Factors involved in the regulation of Long Interspersed-Nuclear-Elements (L1) retrotransposons in the context of Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) generally develops on the background of a chronic liver disease following the accumulation of genetic damage and epigenetic alterations of growth regulatory genes, leading to activation of oncogenes and loss of function of tumour suppressor genes. Recent studies indicate that epigenetic aspects play an important role in the initiation of HCC. This includes dysregulation of repeat elements belonging to the Long Interspersed Nuclear Elements (LINE1 or L1) class. The L1 elements are autonomous mobile elements and upon activation contribute towards genomic instability via insertional mutagenesis. The thesis is aimed at understanding the factors leading to aberrant activation of retrotransposons and regulators of active retrotransposition in the context of HCC. All the liver cancer cell lines (Huh7, HepG2, Hep3B, PLC-PRF/5 and SK-Hep1) supported active retrotransposition in vitro irrespective of their basal L1 expression status or TP53 status. Since, active L1 retrotransposition through ‘Target Primed Reverse Transcription’ (TPRT) involves first DNA strand nicking by ORF2 endonuclease followed by second strand cleavage, we hypothesised that the DNA damage response pathways are involved in regulating the process. To decipher the influence of individual DNA repair pathway elements on the process of active retrotransposition, small molecule inhibitors towards ATM (KU-55933), DNA-PK (NU7441), ATR (VE-821), CHK1 (SRA737) and PARP (Rucaparib) were utilised. Overall, inhibition of ATR (Ataxia Telangiectasia And Rad3-Related Protein), a serine/threonine kinase involved in DNA replication stress and DNA damage signalling increased retrotransposition rate in all the cell lines. In addition, an increase in active retrotransposition was observed in Huh7 cell in presence of subgenomic copy of Hepatitis C Virus (HCV, a prevalent cause of HCC and contributes towards hepatocarcinogenesis by inducing oxidative stress, DNA damage and epigenetic changes in hepatocytes). Interestingly, the rate of retrotransposition remained higher in cells compared to control cell lines even when they were treated with PSI7977 (antiviral agent) successfully eliminating the viral genome from the cells. Hence, HCV upregulated active retrotransposition even beyond viral clearance and thus can contribute towards hepatocarcinogenesis by a ‘hit-and-run’ mechanism. Interrogating publicly available datasets - GSE84346 (RNAseq of Chronic HCV Hepatitis (CHC) patients and controls) and RNAseq data of non-tumour liver from the Cancer Genome Atlas HCC study - confirmed upregulation of L1 transcripts in chronic hepatitis patients liver. Hence, L1s can be activated