Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation

Abstract

Background: Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations.

Methods: TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay.

Results: TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter.

Conclusions: TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them potential therapeutic targets.

Keywords: China; Genetics; Glioma; Neoplasms; Promoter regions; Telomerase.

Figure 1. TERT promoter mutations identified in 799 tumor samples and potential ETS binding sites

Arrows indicate locations of mutations identified (red: potential de novo ETS binding site generated, green: does not generate site).

Figure 2. Correlation of TERT promoter mutations and TERT mRNA expression as well as telomerase activation in gliomas and increase transcriptional activity of the TERT core promoter in a glioma cell line

(A) TERT mRNA expression in both C228T and C250T TERT promoter-mutated gliomas was significantly higher than gliomas without mutation. TERT mRNA expression was measured by RT-qPCR and normalized to GAPDH expression first, and then the expression of TERT of all the samples were normalized to the SF7761 cell line. P values were determined by the Mann-Whitney test. (B) U87-MG cells were transfected with luciferase constructs containing the TERT core promoter (from −424 to +65) with the appropriate variants for 24 h. The luciferase activities were measured and normalized by Renilla luciferase activities. Results are mean±SD of three independent experiments. P values were determined by ANOVA with the Dunnett Test (*P <0.05, ** P<0.01, **** P<0.0001). (C) Both C228T and C250T TERT promoter mutations are associated with telomerase activation in xenografts; (D) Both C228T and C250T TERT promoter mutation upregulate telomerase activity in primary tumor tissues.