Search

Colorectal cancer (CRC) is a high‐incidence malignancy worldwide which still needs better therapy options. Therefore, the aim of the present study was to investigate the responses of normal or malignant human intestinal epithelium to bone morphogenetic protein (BMP)‐9 and to find out whether the application of BMP‐9 to patients with CRC or the enhancement of its synthesis in the liver could be useful strategies for new therapy approaches. In silico analyses of CRC patient cohorts (TCGA database) revealed that high expression of the BMP‐target gene ID1, especially in combination with low expression of the BMP‐inhibitor noggin, is significantly associated with better patient survival. Organoid lines were generated from human biopsies of colon cancer (T‐Orgs) and corresponding non‐malignant areas (N‐Orgs) of three patients. The N‐Orgs represented tumours belonging to three different consensus molecular subtypes (CMS) of CRC. Overall, BMP‐9 stimulation of organoids promoted an enrichment of tumour‐suppressive gene expression signatures, whereas the stimulation with noggin had the opposite effects. Furthermore, treatment of organoids with BMP‐9 induced ID1 expression (independently of high noggin levels), while treatment with noggin reduced ID1. In summary, our data identify the ratio between ID1 and noggin as a new prognostic value for CRC patient outcome. We further show that by inducing ID1, BMP‐9 enhances this ratio, even in the presence of noggin. Thus, BMP‐9 is identified as a novel target for the development of improved anti‐cancer therapies of patients with CRC.

believed to be mainly produced in the liver. The serum levels of BMP-9 were reported to be reduced in newly diagnosed diabetic patients and BMP-9 overexpression ameliorated steatosis in the high fat diet-induced obesity mouse model. Furthermore, injection of BMP-9 in mice enhanced expression of fibroblast growth factor (FGF)21. However, whether BMP-9 also regulates the expression of the related FGF19 is not clear. Because both FGF21 and 19 were described to protect the liver from steatosis, we have further investigated the role of BMP-9 in this context. We first analyzed BMP-9 levels in the serum of streptozotocin (STZ)-induced diabetic rats (a model of type I diabetes) and confirmed that BMP-9 serum levels decrease during diabetes. Microarray analyses of RNA samples from hepatic and intestinal tissue from BMP-9 KO- and wild-type mice (C57/Bl6 background) pointed to basal expression of BMP-9 in both organs and revealed a down-regulation of hepatic Fgf21 and intestinal Fgf19 in the KO mice. Next, we analyzed BMP-9 levels in a cohort of obese patients with or without diabetes. Serum BMP-9 levels did not correlate with diabetes, but hepatic BMP-9 mRNA expression negatively correlated with steatosis in those patients that did not yet develop diabetes. Likewise, hepatic BMP-9 expression also negatively correlated with serum LPS levels. In situ hybridization analyses confirmed intestinal BMP-9 expression. Intestinal (but not hepatic) BMP-9 mRNA levels were decreased with diabetes and positively correlated with intestinal E-Cadherin expression. In vitro studies using organoids demonstrated that BMP-9 directly induces FGF19 in gut but not hepatocyte organoids, whereas no evidence of a direct induction of hepatic FGF21 by BMP-9 was found. Consistent with the in vitro data, a correlation between intestinal BMP-9 and FGF19 mRNA expression was seen in the patients’ samples. In summary, our data confirm that BMP-9 is involved in diabetes development in humans and in the control of the FGF-axis. More importantly, our data imply that not only hepatic but also intestinal BMP-9 associates with diabetes and steatosis development and controls FGF19 expression. The data support the conclusion that increased levels of BMP-9 would most likely be beneficial under pre-steatotic conditions, making supplementation of BMP-9 an interesting new approach for future therapies aiming at prevention of the development of a metabolic syndrome and liver steatosis.

Background: Gene expression signatures correlate genetic alterations with specific clinical features, providing the potential for clinical usage. A plethora of HCC-dependent gene signatures have been developed in the last two decades. However, none of them has made its way into clinical practice. Thus, we investigated the specificity of public gene signatures to HCC by establishing a comparative transcriptomic analysis, as this may be essential for clinical applications. Methods: We collected 10 public HCC gene signatures and evaluated them by utilizing four different (commercial and non-commercial) gene expression profile comparison tools: Oncomine Premium, SigCom LINCS, ProfileChaser (modified version), and GENEVA, which can assign similar pre-analyzed profiles of patients with tumors or cancer cell lines to our gene signatures of interests. Among the query results of each tool, different cancer entities were screened. In addition, seven breast and colorectal cancer gene signatures were included in order to further challenge tumor specificity of gene expression signatures. Results: Although the specificity of the evaluated HCC gene signatures varied considerably, none of the gene signatures showed strict specificity to HCC. All gene signatures exhibited potential significant specificity to other cancers, particularly for colorectal and breast cancer. Since signature specificity proved challenging, we furthermore investigated common core genes and overlapping enriched pathways among all gene signatures, which, however, showed no or only very little overlap, respectively. Conclusion: Our study demonstrates that specificity, independent validation, and clinical use of HCC genetic signatures solely relying on gene expression remains challenging. Furthermore, our work made clear that standards in signature generation and statistical methods but potentially also in tissue preparation are urgently needed.

Numerous gene expression profiling data on liver diseases were generated and stored in public databases. Only few were used for additional analyses by the hepatology research community. This may mostly be due to limited bioinformatics knowledge of most biomedical research personnel. In order to support an easy translation of bioinformatics data into translational hepatology research, we created Hepamine, a liver disease gene expression, visualization platform and data-mining resource. Microarray data were obtained from the NCBI GEO database. Pre-analysis of expression data was performed using R statistical software and the limma microarray analysis package from the Bioconductor repository. We generated Hepamine, a web-based repository of pre-analyzed microarray data for various liver diseases. At its initial release Hepamine contains 13 gene expression datasets, 20 microarray experiments and approximately 400 000 gene expression measurements. A self-explanatory website offers open and easy access to gene expression profiles. Results are furthermore visualized in simple three-color tables indicating differential expression. All data were linked to common functional and genetic databases particularly through the DAVID bioinformatics suite. Hepamine provides comprehensive data and easy access to hepatologic gene expression data even without in depth bioinformatics or microarray profiling experience. http://www.hepamine.de.

ObjectiveTGF-β2 (TGF-β, transforming growth factor beta), the less-investigated sibling of TGF-β1, is deregulated in rodent and human liver diseases. Former data from bile duct ligated and MDR2 knockout (KO) mouse models for human cholestatic liver disease suggested an involvement of TGF-β2 in biliary-derived liver diseases.DesignAs we also found upregulated TGFB2 in liver tissue of patients with primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), we now fathomed the positive prospects of targeting TGF-β2 in early stage biliary liver disease using the MDR2-KO mice. Specifically, the influence of TgfB2 silencing on the fibrotic and inflammatory niche was analysed on molecular, cellular and tissue levels.ResultsTgfB2-induced expression of fibrotic genes in cholangiocytes and hepatic stellate cellswas detected. TgfB2 expression in MDR2-KO mice was blunted using TgfB2-directed antisense oligonucleotides (AON). Upon AON treatment, reduced collagen deposition, hydroxyproline content and αSMA expression as well as induced PparG expression reflected a significant reduction of fibrogenesis without adverse effects on healthy livers. Expression analyses of fibrotic and inflammatory genes revealed AON-specific regulatory effects on Ccl3, Ccl4, Ccl5, Mki67 and Notch3 expression. Further, AON treatment of MDR2-KO mice increased tissue infiltration by F4/80-positive cells including eosinophils, whereas the number of CD45-positive inflammatory cells decreased. In line, TGFB2 and CD45 expression correlated positively in PSC/PBC patients and localised in similar areas of the diseased liver tissue.ConclusionsTaken together, our data suggest a new mechanistic explanation for amelioration of fibrogenesis by TGF-β2 silencing and provide a direct rationale for TGF-β2-directed drug development.

Aberrant activation of the PI3K/AKT/mTOR and Ras/Mitogen-Activated Protein Kinase pathways is a hepatocarcinogenesis hallmark. In a subset of hepatocellular carcinomas (HCC), PI3K/AKT/mTOR signaling dysregulation depends on PIK3CA mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressorRASSF1A. To evaluate a possible co-carcinogenic effect of PIK3CA activation andRASSF1Aknockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wildtype mice by hydrodynamic tail vein injection combined with Sleeping Beauty–mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce hepatocellular carcinomas in mice irrespective ofRASSF1Amutational background. The related tumors displayed a lipogenic phenotype with upregulation of Fatty acid synthase and Stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the Galectin-1 inhibitor OTX-008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.Graphical AbstractHydrodynamic tail vein injection of Phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutant forms E545K and H1047R induces stepwise hepatocarcinogenesis in mice, independent of Ras association domain-containing protein 1 (RASSF1A) status. Gene expression analyses revealed an early increase in Galectin-1, which regulates the lipogenic enzyme Stearoyl-CoA desaturase-1 (SCD1). PIK3CA- and Galectin1 inhibitors act synergistically, pointing at novel therapeutic strategies.

The cyclin-dependent kinases (CDKs) CDK4 and CDK6 are important regulators of the cell cycle and represent promising targets in cancer treatment. We aimed to investigate the relevance of CDK4/6 in the development of hepatocellular carcinoma (HCC) and the potential of ribociclib, a novel orally available CDK4/6 inhibitor, as a treatment for HCC. The effect of ribociclib was assessed in native and sorafenib-resistant HCC cell lines using viability assays, colony formation assays and FACS-based analyses. The expression of potential biomarkers of ribociclib response was assessed in cell lines and primary human hepatocytes using Western blotting. In addition, the prognostic relevance of the cyclin D-CDK4/6-retinoblastoma protein (Rb) pathway was assessed by analysing mRNA expression data from The Cancer Genome Atlas (TCGA). We found that ribociclib downregulated Rb and caused a profound loss of cell viability by inducing G1 cell cycle arrest in HCC cell lines exhibiting Rb-high/p16-low protein expression profiles, but not in Rb-low/p16-high cells, regardless their sensitivity to sorafenib. siRNA-based Rb silencing decreased cell proliferation, but did not diminish the sensitivity of HCC cells to ribociclib. Furthermore, we found that ribociclib synergized with sorafenib to cause cell death. mRNA analysis of primary human HCC specimens showed that CDK4 expression was correlated with patient survival and that the expression of Rb and the p16-encoding CDKN2A gene were inversely correlated. From our data we conclude that impairment of the cyclin D-CDK4/6-Rb pathway is a frequent feature of HCC and that it is associated with a unfavourable prognosis. We also found that ribociclib exhibits a preferential antineoplastic activity in Rb-high HCC cells. Our results warrant further investigation of Rb and p16 expression as markers of HCC sensitivity to ribociclib.

Despite multiple publications, molecular signatures predicting the course of hepatocellular carcinoma (HCC) have not yet been integrated into clinical routine decision-making. Given the diversity of published signatures, optimal number, best combinations, and benefit of functional associations of genes in prognostic signatures remain to be defined. We investigated a vast number of randomly chosen gene sets (varying between 1 and 10,000 genes) to encompass the full range of prognostic gene sets on 242 transcriptomic profiles of patients with HCC. Depending on the selected size, 4.7 to 23.5% of all random gene sets exhibit prognostic potential by separating patient subgroups with significantly diverse survival. This was further substantiated by investigating gene sets and signaling pathways also resulting in a comparable high number of significantly prognostic gene sets. However, combining multiple random gene sets using "swarm intelligence" resulted in a significantly improved predictability for approximately 63% of all patients. In these patients, approx. 70% of all random 50-gene containing gene sets resulted in equal and stable prediction of survival. For all other patients, a reliable prediction seems highly unlikely for any selected gene set. Using a machine learning and independent validation approach, we demonstrated a high reliability of random gene sets and swarm intelligence in HCC prognosis. Ultimately, these findings were validated in two independent patient cohorts and independent technical platforms (microarray, RNASeq). In conclusion, we demonstrate that using "swarm intelligence" of multiple gene sets for prognosis prediction may not only be superior but also more robust for predictive purposes. KEY MESSAGES: Molecular signatures predicting HCC have not yet been integrated into clinical routine Depending on the selected size, 4.7 to 23.5% of all random gene sets exhibit prognostic potential; independent of the technical platform (microarray, RNASeq) Using "swarm intelligence" resulted in a significantly improved predictability In these patients, approx. 70% of all random 50-gene containing gene sets resulted in equal and stable prediction of survival Overall, "swarm intelligence" is superior and more robust for predictive purposes in HCC.

Current staging systems of cancer are mainly based on the anatomical extent of disease. They need refinement by biological parameters to improve stratification of patients for tumor therapy or surveillance strategies. Thanks to developments in genomic, transcriptomic, and big-data technologies, we are now able to explore molecular characteristics of tumors in detail and determine their clinical relevance. This has led to numerous prognostic and predictive gene expression signatures that have the potential to establish a classification of tumor subgroups by biological determinants. However, only a few gene signatures have reached the stage of clinical implementation so far. In this review article, we summarize the current status, and present and future challenges of prognostic gene signatures in three relevant cancer entities: breast cancer, colorectal cancer, and hepatocellular carcinoma.

Aberrant activation of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR and Ras/mitogen-activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/AKT/mTOR signaling dysregulation depends on phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain-containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wild-type mice by hydrodynamic tail vein injection combined with sleeping beauty-mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl-CoA desaturase-1 (SCD1). Galectin-1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co-inhibitory treatment with PIK3CA inhibitors and the galectin-1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.

Non-alcoholic fatty liver disease (NAFLD) shows an increasing prevalence and is associated with the development of liver fibrosis and cirrhosis as the major risk factors of liver-related mortality in this disease. The therapeutic possibilities are limited and restricted to life style intervention, since specific drugs for NAFLD are unavailable so far. TNFα has been implicated as a major pathogenic driver of NAFLD. TNFα-mediated liver injury occurs mainly via TNF-receptor-1 (TNFR1) signaling, whereas TNFR2 mediates protective pathways. In this study, we analyzed the therapeutic effects of a novel antibody, which selectively inhibits TNFR1 while retaining protective TNFR2 signaling in a high-fat diet (HFD) mouse model of NAFLD. Mice were fed with HFD for 32 weeks and treated with anti-TNFR1-antibody or control-antibody for the last 8 weeks. We then investigated the mechanisms of TNFR1 inhibition on liver steatosis, inflammatory liver injury, insulin resistance and fibrosis. Compared to control-antibody treatment, TNFR1 inhibition significantly reduced liver steatosis and triglyceride content, which was accompanied by reduced expression and activation of the transcription factor SREBP1 and downstream target genes of lipogenesis. Furthermore, inhibition of TNFR1 resulted in reduced activation of the MAP kinase MKK7 and its downstream target JNK, which was associated with significant improvement of insulin resistance. Apoptotic liver injury, NAFLD activity and alanine aminotransferase (ALT) levels, as well as liver fibrosis significantly decreased by anti-TNFR1 compared to control-antibody treatment. Thus, our results suggest selective TNFR1 inhibition as a promising approach for NAFLD treatment.

It was previously shown that Bone Morphogenetic Protein (BMP)-9 is constitutively produced and secreted by hepatic stellate cells (HSC). Upon acute liver damage, BMP-9 expression is transiently down-regulated and blocking BMP-9 under conditions of chronic damage ameliorated liver fibrogenesis in C57BL/6 mice. Thereby, BMP-9 acted as a pro-fibrogenic cytokine in the liver but without directly activating isolated HSC in vitro. Lipopolysaccharide (LPS), an endotoxin derived from the membrane of Gram-negative bacteria in the gut, is known to be essential in the pathogenesis of diverse kinds of liver diseases. The aim of the present project was therefore to investigate how high levels of BMP-9 in the context of LPS signalling might result in enhanced liver damage. For this purpose, we stimulated human liver sinusoidal endothelial cells (LSEC) with LPS and incubated primary human liver myofibroblasts (MF) with the conditioned medium of these cells. We found that LPS led to the secretion of factors from LSEC that upregulate BMP-9 expression in MF. At least one of these BMP-9 enhancing factors was defined to be IL-6. High BMP-9 in turn, especially in combination with LPS stimulation, induced the expression of certain capillarization markers in LSEC and enhanced the LPS-mediated induction of pro-inflammatory cytokines in primary human macrophages. In LSEC, pre-treatment with BMP-9 reduced the LPS-mediated activation of the NfkB pathway, whereas in macrophages, LPS partially inhibited the BMP-9/Smad-1 signaling cascade. In vivo, in mice, BMP-9 led to the enhanced presence of F4/80-positive cells in the liver and it modulated the LPS-mediated regulation of inflammatory mediators. In summary, our data point to BMP-9 being a complex and highly dynamic modulator of hepatic responses to LPS: Initial effects of LPS on LSEC led to the upregulation of BMP-9 in MF but sustained high levels of BMP-9 in turn promote pro-inflammatory reactions of macrophages. Thereby, the spatial and timely fine-tuned presence (or absence) of BMP-9 is needed for efficient wound-healing responses in the liver.

The role of therapeutic drug monitoring (TDM) in ustekinumab (UST) therapy for Crohn's disease (CD) has not been established, as only few studies have analyzed the relationship between UST serum concentrations and clinical outcome. In this pilot study, we retrospectively examined the potential of UST-concentrations (cUST) 8 weeks after induction (cUSTw8) to predict clinical response at week 16. Serum samples and clinical data from patients (n = 72) with moderate to severely active CD who received intravenous induction with UST were retrospectively analyzed. cUST were quantitated using liquid chromatography-tandem mass spectrometry (LC-MSMS). A receiver-operating characteristic (ROC) curve and area under ROC curve (AUROC) was computed to analyze the predictive potential of cUSTw8 for clinical response at week 16 and to determine the minimal therapeutic UST trough concentration. Forty-four patients (61 %) achieved clinical response to UST therapy at week 16. cUSTw8 was moderately effective to predict clinical response with a minimal therapeutic cUSTw8 of 2.0 mg/l (AUC 0.72, p = 0.001). Trough concentrations of UST 8 weeks after induction predict clinical response to therapy in week 16 with moderate sensitivity and specificity. TDM using LC-MSMS could prove beneficial in personalized UST therapy of patients with CD by identifying individuals with subtherapeutic concentrations who might benefit from dose escalation. Therapeutisches Drugmonitoring (TDM) ist ein Bestandteil der personalisierten Therapie bei chronisch entzündlichen Darmerkrankungen (CED). Ustekinumab (UST) als monoklonaler Antikörper gegen die p40-Untereinheit der Interleukine 12 und 23 wurde 2016 in Deutschland zur Therapie des M. Crohn (MC) zugelassen. Therapeutische Konzentrationen für UST wurden bisher nicht definiert, weil kaum „Real-World-Daten“ für die UST-Therapie bei MC existieren, die Zusammenhänge zwischen UST-Serumkonzentrationen (cUST) und klinischen Ergebnissen untersuchten. Diese Pilotstudie untersuchte daher bei Patienten mit M. Crohn und einer neu begonnenen UST-Therapie den Zusammenhang zwischen cUST in Woche 8 nach Induktion (cUSTw8) und dem klinischen Ansprechen in Woche 16. Aus Serumproben von 72 Patienten mit mittelschwerem bis schwerem MC von 4 tertiären CED-Zentren in Baden-Württemberg, die 8 Wochen zuvor eine Induktionstherapie mit ca. 6 mg/kg Körpergewicht UST als intravenöse Infusion erhalten hatten, wurde cUSTw8 mittels Tandem-Massenspektrometrie (LC-MSMS) ermittelt. Zur Bestimmung der prädiktiven Aussagekraft von cUSTw8 hinsichtlich des klinischen Ansprechens in Woche 16 auf cUSTw8 und der minimalen therapeutischen Konzentration wurden retrospektiv klinische Daten ausgewertet und eine Receiver-operating-characteristic(ROC)-Kurve sowie die area under ROC (AUROC) ermittelt. 44 Patienten (61 %) erreichten ein klinisches Ansprechen in Woche 16. Bei Patienten mit klinischem Ansprechen lag cUSTw8 zwischen 0,5 mg/l und 9,5 mg/l, ohne Ansprechen zwischen 0 mg/l und 4,2 mg/l. Die minimale cUSTw8 zur Prädiktion eines klinischen Ansprechens in Woche 16 lag bei 2 mg/l (AUROC 0,72; p = 0,001). Es zeigte sich ein signifikanter Zusammenhang zwischen cUSTw8 und dem späteren klinischen Ansprechen auf die UST-Therapie. Der minimale therapeutische UST-Talspiegel lag in unserer Kohorte bei 2 mg/l. TDM könnte die zielgerichtete UST-Therapie verbessern, indem Patienten mit subtherapeutischen Spiegeln frühzeitig identifiziert werden.

Objectives Hepatocellular carcinoma (HCC) is a leading cancer-related cause of death. Unfortunately, recurrence is common even after curative treatment of early-stage patients, and no adjuvant treatment has yet been established. Aberrant expression of OLFM4 in human cancers has been reported; yet, its specific function during tumor development remains poorly understood, and its role in HCC is unknown. The purpose of this study is to examine the prognostic significance of OLFM4 and its functional relevance in determining recurrence in patients with early-stage HCC. Methods Immunohistochemical staining to assess expression, cellular distribution, and prognostic significance of OLFM4 was performed in a tissue microarray comprising 157 HCC tissues and matched nontumor tissues. In addition, expression of OLFM4-coding mRNA was assessed in a separate patients' cohort. The findings were validated by in vitro functional studies using siRNA directed against OLFM4 to assess its effect on cell motility and proliferation. Results The fraction of HCC samples exhibiting positive OLFM4 staining was higher in comparison with that observed in hepatocytes from matched nontumor tissue (61% vs 39%). However, cytoplasmic-only staining for OLFM4 was associated with vascular invasion (P = 0.048), MMP-7 expression (P = 0.002), and poorer survival (P = 0.008). A multivariate analysis confirmed the independent significance of OLFM4 in determining patients' outcome (5-year survival [58.3% vs 17.3%; HR: 2.135 {95% confidence interval: 1.135-4.015}; P = 0.019]). Correspondingly, inhibition of OLFM4 by siRNA modulated the expression of MMP-7 and E-cadherin, causing inhibition of cell proliferation, motility, and migration. Discussion To the best of our knowledge, we provide the first report on the prognostic significance of OLFM4 in HCC and identify its mechanistic role as crucial mediator of MMP family protein and E-Cadherin in determining cell invasion and metastasis formation.

Mouse models of non-alcoholic fatty liver disease (NAFLD) are required to define therapeutic targets, but detailed time-resolved studies to establish a sequence of events are lacking. Here, we fed male C57Bl/6N mice a Western or standard diet over 48 weeks. Multiscale time-resolved characterization was performed using RNA-seq, histopathology, immunohistochemistry, intravital imaging, and blood chemistry, the results were compared to human disease. Acetaminophen toxicity and ammonia metabolism were additionally analyzed as functional readouts. We identified a sequence of eight key events: formation of lipid droplets, inflammatory foci, lipogranulomas, zonal reorganization, cell death and replacement proliferation, ductular reaction, fibrogenesis, and hepatocellular cancer. Functional changes included resistance to acetaminophen and altered nitrogen metabolism. The transcriptomic landscape was characterized by two large clusters of monotonously increasing or decreasing genes, and a smaller number of ‘rest-and-jump genes’ that initially remained unaltered but became differentially expressed only at week 12 or later. Approximately 30% of the genes altered in human NAFLD are also altered in the present mouse model and an increasing overlap with genes altered in human HCC occurred at weeks 30–48. In conclusion, the observed sequence of events recapitulates many features of human disease and offers a basis for the identification of therapeutic targets.

// Stefan M. Brunner 1 , Timo Itzel 2 , Christoph Rubner 1 , Rebecca Kesselring 1 , Eva Griesshammer 1 , Matthias Evert 2 , Andreas Teufel 3 , Hans J. Schlitt 1 and Stefan Fichtner-Feigl 1, 4 1 Department of Surgery, University Medical Center Regensburg, Regensburg, Germany 2 Institute of Pathology, University Medical Center Regensburg, Regensburg, Germany 3 Department of Internal Medicine I, University Medical Center Regensburg, Regensburg, Germany 4 Department of General and Visceral Surgery, University Medical Center Freiburg, Freiburg, Germany Correspondence to: Stefan M. Brunner, email: stefan.brunner@ukr.de Keywords: B cell, immune infiltrate, tumor microenvironment, gene expression, immunoglobulin Received: March 22, 2017 Accepted: May 22, 2017 Published: August 09, 2017 ABSTRACT Background & Aims: The immunological microenvironment of HCC influences patient outcome, however, the role of B cells remains unclear. This study investigated effects of local B-cell infiltration in HCC cohorts on patient survival and immunological and molecular tumor microenvironment. Results: Unsupervised gene expression analysis of full cancer transcriptomes (N=2158) revealed a highly co-regulated immunological cluster in HCC that mainly contained immunoglobulin fragments. More specifically, in an independent patient cohort (N=242) that compares HCC with non tumorous liver tissue high expression of these B-cell associated genes was associated with better patient outcome (P=0.0149). Conclusively, the immunohistochemical analysis of another independent cohort of resected HCCs (N=119) demonstrated that infiltration of HCCs by CD20 + cells (P=0.004) and CD79a + cells (P=0.038) at the infiltrative margin were associated with prolonged patient survival. Further, the immunoglobulin fragments that were identified in the gene expression analysis were detected at high levels in patients with dense B-cell infiltration. Methods: Gene expression of 2 independent HCC tissue databases was compared using microarrays. Additionally, tissue of resected HCCs was stained for CD20, CD79a and immunoglobulins and analysed for the respective cell numbers separately for tumor, infiltrative margin and distant liver stroma. These findings were correlated with clinical data and patient outcome. Conclusions: Infiltration of HCCs by B cells is associated with prolonged patient survival. Further, a distinct B-cell like immunoglobulin profile of HCCs was identified that goes along with better patient outcome. We suggest that B cells contribute to local tumor control by secreting increased levels of immunoglobulins with antitumor activity.

Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. As a result of globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY receptors represent a highly conserved, stress-activated system involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased expression of NPY5 receptor (Y5R) in HCC, which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peritumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R activation. TGF-β1 was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R activation and function in liver cancer. The TGF-β/NPY/Y5R axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.

Constitutive epimutations of tumor suppressor genes are increasingly considered as cancer predisposing factors equally to sequence mutations. In light of the emerging role of the microenvironment for cancer predisposition, initiation, and progression, we aimed to characterize the consequences of a BRCA1 epimutation in cells of mesenchymal origin. We performed a comprehensive molecular and cellular comparison of primary dermal fibroblasts taken from a monozygous twin pair discordant for recurrent cancers and BRCA1 epimutation, whose exceptional clinical case we previously reported in this journal. Comparative transcriptome analysis identified differential expression of extracellular matrix-related genes and pro-tumorigenic growth factors, such as collagens and CXC chemokines. Moreover, genes known to be key markers of so called cancer-associated fibroblasts (CAFs), such as ACTA2, FAP, PDPN, and TNC, were upregulated in fibroblasts of the affected twin (BRCA1mosMe) in comparison to those of the healthy twin (BRCA1wt). Further analyses detected CAF-typical cellular features, including an elevated growth rate, enhanced migration, altered actin architecture and increased production of ketone bodies in BRCA1mosMe fibroblasts compared to BRCA1wt fibroblasts. In addition, conditioned medium of BRCA1mosMe fibroblasts was more potent than conditioned medium of BRCA1wt fibroblasts to promote cell proliferation in an epithelial and a cancer cell line. Our data demonstrate, that a CAF-like state is not an exclusive feature of tumor-associated tissue but also exists in healthy tissue with tumor suppressor deficiency. The naturally occurring phenomenon of twin fibroblasts differing in their BRCA1 methylation status revealed to be a unique powerful tool for exploring tumor suppressor deficiency-related changes in healthy tissue, reinforcing their significance for cancer predisposition.

Motivation : Co-regulated genes are not identified in traditional microarray analyses, but may theoretically be closely functionally linked [guilt-by-association (GBA), guilt-by-profiling]. Thus, bioinformatics procedures for guilt-by-profiling/association analysis have yet to be applied to large-scale cancer biology. We analyzed 2158 full cancer transcriptomes from 163 diverse cancer entities in regard of their similarity of gene expression, using Pearson’s correlation coefficient (CC). Subsequently, 428 highly co-regulated genes (|CC| ≥ 0.8) were clustered unsupervised to obtain small co-regulated networks. A major subnetwork containing 61 closely co-regulated genes showed highly significant enrichment of cancer bio-functions. All genes except kinesin family member 18B (KIF18B) and cell division cycle associated 3 (CDCA3) were of confirmed relevance for tumor biology. Therefore, we independently analyzed their differential regulation in multiple tumors and found severe deregulation in liver, breast, lung, ovarian and kidney cancers, thus proving our GBA hypothesis. Overexpression of KIF18B and CDCA3 in hepatoma cells and subsequent microarray analysis revealed significant deregulation of central cell cycle regulatory genes. Consistently, RT-PCR and proliferation assay confirmed the role of both genes in cell cycle progression. Finally, the prognostic significance of the identified KIF18B- and CDCA3-dependent predictors ( P = 0.01, P = 0.04) was demonstrated in three independent HCC cohorts and several other tumors. In summary, we proved the efficacy of large-scale guilt-by-profiling/association strategies in oncology. We identified two novel oncogenes and functionally characterized them. The strong prognostic importance of downstream predictors for HCC and many other tumors indicates the clinical relevance of our findings. Contact : andreas.teufel@ukr.de Supplementary information: Supplementary data are available at Bioinformatics online.

Leberspezifische Uberexpression des profibrogenetischer Faktors PDGF-B induziert die Ausbildung einer Leberfibrose. Des weiteren konnte gezeigt werden, dass die leberspezifische Uberexpression von PDGF-B auch eine erhohte Expression von TGF-b und des TGF-b Rezeptors Typ II induziert. Allerdings induziert TGF-b auch die Expression von PDGF-B. Um die Kooperativen Effekte einer PDGF-B und TGF-b Uberexpression zu untersuchen, haben wir PDGF-B und TGF-b hepatozytenspezifisch doppelttransgene Tiere im Vergleich zu jeweils einzelntransgenen und Wildtyptieren untersucht. Wahrend die PDGF-B transgenen Tiere PDGF-B konstitutiv uberexprimieren wird bei den TGF-b transgenen Tiere die TGF-b Uberexpression mittels LPS konditionell ab einem Alter von 4 Wochen induziert, um die kooperativen Effekte von TGF-b bei einer bereits vorhandenen PDGF-B Uberexpression zu analysieren. Dabei wurden alle Versuchstiere parallel mit LPS behandelt, um auch die Effekte der LPS-Applikation zu berucksichtigen. Beobachtung der Tiere erfolgte bis zu einem Alter von 24 Wochen. Bis zu einem Zeitpunkt von 20 Wochen versterben alle PDGF-B/TGF-b doppeltransgenen Tiere, wohingegen die Wildtyp- als auch die PDGF-B einfachtransgenen Tieren uberleben. Die TGF-b einfachtransgenen Tiere versterben lediglich zu 20%. Im weiteren wurden Tiere im Alter von 12 Wochen untersucht, da zu diesem Zeitpunkt noch 60% der PDGF-B/TGF-b doppeltrangenen Tiere lebten. Diese Beobachtungen implizieren, dass eine TGF-b Uberexpression in Kooperation mit einer PDGF-B Uberexpression zu signifikanten physiologischen Veranderungen fuhrt, welche nicht allein auf die Fibrogenese zuruckzufuhren sind. Initial wurden an Tieren der einzelnen Versuchsgruppen im Alter von 12 Wochen Genexpressionsanalysen mittels Microarrayhybridisierungen durchgefuhrt. Die hierarchische Clusternalyse der Microarraydaten zeigte, dass sich die Versuchsgruppen in erster Ebene uber PDGF-B auftrennen und dann in zweiter Ebene es durch TGF-b zu einer jeweils klaren Auftrennung der jeweiligen Versuchsgruppen kommt. Eine Netzwerkanalyse der Versuchsgruppen zeigte, dass sich bei den PDGF-B/TGF-b doppeltransgenen Tieren neben zentralen Netzwerkpunkten wie z.B. Tnf, Akt oder p53 (die sich auch in den PDGF-B bzw. TGF-b einzeltransgenen Tieren finden lassen), sich mit Jun sich ein weiterer zentraler Netzwerkpunkt herauskristallisiert. Zusammenfassend zeigen unsere Ergebnisse, dass zwischen PDGF-B und TGF-b ein komplexes Zusammenspiel existiert, das von signifikanter physiologischer Bedeutung ist. Weiterfuhrende Analysen sollen nun die molekularen Kernnetzwege und darin zentral regulatorische Gene identifizieren. Korrespondierender Autor: Teufel, Andreas E-Mail:andreas.teufel@ukr.de

The CellLineNavigator database, freely available at http://www.medicalgenomics.org/celllinenavigator, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.

Background & Aims Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Mouse models of NAFLD have been used in studies of pathogenesis and treatment, and have certain features of the human disease. We performed a systematic transcriptome-wide analysis of liver tissues from patients at different stages of NAFLD progression (ranging from healthy obese individuals to those with steatosis), as well as rodent models of NAFLD, to identify those that most closely resemble human disease progression in terms of gene expression patterns. Methods We performed a systematic evaluation of genome-wide messenger RNA expression using liver tissues collected from mice fed a standard chow diet (controls) and 9 mouse models of NAFLD: mice on a high-fat diet (with or without fructose), mice on a Western-type diet, mice on a methionine- and choline-deficient diet, mice on a high-fat diet given streptozotocin, and mice with disruption of Pten in hepatocytes. We compared gene expression patterns with those of liver tissues from 25 patients with nonalcoholic steatohepatitis (NASH), 27 patients with NAFLD, 15 healthy obese individuals, and 39 healthy nonobese individuals (controls). Liver samples were obtained from patients undergoing liver biopsy for suspected NAFLD or NASH, or during liver or bariatric surgeries. Data sets were analyzed using the limma R-package. Overlap of functional profiles was analyzed by gene set enrichment analysis profiles. Results We found differences between human and mouse transcriptomes to be significantly larger than differences between disease stages or models. Of the 65 genes with significantly altered expression in patients with NASH and 177 genes with significantly altered expression in patients with NAFLD, compared with controls, only 1–18 of these genes also differed significantly in expression between mouse models of NAFLD and control mice. However, expression of genes that regulate pathways associated with the development of NAFLD were altered in some mouse models (such as pathways associated with lipid metabolism). On a pathway level, gene expression patterns in livers of mice on the high-fat diet were associated more closely with human fatty liver disease than other models. Conclusions In comparing gene expression profiles between liver tissues from different mouse models of NAFLD and patients with different stages of NAFLD, we found very little overlap. Our data set is available for studies of pathways that contribute to the development of NASH and NAFLD and selection of the most applicable mouse models (http://www.nash-profiler.com).