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.     

Ribozyme-mediated telomerase inhibition induces immediate cell loss but not telomere shortening in ovarian cancer cells.

Telomerase is a promising target for human cancer gene therapy. Its inhibition allows telomere shortening to occur in cancer cells, which in turn is thought to trigger delayed senescence and/or apoptosis. We tested whether telomerase inhibition might have additional, immediate effects on tumor cell growth. Ovarian cancer cell lines with widely differing telomere lengths were efficiently transduced with an adenovirus expressing a ribozyme directed against the T motif of the catalytic subunit of human telomerase, hTERT. Three days after transduction, telomerase activity was significantly reduced and massive cell loss was induced in mass cultures from all four ovarian cancer cell lines tested, whereas transduction of telomerase-negative human fibroblasts did not attenuate their growth. The kinetics of induction of cell death in cancer cells was not significantly dependent on telomere length, and telomeres did not shorten measurably before the onset of apoptosis. The data suggest the existence of a "fast-track" mechanism by which diminution of telomerase can interfere with cancer cell growth and induce cell death, presumably by apoptosis. This phenomenon might be a consequence of the telomere capping function provided by telomerase in tumor cells. Uncapping of telomeres by ribozyme-mediated inhibition of telomerase bears therapeutic potential for ovarian cancer.     

Telomerase-independent telomere length maintenance in the absence of alternative lengthening of telomeres-associated promyelocytic leukemia bodies

Immortal tumor cells and cell lines employ a telomere maintenance mechanism that allows them to escape the normal limits on proliferative potential. In the absence of telomerase, telomere length may be maintained by an alternative lengthening of telomeres (ALT) mechanism. All human ALT cell lines described thus far have nuclear domains of unknown function, termed ALT-associated promyelocytic leukemia bodies (APB), containing promyelocytic leukemia protein, telomeric DNA and telomere binding proteins. Here we describe telomerase-negative human cells with telomeres that contain a substantial proportion of nontelomeric DNA sequences (like telomerase-null Saccharomyces cerevisiae survivor type I cells) and that are maintained in the absence of APBs. In other respects, they resemble typical ALT cell lines: the telomeres are highly heterogeneous in length (ranging from very short to very long) and undergo rapid changes in length. In addition, these cells are capable of copying a targeted DNA tag from one telomere into other telomeres. These data show that APBs are not always essential for ALT-mediated telomere maintenance.     

Telomerase activity in cell lines and lymphoma originating from Bloom syndrome.

Bloom syndrome (BS) is characterized by premature aging and high predisposition to various types of cancer. BLM is the causative gene for BS. BLM functions as a DNA helicase in the direction of 3' to 5' and small subsets of telomeres colocalize with BLM protein. We investigated telomerase activity and telomere repeat length in the cells from BS patients. In Epstein-Barr-virus (EBV) transformed lymphoblastoid cell lines and lymphoma cells from BS patients, telomerase activity was detected as in the control and compared. The metastatic tumor from BS patient, which had a 9-bp deletion of p53 DNA showed the strongest telomerase activity. Telomere repeat length in BS cells showed that there is no large difference compared with normal cells. Collectively, the results show that the BLM gene is not a major structural and regulatory factor in maintaining telomere repeat length and telomerase activity.     

Tumour-cell apoptosis after cisplatin treatment is not telomere dependent

Cisplatin is a major chemotherapeutic agent, especially for the treatment of neuroblastoma. Telomeres with their sequence (TTAGGG)n are probable targets for cisplatin intrastrand cross-linking, but the role of telomeres in mediating cisplatin cytotoxicity is not clear. After exposure to cisplatin as single dose or continuous treatment, we found no loss of telomeres in either SHSY5Y neuroblastoma cells (telomere length, approximately 4 kbp), HeLa 229 cells (telomere length, 20 kbp) or in the acute lymphoblastic T cell line 1301 (telomere length, approximately 80 kbp). There was no induction of telomeric single strand breaks, telomeric overhangs were not degraded and telomerase activity was down-regulated only after massive onset of apoptosis. In contrast, cisplatin induced a delayed formation of DNA strand breaks and induced DNA damage foci containing gamma-H2A.X at nontelomeric sites. Interstitial DNA damage appears to be more important than telomere loss or telomeric damage as inducer of the signal pathway towards apoptosis and/or growth arrest in cisplatin-treated tumour cells.     

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo

PURPOSE: The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. EXPERIMENTAL DESIGN: Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. RESULTS: Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. CONCLUSIONS: We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.     

Telomere length and the risk of lung cancer

Telomeres play a key role in the maintenance of chromosome integrity and stability. There is growing evidence that short telomeres induce chromosome instability and thereby promote the development of cancer. We investigated the association of telomere length and the risk of lung cancer. Relative telomere length in peripheral blood lymphocytes was measured by quantitative polymerase chain reaction in 243 lung cancer patients and 243 healthy controls that were frequency-matched for age, sex and smoking status. Telomere length was significantly shorter in lung cancer patients than in controls (mean ± standard deviation: 1.59 ± 0.75 versus 2.16 ± 1.10, P  < 0.0001). When the subjects were categorized into quartiles based on telomere length, the risk of lung cancer was found to increase as telomere length shortened ( P _trend < 0.0001). In addition, when the median of telomere length was used as the cutoff between long and short telomeres, individuals with short telomeres were at a significantly higher risk of lung cancer than those with long telomeres (adjusted odds ratio = 3.15, 95% confidence interval = 2.12–4.67, P  < 0.0001). When the cases were categorized by tumor histology, the effect of short telomere length on the risk of lung cancer was more pronounced in patients with small cell carcinoma than in those with squamous cell carcinoma and adenocarcinoma ( P =  0.001, test for homogeneity). These findings suggest that shortening of the telomeres may be a risk factor for lung cancer, and therefore, the presence of shortened telomeres may be used as a marker for susceptibility to lung cancer. ( Cancer Sci 2008; 99: 1385–1389)     

Constitutional short telomeres are strong genetic susceptibility markers for bladder cancer

Lack of functional telomeres can cause chromosomal aberrations. This type of genetic instability may promote tumorigenesis. We have investigated the association between mean telomere length in buccal cells (assessed with quantitative real-time PCR) and bladder cancer risk in a case-control study. Patients with bladder cancer displayed significantly shorter telomeres than control subjects (P = 0.001). Median telomere length ratio was 0.95 (range 0.53-3.2) for cases and 1.1 (0.51-2.4) for controls. Moreover, the adjusted odds ratio (OR) for bladder cancer was significantly increased in the quartile with the shortest telomere length OR = 4.5 [95% confidence interval (CI) 1.7-12]. It is known that oxidative stress, alkylation or UV radiation increases shortening of telomeres. Therefore, we also analyzed whether environmental and genetic factors associated with DNA damage, i.e. smoking and polymorphisms in the genes involved in the metabolism of genotoxic carcinogens (EPHX1, GSTA1, GSTM1, GSTP1, GSTT1, NAT1, NAT2 and NQO1) or DNA repair (APE1, NBS1, XPC, XPD, XRCC1, XRCC3 and XRCC4), could modify the association between telomere length and cancer risk. A clear effect of smoking and telomere length could be observed. Current smokers with short telomeres had more than six times as higher risk as non-smokers/former smokers with long telomeres (OR = 6.3, 95% CI 1.7-23). Lack of the biotransformation gene GSTM1 and short telomeres were associated with OR = 6.5 (95% CI 2.4-18), whereas homozygous carriers of 312Asn in the DNA repair gene XPD, with short telomeres, displayed an OR of 17 (95% CI 1.9-150). However, no significant interaction for cancer risk could be proven for telomere length, smoking and susceptibility genotypes of metabolizing and DNA-repairing genes.     

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.     

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.     

The level of telomere dysfunction determines the efficacy of telomerase-based therapeutics in a lung cancer cell line

Telomerase is the ribonucleoprotein enzyme that maintains telomeres of eukaryotic chromosomes. Activation of telomerase is a common feature of the majority of human cancers, and inhibition of this enzyme has been proposed as a novel target for cancer therapeutics. Here, we investigated the effects of telomerase inhibition in the non-small cell lung cancer cell line NCI-H460, using a genetic approach by ectopic expression of dominant-negative (DN)-hTERT. Five clones were selected in which telomerase activity was completely abolished. As a result, telomere erosion was observed leading to proliferation arrest after a lag period of 20-28 population doublings. Although overall telomere length was similar between the different clones as measured by quantitative fluorescence in situ hybridization (Q-FISH), striking differences were found in telomere length of individual chromosomes. In particular, lack of individual telomeres and formation of end-to-end fusions were variable. Interestingly, this level of individual telomere dysfunction was positively correlated with the remaining life span of the different clones in vitro. In addition, the amount of telomere dysfunction induced by DN-hTERT was twice as high compared to the small molecule telomerase inhibitor BIBR1532, which induced growth arrest after >100 population doublings. Thus, pharmacological strategies that aim at inhibition of telomerase in cancer cells should take into account that not only overall telomere shortening, but rapid induction of a high level telomere dysfunction appears to be the crucial surrogate parameter for the development of future telomerase-based therapeutics.