DNA hypermethylation within TERT promoter upregulates TERT expression in cancer

Abstract

Replicative immortality is a hallmark of cancer cells governed by telomere maintenance. Approximately 90% of human cancers maintain their telomeres by activating telomerase, driven by the transcriptional upregulation of telomerase reverse transcriptase (TERT). Although TERT promoter mutations (TPMs) are a major cancer-associated genetic mechanism of TERT upregulation, many cancers exhibit TERT upregulation without TPMs. In this study, we describe the TERT hypermethylated oncological region (THOR), a 433-bp genomic region encompassing 52 CpG sites located immediately upstream of the TERT core promoter, as a cancer-associated epigenetic mechanism of TERT upregulation. Unmethylated THOR repressed TERT promoter activity regardless of TPM status, and hypermethylation of THOR counteracted this repressive function. THOR methylation analysis in 1,352 human tumors revealed frequent (>45%) cancer-associated DNA hypermethylation in 9 of 11 (82%) tumor types screened. Additionally, THOR hypermethylation, either independently or along with TPMs, accounted for how approximately 90% of human cancers can aberrantly activate telomerase. Thus, we propose that THOR hypermethylation is a prevalent telomerase-activating mechanism in cancer that can act independently of or in conjunction with TPMs, further supporting the utility of THOR hypermethylation as a prognostic biomarker.

Keywords: Cancer; Epigenetics; Oncology; Telomeres.

Figure 1. Defining THOR through DNA CpG methylation analysis of the TERT promoter.

(A) Average CpG methylation of the TERT promoter in normal cell lines and tissues (n = 43, blue) and TERT-expressing cancer cell lines (n = 18, red). THOR is a 433-bp region (–140 to –572, relative to the TSS) comprising 52 CpG sites and located adjacently upstream of the common C228T and C250T TPMs (purple triangles). The UTSS encompasses 5 CpG sites within THOR. ATG and TSS are the start codon and TSS of the TERT promoter, respectively. Lollipops represent individual CpG sites. (B) Methylation heatmap generated from unsupervised clustering displays the methylation percentage of each CpG site within THOR for normal cell lines and tissues (n = 43, blue) and TERT-expressing cancer cell lines (n = 18, red). Gray color indicates unavailability of data.

Figure 2. THOR hypermethylation is prevalent in human cancers.

(A) Average DNA CpG methylation of the TERT promoter in normal cell lines and tissues (n = 43, blue) and tumor samples (n = 87, red). (B) Methylation heatmap generated from unsupervised clustering shows the methylation percentage of each CpG site within THOR for normal cell lines and tissues (n = 43) and tumor samples (n = 87). Gray color indicates unavailability of data. (C) Box-and-whisker plot shows the median and distribution of the average THOR methylation levels in normal control samples (n = 80, blue) and samples from various tumor tissue types (n = 1,352; red). *P < 0.05, by Sidak’s multiple comparisons test. (D) Difference in average THOR methylation levels between each pair of normal tumor samples (n = 99; left plot) and distribution of differences in THOR methylation (right plot; median and IQR). ****P < 0.0001, by paired t test. (E) Pie charts display the frequencies of the THOR hypermethylation signature across various tumor types.

Figure 3. Hypermethylation counteracts the repressive effect of rTHOR on TERT promoter activity.

For the data shown, each experiment was performed in triplicate. (A) Schematic representation of the TERT promoter. rTHOR (red) is a transcriptional regulatory element within THOR, located upstream of the TERT core promoter (blue). Normalized fold changes in TERT promoter activity are shown for the specified luciferase constructs transfected into the glioblastoma cell line LN229. The numbers in the plasmid constructs indicate the distance (bp) from the TERT TSS. *P < 0.05, by unpaired t test. (B) Normalized fold changes in hEF1 promoter activity are shown for CpG-free constructs when rTHOR was unmethylated or methylated (in vitro) in the cancer cell lines LN229, HeLa, and HT1080. *P < 0.05 and ***P < 0.001, by unpaired t test.

Figure 4. Coexistence and interplay of TPM and THOR hypermethylation in human cancers.

For the data shown, each experiment was performed in triplicate. (A) Normalized fold changes in TERT promoter activity are shown for the specified luciferase constructs, with the presence and absence of THOR and/or C228T TPM, in the glioblastoma cell line LN229 and the medulloblastoma cell lines ONS76 and UW228. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired t test. (B) TERT expression (mean ± SD, black bars and dots, y axis) and average THOR methylation levels (red dots, y axis) are shown in human primary (1°) and cancer cell lines. TERT regulation–associated characteristics for all cell lines are shown below the graph. (C) Pie charts display the frequencies of TPMs and the THOR hypermethylation signature in TPM-common tumors (gliomas and melanomas) and TPM-independent tumors (prostate, lung, colon, and breast). (D) TERT expression (mean ± SD, black bars and dots, y axis) and THOR methylation levels (red dots, y axis) are shown in a subset of adult gliomas (n = 21). TERT regulation–associated characteristics for these samples are shown below the graph.