TERT promoter mutations in melanoma render TERT expression dependent on MAPK pathway activation

Abstract

The mechanism of telomerase re-activation in cancer had remained elusive until the discovery of frequent mutations in the promoter of the TERT gene that encodes the catalytic reverse transcriptase subunit of telomerase. We investigated the regulation of TERT expression in melanoma cell lines and our results show that promoter mutations render TERT expression dependent on MAPK activation due to oncogenic BRAF or NRAS mutations. Mutations in the TERT promoter create binding sites for ETS transcription factors. ETS1, expressed in melanoma cell lines, undergoes activating phosphorylation by ERK at Thr38 residue as a consequence of constitutively activated MAPK pathway. We demonstrate that ETS1 binds on the mutated TERT promoter leading to the re-expression of the gene. The inhibition of ETS1 resulted in reduced TERT expression. We provide evidence that the TERT promoter mutations provide a direct link between TERT expression and MAPK pathway activation due to BRAF or NRAS mutations via the transcription factor ETS1.

Keywords: BRAF; ETS1; MAPK pathway; melanoma; telomerase.

Figure 1. Distribution of mutations in the TERT promoter

The sporadic and germline mutations found in melanoma are shown in red below the TERT core promoter. The Ets/TCF consensus motifs created by mutations are underlined. The numbering above the sequence relates to the start codon (ATG) of TERT whereas the standardized positions of mutations on chromosome 5 are indicated in the boxes. The transcription start is indicated by an arrow.

Figure 2. TERT expression is dependent on MAPK pathway activation

(A) Levels of TERT, phosphorylated ETS1 (pETS1), total ETS1 (ETS1), phosphorylated ERK (ppERK), total ERK (ERK) and actin were analyzed by western blotting in melanocytes (NHEM) and melanoma cell lines. The TERT promoter mutation status is indicated above the blots. (B) Melanoma cell lines were treated for 48 hrs with 3 μM, 10 μM of U0126 or DMSO (0). Levels of TERT, phosphorylated ERK (ppERK), total ERK (ERK), phosphorylated ETS1 (pETS1), total ETS1 (ETS1) and actin were analyzed by western blotting. (C) Melanoma cell lines were treated for 24 hrs with 3 μM, 10 μM of U0126 or DMSO (0). TERT mRNA levels were quantified by real-time PCR normalized to GAPDH. Values are mean ± s.d. of two experiments assayed in duplicate. The TERT promoter status is indicated underneath the bar-charts.

Figure 3. ETS1 binds to the TERT promoter and control its expression

(A) Luciferase reporter constructs containing wild type TERT promoter sequence (WT) and sequence with mutant allele for the –124T mutant (-124T) were transfected in triplicate into UKRV-Mel21 cell line with and without plasmids with ETS1 sequence. To determine the effect of MEK inhibitors, in parallel experiments cells after 6 hours of transcfection were treated with Trametinib. Cells were harvested 64 hours post transfection and reporter expression was analyzed using the Dual-Luciferase assay system. The constructs without either reporter gene did not show any activity and were used as controls. (B) Melanoma cell lines were transfected without any siRNA (-), a siRNA control (scr), siRNA targetting ETS1 (ets1a and ets1b) or GABPA (gabpa1 and gabpa2). After 72 hrs, levels of TERT, ETS1 GABPA and ACTIN were analyzed by Western Blotting. The TERT promoter status is indicated above the blots. (C) M74 melanoma cell line was transfected as above. 24 hrs later cells were treated with DMSO or 3 μM of U0126. After 48 hrs of treatment, levels of TERT, ETS1 GABPA and ACTIN were analyzed by Western Blotting. The TERT promoter status is indicated above the blots. (D) Melanoma cell lines were formalin fixed and harvested. Chromatin precipitated by anti-ETS1, anti-GABPA or control IgG was reverse-cross-linked, and the obtained genomic fragments were quantified by real-time PCR. Values are presented as fold enrichment over the mean value of control IgG. Error bars represent standard deviations.