hTERT-Targeted Library
INTRODUCTION
Telomeres are located at the distal ends of the chromosomes and the shortening of which with successive cycles of cell division leads to cell senescence and cell death. Human telomerase, a cellular reverse transcriptase, is a ribonucleoprotein enzyme that catalyzes the synthesis and extension of telomeric DNA. It is composed of at least, a template RNA component (hTR; human Telomerase RNA) and a catalytic subunit, the telomerase reverse transcriptase (hTERT). Except germline cells, activated lymphocytes and some stem cell populations, most adult somatic cells do not express hTERT [1]. In cells where telomerase is activated, hTERT synthesizes a TTAGGG sequence from the RNA template that is then added to the end of the shortening chromosome [2], thus saving the cells from death. The above mechanism is cleverly exploited by tumor cells to maintain their immortality [3]. Together with its universal expression, hTERT represents an ideal target for cancer therapy [4,5].
Telomerase represents a prototype of a universal tumor antigen due to both its expression by the vast majority of tumors and its inherent functional involvement in oncogenic transformation. The absence of telomerase is associated with telomere shortening and aging of somatic cells, while high telomerase activity is observed in over 90% of human cancer cells, strongly indicating its key role during tumorigenesis [6]. Several details regarding telomere structure and telomerase regulation have already been elucidated, providing new targets for therapeutic exploitation. Given these attractive features, the identification of epitopes within hTERT, the catalytic subunit of telomerase, has led to the investigation of this tumor antigen as a broadly applicable immunological target [7]. Further support for anti- telomerase approaches comes from recent studies indicating that telomerase is endowed of additional functions in the control of growth and survival of tumor cells that do not depend only on the ability of this enzyme to maintain telomere length. This observation suggests that inhibiting telomerase or its synthesis may have additional anti-proliferative and apoptosis inducing effect, independently of the reduction of telomere length during cell divisions. Here we provide the basic information about the biology of telomeres and telomerase and attempt to present various approaches that are currently under investigation to inhibit its expression and its activity.
In the past decade, research in the field of telomerases has progressed tremendously, especially in relation to cellular immortality and carcinogenesis. As mentioned above, telomerase activation is observed in a vast majority of human cancers, irrespective of tumor type, while most normal tissues contain inactivated telomerase. The role and timing of telomerase activation in carcinogenesis has been revealed by telomerase-knockout mouse studies [8]. Significant telomere erosions and age- and generation-dependent increases in cytogenic abnormalities are exhibited in telomerase-knockout mice, providing evidence that telomere dysfunction with critically short telomeres causes genomic instability. This concept is further supported by studies using telomerase–/– p53–/– double-knockout mice. These mouse cells demonstrate high levels of genomic instability, exemplified by increases in both formation of dicentric chromosomes and susceptibility to oncogenic transformation. These mice exhibit significantly decreased tumor latency and overall survival. Thus, in the absence of genome checkpoint functions, telomere dysfunction accelerates genomic instability, facilitating cancer initiation [9]. According to this concept, the genomic instability caused by telomere dysfunction occurs in the early stages of carcinogenesis, before telomerase activation. Subsequently, telomeres in these initiated cells undergo further progressive shortening, generating rampant chromosomal instability and threatening cell survival. Telomerase activation necessarily occurs at this stage to stabilize the genome and confer unlimited proliferative capacity upon the emerging and evolving cancer cell. In other words, cells that have acquired telomerase activity can obtain the capacity for cancer progression. Eventually, most cancer cells exhibit telomerase activity. This cancer-specific telomerase activity provides an opportunity for us to utilize it for the design of target-specific library.
Continuous effort has been made to uncover the molecular mechanisms of telomerase activation during carcinogenesis. The hTERT gene is regulated by androgens as well as by different oncogenes including Her-2, Ras, c-Myc and Bcl-2, which seem to play an important role in cancer grow and progression. The discovery of the telomerase subunit hTERT [10], a catalytic subunit bearing the enzymatic activity of telomerase, [11] was the starting point for uncovering the cancerspecific activation of telomerase. Numerous studies have demonstrated that hTERT expression is highly specific to cancer cells and tightly associated with telomerase activity, while the other subunits are constitutively expressed both in normal and cancer cells [12]. Therefore, there is no doubt that hTERT expression plays a key role in cancer-specific telomerase activation.
1. hTERT inhibitors
Telomerase is an attractive target for anti-cancer therapeutics due to its requirement for cellular immortalization and expression in human neoplasms [13]. Because telomerase activity is essential for proliferation of most cancer cells, therapeutic strategies have been developed to inhibit its activity. These strategies centre on targeting the active site, hTERT and hTERC expression, core enzyme stability and telomeric DNA [14]. Successful approaches involve a combination of traditional drugs with telomerase inhibitors. Though initially promising, strategies that inhibit telomerase with either small molecules or antisense oligonucleotides have a major limitation, namely the lag time required for telomere shortening before cellular effects are attained. As alternative approaches, immunotherapy and gene therapy have been tailored to exploit, rather than antagonize telomerase expression and/or activity. Several Phase I studies of hTERT immunotherapy have been completed in patients with breast, prostate, lung and other cancers, and clinical and immunological results are encouraging. Immunotherapy induces functional, antitumour T cells in patients in the absence of clinical toxicity. It requires the presence of the catalytic subunit of telomerase, hTERT, to elicit an immune response directed towards hTERT peptide-presenting cells. hTERT promoter-driven gene therapy and mutant telomerase RNA (hTR) gene therapy depend on the innate telomerase activity of cancer cells to drive the expression of pro-apoptotic genes and to synthesize mutated DNA sequences onto telomeres, respectively. In addition, telomestatin, a G- quadruplex binding ligand may exert anti-proliferative effects independently of telomere shortening. Disrupting the functional expression of hTERT is particularly effective in agreement with evidence that hTERT is an antiapoptotic factor in some cancer cells. In addition, approaches that stabilise DNA secondary structures may disrupt telomere maintenance through a variety of routes making them, potentially, very potent in attacking cancer cells.
Compounds currently under development that seek to inhibit hTERT, the reverse transcriptase component of telomerase, include nucleoside analogs and the small molecule BIBR1532 [15]. Compounds inhibiting the RNA component of telomerase, hTERC, include peptide nucleic acids, 2-5A antisense oligonucleotides, and N3'-P5' thio-phosphoramidates. Recently, an oligonucleotide sharing sequence homology with terminal telomeric DNA, termed 'T-oligo', has shown cytotoxic effects in multiple cancers in culture and animal models. Independent of telomerase function, T-oligo is thought to mimic the DNA-damage response a cell normally experiences when the telomere t-loop structure becomes dysfunctional [16].