Supervisors info:
Γαϊτανάκη Αικατερίνη, Καθηγήτρια Φυσιολογίας Ζώων, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Γεωργοπούλου Ουρανία, Διευθύντρια Ερευνών Εργαστηρίου Μοριακής Ιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Summary:
Epigenetic regulatory mechanisms ensure the heritable alterations of cellular states without affecting the nucleotide sequence of the DNA. In multicellular organisms epigenetic control of transcription, forms the basis for the phenotypic and functional diversity of various cell types that carry identical or nearly identical genomes. Epigenetic regulators affect chromatin structure and promoter activity by depositing stable, but reversible, marks on DNA or DNA-associated proteins. In this current study our interest is directed towards the epigenetic factor called LSD-1. It’s an enzyme located in the cellular nucleus, that belongs to the family of Lysine-specific Histone Demethylases. LSD-1 specifically demethylates mono- or dimethylated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) through a redox process. Recently evidences showed that LSD-1 is required for normal differentiation, cell maintenance and homeostasis of metabolism. However, LSD-1 is overexpressed in several types of cancer and thought to be tumor promoting.
Hepatocellular carcinoma is a heterogeneous group of a tumor that vary in risk factors and genetic and epigenetic alteration events as shown above. Multiple factors including viruses, chemicals, and inborn and acquired metabolic diseases are responsible for its development. More specifically, HCC is closely associated with hepatitis C virus. Liver injury caused by viral factors affects many cellular processes such as cell signaling, apoptosis, and DNA repair. HCV-induced HCC development is a multi-step process that may progress over 20-40 years and involves: establishment of chronic HCV infection, chronic hepatic inflammation, progressive liver fibrosis, initiation of neoplastic clones accompanied by irreversible somatic genetic/epigenetic alterations. It is well certified that HCV virus hijacks host lipid metabolic mechanisms to promote its replication and to ensure the assembly of its viral lipoparticles. Viral structural and nonstructural proteins interfere in lipid synthesis and very-low-density lipoprotein secretion. These HCV related proteins also inhibit fatty acid oxidation and enhance fatty acid release from adipocytes, and this finally results in hepatic steatosis.
So far, correlations are established between LSD-1 and HCC, HCC and HCV virus. In this study under the prism of lipid metabolism, we sought to explore possible interactions between epigenetic factor LSD-1 and HCV virus in cancerous hepatic cell lines. Therefore, we investigated how HCV virus affects LSD-1 expression when it infects hepatic cells, and also how LSD-1 overexpression, in this case, affects HCV’s establishment. Based on our results we have concluded that HCV under conditions of endogenous LSD-1 expression, showcases the ability to increase the levels of LSD-1 in order to promote lipogenesis through the upregulation of the expression of genes likes FAS. LSD-1 can induce such lipogenic genes by activating SREBP transcription factors. By proceeding to electroporation with two types of replicons - JCR2A (Full Length HCV Genome) and PFKWT (NS3-NS5B Proteins) - we specified which type of viral protein is possibly responsible for this effect. Many studies have shown that Core protein is involved in the increased rates of lipogenesis and our data show that: a) under the presence of Core protein the rates of HCV replication are higher and b) a possible way through which it succeeds in this role is by increasing the expression of LSD-1.
Also, we investigated how two different types of viral proteins, NS5A and Core, affect LSD-1 expression and our results show that they have a reverse correlation to LSD-1. NS5A reduces the levels of LSD-1 expression while Core increases them. NS5A is involved in the assembly of the viral particles, so possibly HCV virus during this stage of its cycle doesn’t require the uptake of lipids, in contract with Core that is responsible for formation of the viral capsid where lipid are needed.
At last, according to Abdulla et. al., 2014, LSD-1 overexpression creates a dominant negative effect where it significantly decreases triglyceride levels and we confirmed this data by performing Oil red O and Bodipy staining to a clone that overexpresses LSD-1 and also to control cells. We have seen significant decrease in the number and the size of the lipid droplets inside the hepatic cells. When we perfomed electroporation to this clone with JCR2A genome, we observed almost zero levels of HCV replication comparing to the control cells, which implies that the virus tries but is unable to overcome the effect that the overexpression of LSD-1 creates, even in the presence of Core protein.
Our study has opened new pathways that can lead to a better understanding of the interaction of the virus with the host cell. It would therefore be, of interest to question the effect of LSD-1 inhibitors with respect to viral proliferation at both viral load and host immune response levels, so as to become a valuable tool in future for a better antiviral therapy for the treatment of hepatitis C and HCC.
Keywords:
Epigenetics, LSD-1, Lipid Droplets, Liver, Steatosis, Liver Cancer
