis and in murine and human CCA. The opposite occurred with c-Myc, MafG, and c-Maf expression. MATα1 interacts mainly with Mnt in normal liver, but this switches to c-Maf, MafG, and c-Myc in cholestatic livers and CCA. Promoter regions of these genes have E-boxes that are bound by MATα1 and Mnt in normal liver and benign bile duct epithelial cells that switched to c-Myc, c-Maf, and MafG in cholestasis and CCA cells. E-box positively regulates c-Myc, MafG, and c-Maf, but it negatively regulates MAT1A. MATα1 represses, whereas c-Myc, MafG, and c-Maf enhance, E-box-driven promoter activity. Knocking down MAT1A or overexpressing MafG or c-Maf enhanced CCA growth and invasion in vivo. CONCLUSION: There is a novel interplay between MATα1, c-Myc, and Maf proteins, and their deregulation during chronic cholestasis may facilitate CCA oncogenesis. (Hepatology 2016;64:439-455).

Methionine adenosyltransferase 2B-GIT1 complex serves as a scaffold to regulate Ras/Raf/MEK1/2 activity in human liver and colon cancer cells.

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.

S-Adenosylmethionine and methylthioadenosine inhibit ß-catenin signaling by multiple mechanisms in liver and colon cancer.

S-Adenosylmethionine (SAMe), the principal methyl donor that is available as a nutritional supplement, and its metabolite methylthioadenosine (MTA) exert chemopreventive properties against liver and colon cancer in experimental models. Both agents reduced ß-catenin expression on immunohistochemistry in a murine colitis-associated colon cancer model. In this study, we examined the molecular mechanisms involved. SAMe or MTA treatment in the colitis-associated cancer model lowered total ß-catenin protein levels by 47 and 78%, respectively. In an orthotopic liver cancer model, increasing SAMe levels by overexpressing methionine adenosyltransferase 1A also reduced total ß-catenin levels by 68%. In both cases, lower cyclin D1 and c-Myc expression correlated with lower ß-catenin levels. In liver (HepG2) and colon (SW480, HCT116) cancer cells with constitutively active ß-catenin signaling, SAMe and MTA treatment inhibited ß-catenin activity by excluding it from the nuclear compartment. However, in liver (Huh-7) and colon (RKO) cancer cells expressing wild-type Wnt/ß-catenin, SAMe and MTA accelerated ß-catenin degradation by a glycogen synthase kinase 3-ß-dependent mechanism. Both agents lowered protein kinase B activity, but this was not mediated by inhibiting phosphoinositide 3-kinase. Instead, both agents increased the activity of protein phosphatase 2A, which inactivates protein kinase B. The effect of MTA on lowering ß-catenin is direct and not mediated by its conversion to SAMe, as blocking this conversion had no influence. In conclusion, SAMe and MTA inhibit Wnt/ß-catenin signaling in colon and liver cancer cells regardless of whether this pathway is aberrantly induced, making them ideal candidates for chemoprevention and/or chemotherapy in these cancers.

Methionine adenosyltransferase 2B, HuR, and sirtuin 1 protein cross-talk impacts on the effect of resveratrol on apoptosis and growth in liver cancer cells.

Resveratrol is growth-suppressive and pro-apoptotic in liver cancer cells. Methionine adenosyltransferase 2B (MAT2B) encodes for two dominant variants V1 and V2 that positively regulate growth, and V1 is anti-apoptotic when overexpressed. Interestingly, crystal structure analysis of MAT2B protein (MATß) protomer revealed two resveratrol binding pockets, which raises the question of the role of MAT2B in resveratrol biological activities. We found that resveratrol induced the expression of MAT2BV1 and V2 in a time- and dose-dependent manner by increasing transcription, mRNA, and protein stabilization. Following resveratrol treatment, HuR expression increased first, followed by SIRT1 and MAT2B. SIRT1 induction contributes to increased MAT2B transcription whereas HuR induction increased MAT2B mRNA stability. MATß interacts with HuR and SIRT1, and resveratrol treatment enhanced these interactions while reducing the interaction between MATß and MATα2. Because MATß lowers the Ki of MATα2 for S-adenosylmethionine (AdoMet), this allowed steady-state AdoMet level to rise. Interaction among MATß, SIRT1, and HuR increased stability of these proteins. Induction of MAT2B is a compensatory response to resveratrol as knocking down MAT2BV1 potentiated the resveratrol pro-apoptotic and growth-suppressive effects, whereas the opposite occurred with V1 overexpression. The same effect on growth occurred with MAT2BV2. In conclusion, resveratrol induces HuR, SIRT1, and MAT2B expression; the last may represent a compensatory response against apoptosis and growth inhibition. However, MATß induction also facilitates SIRT1 activation, as the interaction stabilizes SIRT1. This complex interplay among MATß, HuR, and SIRT1 has not been previously reported and suggests that these proteins may regulate each other's signaling.

MAT2B-GIT1 interplay activates MEK1/ERK 1 and 2 to induce growth in human liver and colon cancer.

UNLABELLED: Methionine adenosyltransferase 2B (MAT2B) encodes for two variant proteins (V1 and V2) that promote cell growth. Using in-solution proteomics, GIT1 (G Protein Coupled Receptor Kinase Interacting ArfGAP 1) was identified as a potential interacting partner of MAT2B. Here, we examined the functional significance of this interplay. Coimmunoprecipitation experiments examined protein interactions. Tissue expression levels of proteins were examined using immunohistochemistry and western blotting. Expression levels of proteins were varied using transient knockdown or overexpression to observe the effect of alterations in each protein on the entire complex. Direct interaction among individual proteins was further verified using in vitro translated and recombinant proteins. We found both MAT2B variants interact with GIT1. Overexpression of V1, V2, or GIT1 activated mitogen-activated protein kinase kinase 1 (MEK1) and extracellular signal-regulated kinase (ERK), raised cyclin D1 protein level, and increased growth, whereas the opposite occurred when V1, V2, or GIT1 was knocked down. MAT2B and GIT1 require each other to activate MEK1/ERK and increase growth. MAT2B directly interacts with MEK1, GIT1, and ERK2. Expression level of V1, V2, or GIT1 directly influenced recruitment of GIT1 or MAT2B and ERK2 to MEK1, respectively. In pull-down assays, MAT2B directly promoted binding of GIT1 and ERK2 to MEK1. MAT2B and GIT1 interact and are overexpressed in most human liver and colon cancer specimens. Increased expression of V1, V2, or GIT1 promoted growth in an orthotopic liver cancer model, whereas increased expression of either V1 or V2 with GIT1 further enhanced growth and lung metastasis. CONCLUSION: MAT2B and GIT1 form a scaffold, which recruits and activates MEK and ERK to promote growth and tumorigenesis. This novel MAT2B/GIT1 complex may provide a potential therapeutic gateway in human liver and colon cancer. (HEPATOLOGY 2012).

Effects of S-adenosylmethionine and methylthioadenosine on inflammation-induced colon cancer in mice.

Chronic inflammation is an underlying risk factor for colon cancer. Tumor necrosis factor alpha (TNF-α) plays a critical role in the development of inflammation-induced colon cancer in a mouse model. S-adenosylmethionine (SAMe) and its metabolite methylthioadenosine (MTA) can inhibit lipopolysaccharide-induced TNF-α expression in macrophages. The aim of this work was to examine whether SAMe and MTA are effective in preventing inflammation-induced colon cancer and if so identify signaling pathways affected. Balb/c mice were treated with azoxymethane (AOM) and dextran sulfate sodium to induce colon cancer. Two days after AOM treatment, mice were divided into three groups: vehicle control, SAMe or MTA. Tumor load, histology, immunohistochemistry, gene and protein expression were determined. SAMe and MTA treatment reduced tumor load by ∼40%. Both treatments raised SAMe and MTA levels but MTA also raised S-adenosylhomocysteine levels. MTA treatment prevented the induction of many genes known to play pathogenetic roles in this model except for TNF-α and inducible nitric oxide synthase (iNOS). SAMe also had no effect on TNF-α or iNOS and was less inhibitory than MTA on the other genes. In vivo, both treatments induced apoptosis but inhibited proliferation, ß-catenin, nuclear factor kappa B activation and interleukin (IL) 6 signaling. Effect of SAMe and MTA on IL-6 signaling was examined using Colo 205 colon cancer cells. In these cells, SAMe and MTA inhibited IL-6-induced IL-10 expression. MTA also inhibited IL-10 transcription and signal transducer and activator of transcription 3 activation. In conclusion, SAMe and MTA reduced inflammation-induced colon cancer and inhibited several pathways important in colon carcinogenesis.

Inhibition of human methionine adenosyltransferase 1A transcription by coding region methylation.

Two genes (MAT1A and MAT2A) encode for the essential enzyme methionine adenosyltransferase (MAT). MAT1A is silenced in hepatocellular carcinoma (HCC), and absence of MAT1A leads to spontaneous development of HCC in mice. Previous report correlated promoter methylation to silencing of MAT1A but definitive proof was lacking. Here we investigated the role of methylation in regulating MAT1A expression. There are three MspI/HpaII sites from -1,913 to +160 of the human MAT1A gene (numbered relative to the translational start site) at position -977, +10, and +88. Bisulfite treatment and DNA sequencing, and Southern blot analysis showed that methylation at +10 and +88, but not -977, correlated with lack of MAT1A expression. MAT1A promoter construct methylated at -977, +10 or +88 position has 0.7-fold, 3-fold, and 1.6-fold lower promoter activity, respectively. Methylation at -977 and +10 did not inhibit the promoter more than methylation at +10 alone; while methylation at +10 and +88 reduced promoter activity by 60%. Mutation of +10 and +88 sites also resulted in 40% reduction of promoter activity. Reactivation of MAT1A correlated with demethylation of +10 and +88. In vitro transcription assay showed that methylation or mutation of +10 and +88 sites reduced transcription. In conclusion, our data support the novel finding that methylation of the MAT1A coding region can inhibit gene transcription. This represents a key mechanism for decreased MAT1A expression in HCC and a target for therapy. To our knowledge, this is the first example of coding region methylation inhibiting transcription of a mammalian gene.

A mouse model of cholestasis-associated cholangiocarcinoma and transcription factors involved in progression.

BACKGROUND & AIMS: Cholestasis contributes to hepatocellular injury and promotes liver carcinogenesis. We created a mouse model of chronic cholestasis to study its effects on progression of cholangiocarcinoma and the oncogenes involved. METHODS: To induce chronic cholestasis, Balb/c mice were given 2 weekly intraperitoneal injections of diethylnitrosamine (DEN); 2 weeks later, some mice also received left and median bile duct ligation (LMBDL) and, then 1 week later, were fed DEN, in corn oil, weekly by oral gavage (DLD). Liver samples were analyzed by immunohistochemical and biochemical assays; expression of Mnt and c-Myc was reduced by injection of small inhibitor RNAs. RESULTS: Chronic cholestasis was induced by DLD and accelerated progression of cholangiocarcinoma, compared with mice given only DEN. Cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma developed in the DLD group at weeks 8, 12, 16, and 28, respectively. LMBDL repressed expression of microRNA (miR)-34a and let-7a, up-regulating Lin-28B, hypoxia-inducible factor (HIF)-1α, HIF-2α, and miR-210. Up-regulation of Lin-28B might inhibit let-7a, which is associated with development of cystic hyperplasias, cystic atypical hyperplasias, cholangiomas, and cholangiocarcinoma. Knockdown of c-Myc reduced progression of cholangiocarcinoma, whereas knockdown of Mnt accelerated its progression. Down-regulation of miR-34a expression might up-regulate c-Myc. The up-regulation of miR-210 via HIF-2α was involved in down-regulation of Mnt. Activation of the miR-34a-c-Myc and HIF-2α-miR-210-Mnt pathways caused c-Myc to bind the E-box element of cyclin D1, instead of Mnt, resulting in cyclin D1 up-regulation. CONCLUSIONS: DLD induction of chronic cholestasis accelerated progression of cholangiocarcinoma, which is mediated by down-regulation of miR-34a, up-regulation miR-210, and replacement of Mnt by c-Myc in binding to cyclin D1.

Insulin-like growth factor 1 activates methionine adenosyltransferase 2A transcription by multiple pathways in human colon cancer cells.

We have previously reported that the expression of MAT2A (methionine adenosyltransferase 2A) is increased in human colon cancer and in colon cancer cells treated with IGF-1 (insulin-like growth factor-1), which was required for its mitogenic effect. The aim of the present study was to elucidate the molecular mechanisms of IGF-1-mediated MAT2A induction. Nuclear run-on analysis confirmed that the increase in MAT2A expression lies at the transcriptional level. DNase I footprinting of the MAT2A promoter region revealed a similar protein-binding pattern in colon cancer and IGF-1-treated RKO cells. IGF-1 induced MAT2A promoter activity and increased nuclear protein binding to USF (upstream stimulatory factor)/c-Myb, YY1 (Yin and Yang 1), E2F, AP-1 (activator protein 1) and NF-κB (nuclear factor κB) consensus elements. IGF-1 increased the expression of c-Jun, FosB, MafG, p65, c-Myb, E2F-1 and YY1 at the pre-translational level. Knockdown of p65, MafG, c-Myb or E2F-1 lowered basal MAT2A expression and blunted the inductive effect of IGF-1 on MAT2A, whereas knockdown of YY1 increased basal MAT2A expression and had no effect on IGF-1-mediated MAT2A induction. Consistently, mutation of AP-1, NF-κB, E2F and USF/c-Myb elements individually blunted the IGF-1-mediated increase in MAT2A promoter activity, and combined mutations completely prevented the increase. In conclusion, IGF-1 activates MAT2A transcription by both known and novel pathways. YY1 represses MAT2A expression.

Induction of avian musculoaponeurotic fibrosarcoma proteins by toxic bile acid inhibits expression of glutathione synthetic enzymes and contributes to cholestatic liver injury in mice.

UNLABELLED: We previously showed that hepatic expression of glutathione (GSH) synthetic enzymes and GSH levels fell 2 weeks after bile duct ligation (BDL) in mice. This correlated with a switch in nuclear anti-oxidant response element (ARE) binding activity from nuclear factor erythroid 2-related factor 2 (Nrf2) to c-avian musculoaponeurotic fibrosarcoma (c-Maf)/V-maf musculoaponeurotic fibrosarcoma oncogene homolog G (MafG). Our current aims were to examine whether the switch in ARE binding activity from Nrf2 to Mafs is responsible for decreased expression of GSH synthetic enzymes and the outcome of blocking this switch. Huh7 cells treated with lithocholic acid (LCA) exhibited a similar pattern of change in GSH synthetic enzyme expression as BDL mice. Nuclear protein levels of Nrf2 fell at 20 hours after LCA treatment, whereas c-Maf and MafG remained persistently induced. These changes translated to ARE nuclear binding activity. Knockdown of c-Maf or MafG individually blunted the LCA-induced decrease in Nrf2 ARE binding and increased ARE-dependent promoter activity, whereas combined knockdown was more effective. Knockdown of c-Maf or MafG individually increased the expression of GSH synthetic enzymes and raised GSH levels, and combined knockdown exerted an additive effect. Ursodeoxycholic acid (UDCA) or S-adenosylmethionine (SAMe) prevented the LCA-induced decrease in expression of GSH synthetic enzymes and promoter activity and prevented the increase in MafG and c-Maf levels. In vivo knockdown of the Maf genes protected against the decrease in GSH enzyme expression, GSH level, and liver injury after BDL. CONCLUSION: Toxic bile acid induces a switch from Nrf2 to c-Maf/MafG ARE nuclear binding, which leads to decreased expression of GSH synthetic enzymes and GSH levels and contributes to liver injury during BDL. UDCA and SAMe treatment targets this switch.

Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice.

UNLABELLED: Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. CONCLUSION: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes.

S-adenosylmethionine regulates dual-specificity mitogen-activated protein kinase phosphatase expression in mouse and human hepatocytes.

UNLABELLED: Increased mitogen-activated protein kinase (MAPK) activity correlates with a more malignant hepatocellular carcinoma (HCC) phenotype. There is a reciprocal regulation between p44/42 MAPK (extracellular signal-regulated kinase [ERK]1/2) and the dual-specificity MAPK phosphatase MKP-1/DUSP1. ERK phosphorylates DUSP1, facilitating its proteasomal degradation, whereas DUSP1 inhibits ERK activity. Methionine adenosyltransferase 1a (Mat1a) knockout (KO) mice express hepatic S-adenosylmethionine (SAM) deficiency and increased ERK activity and develop HCC. The aim of this study was to examine whether DUSP1 expression is regulated by SAM and if so, elucidate the molecular mechanisms. Studies were conducted using Mat1a KO mice livers, cultured mouse and human hepatocytes, and 20S and 26S proteasomes. DUSP1 messenger RNA (mRNA) and protein levels were reduced markedly in livers of Mat1a KO mice and in cultured mouse and human hepatocytes with protein falling to lower levels than mRNA. SAM treatment protected against the fall in DUSP1 mRNA and protein levels in mouse and human hepatocytes. SAM increased DUSP1 transcription, p53 binding to DUSP1 promoter, and stability of its mRNA and protein. Proteasomal chymotrypsin-like and caspase-like activities were increased in Mat1a KO livers and cultured hepatocytes, which was blocked by SAM treatment. SAM inhibited chymotrypsin-like and caspase-like activities by 40% and 70%, respectively, in 20S proteasomes and caused rapid degradation of some of the 26S proteasomal subunits, which was blocked by the proteasome inhibitor MG132. SAM treatment in Mat1a KO mice for 7 days raised SAM, DUSP1, mRNA and protein levels and lowered proteosomal and ERK activities. CONCLUSION: DUSP1 mRNA and protein levels are lower in Mat1a KO livers and fall rapidly in cultured hepatocytes. SAM treatment increases DUSP1 expression through multiple mechanisms, and this may suppress ERK activity and malignant degeneration.

Deregulated 14-3-3ζ and methionine adenosyltransferase α1 interplay promotes liver cancer tumorigenesis in mice and humans.

Methionine adenosyltransferase 1A (MAT1A) is a tumor suppressor downregulated in hepatocellular carcinoma and cholangiocarcinoma, two of the fastest rising cancers worldwide. We compared MATα1 (protein encoded by MAT1A) interactome in normal versus cancerous livers by mass spectrometry to reveal interactions with 14-3-3ζ. The MATα1/14-3-3ζ complex was critical for the expression of 14-3-3ζ. Similarly, the knockdown and small molecule inhibitor for 14-3-3ζ (BV02), and ChIP analysis demonstrated the role of 14-3-3ζ in suppressing MAT1A expression. Interaction between MATα1 and 14-3-3ζ occurs directly and is enhanced by AKT2 phosphorylation of MATα1. Blocking their interaction enabled nuclear MATα1 translocation and inhibited tumorigenesis. In contrast, overexpressing 14-3-3ζ lowered nuclear MATα1 levels and promoted tumor progression. However, tumor-promoting effects of 14-3-3ζ were eliminated when liver cancer cells expressed mutant MATα1 unable to interact with 14-3-3ζ. Taken together, the reciprocal negative regulation that MATα1 and 14-3-3ζ exert is a key mechanism in liver tumorigenesis.

Switch from Mnt-Max to Myc-Max induces p53 and cyclin D1 expression and apoptosis during cholestasis in mouse and human hepatocytes.

UNLABELLED: Toxic bile acids induce hepatocyte apoptosis, for which p53 and cyclin D1 have been implicated as underlying mediators. Both p53 and cyclin D1 are targets of c-Myc, which is also up-regulated in cholestasis. Myc and Mnt use Max as a cofactor for DNA binding. Myc-Max typically activates transcription via E-box binding. Mnt-Max also binds the E-box sequence but serves as a repressor and inhibits the enhancer activity of Myc-Max. The current work tested the hypothesis that the switch from Mnt-Max to Myc-Max is responsible for p53 and cyclin D1 up-regulation and apoptosis during cholestasis. Following common bile duct ligation or left hepatic bile duct ligation, the expression of p53, c-Myc, and cyclin D1 increased markedly, whereas Mnt expression decreased. Nuclear binding activity of Myc to the E-box element of p53 and cyclin D1 increased, whereas that of Mnt decreased in a time-dependent fashion. Lithocholic acid (LCA) treatment of primary human hepatocytes and HuH-7 cells induced a similar switch from Mnt to Myc and increased p53 and cyclin D1 promoter activity and endogenous p53 and cyclin D1 expression and apoptosis. Blocking c-Myc induction in HuH-7 cells prevented the LCA-mediated increase in p53 and cyclin D1 expression and reduced apoptosis. Lowering Mnt expression further enhanced LCA's inductive effect on p53 and cyclin D1. Bile duct-ligated mice treated with a lentivirus harboring c-myc small interfering RNA were protected from hepatic induction of p53 and cyclin D1, a switch from Mnt to Myc nuclear binding to E-box, and hepatocyte apoptosis. CONCLUSION: The switch from Mnt to Myc during bile duct ligation and in hepatocytes treated with LCA is responsible for the induction in p53 and cyclin D1 expression and contributes to apoptosis.