Telomerase And Cancer

Cancer Letters 194 (2003) 163–172 www.elsevier.com/locate/canlet

Telomerase and tumorigenesis Kenkichi Masutomia,b, William C. Hahna,b,* a

b

Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Dana 710C, Boston, MA 02115, USA Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA Received 29 August 2002; received in revised form 20 October 2002; accepted 21 October 2002

Abstract The unique biology of telomeres and telomerase plays important roles in many aspects of mammalian cell physiology. Over the past decade, several lines of evidence have confirmed that the maintenance of telomeres and telomerase participate actively in the pathogenesis of human cancer. Specifically, activation of telomerase is strongly associated with cancer, and recent observations confirm that telomeres and telomerase perform important roles in both suppressing and facilitating malignant transformation by regulating genomic stability and cell lifespan. In addition, recent evidence suggests that telomerase activation contributes to tumorigenesis independently of its role in maintaining telomere length. Here we review recent developments in our understanding of the relationships among telomeres, telomerase, and cancer. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Telomerase; Telomerase Reverse Transcriptase (TERT); Telomere; Immortalization; Human Telomerase Reverse Transcriptase (hTERT); Senescence; Tumorigenesis; Malignant transformation; Cancer; Telomere capping; Tumor suppression; Replication; Mouse telomeres; Tumor promotion; Genomic instability; Retinoblastoma; Simian virus 40 (SV40); Human papillomavirus; Senescence

1. Introduction One of the central characteristics of cancer is its complexity. Clinically, cancer encompasses a diverse set of illnesses that share the property of inappropriate cell growth, and biologically, cancer cells harbor thousands of molecular alterations that distinguish them from normal cells [1]. Despite this complexity, it is clear that certain mutations play critical roles in programming the malignant state by imparting or facilitating particular biological phenotypes [2]. Several lines of evidence now firmly implicate * Corresponding author. Tel.: þ1-617-632-2641; fax: þ 1-617632-2375. E-mail address: [email protected] (W.C. Hahn).

dysregulation of the normal homeostasis of telomeres and telomerase as a critical change necessary for the development of cancer [3 – 7]. Telomeres are nucleoprotein structures that terminate eukaryotic chromosomes. One of the primary functions of telomeres is to mark the ends of linear chromosomes as distinct from a broken DNA end and to facilitate chromosome replication [8]. In addition, recent observations implicate the maintenance of telomeres as an important determinant of replicative lifespan in many types of eukaryotic cells [9 –11]. Telomeres are maintained by a specialized reverse transcriptase, the ribonucleoprotein telomerase, which is composed of a ubiquitously expressed RNA subunit, Telomerase RNA Component (TERC) [12],

0304-3835/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 0 4 - 3 8 3 5 ( 0 2 ) 0 0 7 0 3 - 6

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and a protein catalytic subunit, Telomerase Reverse Transcriptase (TERT), whose expression is highly regulated [13,14]. Although other proteins associate with the telomerase holoenzyme [15 – 18], these two subunits comprise the core catalytically active enzyme [19 –21]. Activation of telomerase is strongly correlated with cancer, implicating the interplay of telomerase, telomeres, and telomere maintenance as a critical step in cancer development [3,22]. Here we focus on the roles of telomere maintenance and telomerase activation in the process of malignant transformation. Studies in both human and animal cancer models indicate that telomeres and telomerase serve dual roles in oncogenesis, serving both to suppress and facilitate neoplastic transformation. Understanding the context and mechanisms by which telomeres and telomerase contribute to cancer development will not only provide insight into the complex etiology of cancer but also promises to provide novel targets for cancer therapy.

2. Telomeres and tumor suppression It is a well-established principle that normal human cells exhibit a restricted replicative lifespan when propagated in culture [23]. In fact, several laboratories have identified at least two proliferative barriers that limit the lifespan of human cells. These replicative barriers are called replicative senescence or mortality stage 1 (M1) and crisis or M2 [24]. Since immortalization is required for experimental cell transformation [25,26], several investigators have proposed that the limited replicative capacity of human cells suppresses tumor formation by greatly reducing the available pool of nascent cancer cells [27 – 32]. In human fibroblasts, the retinoblastoma (pRB) and p53 tumor suppressor pathways play critical roles in controlling the onset of cellular senescence. Neutralization of pRB and p53 function, most often accomplished experimentally by the expression of viral oncoproteins such as the Simian virus 40 (SV40) large T antigen [33]or the human papillomavirus (HPV) E6 and E7 oncoproteins [34,35], permits such cells to bypass senescence and extends replicative lifespan. Such post-senescent cells continue to proliferate until they reach crisis, characterized by widespread apoptosis and chromosomal instability

including end-to-end chromosomal fusions and nonreciprocal translocations [36]. Thus, in addition to their roles in regulating cell cycle progression and the response to agents that damage DNA, the p53 and pRB tumor suppressor pathways play important roles in governing cell lifespan. However, loss of function of these pathways, as occurs in most human cancers, fails to immortalize human cells. In addition to these tumor suppressor pathways, the maintenance of telomeres contributes in essential ways to regulating cell lifespan. In primary human cells, telomeres shorten with progressive cell division, and telomere length has been correlated with replicative capacity of human fibroblasts [9,37]. Since most asynchronously proliferating normal human cells fail to display telomerase activity, these observations have led some to propose that a specific telomere length triggers replicative senescence [32]. Although recent evidence suggests that other aspects of telomere structure or telomere-associated proteins may trigger replicative senescence [38 – 40], these observations indicate that the particular telomere status of human cells regulates entry into replicative senescence. In post-senescent cells expressing viral oncoproteins, telomeres continue to shorten with cycles of cell division until these cells reach crisis, where telomere length is critically short [41]. Although introduction of HPV E7 has been reported to elongate telomeres without telomerase activation, long-term cultivation of these cells also eventually results in crisis [42,43]. Studies in both human and murine cells indicate that shortened telomeres in these post-senescent cells no longer retain the capacity to protect chromosomes from degradation and damage, suggesting a plausible mechanism for the chromosomal instability and aneuploidy observed when cells enter crisis [44 –47]. Immortal cells that escape crisis maintain constant telomere lengths and most express telomerase activity [48,49]. Consistent with these observations, introduction of human TERT (hTERT) and activation of telomerase leads to immortalization of some primary human cells and all post-senescent cells [50 –54]. Taken together, these observations indicate that telomeres operate in important ways to regulate cell lifespan at both senescence and crisis. These observations led some to propose that these replicative limits actively suppress tumor formation.

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Recent work has provided support for this notion while elucidating some of the mechanisms responsible for a long-standing paradox in cancer biology. Although the introduction of cooperating oncogenes such as ras and myc or the adenovirus E1A protein and ras into primary murine fibroblasts efficiently converts such cells into tumorigenic cells [55,56], similar experiments in human cells consistently fail to produce immortalized, tumorigenic cell lines [57]. These observations indicate that the biology of murine and human cell transformation differs in significant ways; at least one of these differences is the regulation of telomere length and telomerase activity [58,59]. Telomeres are much longer in the mouse [60], and telomerase is expressed ubiquitously in adult murine somatic tissues [61,62], making it unlikely that appreciable telomere shortening occurs during normal proliferative history of murine cells. Although primary murine cells encounter a proliferative block similar to the senescence observed in cultured human cells with extended passage, this barrier is controlled by the actions of the p53 tumor suppressor pathway rather than by telomere attrition [63]. These observations suggest that the presence of telomerase is responsible for the relative ease with which murine cells are immortalized and rendered tumorigenic. Recent work demonstrates that coexpression of hTERT together with the SV40 Early Region and an oncogenic allele of H-RAS was sufficient to transform a wide variety of human cells [4 – 6,64 –67]. Cells that expressed all three factors are capable of anchorage-independent growth and tumor formation in animal hosts, suggesting that at least one important difference between human and mouse cells is the constitutive presence of active telomerase. Human cells that express only SV40 oncoproteins and H-RAS proliferate only for a short period of time, consistent with a role for telomere attrition in controlling replicative lifespan [4,65]. Importantly, when very early passage primary cells were used, expression of the SV40 Early Region and a mutant HRAS protein sufficed to permit focus formation, suggesting that initially long telomeres permitted adequate cell proliferation for this type of assay [65]. Similarly, introduction of the adenovirus EIA oncoprotein, MDM2, and mutant H-RAS into early passage human fibroblasts suffices to allow growth in soft agar but tumors inevitably activated telomerase

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[111]. Taken together, these studies reinforce the notion that the immortalization of human cells is a prerequisite for the eventual progression to a tumorigenic state and that the telomere attrition serves as a mechanism of tumor suppression. Although human and murine cells differ with respect to their telomere biology, several recent studies using mice in which telomerase activity was eliminated by a targeted deletion of the RNA component, mTerc, provide further evidence that loss of telomere maintenance leads to tumor suppression. In such mice, germline deletion of mTerc results in loss of telomerase activity, and after four to six generations of interbreeding, the telomeres of such mice eventually reach lengths similar to those of cells observed in human cells [68]. Such late generation mTerc null mice are relatively resistant to treatments known to induce skin papillomas, suggesting that loss of telomeres contributes to tumor suppression [69]. Furthermore, when such mTerc null mice were interbred with tumor prone p16INK4A null mice, a marked decrease in the incidence of malignant tumors was observed in the doubly deficient mice [30]. Consistent with these observations, cells from such mice were also relatively resistant to cellular transformation in vitro [30]. Taken together, these results indicate telomere attrition to critically short lengths serves to limit tumor progression both in cell and animal models of transformation.

3. Telomere shortening promotes genomic instability Although telomere shortening appears to limit the replicative lifespan of human cells and suppresses cell transformation, telomere shortening also eventually leads to critically short telomere lengths and the onset of crisis. Although the most of the cells that enter crisis are eliminated by apoptosis [33,49], sporadic cells (, 1 £ 1027) survive crisis and become immortal [33]. As described previously, such immortal cells typically exhibit aneuploidy and extensive nonreciprocal chromosomal translocations providing further support for the notion that telomeres lose their protective function at lengths associated with crisis [48]. Despite exhibiting critical telomere shortening upon entering crisis, cells that survive crisis

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maintain stable telomere lengths [41]. Telomeres in these cells are maintained either by the activation of telomerase [3,41] or a telomerase-independent mechanism of telomere maintenance called alternative lengthening of telomeres (ALT) [70,71]. Thus, critical telomere shortening induces chromosomal instability that promotes the acquisition of mechanisms to maintain stable telomere lengths. This stabilization of telomere length permits immortalization and facilitates further malignant progression. In addition, the genomic instability induced by critical telomere shortening facilitates the accumulation of other genetic alterations that contribute to further malignant progression. For example, transformation of murine embryo fibroblasts (MEF) derived from late generation mice lacking both mTerc and p53 by pairs of oncogenes occurs more efficiently than transformation of MEF derived from early generation doubly deficient mice or from mice deficient only for p53 [46], indicating that the telomere dysfunction induced in this model contributes to malignant transformation. Furthermore, when mice lacking mTerc are interbred with mice heterozygous for p53, the resulting mice develop epithelial malignancies at a substantially higher rate than is observed in p53 heterozygous mice [44]. Correlating with this increase in epithelial cancers, karyotypes from these epithelial tumors are typified by nonreciprocal translocations common in human epithelial malignancies but rare in most murine models of cancer. Recent work indicates that the chromosomal regions involved by these karyotypic abnormalities are syntenic to regions altered in human cancers, suggesting that the genetic instability caused by critical telomere shortening drives changes associated with human epithelial cancers [72]. Similar observations occur in mice doubly deficient for mTerc and the adenomatous polyposis coli (APC) tumor suppressor gene [73]. Strikingly, in human colon cancer specimens, anaphase bridges occur at a time when telomere shortening is likely, suggesting that a similar process occurs in human tumor pathogenesis [73]. It should be noted that in mice, shortened dysfunctional telomeres generate signals dependent upon the activity of the p53 pathway, while in human cells such signals appear to activate both the pRB and p53 pathways [74]. While these observations add further support to the notion that telomere length is regulated

differently in human and murine cells, critical telomere shortening clearly drives chromosomal instability in mammalian cells. Thus, although telomere shortening serves as a tumor suppression function early in cancer development, progressive telomere shortening and critically short telomeres, particularly in the setting of p53 and/or pRb deficiency often observed in later stages of malignant transformation, facilitates tumor progression by two mechanisms: the selection of immortal clones and the acquisition of other genetic alterations associated with epithelial cancers.

4. Telomerase activation and tumor promotion In experimental models, activation of telomerase or ALT during crisis permits cell immortalization and clearly facilitates further steps in malignant progression. Indeed, although immortalization conferred by the expression of hTERT alone fails to transform human cells [75,76], this lifespan extension cooperates with other oncogenes and tumor suppressor inactivation to permit the tumorigenic conversion of primary human cells [4 – 6,64– 66]. In consonance with these findings, the majority of human cancer cell lines and tumor samples express telomerase activity [3,22] while the remainder of these telomerase null, immortal cell lines and tumors often show evidence of ALT [70,71]. Furthermore, inhibition of telomerase via genetic [28,29], anti-sense [77 –79] or pharmacologic methods [80 – 88] inhibits the long-term proliferation of human cancer cell lines and sometimes induces acute cell death [89 – 91]. Taken together, these observations indicate that the telomere maintenance by telomerase or ALT facilitates acquisition of the tumorigenic state through immortalization. Several recent reports suggest, however, that the activation of telomerase serves other equally important functions in promoting tumorigenicity. Unexpectedly, two groups independently found that forced expression of mTert results in mice with an increased propensity to form skin or breast tumors [92,93]. Since these groups used different promoters to drive the expression of the mTert transgene, these observations indicate that such mTert overexpression contributes to tumor development independently of the specific promoter used. Similarly, although ALT

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Fig. 1. The dual role of telomeres and telomerase in cancer development. Telomeres and telomerase function both as a tumor suppression mechanism as well as a crucial factor in allowing cells to become tumorigenic. The contribution of telomeres and telomerase activation to tumor suppression and promotion depends on the genetic context and the proliferative history of the nascent cancer cell.

serves to immortalize human cells, co-expressing an oncogenic allele of H-RAS in some human fibroblasts that exhibit the ALT phenotype and express the SV40 large T and small t antigens failed to permit tumorigenic growth [94]. Such cells were rendered fully transformed with the additional introduction of hTERT or a version of hTERT carrying a carboxyterminal hemagluttinin epitope tag [48], which does not permit telomere elongation [94]. These observations suggest that although catalytic activity is necessary, that telomere elongation alone is not sufficient to explain the contribution of this additional function of hTERT in transformation. These findings may explain the finding that murine tumors exhibit a small but significant increase in telomerase activity [95]. At present the additional mechanism(s) by which telomerase activation contributes to tumorigenicity remain undefined. Several reports indicate that cells expressing catalytically active hTERT are more resistant to apoptosis [96 – 98], and Blackburn and her co-workers have proposed that catalytically active hTERT serves as a physical cap for the telomere [8, 54]. Alternatively, activation of telomerase may

stabilize further karyotypic changes after crisis, facilitating proliferation of an immortal clone [7]. Identifying the molecular mechanisms responsible for these additional transformation-associated functions of telomerase activation will have important implications not only for our understanding of telomere biology but also for the development of telomere- and telomerase-based therapeutics.

5. Integration and implications Thus, telomere shortening and telomerase activation can act both to suppress and to facilitate tumor development depending on the timing and context of these related events. Telomere loss limits cell proliferation and serves as a mechanism for tumor suppression. However, sufficient loss of telomere length eventually leads to genomic disarray that drives tumor formation both through the activation of telomerase and through the generation of other mutations necessary for tumor progression. These opposing roles of telomeres and telomerase operate

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both to suppress and facilitate cancer formation (Fig. 1). Despite these observations, several important questions concerning the role of telomerase in the pathogenesis of human cancer require further study. Initial surveys of telomerase activity using the telomere repeat amplification protocol (TRAP) assay indicated that telomerase activity strongly correlated with malignant disease [3,22]. In addition, a number of laboratories have reported that increased telomerase activity correlates with increased malignant potential and stage [99 – 104] and that genomic instability associated with loss of telomere sequences correlates with a late stage in the development of colonic adenomas [73]. These findings suggest that telomere attrition drives genomic instability and the acquisition of hTERT expression and telomerase activity in the later stages of cancer development. In contrast, other studies, particularly those which used in situ hybridization for hTERT, suggest that hTERT expression is readily detected in a number of pre-cancerous lesions, benign tumors and other disease states [105 – 109]. These observations suggest that the activation of telomerase may occur early in tumor development; perhaps even before sufficient telomere attrition has occurred. Taken together, although it is clear that telomere maintenance and telomerase play critical roles in the pathogenesis of human cancer, further work is necessary to understand the temporal sequence of telomerase activation, telomere shortening, and malignant transformation. Understanding these various roles of telomerase and telomere maintenance in human cancers will facilitate the development of novel therapeutics based on telomere biology. Already preclinical evaluations of telomerase-based pharmacology and immunotherapy show tremendous promise for the development of new treatment strategies [79,81,110]. Further work will clarify which types of cancers and at which stage of disease will present the greatest opportunities to exploit telomere biology therapeutically. Although the time required for telomere shortening and cell death after complete telomerase inhibition suggested that telomere or telomerase-based therapies might be best suited for patients whose cancers were in a minimal disease state [28,29,79,81], recent reports implicating other roles for telomerase in malignant transformation suggest that such therapies will find

useful application in other clinical scenarios. Understanding the unique biology of telomeres and telomerase in cancer development will not only identify targets for drug development but will also facilitate the efficient design of clinical trials to identify effective anti-telomere and anti-telomerasebased therapies.

Acknowledgements We thank J. Boehm for helpful comments. This work was supported in part by grants from the U.S. National Cancer Institute K01 CA94223, the Doris Duke Charitable Foundation, a New Investigator Award from the U.S. Department of Defense DAMD17-01-1-0049, a Dunkin Donuts Rising Star Award and a Kimmel Foundation Scholar Award.

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