Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies

Telomerase upregulation or reactivation is a critical feature in over 90 % of cancers. However, the mechanisms governing hTERT expression in cancer remain incompletely understood. Therefore, understanding how hTERT is activated in cancer cells and how it contributes to further progression of the disease continues to be a major area of research.

hTERT is a 40 kb gene consisting of 15 introns and 16 exons. It is located on the short arm of human chromosome 5 (5p15.33) approximately 1.2 megabases away from the telomere, embedded in a nuclease-resistant chromatin domain [42]. The hTERT promoter is GC rich and lacks both TATA (found in the promoter regions of genes that encode proteins found in both eukaryotes and prokaryotes) and CAAT (which rarely occurs in the promoter region of eukaryotes but is completely absent in prokaryotes) boxes but contains binding sites for multiple transcription factors, suggesting that hTERT expression is under multiple levels of control and may be regulated by different factors in different cellular contexts.

The 260 bp proximal region designated as the hTERT promoter core is responsible for most of its transcriptional activity. It contains at least five GC boxes (GGGCGG), which are binding sites for the zinc finger transcription factor SP1, and are essential for hTERT promoter activity. Two E-boxes (5?-CACGTG-3?), located at positions ?165 and +44 of the nucleotide sequence of hTERT relative to the transcription start site (TSS), provide binding sites for several enhancer binding proteins such as the MYC/MAX/MXD1 family and USF1/2. The E-boxes are not only important for hTERT promoter activation by c-MYC, but also bind to MAD1 and USF1 to mediate hTERT repression. The hTERT promoter core also contains a single TSS that binds the multifunctional transcription factor TFII-I. The transcription of the hTERT promoter is regulated by the action of multiple transcription factors and the telomere chromatin environment. However, it remains unsolved how the interplay between transcription factors and the telomere chromatin milieu controls hTERT transcription. Several transcription factors bind to the hTERT promoter core to activate or repress hTERT transcription. The transcription factors that upregulate transcription include c-MYC, SP1, E-twenty-six (ETS) family members, NF-kB, AP-2 and HIF-1. Transcription factors such as p53 (also known as TP53; represses transcription in an SP1-dependent manner), MAD (transcription factor involved in a network controlling cell cycle progression), WT1, MZF-2, SIP1 and menin have been shown to downregulate hTERT transcription. Most of the transcription factors that upregulate the telomerase gene are widely expressed and cannot fully account for high levels of hTERT expression and activation during tumorigenesis (Fig. 4).

hTERT transcription and promoter mutations. The hTERT gene is tightly repressed in almost all normal cells and tissues. Specific hTERT promoter mutations as part of cancer progression occur leading to increased transcription of hTERT. The transcription of hTERT is regulated by a series of transcription factors (TFs). hTERT promoter mutations create ETS/TCF binding motifs. Each mutation generates a new ETS/TCF binding site. Upregulating TFs such as ETS, c-MYC, SP1 and NF-kB bind to their respective sites and can promote hTERT transcription. Although binding of TFs is essential for hTERT transcription, in addition a permissive chromatin microenvironment is required. Binding of downregulating transcription factors decreases transcription. WT wild type; WT1 Wilms tumor protein1; MZF2 myeloid zinc finger protein 2

Recent observations of two highly recurrent mutations at two sites within the core promoter region of hTERT suggest one possible mechanism for the activation of telomerase in cancer cells. These mutations, which occur at ?124 bp and ?146 bp upstream from the ATG start site, are C?T transitions (at positions 1,295,228 (C228T) and 1,295,250 (C250T) on chromosome 5), and each mutation generates an identical 11 bp nucleotide stretch (5?-CCCCTTCCGGG-3?) containing a consensus binding motif (GGA(A/T)) for ETS transcription factors that can function as transcriptional repressor, activator or both to regulate telomerase expression [3, 4]. However, the molecular mechanisms of telomerase activation by ETS are not clearly understood. It has recently been reported that epidermal growth factor (EGF)-mediated activation of telomerase activity in lung cancer is associated with direct binding of ETS-2 to the hTERT promoter [43]. The recurrent hTERT promoter mutations were first reported as germline mutations from a family of melanoma patients and were later seen through genome sequencing of sporadic melanoma (in 74 % melanomas) and a number of cell lines across numerous cancer types and were associated with increased hTERT promoter activity [3, 4]. These mutations occur in approximately 70 % of melanomas, 80–90 % of glioblastomas, 60 % of hepatocellular carcinomas, 60 % of bladder cancers, 70 % of basal cell carcinomas, 50 % of cutaneous squamous cell carcinomas, up to 30 % of thyroid cancers and approximately 72 % of oligodendrogliomas [4, 6, 44, 45].

Additionally, a less frequent hTERT promoter mutation, ?57 bp upstream from the ATG start site, resulting in an AC transition, and other less frequent but recurrent mutations in cancer are found on chromosome 5 at the following positions: 1,295,228 CA; 1,295,248–1,295,243 CCTT; and 1,295,161 AC [46]. However, when these mutations (?57 AC, ?124 CT, ?146 CT) are introduced into tumor cells (A375 melanoma cells, UAGCC-62 melanoma cells, T24 bladder cancer cells) using a luciferase reporter construct, only a ~1.5- to 2-fold increase in hTERT transcription occurs [4, 45, 47]. Similarly, Huang and colleagues [48] also demonstrated that hTERT promoter mutations C228T and C250T caused a 2.8-fold to 5.3-fold increase in transcription (using a luciferase reporter assay in U87-MG cells) and telomerase activation using the telomere repeat amplification protocol (TRAP) assay in both xenografts and primary tumor tissues. It is not clear whether the observed enhanced hTERT transcription and increased level of hTERT mRNA are actually related to enhanced telomerase functional enzyme activity and TL maintenance in tumor cells.

While hTERT promoter mutations are frequent in multiple non-epithelial cancer types and their distribution is similar in the majority of patients, Chiba and co-workers [49] have emphasized that the impact of hTERT promoter mutations has mostly been studied in already transformed immortal tumor cells with active telomerase maintaining their telomeres. The tumor cells without such mutations also have sufficient telomerase activity to maintain their telomeres. Therefore, they introduced three common hTERT promoter mutations (?57 AC, ?124 CT, ?146 CT) into isogenic human embryonic stem cells (hESCs) using CRISPR/Cas9 genome editing, and observed that in undifferentiated hESCs the presence of ?124 CT caused a 2- to 3-fold increase in hTERT mRNA while neither the ?57 AC nor ?146 CT mutation had any effect on hTERT transcription and none of the three mutations had a major influence on telomerase activity. However, differentiated hESCs (fibroblasts) harboring these mutations continued hTERT transcription (8- to 12-fold increase) relative to normal hESCs, which would downregulate telomerase activity. Furthermore, telomerase activity in differentiated fibroblasts carrying hTERT promoter mutations was comparable to that observed in cancer cell lines. Bell and colleagues [50] proposed that GA-binding protein (GABP), an ETS-binding transcription factor, in conjunction with TERT promoter mutations, drives activation of hTERT. They have shown that C228T and C250T transitions are necessary for hTERT promoter activation, as these generate an ETS motif, which is critically important for the predominant binding of GABP to activate aberrant transcription in cancer cells. However, it is not known whether GABP alone can activate hTERT promoter transcription or if it interacts with other ETS-binding transcription factors. Recently, Li and co-workers [51] have pointed out that hTERT promoter mutations C228T and C250T are functionally different, in that the C250T unlike the C228T mutation is regulated by non-canonical NF-kB signaling, which is required for sustained telomerase activity.

While these non-coding hTERT promoter mutations are the most frequent promoter mutations in cancer, the level and frequency varies with cancer types (Table 1). Some cancers, such as melanoma, pleomorphic dermal sarcoma, myxoid liposarcoma, glioma, urothelial cell carcinoma, carcinoma of the skin and liver cancer, have the highest frequencies of TERT promoter mutations, while low frequencies were noted in gastric cancer, pancreatic cancer, non-small-cell lung cancer and gastrointestinal stromal tumors [6, 45, 48]. One possible explanation for these observations could be that incipient cancer cells, originating from rapidly self-renewing telomerase-competent cells, do not require TERT promoter mutations to regulate TL maintenance. Thus, cancers arising from these rapidly proliferating cells tend to have less frequent hTERT promoter mutations and probably just stably upregulate enzyme activity that is reversibly regulated in normal cells. By contrast, cancer-initiating cells originating from cells with low self-renewing capability may require TERT promoter mutations to overcome the short-telomere-dependent proliferative barrier. However, TERT promoter mutations have not been detected in prostate cancer, a cancer of low self-renewing tissue, suggesting that alterations within the core promoter of the TERT gene do not play an important role in prostate carcinogenesis [52]. The common hTERT promoter mutations have been detected across all stages and grades in most cancers, suggesting that hTERT mutations are generally an early event in the process of carcinogenesis [49]. It will be interesting to determine whether these mutations mostly occur during the period in which cells are undergoing crisis, in order to establish the role of these mutations as early events in the process of malignant transformation.

Frequency spectrum of hTERT promoter mutations across diverse cancer types

Telomerase expression also involves transcriptional, post-transcriptional and epigenetic levels of control, which may occur at any critical steps including transcription, mRNA splicing, hTR and hTERT synthesis and maturation, structural organization of telomerase RNP, nuclear localization of telomerase, post-translational modifications, and recruitment to the telomeres [53]. Epigenetic mechanisms such as chromatin remodeling, DNA methylation and histone modifications for regulation of hTERT transcription have also been described [54, 55]. The expression of hTERT is also regulated by post-transcriptional mechanisms. The process of gene transcription leads to the generation of transcripts (sequence of pre-mRNA produced by transcription) that are further modified into translational forms by several processes such as mRNA capping (5?-cap), 3?-polyadenylation and alternative splicing. Alternative splicing of hTERT mRNA has been shown to be a key post-transcriptional regulatory mechanism [56] but it remains unclear whether telomerase activity is directly associated with hTERT splicing.