Focal adhesion kinase and its epigenetic interactors as diagnostic and therapeutic hints for pediatric hepatoblastoma.

Background: Hepatoblastoma (HB) is the most common pediatric hepatic malignancy. Despite the progress in HB treatment, investigating HB pathomechanisms to optimize stratification and therapies remains a focal point to improve the outcome for high-risk patients. Methods: Here, we pointed to explore the impact of these mechanisms in HB. An observational study was performed on liver samples from a cohort of 17 patients with a diagnosis of HB and two normal liver samples. The in vitro experiments were executed on the Huh6 human HB cell line treated with the FAK inhibitor TAE226. Results: Our results highlight a significant up-regulation of mRNA and protein expression of FAK in livers from HB with respect to normal livers. The increased protein expression of total and Tyr397 phosphorylated FAK (pTyr397FAK) was significantly correlated with the expression of some epigenetic regulators of histone H3 methylation and acetylation. Of note, the expression of pTyr397FAK, N-methyltransferase enzyme (EZH2) and tri-methylation of the H3K27 residue correlated with tumor size and alpha-fetoprotein (AFP) levels. Finally, TAE226 caused a significant reduction of pTyr397FAK, epigenetic regulators, AFP, EPCAM, OCT4, and SOX2, in association with anti-proliferative and pro-apoptotic effects on HB cells. Conclusion: Our results suggest a role of FAK in HB that requires further investigations mainly focused on the exploration of its effective diagnostic and therapeutic translatability.

Environment, Endocrine Disruptors, and Fatty Liver Disease Associated with Metabolic Dysfunction (MASLD).

Ecological theories suggest that environmental factors significantly influence obesity risk and related syndemic morbidities, including metabolically abnormal obesity associated with nonalcoholic fatty liver disease (MASLD). These factors encompass anthropogenic influences and endocrine-disrupting chemicals (EDCs), synergistically interacting to induce metabolic discrepancies, notably in early life, and disrupt metabolic processes in adulthood. This review focuses on endocrine disruptors affecting a child's MASLD risk, independent of their role as obesogens and thus regardless of their impact on adipogenesis. The liver plays a pivotal role in metabolic and detoxification processes, where various lipophilic endocrine-disrupting molecules accumulate in fatty liver parenchyma, exacerbating inflammation and functioning as new anthropogenics that perpetuate chronic low-grade inflammation, especially insulin resistance, crucial in the pathogenesis of MASLD.

Profiling of cell-free DNA methylation and histone signatures in pediatric NAFLD: A pilot study.

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-ß (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

Combination Treatment with Hydroxytyrosol and Vitamin E Improves NAFLD-Related Fibrosis.

Non-alcoholic fatty liver disease (NAFLD)-related liver fibrosis results in the encapsulation of injured liver parenchyma by a collagenous scar mainly imputable to hepatic stellate cells' activation. Approved pharmacological treatments against NAFLD-related fibrosis are still lacking, but natural compounds such as hydroxytyrosol (HXT) and vitamin E (VitE), are emerging as promising therapeutic opportunities. In this study, the potential anti-fibrotic effect of HXT + VitE combination therapy was investigated in vitro and in vivo. In particular, tumor growth factor (TGF)-ß-activated LX-2 cells as an in vitro model, and carbon tetrachloride plus a Western diet as a mice model were employed. The effect of HXT + VitE on fibrosis was also investigated in children with biopsy-proven NAFLD. Our results demonstrated that HXT + VitE caused a reduction of proliferation, migration, contractility, and expression of pro-fibrogenic genes in TGF-ß-activated LX-2 cells. HXT + VitE treatment also antagonized TGF-ß-dependent upregulation of pro-oxidant NOX2 by interfering with nuclear translocation/activation of SMAD2/3 transcription factors. The mouse model of NAFLD-related fibrosis treated with HXT + VitE showed a marked reduction of fibrosis pattern by histology and gene expression. Accordingly, in children with NAFLD, HXT + VitE treatment caused a decrease of circulating levels of PIIINP and NOX2 that was supported over time. Our study suggests that HXT + VitE supplementation may improve NAFLD-related fibrosis.

Hydroxytyrosol Recovers SARS-CoV-2-PLpro-Dependent Impairment of Interferon Related Genes in Polarized Human Airway, Intestinal and Liver Epithelial Cells.

The SARS-CoV-2 pandemic has caused approximately 6.3 million deaths, mainly due to the acute respiratory distress syndrome or multi-organ failure that characterizes COVID-19 acute disease. Post-acute COVID-19 syndrome, also known as long-COVID, is a condition characterized by a complex of symptoms that affects 10-20% of the individuals who have recovered from the infection. Scientific and clinical evidence demonstrates that long-COVID can develop in both adults and children. It has been hypothesized that multi-organ effects of long-COVID could be associated with the persistence of virus RNA/proteins in host cells, but the real mechanism remains to be elucidated. Therefore, we sought to determine the effects of the exogenous expression of the papain-like protease (PLpro) domain of the non-structural protein (NSP3) of SARS-CoV-2 in polarized human airway (Calu-3), intestinal (Caco-2), and liver (HepG2) epithelial cells, and to evaluate the ability of the natural antioxidant hydroxytyrosol (HXT) in neutralizing these effects. Our results demonstrated that PLpro was able to induce a cascade of inflammatory genes and proteins (mainly associated with the interferon pathway) and increase the apoptotic rate and expression of several oxidative stress markers in all evaluated epithelial cells. Noteably, the treatment with 10 µM HXT reverted PL-pro-dependent effects almost completely. This study provides the first evidence that SARS-CoV-2 PLpro remaining in host cells after viral clearance may contribute to the pathogenetic mechanisms of long-COVID. These effects may be counteracted by natural antioxidants. Further clinical and experimental studies are necessary to confirm this hypothesis.

LncOb rs10487505 variant is associated with leptin levels in pediatric non-alcoholic fatty liver disease.

BACKGROUND: Low and high leptin levels are associated with non-alcoholic fatty liver disease (NAFLD). The LncOb rs10487505 variant has been associated with body mass index (BMI), and the C allele was reported as leptin-lowering. We evaluated the association of rs10487505 with leptin levels, liver histology, and surgery-induced weight loss in youths with NAFLD. METHODS: One-hundred five obese youths with NAFLD, of whom 19 undergoing laparoscopic sleeve gastrectomy (LSG), were analyzed for rs10487505 and leptin circulating levels. RESULTS: The G allele frequency was lower in youths with NAFLD than in controls (p = 0.049). No difference was found in anthropometrics, biochemistry and histology between G allele carriers and CC homozygotes, except for leptin levels (p = 0.016). Leptin correlated with body weight, BMI, BMI-z score, waist circumference, insulin resistance/sensitivity, and triglycerides (p ≤ 0.01). A multivariable regression model including body weight and homeostasis model assessment of insulin resistance was a good predictor of plasma leptin (R2 = 0.45), and the addition of genotype to the model increased the R2 to 0.50. Following LSG, leptin levels and body weight were more reduced in G allele carriers (p < 0.05). CONCLUSIONS: LncOb rs10487505 variant was associated with pediatric NAFLD and high leptin levels, and with weight and leptin reduction after LSG in youths. IMPACT: The interplay of environment, genetics and epigenetics is crucial inflating the risk of non-alcoholic fatty liver disease (NAFLD). Several long non-coding RNA (LncRNAs) are found associated with NAFLD pathogenesis. Here, we evaluated the impact of the genetic variant rs10487505 in LncOb which is involved in the regulation of leptin gene expression. The LncOb rs10487505 is associated with increased levels of leptin, but not with liver histology, in youths with NAFLD. The LncOb rs10487505 was also associated with the significant decrease of leptin and body weight after bariatric surgery.

Epigenetic remodelling in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, being the sixth most commonly diagnosed cancer and the fourth leading cause of cancer-related death. As other heterogeneous solid tumours, HCC results from a unique synergistic combination of genetic alterations mixed with epigenetic modifications.In HCC the patterns and frequencies of somatic variations change depending on the nearby chromatin. On the other hand, epigenetic alterations often induce genomic instability prone to mutations. Epigenetics refers to heritable states of gene expression without alteration to the DNA sequence itself and, unlike genetic changes, the epigenetic modifications are reversible and affect gene expression more extensively than genetic changes. Thus, studies of epigenetic regulation and the involved molecular machinery are greatly contributing to the understanding of the mechanisms that underline HCC onset and heterogeneity. Moreover, this knowledge may help to identify biomarkers for HCC diagnosis and prognosis, as well as future new targets for more efficacious therapeutic approaches.In this comprehensive review we will discuss the state-of-the-art knowledge about the epigenetic landscape in hepatocarcinogenesis, including evidence on the diagnostic and prognostic role of non-coding RNAs, modifications occurring at the chromatin level, and their role in the era of precision medicine.Apart from other better-known risk factors that predispose to the development of HCC, characterization of the epigenetic remodelling that occurs during hepatocarcinogenesis could open the way to the identification of personalized biomarkers. It may also enable a more accurate diagnosis and stratification of patients, and the discovery of new targets for more efficient therapeutic approaches.

New Insights on the Nuclear Functions and Targeting of FAK in Cancer.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase over-expressed and activated in both adult and pediatric cancers, where it plays important roles in the regulation of pathogenesis and progression of the malignant phenotype. FAK exerts its functions in cancer by two different ways: a kinase activity in the cytoplasm, mainly dependent on the integrin signaling, and a scaffolding activity into the nucleus by networking with different gene expression regulators. For this reason, FAK has to be considered a target with high therapeutic values. Indeed, evidence suggests that FAK targeting could be effective, either alone or in combination, with other already available treatments. Here, we propose an overview of the novel insights about FAK's structure and nuclear functions, with a special focus on the recent findings concerning the roles of this protein in cancer. Additionally, we provide a recent update on FAK inhibitors that are currently in clinical trials for patients with cancer, and discuss the challenge and future directions of drug-based anti-FAK targeted therapies.

Angiopoietin-2 levels correlates with disease activity in children with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD), a chronic liver disease in children, ranges from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH). We investigated the role of Angiopoietin-2 (Ang-2) as a biomarker for pediatric NAFLD-related liver damage. METHODS: We assessed the plasma levels of Ang-2 and cytokeratin-18 (CK18) fragments and their association with histologic activity in 76 children with NAFLD and 28 controls. RESULTS: The mean plasma levels of Ang-2 and CK18 were higher in children with NAFLD than in age-matched controls (Ang-2 155.4 ± 72.5 vs 7.5 ± 2.3 ng/mL, p < 0.001; CK18 390.4 ± 145.6 vs 193.9 ± 30.8 IU/L, p < 0.001). Ang-2 was significantly increased (p < 0.0001) in children with NASH (N = 41) while CK18 was significantly increased (p = 0.002) in children with fibrosis (N = 47). Ang-2 levels accurately predicted NASH (AUROC 0.911; 95% CI 0.844-0.979; p < 0.0001), while CK18 predicted both NASH (AUROC 0.827; 95% CI 0.735-0.919; p < 0.0001) and fibrosis (AUROC 0.724; 95% CI 0.611-0.837; p = 0.001). Ang-2 and CK18 in combination were good predictors of NASH with a sensitivity of 71.4% and a specificity of 100%. CONCLUSIONS: In conclusion, our data suggested Ang-2 as a suitable biomarker of NASH in the pediatric population. However, our findings need external validation in other cohorts. IMPACT: Several circulating factors have been extensively studied as potential biomarkers for NASH. Angiopoietin-2 circulating levels are increased in children with NAFLD and are associated with NASH. Angiopoietin-2 levels are more efficient than CK18 levels at assessing the most severe form of disease, and the combining of these two biomarkers reached a positive predictive value of 100% for NASH.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetic effects.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors (FAKi), alone or in combination with SOR, using in vitro and in vivo models of HCC. METHODS: The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. RESULTS: TAE226 was the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of FAK nuclear interactome. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation caused a decrease in the nuclear amount of HDAC1/2 and a consequent increase of the histone H3 lysine 27 acetylation, thus counteracting histone H3 lysine 27 trimethylation. CONCLUSIONS: Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduces HCC growth in vitro and in vivo. Also, our data highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising targets for HCC therapy.

Antioxidant activity of Hydroxytyrosol and Vitamin E reduces systemic inflammation in children with paediatric NAFLD.

BACKGROUND: The rise in paediatric non-alcoholic fatty liver disease (NAFLD) is particularly alarming. We recently reported that Hydroxytyrosol (HXT) and Vitamin E (VitE) may improve oxidative stress, insulin resistance, and steatosis in children with biopsy-proven NAFLD. AIM: Here, we investigated if HXT+VitE may reduce systemic inflammation in the above-mentioned patients. METHODS: This study analysed the plasma levels of IL (interleukin)-6, IL-1ß, IL-10, tumour necrosis factor (TNF)-α, 4­hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2'deoxyguanosine (8-OHdG) in children enrolled in the HXT+VitE trial (ClinicalTrials.gov, NCT02842567). RESULTS: Changes in markers of systemic inflammation were found in both placebo (Pla) and HXT+VitE. In particular, after four months, the levels of IL-1ß and TNF-α were reduced in both groups, while IL-6 decreased, and IL-10 increased significantly only in the group treated with HXT+VitE. Children treated with HXT+VitE showed a significant decrease of 4-HNE and 8-OHdG that correlated with the improvement of triglyceride levels. Noticeably, only the 8-OHdG decrease correlated with steatosis amelioration and with the increase of IL-10 levels. CONCLUSION: The treatment with HXT and VitE reduced the NAFLD-related systemic inflammation in children, mainly by an increase of IL-10 circulating levels that occurred in response to DNA damage recovery, ultimately improving steatosis and hypertriglyceridemia.

FAK Signaling in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors' activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings.

The pharmacological treatment of nonalcoholic fatty liver disease in children.

INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in childhood/adolescence. It comprises a broad spectrum of liver disease severity ranging from simple steatosis to steatohepatitis and fibrosis. To date lifestyle modifications, diet and physical activity represent the main option for the management of pediatric NAFLD, but numerous treatments classified depending on the mechanism of action, have been introduced. In keeping with, bariatric surgery, insulin sensitizers, antioxidants, probiotic and dietary supplementations have been evaluated in pediatric clinical trials. AREAS COVERED: This review describes, after a search in PubMed/MEDLINE database, the current pediatric NAFLD non-pharmacological and pharmacological treatments and their effects on biochemical and histological features. We report not only the efficacy of the diet coupled with regular exercise but also advantages of the pharmacological treatments used in combination with lifestyle interventions in pediatric NAFLD. EXPERT OPINION: Since pharmacological and non-pharmacological interventions have demonstrated variable effects in pediatric NAFLD, it is clear that safe and specific and efficient therapeutic strategies have not yet been identified. Therefore, large and long-term clinical trials in children are needed to find a way to reverse the liver tissue damage and the NAFLD-related long-term morbidity and mortality.

Liraglutide Treatment Ameliorates Neurotoxicity Induced by Stable Silencing of Pin1.

Post-translational modulation of peptidylprolyl isomerase Pin1 might link impaired glucose metabolism and neurodegeneration, being Pin1 effectors target for the glucagon-Like-Peptide1 analog liraglutide. We tested the hypotheses in Pin1 silenced cells (SH-SY5Y) treated with 2-deoxy-d-glucose (2DG) and methylglyoxal (MG), stressors causing altered glucose trafficking, glucotoxicity and protein glycation. Rescue by liraglutide was investigated. Pin1 silencing caused increased levels of reactive oxygen species, upregulated energy metabolism as suggested by raised levels of total ATP content and mRNA of SIRT1, PGC1α, NRF1; enhanced mitochondrial fission events as supported by raised protein expression of FIS1 and DRP1. 2DG and MG reduced significantly cell viability in all the cell lines. In Pin1 KD clones, 2DG exacerbated altered mitochondrial dynamics causing higher rate of fission events. Liraglutide influenced insulin signaling pathway (GSK3b/Akt); improved cell viability also in cells treated with 2DG; but it did not revert mitochondrial dysfunction in Pin1 KD model. In cells treated with MG, liraglutide enhanced cell viability, reduced ROS levels and cell death (AnnexinV/PI); and trended to reduce anti-apoptotic signals (BAX, BCL2, CASP3). Pin1 silencing mimics neuronal metabolic impairment of patients with impaired glucose metabolism and neurodegeneration. Liraglutide rescues to some extent cellular dysfunctions induced by Pin1 silencing.