Strategies Targeting Telomerase Inhibition
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- Chen, H., Li, Y. & Tollefsbol, T.O. Mol Biotechnol (2009) 41: 194. doi:10.1007/s12033-008-9117-9
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Telomerase plays a pivotal role in cellular immortality and tumorigenesis. Its activity is normally not detectable in most somatic cells while it is reactivated in the vast majority of cancer cells. Therefore, inhibition of telomerase has been viewed as a promising anticancer approach due to its specificity for cancer cells. Studies so far have shown that telomerase inhibition can inhibit the proliferation of cancer cells or cause apoptosis while it has no effect on most normal cells. Strategies currently being applied to induce telomerase inhibition target virtually all of the major components of the ribonucleoprotein holoenzyme and related cell signal pathways that regulate its activity. These strategies include inhibition of telomerase through targeting at the telomerase reverse transcriptase (TERT) catalytic subunit, the telomerase RNA (TR) component, and associated proteins. Other strategies have been developed to target the proteins associated with telomerase at the telomeric ends of chromosomes such as tankyrase. The specific mechanisms that mediate those inhibition effects include small molecules, antisense RNA, and ribozymes. Although the beneficial evidence of telomerase inhibition is obvious, limitations of strategies remain to be resolved to increase the feasibility of clinical application. This analysis will summarize recent developments of strategies in telomerase inhibition.
KeywordsTelomeraseInhibitionTechniqueMethodTelomerase reverse transcriptase (TERT)Telomerase RNA (TR)
In human cells, a structure referred to as the telomere has been identified to cap the terminal regions of chromosomes which can protect the ends of DNA strands from degradation and fusion. A telomere is composed of telomeric DNA sequence, which is characterized by hexameric 5′-TTAGGG-3′ tandem repeats in humans, a G-rich overhang in its 3′ end, and a multiprotein complex termed telosome or shelterin which can bind to telomeric DNA sequences. Proteins found in this structure include the POT1/TPP1 heterodimer (which can bind to the G-strand overhang), TRF1 and TRF2 (which can bind to double-stranded repeats through their Myb-domain), RAP1 (repressor-activator protein 1) and TIN2 (TRF1-interacting nuclear factor 2, which can bind to TRF1 and TRF2). TANK1 and TANK2 poly(ADP)-ribosylases (also known as tankyrases) have also been indicated to interact with TRF1. Among those proteins, TRF1 and TRF1-associated proteins can negatively regulate telomere length by controlling the access of telomerase to telomeres, while TRF2 and POT1 can protect telomeres from end-to-end chromosome fusions by interacting with DNA-damage signaling and repair factors.
Telomeric DNA sequence will lose up to 150 bp after each cell division due to the end replication problem of linear chromosomes. However, some cells have evolved the telomerase enzyme to overcome this problem. This enzyme can add hexameric 5′-TTAGGG-3′ nucleotide repeats to the 3′ end of telomeric DNA strands to compensate for loss of sequence. Telomerase is a ribonucleoprotein which includes three major components: the telomerase reverse transcriptase (TERT) protein subunit that catalyzes the enzymatic reaction of DNA synthesis, the telomerase RNA (TR) component that serves as a template for TERT, and a protein termed dyskerin which binds to hTR. Although other proteins have been reported to associate with the holoenzyme (such as pontin and reptin which can help the assembly of telomerase), these three components are essential for telomerase activity and telomere lengthening [1–3].
Importance of Telomerase in Cancer and Aging
Telomerase exhibits a high activity in germline and embryonic stem cells which can maintain their immortality, while its activity is low or absent in other stem cells and somatic cells . For those somatic cells and stem cells, the length of their telomeres gradually becomes shorter and shorter with each cellular division. When some of the telomeres reach a certain length, DNA damage signals will be produced by those telomeres and the cells will then undergo growth arrest and cease to divide. This process is referred to as cell replicative senescence . Some cells, such as those with mutations of the p53 and pRB tumor suppressor genes, will continue to proliferate until their telomeres reach a critical length. Eventually, end-to-end chromosome fusions and apoptosis of some of the cells may occur along with genomic instability in a process referred to as cell crisis. Thus, the length of telomeres in normal stem cells and somatic cells has been considered to be a molecular clock important in the aging process . Very few cells (~1 in 107) may escape the crisis by expressing telomerase to maintain telomeres, and this will increase the likelihood of tumorigenesis. Most cancer cells express a high level of telomerase which can satisfy their need for unlimited proliferative capacity while a few type of cancer cells employ alternate lengthening (ALT) of telomeres by recombination to maintain their immortality .
Due to the discrepancy of telomerase activity between cancer cells and normal somatic cells, analysis of telomerase activity has been considered as a potential diagnostic marker of cancer. The increase in telomerase in cancer cells generally occurs very early during tumorigenesis and sensitive techniques such as the Telomere Repeat Amplification Protocol or TRAP assay can detect trace levels of this enzyme which has obvious diagnostic potential in cancer .
Potential of Telomerase Inhibition in Cancer Therapy
Telomerase has been regarded as one of the most promising targets in cancer treatment. Many factors contribute to this such as its high expression level in cancer cells. More importantly, however, cancer cells generally have rather shorter telomeres than normal cells and a more rapid cellular proliferation rate, which means telomerase inhibition strategies would have a greater and more significant impact on the survival of cancer cells while having minimal effects on normal somatic cells. Numerous studies so far have indicated that telomerase inhibition leads to cellular proliferation inhibition or apoptosis of cancer cells [7, 8]. Anticancer approaches to telomerase inhibition are varied and methods ranging from RNA interference of the TERT catalytic subunit to inhibition of the proteins associated with telomerase at the telomeres have proven to have efficacy in cancer therapy. Since the telomeres of the rare normal cells that express telomerase are longer than in most cancer cells and the level of telomerase activity is generally lower in normal telomerase-positive cells as compared to cancer cells, the risks associated with possible telomere shortening in normal cells due to off-target telomere shortening are thought to be relatively minimal. Therefore, the efficacy of telomerase inhibition in inducing loss of viability or apoptosis of cancer cells combined with the relative low risk of inhibition of telomerase to normal cells have moved telomerase research to the forefront of anticancer approaches.
Strategies of Telomerase Inhibition
Methods for Inhibiting the TERT Catalytic Subunit of Telomerase
The hTERT catalytic subunit is the key catalytic component that regulates activity of telomerase. Its expression is tightly controlled which involves a complex transcription network. It cannot usually be detected in normal somatic cells, while it overexpresses in most cancer cells, and its expression level correlates very well with the activity of telomerase. Thus, telomerase inhibition targeted at hTERT has been considered as an ideal direction for cancer treatment.
Nucleoside analogs can mediate telomerase inhibition by blocking the incorporation of dNTPs in telomerase’s reverse transcription (Fig. 1). For instance, AZT (3′-azido-2′,3′-dideoxythymine) can be effective in targeting the active site of TERT but this approach lacks the desired selectivity of many other approaches. Other examples include derivates of AZGTP (7-deaza-2′-deoxygunosine 5′-triphosphate) which may have a stronger inhibitory potential . Small non-nucleosidic synthetic compounds can be quite effective in inhibiting the catalytic activity of the TERT protein component . They can bind to the active site of telomerase and inhibit its function. One compound that has shown promise in this regard is BIBR1532 that inhibits the in vitro processivity of telomerase. The inhibition of TERT activity with BIBR1532 occurs in a dose-dependent manner and higher concentrations of this telomerase inhibitor can be cytotoxic to cancer cells of the hematopoietic system such as HL-60 cells while having little effect on normal cells.
Anticancer immuno-therapeutic approaches have also focused on TERT . These methods involve the use of peptides derived from TERT. The peptides are presented by MHC class I alleles to T lymphocytes. The result is that CD8+ cytotoxic T lymphocytes specific for the TERT-derived antigenic peptides result in effective lysis of cancer cells that express TERT. These immunotherapy approaches directed against the TERT antigen can be carried out in the absence of toxicity and are showing great promise in anticancer research (Fig. 1).
It can be a challenge to identify small molecule compounds that affect the expression of TERT and the use of cell-based reporter systems for the analysis of TERT expression have been developed to enhance these endeavors . For example, the hTERT promoter can be linked to two different reporter genes encoding green fluorescent protein (GFP) and secreted alkaline phosphatase (SEAP). The transfection of these reporter constructs result in stable clones that allow analysis of hTERT expression .
Inhibition of TERT is the goal of many anticancer approaches. Many of the most promising and effective methods for actively knocking down the TERT transcript have been developed, which include ablating its catalytic activity, directing the immune system to lyse telomerase-positive cancer cells, or using expression constructs to identify small molecule components that affect the expression of telomerase.
Telomerase RNA Inhibition as an Anticancer Approach
In addition to antisense sequences directed to the RNA component of telomerase, hammerhead ribozymes and RNAi can be used to inhibit the RNA of this ribonucleoprotein . Both of these methods lead to degradation of the RNA component of telomerase. The effect is rapid cell growth inhibition both in vitro and in vivo independent of telomere length of the target cancer cell. The advantage of this technique is that it greatly reduces the lag period that is often encountered in approaches that are dependent upon the shortening of telomeres to result in cancer cell growth inhibition. Thus, methods to inhibit the RNA component of telomerase by using antisense oligonucleotides, hammerhead ribozymes or RNAi also show great promise in anti-telomerase approaches to cancer therapy (Fig. 2).
Targeting Proteins Associated with Telomerase Activity
More indirect approaches to telomerase inhibition have been developed that do not directly inhibit the TERT or TR components of telomerase, but rather, target proteins that are associated with telomerase activity. An example is tankyrase I, which is a telomeric poly(ADP-ribose) polymerase or PARP that can affect telomerase inhibition in cancer cells (Fig. 2) .
Signaling pathways such as those carried out by MAP kinase can result in stimulation of the hTERT gene. For example, Ets and AP-1 may play a role in MAP kinase signaling to the hTERT gene and inhibition of this pathway could be a novel approach to reducing TERT expression and telomerase activity (Fig. 2) . It is apparent that many additional techniques will be developed to impact the proteins or pathways associated with telomerase activity in cancer cells.
Screening of Telomerase Inhibitors
Finding novel inhibitors of telomerase is an important aspect of increasing the tools that we have for anti-telomerase approaches to cancer therapeutics. A strategy for determining the therapeutic potential of telomerase inhibitors using a screening system in one cell type has been proposed . For example, four completely different compounds, BRACO19, BIBR1532, 2′-O-methyl RNA, and peptide nucleic acids, were chosen for detailing the methods for screening of telomerase inhibitors. Additionally, TRAP assays or assessment of telomere lengths using Southern blot telomere restriction fragment analysis can be applied to determine the effectiveness of telomerase inhibition.
Telomerase Inhibition Combined with Other Chemotherapeutic Agents
Although a number of merits associated with telomerase-targeted treatment have been mentioned, there are concerns in using this strategy. For instance, the lag phase between the time when the telomerase of cancer cells has been inhibited and the shortening of the length of their telomeres may allow cancer cells to develop other mechanisms such as ALT to overcome telomere shortening caused by telomerase inhibition. Another concern is that long-term telomerase-targeted treatment may affect the function of those normal telomerase-positive cells. A solution to these problems is to combine telomerase inhibition with other chemotherapeutic reagents to enhance anticancer effects . For instance, there is an indication that imatinib, a selective inhibitor of the BCR-ABL tyrosine kinase, can enhance the telomerase inhibition carried out using a dominant-negative form of human telomerase (DN-hTERT). In a completely different approach, telomestatin, a natural product and telomerase inhibitor isolated from Streptomyces anulatus, was combined with imatinib, daunorubicin, mitoxantrone, or vincristine and was shown to enhance the sensitivity of these chemotherapeutic agents . Therefore, approaches to telomerase inhibition may also be merged with completely different anticancer approaches such as chemotherapeutic agents to render the most effective modes of cancer therapy.
Telomerase inhibition-based anticancer approaches
Mechanism and consequence
Relative references [numbers]
RNAi-based TERT knockdown
TERT mRNA degradation and gene silencing
Small-interfering RNAs (siRNA)
Oligonucleotide antagonist (biochemical active site inhibitor)
TERT enzymatic inhibition
AZT (3′-azido-2′,3′-dideoxythymidine), BIBR1532
Activate immune system to kill telomerase-positive tumor cells that express TERT
CD8+-induced TERT antigen-expressed cancer cell lysis
hTR degradation and telomerase transcriptase template dysfunction
2′,5′-oligoadenylate (2-5A) antisense system
Interfere telomerase-associated proteins and relative signal pathways
Influence telomerase activity and/or TERT expression
Tankyrase I MAP signal pathway
We apologize to those scientists whose work has not been cited in this article due to limited space. This work was supported by grants from the National Cancer Institute (R01 CA129415) and the Susan G. Komen for the Cure.