Absence of telomerase and shortened telomeres have minimal effects on skin and pancreatic carcinogenesis elicited by viral oncogenes

The telomere-stabilizing enzyme telomerase is induced in tumors and functionally associated with unlimited replicative potential. To further explore its necessity, transgenic mice expressing SV40 or HPV16 oncogenes, which elicit carcinomas in pancreas and skin, respectively, were rendered telomerase-deficient. Absence of telomerase had minimal impact on tumorigenesis, even in terc(-/-) generations (G5-7) exhibiting shortened telomeres and phenotypic abnormalities in multiple organs. Analyses of chromosomal aberrations were not indicative of telomere dysfunction or increased genomic instability in tumors. Quantitative image analysis of telomere repeat intensities comparing biopsies of skin hyperplasia, dysplasia, and carcinoma revealed that telomere numbers and relative lengths were maintained during progression, implicating a means for preserving telomere repeats and functionality in the absence of telomerase.     

Haploinsufficiency of hTERT leads to telomere dysfunction and radiosensitivity in human cancer cells

One of the most consistent differences between cancer cells and normal somatic cells is the continuous expression of telomerase, an enzyme that is important for maintenance of chromosome ends, or telomeres. It is believed that telomerase expression allows cancer cells to maintain their telomeres after many cell divisions and thereby avoid replicative senescence. We have tested this hypothesis by targeting the gene encoding the catalytic subunit of the telomerase holoenzyme, hTERT, in a human cancer cell line. Heterozygous disruption of hTERT led to a reduction in telomerase activity, telomere shortening, activation of DNA damage signaling and the appearance of a subpopulation of cells that displayed features of senescence. Targeted cells were radiosensitive, as compared with parental controls that had two intact hTERT alleles, and expressed a classical marker of senescence after irradiation. These results suggest that telomerase inhibitors might be useful in the sensitization of cancer cells to DNA damaging agents.     

Anthracyclines disrupt telomere maintenance by telomerase through inducing PinX1 ubiquitination and degradation

Telomere maintenance is essential for cancer growth. Induction of telomere dysfunction, for example, by inhibition of telomeric proteins or telomerase, has been shown to strongly enhance cancer cells' sensitivity to chemotherapies. However, it is not clear whether modulations of telomere maintenance constitute cancer cellular responses to chemotherapies. Furthermore, the manner in which anti-cancer drugs affect telomere function remains unknown. In this study, we show that anthracyclines, a class of anti-cancer drugs widely used in clinical cancer treatments, have an active role in triggering telomere dysfunction specifically in telomerase-positive cancer cells. Anthracyclines interrupt telomere maintenance by telomerase through the downregulation of PinX1, a protein factor responsible for targeting telomerase onto telomeres, thereby inhibiting telomerase association with telomeres. We further demonstrate that anthracyclines downregulate PinX1 by inducing this protein degradation through the ubiquitin-proteasome-dependent pathway. Our data not only reveal a novel action for anthracyclines as telomerase functional inhibitors but also provide a clue for the development of novel anti-cancer drugs based on telomerase/telomere targeting, which is actively investigated by many current studies.     

KML001 cytotoxic activity is associated with its binding to telomeric sequences and telomere erosion in prostate cancer cells.

PURPOSE: KML001 (sodium metaarsenite) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in prostate cancer. In this study, we elucidated the mode of action of KML001 and investigated its effects on telomerase and telomeres. EXPERIMENTAL DESIGN: We compared telomere length to KML001 cytotoxic activity in a panel of human solid tumor cell lines. Duration of exposure and concentrations of KML001 that affect telomerase and telomeres were evaluated in relation to established mechanisms of arsenite action such as reactive oxygen species-related DNA damage induction. Binding of KML001 to telomeres was assessed by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: We established a significant inverse correlation (r(2) = 0.9) between telomere length and cytotoxicity. KML001 exhibited activity in tumor cells with short telomeres at concentrations that can be achieved in serum of patients. We found that telomerase is not directly inhibited by KML001. Instead, KML001 specifically binds to telomeric sequences at a ratio of one molecule per three TTAGGG repeats leading to translocation of the telomerase catalytic subunit into the cytoplasm. In prostate cancer cells with short telomeres, KML001 caused telomere-associated DNA damage signaling as shown by gamma-H2AX induction and chromatin immunoprecipitation assays as well as a rapid telomere erosion shown by metaphase fluorescence in situ hybridization. These effects were not seen in a lung cancer cell line with long telomeres. Importantly, arsenification of telomeres preceded DNA lesions caused by reactive oxygen species production. CONCLUSIONS: Sodium metaarsenite is a telomere targeting agent and should be explored for the treatment of tumors with short telomeres.     

Recent advances in telomere biology: implications for human cancer

PURPOSE OF REVIEW: Research into the basic biology of telomeres continues to reveal details relevant to fundamental aspects of human cancer. The goal of this review is to highlight discoveries made within the last year, with emphasis on their relevance to cancer prevention, diagnosis, prognostics, and treatment. RECENT FINDINGS: Increasing evidence indicates that dysfunctional telomeres likely play a causal role in the process of malignant transformation, in at least a fraction of human cancers, by initiating chromosomal instability. Telomeres form protective capping structures composed of telomeric DNA complexed with a multitude of associated proteins, the loss of which can have profound effects on telomeric stability. Critical telomeric shortening can lead to telomere "uncapping" and may occur at the earliest recognizable stages of malignant transformation in epithelial tissues. The widespread activation of the telomere synthesizing enzyme telomerase in human cancers not only confers unlimited replicative potential but also prevents intolerable levels of chromosomal instability. Several details regarding telomere structure and telomerase regulation have recently been elucidated, providing new targets for therapeutic exploitation. Various therapeutic strategies aimed at either telomerase or its telomeric substrate are showing promise and may synergize with established anti-cancer agents. Further support for anti-telomerase approaches comes from recent studies indicating that telomerase may possess additional functions, beyond telomere maintenance, that support the growth and survival of tumor cells. SUMMARY: Substantial progress has been made in understanding the complex relationships that exist between telomeres and cancer. However, important issues, such as transient activation of telomerase in normal cells and the potential for tumor cell immortalization via telomerase independent means, remain to be clarified.     

Telomerase template antagonist GRN163L disrupts telomere maintenance, tumor growth, and metastasis of breast cancer.

PURPOSE: Maintenance of telomeres by telomerase is critical for the continuing proliferation of most advanced cancer cells. Telomerase activity has been detected in the vast majority of cancer cells but not most normal cells, making the enzyme an attractive target for anticancer therapy. The aim of this study was to address the breast cancer translational potential of the novel telomerase inhibitor, GRN163L. EXPERIMENTAL DESIGN: In the present study, we investigated the effects of GRN163L treatment on a panel of breast cancer cells representing different tumor subtypes with varying genetic backgrounds, including ER+, ER-, HER2+, BRCA1 mutant breast tumor cells as well as doxorubicin-resistant cancer cells. To investigate the in vivo effects of GRN163L, we employed a breast cancer xenograft and metastasis model that simulates a clinical situation in which a patient arrives with a primary tumor that may be then treated or surgically removed. RESULTS: GRN163L effectively inhibited telomerase activity in a dose-dependent fashion in all breast cancer cell lines resulting in progressive telomere shortening. A mismatch control oligonucleotide showed no effect on telomerase activity and GRN163L did not significantly affect telomere shortening in normal human mammary epithelial cells or in endothelial cells. Breast cancer cells that exhibited telomerase inhibition also exhibited significant reduction in colony formation and tumorigenicity. Furthermore, GRN163L suppressed tumor growth and lung metastases (P = 0.017) of MDA-MB-231 cells in vivo after 4 weeks of treatment. CONCLUSIONS: These results show in vivo effectiveness of GRN163L in breast cancer and support its promising clinical potential for breast cancer treatment.     

Dysfunctional telomeres promote genomic instability and metastasis in the absence of telomerase activity in oncogene induced mammary cancer

Telomerase is a ribonucleoprotein that maintains the ends of chromosomes (telomeres). In normal cells lacking telomerase activity, telomeres shorten with each cell division because of the inability to completely synthesize the lagging strand. Critically shortened telomeres elicit DNA damage responses and limit cellular division and lifespan, providing an important tumor suppressor function. Most human cancer cells express telomerase which contributes significantly to the tumor phenotype. In human breast cancer, telomerase expression is predictive of clinical outcomes such as lymph node metastasis and survival. In mouse models of mammary cancer, telomerase expression is also upregulated. Telomerase overexpression resulted in spontaneous mammary tumor development in aged female mice. Increased mammary cancer also was observed when telomerase deficient mice were crossed with p53 null mutant animals. However, the effects of telomerase and telomere length on oncogene driven mammary cancer have not been completely characterized. To address these issues we characterized neu proto‐oncogene driven mammary tumor formation in G1 Terc?/? (telomerase deficient with long telomeres), G3 Terc?/? (telomerase deficient with short telomeres), and Terc+/+ mice. Telomerase deficiency reduced the number of mammary tumors and increased tumor latency regardless of telomere length. Decreased tumor formation correlated with increased apoptosis in Terc deficient tumors. Short telomeres dramatically increased lung metastasis which correlated with increased genomic instability, and specific alterations in DNA copy number and gene expression. We concluded that short telomeres promote metastasis in the absence of telomerase activity in neu oncogene driven mammary tumors. © 2011 Wiley Periodicals, Inc.     

Telomerase and telomere dynamics in ageing and cancer: current status and future directions

Abstract This review will focus on the clinical utilities of telomerase for human cancer diagnosis and prognosis. Much attention has been focused on control of telomerase activity in early and late stage tumours. Telomerase stabilisation may be required for cells to escape replicative senescence and to proliferate indefinitely. Because of a very strong association between telomerase and malignancy, both clinicians and pathologists expect this molecule to be a useful diagnostic and prognostic marker and a new therapeutic target. These data have greatly inspired the development of various strategies to target telomere and telomerase for cancer therapy. Finally, evidence is now emerging that G-quadruplex ligands produce rapid senescence and selective cell death. A summary of recent experimental works with new small molecules as potential inhibitors of telomerase is presented. *Supported by an unrestricted educational grant from Pfizer.     

Telomere length maintenance in aging and carcinogenesis

Normal somatic cells have a finite number of divisions, a limited capacity to proliferate. Human telomeres, the long DNA TTAGGG repeats at the ends of chromosomes, are considered a molecular clock marker. The gradual and progressive telomere shortening at each replicative cycle is associated, through the activation of pRB and p53 pathways and genomic instability, to the replicative senescence, a non-dividing state and widespread cell death. Activation of telomere maintenance [telomerase; or alternative lengthening of telomeres mechanisms (ALT), or other adaptive responses] can revert this program. Although not completely known, several mechanisms and modulating agents may be able to up and down-regulate telomere length and its maintenance. Chemopreventive therapies for the up-regulation of telomerase activity, able to prolong the life of cell cultures in a phenotypically youthful state, could have important applications in research and medicine. On the contrary the therapeutic down-regulation of telomerase activity may be used in cancer therapy. Telomerase expression per se is not oncogenic, but telomere shortening and maintenance seem to be crucial events in tumor formation. Thus a particular focus has been pointed out relatively to the immortalization of normal or potential pre-cancerous cells. With the extension of life span the probability to get in contact with carcinogens increases, genetic instability, oncogene activation and/or onco-suppressor gene inactivation (i.e. p53, pRB, ras): the cancer transformation can be then induced in predisposed cells, depending on their genetic context, by the activation of telomere maintenance. Pharmacological intervention may be able to modulate the rate of living, by increasing life span of few specific target cells, or decreasing it in proliferating . Because of the unknown state of the enormous cell number of the human organism, is it safe to extend the human life span by therapeutic agents?     

A human cell line that maintains telomeres in the absence of telomerase and of key markers of ALT

In human somatic cells proliferation results in telomere shortening due to the end replication problem and the absence of adequate levels of telomerase activity. The progressive loss of telomeric DNA has been associated with replicative senescence. Maintenance of telomere structure and function is, therefore, an essential requisite for cells that proliferate indefinitely. Human cells that have acquired the immortal phenotype mostly rely on telomerase to compensate for telomere shortening with cell division. However, a certain percentage of immortalized cell lines and human tumors maintain their telomeres by Alternative Lengthening of Telomeres (ALT), a mechanism not fully understood but apparently based on homologous recombination. Here, we report the isolation of an immortal human cell line that is derived from an ALT cell line but maintains telomeres in the absence of key features of ALT and of telomerase. The properties of these cells suggest that the identification of ALT cells may not be reliably based on known ALT markers. This finding is of relevance for discriminating between the mortal and immortal phenotype among telomerase-negative cells in vitro and in vivo, particularly in regard to the development of pharmacological approaches for cancer treatment based on telomerase inhibition.     

Multiple mechanisms of telomere maintenance exist in liposarcomas.

PURPOSE: Telomeres are specialized nucleoprotein complexes that protect and confer stability upon chromosome ends. Loss of telomere function as a consequence of proliferation-associated sequence attrition results in genome instability, which may facilitate carcinogenesis by generating growth-promoting mutations. However, unlimited cellular proliferation requires the maintenance of telomeric DNA; thus, the majority of tumor cells maintain their telomeres either through the activity of telomerase or via a mechanism known as alternative lengthening of telomeres (ALT). Recent data suggest that constitutive telomere maintenance may not be required in all tumor types. Here we assess the role and requirement of telomere maintenance in liposarcoma. EXPERIMENTAL DESIGN: Tumor samples were analyzed with respect to telomerase activity, telomere length, and the presence of ALT-specific subcellular structures, ALT-associated promyelocytic leukemia nuclear bodies. This multi-assay assessment improved the accuracy of categorization. RESULTS: Our data reveal a significant incidence (24%) of ALT-positive liposarcomas, whereas telomerase is used at a similar frequency (27%). A large number of tumors (49%) do not show characteristics of telomerase or ALT. In addition, telomere length was always shorter in recurrent disease, regardless of the telomere maintenance mechanism. CONCLUSIONS: These results suggest that approximately one half of liposarcomas either employ a novel constitutively active telomere maintenance mechanism or lack such a mechanism. Analysis of recurrent tumors suggests that liposarcomas can develop despite limiting or undetectable activity of a constitutively active telomere maintenance mechanism.     

靶向端粒端粒酶的抗肿瘤治疗研究进展*

端粒是人类染色体末端由重复核酸序列组成的保护性结构,会随着细胞成功的分裂进行性的缩短。超过85% 的肿瘤细胞通过激活在大多数正常体细胞中被抑制的端粒酶来阻止端粒的无限缩短,维持细胞的永生化。肿瘤细胞跟正常细胞相比,有着更短的端粒和被重新激活的端粒酶,这些简单却又特殊的生物学差异促进了靶向端粒/ 端粒酶抗肿瘤治疗的发展。近年来许多成功的治疗药物经过临床前的筛选,在多种肿瘤中取得Ⅰ/ Ⅱ期临床试验的成功,GRN 163L 和GV1001等药物已进入Ⅲ期临床试验。联合传统药物治疗是目前的发展方向,未来靶向端粒/ 端粒酶治疗联合放射治疗可能在取得抗肿瘤疗效叠加的同时,提高治疗的安全性。      

Telomerase is frequently activated in tumors with microsatellite instability

Telomeres are specialized structures at the ends of eukaryotic chromosomes that are required for the complete replication and stability of naturally occurring chromosome ends. Telomere stabilization is critical for the unlimited cellular proliferation that is necessary for tumor formation. While most tumors achieve telomere stabilization through activation of telomerase, a subset of tumors utilize a recombination-based mechanism termed Alternative Lengthening of Telomeres (ALT) to maintain chromosome termini. Tumors utilizing ALT for telomere preservation will likely be refractory to treatment with telomerase inhibitors. Furthermore, tumors carrying mutations that predispose a cell to utilize ALT may activate this pathway when challenged by telomerase inhibition. Mutation of the mismatch repair (MMR) pathway enhances telomerase independent survival in yeast, with the survivors using recombination-based pathways for telomere maintenance. One possibility is that mutation of the MMR pathways alleviates suppression of recombination, thereby abrogating the need for telomerase activation. If true, one might predict an increased frequency of tumors harboring MMR mutation to use ALT for telomere maintenance. Here we characterized tumors with and without MMR mutation for the presence of telomerase activity versus ALT. We found similarly frequent activation of telomerase in tumors with and without MMR mutation, suggesting that human tumors with MMR mutation may respond favorably to treatment with telomerase inhibitors.