SREBP-2-driven transcriptional activation of human SND1 oncogene

Upregulation of Staphylococcal nuclease and tudor domain containing 1 (SND1) is linked to cancer progression and metastatic spread. Increasing evidence indicates that SND1 plays a role in lipid homeostasis. Recently, it has been shown that SND1-overexpressing hepatocellular carcinoma cells present an increased de novo cholesterol synthesis and cholesteryl ester accumulation. Here we reveal that SND1 oncogene is a novel target for SREBPs. Exposure of HepG2 cells to the cholesterol-lowering drug simvastatin or to a lipoprotein-deficient medium triggers SREBP-2 activation and increases SND1 promoter activity and transcript levels. Similar increases in SND1 promoter activity and mRNA are mimicked by overexpressing nuclear SREBP-2 through expression vector transfection. Conversely, SREBP-2 suppression with specific siRNA or the addition of cholesterol/25-hydroxycholesterol to cell culture medium reduces transcriptional activity of SND1 promoter and SND1 mRNA abundance. Chromatin immunoprecipitation assays and site-directed mutagenesis show that SREBP-2 binds to the SND1 proximal promoter in a region containing one SRE and one E-box motif which are critical for maximal transcriptional activity under basal conditions. SREBP-1, in contrast, binds exclusively to the SRE element. Remarkably, while ectopic expression of SREBP-1c or -1a reduces SND1 promoter activity, knocking-down of SREBP-1 enhances SND1 mRNA and protein levels but failed to affect SND1 promoter activity. These findings reveal that SREBP-2 and SREBP-1 bind to specific sites in SND1 promoter and regulate SND1 transcription in opposite ways; it is induced by SREBP-2 activating conditions and repressed by SREBP-1 overexpression. We anticipate the contribution of a SREBPs/SND1 pathway to lipid metabolism reprogramming of human hepatoma cells.This study was supported by Gobierno Vasco grants [IT971-16 and KK2016-00036] and UPV/EHU [UFI11/20 CLUMBER]. S.A., E.A. and H.N.I. were recipients of grants from UPV/EHU and Gobierno Vasco

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease

[EN]Objective Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. Design RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3(-/-)) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. Results RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3(-/-) mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3(-/-) mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor. (PPAR.) was increased in Ripk3(-/-) mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPAR. in controlling fat deposition and fibrosis. Conclusion Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.Main funding is provided by FEDER funds through the COMPETE programme and by national funds through Fundacao para a Ciencia e a Tecnologia to CMPR (grants SAICTPAC/0019/2015-LISBOA-01-0145--FEDER-016405 and PTDC/MED-FAR/29097/2017 -LISBOA-01-0145-FEDER-029097). Additional funding comes from research grant APEF (Portuguese Association for the Study of Liver)/BAYER 2020 to MBA. JG is funded by the Fondation pour la Recherche Medicale (ARF20170938613), the Institute of Cardiometabolism and Nutrition (PAP17NECJG), the Societe Francophone du Diabete (R19114DD) and the Mairie de Paris (Emergences -R18139DD). MBA, PMR, MMP and ALS were investigators or students funded by Fundacao para a Ciencia e a Tecnologia

Adiponectin, leptin, and IGF-1 are useful diagnostic and stratification biomarkers of NAFLD

[EN] Background: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease where liver biopsy remains the gold standard for diagnosis. Here we aimed to evaluate the role of circulating adiponectin, leptin, and insulin-like growth factor 1 (IGF-1) levels as non-invasive NAFLD biomarkers and assess their correlation with the metabolome. Materials and Methods: Leptin, adiponectin, and IGF-1 serum levels were measured by ELISA in two independent cohorts of biopsy-proven obese NAFLD patients and healthy-liver controls (discovery: 38 NAFLD, 13 controls; validation: 194 NAFLD, 31 controls) and correlated with clinical data, histology, genetic parameters, and serum metabolomics. Results: In both cohorts, leptin increased in NAFLD vs. controls (discovery: AUROC 0.88; validation: AUROC 0.83; p < 0.0001). The leptin levels were similar between obese and non-obese healthy controls, suggesting that obesity is not a confounding factor. In the discovery cohort, adiponectin was lower in non-alcoholic steatohepatitis (NASH) vs. non-alcoholic fatty liver (AUROC 0.87; p < 0.0001). For the validation cohort, significance was attained for homozygous for PNPLA3 allele c.444C (AUROC 0.63; p < 0.05). Combining adiponectin with specific serum lipids improved the assay performance (AUROC 0.80; p < 0.0001). For the validation cohort, IGF-1 was lower with advanced fibrosis (AUROC 0.67, p<0.05), but combination with international normalized ratio (INR) and ferritin increased the assay performance (AUROC 0.81; p < 0.01). Conclusion: Serum leptin discriminates NAFLD, and adiponectin combined with specific lipids stratifies NASH. IGF-1, INR, and ferritin distinguish advanced fibrosis.CR was funded by FEDER through the COMPETE program and by national funds through Fundação para a Ciência e a Tecnologia (PTDC/MED-FAR/29097/2017—LISBOA-01- 0145-FEDER-029097) and by European Horizon 2020 (H2020- MSCA-RISE-2016-734719). This work was also supported by Fundação para a Ciência e Tecnologia (PD/BD/135467/2017) and Portuguese Association for the Study of Liver/MSD 2017. JB was funded by Spanish Carlos III Health Institute (ISCIII) (PI15/01132, PI18/01075 and Miguel Servet Program CON14/00129 and CPII19/00008), co-financed by Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (CIBERehd, Spain), La Caixa Scientific Foundation (HR17-00601), Fundación Científica de la Asociación Española Contra el Cáncer, and European Horizon 2020 (ESCALON project: H2020-SC1-BHC-2018-2020)

Metabolic subtypes of patients with NAFLD exhibit distinctive cardiovascular risk profiles

Background and Aims We previously identified subsets of patients with NAFLD with different metabolic phenotypes. Here we align metabolomic signatures with cardiovascular disease (CVD) and genetic risk factors. Approach and Results We analyzed serum metabolome from 1154 individuals with biopsy-proven NAFLD, and from four mouse models of NAFLD with impaired VLDL-triglyceride (TG) secretion, and one with normal VLDL-TG secretion. We identified three metabolic subtypes: A (47%), B (27%), and C (26%). Subtype A phenocopied the metabolome of mice with impaired VLDL-TG secretion; subtype C phenocopied the metabolome of mice with normal VLDL-TG; and subtype B showed an intermediate signature. The percent of patients with NASH and fibrosis was comparable among subtypes, although subtypes B and C exhibited higher liver enzymes. Serum VLDL-TG levels and secretion rate were lower among subtype A compared with subtypes B and C. Subtype A VLDL-TG and VLDL-apolipoprotein B concentrations were independent of steatosis, whereas subtypes B and C showed an association with these parameters. Serum TG, cholesterol, VLDL, small dense LDL5,6, and remnant lipoprotein cholesterol were lower among subtype A compared with subtypes B and C. The 10-year high risk of CVD, measured with the Framingham risk score, and the frequency of patatin-like phospholipase domain-containing protein 3 NAFLD risk allele were lower in subtype A. Conclusions Metabolomic signatures identify three NAFLD subgroups, independent of histological disease severity. These signatures align with known CVD and genetic risk factors, with subtype A exhibiting a lower CVD risk profile. This may account for the variation in hepatic versus cardiovascular outcomes, offering clinically relevant risk stratification.National Institutes of Health (R01DK123763, R01DK119437, HL151328, P30DK52574, P30DK56341, and UL1TR002345); Ministerio de Economía y Competitividad de España (SAF2017-88041-R); Ministerio de Economía y Competitividad de España for the Severo Ochoa Excellence Accreditation (SEV-2016-0644); CIBERehd (Biomedical Research Center in Hepatic and Digestive Diseases) and Netherlands Organization for Applied Scientific Research Program (PMC13 and PMC15); Spanish Carlos III Health Institute (PI15/01132 and PI18/01075); Miguel Servet Program (CON14/00129 and CPII19/00008); Fondo Europeo de Desarrollo Regional, CIBERehd, Department of Industry of the Basque Country (Elkartek: KK-2020/00008); La Caixa Scientific Foundation (HR17-00601); Liver Investigation: Testing Marker Utility in Steatohepatitis consortium funded by the Innovative Medicines Initiative Program of the European Union (777377), which receives support from the European Union’s Horizon 2020 research and innovation programme and EFPIA; Newcastle NIHR Biomedical Research Center; Czech Ministry of Health (RVO-VFN64165/2020); Fondo Nacional De Ciencia y Tecnología de Chile (1191145); and the Comisión Nacional de Investigación, Ciencia y Tecnología (AFB170005, CARE Chile UC); Agencia Nacional de Investigación y Desarrollo (ANID ACE 210009); European Union's Horizon 2020 Research and Innovation Program (825510)

A Novel Serum Metabolomic Profile for the Differential Diagnosis of Distal Cholangiocarcinoma and Pancreatic Ductal Adenocarcinoma

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required.This study was supported by the Centro Internacional sobre el Envejecimiento, Spain (OLD-HEPAMARKER, 0348_CIE_6_E) co-financed with European Union ERDF funds; Carlos III Institute of Health, Spain (PI16/00598, PI16/01126, PI18/01075, PI19/00819) and Miguel Servet Program (CON14/00129) co-financed by European Regional Development Fund; Asociación Española Contra el Cancer, Spain (AECC-Cánceres raros 2017/2020); H2020 ESCALON project: H2020-SC1-BHC-2018-2020; Fundacion La Caixa (Hepacare Project); MCIU/AEI/FEDER, EU (SAF2017-87301-R); Severo Ochoa Excellence Accreditation (SEV-2016-0644). A. Sanchez-Martin and A. Lapitz were supported by pre-doctoral scholarships funded by the Ministry of Science, Innovation and Universities (FPU17/04027) and the Basque Government (PRE_2017_1_0345), respectively, and M.L. Gutiérrez is supported by the "Stop fuga de Cerebros" grant from ROCHE FARMA SA. This work was carried out in the framework of Working Group 5 of the COST Action CA18122, European Cholangiocarcinoma Network, EURO-CHOLANGIO-NET

Serum Metabolites as Diagnostic Biomarkers for Cholangiocarcinoma, Hepatocellular Carcinoma, and Primary Sclerosing Cholangitis

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.Supported by CIBERehd (EHD15PI05), AECC Scientific Foundation (2017/2020), “Fondo de Investigaciones Sanitarias” (Carlos III Health Institute: PI15/01132 and PI18/01075 to J.M.B., PI16/00598 to J.J.G.M., PI16/01845 to B.S., PI16/01126 to M.A.A., PI16/00090 to J.M.), Spanish Ministry of Economy, Industry and Competitiveness (SAF2016‐75197‐R to R.I.R.M., SAF2017‐87301‐R to M.L.M.‐C.), Miguel Servet Programme (CON14/00129 to J.M.B.), “Diputación Foral de Gipuzkoa” (DFG15/010, DFG16/004 to J.M.B.), “Basque Foundation for Innovation and Health Research: EiTB Maratoia” (BIO15/CA/016/BD to J.M.B., BIO15/CA/016/BD to M.L.M.‐C.), Basque Country Department of Health (2013111173 to L.B., 2017111010 to J.M.B., 2013111114 to M.L.M.‐C.), the Andalusian Government (“Consejería de Economía, Innovación, Ciencia y Empleo”: CTS‐6264 and “Consejería de Salud”: PI‐0198‐2016 to J.M.), Junta de Castilla y León, Spain (SA063P17 to J.J.G.M.), European Comission Horizon 2020 project (SEP‐210503876; ESCALON to J.M.B.), and Proyecto Hepacare, Fundación La Caixa (to M.A.A.)