Role of retrotransposons in hepatocellular carcinoma origin and progression – potential biomarker and therapeutic target?

Abstract

Introduction Hepatocellular carcinoma (HCC) has the fourth highest cancer-related mortality worldwide and is associated with a poor 5-year survival. Current therapies are limited and provide short median survival. Thus, there is an urgent need to develop novel therapies. Long interspersed class elements1 (LINE1 or L1) activation has been demonstrated in several cancers including HCC and can inhibit tumour suppressor genes or activate oncogenes. However, the role of L1 in hepatocarcinogenesis is still unknown. Methods L1 expression was evaluated in the RNAseq data of HCC (n=372) from the cancer genome atlas liver hepatocellular carcinoma (TCGA LIHC) study and formalin-fixed paraffin-embedded (FFPE) patient biopsies (n=48) from our own biobank. RNAseq data was analysed for L1 counts and their distribution was assessed in different HCC subclasses based on previously known molecular classifications and associations with clinical parameters were explored. Likewise, FFPE samples were stained for L1orf1p using an automated immunohistochemistry machine and were scored by a pathologist; associations between L1orf1p expression in HCC and clinical parameters like cirrhosis, tumour stage, albumin, bilirubin, alpha fetoprotein (AFP) and survival were explored. The role of L1 was further characterised in different liver cancer cell lines utilising L1 knockdown and overexpression systems. L1 knockdown was achieved using a lentivirus-based shRNA expression vector targeting L1orf1 in Huh-7 cells. The influence of L1-knockdown on functional properties such as proliferation, migration and invasion of the cells were investigated by comparing L1-knockdown cells with wild type and non-targeted controls. RNAseq evaluated the influence of L1-knockdown on whole transcriptome. Transient L1 full-length overexpression and conditional L1orf1p overexpression were used to further validate the influence of L1 on cell signalling pathways. Results L1 expression was elevated in HCC both at transcript and protein level compared to adjacent nontumour tissues. L1 transcripts correlated with high AFP, TP53 mutation, macrovascular invasion and activated TGF-β signalling. Likewise, L1orf1p expression correlated with AFP, activated TGFβ signalling and poorly differentiated tumours. A positive association between L1orf1p and pSMAD3 confirmed the relationship between L1 expression and TGF-β signalling in HCC. L1 knockdown in Huh-7 cells led to decrease in migratory and invasion capacity of the cells compared to control cell lines. Furthermore, gene set enrichment analysis (GSEA) of the RNAseq data demonstrated downregulation of TGF-β pathway in Huh7-L1knockdown cells compared to non- targeting control cells, which was confirmed by Pai1-lucifease reporter assay. Conversely, L1 overexpression (full-length and L1orf1 alone) increased TGF-β signalling as confirmed by Pai1- lucifease reporter assay, RT-qPCR and FACS analysis in HepG2, PLC/PRF-5 and HHL5 cell lines. Conclusion L1 is upregulated in human HCC and associated with high AFP, TP53 mutation and activated TGFβ signalling. Further in vitro studies demonstrated a crosstalk between L1orf1p and TGFβsignalling. Overall, our data demonstrates a causal link between L1orf1p and TGFβ signalling, which presents a novel therapeutic avenue and potential treatment stratification biomarker for HCC.