Frank Staib

Different Efficiency of Heat Shock Proteins (HSP) to Activate Human Monocytes and Dendritic Cells: Superiority of HSP60

One essential immunoregulatory function of heat shock protein (HSP) is activation of the innate immune system. We investigated the activation of human monocytes and monocyte-derived dendritic cells (DC) by recombinant human HSP60, human inducible HSP72, and preparations of human gp96 and HSP70 under stringent conditions, in the absence of serum and with highly purified monocytes. HSP60 induced human DC maturation and activated human DC to secrete proinflammatory cytokines. HSP72 induced DC maturation to a lesser extent, but activated human monocytes and immature DC as efficiently as HSP60 to release proinflammatory cytokines. The independence of the effects of HSP60 and HSP72 from endotoxin or another copurifying bacterial component was shown by the resistance of these effects to polymyxin B, their sensitivity to heat treatment, the inactivity of endotoxin controls at concentrations up to 100-fold above the endotoxin contents of the HSP, and the inactivity of a recombinant control protein. Preparations of HSP70, which consisted mainly of the constitutively expressed HSP73, induced only marginal cytokine release from monocytes. The gp96 preparations did not have significant effects on human monocytes and monocyte-derived DC, indicating that these human APC populations were not susceptible to gp96 signaling under the stringent conditions applied in this study. The biological activities of gp96 and HSP70 preparations were confirmed by their peptide binding activity. These findings show that HSP can differ considerably in the capacity to activate monocyte-derived APC under certain conditions and underline the potential of HSP60 and HSP72 as activation signals for the innate immune system.

Chemotherapy‐induced apoptosis in hepatocellular carcinoma involves the p53 family and is mediated via the extrinsic and the intrinsic pathway

We investigated the downstream mechanisms by which chemotherapeutic drugs elicit apoptosis in hepatocellular carcinoma (HCC). Genomic signatures of HCC cell lines treated with different chemotherapeutic drugs were obtained. Analyses of apoptosis pathways were performed and RNA interference was used to evaluate the role of the p53 family. Endogenous p53, p63 and p73 were upregulated in response to DNA damage by chemotherapeutic drugs. Blocking p53 family function led to chemoresistance in HCC. Stimulation and blocking experiments of the CD95‐, the TNF‐ and the TRAIL‐receptor systems revealed that cytotoxic drugs, via the p53 family members as transactivators, can trigger expression of each of these death receptors and consequently sensitize HCC cells toward apoptosis. Furthermore, our findings demonstrate a link between chemotherapy, the p53 family and the mitochondrial apoptosis pathway in HCC. Chemotherapeutic treatment induces expression of proapoptotic Bcl‐2 family members like Bax and BCL2L11 and the expression of Apaf1, BNIP1, Pdcd8 and RAD. Thus, upon DNA damage, p53, p63 and p73 promote apoptosis via the extrinsic and the intrinsic signaling pathway. In addition, not only proapoptotic genes were upregulated, but also genes known to exert antiapoptotic functions. Bleomycin‐induced upregulation of BCL‐XL/BCLXL1 and MDM2 suggests that it is the ratio of proapoptotic and antiapoptotic proteins that regulates the apoptosis response of HCC cells toward chemotherapy, thereby playing a decisive role between treatment sensitivity vs. drug resistance. The clinical importance of these data is evidenced by our finding that the bleomycin target gene signature can predict the prognosis of patients suffering from HCC.

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.

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.

ΔNp73β is oncogenic in hepatocellular carcinoma by blocking apoptosis signaling via death receptors and mitochondria.

Background & Aims: p73 belongs to the p53 family of transcription factors known to regulate cell cycle and apoptosis. The Trp73 gene has two promoters that drive the expression of two major p73 isoform subfamilies: TA and ΔN. In general, TAp73 isoforms show proapoptotic activities, whereas members of the N-terminally truncated (ΔN) p73 subfamily that lack the transactivation domain show antiapoptotic functions. We found that upregulation of ΔNp73 in hepatocellular carcinoma (HCC) correlated with reduced survival. Here, we investigated the molecular mechanisms accounting for the oncogenic role of ΔNp73 in HCC. Results: ΔNp73ß can directly interfere with the transcriptional activation function of the TA (containing the transactivation domain) isoforms of the p53 family and consequently inhibit transactivation of proapoptotic target genes. Interference of ΔNp73ß with apoptosis-/chemosensitivity takes place at several levels of apoptosis signaling. ΔNp73ß negatively regulates the genes encoding for the death receptors CD95, TNF-R1, TRAIL-R2 and TNFRSF18. Furthermore, ΔNp73β represses the genes encoding caspase-2, -3, -6, -8 and -9. Concomitantly, ΔNp73ß inhibits apoptosis emanating from mitochondria. Conclusions: Thus, ΔNp73 expression in HCC selects against both the death receptor and the mitochondrial apoptosis activity of the TA isoforms. Our data suggest that ΔNp73 isoforms repress apoptosis-related genes of the extrinsic and intrinsic apoptosis signaling pathways thereby contributing to chemoresistance. The clinical importance of these data is evidenced by our finding that the ΔNp73ß target gene signature can predict the prognosis of patients suffering from HCC.

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

BackgroundSystems 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.ResultsWe 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.ConclusionTogether, this database is the first available database providing a comprehensive view and analysis options for published molecular associations on multiple liver diseases.

Snapshot liver transcriptome in hepatocellular carcinoma

Lately, advances in high throughput technologies in biomedical research have led to a dramatic increase in the accessibility of molecular insights at different levels of cancer biology such as genome, epigenome, transcriptome, proteome, and others. Among the diverse biological layers, the transcriptome has been most extensively studied especially due to the successful and broad introduction of the microarray technology. The future prospect of broad disposability of deep sequencing technology will furthermore lead to a more sensitive detection of lowly expressed transcripts and to an increase in the number of newly identified transcripts, but also to increase the discovery and characterization of alternative splicing. A common goal of large scale transcriptome profiling methods is the stratification of patients, eventually leading to personalized prognostic predictions and therapeutic strategies. Also, the observation that diverse etiologies of the mostly underlying liver cirrhosis (alcohol, HBV, HCV a.o.) are represented by different transcriptome profiles may be valuable for the identification of essential tumor targets [3] .I n

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

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.

385 MOLECULAR STAGES OF PDGFB DRIVEN LIVER FIBROSIS: LESONS FROM A TRANSGENIC MOUSE MODEL

underlying pathomechanisms is hampered by the lack of a suitable animal model. To circumvent this problem, we studied hepcidinknockout (KO) mice as a model of iron-overload associated liver disease. Methods: 6 and 12 month-old hepcidin-KO and -wild type (WT) mice fed 3% iron carbonyl-containing diet (Fe-diet) since four weeks of age were compared to age-matched WT and KO animals kept on standard diet. The liver phenotype was quantified serologically as well as morphometrically based on hematoxylin & eosin, Prussian blue and Sirius red stainings. Liver iron content was determined by atomic mass absorption. Liver fibrosis development was determined by collagen RT-PCR and hydroxyproline assay. Results: 6 month Fe-fed hepcidin KO mice (compared to WTs) exhibited (i) increased iron liver contents (2543±114 vs. 1493±136 p < 0.005); (ii) elevated AST and serum iron levels (AST: KO 261±15, WT 142±34 p < 0.05; serum iron: KO 95±3, WT 46±5, p< 0.0005); (iii) increased liver inflammation and hepatocellular apoptosis; (iv) elevated markers of hepatic stellate cell activation (collagen and a-smooth muscle actin mRNA), but no detectable liver fibrosis. 12 month Fe-fed hepcidin KO mice (compared to WTs) also showed increased liver injury markers as well as marked iron accumulation. In addition, they developed liver fibrosis as suggested by collagen RT-PCR, hydroxyproline assay and morphometrical evaluation. As a potential mechanism, hepcidin knockouts fed iron-rich diet exhibited pronounced NFkB activation. Conclusions: Iron-fed hepcidin KOs develop not only a mild chronic liver injury, but also liver fibrosis and therefore represent unique tool to study the mechanism of iron overload-related liver diseases.