Telomerase at the intersection of cancer and aging

Abstract

Although cancer and aging have been studied as independent diseases, mounting evidence suggest that cancer is an aging-associated disease and that cancer and aging share many molecular pathways. In particular, recent studies validated telomerase activation as a potential therapeutic target for age-related diseases, and at the same time, abnormal telomerase expression and telomerase mutations have been associated with many different types of human tumors. Here, we revisit the connection of telomerase to cancer and aging in light of recent findings supporting a role for telomerase not only in telomere elongation, but also in metabolic fitness and Wnt activation. Understanding the physiological impact of telomerase regulation is fundamental considering the therapeutic strategies that are being developed involving telomerase modulation.

Telomerase defects may lead to aging and cancer

Telomeres are repetitive DNA sequences at chromosome ends that are bound by a protective protein complex known as shelterin, which prevents them from eliciting a DNA damage response (DDR) ^{1, 2}. Seminal studies have shown that telomeres shorten with each cell division due in part to the end-replication problem, an inability of the DNA replication machinery to fully replicate DNA ends ^3-6. This is paralleled by the silencing of telomerase, a reverse-transcriptase responsible for de novo telomere extension in most adult tissues. Some adult cell types, such as adult stem cells, have the ability to activate telomerase, particularly in the transient amplifying compartments ⁶. Nevertheless, telomerase expression in stem cells is not sufficient to prevent progressive telomere shortening associated with increasing age ⁷.

The first connection linking telomere length to the aging process came from the observation that human primary fibroblasts had shorter telomeres with increasing donor age and that when telomeres reached a critically short length they resulted in loss of proliferative ability, a terminal condition for cells known as replicative-senescence ⁸. It is now thought that senescence, either triggered by telomere shortening or by other non-telomere related pathways, is a key cellular outcome which may contribute to the aging process, as well as act as a barrier for tumor progression ⁹. In particular, telomere shortening and increased numbers of senescent cells have been found to occur in both proliferative and non-proliferative tissues as they age ^10-12. The importance of cellular senescence in the aging process was recently demonstrated by depletion of senescent cells in the context of an adult organism, the BubR1 progeroid mouse model, which rescued tissue dysfunction and increased organismal health-span (of note, BubR1 mice present an unusually high level of senescent cells and so may not be completely reflective of the natural aging process) ¹³. In a similar manner, telomerase activation strategies have been recently shown to prevent telomere shortening associated with aging, delay organismal aging, and increase both healthspan and longevity ^{14, 15}.

The anti-aging role of telomerase has been demonstrated to be largely mediated by its canonical role in elongating telomeres, which prevents the accumulation of critically short telomeres and loss of tissue homeostasis ^{14, 15}. In particular, telomere shortening in the context of adult stem cell compartments, has been previously demonstrated to cause severe impairment of stem cell mobilization and a subsequent defect in the ability to regenerate tissues ¹⁶, a situation that is similar to that of the so-called human telomere syndromes ^{17, 18}. This is because short/unprotected chromosome ends are recognized as persistent/non-repairable DNA breaks triggering persistent DDR ^18-20, as well as cellular senescence or apoptosis mediated by the p53 pathway.

Short telomeres, and subsequent DDR activation, could occur both in cancer and aging (Fig. 1). On one hand, increased abundance of short telomeres correlates with higher genomic instability and decreased longevity in various organisms, including mice, zebrafish, and yeast ^21-24. In particular, mice deficient for telomerase or for telomere binding proteins are characterized by accelerated age-related defects ^{14, 16, 18, 19, 21, 22, 25-32} with the load of telomere dysfunction correlating with the lifespan of mice ³³. In humans, short telomeres are considered good indicators of an individual’s health status and correlate with both genetic and environmental factors ^{18, 34-37}. Although recent findings strongly support the idea that short telomeres drive several age-related diseases ³⁸ we cannot exclude the possibility that in some situations short telomeres may be a consequence of the disease itself.

Figure 1. Short telomeres in aging and cancer.

Major pathways affected by short telomeres and their impact on aging or cancer. DNA damage and tumor suppressor activity have been shown to impact tissue decline and aging. When DNA damage checkpoints are bypassed, cells with short telomeres could potentially progress to cancer. The role of stem cells with short telomeres in cancer and whether short telomeres could modulate other pathways independently of p53 (such as mitochondrial dysfunction) remains unknown.

Although tumors may arise from cells with short telomeres and chromosomal instability, telomerase activation and telomere maintenance are requisites for the progression of most human tumor types ^39-49. Further linking TERT (telomerase reverse transcriptase, the human telomerase) to cancer are GWAS results showing correlations between particular SNP variants on the 5p15.33 bin (which includes TERT) and a higher cancer risk ^{21, 50-58}. In particular, genetic variants in telomerase-associated genes and in the TERT-CLPTM1L locus are associated with different cancer types ^{50, 59-65}. Although the mechanism by which these variants interfere with telomerase levels/activity is mostly unknown, there are indications that the variants may lead to an increase in the gradual shortening of telomeres over time ^{52, 59}, but these results still need to be confirmed ⁶⁶. On the other hand, two recent studies linked melanoma risk to promoter mutations in the TERT gene associated with increased transcriptional activity of the TERT promoter ^{67, 68}, demonstrating the importance of tightly controlled telomerase expression.

Like cancer, aging encompasses a spectrum of cellular and molecular changes, but in the case of aging, these eventually result in loss of regenerative capacity and tissue dysfunction, either through loss of functional cells or through the accumulation of surviving aberrantly damaged cells, which could result in the appearance of neoplasias. In this review we focus on aging associated with telomere shortening, and on how telomerase could be an important therapeutic target for this process. To support the dual role of telomerase in aging and cancer we highlight recent studies that have demonstrated that expression of telomerase in aged organisms is a valuable tool to counteract tissue degeneration through the protection of short telomeres ⁶⁹, envisioning that controlled telomerase activation under particular settings may delay age-related tumorigenesis.

Telomerase as a key factor that regulates aging

Evolution has developed different barriers against cancer amongst different species. These barriers are related to the ability to cope with DNA damage and the prevention of the accumulation of damaged cells and tissues. Irrespective of its source, damage acts as the basis for the development of dysfunctional tissues, which are a hallmark of age decline as well as the basis for cancer ^{69, 70}.

Patients carrying mutations in genes crucial for telomere maintenance show accelerated aging phenotypes. Such is the case for patients carrying mutations in TERT, TERC or other telomere maintenance genes, which lead to an accelerated aging syndrome known as dyskeratosis congenita (DC) ⁷¹. DC encompasses a spectrum of pathologies including abnormal skin pigmentation, nail dystrophy, leukoplakia and pancytopenia ⁷². In patients carrying mutations in TERT and TERC, the severity of pathologies correlates with the abundance of short telomeres, so the onset of disease is anticipated with increasing generations (a phenomenon known as “genetic anticipation”) ⁷³. Interestingly, human telomere syndromes closely recapitulate the phenotypes of previously generated mouse models for telomerase deficiency. In particular, mice genetically deficient for telomerase or some of the telomere-binding proteins present a plethora of pathologies generally characterized by the loss of tissue regeneration and organ function ^{32, 74}. In addition to the defects in the highly proliferative tissues such as the bone marrow or the skin, mice and humans with telomerase deficiency also present pathologies in more quiescent tissues, such as cardiomyopathy, insulin resistance, and lung and liver fibrosis ^{75, 76}. To date it remains unknown how telomerase deficiency also leads to short telomeres in tissues with a lower proliferative potential ^{77, 78}. In this regard, mitochondrial dysfunction has been recently reported in quiescent tissues (such as the heart and liver) in the context of telomerase deficiency in mice. Several reports described that mt-TERT (TERT that localizes at mitochondria) improves mitochondrial function and protects from oxidative stress ^79-81. In particular, telomerase deficient mice that have been bred for several generations and have an increased abundance of short telomeres present a marked mitochondrial compromise triggered by the suppression of the peroxisome proliferator-activated receptor gamma, coactivator 1 alpha and beta (PGC1α and PGC1β) networks which control, amongst other processes, mitochondrial function and oxidative defense ⁸². Interestingly, this connection between telomere dysfunction and mitochondrial dysfunction is mediated by p53, a common checkpoint to telomere syndromes ⁸³. Additionally, mitochondrial dysfunction in quiescent tissues of telomerase-deficient mice could be initiated by pathways independent of p53 ⁸³. Of note, mitochondrial defects have been described in the first generation of TERT KO mice (G1) ⁸², when telomere length is still conserved, demonstrating that mitochondrial dysfunction could, at least partially, precede or parallel telomere shortening. It has also been recently demonstrated that mitochondrial dysfunction is associated with physiological mouse aging, and reverted by telomerase activation ^{15, 82}.

With the aim of dissecting the role of telomerase activity and telomere length in cancer and aging, various mouse models for telomerase over-expression have been generated (table 1). Transgenic mice that carry the mouse TERT gene under the control of the keratin 5 promoter (K5-mTERT referred hereafter as TgTERT) show increased tissue fitness, however, owing to an increased incidence of spontaneous tumors, these mice do not show an extended median lifespan ⁸⁴. To unmask the potential anti-aging role of telomerase, TgTERT mice were crossed with mice carrying extra copies of the tumor suppressors p53, p16 and Arf (Sp16/SArf/Sp53 mice), which were previously reported to be cancer resistant ¹⁴. In this context, TgTERT/Sp16/SArf/Sp53 showed improved health span and a 40% increase in median longevity compared to wild-type controls, or 26% comparing with the long-lived and healthy Sp16/SArf/Sp53 mice, demonstrating the anti-aging activity of telomerase. A similar scenario occurred when telomerase overexpression was combined with other cancer-protective conditions, such as by subjecting mice to caloric restriction (CR). In this setting, telomerase overexpression synergized with CR to significantly extend mouse lifespan ⁸⁵. This synergy between telomerase and tumor resistance in extending organismal longevity seems to be a naturally occurring strategy, such as in the case of the mole rat or other small animals that are positive for telomerase, present higher tumor suppressor barriers ^{86, 87} and have an unusually increased longevity for their species. Although this synergy could be a strategy in some situations, there are exceptions (such as the American beaver, another long-lived rodent, which has no detectable telomerase activity ⁸⁸), highlighting the complexity of aging.

Table 1. Outcomes of enforced expression of telomerase in mice.

Ref	Model / Telomerase activation	Cancer	Aging	Comments
25	C57Bl/6 Germline K5-mTERT	Stratified epithelia histologically normal More tumors after DMBA+TPA treatment. Skin more sensitive to esters.	Increased wound-healing	High levels of telomerase activity in stratified epithelia do not alter the normal epithelium structure and are not associated with changes in p53, Ras or c-Myc levels.
129	FVB/n strain Germline CAG promoter	Higher incidence of breast carcinoma in all but 1 female of founder A. No differences in males.	n.d.	No susceptibility to spontaneous or DMBA-induced papillomas in mTERT Tg mice. Enforced mTERT expression did not alter the high rate of spontaneous tumor formation in Ink4a/Arf-deficient mice.
130	C57Bl/6 Germline K5-mTERT and K5-mTERT/p53−/−	Higher tumor incidence (spontaneous pre- neoplastic and neoplastic lesions in stratified and non- stratified epithelia)	Lower lifespan in both k5-mTERT or k5-mTERT/p53−/−	Loss of p53 results in a dramatic decrease in the life span of these mice, concomitantly with an increased incidence of tumors, in particular lymphomas.
131	C57Bl/6 Germline Lck-TERT mice	Higher incidence of spontaneous lymphoma.	n.d.	Lck-Tert thymocytes show greater spontaneous and IR-induced chromosomal instability.
84	C57Bl/6 Germline K5-mTERT	More hyperproliferative lesions	Increased maximal lifespan Decreased degenerative lesions (kidney, male germ line)
132	FVB/n strain CMV enhancer/β-actin promoter	n.d.	Enhancing of hair growth through stem cell mobilization (independently of the TERC component)
14	C57Bl/6 Germline K5-mTERT/Sp53 and K5-mTERT/Sp53/SArf/Sp16	Higher tumor incidence (mainly lymphomas) and similar lifespan (K5-mTERT/Sp53 vs K5-mTERT)	Lower tumor incidence and higher lifespan and health-span in K5-mTERT/Sp53/SArf/Sp16 vs K5-mTERT/Sp53 or WT controls
89	G4TERT-ER mice (30–35 week old C57Bl/6) 4-OHT activation late in life	Telomerase activation was not sufficient to promote tumorigenesis.	Extended life and health span	Chromosomal instability was referred.
120	C57Bl/6 (1yr and 2 yrs old) TA-65	No increase in tumor incidence	Extended health. No differences in lifespan	Activation of telomerase is not direct Other studies have described similar telomerase activators in mice and humans (see references: 127, 133)
82	G4 TERT−/− (WW6/C57BL/6) Ad-mTERT (specifically to the liver)	n.d.	Ad-mTERT injection partial rescue PGC-1α/β, Glc-6-P and Pepck expression, accompanied by a 30% increase in glucose levels relative to Ad-GFP controls, in G4-TERT−/− mice
134	C57Bl/6 (18 to 22 g., males and females) Ad-mTERT-GFP (microinjection into the bilateral Dg of mice)	n.d.	Ad-mTERT-GFP led to neurogenesis upregulation, produced antidepressant-like behaviors, and prevented the CMS-induced behavioral modifications
128	CD1 (9–11 weeks old) AGS-499	n.d.	Extended health (neuroprotective effects in NMDA-injected CD-1 mice)	No mechanism of telomerase activation
15	C57Bl/6 (1yr and 2 yrs old) AAV9-mTERT	No increased tumor incidence	Extended life and health span

More recently, two independent studies demonstrated that telomerase activation either in a mouse model of accelerated-aging (late generation TERT-ER mouse model) or in natural-aged mice (1 and 2 year old wild-type mice) is sufficient to delay aging without increasing cancer incidence ^{15, 89}. These studies support the idea that telomere shortening is one of the molecular mechanisms of cellular aging and lifespan modulation, and more notably, they demonstrate that telomerase reactivation in adult (or aged) organisms has a positive impact in delaying aging, which can be separated from its role in cancer when its aberrantly expressed. Future work should focus on understanding the molecular mechanisms by which telomerase delays aging and disease in different organs and tissues. Below we discuss novel pathways and telomerase partners which could be also involved in these processes.

Telomerase regulation in cancer

The role of telomerase in cancer has been extensively studied. Almost all human cancers present activation of telomerase as a hallmark, most likely as a mechanism to allow unlimited cell proliferation of tumor cells ⁹⁰. Although telomerase activation can be an early event in cancer, it is not necessary for cancer initiation ⁹¹. However, telomerase can stimulate tumor progression by ensuring maintenance of telomeres above a critically short length, thus preventing induction of cellular senescence or apoptosis. Several mechanisms have been reported to activate telomerase in cancer, such as different oncogenes including Myc and Wnt ^92-94 which act as transcriptional regulators of telomerase. Additional telomerase activation mechanisms involving alternative splicing or epigenetic alterations have also been described ⁹⁵. Recently, mutations increasing transcriptional activity of the TERT promoter from generation of de novo consensus binding motifs for E-twenty-six (ETS) transcription factors have been described in human melanomas ^{67, 68}. In addition to the canonical role of telomerase in maintaining telomeres above a critical length, telomerase has also been proposed to regulate other pathways, which could have an impact on cancer growth, such as regulation of Wnt targets and metabolism (^{82, 96}). Getting rid of telomerase can also be problematic; the lack of telomerase could lead to increased chromosomal instability, which in turn could be at the basis for cancer initiation when tumor suppressor barriers are bypassed ⁹⁷. Indeed, recent evidence demonstrated that short telomeres alone could lead to genomic instability and cancer ⁹⁸. Thus, the current view is that telomerase deficiency may contribute to the early steps of cancer development by fueling chromosomal instability, while subsequent activation of telomerase may be necessary to allow tumor growth and tumor progression towards more malignant states ⁹⁹.

Loss of function and gain of function mouse models for telomerase have been instrumental in understanding the role of telomerase in cancer. On one hand, telomerase deficient mice (mTR^−/−) are resistant to both induced and spontaneous tumorigenesis ¹⁰⁰, except when telomerase deficient mice were crossed with p53^+/− or p53^{−/− 101, 102}. In this scenario a switch to epithelial carcinogenesis was observed, consistent with the role of telomere shortening in the pathophysiology of human cancers ¹⁰³. Short telomeres could be recognized as DNA double strand (dsDNA) breaks, a deleterious DNA aberration that results in a strong activation of DNA damage repair (DDR) pathways. With an intact DDR and active checkpoints, cells with dsDNA breaks activate a multitude of signaling cascades which conclude in p53 and tumor suppressor activation. This cascade of events culminates in activation of anti-proliferation signals. On the other hand, if tumor suppressors or p53 are bypassed, a common characteristic of tumors, chromosome fusions and genomic instability could converge to give rise to cancer. This potential of telomerase to sustain the growth of tumor cells illustrates the importance of telomerase regulation in adult tissues, and probably explains why most adult cells silence telomerase expression.

Given the importance of telomerase to sustain cancer growth, telomerase inhibitors were considered as potential therapies against tumor malignancy. Recent evidence demonstrates, however, that tumors in which telomerase are lost may well activate different pathways to overcome this situation, such as alternative telomere lengthening ^104-106.

In addition to the canonical role of telomerase in maintaining telomeres, telomerase overexpression has also been shown to influence the regulation of the Wnt pathway, although the physiological relevance and mechanism of this regulation is still debated ^{15, 93, 94, 96, 107}. Nevertheless, given that telomerase activity is aberrantly overexpressed in some cancers, it is possible that Wnt modulation through higher levels of telomerase could contribute to the phenotype of some neoplasias ¹⁰⁸.

Metabolic defects are an important link between cancer and aging. Interestingly, metabolically relevant genes that have been shown to be down-regulated in the presence of short telomeres, such as PGC1α/β, and potentially activated by telomerase re-expression, are also linked to tumor progression ^{109, 110}. Thus, telomerase activation in tumors may also alter cellular metabolism. Further work will be required to refine these complex relationships been telomeres, telomerase and metabolism.

In this regard, transgenic mouse models (e.g., TgTERT mice ¹⁴) have shown that constitutive telomerase over-expression throughout mouse development results in a slightly higher incidence of cancer. Interestingly, telomerase over-expression to similar levels but in the context of the adult organism using a gene therapy strategy, showed beneficial effects delaying aging and extending longevity without increased cancer incidence ¹⁵. This could be related to the fact that the gene-therapy vectors employed (AAV) lead to a loss of TERT expression in highly proliferating cells or tissues. Another explanation could be that AAV preferentially targets post-mitotic cells, which are potentially more resistant to cancer initiation. Alternatively, although the TgTERT mice are the product of single germline integration, they constitutively express telomerase, independently of the replicative potential of a tissue, most likely facilitating proliferation and expansion of cells carrying pathogenic mutations.

Telomerase in stem cells

Stem cells play an important role in the aging process. Stem cell depletion seems to be at the basis of some diseases and could account for accelerated aging syndromes ^111-115. Moreover, conditions that trigger premature aging, such as telomere shortening, also impair the ability of stem cells to regenerate tissues ¹⁶. Indeed, cells with the longest telomeres are enriched at adult stem cell niches both in mice and humans, most likely owing to the fact that these cells have the ability to activate telomerase ^{7, 116}. However, physiological telomerase activation in stem cell compartments is not sufficient to maintain overall telomere length with aging, and telomere shortening and DNA damage accumulation is also a characteristic of aged stem cells ¹¹⁷.

Tumors are thought to be sustained by a subpopulation of cells with stem cell-like properties, the so called cancer initiating cells ^{118, 119}. It will be of interest to address whether these cancer-initiating populations also have the ability to maintain telomeres and activate telomerase activity.

Therapies based on telomerase: therapeutic value and future perspectives

As discussed above, telomerase activation is a potential therapeutic strategy for the treatment of age-related diseases ^{14, 120}. In particular, telomerase activation in adult or old mice by means of a gene therapy strategy was shown to be sufficient to improve metabolic fitness, neuromuscular capacity, and prevent bone loss, as well as significantly increase both median and maximum longevity, without increased cancer incidence. The finding that this strategy of telomerase activation does not lead to cancer could be due to the fact that the vectors used (AAV)9¹²¹ are non-integrative, thus preventing the expansion of clones with telomerase overexpression ¹²². Similarly, telomerase expression in an accelerated model of ageing owing to telomere loss (G4^TERT-ER model) rescued several age phenotypes ⁸⁹, and although higher genomic instability was detected, it did not lead to an increase in tumorigenesis. These studies suggest that telomerase expression could be considered a feasible approach to reverse tissue dysfunction and extend healthy lifespan without increasing cancer incidence. Dedicated studies should be performed in the future, using mice at different ages and comparisons at the same age, to assess the safety potential of these strategies. The actual value of these new therapies will reside in their safety, and a detailed understanding of the telomeric and non-telomeric roles of telomerase in tissue-specific healing and cancer will be crucial for considering telomerase for anti-aging therapies.

Whether these promising results could be translated to humans is unknown. It seems hazardous to use the lack of tumorigenesis in mice as evidence for the safety of pro-telomerase therapies in humans, as it is known that telomerase is differentially regulated in these organisms ^{123, 124}. The fact that human longevity is much longer than that of mice could increase the probability of cancer formation favored by an external telomerase treatment. The opposite argument can be made, however, in that humans are much more resistant to cancer than mice and therefore it is less likely that telomerase activation could lead to cancer in humans compared to mice. Even though the peak of telomerase activity in humans occurs at early stages, as it does in mice,, humans almost completely lose telomerase activity from somatic tissues in the adulthood, contrary to mice where telomerase is found in some somatic tissues ^{125, 126}. As a starting point for translating these findings to the clinic, telomerase activation is likely to be first tested for treatment of the so-called telomere syndromes ¹⁷. In this scenario the use of tissue specific gene-therapy vectors expressing telomerase could be envisaged as a potential solution. Based on those outcomes, it will be easier to assess the feasibility of expanding telomerase activation as a strategy for combating cancer.

Concluding Remarks

The finding that telomerase plays roles in distinct and complementary circuitries have helped reveal its function in cancer and aging. Indeed, a change of paradigm seems to be occurring in telomerase biology, with a switch from viewing telomerase as fueling cancer to reversing aging. Telomerase expression in a background of high levels of tumor suppressors or in aged organisms seems to prevent its expected pro-cancer activity and yet it still functions as an anti-aging factor. Supporting this notion are novel telomerase activators ^{120, 127, 128}, some of which are commercially available, used as anti-aging supplements. Although much of the recent work provides only proof-of-principle that telomerase works for tissue healing, we cannot dismiss that in the future telomerase expression could be used as a safe approach for certain telomere-diseases ¹⁷ or other accelerated aging syndromes.