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Telomeres and cancer: a tale with many endings
70
Telomeres and cancer: a tale with many endings MarõÂa A Blasco
Telomerase activity is necessary to maintain the integrity of telomeres, which in turn prevent chromosome ends from being processed and signaled as damaged DNA. That cancer cells rely on telomerase to maintain functional telomeres and to divide inde®nitely has highlighted the potential for developing novel therapeutic approaches that target telomerase. Addresses Department of Immunology and Oncology, National Centre of Biotechnology, E-28049 Madrid, Spain e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:70±76 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00011-4 Abbreviations ALT alternative lengthening of telomeres APC adenomatous polyposis coli ATM ataxia-telangiectasia mutated DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB double-strand break NHEJ non-homologous end-joining Rb retinoblastoma
Introduction
Telomeres cap the ends of chromosomes by forming a higher-order chromatin structure that protects the 30 end from degradation and DNA repair activities [1±6]. Mammalian telomeres are composed of TTAGGG repeats bound to specialized proteins [3]. As cells proliferate, TTAGGG repeats are lost from telomeres unless they have an active telomerase enzyme Ð a reverse transcriptase that adds TTAGGG repeats onto pre-existent telomeres [7±9]. Most normal somatic cells do not have suf®cient telomerase activity and suffer telomere attrition. When telomeres shorten below a critical length, this results in telomere fusions and cells lose viability. This phenomenon is evident with increasing passages of cultured primary cells and is known as `telomere-induced senescence' [10]. Loss of viability as a result of critically short telomeres has been demonstrated in telomerase-de®cient mice that show degenerative pathologies in various organ systems [5,11±13]. These pathologies are coincidental with decreased proliferative index and/or increased apoptosis of the affected cell types Current Opinion in Genetics & Development 2003, 13:70±76
[5,11±13]. Telomerase re-introduction results in elongation of the population of short telomeres and prevents telomere fusions [14,15], as well as pathologies in these mice [14]. More than 90% of all human tumors reactivate telomerase, suggesting that telomerase is necessary to sustain cancer cell viability [16]. As a consequence, telomerase has been seen universally as a tumor marker as well as an attractive target for therapeutic strategies.
Telomere dysfunction caused by short telomeres
Cancer cells generally have short telomeres and high levels of telomerase activity [16]. Telomerase is able to reverse telomere shortening, thus preventing activation of the DNA-damage responses and enabling the viability of cancer cells, which otherwise would have been arrested in division or undergone apoptosis as a result of telomere damage and catastrophic chromosomal rearrangements [14,15]. In agreement with this, mice de®cient for telomerase activity and with short telomeres are resistant to developing carcinogen-induced skin tumors, coincidental with p53 upregulation [17,18]. If p53 is dysfunctional, however, the DNA-damage response triggered by short telomeres is abrogated and telomerase-de®cient mice show a higher frequency of chromosomal aberrations and a higher incidence of epithelial tumors [17,19]. Therefore, simultaneous absence of telomerase and p53 may favor survival of cancer cells harboring chromosomal aberrations. By contrast, if either Rb/p16 or APC tumor suppressor pathways are abrogated, short telomeres have a negative impact on tumorigenesis [20,21], suggesting that in the mouse, p53 is the main tumor suppressor that mediates cell arrest or apoptosis as a result of telomere dysfunction. Similarly, p53 but not Rb/p16 is important in signaling telomere dysfunction produced by TRF2 disruption in mouse cells [22]. In human cells, however, both pathways are important [22]. Both ATM (ataxiatelangiectasia mutated) and the non-homologous end-joining (NHEJ) machinery have been proposed to signal dysfunctional telomeres [3,6]. Abrogation of the NHEJ protein Ku86, rescues both end-to-end fusions and apoptosis, but not cell-cycle arrest, triggered by critically short telomeres [6]. These ®ndings indicate that a dysfunctional telomere is detected as a double-strand break (DSB) and signaled as such, and predict that mutations that abrogate NHEJ may affect the outcome of telomere dysfunction.
Length-independent telomere dysfunction
Mutation of the genes encoding telomere-binding proteins can disrupt telomeric capping in the absence of www.current-opinion.com
Telomeres and cancer Blasco 71
signi®cant telomere shortening [3,5]. A dominant negative mutant of TRF2, which impairs the binding of TRF2 to TTAGGG repeats, results in telomere fusions with long telomeres at the fusion point [23]. Similarly, mice de®cient for either Ku86 or DNA-PKcs (DNA-dependent protein kinase catalytic subunit) Ð both of which are essential components of the NHEJ machinery for DSB repair Ð also result in end-to-end telomere fusions in the absence of telomere shortening [5,6,24±26]. These ®ndings suggest a role for NHEJ activities in telomere capping, similar to that proposed for TRF2. Indeed, mutants for TRF2 and DNA-PKcs share the outcome that the resulting end-to-end fusions involve telomeres produced by leading-strand synthesis [27], suggesting that these two activities could be required for the post-replicative processing of the G-rich strand, thus contributing to telomere capping. The role of telomere-binding proteins in maintaining telomere function predicts that they may be also important in regulating cellular senescence, as well as cancer and aging. In the case of primary mouse embryonic ®broblasts (MEFs), either telomere shortening to a cri-
tical length in the absence of telomerase (late-generation Terc / cells), or long but dysfunctional telomeres as a result of abrogation of Ku86 (Ku86 / cells), lead to similar frequencies of telomeric fusions [28]. In both cases, dysfunctional telomeres trigger senescence-like arrest and result in decreased immortalization frequencies of MEF [28] (Figure 1). In addition, both Terc- and Ku86-de®cient mice show premature aging phenotypes and are tumor resistant when in a p53 wild-type background, suggesting that dysfunctional telomeres have an impact in both aging and cancer in the mouse [11±13,17± 21,29,30] (Figure 1). A dominant negative mutation of TRF2, which disrupts telomere capping, can in¯uence the average telomere length at which senescence is triggered in human primary cells [31]. Therefore, both telomere length and telomere state in¯uence telomere function, that in turn impacts on senescence and immortalization both in human and mouse cells [28,31].
Functional interactions at the mammalian telomere
p53 abrogation in the context of either telomerase de®ciency or mutation of telomere-binding proteins rescues
Figure 1
Human Fibroblasts
50–70 doublings
Telomere-dependent replicative senescence
No immortalization
Telomere-dependent cell arrest & apoptosis
Tumor suppressor mechanism
Short telomeres Human organism
Aging
Mouse MEF
Ku86–/–
Long but dysfunctional telomeres
G4 Terc–/– Mice
Short telomeres
Decreased immortalization
Premature aging
Tumor resistant
Premature arrest
Decreased immortalization
Premature aging
Tumor resistant
End-to-end fusions
Mice
MEF
Premature arrest
End-to-end fusions
Current Opinion in Genetics & Development
Dysfunctional telomeres either as a result of changes in telomere state or extreme telomere shortening trigger a telomere-dependent senescence-like arrest both in human and mouse cells in culture. Telomere dysfunction also impacts negatively on immortalization of these cells. Dysfunctional telomeres in the context of the organism (i.e. Terc- and Ku86-deficient mice) leads to premature aging phenotypes and increases cancer resistance in a wild-type background. www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:70±76
72 Oncogenes and cell proliferation
proliferative arrest and apoptosis as a result of telomere dysfunction [17,32]. Simultaneous deletion of telomerase and either Ku86 or DNA-PKcs activities, rescues both end-to-end chromosomal fusions and apoptosis produced by critically short telomeres [6,33]. Interestingly, proliferative arrest mediated by short telomeres is not prevented by abrogation of DNA-PKcs [33], as also found recently for other types of DNA damage different from telomere dysfunction [34]. These ®ndings suggest that NHEJ could be upstream of p53 in signaling apoptosis [6,33]. Although it has been described that DNA-PKcs does not phosphorylate p53 in vivo [35], it is possible, that DNA-PKcs may signal through p53 in the particular case of telomere dysfunction. TRF1 and TRF2 also in¯uence telomere length [36,37]. Similarly, simultaneous deletion of telomerase and Ku86 in doubly de®cient Terc/Ku86 mice demonstrated that Ku86 acts as a negative regulator or telomerase-mediated telomere elongation. The study of double Terc / /DNAPKcs / mice, by contrast, showed that DNA-PKcs is required for telomere-length maintenance [33]. In particular, absence of DNA-PKcs leads to a faster rate of telomere loss and to an earlier appearance of phenotypes in telomerase-de®cient mice [33]. These ®ndings suggest that telomere-binding proteins in¯uence both telomere capping and telomere length simultaneously.
A novel role of telomerase in promoting tumorigenesis independently of telomere length
Telomerase activity is upregulated during mouse tumorigenesis despite mice having very long telomeres [38,39]. A selection for cells that are telomerase-positive in mouse tumors could be indicative of a novel role for telomerase in promoting survival independently of telomere length. The study of mice that either lack or have constitutive telomerase activity has provided evidence for this [18,40,41,42,43]. In particular, telomerase-de®cient mice with long telomeres are more tumor resistant than wild-type mice [18]. Further evidence for a role of telomerase in promoting tumor growth independently of telomere length was ®rst obtained from studying mice that express constitutive levels of the catalytic component of mouse telomerase, Tert, in basal keratinocytes [40]. The epidermis of these mice is more responsive to mitogenic stimuli and shows a higher incidence of carcinogen-induced tumors than wild-type controls [40,41]. In addition, these mice are more susceptible to develop neoplasias as they age, and this is further aggravated when in a p53/ genetic background [41]. Interestingly, many human cancers also show high telomerase activity and p53 mutations, suggesting cooperation between high telomerase expression and p53 mutation both in men and mice. Mice with transgenic telomerase expression under a b-actin promoter also have an increased incidence of spontaneous mammary epithelial tumors as they Current Opinion in Genetics & Development 2003, 13:70±76
age [42]. Additional data obtained in epithelial cells and cells of neural origin, in which telomerase is reactivated by forced Tert expression, also indicate that telomerase activity has an active role in promoting growth and survival [44]. The mechanisms by which constitutive telomerase activity may promote both cell growth and survival are still unclear, although they seem to require a catalytically active enzyme [2,44,45]. Yeast lacking telomerase show wide-genome changes of gene expression, supporting the notion that telomerase may have other roles other than that of maintaining telomere length [46]. Telomerase could also have a role in either suppressing or processing DNA damage in the genome, thus favoring cell survival and proliferation (Figure 2) [44,45]. In this regard, telomerase is modi®ed by activities involved in DNA-damage signaling, such as Akt, PI3K, c-Abl, and p53, among others [44]. In summary, telomerase activation could favor tumorigenesis at least by two different mechanisms: by signaling proliferation and promoting growth independently of telomere length, and by `rescuing' critically short telomeres (Figure 2).
Short telomeres and sensitivity to genotoxic agents
Recent ®ndings suggest that telomeres are important biological determinants of sensitivity to DNA-damaging agents [47,48]. In particular, late-generation Terc / mice show an enhanced mortality when irradiated with g-irradiation [47]. Intriguingly, the main DSB repair pathways are not altered signi®cantly in these mice [47,48], suggesting that short telomeres could be interfering directly with proper DSB repair. A correlation between telomere length and radiosensitivity has also been found for cultured cells [49]. In addition, telomere shortening has been shown to alter the chemotherapeutic pro®le of transformed cells and p53 is required for this [50]. Telomeric dysfunction even if telomeres are suf®ciently long, also results in increased radiosensitivity [51]. These ®ndings have important implications for the therapy of cancer, as tumors treated with telomerase inhibitors could lead to telomere loss, thus increasing the sensitivity of these tumors to radiotherapy or to other genotoxic agents.
Inhibiting telomerase in cancer
The dependency of cancer cells on telomerase-mediated rescue of short telomeres presents the possibility of applying telomerase inhibitors to selectively block tumor growth while preserving the viability of normal cells. In turn, a telomerase inhibitor could also impair survival/ growth of tumor cells with long telomeres by interfering with its role in promoting tumorigenesis independently of telomere length [45]. A telomerase inhibitor may also reduce the angiogenic potential of the tumor [52]. www.current-opinion.com
Telomeres and cancer Blasco 73
Figure 2
+Telomerase
Survival signals
Long telomeres
–Telomerase
DNA damage processing?
Progressive telomere loss
Defect in processing DNA damage?
Short telomeres
No survival signals
+Telomerase
Tumor growth DNA-PKcs Ku86
Telomere fusions
Survival signals
Elongation of short telomeres
DNA damage processing?
p53 activation
Cell arrest and/or apoptosis
Tumor growth
Current Opinion in Genetics & Development
Two roles of telomerase in cancer. Telomerase activity allows telomere maintenance and promotes growth of cancer cells that have telomerase activity even when telomeres are sufficiently long. Possible explanations for this is that telomerase may signal survival when present at telomeres or it may have a role in processing or repairing DNA damage in the genome. Those cancer cells that do not have telomerase show progressive telomere loss coupled to cell division, ultimately resulting in critically short telomeres. The absence of telomerase could also impact negatively on tumor survival even before telomeres shorten below a critical length. When telomeres shorten below a critical length, only cancer cells that reactivate telomerase are able to rescue short telomeres and prevent both telomere fusions and loss of cell viability. By contrast, a critically short telomere in the absence of telomerase activity will trigger cell arrest or apoptosis mediated by the p53 DNA damage signaling pathway.
Telomerase inhibitors include antisense oligonucleotides against the telomerase RNA component, dominant negative mutants of the Tert subunit, molecules directed against G-quadruplex formation, as well as small pharmaceutical compounds highly speci®c for telomerase [53,54]. Although the ef®ciency of these inhibitors has yet to be tested in clinical trials of human cancer, there is evidence from both cell-culture models and mouse models to suggest that they would be ef®cient in ceasing tumor growth [55]. In addition, as just discussed above, these inhibitors may be combined with genotoxic agents to potentiate their effects. The ef®ciency of a telomerase-directed drug may depend on the status of p53 in the tumor. However, when telomerase was inhibited in various human cancer cell lines, the status of p53 was irrelevant for the ®nal outcome Ð suggesting that p53-de®ciency is not suf®cient to render human cells permissive to telomere dysfunction [55]. In mouse tumors, inhibition of telomerase using a dominant negative mutant of Tert leads to a rapid selection of www.current-opinion.com
tumor cells that upregulate the endogenous Tert gene, thus compensating for the presence of the inhibitor and allowing growth [56]. Whether this also happens in human tumor cells has yet to be determined. A telomerase inhibitor could also result in the selection of tumor cells that maintain telomeres in the absence of telomerase, a phenomenon termed ALT (alternative lengthening of telomeres) [57]. Although only 5% of human tumors seem to be sustained by ALT, inhibition of telomerase could generate a selection pressure to activate ALT within the tumor, which in turn would no longer be responsive to the inhibitor. Finally, the ef®ciency of a telomerase inhibitor will depend on its toxicity on cells that express telomerase, such as germ cells and stem cells. As these cells have longer telomeres than tumor cells, it has been reasoned that they will not be affected by temporary telomerase inhibition, although there has been no direct evaluation of this. Data from the telomerase knockout model indicate Current Opinion in Genetics & Development 2003, 13:70±76
74 Oncogenes and cell proliferation
that these cells types are not affected in the absence of telomerase until their telomeres shorten below a critical length after several mouse generations [11±13]. Similarly, patients who suffer Dyskeratosis congenita Ð a disease that is characterized by a faster rate of telomere shortening as a result of mutations in either telomerase or in Dyskeryn (required for an active telomerase complex [8]) Ð develop normally during the ®rst years of life before disease manifestation, suggesting that temporary telomerase inhibition during chemotherapy treatment will not have deleterious effects.
Conclusions
It seems clear that to properly evaluate telomere function it is necessary to consider more factors than just telomerase activity and telomere length. Abrogation of telomerebinding proteins can render long telomeres dysfunctional even if telomerase activity is present in the cell. In turn, telomerase activity could be signaling survival of cells with suf®ciently long telomeres. Finally, the outcome of telomere dysfunction depends on the status of p53 or the NHEJ proteins, Ku86 and DNA-PKcs. A further understanding of the complex functional interactions at the telomere is therefore necessary to predict the ef®cacy of therapies on disrupting telomere function in cancer cells.
Acknowledgements
Research at the laboratory of MA Blasco is funded by grants PM97-0133 from the MCYT (Ministry of Science and Technology), 08.1/0030/98 from CAM (Regional Government of Madrid), and by grants EURATOM/991/0201, FIGH-CT-1999-00002 and FIS5-1999-00055, from the European Union, and by the Department of Immunology and Oncology (DIO).
References and recommended reading
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40. Gonzalez-Suarez E, Samper E, Ramirez A, Flores JM, Martin-Caballero J, Jorcano JL, Blasco MA: Increased epidermal tumors and increased wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes. EMBO J 2001, 20:2619-2630. 41. GonzaÂlez-SuaÂrez E, Flores JM, Blasco MA: Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development. Mol Cell Biol 2002, 22:7291-7301. This paper is a continuation of a previous work in which we showed that constitutive transgenic telomerase expression increased the frequency of carcinogen-induced tumors in mice [40]. This implied that telomerase could have a role in promoting tumorigenesis independently of telomere length. In this paper, we demonstrate that constitutive telomerase expression also increases spontaneous tumorigenesis with age. In addition, the authors generate a new mouse model with constitutive telomerase expression in the context of p53 de®ciency. These double mutant mice show an increased frequency of spontaneous tumors at young ages, suggesting cooperation between telomerase activity and p53 mutation in accelerating tumorigenesis. 42. Artandi SE, Alson S, Tietze MK, Sharpless NE, Ye S, Greenberg RA, Castrillon DH, Horner JW, Weiler SR, Carrasco RD, DePinho RA: Constitutive telomerase expression promotes mammary carcinomas in aging mice. Proc Natl Acad Sci USA 2002, 99:8191-8196. The paper describes the generation of a new mouse model with transgenic telomerase expression under a general actin promoter. These mice show an increased incidence of mammary epithelial tumors as they age compared to the wild-type controls. This paper, together with data from papers [40,41], suggest that telomerase has a role in promoting tumorigenesis even if telomeres are suf®ciently long, such as in the context of telomerase-transgenic mice. 43. Oh H, Taffet GE, Youker KA, Entman ML, Overbeek PA, Michael LH, Schneider MD: Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci USA 2001, 98:10308-11033. 44. Mattson MP, Fu W, Zhang P: Emerging roles for telomerase in regulating cell differentiation and survival: a neuroscientist's perspective. Mech Aging Dev 2001, 122:659-671. 45. Blasco MA: Telomerase beyond telomeres. Nat Rev Cancer 2002, 2:627-633. 46. Nautiyal S, DeRisi JL, Blackburn EH: The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 2002, 99:9316-9321. This paper compares genome-wide gene expression changes in yeast containing telomerase activity versus yeast where telomerase is deleted. The fact that many genes show expression changes as a consequence of abrogation of telomerase also supports the notion that telomerase may impact processes other than telomere length maintenance. 47. Goytisolo F, Samper E, MartõÂn-Caballero J, Finnon P, Herrera E, Flores JM, Bouf¯er SD, Blasco MA: Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med 2000, 192:1625-1636. 48. Wong KK, Chang S, Weiler SR, Ganesan S, Chaudhuri J, Zhu C, Artandi SE, Rudolph KL, Gottlieb GJ, Chin L et al.: Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 2000, 26:85-88. 49. McIlrath J, Bouf¯er SD, Samper E, Cuthbert A, Wojcik A, Szumiel I, Bryant PE, Riches AC, Thompson A, Blasco MA et al.: Telomere length abnormalities in mammalian radiosensitive cells. Cancer Res 2001, 61:912-915. 50. Lee KH, Rudolph KL, Ju YJ, Greenberg RA, Cannizzaro L, Chin L, Weiler SR, DePinho RA et al.: Telomere dysfunction alters the chemotherapeutic pro®le of transformed cells. Proc Natl Acad Sci USA 2001, 98:3381-3386. Current Opinion in Genetics & Development 2003, 13:70±76
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51. Finnon P, Wong HP, Silver AR, Slijepcevic P, Bouf¯er SD: Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line. Int J Radiat Biol 2001, 77:1151-1162. 52. Franco S, Segura I, Riese HH, Blasco MA: Decreased B16F10 melanoma growth and impaired vascularization in telomerase-de®cient mice with critically short telomeres. Cancer Res 2002, 62:552-559.
55. Hahn WC, Stewart SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M, Weinberg RA: Inhibition of telomerase limits the growth of human cancer cells. Nat Med 1999, 5:1164-1170.
53. Corey DR: Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 2002, 21:631-637.
56. Sachsinger J, Gonzalez-Suarez E, Samper E, Heicappell R, Muller M, Blasco MA: Telomerase inhibition in RenCa, a murine tumor cell line with short telomeres, by overexpression of a dominant negative mTERT mutant, reveals fundamental differences in telomerase regulation between human and murine cells. Cancer Res 1999, 61:5580-5586.
54. Damm K, Hemmann U, Garin-Chesa P, Hauel N, Kauffmann I, Priepke H, Niestroj C, Daiber C, Enenkel B, Guilliard B et al.: A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 2001, 20:6958-6968.
57. Henson JD, Neumann AA, Yeager TR, Reddel RR: Alternative lengthening of telomeres in mammalian cells. Oncogene 2002, 21:598-610.
Current Opinion in Genetics & Development 2003, 13:70±76
www.current-opinion.com
Telomeres and cancer: a tale with many endings MarõÂa A Blasco
Telomerase activity is necessary to maintain the integrity of telomeres, which in turn prevent chromosome ends from being processed and signaled as damaged DNA. That cancer cells rely on telomerase to maintain functional telomeres and to divide inde®nitely has highlighted the potential for developing novel therapeutic approaches that target telomerase. Addresses Department of Immunology and Oncology, National Centre of Biotechnology, E-28049 Madrid, Spain e-mail: [email protected]
Current Opinion in Genetics & Development 2003, 13:70±76 This review comes from a themed issue on Oncogenes and cell proliferation Edited by Frank McCormick and Kevin Shannon 0959-437X/03/$ ± see front matter ß 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0959-437X(02)00011-4 Abbreviations ALT alternative lengthening of telomeres APC adenomatous polyposis coli ATM ataxia-telangiectasia mutated DNA-PKcs DNA-dependent protein kinase catalytic subunit DSB double-strand break NHEJ non-homologous end-joining Rb retinoblastoma
Introduction
Telomeres cap the ends of chromosomes by forming a higher-order chromatin structure that protects the 30 end from degradation and DNA repair activities [1±6]. Mammalian telomeres are composed of TTAGGG repeats bound to specialized proteins [3]. As cells proliferate, TTAGGG repeats are lost from telomeres unless they have an active telomerase enzyme Ð a reverse transcriptase that adds TTAGGG repeats onto pre-existent telomeres [7±9]. Most normal somatic cells do not have suf®cient telomerase activity and suffer telomere attrition. When telomeres shorten below a critical length, this results in telomere fusions and cells lose viability. This phenomenon is evident with increasing passages of cultured primary cells and is known as `telomere-induced senescence' [10]. Loss of viability as a result of critically short telomeres has been demonstrated in telomerase-de®cient mice that show degenerative pathologies in various organ systems [5,11±13]. These pathologies are coincidental with decreased proliferative index and/or increased apoptosis of the affected cell types Current Opinion in Genetics & Development 2003, 13:70±76
[5,11±13]. Telomerase re-introduction results in elongation of the population of short telomeres and prevents telomere fusions [14,15], as well as pathologies in these mice [14]. More than 90% of all human tumors reactivate telomerase, suggesting that telomerase is necessary to sustain cancer cell viability [16]. As a consequence, telomerase has been seen universally as a tumor marker as well as an attractive target for therapeutic strategies.
Telomere dysfunction caused by short telomeres
Cancer cells generally have short telomeres and high levels of telomerase activity [16]. Telomerase is able to reverse telomere shortening, thus preventing activation of the DNA-damage responses and enabling the viability of cancer cells, which otherwise would have been arrested in division or undergone apoptosis as a result of telomere damage and catastrophic chromosomal rearrangements [14,15]. In agreement with this, mice de®cient for telomerase activity and with short telomeres are resistant to developing carcinogen-induced skin tumors, coincidental with p53 upregulation [17,18]. If p53 is dysfunctional, however, the DNA-damage response triggered by short telomeres is abrogated and telomerase-de®cient mice show a higher frequency of chromosomal aberrations and a higher incidence of epithelial tumors [17,19]. Therefore, simultaneous absence of telomerase and p53 may favor survival of cancer cells harboring chromosomal aberrations. By contrast, if either Rb/p16 or APC tumor suppressor pathways are abrogated, short telomeres have a negative impact on tumorigenesis [20,21], suggesting that in the mouse, p53 is the main tumor suppressor that mediates cell arrest or apoptosis as a result of telomere dysfunction. Similarly, p53 but not Rb/p16 is important in signaling telomere dysfunction produced by TRF2 disruption in mouse cells [22]. In human cells, however, both pathways are important [22]. Both ATM (ataxiatelangiectasia mutated) and the non-homologous end-joining (NHEJ) machinery have been proposed to signal dysfunctional telomeres [3,6]. Abrogation of the NHEJ protein Ku86, rescues both end-to-end fusions and apoptosis, but not cell-cycle arrest, triggered by critically short telomeres [6]. These ®ndings indicate that a dysfunctional telomere is detected as a double-strand break (DSB) and signaled as such, and predict that mutations that abrogate NHEJ may affect the outcome of telomere dysfunction.
Length-independent telomere dysfunction
Mutation of the genes encoding telomere-binding proteins can disrupt telomeric capping in the absence of www.current-opinion.com
Telomeres and cancer Blasco 71
signi®cant telomere shortening [3,5]. A dominant negative mutant of TRF2, which impairs the binding of TRF2 to TTAGGG repeats, results in telomere fusions with long telomeres at the fusion point [23]. Similarly, mice de®cient for either Ku86 or DNA-PKcs (DNA-dependent protein kinase catalytic subunit) Ð both of which are essential components of the NHEJ machinery for DSB repair Ð also result in end-to-end telomere fusions in the absence of telomere shortening [5,6,24±26]. These ®ndings suggest a role for NHEJ activities in telomere capping, similar to that proposed for TRF2. Indeed, mutants for TRF2 and DNA-PKcs share the outcome that the resulting end-to-end fusions involve telomeres produced by leading-strand synthesis [27], suggesting that these two activities could be required for the post-replicative processing of the G-rich strand, thus contributing to telomere capping. The role of telomere-binding proteins in maintaining telomere function predicts that they may be also important in regulating cellular senescence, as well as cancer and aging. In the case of primary mouse embryonic ®broblasts (MEFs), either telomere shortening to a cri-
tical length in the absence of telomerase (late-generation Terc / cells), or long but dysfunctional telomeres as a result of abrogation of Ku86 (Ku86 / cells), lead to similar frequencies of telomeric fusions [28]. In both cases, dysfunctional telomeres trigger senescence-like arrest and result in decreased immortalization frequencies of MEF [28] (Figure 1). In addition, both Terc- and Ku86-de®cient mice show premature aging phenotypes and are tumor resistant when in a p53 wild-type background, suggesting that dysfunctional telomeres have an impact in both aging and cancer in the mouse [11±13,17± 21,29,30] (Figure 1). A dominant negative mutation of TRF2, which disrupts telomere capping, can in¯uence the average telomere length at which senescence is triggered in human primary cells [31]. Therefore, both telomere length and telomere state in¯uence telomere function, that in turn impacts on senescence and immortalization both in human and mouse cells [28,31].
Functional interactions at the mammalian telomere
p53 abrogation in the context of either telomerase de®ciency or mutation of telomere-binding proteins rescues
Figure 1
Human Fibroblasts
50–70 doublings
Telomere-dependent replicative senescence
No immortalization
Telomere-dependent cell arrest & apoptosis
Tumor suppressor mechanism
Short telomeres Human organism
Aging
Mouse MEF
Ku86–/–
Long but dysfunctional telomeres
G4 Terc–/– Mice
Short telomeres
Decreased immortalization
Premature aging
Tumor resistant
Premature arrest
Decreased immortalization
Premature aging
Tumor resistant
End-to-end fusions
Mice
MEF
Premature arrest
End-to-end fusions
Current Opinion in Genetics & Development
Dysfunctional telomeres either as a result of changes in telomere state or extreme telomere shortening trigger a telomere-dependent senescence-like arrest both in human and mouse cells in culture. Telomere dysfunction also impacts negatively on immortalization of these cells. Dysfunctional telomeres in the context of the organism (i.e. Terc- and Ku86-deficient mice) leads to premature aging phenotypes and increases cancer resistance in a wild-type background. www.current-opinion.com
Current Opinion in Genetics & Development 2003, 13:70±76
72 Oncogenes and cell proliferation
proliferative arrest and apoptosis as a result of telomere dysfunction [17,32]. Simultaneous deletion of telomerase and either Ku86 or DNA-PKcs activities, rescues both end-to-end chromosomal fusions and apoptosis produced by critically short telomeres [6,33]. Interestingly, proliferative arrest mediated by short telomeres is not prevented by abrogation of DNA-PKcs [33], as also found recently for other types of DNA damage different from telomere dysfunction [34]. These ®ndings suggest that NHEJ could be upstream of p53 in signaling apoptosis [6,33]. Although it has been described that DNA-PKcs does not phosphorylate p53 in vivo [35], it is possible, that DNA-PKcs may signal through p53 in the particular case of telomere dysfunction. TRF1 and TRF2 also in¯uence telomere length [36,37]. Similarly, simultaneous deletion of telomerase and Ku86 in doubly de®cient Terc/Ku86 mice demonstrated that Ku86 acts as a negative regulator or telomerase-mediated telomere elongation. The study of double Terc / /DNAPKcs / mice, by contrast, showed that DNA-PKcs is required for telomere-length maintenance [33]. In particular, absence of DNA-PKcs leads to a faster rate of telomere loss and to an earlier appearance of phenotypes in telomerase-de®cient mice [33]. These ®ndings suggest that telomere-binding proteins in¯uence both telomere capping and telomere length simultaneously.
A novel role of telomerase in promoting tumorigenesis independently of telomere length
Telomerase activity is upregulated during mouse tumorigenesis despite mice having very long telomeres [38,39]. A selection for cells that are telomerase-positive in mouse tumors could be indicative of a novel role for telomerase in promoting survival independently of telomere length. The study of mice that either lack or have constitutive telomerase activity has provided evidence for this [18,40,41,42,43]. In particular, telomerase-de®cient mice with long telomeres are more tumor resistant than wild-type mice [18]. Further evidence for a role of telomerase in promoting tumor growth independently of telomere length was ®rst obtained from studying mice that express constitutive levels of the catalytic component of mouse telomerase, Tert, in basal keratinocytes [40]. The epidermis of these mice is more responsive to mitogenic stimuli and shows a higher incidence of carcinogen-induced tumors than wild-type controls [40,41]. In addition, these mice are more susceptible to develop neoplasias as they age, and this is further aggravated when in a p53/ genetic background [41]. Interestingly, many human cancers also show high telomerase activity and p53 mutations, suggesting cooperation between high telomerase expression and p53 mutation both in men and mice. Mice with transgenic telomerase expression under a b-actin promoter also have an increased incidence of spontaneous mammary epithelial tumors as they Current Opinion in Genetics & Development 2003, 13:70±76
age [42]. Additional data obtained in epithelial cells and cells of neural origin, in which telomerase is reactivated by forced Tert expression, also indicate that telomerase activity has an active role in promoting growth and survival [44]. The mechanisms by which constitutive telomerase activity may promote both cell growth and survival are still unclear, although they seem to require a catalytically active enzyme [2,44,45]. Yeast lacking telomerase show wide-genome changes of gene expression, supporting the notion that telomerase may have other roles other than that of maintaining telomere length [46]. Telomerase could also have a role in either suppressing or processing DNA damage in the genome, thus favoring cell survival and proliferation (Figure 2) [44,45]. In this regard, telomerase is modi®ed by activities involved in DNA-damage signaling, such as Akt, PI3K, c-Abl, and p53, among others [44]. In summary, telomerase activation could favor tumorigenesis at least by two different mechanisms: by signaling proliferation and promoting growth independently of telomere length, and by `rescuing' critically short telomeres (Figure 2).
Short telomeres and sensitivity to genotoxic agents
Recent ®ndings suggest that telomeres are important biological determinants of sensitivity to DNA-damaging agents [47,48]. In particular, late-generation Terc / mice show an enhanced mortality when irradiated with g-irradiation [47]. Intriguingly, the main DSB repair pathways are not altered signi®cantly in these mice [47,48], suggesting that short telomeres could be interfering directly with proper DSB repair. A correlation between telomere length and radiosensitivity has also been found for cultured cells [49]. In addition, telomere shortening has been shown to alter the chemotherapeutic pro®le of transformed cells and p53 is required for this [50]. Telomeric dysfunction even if telomeres are suf®ciently long, also results in increased radiosensitivity [51]. These ®ndings have important implications for the therapy of cancer, as tumors treated with telomerase inhibitors could lead to telomere loss, thus increasing the sensitivity of these tumors to radiotherapy or to other genotoxic agents.
Inhibiting telomerase in cancer
The dependency of cancer cells on telomerase-mediated rescue of short telomeres presents the possibility of applying telomerase inhibitors to selectively block tumor growth while preserving the viability of normal cells. In turn, a telomerase inhibitor could also impair survival/ growth of tumor cells with long telomeres by interfering with its role in promoting tumorigenesis independently of telomere length [45]. A telomerase inhibitor may also reduce the angiogenic potential of the tumor [52]. www.current-opinion.com
Telomeres and cancer Blasco 73
Figure 2
+Telomerase
Survival signals
Long telomeres
–Telomerase
DNA damage processing?
Progressive telomere loss
Defect in processing DNA damage?
Short telomeres
No survival signals
+Telomerase
Tumor growth DNA-PKcs Ku86
Telomere fusions
Survival signals
Elongation of short telomeres
DNA damage processing?
p53 activation
Cell arrest and/or apoptosis
Tumor growth
Current Opinion in Genetics & Development
Two roles of telomerase in cancer. Telomerase activity allows telomere maintenance and promotes growth of cancer cells that have telomerase activity even when telomeres are sufficiently long. Possible explanations for this is that telomerase may signal survival when present at telomeres or it may have a role in processing or repairing DNA damage in the genome. Those cancer cells that do not have telomerase show progressive telomere loss coupled to cell division, ultimately resulting in critically short telomeres. The absence of telomerase could also impact negatively on tumor survival even before telomeres shorten below a critical length. When telomeres shorten below a critical length, only cancer cells that reactivate telomerase are able to rescue short telomeres and prevent both telomere fusions and loss of cell viability. By contrast, a critically short telomere in the absence of telomerase activity will trigger cell arrest or apoptosis mediated by the p53 DNA damage signaling pathway.
Telomerase inhibitors include antisense oligonucleotides against the telomerase RNA component, dominant negative mutants of the Tert subunit, molecules directed against G-quadruplex formation, as well as small pharmaceutical compounds highly speci®c for telomerase [53,54]. Although the ef®ciency of these inhibitors has yet to be tested in clinical trials of human cancer, there is evidence from both cell-culture models and mouse models to suggest that they would be ef®cient in ceasing tumor growth [55]. In addition, as just discussed above, these inhibitors may be combined with genotoxic agents to potentiate their effects. The ef®ciency of a telomerase-directed drug may depend on the status of p53 in the tumor. However, when telomerase was inhibited in various human cancer cell lines, the status of p53 was irrelevant for the ®nal outcome Ð suggesting that p53-de®ciency is not suf®cient to render human cells permissive to telomere dysfunction [55]. In mouse tumors, inhibition of telomerase using a dominant negative mutant of Tert leads to a rapid selection of www.current-opinion.com
tumor cells that upregulate the endogenous Tert gene, thus compensating for the presence of the inhibitor and allowing growth [56]. Whether this also happens in human tumor cells has yet to be determined. A telomerase inhibitor could also result in the selection of tumor cells that maintain telomeres in the absence of telomerase, a phenomenon termed ALT (alternative lengthening of telomeres) [57]. Although only 5% of human tumors seem to be sustained by ALT, inhibition of telomerase could generate a selection pressure to activate ALT within the tumor, which in turn would no longer be responsive to the inhibitor. Finally, the ef®ciency of a telomerase inhibitor will depend on its toxicity on cells that express telomerase, such as germ cells and stem cells. As these cells have longer telomeres than tumor cells, it has been reasoned that they will not be affected by temporary telomerase inhibition, although there has been no direct evaluation of this. Data from the telomerase knockout model indicate Current Opinion in Genetics & Development 2003, 13:70±76
74 Oncogenes and cell proliferation
that these cells types are not affected in the absence of telomerase until their telomeres shorten below a critical length after several mouse generations [11±13]. Similarly, patients who suffer Dyskeratosis congenita Ð a disease that is characterized by a faster rate of telomere shortening as a result of mutations in either telomerase or in Dyskeryn (required for an active telomerase complex [8]) Ð develop normally during the ®rst years of life before disease manifestation, suggesting that temporary telomerase inhibition during chemotherapy treatment will not have deleterious effects.
Conclusions
It seems clear that to properly evaluate telomere function it is necessary to consider more factors than just telomerase activity and telomere length. Abrogation of telomerebinding proteins can render long telomeres dysfunctional even if telomerase activity is present in the cell. In turn, telomerase activity could be signaling survival of cells with suf®ciently long telomeres. Finally, the outcome of telomere dysfunction depends on the status of p53 or the NHEJ proteins, Ku86 and DNA-PKcs. A further understanding of the complex functional interactions at the telomere is therefore necessary to predict the ef®cacy of therapies on disrupting telomere function in cancer cells.
Acknowledgements
Research at the laboratory of MA Blasco is funded by grants PM97-0133 from the MCYT (Ministry of Science and Technology), 08.1/0030/98 from CAM (Regional Government of Madrid), and by grants EURATOM/991/0201, FIGH-CT-1999-00002 and FIS5-1999-00055, from the European Union, and by the Department of Immunology and Oncology (DIO).
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28. Espejel S, Blasco MA: Identi®cation of telomere-dependent `senescence-like' arrest in mouse embryonic ®broblasts. Exp Cell Res 2001, 276:242-248. The senescence-like arrest shown by MEFs in culture is not likely to be mediated by telomere loss because MEFs have telomerase activity and very long telomeres. In this paper, we demonstrate that it is possible to trigger a telomere-mediated senescence-like arrest in MEFs by either short telomeres in the absence of telomerase (telomerase-de®cient mice) or by deletion of the telomere-binding protein Ku86 (Ku86-de®cient mice). Hence, telomere dysfunction produced by either short telomeres or altered telomere capping leads to senescence-like arrest in mouse cells. 29. Vogel H, Lim DS, Karsenty G, Finegold M, Hasty P: Deletion of Ku86 causes early onset of senescence in mice. Proc Natl Acad Sci USA 1999, 96:10770-10775. 30. Di®lippantonio MJ, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE, Ried T, Nussenzweig A: DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 2000, 404:510-514. 31. Karlseder J, Smogorzewska A, de Lange T: Senescence induced by altered telomere state, not telomere loss. Science 2002, 295:2446-2449. The paper shows that altering the telomere state using a dominant negative mutant of the telomere-binding protein TRF2, it is possible to alter the average length at which telomeres trigger senescence in human cells. Therefore, by altering the telomere state it is possible to induce senescence. 32. Karlseder J, Broccoli D, Dai Y, Hardy S, de Lange T: p53- and ATM-dependent apoptosis induced by telomeres lacking TRF2. Science 1999, 283:1321-1325. 33. Espejel S, Franco S, Sgura A, Gae D, Bailey S, Taccioli G, Blasco MA: Functional interaction between DNA-PKcs and telomerase in telomere length maintenance. EMBO J 2002, 21:6275-6287. Here we describe the generation of mice doubly de®cient for telomerase and DNA-PKcs activities. The results indicate that DNA-PKcs and telomerase functionally interact in maintaining telomere length in mice. In addition, we demonstrate that DNA-PKcs mediates end-to-end fusions and germ-cell apoptosis as a result of critically short telomeres Ð suggesting that DNA-PKcs is important in the processing and signaling of a short telomere as damaged DNA. 34. Wang S, Guo M, Ouyang H, Li X, Cordon-Cardo C, Kurimasa A, Chen DJ, Fuks Z, Ling CC, Li GC: The catalytic subunit of DNA-dependent protein kinase selectively regulates p53-dependent apoptosis but not cell-cycle arrest. Proc Natl Acad Sci USA 2000, 97:1584-1588. 35. Jimenez GS, Bryntesson F, Torres-Arzayus MI, Priestley A, Beeche M, Saito S, Sakaguchi K, Appella E, Jeggo PA, Taccioli GE et al.: DNA-dependent protein kinase is not required for the p53-dependent response to DNA damage. Nature 1999, 400:81-83. 36. Smogorzewska A, van Steensel B, Bianchi A, Oelmann S, Schaefer MR, Schnapp G, de Lange T: Control of human telomere length by TRF1 and TRF2. Mol Cell Biol 2000, 20:1659-1668. 37. Ancelin K, Brunori M, Bauwens S, Koering CE, Brun C, Ricoul M, Pommier JP, Sabatier L, Gilson E: Targeting assay to study the cis functions of human telomeric proteins: evidence for inhibition of telomerase by TRF1 and for activation of telomere degradation by TRF2. Mol Cell Biol 2002, 22:3474-3487. The authors elegantly dissect the roles of TRF1 and TRF2 by tagging these proteins to the telomeres. They show that TRF1 interferes with telomerase-dependent telomere elongation, and that TRF2, however seems to promote telomere degradation by unknown mechanisms. www.current-opinion.com
40. Gonzalez-Suarez E, Samper E, Ramirez A, Flores JM, Martin-Caballero J, Jorcano JL, Blasco MA: Increased epidermal tumors and increased wound healing in transgenic mice overexpressing the catalytic subunit of telomerase, mTERT, in basal keratinocytes. EMBO J 2001, 20:2619-2630. 41. GonzaÂlez-SuaÂrez E, Flores JM, Blasco MA: Cooperation between p53 mutation and high telomerase transgenic expression in spontaneous cancer development. Mol Cell Biol 2002, 22:7291-7301. This paper is a continuation of a previous work in which we showed that constitutive transgenic telomerase expression increased the frequency of carcinogen-induced tumors in mice [40]. This implied that telomerase could have a role in promoting tumorigenesis independently of telomere length. In this paper, we demonstrate that constitutive telomerase expression also increases spontaneous tumorigenesis with age. In addition, the authors generate a new mouse model with constitutive telomerase expression in the context of p53 de®ciency. These double mutant mice show an increased frequency of spontaneous tumors at young ages, suggesting cooperation between telomerase activity and p53 mutation in accelerating tumorigenesis. 42. Artandi SE, Alson S, Tietze MK, Sharpless NE, Ye S, Greenberg RA, Castrillon DH, Horner JW, Weiler SR, Carrasco RD, DePinho RA: Constitutive telomerase expression promotes mammary carcinomas in aging mice. Proc Natl Acad Sci USA 2002, 99:8191-8196. The paper describes the generation of a new mouse model with transgenic telomerase expression under a general actin promoter. These mice show an increased incidence of mammary epithelial tumors as they age compared to the wild-type controls. This paper, together with data from papers [40,41], suggest that telomerase has a role in promoting tumorigenesis even if telomeres are suf®ciently long, such as in the context of telomerase-transgenic mice. 43. Oh H, Taffet GE, Youker KA, Entman ML, Overbeek PA, Michael LH, Schneider MD: Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival. Proc Natl Acad Sci USA 2001, 98:10308-11033. 44. Mattson MP, Fu W, Zhang P: Emerging roles for telomerase in regulating cell differentiation and survival: a neuroscientist's perspective. Mech Aging Dev 2001, 122:659-671. 45. Blasco MA: Telomerase beyond telomeres. Nat Rev Cancer 2002, 2:627-633. 46. Nautiyal S, DeRisi JL, Blackburn EH: The genome-wide expression response to telomerase deletion in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 2002, 99:9316-9321. This paper compares genome-wide gene expression changes in yeast containing telomerase activity versus yeast where telomerase is deleted. The fact that many genes show expression changes as a consequence of abrogation of telomerase also supports the notion that telomerase may impact processes other than telomere length maintenance. 47. Goytisolo F, Samper E, MartõÂn-Caballero J, Finnon P, Herrera E, Flores JM, Bouf¯er SD, Blasco MA: Short telomeres result in organismal hypersensitivity to ionizing radiation in mammals. J Exp Med 2000, 192:1625-1636. 48. Wong KK, Chang S, Weiler SR, Ganesan S, Chaudhuri J, Zhu C, Artandi SE, Rudolph KL, Gottlieb GJ, Chin L et al.: Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation. Nat Genet 2000, 26:85-88. 49. McIlrath J, Bouf¯er SD, Samper E, Cuthbert A, Wojcik A, Szumiel I, Bryant PE, Riches AC, Thompson A, Blasco MA et al.: Telomere length abnormalities in mammalian radiosensitive cells. Cancer Res 2001, 61:912-915. 50. Lee KH, Rudolph KL, Ju YJ, Greenberg RA, Cannizzaro L, Chin L, Weiler SR, DePinho RA et al.: Telomere dysfunction alters the chemotherapeutic pro®le of transformed cells. Proc Natl Acad Sci USA 2001, 98:3381-3386. Current Opinion in Genetics & Development 2003, 13:70±76
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51. Finnon P, Wong HP, Silver AR, Slijepcevic P, Bouf¯er SD: Long but dysfunctional telomeres correlate with chromosomal radiosensitivity in a mouse AML cell line. Int J Radiat Biol 2001, 77:1151-1162. 52. Franco S, Segura I, Riese HH, Blasco MA: Decreased B16F10 melanoma growth and impaired vascularization in telomerase-de®cient mice with critically short telomeres. Cancer Res 2002, 62:552-559.
55. Hahn WC, Stewart SA, Brooks MW, York SG, Eaton E, Kurachi A, Beijersbergen RL, Knoll JH, Meyerson M, Weinberg RA: Inhibition of telomerase limits the growth of human cancer cells. Nat Med 1999, 5:1164-1170.
53. Corey DR: Telomerase inhibition, oligonucleotides, and clinical trials. Oncogene 2002, 21:631-637.
56. Sachsinger J, Gonzalez-Suarez E, Samper E, Heicappell R, Muller M, Blasco MA: Telomerase inhibition in RenCa, a murine tumor cell line with short telomeres, by overexpression of a dominant negative mTERT mutant, reveals fundamental differences in telomerase regulation between human and murine cells. Cancer Res 1999, 61:5580-5586.
54. Damm K, Hemmann U, Garin-Chesa P, Hauel N, Kauffmann I, Priepke H, Niestroj C, Daiber C, Enenkel B, Guilliard B et al.: A highly selective telomerase inhibitor limiting human cancer cell proliferation. EMBO J 2001, 20:6958-6968.
57. Henson JD, Neumann AA, Yeager TR, Reddel RR: Alternative lengthening of telomeres in mammalian cells. Oncogene 2002, 21:598-610.
Current Opinion in Genetics & Development 2003, 13:70±76
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