Cancer is comprised of a diverse set of diseases and disorders that not only stems from almost every tissue but also exhibit a "remarkable heterogeneity in presentation and prognosis" (Hahn, 2003). Despite this massive range of clinical diversity, all human tumours share a restricted set of behaviours that describe the malignant condition. Among the various important hallmarks, unlimited replicative potential and widespread genomic disarray are among the most common characteristics exhibited by all human cancer cells. Up-and-coming evidence implicates "the maintenance and function of the specialized chromosomal terminal structures, termed telomeres, as essential regulators of both cell life span and chromosomal integrity" (Hahn, 2003). Therefore, telomeres and telomerase are designated as hallmarks of cancer as they seem to play a vital role in cancer growth and a great deal of research has been done in recent years to develop a new generation of cancer therapies that targets telomeres and telomerase. The molecular events of telomere and the enzyme telomerase appear to contribute significantly to the malignant transformation and consequently present for vital targets for the selectively killing or inhibition of growth of malignant cells (Hodes, 2001).
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Mammalian telomeres are DNA-protein complexes that cap the ends of eukaryotic chromosomes and confer protection against the loss of DNA (Bearss et al. 2000). The ends of chromosomes pose a distinctive challenge to the DNA replication machinery through the end-replication problem which results in loss of telomeric DNA (Ince and Crum, 2004). The progressive loss of 25-200 DNA base pairs is observed in dividing cells following each cell division ensuing in critically shortened telomeres that are recognized as damaged DNA (Seimiya, 2006). Telomeres are composed of short, tandem repeats of guanine rich sequence motifs (Meyerson, 2000). Telomere consists of tracts of repetitive DNA, TTAGGG/AATCCC double-stranded sequence ending in a single-stranded G-rich 3' overhang, that guard chromosomes from nuclease degradation, inappropriate recombination and loss of essential genes, and also allow the cell to distinguish between double-strand breaks and natural chromosome ends (Hsu and Jin, 2005). Cell death and cellular/replicative senescence takes place when the telomeres reach a critically short length, thereby preventing further DNA-damaging signaling cascades (Baker, 2008).
The proliferative capability of most normal human cells is limited by telomere shortening upon cell division due to the lack of specialized telomere synthesis mechanism, the telomerase enzyme. Telomerase, at first described in the ciliate tetrahymena, is a large ribonucleoprotein reverse transcriptase enzyme on which the de novo synthesis of telomeric DNA depends (Ince and Crum, 2004). Vigorous telomerase activity is noticed in cancer cells that are rendered with unlimited cell division due to the maintenance of telomere length by the addition of repeat units to the telomere (Gellert et al. 2005). The haloenzyme is made up of a functional RNA template (hTR, also called TERC) and a catalytic protein subunit (human telomerase reverse transcriptase; hTERT) that has a reverse transcriptase activity. The protein component, hTERT, is highly regulated and its expression correlates with the telomerase activity proving to be a rate limiting factor of the complex (Hahn et al. 1999). In contrast to this the RNA component is expressed almost everywhere.
The goal of cancer research around the world today possibly is the discovery of a cellular component that is definitely vital for the growth of all cancers but not for the normal cells and has minimal effect on the normal cells (Morin, 1995). Such a factor would prove to be a potential universal target for therapeutic intervention. Since nearly all tumour cells are reliant on the activity of telomerase to maintain the stability of predominantly short individual telomeres, inhibition of this enzyme presents an attractive approach for a mechanism-based anticancer therapy (Zimmermann and Martens, 2007). Telomerase has been elucidated to be the agent that could prove essentially significant in the battle against cancer as studies have reported that telomerase is expressed in more than 85% of all cancers and its activity has been found in almost 90% to 95% of human immortal cell lines (Hsu and Lin, 2005). On the contrary, telomerase is not expressed in normal tissues with the exception of proliferative cells of renewal tissues and its activity is almost repressed or undetectable in majority of somatic cells (Tian et al. 2010). Furthermore, telomerase is expressed in embryonic cells and in adult male germ line cells. Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism that is independent of the telomerase activity and has been observed in the remaining 5% to 10% of the immortal cell lines. Nearly all immortalized cells have readily detectable telomerase activity and express hTERT (Hahn, 1999). At least three applications concerning telomeres and telomerase have been proposed: in cancer diagnosis and prognosis and as a means of monitoring tumour response to therapy; as an aid to tissue engineering; and inhibition as a cancer therapeutic strategy (Kelland, 2001). Recent investigations have unveiled multiple cancer therapy methods targeting telomerase and telomere including immunotherapy, gene therapy, small molecule inhibitors, and signaling pathway inhibitors.
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Figure 1: Multiple molecular targets of telomerase inhibition. Telomerase therapeutics have the potential to become important methodologies for cancer ablation, owing to the plethora of approaches currently available which are ultimately directed towards impeding the cancer growth. These varied approaches are directed at inhibiting all aspects of telomere/telomerase biology such as (1) haltering the transcriptional and translational activation of hTERT and hTR, (2) perturbing telomerase macromolecular structure, assembly and functionality and (3) obstruction of telomere structure and integrity.
Source: Gellert et al, 2005, p.160
Telomerase hTERT poses as a potential target as it has been identified in about 85% of the primary tumours and thereby provides the much needed distinction between the normal and cancer cells (Parkinson and Minty, 2007). Few current approaches to control the expression of tumour involve the employment of hTERT promoter-driven expression of oncolytic adenovirus and/or suicide genes which have been successfully studied in animal models. "Apoptosis-inducing, toxin-encoding, chemotherapeutic sentisitizer, xenoantigen genes, or genes used in gene directed enzyme prodrug therapy (GDEPT)" are among the genes that have been used therapeutically (Hsu and Jin, 2005). These methods have showcased great potential by selectively and effectively killing a wide variety of cancer cells and appear to be silent in normal telomerase-negative cells. In the suicide gene therapy, telomerase-targeted vectors carrying the enzyme encoding gene are transfected into cells resulting in the release of toxins following the addition of pro-drug, for example bacterial cytosine deaminase with the prodrug 5-fluorocytosine. The toxins only seem to target/kill the telomerase positive cells. Another method uses hTERT promoter to drive the expression of proapoptotic genes. Among the important ones are Bax, caspase-6, caspase-8, FAS-associated protein with death domain (FADD), and TRAIL (Olaussen et al. 2006). These genes are known to induce apoptosis selectively in the telomerase-positive tumour cells resulting in a significant decrease in the tumour size, whereas the telomerase-negative cells do not experience the event of apoptosis (Gellert et al. 2005).
The third attempted approach focuses on gene-viral strategies known as oncolytic therapy in which the hTERT promoter controls the replication of the oncolytic virus. Oncolytic viruses can replicate in and lead to the selective lysis of tumour cell with minimum "infection/replication potential" in the adjacent "non-neoplastic tissue" (Hardcastle et al. 2007). Cancer stem cells have shown to have critically shorter telomere lengths compared to the normal stem cells and also have the ability to self-renew (Tian et al. 2010). Therefore, apoptosis is triggered in such cancer stem cells before the normal stem cells by the telomerase inhibitors (Shay and Wright, 2006). The obstacle in this method is to attain viral replication that is completely restricted to the tumour cells as this approach kills the normal as well as the cancer stem cells. In recent times, telomerase-dependent CRAD (Conditionally replicative adenovirus) has been more regularly used that merges the "specificity of hTERT promoter based expression systems with the lytic efficacy of replicative viruses" and is exempt of side effects (Thomas et al. 2005). An example is the adenovirus CNHK300, works by using the hTERT promoter to drive the expression of the adenoviral genes E1A and E1B (Fujiwara et al. 2007).
Telomerase could provide for a prospective candidate for immunotherapy as telomerase
hTERT is almost a universal tumour antigen for CD8+ (Tumour-associated-antigen). A wide variety of tumour cells and HLA alleles are destroyed through the endogenous pathway activated by the hTERT specific cytotoxic T lymphocytyes (Vonderheide, 2008). The CTLs identify the peptides derived from these antigens and can consequently elicit immune responses against the tumour (Shay and Wright, 2002). In vivo studies carried out with hTERT specific CTLs displayed successful destruction of human renal and prostatic cancer cells in mouse models. In this method no lag period is required and also it does not run the risk of toxic side effects (Olaussen et al. 2006). The lag phase is the time period between when telomerase is inhibited and the achievement of critical shortening of the telomeres of tumour cells in order to produce a detrimental effect on the proliferation of cancer cells. However, normal cells that express elevated levels of telomerase may be affected and also cells with ALT may prove resistant towards this form of therapy. The vaccines that have been developed, GRNVAC1 and GV1001, can potentially kill telomerase positive tumour cells (Tian et al. 2010).
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The development of natural or synthetic compounds having the capability of being less detrimental than the contemporary chemotherapies gave birth to small-molecule telomerase inhibitors. Small molecules inhibitors, GRN163/GRN163L and BIBR1532, directly hamper the activity of telomerase leading to the attrition of telomere over consecutive cycles of replication. Moreover, GRN163 has shown substantial repressed cancer cell growth in numerous mouse xenograft models (in vivo), for instance human prostate cancer, multiple melanoma, non-Hodgkins lymphoma and glioblastoma (Shay, 2005). Presently, GRN163 is undergoing phase I/ll clinical trials in patients with chronic lymphocytic leukaemia.
Abolishing the telomerase activity makes the telomeres to become prone to "natural replicative degradation and consequently the cells experience growth arrest" (Baker, 2008). Telomere inhibitors such as telomestatin, TMPyP4, RHPS4 and BRACO-19 destruct the telomere maintenance mechanism by telomerase via the stabilization of the G-quadruples (guanine-rich strands) structures of telomeres. These compounds are the derivatives of anthraquinones, acridines, complex polycyclic systems, cationic porphyrins, triazines, and perylenes. In vivo studies with the drugs RHPS4 and BRACO-19 produced a rather short response time which could be a very promising clinical feature. Also, these drugs have shown positive antitumour activity in combination with taxol (paclitaxel). Compounds such as carbocyanine dyes DODC and a bifuryl compound DB832 have been developed recently that bind with more selectivity and less cytotoxicity to the G-quadruplex. An added notable compound that offers scope for more successful and less toxic cancer therapy is 'T-oligo', oligonucleotides that mimic the terminals (3'-overhang) of the telomeres. T-oligos stimulate a DNA damage response, thereby inhibiting telomerase. In vivo and in vitro studies using human melanoma cells and an 11-mer t-oligo have showcased the potential of T-oligos as an antiproliferative agent.
Among other compounds are nucleoside and non-nucleoside analogs that stall the catalytic activity of the telomerase enzyme (hTERT) and thereby produce an inhibitory effect (Hsu and Jin, 2005). Nucleoside analogs such as Azidothymidine (AZT) produce a reverese transciptase inhibitory effect blocking the inclusion of dNTPs into the neosynthesized DNA. Also, BIBR 1532 (small non-nucleotide synthetic compound) and TNQX are non-competitive inhibitors that target the core components of telomerase: hTR and hTERT directly (Tian et al. 2010). Non-nucleoside inhibitors, isothiazolone derivative TMPI, rhodacyanine FJ5002, EGCG, MST312 and BIBR 1532, cause telomerase inhibition through "binding to the active site of the reverse transcriptase enzyme" and trigger progressive telomere shortening (Olaussen et al. 2006). Pyrimethamine considered being a potent telomerase inhibitor presents as a useful chemopreventative agent. Telomerase inhibition by the activation of cell signalling cascade leads to apoptosis and growth senescence in the tumour cells.
Figure 2: Schematic of mechanisms for telomerase inhibitor-induced, T-oligo-induced, and TTA-induced cytotoxicity of select drugs from each class. Lines indicate inhibition while black arrows show the flow of a signal-cascade and the red arrow indicates upregulation. BIBR1532 inhibits the catalytic unit of telomerase (green) and GRN163/GRN163L inhibits the RNA component (yellow). RHPS4 and BRACO-19 disrupt normal telomere structure (red). T-oligos mimic 3' telomere overhang signals and initiate a signaling cascade similar to that initiated after telomere damage is recognized following exposure to telomerase inhibitors and TTAs.
Source: Baker, 2008, p.73
Results obtained from the exploitation of standard oligodesoxynucleotides have been unsatisfactory with limited stability and bioavailability (Olaussen et al. 2006). Therefore, new methods of antisense gene therapies have provided with a more powerful attempt to inhibiting telomerase specifically. Antisense gene therapies target the hTERT mRNA or telomerase RNA (hTR) based on hammerhead riboenzymes or RNAi. These therapies induce a selective impact on telomerase-positive cells and includes peptide nucleic acids (PNAs), antisense oligonucleotides and chemically modified PNAs, such as 2â€²-O-methyl RNA, 2â€²-O-methoxyethyl RNA, phosphorothioate, etc (Tian et al. 2010). GRN165L is a lipid-modified N3â€²â†’P5â€² thio-phosphoramidate oligonucleotide that inhibits cancer growth through its homology to the template region of the RNA subunit (hTR) of the enzyme. siRNA (short-interfering RNA) "directed against c-Myc activating telomerase through interacting with the TERT gene promoter may have a potential anticancer value" (Tian et al. 2010). The requirement of a lag-period before the destruction of telomerase-positive cells proves to be a major disadvantage of this approach. However, the hammerhead riboenzymes offer great advantages such as high stability, bioavailability and specificity due to which they appear highly potential for future drug development.
Dominant negative hTERT is a mutant hTERT protein that is catalytically inactive due to the introduction of specific changes of amino acids, and yet it remains capable of sequestering hTR (RNA component). DN-hTERT in combination with other chemotherapeutic reagents, for example docetacel, etoposide, cisplatin, ecteinascidin-743, temozolomide and imatinib, aids in increasing the sensitivity to tumour cells (Gellert et al. 2005). On the contrary, DN-hTERT therapy in combination with other antineoplastic therapies such as temozolomide and carmustine enhances resistance to melanoma cells. This particular approach needs to be investigated further and a resolution needs to be found for potential setbacks such as achieving a more efficient cell delivery.
It can be argued that telomerase and telomere provide for attractive cancer targets since they are essentially required for the immortalization of all cancer cells including cancer stem cells (Fleisig and Wong, 2007). In addition, these structures present a relatively safe treatment as major differences occur between the normal and tumour tissues in terms of telomere length, cell kinetics and telomerase expression (Harley, 2008). Several telomerase inhibitors are undergoing active investigation and a few are undertaking clinical trials hinting the development of promising anticancer therapies (Parkinsonhttp://informahealthcare.com/entityImage/?code=200Bâ€Œ, 2003). But numerous issues need to be addressed such as which patient's are most likely to respond to this form of therapy, the lengthy lag period associated with most of the approaches targeting telomerase and telomere. Also, these methods may be more toxic to the normal human somatic cells that do express telomerase and could lead to immediate harmful and unwanted side effects such as hair loss or nausea. The major challenge is to elicit a robust and safe immune response and to overcome the mechanisms of immune-resistance including tumour-derived factors that antagonize cellular immunity and host factors that dampen cellular immune response (Shay and Wright, 2002). Drug resistance may be witnessed in telomerase-independent cancer cells due to the presence of alternative mechanisms that aid in maintaining the telomere. Approaches such as the telomerase template antagonists and small molecule inhibitors would be most suitable for patients with small tumour burden or as an adjuvant therapy along with the conventional drugs. The clinical advancements of these therapies also face the challenge of selection of the most suitable patient population, high quality pharmacodynamic or biological markers to review initial activity, and optimal dose and plan for adjuvant therapies. Another vital concern is the increased genomic instability that could arise in the surviving cells and could eventually lead to more aggressive tumours. However, the preclinical experiments carried out in vitro and in vivo studies have shown encouraging results. It can conclusively be said that further extensive research needs to be carried out and it's more likely that these approaches are likely to enter the domains of cancer chemotherapy.