Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the second most common cause of cancer mortality worldwide [...].

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 (MATβ) 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. MATβ interacts with HuR and SIRT1, and resveratrol treatment enhanced these interactions while reducing the interaction between MATβ and MATα2. Because MATβ lowers the Ki of MATα2 for S-adenosylmethionine (AdoMet), this allowed steady-state AdoMet level to rise. Interaction among MATβ, 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, MATβ induction also facilitates SIRT1 activation, as the interaction stabilizes SIRT1. This complex interplay among MATβ, HuR, and SIRT1 has not been previously reported and suggests that these proteins may regulate each other's signaling.

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.

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).

Hepatic fibrosis is a global health problem currently without effective therapeutic approaches. Even though the ubiquitin-like posttranslational modification of neddylation, that conjugates Nedd8 (neural precursor cell expressed developmentally downregulated) to specific targets, is aberrant in many pathologies, its relevance in liver fibrosis (LF) remained unexplored. Our results show deregulated neddylation in clinical fibrosis and both in mouse bileductligation- and CCl₄ -induced fibrosis. Importantly, neddylation inhibition, by using the pharmacological inhibitor, MLN4924, reduced liver injury, apoptosis, inflammation, and fibrosis by targeting different hepatic cell types. On one hand, increased neddylation was associated with augmented caspase 3 activity in bile-acid-induced apoptosis in mouse hepatocytes whereas neddylation inhibition ameliorated apoptosis through reduction of expression of the Cxcl1 and Ccl2 chemokines. On the other hand, chemokine receptors and cytokines, usually induced in activated macrophages, were reduced after neddylation inhibition in mouse Kupffer cells. Under these circumstances, decreased hepatocyte cell death and inflammation after neddylation inhibition could partly account for reduction of hepatic stellate cell (HSC) activation. We provide evidence that augmented neddylation characterizes activated HSCs, suggesting that neddylation inhibition could be important for resolving LF by directly targeting these fibrogenic cells. Indeed, neddylation inhibition in activated HSCs induces apoptosis in a process partly mediated by accumulation of c-Jun, whose cullin-mediated degradation is impaired under these circumstances.

Conclusion

Neddylation inhibition reduces fibrosis, suggesting neddylation as a potential and attractive therapeutic target in liver fibrosis. (Hepatology 2017;65:694-709).

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.

OBJECTIVE:Non-alcoholic fatty liver disease (NAFLD) is a complex pathology in which several dysfunctions, including alterations in metabolic pathways, mitochondrial functionality and unbalanced lipid import/export, lead to lipid accumulation and progression to inflammation and fibrosis. The enzyme glycine N-methyltransferase (GNMT), the most important enzyme implicated in S-adenosylmethionine catabolism in the liver, is downregulated during NAFLD progression. We have studied the mechanism involved in GNMT downregulation by its repressor microRNA miR-873-5p and the metabolic pathways affected in NAFLD as well as the benefit of recovery GNMT expression. METHODS:miR-873-5p and GNMT expression were evaluated in liver biopsies of NAFLD/NASH patients. Different in vitro and in vivo NAFLD murine models were used to assess miR-873-5p/GNMT involvement in fatty liver progression through targeting of the miR-873-5p as NAFLD therapy. RESULTS:We describe a new function of GNMT as an essential regulator of Complex II activity in the electron transport chain in the mitochondria. In NAFLD, GNMT expression is controlled by miR-873-5p in the hepatocytes, leading to disruptions in mitochondrial functionality in a preclinical murine non-alcoholic steatohepatitis (NASH) model. Upregulation of miR-873-5p is shown in the liver of NAFLD/NASH patients, correlating with hepatic GNMT depletion. Importantly, NASH therapies based on anti-miR-873-5p resolve lipid accumulation, inflammation and fibrosis by enhancing fatty acid β-oxidation in the mitochondria. Therefore, miR-873-5p inhibitor emerges as a potential tool for NASH treatment. CONCLUSION:GNMT participates in the regulation of metabolic pathways and mitochondrial functionality through the regulation of Complex II activity in the electron transport chain. In NAFLD, GNMT is repressed by miR-873-5p and its targeting arises as a valuable therapeutic option for treatment.

Glycine N-methyltransferase (GNMT) is the most abundant methyltransferase in the liver and a master regulator of the transmethylation flux. GNMT downregulation leads to loss of liver function progressing to fibrosis, cirrhosis, and hepatocellular carcinoma. Moreover, GNMT deficiency aggravates cholestasis-induced fibrogenesis. To date, little is known about the mechanisms underlying downregulation of GNMT levels in hepatic fibrosis and cirrhosis. On this basis, microRNAs are epigenetic regulatory elements that play important roles in liver pathology. In this work, we aim to study the regulation of GNMT by microRNAs during liver fibrosis and cirrhosis. Luciferase assay on the 3'UTR-Gnmt was used to confirm in silico analysis showing that GNMT is potentially targeted by the microRNA miR-873-5p. Correlation between GNMT and miR-873-5p in human cholestasis and cirrhosis together with miR-873-5p inhibition in vivo in different mouse models of liver cholestasis and fibrosis [bile duct ligation and Mdr2 (Abcb4)^-/- mouse] were then assessed. The analysis of liver tissue from cirrhotic and cholestatic patients, as well as from the animal models, showed that miR-873-5p inversely correlated with the expression of GNMT. Importantly, high circulating miR-873-5p was also detected in cholestastic and cirrhotic patients. Preclinical studies with anti-miR-873-5p treatment in bile duct ligation and Mdr2^-/- mice recovered GNMT levels in association with ameliorated inflammation and fibrosis mainly by counteracting hepatocyte apoptosis and cholangiocyte proliferation. In conclusion, miR-873-5p emerges as a novel marker for liver fibrosis, cholestasis, and cirrhosis and therapeutic approaches based on anti-miR-873-5p may be effective treatments for liver fibrosis and cholestatic liver disease.

Dysregulation of miRNAs is a hallmark of cancer, modulating oncogenes, tumor suppressors, and drug responsiveness. The multi-kinase inhibitor sorafenib is one of the first-line drugs for advanced hepatocellular carcinoma (HCC), although the outcome for treated patients is heterogeneous. The identification of predictive biomarkers and targets of sorafenib efficacy are sorely needed. Thus, selected top upregulated miRNAs from the C19MC cluster were analyzed in different hepatoma cell lines compared to immortalized liver human cells, THLE-2 as control. MiR-518d-5p showed the most consistent upregulation among them. Thus, miR-518d-5p was measured in liver tumor/non-tumor samples of two distinct cohorts of HCC patients (n = 16 and n = 20, respectively). Circulating miR-518d-5p was measured in an independent cohort of HCC patients receiving sorafenib treatment (n = 100), where miR-518d-5p was analyzed in relation to treatment duration and patient's overall survival. In vitro and in vivo studies were performed in human hepatoma BCLC3 and Huh7 cells to analyze the effect of miR-518d-5p inhibition/overexpression during the response to sorafenib. Compared with healthy individuals, miR-518d-5p levels were higher in hepatic and serum samples from HCC patients (n = 16) and in an additional cohort of tumor/non-tumor paired samples (n = 20). MiR-518d-5p, through the inhibition of c-Jun and its mitochondrial target PUMA, desensitized human hepatoma cells and mouse xenograft to sorafenib-induced apoptosis. Finally, serum miR-518d-5p was assessed in 100 patients with HCC of different etiologies and BCLC-stage treated with sorafenib. In BCLC-C patients, higher serum miR-518d-5p at diagnosis was associated with shorter sorafenib treatment duration and survival. Hence, hepatic miR-518d-5p modulates sorafenib resistance in HCC through inhibition of c-Jun/PUMA-induced apoptosis. Circulating miR-518d-5p emerges as a potential lack of response biomarker to sorafenib in BCLC-C HCC patients.

Targeted therapies and the consequent adoption of "personalized" oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity "broad-spectrum" therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested, many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to be relatively inexpensive, it should help us address stages and types of cancer that lack conventional treatment, and it may reduce relapse risks. A proposed agenda for future research is offered.