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

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.


Human samples
For measurement of telomere fluorescence levels, 93 surgically resected paraffin-embedded HCC tumor tissue samples and corresponding non-tumorous liver tissue samples were analyzed. For measurement of the TERT mRNA expression level, 72 surgically resected frozen HCC tumor tissue samples and corresponding non-tumorous liver tissue samples were analyzed. For methylation-specific PCR analysis, we used 102 samples of HCC tumors, including 24 samples of HCC tumors used in a previous report [1] and 78 samples of HCC tumors used in previous report [2]. The cases were prospectively and consecutively identified at Seoul St. Mary's Hospital of the Catholic University of Korea between 2005 and 2009 (93 paraffin-blocked tissue samples) and between 2011 and 2013 (19 frozen tissue samples), as well as at Korea University Guro Hospital (KU Guro Gene Bank 2013-020) between 2010 and 2013 (53 frozen tissue samples). We designated 93 paraffin-blocked tissues from Seoul St. Mary's Hospital of the Catholic University as the SMH cohort, and a total of 19 frozen tissues from Seoul St. Mary's Hospital of the Catholic University and 53 frozen tissues from Korea University Guro Hospital as the KU cohort, respectively. The histological grade of tumor differentiation was defined according to the Edmondson-Steiner grading system [3].

Quantification of telomere fluorescence level with immunoFISH
Paraffin-embedded sections were deparaffinized with xylene and rehydrated using an ethanol gradient. Antigen retrieval was performed by boiling sections in 100 mM sodium citrate (pH 6.0) for 10 minutes in a microwave oven. After permeabilization with proteinase K (15 μg/mL in phosphate-buffered saline [PBS; pH 7.4]) at 37°C for 20 minutes and 50% formamide treatment, the samples were fixed using 4% formaldehyde followed by air drying. The samples were then denatured for 6 minutes at 90°C followed by hybridization for 2 hours at room temperature in the dark. The hybridization solution contained 70% formamide in 2× saline-sodium citrate buffer, 5% MgCl 2 , 0.25% blocking reagent (Roche, Basel, Switzerland), 15.4 nM of Cy3-labeled telomere peptic nucleic acid (PNA) probe (TelC-Cy3: CCCTAACCCTAACCCTAA; Panagene, Korea), and 18.4 nM of fluorescein amidite (FAM)-labeled centromere PNA probe (Cent-FAM: AAACTAGACAGAAGCATT; Panagene). After the slide had been washed, it was rinsed with PBS containing 4′,6-diamidino-2-phenylindole (DAPI) and then mounted with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA). A centromeric probe was used as an internal control. The telomere fluorescence level refers to the ratio of telomere fluorescence intensity to centromere fluorescence intensity. The same exposure time was used for the quantification of telomere fluorescence level. The telomere fluorescence level was calculated by dividing the telomere fluorescence level of the tumor tissue by that of the corresponding non-tumorous tissue at five random fields. Image analysis was performed using Image-Pro plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Luciferase reporter assay
The pGL3-wild-type TERT promoter plasmid was used as the template to generate mutant constructs through site-directed mutagenesis. The original and converted primer sequences are as follows: TERT promoter with the -124C > T mutation, CCCCGGCCCAGCCCCCTCCGGGCCCTCCCAG to CCCCGGCCCAGCCCCTTCCGGGCCCTCCCAG (109-139 bp upstream of the ATG start site); TERT promoter with the -146C > T mutation, CCCGTCCCGACCCCTCCCGGGTCCCCGGCCC to CCCGTCCCGACCCCTTCCGGGTCCCCGGCCC (131-161 bp upstream of ATG start site); E2F1 for TERTluc mut1, CGCCCAGGACCGCGCTTCCCACGTGGCG to CGCCCAGGACCGCCTTTCCCAC GTGGCG (234-261 bp upstream of ATG start site); E2F1 for TERT-luc mut2, CCGCCTCCTCCGCGCGGACCCCGCCCCG to CCGCCTCCTCCGCCTGGACCCCGCCCCG (158-185 bp upstream of the ATG start site); E2F1 for TERT-luc mut4 (TERT promoter with the rs2853669 variant), GCCCAGGACCGCGCTTCCCACGTGGCGGAGG to GCCCAGGACCGCGCTCCCCACG TGGCGGAGG (230-260 bp upstream of the ATG start site). The bold text indicates the converted sequence. TERT-luc mut3 includes both mutations in TERT-luc mut1 and TERTluc mut2. A total of 20 ng of Renilla luciferase (Promega, Madison, WI, USA) was co-transfected with each sample as an internal control for transfection efficiency. After 48 hours, the luciferase activity was quantified using a Dual-Luciferase ® Reporter assay system (Promega, USA). All experiments were repeated three times.

Immunoblot analysis
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. TERT-specific
[9] The MSP products were subcloned into the pGEM-T easy vector (Promega, USA) in accordance with the manufacturer's instructions. The plasmid DNAs were purified using a plasmid DNA purification kit (Intron Biotechnology, Sungnam, Korea) followed by sequencing (Macrogen, Seoul, Korea).
The TERT promoter methylation levels were determined by methylation specific quantitative PCR (MSqPCR) as previously described [10-12] with certain modifications. Methylation levels of the TERT promoter (the region is from 270 bp upstream of the ATG start site to 31 bp upstream of the ATG start site) were calculated as the fraction of methylated molecules in the total methylated and unmethylated DNA, which was described in a previous report [10,11]. The myogenic differentiation 1 (MYOD1) gene was the internal reference gene [13]; The MYOD1 gene primers (forward primer: CCAACTCCAAATCCCCTCTCTAT; reverse primer: TGATTAATTTAGATTGGGTTTAGAGAAGGA) were located in a region without the CpG nucleotide; thus, the cytosine was converted into uracil using a bisulfite treatment, and the primers for MYOD1 bound only to the bisulfite-modified DNA.

SUPPLEMENTARY REFERENCES
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