The role of transcription factor activating enhancer-binding protein-4 (TFAP4) in the reactivation of telomerase reverse transcriptase (TERT) in hepatocellular carcinoma

Abstract

Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide. Hepatocellular carcinoma (HCC) accounts for 75-85% of primary liver cancer in adults, and hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the major etiological factors, accounting for 80-90% of HCC cases. Even though HCC treatments have developed considerably, our current understanding of the molecular pathogenesis of HCC is limited.

Many cancer types including HCC frequently harbor genetic alterations at the human telomerase reverse transcriptase (TERT) promoter and recruit transcription factors to bind to this region, thereby driving the reactivation of the TERT gene. Moreover, recurrent HBV genome integrations in HCC have been identified at the TERT promoter region. In silico sequence analysis by previous studies identified several putative binding sites of the transcription factor activating enhancer-binding protein-4 (TFAP4) on the TERT promoter. However, the role of TFAP4 in the transcriptional regulation of the TERT promoter remains unclear. This study aimed to characterize the functional role of TFAP4 in human HCC and the mechanistic role of TFAP4 in HBV integration-mediated TERT mRNA transcription.

Using RNA-sequencing and western blot analysis, we demonstrated that TFAP4 was upregulated in both our HCC tumor tissues and HCC cell lines. We further performed gene correlation analysis on TFAP4 and TERT using TCGA database, database from our in-house RNA-sequencing performed on HCC clinical samples, and HCC cell lines, and observed a positive correlation between TFAP4 and TERT at the mRNA level. With dual luciferase reporter assay, we also showed that ectopic expression of TFAP4 led to a significant increase in the promoter activity of TERT. Moreover, the TERT promoter activity was abolished when we created a mutation at nucleotide 111 upstream of the transcription start site by mutagenesis, suggesting that TFAP4 might directly lead to the reactivation of TERT mRNA transcription.

However, knockdown of TFAP4 did not seem to exert much effect on the expression levels of TERT in the HCC cell lines that contain an integrated HBV genome. Silencing of TFAP4 suppressed the level of hepatitis B surface antigen (HBsAg) production, whereas overexpression of TFAP4 did not alter the activities of the promoters for transcribing HBsAg and hepatitis X (HBx) mRNA, as demonstrated by dual luciferase reporter assays. Taken together, we showed that the role of TFAP4 in regulating the telomere expression or activity via HBV DNA integration in HBV-integrated HCC cells might be limited. Instead, our findings suggested that TFAP4 might directly regulate the TERT gene transcription in HCC without HBV integration.