Interaction of BRAF-induced ETS factors with mutant TERT promoter in papillary thyroid cancer

in Endocrine-Related Cancer
Correspondence should be addressed to Y J Park: yjparkmd@snu.ac.kr
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Synergistic effects of BRAFV600E and TERT promoter mutations on the poor clinical outcomes in papillary thyroid cancer (PTC) have been demonstrated. The potential mechanism of this phenomenon has been proposed: MAPK pathway activation by the BRAFV600E mutation may upregulate E-twenty six (ETS) transcription factors, increasing TERT expression by binding to the ETS-binding site generated by the TERT promoter mutation; however, it has not yet been fully proven. This article provides transcriptomic insights into the interaction between BRAFV600E and TERT promoter mutations mediated by ETS factors in PTC. RNA sequencing data on 266 PTCs from The Cancer Genome Atlas and 65 PTCs from our institute were analyzed for gene expression changes and related molecular pathways, and the results of transcriptomic analyses were validated by in vitro experiments. TERT mRNA expression was increased by the coexistence of BRAFV600E and TERT promoter mutations (fold change, 16.17; q-value = 7.35 × 10−12 vs no mutation). In the ETS family of transcription factors, ETV1, ETV4 and ETV5 were upregulated by the BRAFV600E/MAPK pathway activation. These BRAFV600E-induced ETS factors selectively bound to the mutant TERT promoter. The molecular pathways activated by BRAFV600E were further augmented by adding the TERT promoter mutation, and the pathways related to immune responses or adhesion molecules were upregulated by TERT expression. The mechanism of the synergistic effect between BRAFV600E and TERT promoter mutations on cancer invasiveness and progression in PTC may be explained by increased TERT expression, which may result from the BRAF-induced upregulation of several ETS transcription factors.

 

      Society for Endocrinology

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    TERT mRNA expression according to mutational status. TERT mRNA expression levels from RNA sequencing data from TCGA (A and B) and SNU (C and D) database. (A and C) Median expression levels of TERT according to mutational status. (B and D) Each column represents an individual sample.

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    Upregulated expression of ETV1, ETV4, and ETV5 by the BRAFV600E mutation. (A) Heatmap of mRNA expression of ETS according to mutational status in the samples from TCGA database. (B and C) Median expression levels of ETV1, ETV4 and ETV5 according to mutational status in TCGA (B) and SNU (C) database.

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    In vitro validation of changes in TERT and ETS expression by the BRAFV600E mutation and direct binding of ETS factors to the mutant TERT promoter. (A) In human thyroid cell lines expressing either wild-type BRAF (Nthy/WT) or mutant BRAF (Nthy/V600E), TERT, ETV1, ETV4 and ETV5 gene expression levels were quantified by RT-PCR. *P < 0.05 vs. Nthy/WT. (B) BCPAP, KTC-2, SW1736, FRO, 8505C, the human thyroid cancer cell lines harboring both BRAFV600E and TERT promoter mutations, were treated with PLX4720 (a BRAF inhibitor) or PD98059 (an ERK inhibitor), and TERT, ETV1, ETV4 and ETV5 gene expression levels were quantified by RT-PCR. *P < 0.05 vs mock. (C) BCPAP cells were treated with PLX4720 (0.1, 0.5, 1 and 10 μM) and proteins were harvested from total cell lysates 24 h after treatment. Western blot analysis was performed with anti-pERK and anti-ERK. *P < 0.05 vs mock. (D) Chromatin immunoprecipitation (ChIP) assay for ETV1, ETV4 and ETV5 occupancy at TERT promoter in Nthy-ori 3-1 cells without BRAFV600E and TERT promoter mutations and in KTC-2, BCPAP and MDA-T32 cells with both mutations. KTC-2, BCPAP and MDA-T32 cells were treated with PLX4720. The fold enrichment was the fold increase for the signal from each antibody-enriched chromatin relative to a control IgG. Cyclophilin (Cyclo) was used as negative control. All data are expressed as mean ± s.d. *P < 0.05 vs Nthy-ori 3-1, #P < 0.05 vs mock.

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    Transcriptional changes according to mutational status of the BRAFV600E and TERT promoter. (A, B and C) Volcano plots for DEGs according to mutational status of samples from TCGA database: no-mutation vs BRAF-only (A), no-mutation vs BRAF + TERT (B), and BRAF-only vs BRAF + TERT (C). (D) Venn diagram for the overlapping downregulated (green) and upregulated (red) DEGs between groups no-mutation vs BRAF-only and no-mutation vs BRAF + TERT. (E) The degree of changes in the gene expression of common DEGs, expressed as mean ± standard error of the mean. (F) The top 10 most significantly enriched molecular pathways of common DEGs.

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    Transcriptional changes by TERT expression in addition to the BRAFV600E mutation. (A) Each column represents the TERT mRNA expression level of an individual sample of TCGA database. (B, C and D) Volcano plots for DEGs according to BRAFV600E mutation and TERT mRNA expression: no mutation without TERT expression vs BRAFV600E mutation without TERT expression (B), no mutation without TERT expression vs BRAFV600E mutation with TERT expression (C), and BRAFV600E mutation without TERT expression vs BRAFV600E mutation without TERT expression (D). (E) The top 10 most significantly enriched molecular pathways of upregulated DEGs in BRAF-mutated PTCs with TERT expression compared to those without TERT expression. (F) Median expression levels of genes CD28, CTLA4 and VCAM1, which were upregulated DEGs in BRAF-mutated PTCs with TERT expression compared to those without TERT expression.

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