Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase and is essential for telomerase activity. Upregulation of TERT expression and resulting telomerase activity occurs in the large majority of malignancies, including thyroid cancer. This upregulation results in continued cellular proliferation and avoidance of cellular senescence and cell death. In this review we will briefly introduce TERT and telomerase activity as it pertains to thyroid cancer and, highlight the effects of TERT on cancer cells. We will also explore in detail the different TERT regulatory strategies and how TERT is reactivated in thyroid cancer cells, specifically. These regulatory mechanisms include both activating single base pair TERT promoter mutations and epigenetic changes at the promoter, including changes in CpG methylation and histone modifications that affect chromatin structure. Further, regulation includes the allele-specific regulation of the TERT promoter in thyroid cancer cells harboring the TERT promoter mutation. These entail allele-specific transcriptional activator binding, DNA methylation, histone modifications, and mono-allelic expression of TERT. Lastly, TERT copy number alterations and alternative splicing are also implicated. Both amplifications of the TERT locus and increased full-length transcripts and decreased inactive and dominant negative isoforms result in active telomerase. Finally, the clinical significance of TERT in thyroid cancer is also reviewed.
Background: Telomerase reverse transcriptase (TERT) promoter mutations play a role in carcinogenesis and are found in both tumors and cancer cell lines. TERT promoter methylation, transcription factor binding, chromatin remodeling, and alternative splicing are also known to play an integral role in TERT regulation. Methods: Using nanopore Cas9 targeted sequencing, we characterized allele-specific methylation in thyroid cancer cell lines heterozygous for the TERT promoter mutation. Furthermore, using chromatin immunoprecipitation followed by Sanger sequencing, we probed allele-specific binding of the transcription factors GABPA (GA binding protein transcription factor subunit alpha) and MYC, as well as the chromatin marks H3K4me3 and H3K27me3. Finally, using coding single nucleotide polymorphisms and the long-read sequencing, we examined complementary DNA for monoallelic expression (MAE). Results: We found the mutant TERT promoter allele to be significantly less methylated than wild type, while more methylated in the gene body in heterozygous TERT mutant cell lines. We demonstrated that the transcriptional activators GABPA and MYC bind only to the mutant TERT allele. In addition, the activating and repressive chromatin marks H3K4me3 and H3K27me3, respectively, bind mutant and wild-type alleles exclusively. Finally, in heterozygous mutant cell lines, TERT exhibits MAE from the mutant allele only. Conclusions: In summary, by employing new long-read sequencing methods, we were able to definitively demonstrate allele-specific DNA methylation, histone modifications, transcription factor binding, and the resulting monoallelic transcription in cell lines with heterozygous TERT mutations.
Alterations in the genome, including mutations and copy number variation (CNV), can drive cancer progression. The Cancer Genome Atlas (TCGA) project studying papillary thyroid cancer (PTC) identified a number of recurrent arm‐level copy number amplifications, some spanning genes that are also commonly mutated in thyroid cancer. Herein, we focus on the role of TERT and BRAF CNV in PTC, including its relation to mutation status, gene expression, and clinicopathological characteristics. Utilizing TCGA CNV data, we identified focal amplifications and deletions involving the TERT and BRAF loci. TERT amplifications are more frequent in later stage thyroid tumors; in contrast, BRAF amplifications are not associated with stage. Furthermore, TERT amplifications are more frequently found in tumors also harboring TERT mutations, the combination further increasing TERT expression. Conversely, BRAF amplifications are more frequently found in BRAF wildtype tumors, and are more common in the follicular subtype of PTC as well as classic PTCs associated with a high follicular component and a RAS‐like expression profile (assessed by the BRAF/RAS score). This is the first study to examine the TCGA thyroid dataset for gene‐level CNV of TERT and BRAF, and their relationship with mutation status, tumor type and tumor stage. Assessing the differences in patterns of TERT and BRAF amplifications in the context of the mutation status of these genes may provide insight into the differing roles CNV can play depending on tumor type, and may lead to a better understanding of cancer drivers in thyroid cancer.
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