Roles of telomeres and telomerase in cancer, and advances in telomerase-targeted therapies
© The Author(s). 2016
Published: 20 June 2016
Telomeres maintain genomic integrity in normal cells, and their progressive shortening during successive cell divisions induces chromosomal instability. In the large majority of cancer cells, telomere length is maintained by telomerase. Thus, telomere length and telomerase activity are crucial for cancer initiation and the survival of tumors. Several pathways that regulate telomere length have been identified, and genome-scale studies have helped in mapping genes that are involved in telomere length control. Additionally, genomic screening for recurrent human telomerase gene hTERT promoter mutations and mutations in genes involved in the alternative lengthening of telomeres pathway, such as ATRX and DAXX, has elucidated how these genomic changes contribute to the activation of telomere maintenance mechanisms in cancer cells. Attempts have also been made to develop telomere length- and telomerase-based diagnostic tools and anticancer therapeutics. Recent efforts have revealed key aspects of telomerase assembly, intracellular trafficking and recruitment to telomeres for completing DNA synthesis, which may provide novel targets for the development of anticancer agents. Here, we summarize telomere organization and function and its role in oncogenesis. We also highlight genomic mutations that lead to reactivation of telomerase, and mechanisms of telomerase reconstitution and trafficking that shed light on its function in cancer initiation and tumor development. Additionally, recent advances in the clinical development of telomerase inhibitors, as well as potential novel targets, will be summarized.
Cancer is generally an age-related genetic disease, manifesting only when normal cells accumulate genomic instability over a period of time and acquire the capability of replicative immortality. Telomere attrition during successive cell divisions induces chromosomal instability and contributes significantly to genomic rearrangements that can result in tumorigenesis. Telomeres, repetitive (TTAGGG) DNA–protein complexes at the ends of chromosomes, are crucial for the survival of cancer cells. They are maintained by an enzyme called telomerase in the vast majority of tumors. The mechanisms underlying telomere length (TL) maintenance and telomerase expression involve transcriptional, post-transcriptional and epigenetic regulation, and in-depth understanding of these mechanisms may provide novel biomarkers and targets for early detection of disease, determination of disease prognosis, and the development of therapeutics .
Telomeres protect chromosome ends from fusion and from being recognized as sites of DNA damage (Box 1). Dysfunctional telomeres, arising by critical shortening of telomeres in normal somatic cells during progressive cell divisions, elicit DNA damage responses (DDRs) that trigger cellular senescence. Cells that gain oncogenic changes bypass senescence and continue to divide (extended lifespan period) until multiple critically shortened telomeres initiate crisis (a period of complete replicative senescence, chromosome end-to-end fusions, and extensive apoptosis). This leads to breakage–fusion–bridge cycles in which two sister chromatids lacking telomeres fuse together, forming a bridge with a chromatin connection. During anaphase, the sister chromatids are drawn apart owing to movement towards opposite poles, resulting in the formation of uneven derivative chromosomes, leading to genomic instability. The period of crisis results in extensive cell death. However, certain rare cells escape crisis and maintain stable but usually shortened telomere lengths for continued cell growth, eventually progressing to a malignant phenotype. Cancer cells achieve proliferative immortality by activating or upregulating the normally silent human TERT gene (hTERT) that encodes telomerase, a protein with reverse transcriptase activity that complexes with other proteins and a functional RNA (encoded by hTR, also called hTERC) to make a ribonucleoprotein enzyme complex. Rarely, another DNA recombination mechanism termed alternative lengthening of telomeres (ALT) reverses telomere attrition in order to bypass senescence. Although hTERT is usually silenced in almost all somatic cells, it is significantly expressed in ~90 % of human cancers. The details of the underlying mechanisms of hTERT activation are still being elucidated, but they mainly include mutations in the hTERT promoter, alterations in alternative splicing of hTERT pre-mRNA, hTERT amplification, epigenetic changes, and/or disruption of telomere position effect (TPE) machinery .
Recent reports have implicated two cancer-specific hTERT promoter mutations (mainly C˃T transitions) in the activation of telomerase in cancer cells [3, 4]. These mutations, which are located either −124 base pairs (bp) or −146 bp upstream from the TERT translation start site [5, 6], have been found to be associated with increased telomerase activity . Therefore, molecular mechanisms that regulate hTERT expression and telomerase assembly have been subjected to intense investigation. Studies using telomerase inhibition strategies have established that robust hTERT inhibition can lead to progressive telomere shortening and eventually cancer cell death. Several approaches, including use of small-molecule inhibitors, antisense oligonucleotides, immunotherapy, and G-quadruplex stabilizers have been employed to inhibit telomerase function . Currently, many anti-telomerase therapeutics are being evaluated in clinical trials against a variety of cancer types. The following sections will cover recent developments in the area of telomere and telomerase biology, their implications for understanding mechanisms underlying cancer and for the development of cancer therapies, as well as outstanding questions for the field.
Telomeres: organization, function and association with cancer
Apart from DNA end protection, telomeres also perform other important functions such as regulation of gene expression through transcriptional silencing of genes located close to the telomeres, called TPE , or located at long distances from telomeres, termed TPE over long distances (TPE-OLD) . The function of telomeres is tightly regulated and depends on a minimal length of telomeric repeats and the functionality of the associated shelterin protein complexes. In addition, higher-order DNA conformations, such as the T-loop and G-quadruplexes (G-rich four-stranded non-helical structures) are thought to contribute to normal telomere function. Moreover, telomeric chromatin has an important role in telomere maintenance, signaling and regulation of telomere function, but many of the precise structures and molecular mechanisms of human telomeric chromatin are not well understood. However, telomeric regions contain telomeric repeat-containing RNA (TERRA), a long non-coding RNA that is transcribed from telomeric DNA by RNA polymerase II . TERRA has been implicated in telomerase regulation, organization of heterochromatin at telomeres, regulation of gene expression, and in DDR triggered by dysfunctional telomeres . The mammalian cell lines harboring active ALT have higher TERRA levels compared with telomerase-positive cells . However, the exact role of TERRA in activation of the ALT mechanism is not clear .
Although telomerase maintains telomere length in the majority of cancer cells, the ALT mechanism is also employed by 10–15 % of tumors . The ALT pathway utilizes a homologous recombination-based DNA replication mechanism to extend telomere length. The activation of the ALT mechanism is thought to involve loss of chromatin-remodeling factors such as ATRX and DAXX, resulting in reduced compaction of telomeric chromatin, which leads to the production of altered telomeric DNA sequences and activation of a telomere-specific DDR pathway [28, 29], which in turn stimulates homology-directed synthesis of telomeric DNA. Recently, Flynn and colleagues  reported that inhibition of the protein kinase ATR disrupts the ALT mechanism in ALT-positive cancer cells, resulting in cell death. This suggests that ATR inhibitors may be a useful therapeutic intervention for ALT-harboring tumors.
Telomerase: the key telomere length maintenance mechanism
Telomerase is a large ribonucleoprotein complex responsible for progressive synthesis of telomeric DNA repeats (TTAGGG) at the 3′ ends of linear chromosomes, thereby reversing the loss of DNA from each round of replication. Telomerase is a reverse transcriptase that consists of a catalytic protein subunit called telomerase reverse transcriptase (TERT), encoded by the hTERT gene in humans that is positioned at chromosome 5p15.33, and an essential RNA component known as human telomerase RNA (hTR) or human telomerase RNA component (hTERC), encoded by the hTERC gene found on chromosomal region 3q26. hTR acts as a template (carries sequence complementary to one or more copies of telomeric repeats) for the synthesis of telomere DNA, and is also involved in the catalysis, localization and assembly of the telomerase holoenzyme . Recent studies have reported that, in addition to TL maintenance, telomerase is also involved in gene expression regulation, cell proliferation, apoptosis, WNT/β-catenin signaling, NF-kB signaling, MYC-driven oncogenesis, DDR, cell adhesion and migration, and epithelial–mesenchymal transition [32–35]. All these activities of telomerase are thought to contribute significantly to the process of oncogenesis.
TL maintenance by telomerase is a complex multistep process that involves a series of molecular events including hTERT protein transport and trafficking into the nucleus, hTR and hTERT assembly with accessory components in the nucleus, and recruitment to telomeres at the appropriate time during DNA replication. It has been reported that at least hTERT and hTR are essential for the in vitro reverse transcriptase activity of the human telomerase enzyme . However, under in vivo conditions the telomerase holoenzyme also contains four additional proteins—dyskerin, NHP2, NOP10 and GAR1 (localization factor)—associated with the H/ACA class of small nucleolar RNAs that play an important role in the process of pseudouridylation during post-transcriptional modification of RNAs. In addition, a WD-repeat-containing protein 79 called TCAB1 binds to the CAB-box sequence within hTR and directs the telomerase holoenzyme to localize at Cajal bodies bound to the nucleolus . Numerous additional factors such as the chaperones HSP90 and p23, as well as the ATPases pontin and reptin, have also been observed to bind to the two main subunits of telomerase . Many of these factors are thought to be involved in the assembly of a functional telomerase holoenzyme in vivo but the actual mechanisms by which they interact with telomerase remain poorly understood. One working model of human telomerase biogenesis is that dyskerin, pontin and reptin form a scaffold and create an assembly platform for nascent hTR transcripts. Then, the H/ACA motif-binding complex of dyskerin, NHP2 ribonucleoprotein, NOP10 ribonucleoprotein, a nuclear assembly factor ribonucleoprotein (NAF1) and the telomerase ribonucleoprotein (RNP) particle associate. Next, hTR removes NAF1 and attaches GAR1, leading to the formation of a physiologically stable hTR-H/ACA-RNP complex. The hTR 3′-hairpin CAB-box sequence recruits TCAB1, and finally hTERT binds to two structurally independent hTR domains (CR4/CR5), thus generating the catalytically active telomerase RNP . TCAB1, found in Cajal bodies, binds to the CAB box of hTR and guides telomerase to the Cajal bodies, where it remains localized for most of the cell cycle, but the physiological significance of this process is not known.
The role of telomerase in cancer: TERT promoter mutations and telomerase reactivation
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 . 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.
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 . 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 A>C transition, and other less frequent but recurrent mutations in cancer are found on chromosome 5 at the following positions: 1,295,228 C>A; 1,295,248–1,295,243 CC>TT; and 1,295,161 A>C . However, when these mutations (−57 A>C, −124 C>T, −146 C>T) 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  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  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 A>C, −124 C>T, −146 C>T) into isogenic human embryonic stem cells (hESCs) using CRISPR/Cas9 genome editing, and observed that in undifferentiated hESCs the presence of −124 C>T caused a 2- to 3-fold increase in hTERT mRNA while neither the −57 A>C nor −146 C>T 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  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  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.
Frequency spectrum of hTERT promoter mutations across diverse cancer types
Mutation frequency (%)
Renal pelvic carcinoma
Skin basal cell carcinoma
Thyroid cancer (papillary and poorly differentiated carcinomas)
Breast cancer, colorectal cancer, medullary thyroid carcinoma, ovarian cancer, esophageal adenocarcinoma, acute myeloid leukemia, chronic lymphoid leukemia, pancreatic cancer, prostate cancer, testicular carcinoma, uterine cervix cancer
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 . 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  but it remains unclear whether telomerase activity is directly associated with hTERT splicing.
Telomerase as a target for anticancer therapeutics
Completed and ongoing clinical trials of imetelstat in cancer patients
Refractory or relapsed multiple myeloma
Safety and MTD determination
November 2007/July 2011
3 + 3 cohort; dose escalation study
DLT: thrombocytopenia, neutropenia, anemia, aPTT prolongation, fatigue, nausea, anorexia and dizziness.
Recurrent or metastatic breast cancer
Safety, MTD, efficacy in combination with paclitaxel and bevacizumab
July 2008/March 2012
3 + 3 cohort;dose escalation study
DLT: thrombocytopenia, neutropenia.
Solid tumor malignancies
Safety and MTD determination
March 2006/March 2013
3 + 3 cohort;
dose escalation study
DLT: thrombocytopenia, myelosuppression.
NCT 00718601phase I
Safety and MTD determination in combination with bortezomib and dexamethasone
July 2008/October 2011
3 + 3 cohort; dose escalation study
Results not available in public domain.
Refractory chronic lymphoproliferative
Safety, tolerability, dose-limiting toxicities, and MTD
July 2005/March 2013
Sequential dose cohort, open label, escalation trial evaluating one infusion duration of 2 h; weekly intravenous infusion
Results not available in public domain.
Metastatic non-small-cell lung cancer
Safety, DLT, MTD in combination with a standard paclitaxel/carboplatin regimen
July 2007/April 2011
Dose cohorts with a minimum of 3 patients
Patients on imetelstat with short TL showed a trend towards longer median PFS as well as OS. However, imetelstat treatment in patients with long TL had no improvement in median PFS or OS.ADRs: neutropenia and thrombocytopenia.
HER2+ breast cancer
DLT in combination with trastuzumab
January 2011/October 2015
Open label, non-randomized study
Results not available in public domain.
Improved outcome in patients previously treated with imetelstat.
November 2010/December 2015
Imetelstat 2 h intravenous Infusion on day 1 and day 8 of a 28-day cycle
Results not available in public domain.
Safety and efficacy
November 2015/May 2019
Randomized, double blind
patients previously treated with
Safety and efficacy
June 2015/March 2018
Randomized, single-blind, multicenter
Safety and efficacy
December 2010/April 2015
Open label, single group
Eighteen patients, all with positive hematologic response. Positive molecular response in most patients with JAK2 V617F mutation. ADRs: neutropenia, anemia.
October 2012/January 2019
Open label, parallel, active, not recruiting
Complete or partial remission in 21 % patients. Bone marrow fibrosis was reversed in a few patients.
Developmental highlights of oligonucleotide inhibitor imetelstat
Imetelstat has been extensively evaluated for its activity and efficacy against multiple cancer cell lines and in mouse xenograft models in preclinical studies. Imetelstat demonstrated potent inhibitory action against telomerase, causing shortening of telomeres in a large spectrum of cancer cell lines derived from tumors of the bladder, breast, lung, liver, prostrate and pancreas [62–64]. In vivo preclinical studies in mouse models of human tumor xenografts showed that the compound was well tolerated and highly efficacious in inducing telomerase inhibition, leading to reduced tumor growth, prevention of metastasis, and sensitization of tumors to standard chemotherapy . Imetelstat was also found to efficiently prevent glioblastoma tumor growth in a xenograft model by crossing the blood–brain barrier, probably owing to its highly lipophilic nature . Additionally, simultaneous suppression of homologous recombination and telomerase activity in a mouse model of Barrett’s adenocarcinoma with the combination of nilotinib (tyrosine kinase inhibitor) and imetelstat was reported to be more effective compared to either compound alone .
Imetelstat has been undergoing clinical trials for several years, and while some trials have already been completed, some were discontinued (breast and lung cancer, lymphoproliferative disorders and polycythemia vera) because the US FDA put these on hold due to hematological toxicity, but a few are still continuing (Table 2). Recent clinical development of imetelstat includes two studies, one with patients with myelofibrosis, referred to as the Initial MF Study or the IMbark™ study, and one with patients with myelodysplastic syndrome, called the MDS or IMerge™ study (Table 2). Currently, these studies are recruiting targeted patients at various centers in the USA, Europe and Asia.
Completed and ongoing clinical trials of anti-telomerase vaccines: current status
NCT00510133/GRNVAC1 phase II
Acute myelogenous leukemia
July 2007/August 2014
GRNVAC1 was found to be safe and well tolerated.
Positive immune responses in 55 % of patients.
Asterias Biotherapeutics (http://asteriasbiotherapeutics.com/pipeline/ast-vac1/)
NCT01579188/GV1001 phase III
Non-small-cell lung cancer
May 2012/May 2016
NCT00425360/GV1001 phase III
Metastatic pancreatic cancer
Efficacy in combination with chemotherapy
Adding GV1001 vaccination to chemotherapy did not improve overall survival.
NCT01935154/Vx001 phase II
Non-small-cell lung cancer
Vaxon Biotech (http://www.clinicaltrials.gov)
Clinical trial results have demonstrated that GRNVAC1, Vx001 and GV1001 are promising telomerase-targeting vaccines capable of stimulating CD4+ and CD8+ responses in telomerase-positive tumors, showing minimal effects on normal cells and no autoimmunity. Large multicenter studies are required to determine long-term toxicities in patients. However, at present, it is not certain if any of these vaccine candidates will progress to registration studies to get approval for clinical application.
Exploiting telomerase activity to selectively kill cancer cells
A major challenge for anti-telomerase-directed therapy is the long lag period required to observe enough TL attrition to induce cell death. Telomere shortening requires a series of cell division cycles to become apparent, and treatment may have to be given continuously for months to induce therapeutically relevant tumor reduction effects. During this treatment period, most tumor cells will continue to grow, which may require the use of other treatment modalities for successful clinical outcomes. Importantly, with direct telomerase inhibitors, if the patient has hematological or any other toxicities (for example, one concern with imetelstat is the development of hematological toxicities requiring drug holidays), then going off treatment for a few weeks would reverse some of the benefits already obtained—the decision about treatment termination or stopping treatment for a short duration may depend upon the risk–benefit ratio in terms of efficacy and manifested toxicity.
Therefore, novel fast-acting therapeutic agents that can inhibit telomerase activity would be highly desirable. One such strategy is not to target telomerase directly but to introduce a modified nucleoside into cells so that telomerase would preferentially incorporate it into telomeric DNA. An altered nucleotide incorporated into telomeres would not bind to shelterin proteins efficiently and should lead to telomere dysfunction and rapid cell death. Mender and colleagues  have recently demonstrated that, in telomerase-positive cells, 6-thio-2′-deoxyguanosine (6-thio-dG), a nucleoside analogue of 6-thioguanine (an approved drug), is recognized by telomerase and incorporated into telomeres. This results in altered telomere organization and activation of telomere-associated DNA damage signals called telomere dysfunction-induced foci, and rapid cell death. The nucleoside analogue 6-thio-dG has been evaluated against cell lines and in vivo. Treatment with 6-thio-dG resulted in rapid cell death, whereas normal telomerase-silent (telomerase-negative) human fibroblasts and normal human colonic epithelial cells were largely unaffected. In in-vivo studies, 6-thio-dG treatment caused significant reduction in tumor growth rates and was superior to 6-thioguanine treatment. Additionally, mice treated with 6-thio-dG at effective doses for a month did not show any hematological, hepatic or renal side effects. Thus, a telomerase-mediated telomere-disrupting approach may provide a safe and efficacious option for the treatment of cancer .
Conclusions and future perspectives
Telomere maintenance has been extensively studied, and our understanding of the role of telomerase and ALT in cancer has improved remarkably in recent years. It is becoming clear how cancer cells regulate different molecular events involved in telomere maintenance to expand their proliferative capacity. Recent insights into the control of telomerase activity at telomeres, through telomerase–shelterin interactions, by regulating telomerase recruitment or productive substrate engagement at the enzyme active site, have highlighted opportunities for the development of novel diagnostic tools and effective anticancer agents. Furthermore, recent knowledge gained about the mechanisms underlying the non-canonical functions of telomerase has significantly improved our understanding of the role of telomerase in cancer progression. However, further research efforts are needed to obtain an in-depth understanding of hTERT activation in the initial stages of carcinogenesis, and the various genetic and epigenetic mechanisms involved in its regulation. While the recurrent hTERT promoter mutations are highly frequent in many cancers and play a pivotal role in the induction of telomerase reactivation in cancer cells, much remains to be learned about the sufficiency or necessity of hTERT promoter mutations in cancer initiation and progression. It is still not established whether telomerase expression has any oncogenic characteristics or is simply required for the maintenance of sustained tumor growth (that is, whether it is permissive). Moreover, there are many other unresolved questions regarding telomeres and telomerase function that deserve further investigation (Box 2). Although target-based compounds have greatly benefited patients who have tumors with specific oncogenic mutations, such as EGFR mutation, HER2 amplification, or mutations resulting in ALK expression or KIT expression, the vast majority of common tumors remain less responsive to these target-based drugs. Therefore, novel targeted interventions are required and telomerase inhibition remains a promising strategy for cancer treatment. Recent advances in telomere biology are beginning to unravel potential new telomerase targets (Box 3) for the design of novel molecules targeting the activity of this key enzyme.
Clinical trials with telomerase inhibitors have established telomerase as a viable target, but the time lag between drug administration and clinical response is long. Continued treatment is required for successful clinical outcome, which may lead to severe toxicity in patients. Therefore, a major challenge is to develop a telomerase inhibitor that rapidly kills telomerase-positive tumor cells while sparing normal telomerase-carrying cells.
Box 1. Major historical research milestones in telomere and telomerase biology
Telomeres: Discovered by American geneticist Hermann J. Muller working on Drosophila melanogaster in 1938. He observed that the ends of irradiated chromosomes were resistant to mutagenic X-rays and did not undergo deletions or inversions due to the presence of cap-like structures that he called “telomeres” .
A crucial role for telomeres in chromosomal integrity: Elucidated by Barbara McClintock in 1941. She described that rupture of the chromosomes resulted in the formation of dicentric chromosomes due to fusion of their ends, and demonstrated that damaged ends of the chromosomes could be restored .
Cellular immortality in culture: Alex Carrel, recipient of the 1912 Nobel Prize in physiology, working at the Rockefeller Institute demonstrated that chick heart tissue culture cells can be maintained in long-term cultures by replenishing with fresh culture medium. He hypothesized that the lifespan of cultured tissues could be extended indefinitely and that the tissues should intrinsically be able to maintain permanent life in vitro under ideal culture conditions. Later, Carrel's associates showed a continuous culture of chick heart cells from 1912 to 1946, and the idea of cell immortality as an intrinsic property was widely accepted by the scientific community. However, it was then discovered that the use of chick embryo extract to culture these cells was actually re-seeding fetal cells and thus the immortality reported by the Carrel laboratory has been largely discounted.
The concept of normal cell immortality challenged: Leonard Hayflick, in 1961 at the Wistar Institute, demonstrated that normal human fetal cells in culture could divide only 40 to 60 times, and after that they underwent aging at the cellular level (then called phase III and now replicative senescence) .
End replication problem: In 1971, James Watson, the co-discoverer of the DNA double helix, suggested that there was an “end replication problem” due to the mechanism governing semi-conservative DNA replication. Watson predicted, based on the asymmetry of how linear duplex DNA is copied, that each cell division would result in the extreme termini of chromosomes being lost. This would be incompatible with long-term maintenance of the genome owing to progressive chromosome shortening with each replication cycle, eventually reaching a critical point leading to cell senescence or death. In addition, he postulated the existence of a protective mechanism to prevent chromosomal shortening .
Hypothesis about cellular aging: Also in 1971, Alexsey Olovnikov, a Russian scientist, hypothesized that there could be a problem with the ends of chromosomes. He postulated that progressive shortening of the telomere would eventually run into essential genes, leading to cellular aging and perhaps contributing to human aging .
Tetrahymena thermophila telomeres tandem repeat sequences: In 1978, Elizabeth Blackburn and Joseph Gall carried out sequencing experiments for the DNA of the Tetrahymena thermophila minichromosome and reported that telomeres contained 20–70 tandem copies of a simple hexanucleotide with the sequence 5′-CCCCAA-3′ on one strand and 5′-TTGGGG-3′ on the complementary strand .
Telomerase: Blackburn and Carol Greider, at Berkeley in 1985, identified an enzymatic activity capable of extending telomeric sequences. The enzyme was named terminal telomere transferase but is now known as telomerase . Along with Jack W. Szostak they received the 2009 Nobel Prize for their discovery that telomeres are protected from progressive shortening by the enzyme telomerase.
Human telomerase: Gregg Morin, in 1989 at Yale University, was the first to report telomerase activity in crude HeLa cell extracts. He also demonstrated that human telomeres consisted of the repeated sequence TTAGGG . In 1994, Jerry Shay and colleagues showed telomerase activity in ~90 % of human cancers and cell lines , and in 1998 the same team demonstrated that introduction of hTERT (the catalytic protein reverse transcriptase component of telomerase) into normal human cells was sufficient to immortalize cells .
Box 2. Key outstanding research issues in cancer telomere and telomerase biology
Determining mechanism(s) of escape from crisis: Cells in crisis undergo tremendous genomic instability due to bridge–fusion–breakage cycles. However, many molecular details remain unclear. What are the molecular features of cells that escape crisis? Do they have hTERT promoter mutations? Why do incipient cancer cells during crisis acquire stem cell-like properties?
hTERT promoter mutations: Some believe that hTERT promoter mutations drive carcinogenesis, while others believe that promoter mutations are only permissive for tumor growth maintenance. How these widespread hTERT promoter mutations regulate hTERT expression during cellular transformation is not fully understood.
Telomerase holoenzyme assembly: Although there has recently been progress on determining the yeast and ciliate telomerase structure, the processes of assembly and function of telomerase in human cancer cells remain poorly understood.
Recruitment of human telomerase to telomeres in cancer cells: The recruitment of telomerase to telomeres is highly regulated and occurs only after the replication fork remodels protected DNA 3′ ends during the S phase of the cell cycle. It involves protein–protein interactions between the shelterin complex components TPP1 and POT1 and the DAT domain of hTERT. TPP1 contains an N-terminal OB-fold domain containing a patch of amino acids termed the Tel patch that directly interacts with the telomerase DAT domain . However, the signaling pathways that regulate telomerase recruitment in human cancer cells are not clearly understood. The telomerase recruitment process is likely regulated by as yet unknown signaling pathways.
Shelterin protein complex: Emerging evidence suggests a crucial role for shelterin components in cancer progression, but how these components are regulated during different stages of cancer development is not well understood.
Alternative lengthening of telomeres: Recently, knowledge about ALT has increased significantly. The chromatin remodeling factor ATRX acts as a suppressor of ALT in normal cells and mutations in ATRX and DAXX contribute to activation of ALT [28, 91]. However, knockdown of ATRX is not sufficient to trigger the ALT pathway in telomerase-positive cell lines or to directly activate ALT in normal somatic cells, implying the existence of other necessary contributing factors involved in activation of ALT in cancer cells [92, 93]. Thus, many key questions remain unanswered, such as why ALT is more frequent in certain cancer subtypes? How does ATRX/DAXX repress ALT and what is the molecular basis of its activation in cancer cells with wild-type ATRX/DAXX? What is the function of variant DNA repeats in ALT? How does RAD51 interact with the 5′ overhang of ALT telomeric DNA to facilitate its invasion into homologous DNA, and how are shelterin proteins organized in ALT telomeres? Answers to these questions may facilitate development of mechanism-based inhibitors for ALT-positive cancers.
Box 3. Telomere biology-based potential novel targets for the development of anticancer agents
Inhibition of the Tel patch to block telomerase recruitment to telomeres: The “Tel patch”, a specific amino acid sequence in the OB-fold domain of shelterin complex protein TPP1, is involved in telomerase binding, recruitment, enzyme processivity and telomere elongation. Thus, inhibition of telomerase recruitment may result in cell death .
Inhibition of telomerase non-canonical function mediators: In addition to telomere maintenance, telomerase may also be involved in other important activities such as regulating gene expression, mitochondrial activity, cell proliferation, apoptosis, epithelial–mesenchymal transitions and DNA damage repair. These non-canonical putative telomerase functions may be mediated through a network of “feed forward signaling loops” . Interventions targeting the molecules involved in non-telomeric functions of telomerase may be a rational approach for cancer treatment.
Inhibition of TRF1 shelterin protein: TRF1 is overexpressed in many cancer types and plays a central role in controlling replication of telomeric DNA. The genetic abrogation of TRF1 leads to a marked reduction in lung carcinoma tumor growth in the K-Ras G12V lung cancer mouse model due to acute telomere uncapping independent of telomere length . However, it is not clear what effects targeting shelterin proteins would have on normal cells.
Inhibition of ATM kinase: The ATM kinase plays a crucial role in the cellular response to telomere dysfunction-mediated DNA damage and subsequent repair pathways. ATM has recently been shown to be required for the addition of telomeric DNA repeats to telomeres and telomere elongation by telomerase in human cells. Blocking ATM inhibits telomere elongation and inhibition of PARP1, which activates ATM and increases telomere elongation . ATM may regulate telomerase access to telomeres through interaction with TRF1 .
Inhibition of alternative splicing of mRNA of hTERT: The human TERT gene produces numerous alternatively spliced variants with a few isoforms capable of producing full-length catalytically active telomerase. A fuller understanding of the process of alternative splicing may lead to the development of molecules to inhibit the generation of full-length telomerase and be a new approach to telomerase therapy in cancer .
Inhibition of TERT RNA-binding domain (tTRBD): TERT protein binds to the template boundary element of TR (TERC), crucial for the recognition of the precise telomere sequence to be reverse transcribed by TERT . This is a potential intervention target, but this discovery needs to be established in human cells.
ALT, alternative lengthening of telomeres; bp, base pairs; DAT, dissociates the activities of telomerase; DDR, DNA damage response; FDA, Food and Drug Administration; GM-CSF, granulocyte–monocyte colony-stimulating factor; hESC, human embryonic stem cell; kb, kilobases; OB, oligonucleotide/oligosaccharide-binding; TERRA, telomeric repeat-containing RNA; T-loop, telomeric loop; TL, telomere length; TPE, telomere position effect; TSS, transcription start site
The laboratory of JWS is supported by the National Cancer Institute (Lung SPORE P50CA70907), RO1AG001228 from the National Institutes of Health, and a distinguished chair from the Southland Financial Foundation in Geriatrics Research. This work was performed in laboratories constructed with support from National Institutes of Health grant C06 RR30414.
MAJ wrote manuscript and SAA drew the figures; MHA provided inputs into manuscript writing and JWS edited and finalized the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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