The TERT promoter SNP rs2853669 decreases E2F1 transcription factor binding and increases mortality and recurrence risks in liver cancer

Abstract

A common single-nucleotide polymorphism in the telomerase reverse transcriptase (TERT) promoter, rs2853669 influences patient survival rates and the risk of developing cancer. Recently, several lines of evidence suggest that the rs2853669 suppresses TERT promoter mutation-mediated TERT expression levels and cancer mortality as well as recurrence rates. However, no reports are available on the impact of rs2853669 on TERT expression in hepatocellular carcinoma (HCC) and its association with patient survival. Here, we found that HCC-related overall and recurrence-free survival rates were not associated with TERT promoter mutation individually, but rs2853669 and the TERT promoter mutation in combination were associated with poor survival rates. TERT mRNA expression and telomere fluorescence levels were greater in patients with HCC who had both the combination. The combination caused TERT promoter methylation through regulating the binding of DNA methyltransferase 1 and histone deacetylase 1 to the TERT promoter in HCC cell lines. The TERT expression level was significantly higher in HCC tumor with a methylated promoter than in that with an unmethylated promoter. In conclusion, we demonstrate a substantial role for the rs2853669 in HCC with TERT promoter mutation, which suggests that the combination of the rs2853669 and the mutation indicate poor prognoses in liver cancer.

Keywords: A TERT transcription repressor; TERT promoter mutation; mechanism for regulation of SNP-dependent TERT promoter activity; risk of hepatocellular carcinoma (HCC)-related mortality and recurrence; single-nucleotide polymorphism at telomerase reverse transcriptase (TERT) promoter.

Figure 1. The variant rs2853669 at the TERT promoter is associated with a high risk of death and cancer recurrence in hepatocellular carcinoma (HCC) patients with a TERT promoter mutation

A. A representative map describing the SNP rs2853669 (−245T > C), −146C > T mutation, and −124C > T mutation in the human TERT gene. ChIP, Chromatin immunoprecipitation; MSP product, methylation specific PCR product. B. Kaplan–Meier analysis of differences in overall survival and C, D. recurrence-free survival based on the presence of the rs2853669 variant and TERT promoter mutation status. HCC patients included the SMH cohort (B, C) and KU cohort (D) *P < 0.05; **P < 0.01.

Figure 2. The variant rs2853669 at the TERT promoter is associated with an increased TERT promoter activity in HCC cell lines, and a high TERT mRNA expression level and long telomere lengths in HCC tumors

A–C. Quantification of TERT promoter activity in hepatocellular carcinoma (HCC) cell lines (A), TERT expression (B) and telomere fluorescence levels (C) in HCC tumor tissues based on the presence of the variant rs2853669 (single-nucleotide polymorphism (SNP)) only, the mutation only (−124C > T or −146C > T), and a combination of both (−124C > T + SNP or −146C > T + SNP). The data in A are presented as the means ± SEM (n = 4). The horizontal bar in B and C show the median. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. E2F1 is a TERT transcriptional repressor in Huh7 cells without rs2853669

A. Domain structure of wild-type E2F1 (E2F1 WT) and dominant-negative E2F1 (DN-E2F1); the immunoblot assay, qPCR, and the luciferase assay using a 3x Flag pCMV-10 empty vector, 3x Flag E2F1, and 3x Flag DN-E2F1. DN-E2F1 is mutated in the DNA binding domain. B. Luciferase assay using the WT TERT promoter (TERT-WT-luc), TERT promoter with a mutated E2F1-binding site (−247 bp upstream of ATG) (TERT-mut1-luc), TERT promoter with a mutated E2F1-binding site (−171 bp upstream of ATG) (TERT-mut2-luc), TERT promoter with mutated E2F1-binding sites (−247 bp upstream and −171 bp upstream of ATG) (TERT-mut3-luc), and TERT promoter with rs2853669 (−245 bp upstream of ATG) (TERT-mut4-luc). The data are shown as the mean ± SEM, **P < 0.01, *** P < 0.005.

Figure 4. Epigenetic changes in the TERT promoter are involved in increased TERT transcription levels in Huh7 cells

A. Immunofluorescence staining of E2F1 (red), DNMT1 (green), and DAPI (blue) in Huh7 cell lines. Scale bar, 20 μm. The number-labeled boxes indicate the areas of protein co-localization (yellow). DAPI, 4′,6-diamidino-2-phenylindole. B. Huh7 cell nuclear extracts or immunoprecipitated products (IP) generated using control IgG and E2F1 antibodies were subjected to immunoblot analysis (IB) using the antibodies indicated on the right. C. Immunoblot assay and ChIP experiments using the TERT promoter from each 3x Flag empty vector- or 3x Flag DN-E2F1-ectopic expressed Huh7 cell line. Pol II, RNA polymerase II; H3Ac and H4Ac, acetylated histones H3 and H4. The data are shown as the mean ± SEM, **P < 0.01, *** P < 0.005.

Figure 5. E2F1 does not repress TERT transcription in HepG2 cells with rs2853669

A. ChIP experiments using TERT promoter from each 3x Flag empty vector–, 3x Flag E2F1–, or 3x Flag DN-E2F1–ectopic expressed HepG2 cell line. Primers for Minichromosome maintenance complex component 3 (MCM3) were used as an internal control. B, C. Luciferase assay (B) and qPCR (C) using 3x Flag empty vector–, 3x Flag E2F1–, or 3x Flag DN-E2F1–ectopic expressed HepG2 cells. The data are shown as the mean ± SEM, *P < 0.05, ** P < 0.01.

Figure 6. Blocking DNMT1 activity increases TERT transcription levels in Huh7 cells without rs2853669

A, B. MS-PCR using the primer for the TERT CpG island MSP targeting site, qPCR, and the ChIP experiment using 5-aza-dC-treated Huh7 cells (A), and a 3x Flag empty vector–, 3x Flag E2F1–, or 3x Flag E2F1 combined with 5-aza-dC-treated Huh7 cells (B) The data are shown as the mean ± SEM, *P < 0.05, ** P < 0.01, *** P < 0.005. MS-PCR, methylation specific PCR.

Figure 7. E2F1 and ETS2 regulate the rs2853669 variant-mediated TERT expression in HCC cells

A, B. ETS2 ChIP experiments using the TERT promoter after ectopic expression of each 3x Flag empty vector and 3x Flag E2F1 (A) and a 3x Flag empty vector and 3x Flag DN-E2F1 (B) in Huh7 and HepG2 cells. C. Immunoblot assay and qPCR after siRNA-mediated ETS2 knockdown in Huh7 and HepG2 cells. D. MS-PCR using the primer for the TERT CpG island MSP targeting site and ChIP experiment using siControl (siCon)- and siETS2-treated HepG2 cells. The data are shown as the mean ± SEM, **P < 0.01, *** P < 0.001. MS-PCR, methylation specific PCR.

Figure 8. The rs2853669 variant combined with the −124C>T mutation is associated with TERT promoter methylation in HCC tumors

A. Quantification of TERT mRNA levels in HCC tumors with a low-methylated- or high-methylated-TERT promoter. Horizontal bars indicate the median value. Samples were separated into 2 groups based on the median methylation level of TERT promoter. n = 102. B. Quantification of TERT protein expression in HCC tumors with a low-methylated- or high-methylated-TERT promoter. Horizontal bars indicate the median value. Samples were separated into 2 groups based on the median methylation level of TERT promoter. n = 42. C. Quantitation of TERT promoter methylation in HCC tumors without rs2853669 (−245T > C) and the −124C > T mutation or with the SNP rs285366 only, the −124C > T mutation only, or combination of both (−124C > T + SNP). D. Linear regression analysis of TERT promoter methylation levels (%) and TERT mRNA levels. R, Spearman's rank correlation coefficient. E. Molecular model illustrating the potential function of rs2853669 and −124C > T mutation in regulating TERT transcription.