María Luz Martínez-Chantar

Spontaneous oxidative stress and liver tumors in mice lacking methionine adenosyltransferase 1A

In mammals, methionine metabolism occurs mainly in the liver via methionine adenosyltransferase‐catalyzed conversion to S‐adenosylmethionine. Of the two genes that encode methionine adenosyltransferase(MAT1A and MAT2A), MAT1A is mainly expressed in adult liver whereas MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic S‐adenosylmethionine content and hyperplasia and spontaneously develop nonalcoholic steatohepatitis. In this study, we examined whether chronic hepatic Sadenosylmethionine deficiency generates oxidative stress and predisposes to injury and malignant transformation. Differential gene expression in MAT1A knockout mice was analyzed following the criteria of the Gene Ontology Consortium. Susceptibility of MAT1A knockout mice to CCl4‐induced hepatotoxicity and malignant transformation was determined in 3‐ and 18month‐old mice, respectively. Analysis of gene expression profiles revealed an abnormal expression of genes involved in the metabolism of lipids and carbohydrates in MAT1A knockout mice, a situation that is reminiscent of that found in diabetes, obesity, and other conditions associated with nonalcoholic steatohepatitis. This aberrant expression of metabolic genes in the knockout mice was associated with hyperglycemia, increased hepatic CYP2E1 and UCP2 expression and triglyceride levels, and reduced hepatic glutathione content. The knockout animals have increased lipid peroxidation and enhanced sensitivity to CCl4‐induced liver damage, which was largely due to increased CYP2E1 expression because diallyl sulfide, an inhibitor of CYP2E1, prevented CCl4‐induced liver injury. Hepatocellular carcinoma developed in more than half of the knockout mice by 18 months of age. Taken together, our findings define a critical role for S‐adenosylmethionine in maintaining normal hepatic function and tumorigenesis of the liver.

Salermide, a Sirtuin inhibitor with a strong cancer-specific proapoptotic effect

Sirtuin 1 (Sirt1) and Sirtuin 2 (Sirt2) belong to the family of NAD+ (nicotinamide adenine dinucleotide-positive)-dependent class III histone deacetylases and are involved in regulating lifespan. As cancer is a disease of ageing, targeting Sirtuins is emerging as a promising antitumour strategy. Here we present Salermide (N-{3-[(2-hydroxy-naphthalen-1-ylmethylene)-amino]-phenyl}-2-phenyl-propionamide), a reverse amide with a strong in vitro inhibitory effect on Sirt1 and Sirt2. Salermide was well tolerated by mice at concentrations up to 100 μM and prompted tumour-specific cell death in a wide range of human cancer cell lines. The antitumour activity of Salermide was primarily because of a massive induction of apoptosis. This was independent of global tubulin and K16H4 acetylation, which ruled out a putative Sirt2-mediated apoptotic pathway and suggested an in vivo mechanism of action through Sirt1. Consistently with this, RNA interference-mediated knockdown of Sirt1, but not Sirt2, induced apoptosis in cancer cells. Although p53 has been reported to be a target of Sirt1, genetic p53 knockdowns showed that the Sirt1-dependent proapoptotic effect of Salermide is p53-independent. We were finally able to ascribe the apoptotic effect of Salermide to the reactivation of proapoptotic genes epigenetically repressed exclusively in cancer cells by Sirt1. Taken together, our results underline Salermides promise as an anticancer drug and provide evidence for the molecular mechanism through which Sirt1 is involved in human tumorigenesis.

Sirtuin 1 regulation of developmental genes during differentiation of stem cells

The longevity-promoting NAD+–dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.

Schwann cell autophagy, myelinophagy, initiates myelin clearance from injured nerves

Schwann cells degrade myelin after injury by a novel form of selective autophagy, myelinophagy, which is positively regulated by the JNK/c-Jun pathway and is defective in the injured central nervous system.

Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation

Hu antigen R (HuR) is a central RNA‐binding protein regulating cell dedifferentiation, proliferation, and survival, which are well‐established hallmarks of cancer. HuR is frequently overexpressed in tumors correlating with tumor malignancy, which is in line with a role for HuR in tumorigenesis. However, the precise mechanism leading to changes in HuR expression remains unclear. In the liver, HuR plays a crucial role in hepatocyte proliferation, differentiation, and transformation. Here, we unraveled a novel mean of regulation of HuR expression in hepatocellular carcinoma (HCC) and colon cancer. HuR levels correlate with the abundance of the oncogene, murine double minute 2 (Mdm2), in human HCC and colon cancer metastases. HuR is stabilized by Mdm2‐mediated NEDDylation in at least three lysine residues, ensuring its nuclear localization and protection from degradation. Conclusion: This novel Mdm2/NEDD8/HuR regulatory framework is essential for the malignant transformation of tumor cells, which, in turn, unveils a novel signaling paradigm that is pharmacologically amenable for cancer therapy. (Hepatology 2012)

Neoangiogenesis-related genes are hallmarks of fast-growing hepatocellular carcinomas and worst survival. Results from a prospective study

Objective The biological heterogeneity of hepatocellular carcinoma (HCC) makes prognosis difficult. We translate the results of a genome-wide high-throughput analysis into a tool that accurately predicts at presentation tumour growth and survival of patients with HCC. Design Ultrasound surveillance identified HCC in 78 (training set) and 54 (validation set) consecutive patients with cirrhosis. Patients underwent two CT scans 6 weeks apart (no treatment in-between) to determine tumour volumes (V0 and V1) and calculate HCC doubling time. Baseline-paired HCC and surrounding tissue biopsies for microarray study (Agilent Whole Human Genome Oligo Microarrays) were also obtained. Predictors of survival were assessed by multivariate Cox model. Results Calculated tumour doubling times ranged from 30 to 621 days (mean, 107±91 days; median, 83 days) and were divided into quartiles: ≤53 days (n=19), 54–82 days (n=20), 83–110 days (n=20) and ≥111 days (n=19). Median survival according to doubling time was significantly lower for the first quartile versus the others (11 vs 41 months, 42, and 47 months, respectively) (p<0.0001). A five-gene transcriptomic hepatic signature including angiopoietin-2 (ANGPT2), delta-like ligand 4 (DLL4), neuropilin (NRP)/tolloid (TLL)-like 2 (NETO2), endothelial cell-specific molecule-1 (ESM1), and nuclear receptor subfamily 4, group A, member 1 (NR4A1) was found to accurately identify rapidly growing HCCs of the first quartile (ROC AUC: 0.961; 95% CI 0.919 to 1.000; p<0.0001) and to be an independent factor for mortality (HR: 3.987; 95% CI 1.941 to 8.193, p<0.0001). Conclusions The hepatic five-gene signature was able to predict HCC growth in individual patient and the consequent risk of death. This implies a role of this molecular tool in the future therapeutic management of patients with HCC. Trial registration number ClinicalTrials.gov Identifier: NCT01657695.

Insulin-like growth factor-I improves intestinal barrier function in cirrhotic rats

Background and aims: In liver cirrhosis, disruption of the intestinal barrier facilitates bacterial translocation and spontaneous bacterial peritonitis. Insulin-like growth factor I (IGF-I) is an anabolic hormone synthesised by hepatocytes that displays hepatoprotective activities and trophic effects on the intestine. The aim of this study was to investigate the effect of IGF-I on intestinal barrier function in cirrhotic rats. Methods: In rats with carbon tetrachloride induced cirrhosis, we investigated the effect of IGF-I therapy on: (a) portal pressure; (b) intestinal histology and permeability to endotoxin and bacteria; (c) intestinal expression of cyclooxygenase 2 (COX-2) and tumour necrosis factor α (TNF-α), two factors that influence in a positive and negative manner, respectively, the integrity of the intestinal barrier; (d) intestinal permeability to 3H-mannitol in rats with bile duct ligation (BDL); and (e) transepithelial electrical resistance (TER) of polarised monolayers of rat small intestine epithelial cells. Results: IGF-I therapy reduced liver collagen expression and portal pressure in cirrhotic rats, induced improvement in intestinal histology, and caused a reduction in bacterial translocation and endotoxaemia. These changes were associated with diminished TNF-α expression and elevated COX-2 levels in the intestine. IGF-I reduced intestinal permeability in BDL rats and enhanced barrier function of the monolayers of epithelial intestinal cells where lipopolysaccharide (LPS) caused a decrease in TER that was reversed by IGF-I. This effect of IGF-I was associated with upregulation of COX-2 in LPS treated enterocytes. Conclusions: IGF-I enhances intestinal barrier function and reduces endotoxaemia and bacterial translocation in cirrhotic rats. IGF-I therapy might be useful in the prevention of spontaneous bacterial peritonitis in liver cirrhosis.

SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling

Sirtuin1 (SIRT1) regulates central metabolic functions such as lipogenesis, protein synthesis, gluconeogenesis, and bile acid (BA) homeostasis through deacetylation. Here we describe that SIRT1 tightly controls the regenerative response of the liver. We performed partial hepatectomy (PH) to transgenic mice that overexpress SIRT1 (SIRT). SIRT mice showed increased mortality, impaired hepatocyte proliferation, BA accumulation, and profuse liver injury after surgery. The damaging phenotype in SIRT mice correlated with impaired farnesoid X receptor (FXR) activity due to persistent deacetylation and lower protein expression that led to decreased FXR‐target gene expression; small heterodimer partner (SHP), bile salt export pump (BSEP), and increased Cyp7A1. Next, we show that 24‐norUrsodeoxycholic acid (NorUDCA) attenuates SIRT protein expression, increases the acetylation of FXR and neighboring histones, restores trimethylation of H3K4 and H3K9, and increases miR34a expression, thus reestablishing BA homeostasis. Consequently, NorUDCA restored liver regeneration in SIRT mice, which showed increased survival and hepatocyte proliferation. Furthermore, a leucine‐enriched diet restored mammalian target of rapamycin (mTOR) activation, acetylation of FXR and histones, leading to an overall lower BA production through SHP‐inhibition of Cyp7A1 and higher transport (BSEP) and detoxification (Sult2a1) leading to an improved liver regeneration. Finally, we found that human hepatocellular carcinoma (HCC) samples have increased presence of SIRT1, which correlated with the absence of FXR, suggesting its oncogenic potential. Conclusion: We define SIRT1 as a key regulator of the regenerative response in the liver through posttranscriptional modifications that regulate the activity of FXR, histones, and mTOR. Moreover, our data suggest that SIRT1 contributes to liver tumorigenesis through dysregulation of BA homeostasis by persistent FXR deacetylation. (Hepatology 2014;59:1972–1983)

Mitochondrial GSH determines the toxic or therapeutic potential of superoxide scavenging in steatohepatitis

BACKGROUND & AIMS Steatohepatitis (SH) is associated with mitochondrial dysfunction and excessive production of superoxide, which can then be converted into H(2)O(2) by SOD2. Since mitochondrial GSH (mGSH) plays a critical role in H(2)O(2) reduction, we explored the interplay between superoxide, H(2)O(2), and mGSH in nutritional and genetic models of SH, which exhibit mGSH depletion. METHODS We used isolated mitochondria and primary hepatocytes, as well as in vivo SH models showing mGSH depletion to test the consequences of superoxide scavenging. RESULTS In isolated mitochondria and primary hepatocytes, superoxide scavenging by SOD mimetics or purified SOD decreased superoxide and peroxynitrite generation but increased H(2)O(2) following mGSH depletion, despite mitochondrial peroxiredoxin/thioredoxin defense. Selective mGSH depletion sensitized hepatocytes to cell death induced by SOD mimetics, and this was prevented by RIP1 kinase inhibition with necrostatin-1 or GSH repletion with GSH ethyl ester (GSHee). Mice fed the methionine-choline deficient (MCD) diet or MAT1A(-/-) mice exhibited reduced SOD2 activity; in vivo treatment with SOD mimetics increased liver damage, inflammation, and fibrosis, despite a decreased superoxide and 3-nitrotyrosine immunoreactivity, effects that were ameliorated by mGSH replenishment with GSHee, but not NAC. As a proof-of-principle of the detrimental role of superoxide scavenging when mGSH was depleted transgenic mice overexpressing SOD2 exhibited enhanced susceptibility to MCD-mediated SH. CONCLUSIONS These findings underscore a critical role for mGSH in the therapeutic potential of superoxide scavenging in SH, and suggest that the combined approach of superoxide scavenging with mGSH replenishment may be important in SH.

S-adenosylmethionine and proliferation: new pathways, new targets.

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.