Early and differential diagnosis of intrahepatic cholangiocarcinoma (iCCA) and hepatocellular carcinoma (HCC) by noninvasive methods represents a current clinical challenge. The analysis of ...low‐molecular‐weight metabolites by new high‐throughput techniques is a strategy for identifying biomarkers. Here, we have investigated whether serum metabolome can provide useful biomarkers in the diagnosis of iCCA and HCC and could discriminate iCCA from HCC. Because primary sclerosing cholangitis (PSC) is a risk factor for CCA, serum metabolic profiles of PSC and CCA have also been compared. The analysis of the levels of lipids and amino acids in the serum of patients with iCCA, HCC, and PSC and healthy individuals (n = 20/group) showed differential profiles. Several metabolites presented high diagnostic value for iCCA versus control, HCC versus control, and PSC versus control, with areas under the receiver operating characteristic curve (AUC) greater than those found in serum for the nonspecific tumor markers carbohydrate antigen 19‐9 (CA 19‐9) and alpha‐fetoprotein (AFP), commonly used to help in the diagnosis of iCCA and HCC, respectively. The development of an algorithm combining glycine, aspartic acid, SM(42:3), and SM(43:2) permitted to accurately differentiate in the diagnosis of both types of tumors (biopsy‐proven). The proposed model yielded 0.890 AUC, 75% sensitivity, and 90% specificity. Another algorithm by combination of PC(34:3) and histidine accurately permitted to differentiate PSC from iCCA, with an AUC of 0.990, 100% sensitivity, and 70% specificity. These results were validated in independent cohorts of 14‐15 patients per group and compared with profiles found in patients with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. Conclusion: Specific changes in serum concentrations of certain metabolites are useful to differentiate iCCA from HCC or PSC, and could help in the early diagnosis of these diseases.
Background & Aims Fibroblast growth factor 21 (FGF21) is an hepatic protein that plays a critical role in metabolism, stimulating fatty acid oxidation in liver and glucose uptake in fat. Systemic ...administration to obese rodents and diabetic monkeys leads to improved glucose homeostasis and weight loss. In rodents, FGF21 increases with fasting and consumption of a ketogenic diet (KD). In humans, FGF21 correlates with body mass index (BMI), but studies evaluating other parameters show inconsistent results. We examined FGF21 serum levels in lean and obese individuals and in response to dietary manipulation. We also evaluated FGF21 serum levels and liver messenger RNA (mRNA) expression in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Methods Serum FGF21 was measured after an overnight fast in individuals with BMI ranging from normal to obese. Volunteers fasted for 16 or 72 hours and then ate a standard meal. Another group consumed KD for 12 days. Serum FGF21 and hepatic mRNA expression were measured in obese individuals with NAFLD or NASH. Results There was a positive correlation between BMI and FGF21. There was no change in FGF21 in response to a short fast or KD. A nonstatistically significant fall in FGF21 levels was seen after a 72-hour fast. Hepatic FGF21 mRNA expression was significantly elevated in NAFLD, which correlated with a substantial increase in serum FGF21. In NASH, serum FGF21 but not liver mRNA was increased. Conclusions FGF21 correlates with BMI and may be a novel biomarker for NAFLD, but is not nutritionally regulated in humans.
Background and Purpose
The expression of the human organic cation transporter‐1 (hOCT1, gene SLC22A1) is reduced in hepatocellular carcinoma (HCC). The molecular bases of this reduction and its ...relationship with the poor response of HCC to sorafenib were investigated.
Experimental Approach
HCC transcriptomes from 366 samples available at TCGA were analysed. Alternative splicing was determined by RT‐PCR. The role of miRNAs in SLC22A1 downregulation was investigated. Expression of Oct1 was measured in rodent HCC models (spontaneously generated in Fxr‐/‐ mice and chemically‐induced in rats). hOCT1 was overexpressed in human hepatoma cells (HuH7 and HepG2). Sorafenib and regorafenib uptake was determined by HPLC‐MS/MS.
Key Results
hOCT1 overexpression enhanced sorafenib, but not regorafenib, quinine‐inhibitable uptake by hepatoma cells. In rodent HCC, Oct1 was downregulated, which was accompanied by impaired sorafenib uptake. In mice with s.c.‐implanted HCC, sorafenib inhibited the growth of hOCT1 overexpressing tumours. In human HCC, hOCT1 expression was inversely correlated with SLC22A1 promoter methylation, whereas demethylation with decitabine enhanced hOCT1 expression in hepatoma cells. Increased proportion of aberrant hOCT1 mRNA variants was found in HCC samples. In silico analysis identified six miRNAs as candidates to target hOCT1 mRNA. When overexpressed in HepG2 cells a significant hOCT1 mRNA decay was induced by hsa‐miR‐330 and hsa‐miR‐1468. Analysis of 39 paired tumour/adjacent samples from TCGA revealed that hsa‐mir‐330 was consistently upregulated in HCC.
Conclusion and Implications
Impaired hOCT1 expression/function in HCC, in part due to epigenetic modifications, plays an important role in the poor pharmacological response of this cancer to sorafenib.
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).
Although the multi‐tyrosine kinase inhibitor sorafenib is useful in the treatment of several cancers, cholangiocarcinoma (CCA) is refractory to this drug. Among other mechanisms of chemoresistance, ...impaired uptake through human organic cation transporter type 1 (hOCT1) (gene SLC22A1) has been suggested. Here we have investigated the events accounting for this phenotypic characteristic and have evaluated the interest of selective gene therapy strategies to overcome this limitation. Gene expression and DNA methylation of SLC22A1 were analyzed using intrahepatic (iCCA) and extrahepatic (eCCA) biopsies (Copenhagen and Salamanca cohorts; n = 132) and The Cancer Genome Atlas (TCGA)‐CHOL (n = 36). Decreased hOCT1 mRNA correlated with hypermethylation status of the SLC22A1 promoter. Treatment of CCA cells with decitabine (demethylating agent) or butyrate (histone deacetylase inhibitor) restored hOCT1 expression and increased sorafenib uptake. MicroRNAs able to induce hOCT1 mRNA decay were analyzed in paired samples of TCGA‐CHOL (n = 9) and Copenhagen (n = 57) cohorts. Consistent up‐regulation in tumor tissue was found for miR‐141 and miR‐330. High proportion of aberrant hOCT1 mRNA splicing in CCA was also seen. Lentiviral‐mediated transduction of eCCA (EGI‐1 and TFK‐1) and iCCA (HuCCT1) cells with hOCT1 enhanced sorafenib uptake and cytotoxic effects. In chemically induced CCA in rats, reduced rOct1 expression was accompanied by impaired sorafenib uptake. In xenograft models of eCCA cells implanted in mouse liver, poor response to sorafenib was observed. However, tumor growth was markedly reduced by cotreatment with sorafenib and adenoviral vectors encoding hOCT1 under the control of the BIRC5 promoter, a gene highly up‐regulated in CCA. Conclusion: The reason for impaired hOCT1‐mediated sorafenib uptake by CCA is multifactorial. Gene therapy capable of selectively inducing hOCT1 in tumor cells can be considered a potentially useful chemosensitization strategy to improve the response of CCA to sorafenib.
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.
NFATc4: New hub in NASH development Martinez-Chantar, Maria L.; Foti, Michelangelo
Journal of hepatology,
December 2020, 2020-12-00, 20201201, Letnik:
73, Številka:
6
Journal Article
Background and Aims
G protein–coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l‐α‐lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked ...to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown.
Approach and Results
We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up‐regulated in patients with NASH. LPI induced adenosine monophosphate–activated protein kinase activation of acetyl–coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing β‐oxidation. The inhibition of GPR55 and ACCα blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high‐fat diet and in mice fed a methionine‐choline–deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC.
Conclusions
The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.
Axonal myelination is essential for rapid saltatory impulse conduction in the nervous system, and malformation or destruction of myelin sheaths leads to motor and sensory disabilities. DNA ...methylation is an essential epigenetic modification during mammalian development, yet its role in myelination remains obscure. Here, using high-resolution methylome maps, we show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine (SAMe), the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process. Our studies also point to a possible role of SAMe in establishing the aberrant DNA methylation patterns in a mouse model of diabetic neuropathy, implicating SAMe in the pathogenesis of this disease. These critical observations establish a link between SAMe and DNA methylation status in a defined biological system, providing a mechanism that could direct methylation changes during cellular differentiation and in diverse pathological situations.
•Schwann cell myelination is characterized by global DNA demethylation•Activation of critical myelination-related genes correlates with DNA demethylation•SAMe regulates DNA methylation dynamics during Schwann cell myelination•Aberrant DNA methylation in diabetic neuropathy could be determined by SAMe levels
Using high-resolution methylome maps, Varela-Rey et al. show that DNA methylation could play a key gene regulatory role in peripheral nerve myelination and that S-adenosylmethionine, the principal methyl donor in cytosine methylation, regulates the methylome dynamics during this process.
LKB1, originally considered a tumor suppressor, plays an important role in hepatocyte proliferation and liver regeneration. Mice lacking the methionine adenosyltransferase (MAT) gene MAT1A exhibit a ...chronic reduction in hepatic S‐adenosylmethionine (SAMe) levels, basal activation of LKB1, and spontaneous development of nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). These results are relevant for human health because patients with liver cirrhosis, who are at risk to develop HCC, have a marked reduction in hepatic MAT1A expression and SAMe synthesis. In this study, we isolated a cell line (SAMe‐deficient SAMe‐D) from MAT1A knockout (MAT1A‐KO) mouse HCC to examine the role of LKB1 in the development of liver tumors derived from metabolic disorders. We found that LKB1 is required for cell survival in SAMe‐D cells. LKB1 regulates Akt‐mediated survival independent of phosphoinositide 3‐kinase, adenosine monophosphate protein–activated kinase (AMPK), and mammalian target of rapamycin complex (mTORC2). In addition, LKB1 controls the apoptotic response through phosphorylation and retention of p53 in the cytoplasm and the regulation of herpesvirus‐associated ubiquitin‐specific protease (HAUSP) and Hu antigen R (HuR) nucleocytoplasmic shuttling. We identified HAUSP as a target of HuR. Finally, we observed cytoplasmic staining of p53 and p‐LKB1(Ser428) in a NASH‐HCC animal model (from MAT1A‐KO mice) and in liver biopsies obtained from human HCC derived from both alcoholic steatohepatitis and NASH. Conclusion: The SAMe‐D cell line is a relevant model of HCC derived from NASH disease in which LKB1 is the principal conductor of a new regulatory mechanism and could be a practical tool for uncovering new therapeutic strategies. (HEPATOLOGY 2010)
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