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Characterisation of early cellular and molecular alterations leading to inflammation and HCC using a mouse model with liver cell-specific NEMO ablation

Final Report Summary - KONDEVANS2010 (Characterisation of early cellular and molecular alterations leading to inflammation and HCC using a mouse model with liver cell-specific NEMO ablation)

Liver cancer is one of the most common cancers world-wide, both in terms of incidence and cancer-related mortality. Mice with liver parenchymal cell (LPC)-specific deletion of NEMO are used as a genetic model to study the development of chronic liver inflammatory diseases that lead to hepatocellular carcinoma. This project was designed to accomplish two parallel goals:
1. To identify cellular and molecular alterations in the liver of NEMO LPC-KO mice that could be responsible for the disease development. This was proposed to be achieved through the targeted investigation of cellular processes, such as mitochondrial and ER functions or autophagy, which could be defective in NEMO-deficient hepatocytes, thus leading to early cellular dysfunction, as well as through non-biased high throughput approaches.
2. To help the researcher’s career development by giving him the opportunity to acquire expertise in an experimental model/methodology (mouse genetics) and scientific field (NF-kB signalling, inflammation-associated carcinogenesis, cell death and liver biology/chronic liver diseases) that he had no previous expertise. In addition, this project would train the researcher in terms of management and networking skills so that he can eventually reach scientific independence.

During the reporting period, the researcher undertook the following experimental approaches:
1. To identify subcellular changes before and after the onset of liver inflammation in NEMO LPC-KO mice, he performed detailed immuno-histochemical (IHC), immunoblotting and electron microscopic analyses on liver samples from 2- and 8-week-old mice, as well as in vitro experiments on primary hepatocytes and mouse embryonic fibroblasts.
2. Based on suggestions from published literature, the potential contribution of autophagy and oxidative stress in hepatocyte death, hepatitis and HCC formation in NEMO LPC-KO mice was investigated by generating double transgenic mice. In addition, a phenotypic comparison in the liver pathology between NEMO and ATG16L1 LPC-KO was performed. ATG16L1 is an essential protein for autophagy and the mice lacking it were used as positive control for defective autophagy.
3. A detailed comparison between NEMO and p65/RelB/cRel LPC-KO (NF-kB LPC-KO) mice was performed at key time-points to determine the NF-kB signalling dependence of the chronic liver pathology of NEMO LPC-KO mice.
4. A gene expression profile analysis was performed in liver samples from 2-week-old mice with various genotypes to obtain an overview of the processes that are deregulated early on in the NEMO LPC-KO livers.

The main results that were obtained during the reporting period, including their potential impact in terms of scientific publications, are outlined below.
1. The morphologic and functional analysis of mitochondria, cell responses to ER stress and autophagy provided no concrete evidence that the defective function of these organelles could be the primary reason for the hepatocellular death observed in NEMO LPC-KO mice.
2. As reported for many chronic inflammatory diseases, we observed a progressive accumulation of p62 in hepatocytes of NEMO LPC-KO mice. Although this could reflect a defect in autophagy, our genetic and in vitro approaches suggest that this is not the case. In addition, although ATG16L1 LPC-KO mice exhibit similar aspects to NEMO LPC-KO mice, they do not phenocopy the latter and the double LPC-KO mice show exacerbated liver pathology. Altogether, these results clearly suggest that NEMO and autophagy/p62 regulate crucial but independent survival pathways for liver pathophysiology. These data are currently finalised and will be submitted for publication.
3. Phenotypic characterisation of NF-kB LPC-KO mice showed that they exhibit significantly lower liver damage, compensatory proliferation and hepatitis compared to NEMO LPC-KO mice. These results are surprising as they indicate that although canonical NF-kB signalling is inhibited in NEMO-deficient hepatocytes, the phenotype of NEMO LPC-KO mice is largely independent of its role in NF-kB activation. These data are included in a manuscript that is currently under revision.
4. Gene Set Enrichment Analysis of the gene expression profiling results showed that pathways, such as xenobiotic metabolism and cell cycle/proliferation/cancer, were enriched in the genes whose expression was upregulated in NEMO LPC-KO mice. Selected genes are currently being analysed for their contribution to NEMO LPC-KO pathology. Although the expression of death receptor TRAIL-R was upregulated early in NEMO LPC-KO mouse livers, NEMO-TRAIL-R LPC-KO mice were not protected from cell death, as well as the NEMO LPC-KO mice that lack all three death receptors (TNF-R1, TRAIL-R, Fas) from their hepatocytes. These data suggest that stimuli other than the death ligands can also induce liver damage in the absence of the latter. These data are accepted for publication in Cell Death Differentiation and the researcher is one of the co-authors.

During the reporting period, the researcher took also steps towards his goal for scientific independence. More specifically, he became familiar with some aspects of managing a research group by training one technician to process samples for electron microscopy, and supervised one Master’s and one Batchelor’s thesis. As the host group is part of a public institution and organises twice a year a Master’s practical course on modern mouse genetics, the researcher has been giving a lecture on ‘inflammatory liver diseases’, while he has been involved in peer-reviewing of manuscripts submitted for publication to international journals. Additionally, he has become a member of European Association for the Study of the Liver (EASL) and participated in its annual conference, including ILC2014 as a selected speaker. Moreover, he has exercised his networking skills by establishing collaborations with members of the EU-funded research consortium INFLA-CARE and a pharmaceutical company. Finally, he has also been involved in intra-laboratory collaborations providing and gaining expertise on various research projects. This has earned him co-authorships in thus far two publications, which are listed also in the final report.