Random gene sets in predicting survival of patients with hepatocellular carcinoma.

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.

Comparison of Gene Expression Patterns Between Mouse Models of Nonalcoholic Fatty Liver Disease and Liver Tissues From Patients.

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).

Increased liver carcinogenesis and enrichment of stem cell properties in livers of Dickkopf 2 (Dkk2) deleted mice.

Dkk2 a antagonist of the Wnt/ß-catenin-signaling pathway was shown to be silenced in diverse cancers. More recent data indicate that Dkk family members may also possess functions independent of Wnt-signaling during carcinogenesis. The detailed biological function of Dkks and its relevance for liver cancer is unknown. We analyzed the effects of a genetic deletion of Dkk2 (Dkk2-/-) in a hepatocarcinogenesis model using DEN/Phenobarbital. Untreated Dkk2-/- animals, showed considerable atypia with variation of hepatocyte size and chromatin density. In livers of Dkk2-/- mice nodule formation was seen at 9 months of age with focal loss of trabecular architecture and atypical hepatocytes and after DEN induction Dkk2-/- mice developed significantly more liver tumors compared to controls. Whole transcriptome analysis of untreated Dkk2-/- liver tissue revealed a Dkk2-dependent genetic network involving Wnt/ß-Catenin but also multiple additional oncogenic factors, such as e.g. Pdgf-b, Gdf-15 and Hnf4a. Dkk2-/- tumor cells showed a significant deregulation of stemness genes associated with enhanced colony forming properties. Integration of the Dkk2-/- signature into human data was strongly associated with patients survival. Dkk2 deletion results in alterations of liver morphology leading to an increased frequency of liver cancer. The associated genetic changes included factors not primarily related to Wnt/ß-Catenin-signaling and correlated with the clinical outcome of HCC-patients.

Smad7 regulates compensatory hepatocyte proliferation in damaged mouse liver and positively relates to better clinical outcome in human hepatocellular carcinoma.

Transforming growth factor ß (TGF-ß) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-ß role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-ß inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-ß effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-ß-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-ß actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

Translating bioinformatics in oncology: guilt-by-profiling analysis and identification of KIF18B and CDCA3 as novel driver genes in carcinogenesis.

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. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CellMinerHCC: a microarray-based expression database for hepatocellular carcinoma cell lines.

BACKGROUND & AIMS: Therapeutic options for hepatocellular carcinoma (HCC) still remain limited. Development of gene targeted therapies is a promising option. A better understanding of the underlying molecular biology is gained in in vitro experiments. However, even with targeted manipulation of gene expression varying treatment responses were observed in diverse HCC cell lines. Therefore, information on gene expression profiles of various HCC cell lines may be crucial to experimental designs. To generate a publicly available database containing microarray expression profiles of diverse HCC cell lines. METHODS: Microarray data were analyzed using an individually scripted R program package. Data were stored in a PostgreSQL database with a PHP written web interface. Evaluation and comparison of individual cell line expression profiles are supported via public web interface. RESULTS: This database allows evaluation of gene expression profiles of 18 HCC cell lines and comparison of differential gene expression between multiple cell lines. Analysis of commonly regulated genes for signaling pathway enrichment and interactions demonstrates a liver tumor phenotype with enrichment of major cancer related KEGG signatures like 'cancer' and 'inflammatory response'. Further molecular associations of strong scientific interest, e.g. 'lipid metabolism', were also identified. CONCLUSIONS: We have generated CellMinerHCC (http://www.medicalgenomics.org/cellminerhcc), a publicly available database containing gene expression data of 18 HCC cell lines. This database will aid in the design of in vitro experiments in HCC research, because the genetic specificities of various HCC cell lines will be considered.

CellLineNavigator: a workbench for cancer cell line analysis.

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.

Genetic signatures shared in embryonic liver development and liver cancer define prognostically relevant subgroups in HCC.

Multiple activations of individual genes during embryonic liver and HCC development have repeatedly prompted speculations about conserved embryonic signatures driving cancer development. Recently, the emerging discussion on cancer stem cells and the appreciation that generally tumors may develop from progenitor cells of diverse stages of cellular differentiation has shed increasing light on the overlapping genetic signatures between embryonic liver development and HCC. However there is still a lack of systematic studies investigating this area. We therefore performed a comprehensive analysis of differentially regulated genetic signaling pathways in embryonic and liver cancer development and investigated their biological relevance.Genetic signaling pathways were investigated on several publically available genome wide microarray experiments on liver development and HCC. Differentially expressed genes were investigated for pathway enrichment or underrepresentation compared to KEGG annotated pathways by Fisher exact evaluation. The comparative analysis of enrichment and under representation of differentially regulated genes in liver development and HCC demonstrated a significant overlap between multiple pathways. Most strikingly we demonstrated a significant overlap not only in pathways expected to be relevant to both conditions such as cell cycle or apoptosis but also metabolic pathways associated with carbohydrate and lipid metabolism. Furthermore, we demonstrated the clinical significance of these findings as unsupervised clustering of HCC patients on the basis of these metabolic pathways displayed significant differences in survival.These results indicate that liver development and liver cancer share similar alterations in multiple genetic signaling pathways. Several pathways with markedly similar patterns of enrichment or underrepresentation of various regulated genes between liver development and HCC are of prognostic relevance in HCC. In particular, the metabolic pathways were identified as novel prognostically relevant players in HCC development.

Identification of RARRES1 as a core regulator in liver fibrosis.

Genetic factors contribute to progression and modulation of hepatic fibrosis. High throughput genomics/transcriptomics approaches aiming at identifying key regulators of fibrosis development are tainted with the difficulty of separating essential biological "driver" from modifier genes. We applied a comparative transcriptomics approach and investigated fibrosis development in different organs to identify overlapping expression changes, since these genes may be part of core pathways in fibrosis development. Gene expression was analysed on publicly available microarray data from liver, lung and kidney fibrosis. RARRES1, AGER and S100A2 were differentially regulated in all fibrosis experiments. RARRES1 was extensively analysed by means of advanced bioinformatics analyses and functional studies. Microarray and Western Blot analysis of a standard liver fibrosis model (CCl(4)) demonstrated an early induction of RARRES1 mRNA and protein expression. In addition, quantitative RT-PCR in tissue samples from patients with advanced liver fibrosis showed higher expression as compared to non-fibrotic biopsies. Microarray analysis of RARRES1 overexpressing cells identified an enrichment of a major signature associated with fibrosis. Furthermore, RARRES1 expression increased during in vitro activation of hepatic stellate cells. To further verify the pro-fibrogenic role across organs, we demonstrated an increase in RARRES1 expression in a rat lung fibrosis model induced by adenoviral TGF-ß1 induction. We have performed a comparative transcriptomics analysis in order to identify core pathways of liver fibrogenesis, confirmed a candidate gene and enlightened the up- and downstream mechanisms of its action leading to fibrosis across organs and species.

Liver-specific overexpression of matrix metalloproteinase 9 (MMP-9) in transgenic mice accelerates development of hepatocellular carcinoma.

Matrix metalloproteinase-9 (MMP-9) plays a central role in tumor invasion and development of metastases. Expression of MMP-9 had been shown in human hepatocellular carcinomas (HCCs). However, it remained unclear whether MMP-9 could influence development of HCC. In order to address this issue, we generated transgenic mice overexpressing MMP-9 in the liver. In order to avoid embryonic lethality a Cre-lox system was utilized for conditional overexpression of MMP-9 under control of an albumin enhancer and promoter. Induction of MMP-9 overexpression in transgenic mice was achieved by i.v. injection of an adenovirus coding for the Cre recombinase. Initiation of liver carcinogenesis was achieved by injection of diethylnitrosamine (DEN) followed by Phenobarbital administration in drinking water. Transgene expression was induced at the age of 6 wk. Four and six months later mice were sacrificed and examined macroscopically and microscopically in a blinded manner. Alb/Cre/MMP-9-transgenic mice showed liver specific overexpression of MMP-9-mRNA and protein after induction. At the age of 6 months livers of transgenic mice showed 15.7 ± 11.6 tumors (mean ± SD) in contrast to wildtype mice with only 7.9 ± 11.0 tumors (P < 0.03). By histopathology examination of the livers HCCs were identified in 42% of the transgenic mouse livers but only 8% in wildtype animals. In summary, we established a novel MMP-9 transgenic mouse model, and report on a significantly increased susceptibility of MMP-9 transgenic mice to chemically induced carcinogenesis. This is the first in vivo proof that MMP-9 overexpression promotes liver tumor development.

The functional cancer map: a systems-level synopsis of genetic deregulation in cancer.

BACKGROUND: Cancer cells are characterized by massive dysegulation of physiological cell functions with considerable disruption of transcriptional regulation. Genome-wide transcriptome profiling can be utilized for early detection and molecular classification of cancers. Accurate discrimination of functionally different tumor types may help to guide selection of targeted therapy in translational research. Concise grouping of tumor types in cancer maps according to their molecular profile may further be helpful for the development of new therapeutic modalities or open new avenues for already established therapies. METHODS: Complete available human tumor data of the Stanford Microarray Database was downloaded and filtered for relevance, adequacy and reliability. A total of 649 tumor samples from more than 1400 experiments and 58 different tissues were analyzed. Next, a method to score deregulation of KEGG pathway maps in different tumor entities was established, which was then used to convert hundreds of gene expression profiles into corresponding tumor-specific pathway activity profiles. Based on the latter, we defined a measure for functional similarity between tumor entities, which yielded to phylogeny of tumors. RESULTS: We provide a comprehensive, easy-to-interpret functional cancer map that characterizes tumor types with respect to their biological and functional behavior. Consistently, multiple pathways commonly associated with tumor progression were revealed as common features in the majority of the tumors. However, several pathways previously not linked to carcinogenesis were identified in multiple cancers suggesting an essential role of these pathways in cancer biology. Among these pathways were 'ECM-receptor interaction', 'Complement and Coagulation cascades', and 'PPAR signaling pathway'. CONCLUSION: The functional cancer map provides a systematic view on molecular similarities across different cancers by comparing tumors on the level of pathway activity. This work resulted in identification of novel superimposed functional pathways potentially linked to cancer biology. Therefore, our work may serve as a starting point for rationalizing combination of tumor therapeutics as well as for expanding the application of well-established targeted tumor therapies.

Liver specific overexpression of platelet-derived growth factor-B accelerates liver cancer development in chemically induced liver carcinogenesis.

A genetic basis of hepatocellular carcinoma (HCC) has been well-established and major signaling pathways, such as p53, Wnt-signaling, transforming growth factor-ß (TGF-ß) and Ras pathways, have been identified to be essential to HCC development. Lately, the family of platelet-derived growth factors (PDGFs) has shifted to the center of interest. We have reported on spontaneously developing liver fibrosis in PDGF-B transgenic mice. Since HCC rarely occurs in healthy liver, but dramatically increases at the cirrhosis stage of which liver fibrosis is a preliminary stage, we investigated liver cancer development in chemically induced liver carcinogenesis in these mice. HCC induction was performed by treatment of the mice with diethylnitrosamine and phenobarbital. At an age of 6 months, the tumor development of these animals was analyzed. Not only the development of dysplastic lesions in PDGF-B transgenic mice was significantly increased but also their malignant transformation to HCC. Furthermore, we were able to establish a key role of PDGF-B signaling at diverse stages of liver cancer development. Here, we show that development of liver fibrosis is likely through upregulation of TGF-ß receptors by PDGF-B. Additionally, overexpression of PDGF-B also leads to an increased expression of ß-catenin as well as vascular endothelial growth factor and platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), all factors with established roles in carcinogenesis. We were able to extend the understanding of key genetic regulators in HCC development by PDGF-B and decode essential downstream signals.

Microarray-based gene expression analysis of hepatocellular carcinoma.

Microarray studies have successfully shed light on various aspects of the molecular mechanisms behind the development of hepatocellular carcinoma (HCC), such as the identification of novel molecular subgroups and the genetic profiles associated with metastasis and venous invasion. These experiments, mainly comprising genome wide profiling, potentially represent the basis of novel targeted therapeutic strategies in HCC. In response, we summarize the multiple reported expression profiles in HCC associated with HCC development, novel subgroups, venous invasion and metastasis.

Liver-specific Ldb1 deletion results in enhanced liver cancer development.

BACKGROUND & AIMS: LIM-domain-binding (Ldb) proteins have been demonstrated to be essential not only to key embryonic developmental processes but also to carcinogenesis. We have previously demonstrated Ldb1 to be of high biological and developmental relevance, as a targeted deletion of the Ldb1 gene in mice results in an embryonic lethal and pleiotropic phenotype. METHODS: We have now established a liver-specific Ldb1 knock out to investigate the role of Ldb1 in carcinogenesis, in particular in hepatocellular carcinoma (HCC) development, in vivo. RESULTS: These mice demonstrated a significantly enhanced growth of liver cancer by means of tumor size and number, advocating for an essential role of Ldb1 in HCC development. In addition, proliferation and resistance against apoptosis were increased. In order to identify the functional disturbances due to a lack of Ldb1, we performed a 15k mouse gene microarray expression analysis. We found the Myc oncogene to be regulated in the microarray analysis and were able to further confirm this regulation by demonstrating an over-expression of its downstream target Cyclin D1. Furthermore, we were able to demonstrate a down-regulation of the tumor suppressor p21. Finally, the liver stem cell marker EpCAM was also identified to be over expressed in Ldb1(-/-) knock out mice. CONCLUSIONS: We have established a significant role of Ldb1 in cancer development. Furthermore, we provided evidence for a myc/cyclin D1, p21, and EpCAM-dependent signalling to be key downstream regulators of this novel concept in HCC development.

Library of molecular associations: curating the complex molecular basis of liver diseases.

BACKGROUND: Systems biology approaches offer novel insights into the development of chronic liver diseases. Current genomic databases supporting systems biology analyses are mostly based on microarray data. Although these data often cover genome wide expression, the validity of single microarray experiments remains questionable. However, for systems biology approaches addressing the interactions of molecular networks comprehensive but also highly validated data are necessary. RESULTS: We have therefore generated the first comprehensive database for published molecular associations in human liver diseases. It is based on PubMed published abstracts and aimed to close the gap between genome wide coverage of low validity from microarray data and individual highly validated data from PubMed. After an initial text mining process, the extracted abstracts were all manually validated to confirm content and potential genetic associations and may therefore be highly trusted. All data were stored in a publicly available database, Library of Molecular Associations http://www.medicalgenomics.org/databases/loma/news, currently holding approximately 1260 confirmed molecular associations for chronic liver diseases such as HCC, CCC, liver fibrosis, NASH/fatty liver disease, AIH, PBC, and PSC. We furthermore transformed these data into a powerful resource for molecular liver research by connecting them to multiple biomedical information resources. CONCLUSION: Together, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.

A systems biology perspective on cholangiocellular carcinoma development: focus on MAPK-signaling and the extracellular environment.

BACKGROUND/AIMS: Multiple genes have been implicated in cholangiocellular carcinoma (CCC) development. However, the overall neoplastic risk is likely associated with a much lower number of critical physiological pathways. METHODS: To investigate this hypothesis, we extracted all published genetic associations for the development of CCC from PubMed (genetic association studies, but also studies associating genes and CCC in general, i.e. functional studies in cell lines, genetic studies in humans, knockout mice etc.) and integrated CCC microarray data. RESULTS: We demonstrated the MAPK pathway was consistently enriched in CCC. Comparing our data to genetic associations in HCC often successfully treated by a multityrosine kinase inhibitor, sorafenib, we demonstrated a similar overrepresentation of MAPK. In contrast, most cancer-related genetic studies focusing on genes related to transcription and cell cycle control, we consistently found genes coding for products in the extracellular environment to be significantly enriched. Thus, CCC must be regarded as developing in the context of an altered extracellular environment. CONCLUSIONS: Our study suggests the liver microenvironment holds essential functions and structures key to CCC progression. Furthermore, we identified the MAPK signaling pathway consistently enriched, pointing towards a critical role in CCC development. These data may provide a rationale for treatment of CCC with sorafenib.

Knockout of myeloid cell leukemia-1 induces liver damage and increases apoptosis susceptibility of murine hepatocytes.

UNLABELLED: Myeloid cell leukemia-1 (Mcl-1) is an antiapoptotic member of the Bcl-2 protein family. It interacts with proapoptotic Bcl-2 family members, thereby inhibiting mitochondrial activation and induction of apoptosis. Mcl-1 is essential for embryonal development and the maintenance of B cells, T cells, and hematopoietic stem cells. We have recently shown that induction of Mcl-1 by growth factors rescues primary human hepatocytes from CD95-mediated apoptosis. This prompted us to further analyze the relevance of Mcl-1 for hepatocellular homeostasis. Therefore, we generated a hepatocyte-specific Mcl-1 knockout mouse (Mcl-1(flox/flox)-AlbCre). Deletion of Mcl-1 in hepatocytes results in liver cell damage caused by spontaneous induction of apoptosis. Livers of Mcl-1(flox/flox)-AlbCre mice are smaller compared to control littermates, due to higher apoptosis rates. As a compensatory mechanism, proliferation of hepatocytes is enhanced in the absence of Mcl-1. Importantly, hepatic pericellular fibrosis occurs in Mcl-1 negative livers in response to chronic liver damage. Furthermore, Mcl-1(flox/flox)-AlbCre mice are more susceptible to hepatocellular damage induced by agonistic anti-CD95 antibodies or concanavalin A. CONCLUSION: The present study provides in vivo evidence that Mcl-1 is a crucial antiapoptotic factor for the liver, contributing to hepatocellular homeostasis and protecting hepatocytes from apoptosis induction.

The longevity assurance homologue of yeast lag1 (Lass) gene family (review).

The Lass gene family contains a group of highly conserved genes that are found in eukaryotic species. The founding member, lag1, was discovered in a screen for yeast longevity genes. Subsequently, lag1 homologs were discovered in other organisms including six mammalian paralogs. All Lass genes encode a highly conserved Lag1 domain and many also have an additional Hox domain. Lass proteins are ceramide synthases and therefore are critical for ceramide biosynthesis. Ceramide synthase is also a critical enzyme in the sphingolipid biosynthetic pathway. As ceramide and sphingolipids are key intermediates in diverse cellular processes such as cell growth, apoptosis, and stress response and may also play a role in cancer development, the function of Lass proteins is of great interest. In this review, we summarize the state of knowledge regarding Lass protein structure, biological function, and their emerging role in cancer development.

Spontaneous hepatic fibrosis in transgenic mice overexpressing PDGF-A.

Platelet derived growth factor (PDGF) plays a central role in repair mechanisms after acute and chronic tissue damage. To further evaluate the role of PDGF-A in liver fibrogenesis in vivo, we generated transgenic mice with hepatocyte-specific overexpression of PDGF-A using the CRP-gene promoter. Transgenic but not wildtype mice showed expression of PDGF-A mRNA in the liver. Hepatic PDGF-A overexpression was accompanied by a significant increase in hepatic procollagen III mRNA expression as well as TGF-beta1 expression. Liver histology showed increased deposition of extracellular matrix in transgenic but not in wildtype mice. PDGF-A-transgenic mice showed positive sinusoidal staining for alpha-SMA indicating an activation of hepatic stellate cells. Since the profibrogenic effect of PDGF-A was accompanied by increased TGF-beta1 protein concentration in the liver of transgenic mice, it can be postulated that PDGF-A upregulates expression of TGF-beta1 which is a strong activator of hepatic stellate cells. Thus, these results point towards a fibrosis induction by PDGF-A via the TGF-beta1 signalling pathway. In conclusion, expression and functional analysis of PDGF-A in the liver of transgenic mice suggest a relevant profibrogenic role of PDGF-A via TGF-beta1 induction. Counteracting PDGF-A may therefore be one of the effects of tyrosine kinase inhibitors which showed protective effects in animal models of liver fibrosis.

TGF-beta superfamily signaling is essential for tooth and hair morphogenesis and differentiation.

Members of the transforming growth factor beta (TGF-beta) superfamily of signaling molecules are involved in the regulation of many developmental processes that involve the interaction between mesenchymal and epithelial tissues. Smad7 is a potent inhibitor of many members of the TGF-beta family, notably TGF-beta and activin. In this study, we show that embryonic overexpression of Smad7 in stratified epithelia using a keratin 5 promoter, results in severe morphogenetic defects in skin and teeth and leads to embryonic and perinatal lethality. To further analyze the functions of Smad7 in epithelial tissues of adult mice, we used an expression system that allowed a controlled overexpression of Smad7 in terms of both space and time. Skin defects in adult mice overexpressing Smad7 were characterized by hyper-proliferation and missing expression of early markers of keratinocyte differentiation. Upon Smad7-mediated blockade of TGF-beta superfamily signaling, ameloblasts failed to produce an enamel layer in incisor teeth. In addition, TGF-beta blockade in adult mice altered the pattern of thymic T cell differentiation and the number of thymic T cells was significantly reduced. This study shows that TGF-beta superfamily signaling is essential for development of hair, tooth and T-cells as well as differentiation and proliferation control in adult tissues.