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Telomeres and Telomerase in cancer

The Science that could lead to a medical breakthrough?

Masters Paper

By: Yasamin Badiei

April 21st, 2010

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Abstract The telomeres, which are special protective structures at the end of eukaryotic chromosomes that help stabilize our genetic information and allow for our cells to divide, may hold some secrets to how we develop cancer. This topic has gained extensive coverage in late cancer research due to mounting evidence that loss of telomere function, either by altering telomere-binding proteins or by critical shortening of telomeric sequences, is associated with loss of cell viability through induction of apoptosis and the activation of tumor suppressor mechanisms. Further identification of an extraordinary enzyme named telomerase that acts on telomeres and is thought to be required for the maintenance of many human cancers has sparked much speculation that drugs able to inhibit the enzyme might combat a wide array of malignancies. In this review, recent findings in telomere and telomerase biology, their role in tumorigenesis and insights that could lead to drug development shall be the focus of this paper. Table of Contents Abstract………………………………………………….. ..............................................................................................2 I. Introduction....................................................................................................................................................................3 II.Telomere Biology and link to cancer……………………………….............................................................................3 A Telomeres and the protection of chromosome ends.……...……….………………………………………………….4 B ‘End-replication’ problem and the discovery of telomerase…..………………………………………………………5 C Replicative senescence: A Tumor suppressor mechanism?...........................................................................................8 III Telomerase Biology and link to cancer……… ..........................................................................................................10 A Telomerase RNA (TR) …………………………………………….…………... .......................................................11 B Telomerase Reverse Transcriptase (TERT)…………………………………………………………………………..13 C The Implication of Human Telomerase Reverse Transcriptase (hTERT) in cancer……………………………….....16 IV Molecular-based approaches for Cancer Therapeutics..………………………………………………………..........17 A Telomerase inhibition: targeting telomerase enzyme components…………………………………………………...18 B Telomerase inhibition: Expression of dominant-negative hTR and hTERT mutants………………………………...21 C Potential drawbacks of telomerase inhibitors…………………………………………………………………………21 D Telomere inhibition: an alternative therapeutic intervention?.......................................................................................22 V. Conclusion ………………….......................................................................................................................................22 References.........................................................................................................................................................................24

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I Introduction

Nature does not favor DNA with free ends. Hence viruses and bacteria have avoided this by circularizing their DNA, however organisms which persisted with linear DNA had to develop special sequences that preserved their chromosome ends. These structures, the telomeres, have their roots in experiments carried out in the 1930s by the two geneticists Barbara McClintock and Hermann J. Muller who working separately and with different organisms realized that chromosomes bore a special component at their ends that provided stability which they named "telomere" from the Greek for "end" (telos) and "part" (meros) (Blackburn et al. 2006). It was not until in 1978, however, that the precise makeup of the telomere was determined by Elizabeth H. Blackburn and Joseph G. Gall who found that the telomeres in Tetrahymena thermophilia, a ciliated protozoan, whose chromosome ends contained an extremely short, simple sequence of nucleotides TTGGGG — repeated over and over. Telomeric DNAs from a variety of eukaryotes have now been characterized and are shown to mostly have a high G content in the strand that runs in the 5’→3’ direction. In humans and other vertebrates the double stranded repeat array is TTAGGG which gives rise to 4-15 kb of repetitive non-coding DNA in humans and up to 50 kb in mice (Tropp et al. 2008).

From recharge biomedical clinic: www.rechargebiomedical.com/images/telomerase

Figure 1: Telomeres shown at end of chromosome II Telomere biology and link to cancer The structure and function of telomeres has been explored to some detail in the current literature. In terms of their structure telomeres are nucleoprotein complexes that consist of many double-stranded TTAGGG repeats, a 3’ single-stranded (ss) overhang, as well as many associated telomere-binding proteins. As chromosome “caps”, telomeres are thought to have three critical functions: (A) to protect chromosome ends from being recognized as DNA damage, (B) to serve as a buffer zone to protect coding genes against the “end-replication” problem, and (C) to serve as a “mitotic clock” for dividing cells which shall all be explored below in further detail.

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A Telomeres and the protection of chromosome ends As nucleoprotein structures, telomeres protect the ends of eukaryotic chromosomes through specialized three-dimensional DNA-protein structures that prevent the telomeres from being recognized as DNA double-strand breaks and prevent the activation of cellular DNA damage response pathways (DDR). If the latter were to occur cells would permanently arrest in the cell cycle, and attempts to “repair” the chromosome ends would have devastating consequences for genome integrity. One structure believed to contribute to the telomeres end protection function is the t-loop, in which the single-strand G-rich 3’-overhang at the end of the telomere has been proposed to invade the double-stranded (ds) telomeric DNA, base pairing with the C-strand and displacing the G-strand so that it becomes inserted as a “D-loop” into the ds telomeric sequences (Figure 2). The name t-loop hence stems from the over-all structure formed since the strand invasion takes place at a distance from the physical end of the telomeres (Griffith et al 1999). The key feature of the t-loop is that the end of the telomere is tucked in away from DNA damage checkpoints.

Figure 2: Formation of t-loop structure and formation of the shelterin complex which protects the telomere. TRF1 and TRF2 are shown binding the double-stranded telomere whereas POT1 and TPP1 associate with the single stranded overhang (De Lange et al. 2009).

DDR are activated by double-strand breaks through two-independent signaling pathways: the ATM (ataxia telangiectasia mutated) kinase pathway activated directly by DNA ends, and the ATR (ataxia telangiectasia and Rad3 related) kinase pathway activated by the single-stranded DNA formed when the 5’end of a double-strand becomes shortened. Mammalian telomeres are composed of a six-subunit protein complex called shelterin that are comprised of the three subunits TRF1, TRF2, and POT1 which specifically recognize the TTAGGG repeats of the telomeres; and are interconnected by the three additional shelterin proteins, TIN2, TPP1, and Rap1. Double-stranded telomere

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sequences are bound directly by the two sequence-specific DNA binding proteins: telomeric repeat binding factors 1 (TRF1) and 2 (TRF2) that bind DNA through their C-terminus domains. TRF1 and TRF2 differ in their N-termini, which are rich in either acidic residues (TRF1) or basic residues (TRF2) and are thought to play different roles such that TRF1 serves as a negative modulator of telomere function (van Steensel et al. 1997) and helps facilitate DNA replication (Sfeir et al. 2009); whereas the repression of the ATM kinase pathway at telomeres is thought to occur through TRF2 which was has been shown in vivo to participate in t-loop formation and is hence required to cap and protect the chromosomes (Stansel et al. 2001). To illustrate, the expression of a dominant-negative allele of TRF2 in cultured human cells led to the loss of the protective capped structure of telomeres and was found to rapidly trigger responses typical of those induced by ds breaks. Such responses include the induction of apoptosis through the ATM/p53-dependent DNA damage checkpoint pathway (Karlseder et al. 1999), the loss of the 3' ss overhang and induction of end-to-end chromosome fusions (van Steensel et al. 1998). TRF1 and TRF2 each interact with a common factor TRF1-interacting nuclear factor (TIN2) that acts as a central component in the shelterin complex and which further interacts with TPP1 as illustrated in Figure 2. Both TPP1 and POT1 are oligosaccharide binding (OB)-fold containing proteins that serve a role in protecting the single-stranded overhang of the telomere by binding to it (Artandi et al. 2010). For instance, the second threat of the ATR signaling pathway is handled by POT1 as the deletion of the two POT1 genes in mice results in a telomere damage response induced by DNA damage at telomeres and the phosphorylation of the ATR target (Hockemeyer et al. 2006). In addition by interacting with the single-stranded telomeric DNA, the shelterin subunits POT1 and TPP1 are thought to play a role in determining the length of the telomere and its enzymatic regulation as shall be explored further along the paper.

Shelterin-related complexes are also found at telomeres in other eukaryotes; for

instance, POT1-like proteins are present in nearly all eukaryotes, a TRF1/2 like protein is found in fission yeast, and Rap1 is present in fungi. In contrast, the shelterin subunits TIN2 and TPP1 have so far only been found in vertebrates. Thus, shelterin appears to be in general built up from a duplex telomeric DNA-binding protein, a single-stranded DNA (ssDNA)-binding protein, and Rap1 (Figure 2). The one exception to this rule, however, may be Saccharomyces cerevisiae which lacks a TRF-like protein and uses instead a highly diverged Rap1 ortholog that binds double-stranded telomeric DNA directly unlike in mammals in which it is recruited to telomeres through its interaction with TRF1 (Artandi et al. 2010). B ‘End-replication problem’ and the discovery of telomerase During each round of cell division, 50-200 base pairs are lost from the ends of linear human chromosomes. This end replication problem occurs because DNA replication machinery is unable to replicate completely the 3’ ends of chromosomal DNA during each cell cycle which leads to the loss of the 5’ end of one daughter DNA strand since DNA polymerases can only synthesize DNA in the 5' 3' direction. The leading strand can, in principle, be continuously synthesized while the lagging strand can be synthesized only by using short, RNA primers (8-12 bp). After extension, the RNA primers are removed and the gaps are filled in by DNA polymerase. Removal of the 5'-most RNA primer generates an 8–12-base gap however, and failure to fill in this gap

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leads to a small loss of DNA in each round of DNA replication (Figure 3). The presence of the non-coding telomeric repeats hence provides a buffer that prevents the loss of genetic information during each cycle of replication. Unless something is done to restore the lost sequence, this processing step will cause the telomere to become shorter every time the cell divides.

Figure 3: End-replication problem illustration

To counter this problem, a special type of DNA polymerase known as telomerase was first classified in Tetrahymena by Greider and Blackburn, and who for this work were awarded the 2009 Nobel Prize in medicine (Greider et al. 1985). Telomerase can add telomeric repeats to the end of the chromosome to elongate telomeres in cells that possess its activity (Figure 4). The enzyme is a ribonucleoprotein that consists of a

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catalytic protein component and an essential RNA component. It acts as reverse transcriptase, or a RNA-dependent DNA polymerase, with the RNA moiety acting as a template that is complementary to the telomeric repeat sequence. This template region is copied using conventional Watson-Crick base-pairing to specify the telomeric sequence such that the telomerase reaction proceeds by the sequential addition of deoxynucleotide triphosphates onto the 3’end (the GT-rich single-stranded overhang) present at the extreme terminus of the telomeric DNA sequence. In this chromosomal setting, the access of telomerase to the 3’ end is regulated by the telomere-binding proteins POT1 and TPP1. For instance when POT1 is over expressed it is found to inhibit telomerase activity and lead to telomere shortening (De Lange et al. 2005, Loayaza et al. 2003) due to its ss-DNA binding activity which could block telomerase from gaining access to the 3’ telomere terminus. In contrast POT1 and TPP1 were found to form a complex in vivo that enhances the telomerase enzyme’s processivity during telomeric extension (Wang et al. 2007) suggesting a switch in function for the proteins. Conventional synthesis of the complementary C-rich strand by DNA polymerase then completes the replication of chromosomal ends, thus compensating for the shortening that would otherwise occur.

From the National Institutes of Health resource for stem cell research

http://stemcells.nih.gov/info/2001report/appendixC.asp

Figure 4: Effect of telomerase on elongation of telomere

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C Replicative Senescence: A Tumor suppressor mechanism? Telomeres have been proposed to act as a ‘mitotic clock’ that counts the number of divisions a cell has undergone and halts any further division after a predetermined number of cell divisions. Normal cells have been known to possess a finite replicative capacity in vivo after which they remain metabolically active but cease to proliferate; this period of arrest is referred to as M1 (mortality 1) or cellular senescence and is triggered by tumor repressor pathways (such as p53- and Rb-dependent checkpoints). Cellular senescence can be thus bypassed by repression of tumor suppressor proteins, activation of oncogenes, or other mutations. By escaping senescence, rare cells can continue to divide and their telomeres shorten until they reach a critical stage referred to as M2 (crisis) or cell death (Artandi et al. 2009). At this stage many chromosomes have undergone massive chromosomal instability resulting from end-to-end chromosomal fusions or non-disjunctions during mitosis, leading to genetic catastrophe and cell death from which only rare survivors can emerge through possible telomerase reactivation. An efficient way of keeping cells healthy is thought to be by triggering such programmed cell death (apoptosis) of damaged cells and replacing them with new healthy ones. However Guarante has argued that using cell turnover to repair tissues could lead to mistakes in copying DNA and harmful mutations so that limiting the number of cell divisions would provide an independent way of preventing malignancies at the expense of the development of other age-related diseases (Guarante et al. 2008). This trade-off has led to the hypothesis that a limited proliferative capacity plays a role in both ageing and tumor suppression (Stewart et al. 2006). In order for a normal cell to develop into a cancer cell, it would require many cell divisions and bypassing the two telomere-based growths check points (M1/ senescence and M2/crisis) to help protect the cells from obtaining the unlimited proliferative potential that is often a hallmark of cancer cells. A variety of anticarcinogenic mechanisms can act to prevent this and trigger cessation of cell proliferation. Telomere shortening is one such mechanism which is thought to lead to the loss of the structural integrity of the telomere nucleoprotein and the activation of critical tumor suppressor pathways such as p53 and Rb (Shay 2005). Since telomerase enzyme, which is used in the elongation of telomeres, is expressed during early development and remains fully active in specific germ-line cells (e.g. renewal cells such as bone marrow cells, epidermal and intestinal crypt cells) but is undetectable in most normal somatic cells; in all non-reproductive cells progressive telomere shortening is observed and telomeres become progressively short until cell division is blocked. This is known as replicative senescence. It seems, however, that telomeres can control replicative lifespan through mechanisms that are more complex than the simple maintenance of telomere length above a critical threshold level. Instead it seems that the maintenance of a properly functional or capped telomere molecular structure, such as the t-loop, is critical for continued cellular proliferation and for the avoidance of senescence. For instance, the eventual growth arrest of cultured cells is thought to occur when some of the telomeres become sufficiently short to compromise their interaction with specific telomere-binding proteins such as TRF2 (van Steensel et al. 1998). Hence the overexpression of telomere-binding protein mutants such as the dominant negative form of TRF2 in vivo was found to result in t-loop loss or ‘uncapped’ telomeres, and as a result induce senescence in wild-type human fibroblasts (Karlseder et al. 2002) as well as result in the activation of the ATM

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and ATR kinases by eliciting a classical DNA damage response (Karlseder et al. 1999). At the same time, however, there’s good evidence that in the absence of p53 checkpoint activation (such as in the case of genetic disorders such as dyskeratosis congenital), short telomeres can instead lead to genomic instability which can contribute to cancer formation. As such telomere shortening has thus been described as a two-staged sword, both preventing and contributing to tumorigenesis. It is argued by Guarante that in the absence of rare genetic defects, the balance between preventing cancer and contributing to tumorigenesis played by short telomeres is thought to be heavily in favor of prevention (2008). This is in contrast to cells that have bypassed cell cycle checkpoint pathways, in which telomeres not only continue to shorten, but also lead to genomic instability and cancer progression (Figure 5). The latter is consistent with studies in which cancer cells were found to have shorter telomeres than in normal cells (De Lange et al. 1999, Meeker et al. 2002, Zheng et al. 2009).

Figure 5: Critical telomere shortening results in a DNA damage response that activates the p53 tumor suppressor protein which induces replicative senescence. If p53 is mutated or deleted however, these responses to telomere dysfunction are tolerated and these broken ends drive development of carcinomas (which explains the widespread gene copy number changes observed in human cancers) (Artandi and DePinho 2010).

Telomere length itself, however, is not yet proven to be a good indicator of malignancy as this has been contradicted in a study in which no statistically significant association was found between leukocyte telomere lengths and advanced prostate cancer (Mirabello et al. 2009). Although this might be attributed to the considerable variability of telomere length within the cells and tissues of an individual and among groups of similar individuals based on health status, it is hence thought that the various techniques used for measuring telomere length may not be reliable or reproducible within small

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ranges. In contrast with the Mirabello study, cell senescence is furthermore not necessarily dictated by the intrinsic shortening of telomeres alone (caused by the end-replication problem); but is known to be initiated as well by other stress signals that can lead to irreversible cell cycle arrest including oxidative damage, overexpression of oncoproteins, chromatin changes and DNA damage (stress-induced senescence) that further induce telomere shortening. This can occur by oxidative intermediates that can migrate along the DNA and produce damage at the triplet GGG rich telomeric sequences. This is due to the fact that not only are telomeres more sensitive to oxidative damage, but DNA damage at telomeres is repaired with less efficiency due to the fundamental nature of telomeric proteins in hiding telomeres from being recognized as damaged DNA needing repair (Kruk et al. 1995). In addition it is thought that tumor cells might typically display telomeric lengths that are shorter than neighboring normal cells since telomerase activation is thought to be a relatively late event in the transformation process that, once achieved, may stabilize existing telomere lengths but fails to restore telomeres to the lengths seen in the nearby normal cells (Stewart 2006). However at the same time it is known that cells that escape senescence and become immortalized generally have their telomere length stabilized and extended in tumor cells not only by the reactivation or up-regulation of telomerase enzyme, but also through less understood molecular mechanisms that are telomerase-independent and rely on homologous recombination of telomeric repeats instead. These mechanisms are generally referred to as alternative lengthening of telomeres (ALT) and ALT-positive cells are typically recognized by very long telomeres (>40 kb) as well as the presence of a large variation in telomere length within the same nucleus (Boukamp et al. 2007). To sum up a central question that is raised is whether the short telomeres that induce replicative senescence can act as an initial protection mechanism in the absence of other genetic and epigenetic alterations or if they actually drive genomic instability leading to cancer. From the current findings in the literature it is deduced that although a few short telomeres can lead to M1 senescence and may be thought of as an antitumor mechanism; in cells that have bypassed critical cell cycle checkpoint pathways (initiated by telomere dysfunction) telomeres not only continue to shorten but may also lead to genomic instability and cancer. As mentioned earlier, M1 senescence may be bypassed as a result of disabled tumor suppressor pathways (e.g. p53 and pRb) caused by mutations; however ultimately the continuing cell division in the absence of telomerase leads to terminal telomere shortening and reaching the crisis or M2 stage which is thought to be the one critical and rate-limiting step in the evolution of most malignancies (De Lange, Blackburn et al. 2006). Clinically significant tumors must hence have a mechanism for telomere maintenance to have the unlimited proliferative capacity required of having at least four to six mutations (if not more) in order to form cancerous cells and this is thought to be brought about through oncogenic mutations which reactivate telomerase enzyme in tumor cells as we shall explore further. III Telomerase biology and link to cancer The telomerase enzyme is a ribonucleoprotein (RNP) that consists of two essential components: a catalytic protein component, telomerase reverse transcriptase (TERT) or hTERT in humans, and an essential RNA component, telomerase RNA (TR)

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or as sometimes referred to as TERC or hTR in humans. Telomerase reverse transcribes the template sequence of its RNA moiety into telomeric repeats thus solving the end replication problem as discussed before. The holoenzyme adds the TTAGGG repeats onto the telomere by elongation and translocation. TERT catalyzes the elongation by adding the repeats complementary to the template region of the TR followed by the complex pausing and then translocation (Figure 4). This telomeric reaction is mediated and regulated by multiple activities including telomere binding proteins, protein kinases, DNA helicases and nucleases. Some of these proteins have dual functions in telomere maintenance and in the repair of double strand breaks during non-homologous end-joining (Lundland 2000). Although the enzymatic reaction and the maintenance of telomeres by telomerase are conserved in most eukaryotes; some of the structural components of telomerase are found to vary between different eukaryotes with features having important features being conserved (Figure 7). In this section we shall explore the structural and functional features of TR and TERT while providing a review of additional protein components of the enzyme that are required in human cancer cells as well as end by discussing the specific implications of hTERT in human tumors. A Telomerase RNA (TR) In 1989, the 159 nucleotide Tetrahymena TR was cloned by Elizabeth H. Blackburn and Carol W. Greider revealing a nine nucleotide-stretch complementary to the telomeric repeat sequence in this organism. This sequence was referred to as telomerase RNA template and revealed the identity of telomerase as a reverse transcriptase (Greider and Blackburn 1989). In addition to providing the template for telomeric repeat synthesis, the TR may fulfill at least two other major functions: it provides binding sites for telomerase protein subunits and it may actively participate in the telomerase reaction cycle. This was deduced upon comparing the secondary structures of TR subunits from different species which have highly variable telomerase RNA components in both sequence and size (Zappulla and Cech 2004). For instance TR’s sizes range from 146-200 nucleotides in ciliates (Romeo and Blackburn 1991) to approximately 1544 in budding yeasts (Tzfati et al 2000). The TR’s sequences were aligned among closely related organisms, and secondary structure models were derived for the different groups by phylogenetic comparison revealing that certain secondary-structural elements are conserved and suggesting that important functions are associated with them. For instance as shown in Figure 6 the vertebrates TRs consist of four conserved structural elements: the pseudoknot, the CR4-CR5, the Box H/ACA snoRNAs and the C7 domains and it’s thought that these unique structures might play some vertebrate-specific roles for telomerase function (Chen et al. 2000). Larger vertebrates and yeast RNAs have diverged apparently by acquiring additional RNA structural domains during evolution that serve as binding sites for species-specific telomeric proteins such as p43 protein (ciliate), Sm protein (yeast), Ku protein (yeast), and dyskerin complex (vertebrate). The proposed secondary structures of TRs from most species are found to however share a similar structural configuration near the template region. The template is flanked 5’ upstream by a long-range base-pairing element, referred to as the template boundary or helix I or P1 region, and 3’ by a downstream element referred to as the pseudoknot domain; both of which are conserved in most species (Figure 7).

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Figure 6: The proposed secondary structures of the vertebrates (A) human and (B) sharpnose shark telomerase RNAs were determined by phylogenetic comparative analysis. Invariant nucleotides are shown with red letters in bold. The four conserved structural domains described in the text are shaded in gray and labeled as the pseudoknot domain containing the template region, the CR4-CR5, Box H/ ACA, and C7 domains while the conserved nucleotides are boxed (Chen et al. 2000).

The fact that these secondary structures are conserved suggests important functions associated with them. The conserved template domain consists of the pseudoknot structure, a primer alignment region, and a region of the RNA that specifies the template’s 5’ boundary. Hence the conservation of the template sequence in a single stranded region allows it to be accessible for annealing and base pairing with the telomere 3’ end and for residing in the active site of the catalytic TERT domain; while having a defined 5’ boundary helps determine where polymerization stops and/or translocation occurs and helps in maintaining the enzyme’s processivity. It is thought that shorter or ‘uncapped’ telomeres are better substrates for telomerase than longer telomeres; the latter

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of which may be in an inaccessible state for telomerase access such that telomere shortening due to incomplete end-replication may increase the accessibility of a telomere to telomerase and again favor its re-elongation. This is also implies that the telomere actually has to look like a DNA break for telomerase to assemble to it and thereby recap the telomere (Blackburn 2000). The evolutionary conservation of the pseudoknot structure is thought to be important for this assembly of the telomerase enzyme. Within the pseudoknot domain, the RNA template is adjacent to the 5’ end of the pseudoknot structure which is the binding site for the TERT protein in vertebrate and yeast RNA. This specific RNA-protein interaction is thought to allow the correct positioning of the RNA template in the catalytic pocket of the TERT protein (De Lange, Blackburn 2006).

Figure 7: Comparison of Ciliate and Vertebrate TR Structures. The conservation of the pseudoknot and the helix 1 regions flanking the template domain is illustrated. Vertebrates are likely to have become more evolved from the ancestor TR lineage than ciliates as they have acquired additional RNA structural domains (Chen et al. 2000). B Telomerase Reverse Transcriptase (TERT) Reverse transcription of the template sequence is catalyzed by the reverse transcriptase subunit (TERT). In contrast to the TR, sequence analysis of TERT proteins from distantly related organisms reveals several regions of conserved sequence motifs. These motifs can be grouped into three distinct regions: the amino-terminal domain, reverse transcriptase (RT) domain, and carboxyl-terminal domain. Motifs in the RT domain are universally conserved among all reverse transcriptases whereas motifs in the amino-terminal and carboxyl-terminal domains are unique to telomerase and are not as conserved as the RT domain (Figure 8).

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Figure 8: Alignment of the telomerase reverse transcriptase (TERT) components of human, mouse and S. cerevisiae telomerase (Nakamura 1998). The structure of the full-length catalytic subunit of the Tribolium castaneum active telomerase was solved by Gillis and was shown to consist of the three highly conserved domains, organized into a ring-like structure that places motifs implicated in substrate binding and catalysis in the interior of the ring (see Figure 9). The structure also showed that TERT resembles, both structurally and functionally, other reverse transcriptases such as the HIV reverse transcriptase, viral RNA polymerases and the B-family DNA polymerases which have been compared to that of a right hand with fingers (Gillis 2008). The TERT three distinct domains are: an RNA-binding domain (TRBD), the reverse transcriptase (RT) domain representing the finger domain and the carboxyl-terminal extension (CTE) or the thumb domain. Each of these domains is thought to have distinct functional roles including catalysis, RNA binding, enzyme processivity and recruitment to the telomere.

The high affinity RNA binding domain (TRBD) has been identified within the Tetrahymena TERT N-terminal extension such that specific interactions between this region of the TERT protein and the TER are thought to be necessary for the functional assembly of the telomerase complex (O’Connor 2005). This is a confirmation to similar results obtained showing that mutations in the corresponding amino-terminal region in the S.cerevisiae TERT Est2p interfered with binding to the yeast telomerase RNA (Friedman et al. 1999). The reverse transcriptase (RT) domain, on the other hand, is described as a mixture of α-helices and β-strands organized into two sub-domains that are most similar to the ‘fingers’ and ‘palm’ and has its catalytic activity due to the presence of three conserved aspartic acid residues within the active site, present in the palm, which coordinate the two active-site metal ions (Mg+2 or Mn+2) found in all polymerase (Gills et al. 2008). Since elongation of the telomerase complex is thought to occur by the formation of a nucleic acid heteroduplex of DNA and RNA at the active site; the reverse transcriptase motifs has in addition the conserved residues of lysine and asparagine that are found within the spiral and are involved in nucleotide binding so as to facilitate the formation of the nucleic acid heteroduplex and the alignment of the 3' end of the DNA towards the active site for nucleotide addition (see Figure 10). Telomerase repeat addition processivity is also attributed to the IFD (insertion in fingers domain) motif present in the reverse transcriptase in addition to the carboxy-terminal extension (CTE) which is proposed to constitute the ‘thumb’ domain of telomerase analogous to that found in other reverse transcriptases. For instance, in humans, the hTERT C-terminus domain was shown to be important for the successive nucleotide addition within a single 6-base telomeric repeat (type I processivity) and for the repetitive addition of multiple telomeric

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repeats (type II processivity) that are characteristic of telomerase processivity as demonstrated by hTERT C-terminal domain mutants that had reduced processivity (Huard et al. 2003). (A) (B)

www.doe-mbi.ucla.edu Figure 9: (A) The ring structure of TERT and the separate domains within the enzyme. The TRBD domain (purple) is mostly helical and binds the RNA while the palm (red) and fingers (orange) motifs combine to form the reverse transcriptase domain which contains the active site and supports DNA positioning. (B) A closer look at the ring structure of TERT showing the RNA binding loop (green) and the active site (cyan) positioned on opposite ends of the ring.

Figure 10: Model of Euplotes telomerase (a ciliate distantly related to Tetrahymena) as an RNA-reverse transcriptase complex. The formation of the TERT-TER-DNA heteroduplex is shown at the active site. (The p123 region is homologous to the Est2p-binding site it yeast) (Lingner et al. 1997).

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C The Implication of Human Telomerase Reverse Transcriptase (hTERT) in cancer

Even though telomerase is composed of an RNA moiety and several proteins, as described earlier, the only essential protein essential for the enzyme’s catalytic activity is hTERT. Hence while hTR is expressed in normal cells, it is the presence of hTERT that confers telomerase activity in human cancer cells. The identification that hTERT, which is the rate-limiting component for telomerase activity, is expressed by >85% of all human cancers but is absent in normal cells confirms the suggestion made in the beginning that telomerase activity is up regulated in cancer cells and further provides evidence of a causal link between telomere maintenance by telomerase and the immortalization of tumor cells (Kim et al. 1994). Moreover the fact that the formation and development of human tumors usually involves the activation of oncogenes that control cell cycle or cell death and knowing that gene amplification is one of the most common mechanisms for oncogene activation; the observation that hTERT gene amplification was frequently found in human tumors indicates that this genetic event may be an important driving force to activate telomerase in at least some types of cancer. From that we can deduce that telomerase and hTERT are most likely candidate oncogenes for the formation of cancers and that the amplification of the hTERT gene is the likely culprit in the oncogenic process. However, the fact that hTERT amplification occurs in a minority (up to 30%) of human tumors while telomerase is widely activated in various malignancies clearly indicates that many other elements play a significant role in the regulation of hTERT or telomerase expression (Krupp and Parwaresch 2003). The exact mechanism for hTERT activation is yet unclear, however in a study a strong association has been found between increased hTERT copy numbers and the decreased expression of the p53 tumor suppressor protein in breast cancer which is again consistent with our understanding of the DNA damage response being initiated by the repression of tumor suppressor proteins (Zhang et al. 2000). In a further study that links hTERT to cancer, Hahn et al. demonstrated that a normal cell can be converted to a fully tumorigenic cell through the introduction of a clone of hTERT gene, inactivation of the p53 and Rb tumor suppressor pathways and mitogenic simulation through oncogenic H-Ras expression (1999). These factors needed to be all present which indicates that hTERT can immortalize cells, but it does not on its own lead to cancer. This role of hTERT in leading to the immortalization of cancer cells was further validated in other studies which showed that the inhibition of hTERT activity through the injection of a dominant-negative allele of hTERT (DN-hTERT) in already immortalized cells resulted in the progressive loss of telomeric repeats during successive rounds of DNA replication until telomeres became critically shortened, as observed in crisis, and was followed by cell death (see Figure 11). At the same time the RNAi-directed knockdown of hTERT in normal cells also led to the loss of the single-stranded overhang of telomeres and as such led to an accelerated entrance into senescence which suggests that telomerase expression is required to rebuild the overhang and allow the proper telomere assembly and function even when telomeres are long (Stewart et al. 2006).

In a recent study that adds complexity, however, it is suggested that the disruption

of telomerase activity in normal human cells slows cell proliferation and restricts the cellular lifespan by altering the maintenance of the 3′ single-stranded telomeric overhang without changing the rate of overall telomere shortening. These observations indicate that telomerase and telomere structure are dynamically regulated in normal human cells in

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contrast to the previously widely held view and further suggests that telomere length alone is unlikely to trigger entry into replicative senescence (Masutomi et al. 2003). This is as mentioned before reflects the indecisiveness regarding the role of telomere length in leading to cancer in the current literature.

In brief the fact that telomerase and specifically its catalytic subunit are

implicated in cancer suggests the use of research ways to inhibit its activation so as to prevent cancer proliferation. Further efforts in trying to better understand the molecular structure and the regulation of the telomerase enzyme would hence prove fruitful for such a purpose.

Figure 11: Telomere length dictates entrance into crisis. Introduction of DN-hTERT into immortal telomerase-positive cells resulted in a growth arrest reminiscent of crisis that was characterized by telomere loss followed by cell death. Cells that maintain their telomeres through the ALT mechanism (see previous discussion) were unaffected by introduction of the DN-hTERT allele (Stewart 2006). IV. Molecular-based approaches for Cancer Therapeutics Cancer remains the major cause of death in the United States for those under the age of 85 (98% of the population) despite substantial progress in understanding its molecular mechanisms and the development of an array of powerful treatments (De Lange, Blackburn et al. 2006). The discovery of validated targets and new drugs is therefore a high priority. Since telomerase is expressed during embryonic development but is repressed in most adult tissues; the fact that telomerase activity (or i.e. hTERT) has been detected in approximately 85% of the malignant human tumors tested provided evidence that up-regulated telomerase activation is the norm in human tumors and hence it is logical to propose that telomerase inhibition might be useful to block cancer proliferation. Telomere maintenance is also critical for constitutive proliferation of malignant cells and hence both telomerase and telomeres could become potential targets

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for drug therapy. The rational for exploring telomerase as a therapeutic target is that the disruption of telomerase activity would prevent cells from maintaining their telomeres and thus leads to telomere shortening and senescence. However as we may later see this does not necessarily occur or might rather be an eventual process rather than a rapid one (Figure 15). Telomerase could be thus thought of as a universal tumor marker and it might be of potential interest to use its catalytic subunit hTERT as a screen for the early detection of human cancer, prior to progression to a high-grade malignant status. The most frequently used method for that purpose would be the Telomere Repeat Amplification Protocol (TRAP assay) as described by Kim et al. due its high sensitivity and usefulness for the identification of low levels of telomerase activity. However TRAP activity can only be determined from fresh tissue and might provide false results if telomerase-positive non-tumor cells are included. The detection of hTERT can be alternatively achieved thus using PCR, which is even more sensitive than the TRAP assay and is not susceptible to the problems inherent in enzyme assays. However, PCR is time consuming hence developing a more feasible method of rapid analysis will be important should TERT be used as a screening tool (Kirkpatrick et al. 2001). A Telomerase inhibition: targeting telomerase enzyme components Telomerase activity is typically inhibited in cancer cells by the expression of dominant-negative mutants of the hTERT gene (as shown in previous studies), or through the direct targeting of telomerase enzyme components by the use of anti-sense oligonucleotides, specific siRNAs or ribozymes directed against hTR or hTERT the latter of which we shall discuss first.

One of the oldest and most commonly used classes of telomerase inhibitory agents is antisense DNA oligonucleotides which have been used since the 1990’s to inactivate gene expression at the transcriptional level typically by using short oligonucleotides or mimics that block the translation of mRNA into a functional protein. The way this works is that antisense technology utilizes nucleotides with sequence complementarities to the corresponding template region of human telomerase which can hybridize and thus compete with the 3’ end of telomeric DNA for telomerase substrate binding. In addition these molecules may be designed to occupy the ribosome binding site hence preventing the ribosome from binding the target mRNA, halting translation and producing a non-functional or truncated protein in the process. An advantage of this approach is the potential affinity and selectivity that oligonucleotide mimics have for targeting nucleic acids. However there have been numerous modifications of antisense oligomers reflecting efforts to enhance their cellular up-take, potency, half-life and hybridization properties. For instance some modifications help recruit the activity of RNase H enzyme which degrades the RNA strand of a formed RNA-DNA duplex so as to release the antisense DNA molecule and allow it to become free in order to bind other mRNA molecules (Cunningham et al. 2006). Other modifications take into account that natural oligonucleotides (DNA, RNA) have limited potential as therapeutic agents due to their poor in vivo stability because of the phosphodiester backbone’s susceptibility to enzymatic cleavage by cellular nucleases. To overcome this general problem, there have been significant advances in the discovery and development of analogues bearing structural features which improve the pharmacological properties of natural oligonucleotides; the most potent of which include phosphorothioate DNA (PS-DNA)

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(replacement of one non-bridging phosphate oxygen with sulphur), 2’-O-alkyl RNA (not shown) and peptide nucleic acids (PNA) (Figure 12). Peptide nucleic acids (PNA) are molecules in which the antisense bases are connected to various peptide backbones and are known to improve the half-life of antisense oligomers and enhance their hybridization properties (Figure 13). This is attributed to the higher binding affinity that PNAs have to

Figure 12: Examples of modifications made to traditional antisense oligonucleotides with the susceptible phosphodiester backbone. complementary sequence relative to analogous DNA or RNA which is generally due to the lack of electrostatic repulsion between the neutral PNA backbone and the negatively charged phosphodiester backbone of nucleic acids. Furthermore PNAs are thought to possess higher biological and chemical stability due to the unnatural amide backbone that is not recognized by nucleases or proteases (Krupp and Parwaresch 2006).

Figure 13: Examples of peptide nucleic acids (PNAs) molecules (Cummingham 2006).

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RNA interference phenomenon (RNAi) or RNAi-mediated gene knockout is another inhibition method which involves the use of short double-stranded RNA molecules to activate a pathway that results in the degradation of the homologous mRNA target. For instance small interfering RNAs (siRNAs) were shown to inhibit telomerase activity by expressing siRNAs constructs in vitro using plasmids or viral expression vectors that acted against hTR or hTER in a dose-dependent manner (Kosciolek et al. 2003). These results came a bit of a surprise considering that RNAi are known to be confined to the cytoplasm whereas telomerase’s site of action is in the nucleus, however; since telomerase is manufactured in the cytoplasm, it is thought that it remains in the cytoplasm for at least long enough for RNA interference to cause hTR or hTERT mRNA degradation. Also interestingly siRNA targets hTR inhibited cancer cells not by causing telomere shortening but by inducing changes in the global gene expression profile (Li et al. 2005). To further evaluate siRNA effectiveness against telomerase as an anticancer agent, it will be necessary to achieve more complete inhibition as shown by Kosciolek et al. in which beyond a certain concentration of siRNA, increases in siRNA concentration did not increase inhibition (Figure 14). Meanwhile ribozymes, catalytic RNA molecules with specific endoribonuclease activity, which also have the ability to cleave specific sites of target mRNAs, could be further adopted to be used as human gene therapeutic agents.

Figure 14: siRNAs decrease telomerase activity in HCT-15 human colon carcinoma cells. Note that this decrease does not extend beyond the 71 nm concentration of siRNA. (Kosciolek et al. 2003). Other interesting inhibition techniques include telomerase immunotherapy through which vaccination is performed with hTERT peptides in a variety of advanced tumor patients. This is done in order to induce functional cytotoxic T cells (CTL) to recognize hTERT-specific peptides that are expressed on the cell surface of only tumor cells hence killing the hTERT-positive tumor cells (Boukamp et al. 2006). Furthermore, other recent approaches are based on gene therapy strategies such as the suicide gene therapy in which telomerase-targeted vectors carrying the enzyme encoding gene are transferred into cells so that toxins are released killing only the telomerase positive cells.

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A related method, oncolytic virus therapy, uses the hTERT promoter to control the replication of the oncolytic virus such that these viral vectors only replicate in and lyses tumor cells selectively (Xiaoping et al. 2010). B Telomerase inhibition: Expression of dominant-negative hTR and hTERT mutants Similar to actively destroying hTR and hTER transcripts, the expression of mutant forms of either hTR or hTERT would effectively disrupt telomerase activity (as described earlier). As shown in the previous section, strategies that target the RNA subunit of telomerase with antisense RNA (or peptide nucleic acids) diminish cellular telomerase activity and induce some changes in cellular growth; however, those approaches did not consistently lead to complete inhibition of telomerase activity, and the specific effects of such agents on both telomere shortening and cell death were difficult to assess. In response one promising therapeutic development was the creation of dominant negative hTERT protein mutants (DN-TERT) in which the catalytic site was inactivated by substituting the aspartic acid and valine amino acid residues 710 and 711 with alanine and isoleucine. This not only resulted in telomere shortening but also in growth arrest and death hence indicating complete inhibition of telomerase activity in many distinct immortal and malignant cells (Hahn 1999). This encouraging finding hence further provides evidence that DN-TERT may be used as a therapeutic telomerase inhibitor for a wide range of human malignancies. C Potential drawbacks of telomerase inhibitors One important step in the preclinical evaluation of the use of these inhibitors is the use of animal studies in conducting the experiments. As telomeres are known to be longer in mice than humans and telomerase is hence thought to be less tightly regulated in mouse than in man, telomerase gene therapy may however be difficult to prove conclusively in mouse models. In addition, despite the benefits that could be garnered from the use of these inhibitors in treating cancer, there are a few risks worth mentioning. For instance, although telomerase is not significantly expressed in normal somatic cells (not taking into consideration the contradicting results by Masutomi et al.), it is unknown if other cells that do express telomerase such as stem cells, intestinal crypt cells, and cells lining the epithelium are affected by such telomerase inhibition treatment. It is thought, however, that such adverse effects would be minimal considering that normal cells express telomerase infrequently and at lower levels as compared to cancer cells and that the telomeres are also generally of greater length in the rare telomerase-positive cells which makes these cells less susceptible than to telomerase inhibition. It has yet to be confirmed experimentally however if these risks can be ruled out. Another concern is also that treatment with telomerase inhibitors may incur a clinically time lag time before telomeric uncapping initiates cell senescence or apoptosis or may be ineffective in case of cells that maintain their telomeres using the ALT pathway (Figure 15). The lag time in treatment may be of less concern considering the recent numerous reports of rapid apoptotic responses reported upon telomerase inhibition; however there are concerns that eliminating telomerase activity may promote the cell’s reliance on the ALT pathway and hence further efforts should be made to examine the relationship between telomerase inhibition and induction of ALT. As such it is feasible that telomerase inhibitors could

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not be used as stand-alone pharmaceuticals but as part of a coordinated treatment strategy.

Figure 15: The possible outcomes of telomerase inhibition (Cunningham 2006). D Telomere inhibition: an alternative therapeutic intervention? In addition to telomerase-based approaches, it is argued by Boukamp that telomeres may possibly be better targets for cancer therapeutics. Because the telomere 3’ overhang is the substrate in the telomere elongation reaction and since it is known that the G-rich telomere structures can fold into four-stranded G-quadruplex structures that impede the telomerase’s access to the 3’ overhang; the use of small molecules that can selectively stabilize the telomeric G-quadruplex structures can cause telomere shortening and senescence (2006). Examples of such molecules include telomestatin, a G-quadruplex-stabilizing agent that was described as a potent telomerase inhibitor and was shown to cause cell death in cancerous but not normal cells (Tahara et al. 2005). Telomestatin is thought to function by provoking the disassociation of POT1 and TRF2 proteins causing telomere uncapping. In particular the TRF2 protein which is responsible for the stabilization of the t-loop structure and capping of the telomeres is thought to be implicated in the mechanism for this inhibition as it was shown before (on page 3) that inhibition of this protein by over expressing a dominant-negative mutant resulted in the activation of DNA damage pathways and hence would eventually lead to cell senescence and apoptosis. Conclusion In conclusion, understanding how telomeres function and how telomerase exerts its protective effects will be necessary if telomerase-based drug therapy is to be applied effectively for medical use in the future. Therefore a number of questions must be first

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answered, for instance, researchers need to determine which normal cells (beyond the few already identified) make telomerase, and they need to assess the importance of the enzyme to those cells. For instance, if telomerase is proven to be crucial in these cells, drugs that interfere with it might in fact prove unacceptably toxic. At the same time investigators must also demonstrate that inhibition of telomerase can destroy telomerase-producing tumors as expected and not develop some kind of resistance caused by cells resorting to alternative recombination mechanisms for the elongation of telomeres. Finally, to develop agents that will block telomerase in the human body investigators must also have a better understanding of exactly how the telomerase enzyme functions and is regulated at the molecular level in tumor cells.

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