Implications of telomere-independent activities of telomerase reverse transcriptase in human cancer

Authors


Abstract

Telomerase plays a pivotal role in the pathology of cancer by maintaining genome integrity, controlling cell proliferation, and regulating tissue homeostasis. Experimental data from genetically modified mice and human premature aging diseases clearly indicate that intact telomere function is crucial for cell proliferation and survival, whereas dysfunctional telomeres can lead to either cancer or aging pathologies, depending on the integrity of the cellular stress response pathways. The canonical function of telomerase reverse transcriptase is the synthesis of telomeric DNA repeats and the maintenance of telomere length. However, accumulating evidence indicates that telomerase reverse transcriptase may also exert some fundamental biological functions independently of its enzymatic activity in telomere maintenance. More recent studies have demonstrated that telomerase reverse transcriptase can act as a transcriptional modulator in the nucleus and exhibits RNA-dependent RNA polymerase activity in the mitochondria. Telomerase activation may have both telomere-dependent and telomere-independent implications for tumor progression. Many excellent reviews have described critical roles of telomere and telomerase in human cancer; this minireview will focus on the role of telomerase in cancer progression, with a special emphasis on the nontelomeric function of telomerase.

Abbreviations
ALT

alternative lengthening of telomeres

EMT

epithelial–mesenchymal transition

mTERT

mouse telomerase reverse transcriptase

mTR

mouse telomerase RNA

NF-κB

nuclear factor-κB

RMRP

mitochondrial RNA-processing endoribonuclease

ROS

reactive oxygen species

TERT

telomerase reverse transcriptase

VEGF

vascular endothelial growth factor

Introduction

The ends of linear chromosomes are special functional complexes consisting of tandem DNA repeat sequences and associated proteins, constituting a specialized heterochromatic structure known as the telomere [1]. Mammalian telomeres are formed by tandem repeats of TTAGGG sequences that are bound by a specialized protein complex. Telomeres not only protect the ends of linear chromosomes from degradation and repair activities, and ensure genome stability, but are also critical components involved in a number of fundamental cellular processes, such as cellular senescence and immortalization, epigenetic programming, and metabolism [2-4]. Normal cells must bypass the two tumor suppression barriers (senescence and apoptosis) and evolve progressively through the multistep processes of tumorigenesis to become malignant. The activation of telomerase plays a fundamental role in bypassing the cellular senescence and crisis barriers, leading to replicative immortality and oncogenic transformation [2, 5, 6].

Telomerase is essentially composed of the RNA component (telomerase RNA), which serves as a template for telomeric DNA synthesis, and the catalytic subunit [telomerase reverse transcriptase (TERT)] [7]. Telomerase activity is repressed during embryonic differentiation, and, indeed, is absent in most types of normal human somatic cells with limited proliferative capacity. However, the enzyme remains active in some tissues, such as male germ cells, stem cell populations, and activated lymphocytes, and is activated in > 90% of cancerous cells through genetic and epigenetic alterations during the cellular immortalization and transformation process [8, 9]. The maintenance of telomere length and integrity is a crucial determinant of the normal cell lifespan and the replicative immortality of cancer cells.

Telomerase activation is viewed as one of the six key events common to cancer development [10, 11]. Understanding the biological functions of telomerase and the mechanisms of telomerase action would provide opportunities for better therapeutic and prognosis strategies in cancer management.

Telomerase activation and replicative immortality

Functional telomeres are involved in several essential biological functions: they protect chromosomes from recombination and end-to-end fusion, thereby preventing unwanted DNA damage signaling and genome instability; and they provide a means for the complete replication of chromosomes in proliferating cells and for control of the replicative capacity of human cells [1-3]. Dysfunctional telomeres can lead to either cancer or aging pathologies, depending on the integrity of the DNA damage response [12, 13].

Owing to the ‘end replication problem’, telomeres shorten with each cell division [4]. At the Hayflick limit [14], critically shortened telomeres trigger the first proliferative barriers in a process known as replicative senescence or mortality stage 1. By inactivating critical cell cycle checkpoint genes or tumor suppressors, or activating certain oncogenes, cells that bypass replicative senescence continue to divide and suffer further telomere loss, until they reach a second proliferative barrier, called crisis or mortality stage 2. These cells show massive cell death resulting from chromosome instability. Rare survivor cells that escape from crisis are able to maintain telomere length, most commonly by activating telomerase, the specialized cellular reverse transcriptase that adds the telomeric repeats onto the ends of chromosomes [15].

The idea that cellular senescence and crisis driven by telomere dysfunctions or other insults are two important proliferative barriers that prevent neoplastic growth has been well accepted [16]. Importantly, the massive genetic instability that occurs during the crisis stage may provide the constellation of genetic alterations required for malignant transformation. In addition to the gains and losses of oncogenes and tumor suppressor genes, one of the critical events during crisis is the activation of telomerase [5, 9]. The maintenance of telomere length by telomerase provides transformed cells with unlimited proliferation potential, which represents a critical step in oncogenic transformation. The maintenance of telomere length and integrity is achieved by the coordinated regulation of telomeric protein complexes and telomerase [17, 18].

Telomere maintenance not only determines the proliferation capacity of cells but also affects a number of cellular processes, such as the DNA damage checkpoint machinery, stem cell biology, mitochondrial function, the stress response, metabolism, and epigenetic regulation of telomeric chromatin [1, 13, 19, 20]. Accordingly, the activation of telomerase may profoundly affect the physiology of cells. There are thousands of publications and numerous review articles documenting the important roles of telomere maintenance and telomerase activation in tumor biology [5, 8, 12]. However, recent progress indicates that telomerase has additional functions beyond telomere length maintenance.

Telomere-independent activities of TERT

In humans, telomerase is active early in embryonic development to offset telomere loss during the rapid proliferation necessary for tissue growth and differentiation, and is turned off in the majority of adult tissues. Telomerase is upregulated or reactivated in > 90% of cancerous cells [21]. Telomerase activation is a crucial prerequisite for immortalization, and plays an important role during the malignant progression of cancer cells. However, the mechanisms of action of telomerase in cancer remain incompletely understood. Increasing evidence is emerging to implicate telomerase in cancer not only by maintaining telomeres but also via mechanisms independent of telomere maintenance [20, 22].

Mounting experimental data have suggested that the ectopic expression of TERT [mouse TERT (mTERT) or human TERT in transgenic mice and in human cells exerts telomere-independent effects on cellular transformation, proliferation, stem cell biology, mitochondrial function, cell survival, the DNA damage response, chromatin remodeling, and the regulation of gene expression [22]. However, the molecular mechanisms of the functions of TERT beyond telomeres are poorly understood. Several recent findings indicate that TERT may indeed possess biochemical activities other than telomeric DNA synthesis and telomere maintenance.

Previously, two groups have independently reported that overexpression of TERT activates epidermal stem cell proliferation and mobilization in a transgenic mouse model. This effect is accompanied by increased keratinocyte proliferation, enhanced hair growth, and augmented skin hyperplasia, independently of its reverse transcriptase activity [23, 24]. In a follow-up study, Artandi's group performed a genome-wide transcriptional profiling experiment analyzing the response of mouse skin to acute changes in TERT levels [25]. In this analysis, they found that the acute withdrawal of TERT resulted in a rapid change in the expression of genes involved in epithelial development, signal transduction, and cytoskeleton/adhesion. Statistical analysis using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembled the responses mediated by Myc and Wnt, two important factors in stem cell function and cancer. This finding suggested that the role of TERT in activating stem cells might be mediated by the transcriptional regulation of a developmental program converging on the Myc and Wnt pathways. Indeed, the authors demonstrated that TERT can function as a transcriptional modulator of the Wnt–β-catenin signaling pathway [26]. They showed that TERT physically occupies the promoters of Wnt target genes, and functions as a cofactor in a Wnt–β-catenin transcriptional complex through an interaction with the SWI/SNF-related chromatin remodeling protein BRG1. This finding that TERT is directly involved in the transcriptional regulation of Wnt target genes by interaction with the Wnt–β-catenin transcriptional complex may provide mechanistic evidence for a nontelomeric function of TERT in stem cell behavior and cancer.

Controversially, however, Strong et al. have characterized both mTERT−/+ and mTERT−/− mice in comparison with mouse telomerase RNA (mTR)−/+ and mTR−/− mice. They observed indistinguishable phenotypes, characterized by a loss of tissue renewal capacity with progressive breeding of heterozygotes, between the mice deficient in mTERT and mTR, but no apparent Wnt pathway defects were observed in the mTERT−/− mice [27]. Similarly, Vidal-Cardenas and Greider [28] critically examined the gene expression profiles in first-generation mTR−/− and mTERT−/− mice containing long telomeres, and did not identify any genes that were differentially expressed between the mTR−/− G1 mice and mTERT−/− G1 mice as compared with wild-type mice under normal physiological conditions.

These apparent discrepancies regarding the involvement of TERT in the regulation of gene expression and the Wnt pathway may result from differences in the strain background or experimental setting. Most observations that have suggested a role for TERT in the regulation of gene expression have been based on the overexpression or downregulation of TERT in a transient fashion. The question raised is whether the observed telomere-independent effects of TERT on gene expression or the Wnt pathway are physiologically relevant. The TERT gene is subject to complex regulation by many transcription factors involved in several intracellular and extracellular signaling pathways, depending on the cell type and tissue environments. Thus, it is conceivable that the impact of TERT on gene expression, Wnt signaling or other telomere-independent activities may become biologically relevant under particular physiopathological conditions.

A recent finding further supports the concept that hTERT possesses telomere-independent biochemical properties. It has recently been shown that hTERT interacts with the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP) [29]. RMRP is a 267-nucleotide noncoding RNA, and is a small nucleolar RNA found in mitochondria. The gene is mutated in the inherited pleiotropic syndrome cartilage–hair hypoplasia [30]. Significantly, this study demonstrated that hTERT and RMRP form a distinct ribonucleoprotein complex with RNA-dependent RNA polymerase activity, and produce double-stranded RNAs that can be processed into small interfering RNA in a Dicer-dependent manner [29]. Thus, it is possible that the TERT–RMRP complex may generate other small interfering RNAs that regulate gene expression at the post-transcriptional level. In support of this hypothesis, gene profiling experiments by several independent investigators have revealed several hundred genes whose expression has been found to be affected by the downregulation or upregulation of TERT [22]. Interestingly, Santos et al. [31] have recently demonstrated that mitochondrial hTERT binds various mitochondrial RNAs and acts as a human telomerase RNA-independent reverse transcriptase in the mitochondria. Therefore, TERT can be targeted to different cellular locations to exert distinct biological activities.

Santos et al. [32-34] have performed a series of studies to define the role of TERT in the mitochondria. Compelling evidence has confirmed the presence of TERT in the mitochondria. TERT contains a mitochondrial localization signal peptide at its N-terminus that has been shown to target TERT to the mitochondria. It is conceivable that TERT may affect signaling in the mitochondria, favoring cellular survival over stress-induced cell death. Experimental data suggest that hTERT overexpression alleviates intracellular reactive oxygen species (ROS) production, improves mitochondrial function and inhibits ROS-mediated apoptosis in cancer cells [33]. However, whether TERT is required for mitochondrial function under normal physiological conditions, whether TERT is transiently targeted to this organelle under stress conditions and, similarly, whether the metabolic compromise and mitochondrial defects observed in TERT knockout mice are also associated with a direct role of TERT in mitochondrial function or regulation remain to be elucidated [13].

Telomere-independent roles of telomerase in cancer progression

Previous studies have shown that levels of hTERT expression correlate with advanced disease and unfavorable prognosis in different types of human cancer [8, 35, 36]. In mouse models, the upregulation of telomerase is observed in a spectrum of developing tumor types, despite ample telomere reserves in mouse cells [5]. mTERT transgenic mice manifest an increased incidence of spontaneous and carcinogen-induced cancer [37]. The enforced expression of hTERT in combination with oncogenic RAS and the SV40 early region induce the tumorigenic conversion of fibroblasts, kidney epithelial cells, and mammary epithelial cells [38-40]. In addition, studies in both human and mouse cell culture models clearly indicate that the presence of long telomeric tracts alone does not necessarily guarantee the oncogenic transformation of cells. Some telomerase-negative immortalized cell lines and cancers use a homologous recombination-mediated mechanism of telomere elongation known as alternative lengthening of telomeres (ALT) [41]. The coexpression of oncogenic H-RAS with the SV40 large and small T antigens in ALT fibroblasts yielded inefficient tumor formation in mice. However, cotransfection of human hTERT with the oncogenic H-RAS and SV40 large and small T antigens resulted in highly efficient malignant transformation of human ALT fibroblasts in vivo [39, 40]. Together, these observations strongly support the existence of telomere-independent roles of telomerase in cancer progression. However, the pathways and molecular mechanisms by which telomerase promotes cancer, apart from its critical role in telomere maintenance, remain incompletely understood.

Cancer progression is a complex morphogenetic program that involves integrated multistep events, such as proliferation, survival, matrix degradation, induction of cell polarity, migration, angiogenesis, and metastasis, which require the coordinated regulation of gene expression [11]. A number of studies have reported that telomerase has a regulatory function in the expression of genes involved in diverse cellular processes, such as the cell cycle, metabolism, differentiation, cell signaling, cell survival, and angiogenesis [22]. Changes in gene expression associated with telomerase activation may also recondition the tumor microenvironment to be favorable for the invasive growth and metastasis of cancer cells. In this regard, it has been reported that vascular endothelial growth factor (VEGF), an endothelial mitogen and key regulator of angiogenesis, induces hTERT expression and telomerase activity [42, 43]. Conversely, our work has shown that hTERT induces VEGF expression at the transcriptional level independently of telomerase activity [44]. It is possible that there is a positive regulatory feedback loop between hTERT and VEGF, which may contribute to the collaborative roles of hTERT and VEGF in tumorigenesis.

A recent study demonstrated that telomerase can directly regulate nuclear factor-κB (NF-κB)-dependent transcription by binding to the NF-κB p65 subunit and recruitment to promoters of NF-κB target genes, such as those encoding interleukin-6 and tumor necrosis factor-α, cytokines that are critical for inflammation and cancer progression [45]. Given that NF-κB is activated in a large number of human cancers and regulates several genes that are important for cell proliferation, resistance to apoptosis, and invasion, this study revealed an important role for telomerase as a transcriptional modulator of the NF-κB signaling pathway that may contribute to cancer development and progression.

Epithelial–mesenchymal transition (EMT) is a developmental regulatory program employed by cancer cells that has been implicated as a critical player in tumor development. Through EMT, transformed epithelial cells can acquire the capacity to invade, resist apoptosis, and disseminate during the course of cancer invasion and metastasis [46]. Recently, Liu et al. have demonstrated a potential role of hTERT in EMT [47]. Using gastric cancer as a model, they showed that the ectopic expression of hTERT promotes EMT and the stemness of gastric cancer cells. Conversely, the downregulation of hTERT expression by small interfering RNAs suppressed transforming growth factor-β1 and β-catenin-mediated EMT. Furthermore, they showed that hTERT interacts with β-catenin, enhances its nuclear localization and transcriptional activity, and occupies the promoter of the β-catenin target vimentin [47]. Interestingly, all of these effects of hTERT were found to be independent of its telomere-lengthening function and telomerase activity. In addition, hTERT and EMT marker expression are positively correlated in gastric cancer samples, and hTERT promoted cancer cell colonization in a mouse model [47].

Okamoto et al. [48] recently showed that hTERT forms a complex with the nucleolar GTP-binding protein nucleostemin/GNL3L and BRG1, which operate in a telomere-independent manner to drive transcriptional programs essential for the maintenance of the state of tumor-initiating cells or cancer stem cells.

Collectively, these studies indicate that hTERT may have multiple telomere-independent functions in gene expression, and in regulation of EMT and the stemness of cancer cells, thereby promoting cancer metastasis and recurrence.

Concluding remarks

It has been well documented that telomerase plays a fundamental role in cancer development by maintaining telomere homeostasis and the infinite replicative potential of cancer cells. Recent compelling evidence supports telomere-independent mechanisms of TERT in several essential cellular functions, including the regulation of gene expression, mitochondrial function, cell survival, cell transformation, and EMT, which may provide transformed cells with cancer-specific capacities at multiple stages of tumor development (Fig. 1). The finding that telomerase acts as a transcriptional modulator of the Wnt–β-catenin and NF-κB signaling pathways, or as an RNA-dependent RNA polymerase, provides important insights for the molecular dissection of the regulatory function and biochemical properties of TERT. A future challenge is to elucidate the signaling pathways downstream of telomerase in both the telomere-dependent and the telomere-independent mechanisms. The identification of novel hTERT-interacting proteins and an understanding of the molecular mechanisms controlling the expression, modification and subcellular targeting of hTERT will provide new and important insights into the role of telomerase in cancer, and help with the development of specific strategies for the therapeutic manipulation of telomerase in human cancer.

Figure 1.

The functions of hTERT in cancer progression. hTERT functions as the catalytic component of telomerase in maintaining telomere homeostasis, and also forms complexes with different cellular factors involved in several fundamental cellular functions in a telomere-independent fashion, which may provide transformed cells with cancer-specific capacities during multiple stages of tumor development. IL, interleukin.

Acknowledgements

We thank X. Wang, D. Xu and J. Wong for comments on the draft of this review. The work in the author's laboratory was supported in part by grants from the National Natural Science Foundation of China (31071200, 31171320, 31201038) and the National Basic Research Program of China (2012CB911203).

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