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), whose catalytic subunit is encoded by the telomerase reverse transcriptase (TERT) gene. However, until recently, the underlying mechanisms for telomerase activation in cancer cells were largely unknown.
Recurrent transcription activating mutations of the TERT promoter were first described in melanoma and subsequently in other tumor types (
). More recently it was shown that a region of the TERT promoter upstream of the transcription start site is methylated in malignant telomerase-expressing pediatric brain tumors but not in telomerase-negative normal brain tissue or low-grade tumors (
). To determine whether epigenetic modifications may play a role in telomerase expression in this melanoma subtype, we analyzed the DNA methylation profile of a CpG-rich region of the TERT promoter, shown previously to be differentially methylated between normal and malignant tissues (
), in 13 melanomas (3 arising in GCN, 7 conventional, and 3 spitzoid) and 10 benign or borderline melanocytic tumors (1 GCN, 3 GCN with nodular proliferation, and 6 borderline spitzoid melanocytic neoplasms) from 23 pediatric and adult patients. The human investigations were performed after approval by local institutional review boards. Written, informed patient consent was waived because the research involved no more than minimal risk to the subjects. The status of the TERT promoter, BRAF and NRAS mutations, and kinase fusions was available for the spitzoid tumors and a subset of melanoma samples from our prior studies (
Supplementary Table S1 (online) and Figure 1 summarize the demographic and outcome data and the primary driver oncogene for the 23 study subjects. PCR–Sanger sequencing identified a hotspot TERT promoter mutation (4 C228T [chr5:1,295,228], 3 C250T [chr5: 1,295,250], and 2 CC242/243TT [chr5: 1,295,242-3]) in 9 of 13 melanoma samples (6/7 conventional; 3/3 spitzoid; 0/3 melanomas in GCN) but not in the 10 benign or borderline melanocytic neoplasms (Figure 1). The DNA methylation status of a region of the TERT promoter, from 482 bp to 667 bp upstream of the ATG start site (chr5: 1295586−1295771 [GRCh37/hg19]) (see Supplementary Figure S1, online), encompassing 26 CpG sites, was assayed by next-generation bisulfite sequencing (see Supplementary Methods, online). For each CpG site, the methylation ratio (beta value) was measured in the range of 0 to 1 (see Supplementary Table S2, online). The methylation status was defined as follows: >0.7, methylated (Figure 1, red); 0.5 to 0.7, partially methylated (orange); 0.3 to <0.5, partially unmethylated (cyan); and <0.3, unmethylated (blue). Supplementary Table S3 (online) shows the total number of methylated Cs and unmethylated Cs in the sequenced region for each sample. Remarkably, almost all 26 CpG sites in the sequenced region were highly methylated in the three melanomas arising in GCN (S1, S2, S21) and in the one conventional melanoma bearing wild-type TERT promoter (S22), whereas the CpG sites remained predominantly unmethylated in the 9 mutant TERT promoter melanomas and the 10 benign or borderline melanocytic neoplasms (Figure 1).
Next, we evaluated the association of TERT promoter CpG methylation with telomerase expression by TERT mRNA in situ hybridization and by gene expression analysis (Supplementary Methods). TERT mRNA in situ hybridization revealed distinct, intracellular punctate signals in melanomas arising in GCN (Figure 2c and f) but not in the proliferative nodules in GCN (Figure 2i and l). The TERT promoter methylation level was calculated as the log 2 ratio of the total number of methylated Cs versus the total number of unmethylated Cs in the sequenced region (logit [beta value]). The TERT expression level was measured by using RNA sequencing data available for a subset of samples. An association analysis revealed a strong correlation between TERT promoter methylation and TERT expression level (P = 0.0422, adjusted r2 = 0.5145; see Supplementary Figure S2, online).
Our data demonstrate that epigenetic modification through TERT promoter CpG methylation is an alternative pathway for TERT reactivation in melanoma. Although epigenetic remodeling by promoter methylation is generally considered a signature of gene silencing, TERT expression is paradoxically increased by promoter methylation (
) from binding to the target DNA-binding sites in the region (Supplementary Figure S1). Also, even when the promoter is largely methylated, a small region of the core promoter upstream of the transcription start site remains unmethylated to allow for the continued transcriptional activity of TERT (
). A much more frequent change in these nevi than melanoma is the development of clonal proliferations often in the form of nodules (Figure 2g, h, j, and k), which may suggest or mimic melanoma on clinical or histologic grounds (
). The differential pattern for TERT promoter methylation and telomerase expression between melanomas in GCN and proliferative nodules demonstrated in our study is consistent with the benign clinical course of proliferative nodules compared with the invariably aggressive behavior of melanoma arising in GCN. Further studies in a larger number of patients are needed to determine the potential diagnostic value of TERT promoter methylation assays for ambiguous proliferative lesions within GCN.
In our cohort, TERT promoter hypermethylation or promoter mutations occurred in all melanoma samples but in none of the benign or borderline melanocytic lesions, suggesting that a panel incorporating TERT promoter methylation and mutation assays may help discriminate between benign/borderline and overtly malignant melanocytic neoplasms. Future studies need to assess the potential use of these assays for diagnostic or prognostic purposes in the clinic.
In summary, we demonstrate that in subsets of malignant melanoma, TERT is upregulated epigenetically by a methylation-dependent mechanism. These findings have potential therapeutic implications. TERT promoter CpG hypermethylation is a reversible phenomenon. Treatment with DNA demethylating agents reduced TERT expression and telomerase activity in telomerase-positive cell lines (
). Together, these findings provide a rationale for developing a therapeutic strategy in preclinical studies through epigenetic modifications at TERT promoter regulatory sites in melanomas with the CpG island methylator phenotype.
Conflict of Interest
The authors state no conflict of interest.
This research was supported in part by the National Cancer Institute of the National Institutes of Health under Award Number P30CA021765 and by ALSAC.