LGR5 is associated with tumor aggressiveness in papillary thyroid cancer.

PurposeLeucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a cancer stem cell marker and a down-stream target in Wnt/β-catenin signaling. In human papillary thyroid cancer (PTC), over activation of Wnt/β-catenin has been associated with tumor aggressiveness.Patients and methodsUsing established human cell lines (TPC-1, KTC-1, Nthy-ori-3-1), we report LGR5 and R-spondin (RSPO1-3) overexpression in PTC and manipulate LGR5 and Wnt/β-catenin signaling via both pharmacologic and genetic interventions. We test the association of LGR5 tumor expression with markers of PTC aggressiveness using a Discovery Cohort (n = 26 patients) and a Validation Cohort (n = 157 patients). Lastly, we explore the association between LGR5 and the BRAFV600E mutation (n = 33 patients).ResultsOur results reveal that LGR5 and its ligand, RSPO, are overexpressed in human PTC, whereby Wnt/β-catenin signaling regulates LGR5 expression and promotes cellular migration. In two separate cohorts of patients, LGR5 and RSPO2 were associated with markers of tumor aggressiveness including: lymph node metastases, vascular invasion, increased tumor size, aggressive histology, advanced AJCC TNM stage, microscopic extra thyroidal extension, capsular invasion, and macroscopic invasion. As a biomarker, LGR5 positivity predicts lymph node metastasis with 95.5% sensitivity (95% CI 88.8%-98.7%) and 61% specificity (95% CI: 48.4%-72.4%) and has a negative predictive value (NPV) of 91.3% (95% CI 79.2%-97.5%) for lymph node metastatic disease. In human PTC, LGR5 is also strongly associated with the BRAFV600E mutation (p = 0.005).ConclusionWe conclude that overexpression of LGR5 is associated with markers of tumor aggressiveness in human PTC. LGR5 may serve as a future potential biomarker for patient risk stratification and loco regional metastases in PTC

Upregulation of MIR21 and Repression of GRHL3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4-5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH

Alcohol Activates the Hedgehog Pathway and Induces Related Procarcinogenic Processes in the Alcohol-Preferring Rat Model of Hepatocarcinogenesis

Alcohol consumption promotes hepatocellular carcinoma (HCC). The responsible mechanisms are not well understood. Hepatocarcinogenesis increases with age and is enhanced by factors that impose a demand for liver regeneration. Because alcohol is hepatotoxic, habitual alcohol ingestion evokes a recurrent demand for hepatic regeneration. The alcohol-preferring (P) rat model mimics the level of alcohol consumption by humans who habitually abuse alcohol. Previously, we showed that habitual heavy alcohol ingestion amplified age-related hepatocarcinogenesis in P-rats, with over 80% of alcohol-consuming P rats developing HCCs after 18 months of alcohol exposure, compared to only 5% of water-drinking controls

Cross-platform genetic discovery of small molecule products of metabolism and application to clinical outcomes

Circulating levels of small molecules or metabolites are highly heritable, but the impact of genetic differences in metabolism on human health is not well understood. In this cross-platform, genome-wide meta-analysis of 174 metabolite levels across six cohorts including up to 86,507 participants (70% unpublished data), we identify 499 (362 novel) genome-wide significant associations (p<4.9×10 -10 ) at 144 (94 novel) genomic regions. We show that inheritance of blood metabolite levels in the general population is characterized by pleiotropy, allelic heterogeneity, rare and common variants with large effects, non-linear associations, and enrichment for nonsynonymous variation in transporter and enzyme encoding genes. The majority of identified genes are known to be involved in biochemical processes regulating metabolite levels and to cause monogenic inborn errors of metabolism linked to specific metabolites, such as ASNS (rs17345286, MAF=0.27) and asparagine levels. We illustrate the influence of metabolite-associated variants on human health including a shared signal at GLP2R (p.Asp470Asn) associated with higher citrulline levels, body mass index, fasting glucose-dependent insulinotropic peptide and type 2 diabetes risk, and demonstrate beta-arrestin signalling as the underlying mechanism in cellular models. We link genetically-higher serine levels to a 95% reduction in the likelihood of developing macular telangiectasia type 2 [odds ratio (95% confidence interval) per standard deviation higher levels 0.05 (0.03-0.08; p=9.5×10 -30 )]. We further demonstrate the predictive value of genetic variants identified for serine or glycine levels for this rare and difficult to diagnose degenerative retinal disease [area under the receiver operating characteristic curve: 0.73 (95% confidence interval: 0.70-0.75)], for which low serine availability, through generation of deoxysphingolipids, has recently been shown to be causally relevant. These results show that integration of human genomic variation with circulating small molecule data obtained across different measurement platforms enables efficient discovery of genetic regulators of human metabolism and translation into clinical insights.M.P. was supported by a fellowship from the German Research Foundation (DFG PI 1446/2-1). C.O. was founded by an early career fellowship at Homerton College, University of Cambridge. L. B. L. W. acknowledges funding by the Wellcome Trust (WT083442AIA). J.G. was supported by grants from the Medical Research Council (MC_UP_A090_1006, MC_PC_13030, MR/P011705/1 and MR/P01836X/1). Work in the Reimann/Gribble laboratories was supported by the Wellcome Trust (106262/Z/14/Z and 106263/Z/14/Z), UK Medical Research Council (MRC_MC_UU_12012/3) and PhD funding for EKB from MedImmune/AstraZeneca. Praveen Surendran is supported by a Rutherford Fund Fellowship from the Medical Research Council grant MR/S003746/1. A. W. is supported by a BHF-Turing Cardiovascular Data Science Award and by the EC-Innovative Medicines Initiative (BigData@Heart). J.D. is funded by the National Institute for Health Research [Senior Investigator Award] [*]. The EPIC-Norfolk study (https://doi.org/10.22025/2019.10.105.00004) has received funding from the Medical Research Council (MR/N003284/1 and MC-UU_12015/1) and Cancer Research UK (C864/A14136). The genetics work in the EPIC-Norfolk study was funded by the Medical Research Council (MC_PC_13048). Metabolite measurements in the EPIC-Norfolk study were supported by the MRC Cambridge Initiative in Metabolic Science (MR/L00002/1) and the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372. We are grateful to all the participants who have been part of the project and to the many members of the study teams at the University of Cambridge who have enabled this research. The Fenland Study is supported by the UK Medical Research Council (MC_UU_12015/1 and MC_PC_13046). Participants in the INTERVAL randomised controlled trial were recruited with the active collaboration of NHS Blood and Transplant England (www.nhsbt.nhs.uk), which has supported field work and other elements of the trial. DNA extraction and genotyping was co-funded by the National Institute for Health Research (NIHR), the NIHR BioResource (http://bioresource.nihr.ac.uk) and the NIHR [Cambridge Biomedical Research Centre at the Cambridge University Hospitals NHS Foundation Trust] [*]. Nightingale Health NMR assays were funded by the European Commission Framework Programme 7 (HEALTH-F2-2012-279233). Metabolon Metabolomics assays were funded by the NIHR 26 BioResource and the National Institute for Health Research [Cambridge Biomedical Research Centre at the Cambridge University Hospitals NHS Foundation Trust] [*]. The academic coordinating centre for INTERVAL was supported by core funding from: NIHR Blood and Transplant Research Unit in Donor Health and Genomics (NIHR BTRU-2014-10024), UK Medical Research Council (MR/L003120/1), British Heart Foundation (SP/09/002; RG/13/13/30194; RG/18/13/33946) and the NIHR [Cambridge Biomedical Research Centre at the Cambridge University Hospitals NHS Foundation Trust] [*].The academic coordinating centre would like to thank blood donor centre staff and blood donors for participating in the INTERVAL trial. This work was supported by Health Data Research UK, which is funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation and Wellcome. *The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care. UK Biobank: This research has been conducted using the UK Biobank resource under Application Number 44448

Paracrine Hedgehog Signaling Drives Metabolic Changes in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) typically develop in cirrhosis, a condition characterized by Hedgehog (Hh) pathway activation and accumulation of Hh-responsive myofibroblasts (MF). Although Hh signaling generally regulates stromal-epithelial interactions that support epithelial viability, the role of Hh-dependent MF in hepatocarcinogenesis is unknown. Here we used human HCC samples, a mouse HCC model, and hepatoma cell/MF co-cultures to examine the hypothesis that Hh signaling modulates MF metabolism to generate fuels for neighboring malignant hepatocytes. The results identify a novel paracrine mechanism whereby malignant hepatocytes produce HH-ligands to stimulate glycolysis in neighboring MF, resulting in release of MF-derived lactate that the malignant hepatocytes use as an energy source. This discovery reveals new diagnostic and therapeutic targets that might be exploited to improve the outcomes of cirrhotic patients with HCC

Hedgehog Signaling Antagonist Promotes Regression of Both Liver Fibrosis and Hepatocellular Carcinoma in a Murine Model of Primary Liver Cancer

Chronic fibrosing liver injury is a major risk factor for hepatocarcinogenesis in humans. Mice with targeted deletion of Mdr2 (the murine ortholog of MDR3) develop chronic fibrosing liver injury. Hepatocellular carcinoma (HCC) emerges spontaneously in such mice by 50–60 weeks of age, providing a model of fibrosis-associated hepatocarcinogenesis. We used Mdr2−/− mice to investigate the hypothesis that activation of the hedgehog (Hh) signaling pathway promotes development of both liver fibrosis and HCC

Deletion of Wntless in myeloid cells exacerbates liver fibrosis and the ductular reaction in chronic liver injury

Background: Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro-and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice)