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Background & Aims: GD2, a member of the ganglioside (GS) family (sialic acid-containing glycosphingolipids), is a potential biomarker of cancer stem cells (CSC) in several tumours. However, the possible role of GD2 and its biosynthetic enzyme, GD3 synthase (GD3S), in intrahepatic cholangiocarcinoma (iCCA) has not been explored., Methods: The stem-like subset of two iCCA cell lines was enriched by sphere culture (SPH) and compared to monolayer parental cells (MON). GS profiles were evaluated by chromatography, after feeding with radioactive sphingosine. Membrane GD2 expression was evaluated by FACS, and the expression of enzymes of GS biosynthesis was analysed by RT-qPCR. The modulation of stem features by GS was investigated in vitro and in vivo using GD3S-overexpressing cells and corroborated by global transcriptomic analysis., Results: GS composition was markedly different comparing SPH and MON. Among complex GS, iCCA-SPH showed increased GD2 levels, in agreement with the high expression levels of GD3 and GM2/GD2 synthases. iCCA cells overexpressing GD3S had higher sphere-forming ability, invasive properties and drug resistance than parental cells. NOD/SCID mice implanted with CCLP1 cells overexpressing GD3S developed larger tumours than control cells. By global transcriptomic analysis, ontology investigation identified 74 processes shared by the iCCA-SPH and GD3S-transfected cells, with enrichment for development and morphogenesis processes, MAPK signalling and locomotion. In a cohort of patients with iCCA, GD3S expression was correlated with lymph node invasion, indicating a possible relevance of GD3S in the clinical setting., Conclusions: The profile of GS derivatives regulates the stem-like properties of iCCA cells., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background & Aims: Among the reprogrammed metabolic pathways described in cancer stem cells, aberrant lipid metabolism has recently drawn increasing attention. Our study explored the contribution of fatty acids (FA) in the regulation of stem-like features in intrahepatic cholangiocarcinoma (iCCA)., Methods: We previously identified a functional stem-like subset in human iCCA by using a three-dimensional sphere (SPH) model in comparison to parental cells grown as monolayers (MON). In this study, quantification of intracellular free FA and lipidomic analysis (triacylglycerol [TAG] composition, de novo synthesis products) was performed by Liquid chromatography-mass spectrometry (LC-MS); quadrupole time-of-flight liquid chromatography/mass spectrometry (Q-TOF LC/MS), respectively, in both SPH and MON cultures., Results: Stem-like SPH showed a superior content of free FA (citric, palmitic, stearic, and oleic acids) and unsaturated TAG. Molecularly, SPH showed upregulation of key metabolic enzymes involved in de novo FA biosynthesis (AceCS1, ACLY, ACAC, FASN, ACSL1) and the mTOR signalling pathway. In patients with iCCA (n = 68), tissue expression of FASN , a key gene involved in FA synthesis, correlated with 5-year overall survival. Interference with FASN activity in SPH cells through both specific gene silencing (siRNA) or pharmacological inhibition (orlistat) decreased sphere-forming ability and expression of stem-like markers. In a murine xenograft model obtained by injection of iCCA-SPH cells, FASN inhibition by orlistat or injection of FASN -silenced cells significantly reduced tumour growth and expression of stem-like genes., Conclusion: Altered FA metabolism contributes to the maintenance of a stem-like phenotype in iCCA. FASN inhibition may represent a new approach to interfere with the progression of this deadly disease., Impact and Implications: Recent evidence indicates that metabolic disorders correlate with an increased susceptibility to intrahepatic cholangiocarcinoma (iCCA). Our investigation emphasises the pivotal involvement of lipid metabolism in the tumour stem cell biology of iCCA, facilitated by the upregulation of crucial enzymes and the mTOR signalling pathway. From a clinical perspective, this underscores the dual role of FASN as both a prognostic indicator and a therapeutic target, suggesting that FASN inhibitors could enhance patient outcomes by diminishing stemness and tumour aggressiveness. These findings pave the way for novel therapeutic strategies for iCCA and shed light on its relationship with metabolic disorders such as diabetes, obesity, metabolic syndrome, and metabolic dysfunction-associated steatotic liver disease., (© 2024 The Author(s).)

Preliminary evidence in the literature suggests a high prevalence of malnutrition (undernutrition) in patients with severe acquired brain injuries (sABI), with an expected negative impact on clinical outcomes and pressure ulcers (PUs) in particular. In a retrospective cohort study on patients discharged from intensive care units (ICU) and admitted to an intensive rehabilitation unit (IRU), the risk of malnutrition was systematically assessed, in addition to standard clinical procedures (including PUs evaluation), using two different tools: the Malnutrition Universal Screening Tool (MUST) and the Controlling Nutritional Status (CONUT) tool. Eighty-eight patients were included in the analysis. A high proportion (79.5%) of patients with sABI suffered from PUs, being older and more frequently men, with a longer ICU stay between the event and admission to IRU, and a greater MUST score. At discharge, when compared to patients whose PUs had healed, those with persisting PUs were more often men and had the worst cognitive performance at admission. As for nutritional risk, the baseline CONUT score was identified as an independent negative predictor of PUs at discharge by the logistic regression model. In conclusion, the assessment of nutritional risk using simple standard tools may be useful in the clinical evaluation of sABI patients with PUs.

Background and Aims: Cholangiocarcinoma (CCA) is characterized by high resistance to chemotherapy and poor prognosis. Several oncogenic pathways converge on activation of extracellular signal-regulated kinase 5 (ERK5), whose role in CCA has not been explored. The aim of this study was to investigate the role of ERK5 in the biology of CCA., Approach and Results: ERK5 expression was detected in two established (HuCCT-1 and CCLP-1) and two primary human intrahepatic CCA cell lines (iCCA58 and iCCA60). ERK5 phosphorylation was increased in CCA cells exposed to soluble mediators. In both HuCCT-1 and CCLP-1 cells, ERK5 was localized in the nucleus, and exposure to fetal bovine serum (FBS) further increased the amount of nuclear ERK5. In human CCA specimens, ERK5 mRNA expression was increased in tumor cells and positively correlated with portal invasion. ERK5 protein levels were significantly associated with tumor grade. Growth, migration, and invasion of CCA cells were decreased when ERK5 was silenced using specific short hairpin RNA (shRNA). The inhibitory effects on CCA cell proliferation, migration and invasion were recapitulated by treatment with small molecule inhibitors targeting ERK5. In addition, expression of the angiogenic factors VEGF and angiopoietin 1 was reduced after ERK5 silencing. Conditioned medium from ERK5-silenced cells had a lower ability to induce tube formation by human umbilical vein endothelial cells and to induce migration of myofibroblasts and monocytes/macrophages. In mice, subcutaneous injection of CCLP-1 cells silenced for ERK5 resulted in less frequent tumor development and smaller size of xenografts compared with cells transfected with nontargeting shRNA., Conclusions: ERK5 is a key mediator of growth and migration of CCA cells and supports a protumorigenic crosstalk between the tumor and the microenvironment., (© 2021 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Defective mitosis with chromosome missegregation can have a dramatic effect on genome integrity by causing DNA damage, activation of the DNA damage response (DDR), and chromosomal instability. Although this is an energy-dependent process, mechanisms linking DDR to cellular metabolism are unknown. Here we show that checkpoint kinase 2 (CHK2), a central effector of DDR, regulates cellular energy production by affecting glycolysis and mitochondrial functions. Patients with hepatocellular carcinoma (HCC) had increased CHK2 mRNA in blood, which was associated with elevated tricarboxylic acid cycle (TCA) metabolites. CHK2 controlled expression of succinate dehydrogenase (SDH) and intervened with mitochondrial functions. DNA damage and CHK2 promoted SDH activity marked by increased succinate oxidation through the TCA cycle; this was confirmed in a transgenic model of HCC with elevated DNA damage. Mitochondrial analysis identified CHK2-controlled expression of SDH as key in sustaining reactive oxygen species production. Cells with DNA damage and elevated CHK2 relied significantly on glycolysis for ATP production due to dysfunctional mitochondria, which was abolished by CHK2 knockdown. This represents a vulnerability created by the DNA damage response that could be exploited for development of new therapies. SIGNIFICANCE: This study uncovers a link between a central effector of DNA damage response, CHK2, and cellular metabolism, revealing potential therapeutic strategies for targeting hepatocellular carcinoma., (©2021 American Association for Cancer Research.)

Introduction: The Mediterranean Diet (MD) is useful in the prevention of overweight, obesity and metabolic disease. High Quality-Extra Virgin Olive Oil (HQ-EVOO), an essential component of this diet, exerts protective effects against chronic diseases. Gut Microbiota (GM), recognized as a key factor in driving metabolic activities, is involved in the regulation of host immunity. Lactic Acid Bacteria (LAB) and their probio-active cellular substances produce beneficial effects in the gastrointestinal tract. Materials and Methods: Eighteen overweight/obese subjects (cases, BMI ≥25 kg/m 2 ) and 18 normal weight controls (BMI 18.5-24.9 kg/m 2 ) were fed with MD enriched with 40 g/die HQ-EVOO for three months. Feces and blood samples were collected at time 0 (T0) and after three months (T1) for LAB composition, oxidative stress, metabolic and inflammation parameter determinations. Results: Myeloperoxidase and 8-hydroxy-2-deoxyguanosine, markers of inflammation and oxidative stress, were significantly decreased after MD rich in HQ-EVOO both in controls and in cases. Proinflammatory cytokines levels were significantly decreased in cases in comparison to controls, while IL-10 and adiponectin were significantly increased in cases. LAB's rpo B copies/ng of DNA increased 55.6 folds in cases compared to their baseline after MD rich in HQ-EVOO. MD rich in HQ-EVOO increased adiponectin and IL-10 concentration in overweight/obese subjects and decreased oxidative stress and inflammation parameters and at the same time, increased LAB number in GM. Discussion: Our results indicate that MD rich in HQ-EVOO induces an increase of LAB in GM and could have a potential role in the prevention of inflammation. Clinical Trial Registration: www.ClinicalTrials.gov, identifier NCT03441802., (Copyright © 2019 Luisi, Lucarini, Biffi, Rafanelli, Pietramellara, Durante, Vidali, Provensi, Madiai, Gheri, Masini and Ceccherini.)

Objective: Chromosomal instability (CIN) is the most common form of genomic instability, which promotes hepatocellular carcinoma (HCC) progression by enhancing tumour heterogeneity, drug resistance and immunity escape. CIN per se is an important factor of DNA damage, sustaining structural chromosome abnormalities but the underlying mechanisms are unknown., Design: DNA damage response protein checkpoint kinase 2 (Chk2) expression was evaluated in an animal model of diethylnitrosamine-induced HCC characterised by DNA damage and elevated mitotic errors. Chk2 was also determined in two discrete cohorts of human HCC specimens. To assess the functional role of Chk2, gain on and loss-of-function, mutagenesis, karyotyping and immunofluorescence/live imaging were performed by using HCT116, Huh7 and human hepatocytes immortalised with hTERT gene (HuS)., Results: We demonstrate that mitotic errors during HCC tumorigenesis cause lagging chromosomes/DNA damage and activation of Chk2. Overexpression/phosphorylation and mislocalisation within the mitotic spindle of Chk2 contributes to induce lagging chromosomes. Lagging chromosomes and mitotic activity are reversed by knockdown of Chk2. Furthermore, upregulated Chk2 maintains mitotic activity interacting with Aurora B kinase for chromosome condensation and cytokinesis. The forkhead-associated domain of Chk2 is required for Chk2 mislocalisation to mitotic structures. In addition, retinoblastoma protein phosphorylation contributes to defective mitoses. A cohort and independent validation cohort show a strong cytoplasm to nuclear Chk2 translocation in a subset of patients with HCC., Conclusions: The study reveals a new mechanistic insight in the coinvolvement of Chk2 in HCC progression. These findings propose Chk2 as a putative biomarker to detect CIN in HCC providing a valuable support for clinical/therapeutical management of patients., Competing Interests: Competing interests: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Background: The accuracy of non-invasive methods for the quantification of liver fibrosis in primary biliary cholangitis (PBC) is still debated., Aims: To determine the histo-morphometric measurement of fibrotic tissue and to explore the possible association between indirect markers (APRI, FORNS, FIB-4, and Lok) and morphometry., Methods: Retrospective analysis of medical data from patients with PBC, on whom needle liver biopsy was performed as part of the diagnostic assessment. One section of each biopsy stained with Sirius red was used for calculating the percentage of collagen. Quantitative measure of fibrotic tissue (fibrosis morphometry) was calculated as a percentage of collagen content by digital image analysis. Morphometry results were divided into four groups reflecting Ludwig's staging and compared with values for indirect serum markers., Results: 50 PBC patients were enrolled (86% females, mean age 57 ± 12.30 years), 19 were Ludwig's stage I (38%), 14 stage II (28%), 12 stage III (24%), and five stage IV (10%). Morphometry results were significantly different among Ludwig stages (p<0.05). No significant differences were found for indirect serum markers. A significant correlation was found between morphometry results and indirect serum markers tested (p<0.05)., Conclusion: In our cohort, the histo-morphometric values of fibrotic tissue increased progressively with Ludwig's stages of PBC, while non-invasive markers did not., (Copyright © 2015 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Several actin-binding proteins have been shown to be altered in metastatic cell lines and tumours and, in particular, Myristoylated Alanine-Rich protein Kinase C substrate (MARCKS) has been implicated in the pathogenesis of various highly metastatic epithelial malignancies. Considering that a large percentage of deaths due to colorectal cancer (CRC) are consequent to hepatic metastasization, aim of this study was to elucidate the involvement and mechanism of MARCKS in CRC by employing in vitro and in vivo approaches. Loss-of and-gain-on function approaches of MARCKS were employed in two human CRC cell lines: Clone A cells expressing MARCKS and LoVo cells known to have a frameshift mutation of MARCKS i.e. typically for MSI-H CRC. The data unveiled that altering MARCKS expression suppresses cell motility and invasion in human colon carcinoma cells when conditioned medium of liver-specific stromal cells (hepatic stellate cells) was used as chemoattractant. Depletion or re-expression of MARCKS inhibited proliferation with a reduction in expression of the mitotic regulator Aurora B kinase (AURKB), whereas AURKB-depletion did not modify MARCKS expression. In murine colon carcinoma CT26 cells, shRNA MARCKS-depletion reduced motility and invasion, and induced an aberrant, prolonged mitotic process. Significantly less metastases were produced in a syngeneic model of colon metastasis by MARCKS-depleted CT26 in comparison to CT26-tumour challenged mice. In conclusion, MARCKS plays an articulated role in the progression of colorectal cancer and might represent a suitable target to interfere and overcome the invasive behaviour of colon carcinoma cells at primary and distant sites., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

1. To examine whether posture-induced changes in central volume affect brain natriuretic peptide secretion, plasma levels of human brain natriuretic peptide-32-like immunoreactivity (hBNP-32-li) were measured by radioimmunoassay in 11 healthy subjects and 20 patients with essential hypertension after 15 min supine, 15 min sitting and 15 min with the legs raised at 60 degrees, together with plasma atrial natriuretic peptide concentration, plasma renin activity and plasma aldosterone concentration. 2. In the supine position, the plasma hBNP-32-li level was 1.57 +/- 0.10 fmol/ml in healthy subjects and significantly higher in hypertensive patients (2.39 +/- 0.13 fmol/ml, P < 0.001). In both groups, plasma hBNP-32-li level significantly (P < 0.001) decreased when sitting (normotensive, 1.22 +/- 0.08 fmol/ml; hypertensive, 1.85 +/- 0.15 fmol/ml, P < 0.001 versus normotensive) and increased again after leg raising (normotensive, 2.13 +/- 0.12 fmol/ml; P < 0.002 versus resting; hypertensive, 2.84 +/- 0.16 fmol/min, P < 0.001 versus resting, P < 0.025 versus normotensive). 3. The plasma atrial natriuretic peptide concentration showed similar behaviour to the plasma hBNP-32-li, whereas plasma renin activity and plasma aldosterone concentration increased during sitting and decreased during leg raising in both healthy subjects and hypertensive patients, who had significantly higher plasma aldosterone levels when supine and sitting.(ABSTRACT TRUNCATED AT 250 WORDS)