**4. Regulation of cancer stemness by telomerase**

A further means by which non-CSCs acquire cancer stemness traits is through the EMT process. Physiologically, EMT causes cells to change from a stationary epithelial to a motile mesen‐ chymal morphology, thereby allowing for wound healing, tissue regeneration and organ fibrosis in adults and cell migration and tissue remodelling in developing embryos [50]. In the context of epithelium-derived carcinoma, however, the reactivation of the EMT programme contributes to the evolution of primary tumours towards increasingly aggressive phenotypes. The complex molecular, cellular and morphological alterations linked to pathological EMT are generally mediated by the joint action of the signals from the tumour microenvironment that induce the EMT programme (for example, TGF-β signalling, inflammatory cytokines, and hypoxia), the transcription factors that coordinate the EMT programme (for example, SNAI-1, SNAI-2, ZEB-1, ZEB-2, TWIST-1, and TWIST-2), and the effector proteins that execute the EMT programme (for example, low levels of E-cadherin and high levels of vimentin, N-cadherin, fibronectin, CD44, and MMPs) [51]. Such cooperation between the cell-extrinsic signals and the cell-intrinsic regulators is fully important and primarily responsible for endowing epithelial tumour cells with CSC-like properties, ranging from cell motility to invasiveness to cell survival, which are indispensable to metastatic dissemination from the primary tumour site, secondary tumour growth at a distant site, and resistance to therapy, respectively. Although CSCs originating from bulk tumour cells within epithelium-derived carcinomas achieve their final stemness state possibly *via* EMT, the degree to which they resemble or depart

from CSCs originating from adult stem cells has yet to be fully explored.

lation, not by glycolysis [57, 58].

192 Telomere - A Complex End of a Chromosome

Metabolic reprogramming is also an obvious mechanism of intervening and redirecting the cell fate of differentiated (normal or non-CSC tumour) cells. Traditionally, energy metabolism was widely accepted as a passive process that generated ATP and building blocks to meet the demands of the specialised cell types of the body in response to extra- and/or intracellular signals. Today, however, the modulation of energy metabolism and build-up of oncogenic metabolites are viewed as the harbingers of cancer stemness [52]. Typically, cancer cells are dependent more on glycolysis for energy production, even in the presence of sufficient oxygen to support oxidative phosphorylation. This phenomenon of aerobic glycolysis is commonly referred to as the Warburg effect and appears to fulfil the requirement of proliferating cancer cells to rapidly yield ATP and to provide anabolic substrates (such as amino acids, nucleotides, and phospholipids) for their daughter cells [53]. Besides, increased lactate generation during aerobic glycolysis provokes the acidification of the tumour microenvironment, ultimately giving rise to motile, invasive/metastatic and drug-resistant cells [54]. In agreement with this, a recent report confirmed and substantiated the need for a metabolic switch to glycolysis in the emergence of EMT-driven CSC-like characteristics in basal-like breast cancer cells [55]. Another report utilising nasopharyngeal carcinoma as a model system established that behaviourally-selected and accordingly-assayed CSCs, as distinct from their differentiated progenies, exhibit a metabolic shift from oxidative phosphorylation to glycolysis for ATP supply [56]. Nevertheless, contradictory evidence on the metabolic profile of CSCs has also been presented; two independent research groups reported that the bioenergetic and biosyn‐ thetic demands of quiescent/slow-cycling CSCs are likely to be met by oxidative phosphory‐

Most adult somatic cells do not or only transiently express telomerase and undergo telomere shortening with every cell division until the cell eventually dies. Most tumour cells, including CSCs, however, display high levels of telomerase activity and possess the ability to continually regenerate their telomeres [65]. As a result, telomerase upregulation/reactivation serves as an important mechanism for CSCs to attain indefinite (or at least extremely long) replicative lifespans. In fact, in reality, telomerase undertakes roles that significantly diverge from its normal role in elongating telomeres, as suggested by contemporary research on manipulation of telomerase expression and/or function in cells representing potential targets for oncogenic transformation and cellular (nuclear and metabolic) reprogramming. Central to the extratelo‐ meric roles of telomerase (particularly of TERT) is its interaction with key downstream components of the main embryonic/oncogenic signalling pathways or with other macromo‐ lecules (such as DNA and transcription factors) by which gene expression is regulated. The presence of a few to several hundred copies of TERT, which are not assembled into telomerase, in human immortalised cell lines reasonably provides a molecular basis for the formidable power of TERT as a transcriptional cofactor in oncogenic transformation and cellular (nuclear and metabolic) reprogramming, irrespectively of its TER-dependent DNA polymerase activity [66]. A very recent systemic review of the literature by us disclosed that most of the noncanonical responsibilities of telomerase identified so far strongly relate to the control of cancer stemness traits [67]. Telomerase/TERT-controlled aspects of the CSC phenotype involve proliferation, survival, therapy resistance, induced pluripotency, motility, glycolytic metabo‐ lism, and niche establishment and integrity (Figure 2). Equally strikingly, there seems to be a positive feedback loop between a number of gene products targeting TERT and TERT expression itself, plausibly amplifying the effects of central oncogenes and oncogenic path‐ ways associated with the generation and/or maintenance of cancer stemness traits in a cellautonomous manner. Although some of the observed cell-intrinsic/microenvironmental changes may require a catalytically active enzyme, there are several examples of oncogenic alterations brought about by catalytically inactive telomerase, as in the case of alternatively spliced (AS) TERT variants. To date, as many as twenty different AS TERT variants have been identified [68]. These variants tend to occur more frequently in cancer cells than in normal cells, indicating that they may be evolutionarily favoured in the context of pathology.

#### **4.1. Stimulation of CSC proliferation**

Given their role in the expansion of a tumour cell population, CSCs must display extensive proliferative capacity. Cell proliferation is both a matter of progression through the cell cycle and an issue necessitating cell growth (biosynthesis). There is a wealth of information in the literature on the promotive role of telomerase, independent of its telomere-elongating function, in cell proliferation. In an early study of the association between telomerase and cell prolifer‐ ation, telomerase was shown to support the proliferation of human mammary epithelial cells through elevated EGFR signalling (even though it is quite ambiguous whether this effect is telomere length-independent or not) [69]. Moreover, TERT confers CSC characteristics to glioma cells by inducing EGFR expression, disconnectedly from its role in telomere biology [70]. Interestingly, telomerase upregulation was found to be closely linked to EGFR expression in actively proliferating normal human epithelial cells [71]. These observations imply the existence of a feed-forward loop that involves telomerase/TERT and EGFR. A plausible mechanism linking the EGFR‒telomerase axis to cancer is that aberrant EGFR signalling may render CSCs less dependent on exogenous mitogens/growth factors and reinforce the persis‐ tent expression of telomerase in CSCs, thus playing a critical role in tumour development and progression.

Expanding these findings, one research group demonstrated that TERT promotes the prolif‐ eration of mammalian tissue progenitor cells *via* transcriptional control of a MYC- and Wntrelated developmental program [72]. To be more precise, TERT physically occupies the promoters of Wnt/β-catenin target genes, including those encoding cyclin D1 and MYC [73]. Cyclin D1 is a cell cycle control protein with oncogenic potential and has both enzymatic and nonenzymatic activities which are of great significance in tumour cells [74]. An additional molecular component involved in cyclin D1 expression in proliferating cells is nucleolar

On the Far Side of Telomeres: The Many Roles of Telomerase in the Acquisition and Retention of Cancer Stemness http://dx.doi.org/10.5772/65762 195

in human immortalised cell lines reasonably provides a molecular basis for the formidable power of TERT as a transcriptional cofactor in oncogenic transformation and cellular (nuclear and metabolic) reprogramming, irrespectively of its TER-dependent DNA polymerase activity [66]. A very recent systemic review of the literature by us disclosed that most of the noncanonical responsibilities of telomerase identified so far strongly relate to the control of cancer stemness traits [67]. Telomerase/TERT-controlled aspects of the CSC phenotype involve proliferation, survival, therapy resistance, induced pluripotency, motility, glycolytic metabo‐ lism, and niche establishment and integrity (Figure 2). Equally strikingly, there seems to be a positive feedback loop between a number of gene products targeting TERT and TERT expression itself, plausibly amplifying the effects of central oncogenes and oncogenic path‐ ways associated with the generation and/or maintenance of cancer stemness traits in a cellautonomous manner. Although some of the observed cell-intrinsic/microenvironmental changes may require a catalytically active enzyme, there are several examples of oncogenic alterations brought about by catalytically inactive telomerase, as in the case of alternatively spliced (AS) TERT variants. To date, as many as twenty different AS TERT variants have been identified [68]. These variants tend to occur more frequently in cancer cells than in normal

cells, indicating that they may be evolutionarily favoured in the context of pathology.

Given their role in the expansion of a tumour cell population, CSCs must display extensive proliferative capacity. Cell proliferation is both a matter of progression through the cell cycle and an issue necessitating cell growth (biosynthesis). There is a wealth of information in the literature on the promotive role of telomerase, independent of its telomere-elongating function, in cell proliferation. In an early study of the association between telomerase and cell prolifer‐ ation, telomerase was shown to support the proliferation of human mammary epithelial cells through elevated EGFR signalling (even though it is quite ambiguous whether this effect is telomere length-independent or not) [69]. Moreover, TERT confers CSC characteristics to glioma cells by inducing EGFR expression, disconnectedly from its role in telomere biology [70]. Interestingly, telomerase upregulation was found to be closely linked to EGFR expression in actively proliferating normal human epithelial cells [71]. These observations imply the existence of a feed-forward loop that involves telomerase/TERT and EGFR. A plausible mechanism linking the EGFR‒telomerase axis to cancer is that aberrant EGFR signalling may render CSCs less dependent on exogenous mitogens/growth factors and reinforce the persis‐ tent expression of telomerase in CSCs, thus playing a critical role in tumour development and

Expanding these findings, one research group demonstrated that TERT promotes the prolif‐ eration of mammalian tissue progenitor cells *via* transcriptional control of a MYC- and Wntrelated developmental program [72]. To be more precise, TERT physically occupies the promoters of Wnt/β-catenin target genes, including those encoding cyclin D1 and MYC [73]. Cyclin D1 is a cell cycle control protein with oncogenic potential and has both enzymatic and nonenzymatic activities which are of great significance in tumour cells [74]. An additional molecular component involved in cyclin D1 expression in proliferating cells is nucleolar

**4.1. Stimulation of CSC proliferation**

194 Telomere - A Complex End of a Chromosome

progression.

**Figure 2. Emergence and persistence of cancer stemness by telomerase/TERT (figure adapted from [67] with permis‐ sion).** Clearly and unmistakably, telomerase/TERT is a powerful regulator of many aspects of the CSC phenotype, in‐ cluding: (a) proliferation, survival, therapy resistance; and (b) induced pluripotency, motility, glycolytic metabolism, niche establishment and integrity. This multifaceted ribonucleoprotein complex exerts its telomere-independent tu‐ mour-promoting effects partly by diverting and co-opting developmental signalling pathways and modulating gene expression. A cross symbol denotes an inhibition (blockage). A dashed arrow indicates that a given cancer stemness trait is not a direct consequence of the process shown in the preceding box, but of the inhibition of that process. AG, angiogenesis; DDR, DNA damage response; dsDNA, double-stranded DNA; dsRNA, double-stranded RNA; GSH, glutathione; INF, inflammation; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; rDNA, ribosomal DNA; RMRP, RNA component of mitochondrial RNA-processing endoribonuclease; ROS, reactive oxygen species; siRNAs, small in‐ terfering RNAs

antigen p120 (NOL1), as suggested by the results of a very recent study [75]. In this study, telomerase was found to interact, in a TER-dependent fashion, with NOL1 and activate the transcription of the gene coding for cyclin D1. The relationship between TERT and cyclin D1 expression was corroborated also by other scientists [76–79]. Further support for TERT involvement in the stimulation of Wnt signalling-mediated cell proliferation was evidenced by an independent approach employing the Δ4‒13 AS variant of human TERT that is devoid of reverse transcriptase activity [80]. In this approach, ectopic expression and small interfering RNA (siRNA)-mediated knockdown of the Δ4‒13 AS variant in ALT cells, transformed telomerase-positive cell lines and telomerase-negative normal cells unquestionably proved that the proliferative effect of TERT is not coupled to telomerase activity. Because β-catenin is known to modulate TERT expression in stem cells and tumour cells [81], it is tempting to speculate that telomerase and Wnt/β-catenin signalling may act together in a positive feedback circuit to actively encourage the proliferation of CSCs.

Intriguingly, one group failed to find evidence that TERT promotes Wnt signalling in human breast cancer cells, indicating that TERT's effect on Wnt signalling is possibly context- and cell type-dependent [82]. Their findings are in the same direction as those from a former study on telomerase-null mouse models, where TERT loss-of-function in a physiological setting was reported to have no evident effects on Wnt signalling [83]. An alternative mechanism of action of telomerase on cell proliferation, as deduced by reverse genetics in human mammary epithelial cells, is that TERT-induced cell proliferation may result primarily from decreased levels of the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP), not from increased Wnt signalling [78]. TERT and RMRP form a definite ribonucleoprotein complex that exhibits RNA-dependent RNA polymerase activity and, using RMRP as a template, produces double-stranded RNAs that can be later processed into siRNAs in a Dicerdependent fashion [84]. siRNAs regulate gene expression at the posttranscriptional level as well as at the level of chromatin structure; therefore, it is reasonable to question whether their mutations or altered expression correlate with human cancers.

Aside from activating Wnt signalling and regulating gene expression (in the presence of RMRP), telomerase also stimulates ribosomal biogenesis through increased Pol I-directed ribosomal DNA transcription, exerting a positive influence on cell cycle and proliferation dynamics [85]. This may ultimately improve the protein synthesis capacity of CSCs for unrestrained growth. The molecular mechanism behind telomerase-induced ribosomal biogenesis was investigated in a very recent report where a MYC-driven oncogenesis model was proposed [79].

#### **4.2. Promotion of CSC survival**

Apoptosis is a form of cell death induced by miscellaneous stimuli and mediated by a subset of cysteine proteases termed caspases. A cancer cell's ability to evade death signals, thus preventing self-destruction by the activation of an apoptotic programme, is regarded as one of the hallmarks of cancer [86]. Several pieces of information suggest that telomerase exerts antiapoptotic effects in cancer cells through telomere-independent mechanisms. In the case of CSCs, telomerase-mediated inhibition of apoptosis may contribute to the enhanced and

continued survival of these cells in tumours. In keeping with its cytoprotective function, TERT was revealed to inhibit cell death by blocking the death receptor-initiated (or extrinsic) apoptotic pathway in acute promyelocytic cells [87]. Similarly, yet in a mechanistically different way, TERT was also shown to block the mitochondrion-initiated (or intrinsic) apoptotic pathway in colon and cervical carcinoma cell lines [88]. A more thorough prob‐ ing of the molecular mechanism behind telomerase-mediated suppression of the intrinsic apoptotic pathway found that TERT overexpression upregulates the expression of the antiapoptotic mitochondrial protein BCL-2, downregulates the expression of some proapop‐ totic mitochondrial proteins (for example, BAX) and reduces the activation of some caspas‐ es (for example, caspase-9) in ovarian surface epithelial cells [89]. Given the capacity of BCL-2 to increase telomerase activity in human colorectal and cervical carcinoma cell lines [90], it is conceivable that telomerase and BCL-2 are engaged in a positive feedback loop that impedes apoptosis. In this connection, it is well to add that the introduction of a constitutively expressed TERT construct into colon carcinoma and Burkitt's lymphoma cell lines was, independently of telomerase activity, associated with the reversion of a transcriptional programme coordinated by p53, a potent and common activator of both the intrinsic and extrinsic apoptotic pathways [91].

Apart from suppressing mitochondrion-initiated cell death, overexpression of TERT was also found to suppress, in a telomere-independent manner, endoplasmic reticulum (ER) stressinduced cell death in murine primary neural cells and human cancer cell lines [92]. ER stress arises as a result of perturbations in ER function and elicits the unfolded protein response (UPR), a conserved signal transduction pathway for dealing with misfolded proteins. When the UPR-induced mechanisms fail to alleviate ER stress, both the intrinsic and extrinsic apoptotic pathways may become activated [93]. Reciprocally, specific activation of ER stress was demonstrated to upregulate TERT expression in a breast cancer cell line [94]. It therefore seems reasonable to suggest that TERT and ER stress are involved in a dynamic interplay supporting CSC survival in abnormal metabolic conditions such as glucose starvation.

#### **4.3. Induction of pluripotency**

antigen p120 (NOL1), as suggested by the results of a very recent study [75]. In this study, telomerase was found to interact, in a TER-dependent fashion, with NOL1 and activate the transcription of the gene coding for cyclin D1. The relationship between TERT and cyclin D1 expression was corroborated also by other scientists [76–79]. Further support for TERT involvement in the stimulation of Wnt signalling-mediated cell proliferation was evidenced by an independent approach employing the Δ4‒13 AS variant of human TERT that is devoid of reverse transcriptase activity [80]. In this approach, ectopic expression and small interfering RNA (siRNA)-mediated knockdown of the Δ4‒13 AS variant in ALT cells, transformed telomerase-positive cell lines and telomerase-negative normal cells unquestionably proved that the proliferative effect of TERT is not coupled to telomerase activity. Because β-catenin is known to modulate TERT expression in stem cells and tumour cells [81], it is tempting to speculate that telomerase and Wnt/β-catenin signalling may act together in a positive feedback

Intriguingly, one group failed to find evidence that TERT promotes Wnt signalling in human breast cancer cells, indicating that TERT's effect on Wnt signalling is possibly context- and cell type-dependent [82]. Their findings are in the same direction as those from a former study on telomerase-null mouse models, where TERT loss-of-function in a physiological setting was reported to have no evident effects on Wnt signalling [83]. An alternative mechanism of action of telomerase on cell proliferation, as deduced by reverse genetics in human mammary epithelial cells, is that TERT-induced cell proliferation may result primarily from decreased levels of the RNA component of mitochondrial RNA-processing endoribonuclease (RMRP), not from increased Wnt signalling [78]. TERT and RMRP form a definite ribonucleoprotein complex that exhibits RNA-dependent RNA polymerase activity and, using RMRP as a template, produces double-stranded RNAs that can be later processed into siRNAs in a Dicerdependent fashion [84]. siRNAs regulate gene expression at the posttranscriptional level as well as at the level of chromatin structure; therefore, it is reasonable to question whether their

Aside from activating Wnt signalling and regulating gene expression (in the presence of RMRP), telomerase also stimulates ribosomal biogenesis through increased Pol I-directed ribosomal DNA transcription, exerting a positive influence on cell cycle and proliferation dynamics [85]. This may ultimately improve the protein synthesis capacity of CSCs for unrestrained growth. The molecular mechanism behind telomerase-induced ribosomal biogenesis was investigated in a very recent report where a MYC-driven oncogenesis model

Apoptosis is a form of cell death induced by miscellaneous stimuli and mediated by a subset of cysteine proteases termed caspases. A cancer cell's ability to evade death signals, thus preventing self-destruction by the activation of an apoptotic programme, is regarded as one of the hallmarks of cancer [86]. Several pieces of information suggest that telomerase exerts antiapoptotic effects in cancer cells through telomere-independent mechanisms. In the case of CSCs, telomerase-mediated inhibition of apoptosis may contribute to the enhanced and

circuit to actively encourage the proliferation of CSCs.

196 Telomere - A Complex End of a Chromosome

mutations or altered expression correlate with human cancers.

was proposed [79].

**4.2. Promotion of CSC survival**

Restoration of the molecular circuitry that forms the necessary base of pluripotency in embryonic stem cells (ESCs) strongly correlates with the gaining and retention of cancer stemness. In ESCs, this circuitry is made up of special transcription factors and function as a repressor of differentiation. Takahashi and Yamanaka were the first to demonstrate that a cocktail of four transcription factors (namely OCT-3/4, SOX-2, MYC, and KLF4) are necessa‐ ry and sufficient for nuclear reprogramming into an ESC-like state [95]. In CSCs, the socalled Yamanaka factors, besides driving the induction of pluripotency, are additionally involved in inhibiting apoptosis [96]. A valued piece of work documented that TERT forms a ternary complex with the nucleolar GTP-binding protein NS/GNL3L and the chromatin remodelling factor BRG1 and that the resulting NS/GNL3L TERT BRG1 complex is re‐ quired for NS/GNL3L-induced upregulation of the nuclear reprogramming factors OCT-3/4, MYC, and KLF-4 [97]. The likely part played by TERT/telomerase in contributing to the pluripotent character of CSCs is also congruous with the later finding that siRNA-mediat‐ ed hTERT depletion in gastric CSCs downregulates the induced pluripotency-associated transcription factor OCT-4 [98].

#### **4.4. Increase of CSC motility and invasiveness**

Migrating CSCs and EMT-phenotypic cells have the ability to disseminate from their primary site and are thus present in the invasive front of tumours. The initial evidence for telomerase/ TERT participation in cell migration came from experiments measuring the *in vitro* migration rate of telomerase-positive progenitor cells and cancer cell lines [99, 100]. Later experiments aiming at uncovering the molecular mechanism behind this positive trend showed that telomerase reconstitution boosts cell migration through the activation of Rho family members and the SDF-1‒CXCR4 axis [101]. With respect to CSC motility, expression of the chemokine receptor CXCR4 may enable CSCs to migrate along a gradient of the ligand SDF-1 and thus help facilitate their spread. Therapeutic strategies intended to interfere with the SDF-1‒CXCR4 axis can possibly have useful clinical relevance and application in the prevention of metastatic disease.

Differentiated epithelial cells that have undergone EMT may as well exhibit augmented motility and invasiveness leading to metastasis. The ternary complex containing TERT, BRG1, and NS or GNL3L (see section 4.3) acts in a telomere-independent mode to activate the EMT programme *via* NS/GNL3L-induced upregulation of vimentin, SNAI, and TWIST, three of the mesenchymal cell markers, in genetically defined cancer cells [97]. TERT additionally stimu‐ lates EMT in gastric cancer cells through directly regulating the expression of Wnt/β-catenin target genes like those coding for vimentin and SNAI-1 [98]. Equally important is the fact that TERT, in a telomere-independent manner, regulates the expression of several MMP family members, such as MMP-9, *via* the NF-κB pathway [102]. MMPs are the key mediators pro‐ moting extracellular matrix (ECM) degradation and remodelling, both of which pave the way for EMT and subsequent metastasis. The indirect involvement of TERT in dissemination was also highlighted by a separate set of data which documented that changes in the motility and invasiveness of malignant cells are likely to result from the TERT-induced upregulation of the metastasis-implicated proteins RhoC and MMP-9 [103]. Interestingly, MMP-9 silencing was shown to downregulate TERT expression *via* ITGB1-mediated FAK signalling in glioma xenograft cells [104]. It is worth mentioning here that a very recent report found that ITGB1 itself is regulated by TERT and that TERT may promote the invasion and metastasis of gastric cancer cells by enhancing ITGB1 protein levels [105]. Collectively, these findings reinforce the notion that there is an indirect, metastasis-favouring interaction between TERT, MMP-9 and ITGB1 in cancer cells. Another study discovered that TERT upregulates the levels of MAC2BP, a metastasis-related secreted ECM glycoprotein, in gastric cancer cells [106]. MAC2BP is believed to support metastasis through interacting with galectins and altering cell‒cell and cell‒matrix adhesion properties [107].

Another contribution to knowledge came from a very recent report in which TERT was found to stimulate the expression of oncomiRs, including miR-21, in human leukaemia and HeLa cell lines [108]. Extant research identifies miR-21 as being among the most frequently upregulated miRNAs in epithelial cell-derived solid tumours [109] and also as having a

decisive role in the conservation of CSC phenotype *via* the AKT and ERK1/2 signalling pathways targeting PTEN [110]. The centrality of miR-21 to cancer stemness was con‐ firmed in a recent study on the antisense oligonucleotide-mediated inhibition of miR-21 in two different anaplastic thyroid carcinoma (ATC) cell lines, where the knockdown of miR-21 disturbed the stemness state of ATC cells, as assessed by a decreased expression of the genes encoding OCT-4 and ABCG2 [111].

#### **4.5. Modulation of energy metabolism**

ed hTERT depletion in gastric CSCs downregulates the induced pluripotency-associated

Migrating CSCs and EMT-phenotypic cells have the ability to disseminate from their primary site and are thus present in the invasive front of tumours. The initial evidence for telomerase/ TERT participation in cell migration came from experiments measuring the *in vitro* migration rate of telomerase-positive progenitor cells and cancer cell lines [99, 100]. Later experiments aiming at uncovering the molecular mechanism behind this positive trend showed that telomerase reconstitution boosts cell migration through the activation of Rho family members and the SDF-1‒CXCR4 axis [101]. With respect to CSC motility, expression of the chemokine receptor CXCR4 may enable CSCs to migrate along a gradient of the ligand SDF-1 and thus help facilitate their spread. Therapeutic strategies intended to interfere with the SDF-1‒CXCR4 axis can possibly have useful clinical relevance and application in the prevention of metastatic

Differentiated epithelial cells that have undergone EMT may as well exhibit augmented motility and invasiveness leading to metastasis. The ternary complex containing TERT, BRG1, and NS or GNL3L (see section 4.3) acts in a telomere-independent mode to activate the EMT programme *via* NS/GNL3L-induced upregulation of vimentin, SNAI, and TWIST, three of the mesenchymal cell markers, in genetically defined cancer cells [97]. TERT additionally stimu‐ lates EMT in gastric cancer cells through directly regulating the expression of Wnt/β-catenin target genes like those coding for vimentin and SNAI-1 [98]. Equally important is the fact that TERT, in a telomere-independent manner, regulates the expression of several MMP family members, such as MMP-9, *via* the NF-κB pathway [102]. MMPs are the key mediators pro‐ moting extracellular matrix (ECM) degradation and remodelling, both of which pave the way for EMT and subsequent metastasis. The indirect involvement of TERT in dissemination was also highlighted by a separate set of data which documented that changes in the motility and invasiveness of malignant cells are likely to result from the TERT-induced upregulation of the metastasis-implicated proteins RhoC and MMP-9 [103]. Interestingly, MMP-9 silencing was shown to downregulate TERT expression *via* ITGB1-mediated FAK signalling in glioma xenograft cells [104]. It is worth mentioning here that a very recent report found that ITGB1 itself is regulated by TERT and that TERT may promote the invasion and metastasis of gastric cancer cells by enhancing ITGB1 protein levels [105]. Collectively, these findings reinforce the notion that there is an indirect, metastasis-favouring interaction between TERT, MMP-9 and ITGB1 in cancer cells. Another study discovered that TERT upregulates the levels of MAC2BP, a metastasis-related secreted ECM glycoprotein, in gastric cancer cells [106]. MAC2BP is believed to support metastasis through interacting with galectins and altering cell‒cell and

Another contribution to knowledge came from a very recent report in which TERT was found to stimulate the expression of oncomiRs, including miR-21, in human leukaemia and HeLa cell lines [108]. Extant research identifies miR-21 as being among the most frequently upregulated miRNAs in epithelial cell-derived solid tumours [109] and also as having a

transcription factor OCT-4 [98].

198 Telomere - A Complex End of a Chromosome

cell‒matrix adhesion properties [107].

disease.

**4.4. Increase of CSC motility and invasiveness**

Apparently, genetic, epigenetic and microenvironmental changes that regulate the transition to a CSC-like state cannot occur without the presence of a favourable metabotype. In general, stimulation of aerobic glycolysis promotes metabolic reprogramming, whereas inhibition of glycolytic enzymes impairs metabolic reprogramming. In harmony with the concept that metabolism is involved in the control of cancer stemness, a microarray-based gene expression profiling study elucidated that ribozyme-mediated targeting of telomerase in murine mela‐ noma cells downregulates the expression of more than a few glycolytic pathway genes such as those coding for phosphofructokinase and aldolase C [112]. Additionally, a very recent report showed that siRNA-mediated knockdown of TERT in human lymphoma cells lowers the expression of MYC-regulated target genes such as those coding for the glycolytic enzymes lactate dehydrogenase, hexokinase 2, and pyruvate kinase M2 isoform [79]. Due to the fact that MYC is a well-established oncogenic transcription factor activating TERT expression [113], a feed-forward mechanism for the rewiring of glucose metabolism in CSCs is likely to prevail between TERT and MYC.

#### **4.6. Contribution to therapy resistance**

CSCs are notorious for their resistance to existing cancer treatment regimens, including radiotherapy and chemotherapy. Both cell-intrinsic and microenvironmental factors appear to contribute to the emergence of therapy resistance in CSCs. Radiotherapy works by directing ionising radiation toward tumours to induce the generation of reactive oxygen species (ROS) which react with and cause damaging of DNA. By the same token, a number of chemothera‐ peutic agents such as platinum-based antitumour drugs are known to bind to and cause crosslinking of DNA. In this regard, implementation by CSCs of fast and efficient DNA repair mechanisms as well as potent antioxidant/scavenger systems may prove vital to circumvent the deleterious effects of irradiation and several classes of antitumour compounds.

Growing evidence points to a role for telomerase in modulating DDR and contributing to DNA repair. In a prior report, TERT was proposed to, independently of its effect on telomere length, set in motion a transcriptional programme leading to enlarged ribonucleotide (NTP) pools, enhanced DNA repair, and increased chromosomal stability [114]. A circumstantial investi‐ gation into the enhanced DNA repair capability of telomerase-expressing cells suggested that TERT/telomerase increases DNA end-joining repair and accelerates nucleotide excision repair through recruiting proteinaceous factors to sites where DNA damage is occurring [115]. The aforementioned findings are consistent with a newer report which showed that TERT expres‐ sion affords a means of protecting human transformed cells against double-stranded DNA- damaging drugs and increases their endurance to chromosomal instability [116]. Specifically expressing TERT mutants lacking catalytic activity in ALT cells, the authors of the same report reached the conclusion that the observed cytoprotective effect of telomerase is distinct from its function in telomere biosynthesis. In a separate study providing evidence for an epigenetic component to telomerase-induced treatment resistance, stable short hairpin RNA (shRNA) mediated suppression of TERT expression was demonstrated to, in a telomere lengthindependent way, diminish the response of human fibroblasts to DNA double strand breaks, most likely through a mechanism altering the overall state (that is to say, configuration) of chromatin [117].

Telomerase upregulation/reactivation also seems to be involved in counteracting oxidative stress-induced intracellular injury that often follows therapy, as evident from several experi‐ mental studies. The initial study examining the extratelomeric function of telomerase under oxidative stress found that mitochondrially-localised TERT decreases cellular peroxide levels and mitochondrial superoxide production, increases mitochondrial membrane potential and protects mitochondrial DNA from oxidative damage in human lung fibroblasts [118]. The observation that telomerase provides resistance to oxidative stress was validated and extended in an ensuing study where TERT was shown to bind to mitochondrial DNA and accordingly protect it and its function against damage [119]. An alternative explanation for telomeraseinduced resistance to oxidative stress came from a more recent study in which TERT overex‐ pression in cancer cells was demonstrated to alleviate basal ROS levels and intracellular ROS production through potentiating the effects of endogenous antioxidants or free radical scavengers such that the proportion of reduced to oxidised glutathione (GSH/GSSG) is increased and peroxiredoxin is replenished in the interior of the cell [120]. Apart from serving to keep mitochondrial DNA damage-free, mitochondrially-localised telomerase also guards nuclear DNA against oxidative attack through decreasing mitochondrial ROS production [121]. Finally, it is appropriate to mention that the β-deletion variant, a catalytically defective AS variant of TERT, localises to both mitochondria and the nucleus and, distinct from the canonical role of TERT in telomere extension, protects three basal breast cancer cell lines from cisplatin-induced apoptosis, endowing breast tumours with chemotherapy resistance [122].

#### **4.7. Establishment and integrity of the CSC niche**

The tumour microenvironment (TME) is an umbrella term that encompasses all cellular and non-cellular components surrounding a tumour. These components include tumour-adjacent stromal cells (for example, endothelial cells and fibroblasts), diverse effectors of the immune system (for example, lymphocytes and mesenchymal stem cells), ECM elements, proteases, and networks of cytokines, growth factors and other soluble factors. Specifically, both the immediate TME (cell‒cell and cell‒matrix connections) and the extended TME (for example, vascular bed) are thought to be implicated in tumour progression. The TME is also capable of creating a niche for CSCs, in which they remain in an undifferentiated state until stimulated to differentiate into non-CSC tumour cells and form tumour bulk. Modulation of gene expression and metabolism by telomerase in CSCs may recondition the CSC niche in favour of the hypermalignant (that is to say, highly metastatic, therapy-resistant) nature of these cells. It is in this regard that a recent study demonstrated that telomerase binds to p65 and localises to promoters of NF-κB target genes, such as those encoding IL-6, TNF-α, and IL-8, proinflam‐ matory cytokines that are the critical triggers of inflammatory responses [123]. Inflammation is considered an enabling characteristic of cancer for the reason that it supplies bioactive molecules (for example, growth factors and EMT-inducing ligands) to the TME and primes cells to release ROS and other chemicals that drive the mutagenesis, and hence genetic evolution, of nearby tumour cells toward hypermalignancy [86]. Since NF-κB is known to transcriptionally upregulate telomerase levels [124], this finding implies that a positive feedback loop between telomerase and NF-κB may explain the grounds for the coexistence of chronic inflammation and sustained telomerase activity in neoplastic lesions.

damaging drugs and increases their endurance to chromosomal instability [116]. Specifically expressing TERT mutants lacking catalytic activity in ALT cells, the authors of the same report reached the conclusion that the observed cytoprotective effect of telomerase is distinct from its function in telomere biosynthesis. In a separate study providing evidence for an epigenetic component to telomerase-induced treatment resistance, stable short hairpin RNA (shRNA) mediated suppression of TERT expression was demonstrated to, in a telomere lengthindependent way, diminish the response of human fibroblasts to DNA double strand breaks, most likely through a mechanism altering the overall state (that is to say, configuration) of

Telomerase upregulation/reactivation also seems to be involved in counteracting oxidative stress-induced intracellular injury that often follows therapy, as evident from several experi‐ mental studies. The initial study examining the extratelomeric function of telomerase under oxidative stress found that mitochondrially-localised TERT decreases cellular peroxide levels and mitochondrial superoxide production, increases mitochondrial membrane potential and protects mitochondrial DNA from oxidative damage in human lung fibroblasts [118]. The observation that telomerase provides resistance to oxidative stress was validated and extended in an ensuing study where TERT was shown to bind to mitochondrial DNA and accordingly protect it and its function against damage [119]. An alternative explanation for telomeraseinduced resistance to oxidative stress came from a more recent study in which TERT overex‐ pression in cancer cells was demonstrated to alleviate basal ROS levels and intracellular ROS production through potentiating the effects of endogenous antioxidants or free radical scavengers such that the proportion of reduced to oxidised glutathione (GSH/GSSG) is increased and peroxiredoxin is replenished in the interior of the cell [120]. Apart from serving to keep mitochondrial DNA damage-free, mitochondrially-localised telomerase also guards nuclear DNA against oxidative attack through decreasing mitochondrial ROS production [121]. Finally, it is appropriate to mention that the β-deletion variant, a catalytically defective AS variant of TERT, localises to both mitochondria and the nucleus and, distinct from the canonical role of TERT in telomere extension, protects three basal breast cancer cell lines from cisplatin-induced apoptosis, endowing breast tumours with chemotherapy resistance [122].

The tumour microenvironment (TME) is an umbrella term that encompasses all cellular and non-cellular components surrounding a tumour. These components include tumour-adjacent stromal cells (for example, endothelial cells and fibroblasts), diverse effectors of the immune system (for example, lymphocytes and mesenchymal stem cells), ECM elements, proteases, and networks of cytokines, growth factors and other soluble factors. Specifically, both the immediate TME (cell‒cell and cell‒matrix connections) and the extended TME (for example, vascular bed) are thought to be implicated in tumour progression. The TME is also capable of creating a niche for CSCs, in which they remain in an undifferentiated state until stimulated to differentiate into non-CSC tumour cells and form tumour bulk. Modulation of gene expression and metabolism by telomerase in CSCs may recondition the CSC niche in favour of the hypermalignant (that is to say, highly metastatic, therapy-resistant) nature of these cells.

chromatin [117].

200 Telomere - A Complex End of a Chromosome

**4.7. Establishment and integrity of the CSC niche**

Furthermore, it was reported that TERT activates the transcription of VEGF, an endothelial mitogen and master orchestrator of angiogenesis, independently of telomerase activity in HeLa cells [125]. Further dissection of the underlying regulatory mechanism led to the conclusion that TERT upregulates VEGF expression through its interaction with the specificity protein 1 (Sp1) transcription factor [126]. Angiogenesis is the process of growth or formation of fresh blood vessels from the pre-existing vasculature, and its induction is widely considered an essential attribute of tumour growth as well as metastasis as solid tumours larger than 1 cm3 have to develop their own blood supply to circumvent necrotic cell death. Given the prior discovery that VEGF stimulates the production of TERT [127], it may be that there is a positive feedback circuit between TERT and VEGF. This regulation may account for the combined and continuing contribution of these two proteins to the maintenance of CSCs in solid tumours. Moreover, siRNA-mediated knockdown of TERT was shown to downregulate the prostaglan‐ din-synthesising enzyme COX-2 in pancreatic cancer cells [128]. COX-2, like VEGF, is a proangiogenic factor that has the potential to establish a selective niche favourable to the preservation of CSCs. Subsequent studies showed that COX-2 stimulates the expression of TERT in cervical cancer cells [129]. Collectively, these data indicate that a feed-forward regulation, which could be important in carcinoma growth and progression, occurs between TERT and COX-2.
