Current Pharmaceutical Design - Volume 20, Issue 41, 2014

The discovery of new cancer-related targets and the development of innovative therapeutic interventions are mainly based on the identification of genes involved in pathways selectively deregulated in cancer cells. The unlimited replicative potential is one of the principal hallmarks of cancer and it requires the activation of telomere maintenance mechanisms (TMMs). Two TMMs are currently known in human cancer, namely telomerase activity and the alternative lengthening of telomere (ALT) mechanisms. Although both TMM appear to be equivalent in their ability to support immortalization, their contribution to tumor growth and survival, and consequently patients’ prognosis, may differ. In the opening review article of the present hot topic issue of Current Pharmaceutical Design, Reddel R. [1] outlines the opportunities and challenges presented by telomeres and TMMs for the clinical management of cancer. Different methods currently used to assess the presence of individual TMM in clinical tumor specimens and the information collected thus far concerning the diagnostic, prognostic and therapeutic potential of TMM are also deeply discussed. Telomeres are specialized DNA-protein structures located at the end of eukaryotic chromosomes. They are essential for continued cell proliferation. Indeed, telomere attrition, which occurs within each cell division, represents a molecular clock that counts the number of times a cell can divide and determines its entry into senescence [1]. Other than acting as a mitotic clock, telomeres play an important role in the maintenance of genomic integrity. As pointed out by Feijoo P. et al. [2], telomere erosion, in a context of impaired cell cycle checkpoint, may constitute an important mutator mechanism during tumoriogenesis. Telomere-driven chromosome instability and the consequent fusion-bridgebreakage cycles represent a mechanism that drives the formation of the unbalanced chromosome rearrangements responsible for the changes in gene dosage as well as the aberrations in the karyotype (e.g., aneuploid, polyploid) that are relevant events on the way toward cancer development [2]. Saretzki G. reports on accumulating evidence showing that telomerase is interconnected with different pathways involved in cell proliferation, thus favoring tumor cell survival independently of its activity at telomere ends (i.e., extra-telomeric functions). These telomere-independent activities of telomerase influence essential cellular processes, such as gene expression, signaling pathways, mitochondrial function as well as cell survival and stress resistance. Understanding these different mechanisms and their complexity in cancer cells might help to design more effective cancer therapies in the future [3]. At present, scanty information is available regarding the molecular underpinnings leading to the establishment of a specific TMM in tumors, but there are indications that a complex network of regulatory factors might be involved. Owing to their documented role in cancer development and progression, it has been reported that the deregulated expression levels of specific microRNAs (i.e., small non-coding RNAs that negatively regulate gene expression) may likely contribute to the activation of a TMM rather than the other during tumorigenesis or to play a role in any aspect of telomere biology. In this context, Santambrogio F. et al. [4] report on available data concerning microRNAs that have been shown to impair telomerase activity or to affect telomere functions in cancer cells. Such a research field is still in its infancy but available data indicate that microRNA-based approaches are promising tools for anticancer therapeutic interventions. Consequently, the possibility to identify microRNAs specifically associated to a TMM may provide useful and innovative therapeutic tools or targets to interfere with the unlimited replicative potential of tumor cells [4]. Since telomerase is ubiquitously expressed in a wide range of human tumors, it has been considered as a good target for anti-cancer therapies. Crees Z. et al. [5], Romaniuk A. et al. [6] and Uziel O. and Lahav M. [7] describe the approaches developed during the last decades to inhibit telomerase, including strategies aimed to interfere with the enzyme’s catalytic activity, the expression of its core subunits and the signalling pathways responsible for the transcriptional regulation and post-transcriptional/translational modifications of the enzyme components. Overall, accumulating evidence from preclinical studies on the effects of telomerase inhibition in human cancer has provided persuasive arguments to indicate that the enzyme is a well-validated cancer target and an ideal tumor-associated antigen [5-7]. Specifically, the interference with telomerase activity or the expression of its components results in the decline of tumor growth as a consequence of progressive telomere erosion, telomere uncapping and/or impairment of the extra-telomeric and pro-survival functions of the enzyme. In contrast, although ALT phenotype is thought to be driven by a recombination–based mechanism, factors that can act as the main engine of this pathway have not yet been fully enumerated. Consequently, inhibitors that could specifically target such a TMM in cancer have not been reported thus far. Whether or not anti-telomerase therapies may be hampered by the emergence of possible adaptive responses is still a matter of debate. It is plausible to hypothesize that prolonged treatment with telomerase inhibitors may exert a selective pressure for the emergence of cells that could become resistant to treatment by activating ALT mechanisms. This notion, together with the evidence that ALT is activated in a significant fraction of solid tumors and that both TMM may coexist within the same tumor, suggests that ALT may exert an unprecedented role in tumor biology and may impinge on the clinical efficacy of telomerase inhibitors and on patients’ outcome [1]. As highlighted by Draskovic I. and Londoño-Vallejo A. [8], the development of ALT-specific therapeutic interventions is of pivotal importance and a better understanding of the molecular details responsible for the ALT-associated recombination activity is urgently warrented. In this context, strategies aimed at preventing telomere recruitment to APBs (the platform where the ALT-associated recombination events seem to occur), strand invasion and annealing (the first molecular events in the recombination process), DNA synthesis and the final template/substrate resolution of the ALTmediated telomere elongation reaction have been suggested to be useful for the identification of potential targets specifically relevant to ALT [8]. Other than interfering with telomerase activity/expression, additional therapeutic opportunities at telomeric level have been envisaged, such as the targeting of telomere-associated proteins (e.g., tankyrase) or the stabilization of telomeric G4 structures. As pointed out by Haikarainen T. et al. [9], tankyrase is a poly(ADP)-ribosylase that plays a pivotal role in several cellular processes, including telomere length regulation, glucose metabolism and cell cycle progression, thus emerging as a promising target for different pathological conditions, including cancer. In this context, the development of tankyrase inhibitors has recently received much attention and several small molecules with the characteristics of lead compounds to be used for proof-of-concept studies in cancer and other tankyrase-related disease have been described [9]. Due to their distinctive structural features likely connected with varied cellular functions, non-B DNA conformations, such as G-quadruplexes (G4), represent attractive targets for drug design. Sissi C. and Palumbo M. [10] report that the elucidation of the telomeric G4 structures has led to the rational development of effective G4-stabilizing agents belonging to different chemical classes and including, among others, natural products (e.g., telomestatin, sanguinarine), polycyclic compounds (e.g., perylenes, naphthalene diimides) and porphyrines (e.g. TMPyP4). Due to the inability of telomerase to extend a G4-folded telomeric substrate, G4 stabilizing agents have been primarily considered as indirect telomerase inhibitors [5-7]. However, it has been reported that G4 ligands may trigger telomere uncapping/dysfunctions, resulting in a rapid induction of programmed cell death in a variety of tumors, as a consequence of their interference with the complex architecture of telomeres [5-7]. This evidence clearly suggests that the anticancer effect of G4 ligands may be largely independent of the presence of active telomerase and, consequently, represent attractive therapeutic agents also for ALT-positive tumor cells. Finally, several findings indicated that interference with telomerase expression/function as well as the stabilization of telomeric G4 structures lead to increased sensitivity of tumor cells to conventional anticancer agents and radiation [2,7]. Nevertheless, telomerase-based cancer therapeutics is moving very slowly to the clinical setting. In this context, active immunotherapy (e.g., GV1001, GRNVAC1, Vx-001) and the unique antagonist (i.e., Imetelstat/GRN163L) of human telomerase are the only telomerase-based therapeutic approaches that have thus far entered clinical trials to defeat cancer [5-7]. The purpose of this special issue, thanks to the contribution of eminent experts in the field, is to give an up-to-date view of the present knowledge about the tight link between telomere biology/maintenance and cancer and how such a network could be exploited to identify novel targets for the development of rationally conceived anticancer therapies.

The presence of immortal cell populations with an up-regulated telomere maintenance mechanism (TMM) is an almost universal characteristic of cancers, whereas normal somatic cells are unable to prevent proliferation-associated telomere shortening and have a limited proliferative potential. TMMs and related aspects of telomere structure and function therefore appear to be ideal targets for the development of anticancer therapeutics. Such treatments would be targeted to a specific cancer-related molecular abnormality, and also be broad-spectrum in that they would be expected to be potentially applicable to most cancers. However, the telomere biology of normal and malignant human cells is a relatively young research field with large numbers of unanswered questions, so the optimal design of TMM-targeted therapeutic approaches remains unclear. This review outlines the opportunities and challenges presented by telomeres and TMMs for clinical management of cancer.

Most cancer genomes show abnormalities in chromosome structure and number, two types of aberrations that could share a common mechanistic origin through proliferation-dependent loss of telomere function. Impairment of checkpoints that limit cell proliferation when telomeres are critically short might allow unrestrained cell division. The resulting uncapped chromosomes can fuse to each other, forming unstable configurations that can bridge during mitosis. Chromatin bridges can break to generate new broken ends that will then fuse with other broken ends. Successive events of break and fusion will continuously generate unbalanced chromosomal rearrangements, leading to gene-copy gains and losses. However, chromosome bridges do not always break. Evidence has recently been obtained to suggest that telomere-dependent chromosome bridges remaining unbroken can hinder cytokinesis and yield tetraploid cells. This might constitute an unstable intermediate in tumorigenesis, as progressive losses of individual chromosomes due to geometrical defects during cell division result in subtetraploid karyotypes. Additionally, the presence of short dysfunctional telomeres in cells can also cause these cells to become sensitive to mutagens, and particularly to radiation exposure. Human individuals exhibit differences in their sensitivity to radiation, which can be relevant for choice of therapy. Telomere function may well be involved in cellular and organism responses to ionizing radiation. Since eroded telomeres are sensed and act as double-strand breaks, they can interact with radiation-induced breaks, sharply increasing the possibility of misjoining. Altogether, this scenario provides certain clues to understanding the important role of telomeres in maintaining genomic integrity.

Telomerase activity is essential for human cancer cells in order to maintain telomeres and provide unlimited proliferation potential and cellular immortality. However, additional non-telomeric roles emerge for the telomerase protein TERT that can impact tumourigenesis and cancer cell properties. This review summarises our current knowledge of non-telomeric functions of telomerase in human cells, with a special emphasis on cancer cells. Non-canonical functions of telomerase can be performed within the nucleus as well as in other cellular compartments. These telomereindependent activities of TERT influence various essential cellular processes, such as gene expression, signalling pathways, mitochondrial function as well as cell survival and stress resistance. Emerging data show the interaction of telomerase with intracellular signalling pathways such as NF-ΚB and WNT/β-catenin; thereby contributing to inflammation, epithelial to mesenchymal transition (EMT) and cancer invasiveness. All these different functions might contribute to tumourigenesis, and have serious consequences for cancer therapies due to increased resistance against damaging agents and prevention of cell death. In addition, TERT has been detected in non-nuclear locations such as the cytoplasm and mitochondria. Within mitochondria TERT has been shown to decrease ROS generation, improve respiration, bind to mitochondrial DNA, increase mitochondrial membrane potential and interact with mitochondrial tRNAs. All these different non-telomere-related mechanisms might contribute towards the higher resistance of cancer cells against DNA damaging treatments and promote cellular survival. Understanding these different mechanisms and their complexity in cancer cells might help to design more effective cancer therapies in the future.

The activation of telomere maintenance mechanisms, which rely on telomerase reactivation or on a recombination-based process known as alternative lengthening of telomeres, guarantees a limitless proliferative potential to human tumor cells. To date, the molecular underpinnings that drive the activation of telomere maintenance mechanisms during tumorigenesis are poorly understood, but there are indications that complex signaling networks might be involved. Since telomerase activity has been mainly detected in tumors of epithelial origin and the alternative lengthening of telomere mechanisms is more frequently expressed in mesenchymal and neuroepithelial cancers, it could be hypothesized that cell-type specific mechanisms can favor their activation during tumor development. In this context, microRNAs – small non coding RNAs that regulate gene expression at post-transcriptional level - have emerged as key players in the development and progression of human cancers, being involved in the control of all the typical features of cancer cells, including the limitless replicative potential. In the present review, we will summarize the recent findings concerning the identification and biological validation of microRNAs which may play a role in the regulation of telomere biology as well as of the mechanisms that govern telomere maintenance.

Cancer is a leading cause of death worldwide and an estimated 1 in 4 deaths in the United States is due to cancer. Despite recent advances in cancer treatment, adverse effects related to cancer therapy remain a limiting factor for many patients. The ideal cancer treatment would selectively target cancerous cells while sparing normal, healthy cells to offer maximal therapeutic benefit while minimizing toxicity. Telomeres are structurally unique DNA sequences at the end of human chromosomes, which play an integral role in the cellular mortality of normal cells. As telomeres shorten with successive cellular divisions, cells develop chromosomal instability and undergo either apoptosis or senescence. In many cancers, this apoptosis or senescence is avoided as normal telomere length is maintained by a ribonucleoprotein reverse transcriptase called telomerase. Telomerase is expressed in more than 85% of all cancers and confers cancerous cells with a replicative immortality, which is a hallmark of malignant tumors. In contrast, telomerase activity is not detectable in the majority of normal somatic cell populations. Therefore, the targeting of telomerase and telomere maintenance mechanisms represent a potentially promising therapeutic approach for various types of cancer. This review evaluates the roles of GRN163L, T-oligo and small molecule G-quadruplex stabilizers as potential anticancer therapies by targeting telomerase and other telomere maintenance mechanisms.

Telomerase is a specialized enzymatic complex responsible for the synthesis of telomeric repeats 5'-TTAGGG-3' localized at the ends of eukaryotic chromosomes. This mechanism prevents shortening of telomeres after each cell division. The enzyme is detected in about 85% of human tumors, but it is not expressed in normal cells or its expression is significantly lower. Consequently, it provides the cancer cells immortality. Thus, since showing cancer cell specificity (to a certain extent), the enzyme became a target for an adjuvant cancer therapy. So far, in vitro studies and preclinical studies seem to be promising. This work focuses on the pathways and mechanisms that are targeted in order to eliminate telomerase with consequence of cancer cell death. The anti-telomerase strategy may be beneficial especially in the context of sensitization of tumor cell to chemotherapeutic agents. We also indicate potential side effects and consequences of telomerase downregulation that should be considered when anti-telomerase strategy is undertaken. Alternatively, we also emphasize potential useful application of telomerase induction.

The telomere-telomerase system has a unique role in the biology of cancer. Telomere maintenance, mostly affected by the up regulation of telomerase activity, is a prerequisite for perpetuation of malignant cells. This fundamental biologic feature defines telomere maintenance as an attractive therapeutic target for most types of cancer. This review summarizes some critical aspects of telomere biology with special emphasis on the connection to anticancer therapy. In particular, the effects on the telomere - telomerase system of conventional anticancer treatments, including various cytotoxic drugs, targeted biological agents and radiotherapy, and their possible combination with telomerase-directed therapy are discussed. Several potential problems, including side effects and complications inherent to perturbations of telomere biology in normal cells, are also highlighted. In spite of significant progress in this field, there are still several issues that have to be addressed and ultimately resolved in order to obtain a better characterization of the pros and cons of telomerasedirected therapies and, consequently, their clinical relevance.

Telomeres are essential for cell proliferation and tumor cell immortalization requires the presence of a telomere maintenance mechanism. Thus, interfering with this mechanism constitutes a potential means to impede cell proliferation and tumor progression. Many cancer cells rely on telomerase activity to ensure indefinite proliferation capacity and developing therapeutic approaches that target telomerase has attracted much attention in the last couple of decades. Nevertheless, a non-negligible proportion of tumors utilize telomerase- independent, alternative mechanisms to lengthen telomeres (ALT). Here we briefly discuss both our current understanding of ALT mechanisms and the potential to develop a therapeutic approach targeting ALT.

Several cellular signaling pathways are regulated by ADP-ribosylation, a posttranslational modification catalyzed by members of the ARTD superfamily. Tankyrases are distinguishable from the rest of this family by their unique domain organization, notably the sterile alpha motif responsible for oligomerization and ankyrin repeats mediating protein-protein interactions. Tankyrases are involved in various cellular functions, such as telomere homeostasis, Wnt/β-catenin signaling, glucose metabolism, and cell cycle progression. In these processes, Tankyrases regulate the interactions and stability of target proteins by poly (ADP-ribosyl)ation. Modified proteins are subsequently recognized by the E3 ubiquitin ligase RNF146, poly-ubiquitinated and predominantly guided to 26S proteasomal degradation. Several small molecule inhibitors have been described for Tankyrases; they compete with the co-substrate NAD+ for binding to the ARTD catalytic domain. The recent, highly potent and selective inhibitors possess several properties of lead compounds and can be used for proof-of-concept studies in cancer and other Tankyrase linked diseases.

The discovery of novel nucleic acid folding architectures bears a twofold interest related to the structural properties of unprecedented forms and to their functional significance. In addition, physiologically and pathologically important processes can be impaired by endogenous or xenobiotic ligands interacting with specific target sequences. In this paper we will focus on recent advances in the study of telomeric G-quadruplex DNA and RNA structures and the rational design and development of synthetic ligands aimed at pharmacological applications.