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Abstract

The liver is the site of the sixth most common form of primary cancer - represented mainly by hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). Although the recent increment of knowledge on immunological, metabolic, and genetic mechanisms - from a systemic to a single cell level approach - led to consistent implementation of the therapeutic management of liver diseases and improved quality of life in patients, new challenges became apparent in the development of arising therapeutic strategies for pathologies accompanied by chronic inflammation, like liver cancer. Elevated endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been observed in precancerous diseases associated with the development of liver cancer, such as hepatic viral infection and nonalcoholic steatohepatitis (NASH)1 . In the context of liver diseases, the inositol requiring enzyme 1 (IRE1 and the protein kinase R (PKR)–like ER kinase (PERK) branches of UPR have been intensively investigated, whereas the role of activating transcription factor 6 (ATF6) in hepatic diseases has remained elusive2 . In this study, by employing different genetically modified mouse models and cell lines, I tried to examine and illustrate the role of ATF6 in hepatic tumorigenesis. In the first place, I analyzed the publicly available databases of liver cancer, the liver biopsy from healthy donors and NASH-diagnosed patients, para-tumor and tumor tissue from liver cancer patients, and tissues from liver cancer mouse models for the expression of ATF6 at both mRNA and protein levels. Strikingly, I detected a significant increase in ATF6 mRNA and protein expression in the diseased areas compared to their corresponding controls. Moreover, by doing immunohistochemistry, I identified the activation of ATF6 in the diseased tissues, indicated by the nuclear localization of ATF6. Based on these observations, I worked on the generation of hepatocyte-specific nuclear-ATF6 (nATF6) overexpression mice. In a mouse model of hepatocyte-specific activation of the ATF6 branch of UPR, I observed that transgenic homozygous mice die shortly after birth, whereas their heterozygous counterparts can survive for more than one year instead, suggesting a dose-effect. Heterozygous mice develop hepatomegaly, liver damage, and cholestasis at their young ages. Strikingly, the heterozygous mice progress to liver cancer with a tumor incidence of 100% at 12 months. To investigate the underlying mechanisms of the pro-tumorigenic effects by persistent ATF6 activation, I performed RNA-seq, proteomic and metabolic analysis on the liver of the heterozygous animals. I found out that ATF6 is intensively involved in the regulation of hepatic glucose, lipid, and amino acid metabolism. The sustained activation of the ATF6 arm of UPR in hepatocytes induces hepatocyte cell death and shifts the cellular metabolism to support the energy and building blocks requirements for compensatory proliferation. The high rate of hepatocyte turnover and constant ER stress lead to oxidative stress and hepatic inflammation, resulting in hepatic tumor onset. Meanwhile, the metabolic switch in hepatocytes deprives nutrients in the surrounding environment and further suppresses the anti-tumor function of immune cells. In the end, I generated hepatocyte-specific ATF6 knockout mice, and I challenged this mouse model with different carcinogenic treatments. Surprisingly, I found ATF6 knockout confers general hepato-protection to mice in response to these treatments, indicating a potential clinical application of ATF6 inhibition in anti-tumor therapies