Dissertation committee:
Γεωργοπούλου Ουρανία, Διευθύντρια Ερευνών, Ερηαστήριο Μοριακής Ιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Διαλλινάς Γεώργιος, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Κοσκίνας Ιωάννης, Καθηγητής, Τμήμα ιατρικής, ΕΚΠΑ
Παπαζαφείρη Παναγιώτα, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Στραβοπόδης Δημήτριος, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Τσιτσιλώνη Ουρανία, Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Φωκά Πελαγία, Κύρια Ερευνήτρια, Εργαστήριο Μοριακής Ιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Summary:
Liver cancer is one of the most lethal cancers and HCV infection is currently one of the leading causes of chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC). There are 1.5 million new cases of hepatitis C each year, and estimates for 2022 indicate that there were about 58 million virus carriers globally. Furthermore, in 2019 alone, there were 290,000 patient fatalities from hepatitis C, primarily due to cirrhosis and HCC. Direct-acting antivirals successfully treat over 95% of patients; however, the risk of developing HCC is elevated, particularly when fibrosis is present. This is primarily because of epigenetic modifications that result in post-translational modifications of proteins and consequently chronic diseases such as fibrosis, inflammation, and steatosis. The necessity to clarify the currently understood epigenetic pathways induced by HCV leading to chronic diseases is underscored, given the slow progression of liver disease and the absence of symptoms for many years after the initial event of infection. The cellular factors that dictate the course of HCV infection are still unresolved mostly because they are rarely looked at on the onset of infection since they do not exhibit symptoms. In addition, the heterogeneity of the numerous viral strains makes it challenging to draw firm conclusions.HCV, in order to use the host cell as a factory for replication, recruits its proteins to change cellular function and produce an environment that is favorable to its reproduction. Lysine-specific demethylases 1 and 2 (LSD1 and LSD2), which function by demethylating methylated lysine residues, are a class of epigenetic factors that regulate numerous cellular activities and may mediate changes in cellular function for HCV replication. Beyond histones, LSDs also target non-histone proteins like p53 to modify their post-translational activity. LSD1 enzymatic activity and normal expression abnormalities have been linked to a number of cancers, including HCC. Lately, the role of LSD1 in the successful establishment of infections by several viruses was examined, including hepatitis B and herpes simplex virus type 1, as well as in the suppression of others, such influenza type A or vesicular stomatitis virus.LSD2, the homolog protein of LSD1, has been studied to a much lesser extent than the first. There are data supporting that it participates in the lipid metabolism of hepatocytes and in the control of the transcriptional machinery as well as in carcinogenic pathways. To date, LSD2 has not been associated with viral infections.Taking into account all the above, the first aim of this study was to investigate the endogenous levels of LSDs during HCV infection and any potential effects on fundamental cellular processes that can result in adverse changes in cellular phenotype. At the same time, we intended to examine the potential involvement of LSDs in the progression of HCV infection as well as the mechanisms involved.It is well established that HCV increases matrix metalloproteinases (MMPs), proteases that degrade extracellular matrix components. The activity of these proteins is regulated by cytokines and pro-inflammatory regulators and by the extracellular matrix metalloproteinase inducer (EMMPRIN), a transmembrane glycoprotein which participates in the extracellular matrix remodeling, cell differentiation and energy metabolism. EMMPRIN overexpression and hyperglycosylation have been shown to be closely associated with HCC metastasis. It has been observed that intrahepatic metastases occur frequently during HCC, significantly worsening the prognosis for patients. Furthermore, current studies demonstrate that the pre-metastatic events begin considerably earlier than the time a metastasis is found. Viruses like HCV accelerate cell motility. Thus, testing a putative LSD1-EMMPRIN-MMPs axis and its role in the development of HCV-induced metastatic cancer was also of great concern of this study. In our experiments, we used Huh7.5 cell line, as well as viral stocks of HCV-2a and -3a strains which differ significantly in terms of their function, rate of replication, and impact on cellular phenotype. Infections were conducted under the modulation of LSD1 or LSD2 endogenous expression by genetic engineering methods, in order to ivestigate their role in HCV infection. It was found that the overexpression of LSD1 inhibits viral replication while its elimination greatly increases it, indicating that LSD1 has a detrimental function in the development of a successful viral infection. The aforementioned findings were also verified in triple co-cultures of hepatocytes with hepatic sinusoidal cells, which was used to simulate the in vivo microenvironment. On the other hand, bypassing the entry of viruses upon LSD1 overexpression by direct transfection with HCV RNA, showed no inhibition of viral replication. This led us to the conclusion that, in the presence of LSD1 overexpression, viral replication can only occur if the HCV virus entry stage is omitted. The kinetics of infection did not seem to be significantly affected by LSD2 overexpression.Interferon-induced transmembrane protein 3 (IFITM3), which is located on the membrane of endosomes and lysosomes, participates in the host defense against viruses that follow the endocytosis pathway by directing incoming viruses into lysosomes. Interestingly, its antiviral properties are triggered by LSD1 demethylating the monomethylated K88 residue. It was observed that IFITM3 expression was increased by HCV-infection, independent of LSD1 levels, emphasizing its function as an HCV defensive mechanism. In fact, in LSD1 overexpression, IFITM3 levels remained elevated even 24 hours after infection. Furthermore, in overexpression conditions, inactive IFITM3 was reduced after infection, while in LSD1 overexpression it was diminished, indicating increased antiviral activity of IFITM3.It is well established that the presence of intracellular lipids is essential to the life cycle of HCV and that modifying lipid metabolism is necessary to produce an intracellular milieu rich in lipids, which in turn facilitates HCV replication, assembly and secretion from the cell. The activation of de novo fatty acid biosynthesis and the production of lipid droplets (LDs), which are essential for viral replication, are essential for a successful infection. Experiments using LD staining demonstrated a significant decrease in LD numbers upon LSD1 overexpression, even in HCV-infected cells, possibly impeding viral assembly.Regarding the later stages of infection, we showed that HCV causes a gradual hyperglycosylation of EMMPRIN within the cell, most likely as a result of long-term LSD1 oscillations induced by long-term HCV infection that promotes EMMPRIN hyperglycosylation. Concerning the induction of MMPs, the ratio of high/low glycosylated EMMPRIN is of importance. The current investigation indicated the existence of an LSD1-EMMPRIN-MMPs axis that may be enhanced by HCV infection, inducing pro-metastatic mechanisms.This thesis highlighted the function of LSD1 in HCV infection as well as the regulation of LSD2, which has not previously been investigated in viral infections. Furthermore, the detrimental role of LSD1 occurs at two distinct phases of the viral cycle. Ultimately, it was discovered that LSD1 is involved in pre-metastatic processes via EMMPRIN, triggered by HCV. These findings serve as a basis for a more thorough investigation of the mechanisms underlying the development of hepatocarcinogenesis and the link between HCV infection and chronic liver disease.
Keywords:
epigenetics, viral hepatitis, hepatitis C, hepatitis B, hepatocellular carcinoma, liver cancer, metastasis, extracellular matrix, metalloproteinases, LSD1, LSD2, EMMPRIN, endosomes, lysosomes, epigenetic signature, hepatocarcinogenesis.
