Systems Analysis of TNF and TRAIL Signalling Pathways in Hepatocytes
Lead Research Organisation:
University College London
Department Name: Oncology
Abstract
Systems level understanding of complex biological pathways and networks requires knowledge of its units, structures and temporal processes. Hepatocellular carcinoma (HCC) is one of the commonest cancers world-wide. HCC is induced by inflammation, interfering with induction of apoptosis. As a result, therapeutic options for HCC are limited due to resistance to current therapies. Tumour necrosis factor (TNF) and the TNF-related apoptosis-inducing ligand (TRAIL) are two cytokines which differentially trigger inflammatory and apoptotic signalling in liver cells. Co-treatment with inhibitors of TNF, chemotherapeutic agents or irradiation results in TRAIL sensitisation of primarily TRAIL-resistant HCC cell lines whereas normally differentiated hepatocytes remain resistant. Within the Era SysBioPlus consortium we propose to investigate the dynamic regulation and threshold of TNF- and TRAIL-induced inflammatory and apoptotic signalling pathways in primary and transformed hepatocytes and to build topology-based models to identify critical points for pathway regulation. Our group will focus entirely on the analysis and perturbation of TNF- and TRAIL-induced signalling pathways primary human hepatocytes.
Technical Summary
Systems level understanding of complex biological pathways and networks requires knowledge of its units, structures and temporal processes. Hepatocellular carcinoma (HCC) is one of the commonest cancers world-wide. HCC is induced by inflammation, interfering with induction of apoptosis. As a result, therapeutic options for HCC are limited due to resistance to current therapies. Tumour necrosis factor (TNF) and the TNF-related apoptosis-inducing ligand (TRAIL) are two cytokines which differentially trigger inflammatory and apoptotic signalling in liver cells. Co-treatment with inhibitors of TNF, chemotherapeutic agents or irradiation results in TRAIL sensitisation of primarily TRAIL-resistant HCC cell lines whereas normally differentiated hepatocytes remain resistant. Within the EraSysBioPlus consortium we propose to investigate the dynamic regulation and threshold of TNF- and TRAIL-induced inflammatory and apoptotic signalling pathways in primary and transformed hepatocytes and to build topology-based models to identify critical points for pathway regulation. Our group at Imperial College will focus entirely on the analysis and perturbation of TNF- and TRAIL-induced signalling pathways in primary human hepatocytes.
Planned Impact
Systems biology is one of the key new strategic areas of research that will lead to a deeper understanding of complex physiological processes. By collaborating with two internationally leading groups on this new field of research in the context of the EraSysBioPlus programme the current project shall add to the international positioning and visibility of Imperial College specifically and the UK in general as a major contributor to this important field of research. The successful completion of this research project shall contribute to the UK's biotechnological output by investigating the physiological process of inflammatory and apoptotic signal transduction in normal human hepatocytes. By comparison to a similar set of experiments done by our collaboration partners in other countries we shall identify potential drug targets and/or biomarkers which may in future be developed on the basis of intellectual property generated at Imperial College and collaborating institutions. Thereby, the UK's educational system may benefit from the return which we aim to generate from successful outlicensing and development of drugs/biomarkers from these findings, as has been achieved by the applicant in the past. By publishing our results in scientific journals with international visibility we also intend to maintain and reinforce the excellent reputation of Imperial College as one of the UK's leading higher education institutions. Hence both, Imperial College and the UK higher education system may both be beneficiaries of this research. The core apoptotic pathway of cell death is conserved between animal species as diverse as C. elegans and H. sapiens. The results we aim to obtain by performing the research proposed here are likely to be of fundamental importance not only in humans but also in many other species, including farmed and non-farmed animals. Thereby it may have an impact on animals health. With respect to farmed animals this is a market of considerable size. Therefore, it may be of benefit to test the efficacy of a given new drug or drug combination also in farmed and/or non-farmed animal species. Thus, the UK's farming and animal health industries may be beneficiaries of this research project.
People |
ORCID iD |
| Henning Walczak (Principal Investigator) |
Publications
Hartwig T
(2017)
The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2.
in Molecular cell
Shimizu Y
(2017)
The Linear ubiquitin chain assembly complex acts as a liver tumor suppressor and inhibits hepatocyte apoptosis and hepatitis.
in Hepatology (Baltimore, Md.)
| Description | We have established the culturing of primary human hepatocytes (PHH) to enable the analysis of the crosstalk between TNF- and TRAIL-induced signalling pathways (WP1). To do so, PHH were obtained from Clonetics (Lonza, UK) and validation experiments were performed with isoleucine zipper (iz)-TRAIL, a highly active recombinant form of soluble human TRAIL, both alone and in the presence of a kinase inhibitor (PIK-75) that we previously identified as a highly potent TRAIL sensitizer for human cancer cells. We used the Cell Titer Glo(R) assay to assess cell viability. In addition, we determined release of liver enzymes such as AST, as well as presence of cleaved cytokeratin-18 (CK18) in the cells' supernatant to cell death. These results revealed that PIK-75 in combination with izTRAIL did not kill PHH, whereas the TRAIL-related death ligand CD95L readily induced death of PHH already in the absence of PIK-75. Using the RNAi-mediated suppression of the expression of PI3K p110_ we were able to successfully establish key protocols for transient RNAi in PHH. Using these protocols have now performed RNAi-mediated suppression of expression of six proteins which we had identified in a previously performed genome-wide RNAi TRAIL sensitiser screen as factors whose presence keeps cancer cells resistant to the apoptosis-inducing potential of TRAIL. We also performed knockdown of factors which we recently identified as decisive for the balance of TNF-induced gene-activatory and cell-death-inducing capacity (Haas et al., Molecular Cell, 2009; Gerlach et al., Nature 2011). Expression of these factors was successfully suppressed by transient RNAi in primary PHH. We recently isolated RNA according to the established SOPs from PHH that were either treated with PIK-75 or in which one of these factors was knocked down by RNAi before and after treatment with TRAIL and/or TNF. Currently we are performing quality control assays on the obtained RNA. If this quality control turns out to be positive, the obtained RNA is ready for analysis by NGS (RNASeq). |
| Exploitation Route | By developing novel treatments for auto-immune diseases based on the concepts put forward in our publications, especially the Article published in Nature in 2011 (Gerlach et al., Nature 2011). |
| Sectors | Agriculture, Food and Drink,Digital/Communication/Information Technologies (including Software),Healthcare,Pharmaceuticals and Medical Biotechnology |
| Description | Apart from the internationally visible scientific publications as listed our findings have also been used to create new software for determining signalling pathway crosstalk for primary human hepatocytes. |
| First Year Of Impact | 2014 |
| Sector | Digital/Communication/Information Technologies (including Software),Healthcare,Pharmaceuticals and Medical Biotechnology |
| Impact Types | Cultural |
| Description | ERC Advanced Grant |
| Amount | € 2,367,627 (EUR) |
| Organisation | European Research Council (ERC) |
| Sector | Public |
| Country | Belgium |
| Start | 04/2012 |
| End | 03/2017 |
| Description | Wellcome Trust Senior Investigator Award |
| Amount | £2,500,000 (GBP) |
| Organisation | Wellcome Trust |
| Sector | Charity/Non Profit |
| Country | United Kingdom |
| Start | 01/2013 |
| End | 12/2018 |
| Description | Systems analysis for TNF and TRAIL signalling towards cell death and inflammation |
| Organisation | German Cancer Research Center |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | We have established the culturing of primary human hepatocytes (PHH) to enable the analysis of the crosstalk between TNF- and TRAIL-induced signalling pathways (WP1). To do so, PHH were obtained from Clonetics (Lonza, UK) and validation experiments were performed with isoleucine zipper (iz)-TRAIL, a highly active recombinant form of soluble human TRAIL, both alone and in the presence of a kinase inhibitor (PIK-75) that we previously identified as a highly potent TRAIL sensitizer for human cancer cells. We used the Cell Titer Glo(R) assay to assess cell viability. In addition, we determined release of liver enzymes such as AST, as well as presence of cleaved cytokeratin-18 (CK18) in the cells' supernatant to cell death. These results revealed that PIK-75 in combination with izTRAIL did not kill PHH, whereas the TRAIL-related death ligand CD95L readily induced death of PHH already in the absence of PIK-75. Using the RNAi-mediated suppression of the expression of PI3K p110_ we were able to successfully establish key protocols for transient RNAi in PHH. Using these protocols have now performed RNAi-mediated suppression of expression of six proteins which we had identified in a previously performed genome-wide RNAi TRAIL sensitiser screen as factors whose presence keeps cancer cells resistant to the apoptosis-inducing potential of TRAIL. We also performed knockdown of factors which we recently identified as decisive for the balance of TNF-induced gene-activatory and cell-death-inducing capacity (Haas et al., Molecular Cell, 2009; Gerlach et al., Nature 2011). Expression of these factors was successfully suppressed by transient RNAi in primary PHH. We recently isolated RNA according to the established SOPs from PHH that were either treated with PIK-75 or in which one of these factors was knocked down by RNAi before and after treatment with TRAIL and/or TNF. Currently we are performing quality control assays on the obtained RNA. If this quality control turns out to be positive, the obtained RNA is ready for analysis by NGS (RNASeq). |
| Collaborator Contribution | They provided all the RNA interference, sequencing and bioinformatics knowledge. |
| Impact | Information of the public about cell death and inflammation mechanisms and how they impact health. New avenues of research into cell death and inflammation. Papers as listed elsewhere. |
| Start Year | 2006 |