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

PhD thesisHepatocellular 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 activatedNewcastle University NUORS fellowship and JGW Patterson special gran

Impact of retrotransposon protein L1 ORF1p expression on oncogenic pathways in hepatocellular carcinoma: the role of cytoplasmic PIN1 upregulation

BACKGROUND: Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood. METHODS: We analysed L1 expression in the TCGA-HCC RNAseq dataset (n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems. RESULTS: We observed positive associations between L1 and activated TGFβ-signalling, TP53 mutation, alpha-fetoprotein and tumour invasion. IHC confirmed a positive association between pSMAD3, a surrogate for TGFβ-signalling status, and L1 ORF1p (P < 0.0001, n = 32). Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p. DISCUSSION: Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

Previously, we discovered that deletion of c-Rel in the Em-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-κB subunit in B-cell malignancies. Here we report that Em-Myc/cRel−/− cells have an unexpected and major defect in the CHK1 pathway. Total and phospho proteomic analysis revealed that Em-Myc/cRel−/− lymphomas highly resemble wild-type (WT) Em-Myc lymphomas treated with an acute dose of the CHK1 inhibitor (CHK1i) CCT244747. Further analysis demonstrated that this is a consequence of Em-Myc/cRel−/ − lymphomas having lost expression of CHK1 protein itself, an effect that also results in resistance to CCT244747 treatment in vivo. Similar down-regulation of CHK1 protein levels was also seen in CHK1i resistant U2OS osteosarcoma and Huh7 hepatocellular carcinoma cells. Further investigation revealed that the deubiquitinase USP1 regulates CHK1 proteolytic degradation and that its down-regulation in our model systems is responsible, at least in part, for these effects. We demonstrate that treating WT Em-Myc lymphoma cells with the USP1 inhibitor ML323 was highly effective at reducing tumour burden in vivo. Targeting USP1 activity may thus be an alternative therapeutic strategy in MYC-driven tumours