Role and regulation of the metalloproteinase ADAM8 in liver inflammation and hepatocellular carcinoma

Awan, Tanzeela; Ludwig, Andreas (Thesis advisor); Pradel, Gabriele (Thesis advisor); Zenke, Martin (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

The liver is the main site for the metabolism of lipids, proteins, and carbohydrates and is constantly exposed to the gut-derived metabolites such as lipopolysaccharides (LPS) and toxins which could normally trigger an immune response in the liver resulting in liver inflammation. LPS is also involved in the development and progression of chronic liver injury which includes viral hepatitis, alcoholic liver disease, and non-alcoholic fatty liver disease (NAFLD). NAFLD includes a spectrum of diseases ranging from simple steatosis to a progressive form of the disease that is non-alcoholic steatohepatitis which can further progress to cirrhosis and hepatocellular carcinoma (HCC). During the past decades, ADAM family members have been discussed vastly for their role in the development of inflammation and cancer. ADAM8 is one of the important members of the ADAM family that is strongly associated with inflammation and metastasis, not only via its catalytic activity but also via interaction with other cell surface proteins. The present study aimed to establish and explore the relationship of ADAM8 with liver inflammation and liver carcinoma. In inflammatory mouse models such as NAFLD, LPS, bile duct ligation (BDL) and after partial hepatectomy the mRNA expression of ADAM8 was upregulated compared to healthy controls. The enhanced mRNA expression of ADAM8 in the tested mouse models was associated with elevated expression of TNFα and IL-6. In parallel, the regulation of ADAM8 expression was studied in-vitro using different cultured liver cell types. ADAM8 expression was highly upregulated on mRNA and protein levels in human and murine hepatocyte cell lines, endothelial cell lines and liver stellate cells and also in primary murine hepatocytes, under NAFLD conditions. ADAM8 expression was then silenced in murine cells by using siRNA and in human cells using shRNA. The induction of released TNFα, IL-6, IL-8/KC, and CX3CL1 showed a positive association with ADAM8 expression in almost all cell types. Moreover, ADAM8 KD also reduced the mRNA expression of αSMA in liver stellate cells. The role of ADAM8 for the response to LPS was studied by using ADAM8 KD liver cells and ADAM8 knockout (KO) mice. Primary murine hepatocytes were also obtained from healthy control (WT) and ADAM8 KO mice and treated with LPS. LPS treatment increased the expression of ADAM8 in primary hepatocytes from control mice and in hepatocyte and endothelial cell lines. Along with this, the expression and release of TNFα and IL-6 were also increased and this response was reduced upon ADAM8 silencing. For in-vivo investigations, healthy controls and ADAM8 KO mice were either treated with saline or LPS to induce acute liver inflammation. LPS treatment elevated the mRNA expression of ADAM8, TNFα and IL-6 in healthy controls. ADAM8 KO appeared to reduce the expression of IL-6 only but this was not significant due to high variability among the animals. The release of TNFα and IL-6, and the liver enzyme levels were not much induced by LPS treatment. This may be explained by these circumstances that that this was a mild inflammation in the liver which could only elevate the mRNA expression of cytokines. The expression of ADAM8 is also highly upregulated in various cancers, which is correlated often with poor prognosis. In the present study, ADAM8 expression was found enhanced in HCC liver tissues. The comparison of ADAM8 expression in hepatocyte cell lines (hepatoma cell lines; HepG2 & Hepa1-6) and primary hepatocytes showed that ADAM8 is expressed at a much higher level in hepatoma cell lines. ADAM8 KD in hepatoma cell lines decreased cell proliferation, cell migration and cell invasion while ADAM8 overexpression increased all these cellular activities. Endothelial cells also showed decreased cell proliferation, migration and invasion upon ADAM8 KD. Additionally, tube formation capability of endothelial cells was reduced after ADAM8 KD. The apoptosis was highly induced in ADAM8 KD hepatoma cells and decreased upon ADAM8 overexpression. Furthermore, a positive relationship of ADAM8 was established with β1 integrin expression and activation of downstream signalling molecules including FAK, Src kinase, MAPK and Rho GTPase using hepatoma cell lines. These results indicate the critical role of ADAM8 in regulating metastasis via activating the β1-integrin-FAK-Rho axis in HCC cells. ADAM8 was also found essential for angiogenesis and targeting ADAM8 could not only control metastasis and proliferation but also angiogenesis in HCC.

Institutions

• Department of Cellular and Applied Infection Biology [164020]
• Department of Biology [160000]
• [528500-2]