**1. Introduction**

Telomeres are specialised structures that define the very ends of linear eukaryotic chromo‐ somes and provide for their stability by protecting against degradation or end-to-end fusion. In mammals, telomeres are localised throughout the nucleus and associated with the nuclear matrix. Telomeric DNA of human cells is composed of a long (5 to 15 kb) stretch of the repeating hexanucleotide sequence 5'‒TTAGGG‒3' on one strand (the G-rich strand) and the comple‐ mentary 5'‒CCCTAA‒3' on the other (the C-rich strand). The G-rich strand has a short (35 to

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600 nt) single-stranded overhang at its 3' end (the G-overhang) which folds back and base pairs with the C-rich strand, forming a T-loop [1]. In humans, hexameric telomere repeats act as binding sites for various telomere-binding proteins collectively termed the shelterin complex, a dynamic ensemble of interactions that allows the cell to distinguish between natural chromosome ends and DNA double-strand breaks, preventing the cell's DNA damage response (DDR) from improper activation [2]. Telomeres undergo progressive shortening with each cell division as a result of incomplete lagging strand synthesis, less widely known endprocessing events, and oxidative damage [3, 4]. This so-called telomere erosion operates as a kind of mitotic clock that determines ageing of the whole organism and suppresses malignant transformation of its constituent cells. The biological function of telomeres is heavily regulated and relies on both a minimal length of telomeric DNA and the proper functioning of the associated shelterin complex. A unique enzyme termed telomerase assists in replicating linear chromosomes through *de novo* synthesis of telomeric repeats, thereby counteracting the progressive telomere erosion that would otherwise occur in its partial or complete absence. In addition to its role in telomere length homeostasis, telomerase also performs telomere lengthindependent functions such as modulation of gene expression. In a pathological context, telomerase's new talents are intimately related to tumour development and progression to metastatic disease. This chapter summarises the newly discovered extracurricular activities of telomerase and describe how these are involved in regulating cancer stemness, the stem-like component of human tumours.

#### **2. Telomerase and the cancer connection**

Telomerase is a conserved RNA-dependent DNA polymerase canonically responsible for the maintenance of telomere length above a critical threshold. Human telomerase is primarily localised in the nucleus, as deducible from its role in telomere biology, but it can also be found in other cellular compartments such as the cytosol and mitochondria [5]. Telomerase is ribonucleoprotein in nature and consists minimally and essentially of a protein catalytic subunit (telomerase reverse transcriptase, TERT) and a large RNA subunit (telomerase RNA, TER). Active human telomerase has a bilobal architecture where one TERT subunit and one TER subunit participate in the formation of each lobe and a hinge region connects the two lobes [6]. This conformationally flexible, dimeric structure of the human enzyme undoubtedly has profound functional implications with respect to the catalytic cycle. Firstly, during the synthesis of telomeric DNA by telomerase, the 3′ end of the G-overhang is positioned in the active site of TERT and aligned by base pairing with the 3' end of the RNA template in TER. Secondly, TERT catalyses the addition of deoxyribonucleotides to the chromosome substrate through reverse transcribing TER into hexameric telomere repeats until the 5' end of the RNA template is reached. Lastly, telomerase translocates and realigns with the newly synthesised 3' end of the chromosome substrate to restart the catalytic cycle [7, 8]. In spite of the fact that TERT and TER are the two subunits that provide the catalytic core of telomerase, there are several other molecules that associate with telomerase and are involved in its biogenesis, trafficking, recruitment, and activation. Some of the most well-known telomerase-associated proteins include the nucleolar protein dyskerin [9], the three other nucleolar proteins NOP10, NHP2 and GAR1 [10], the two AAA+ ATPases pontin and reptin [11], and the WD40-repeat protein TCAB1 [12]. It should be noted that not all cells necessarily rely upon telomerase to maintain telomere length. Some telomerase-negative immortalised cell lines and tumours are able to elongate their telomeres by the much rarer alternative lengthening of telomeres (ALT) pathway. In contrast to telomerase, which utilises an RNA template to *de novo* synthesise telomeric repeats, the ALT pathway utilises a DNA template for DNA copying in an inter- or intramolecular recombination event [13].

Cancer is usually an age-related genetic disease, manifesting only when normal cells develop genomic instability over a reasonable period of time and acquire unlimited replicative potential that leads to the generation of macroscopic tumours. Telomerase upregulation/ reactivation is observed in at least 85% of advanced human tumours, strongly suggesting a crucial role during human tumour pathogenesis [14, 15]. The most widely accepted multistep model of general tumourigenesis for explaining the part played by telomerase in telomere maintenance and cellular immortalisation is provided in section 3.2. Besides being found in primary tumours, telomerase activity is also detected in circulating tumour cells in, for instance, breast [16], ovarian [17] and prostate [18] cancers. Telomerase is upregulated/ reactivated in premalignant cells by five common mechanisms: (i) increased transcriptional activation of *TERT* and/or *TER*; (ii) loss of transcriptional repressors of *TERT*; (iii) mutations in the *TERT* gene promoter/enhancer region (which result in the transactivation of this gene); (iv) several kinases (which phosphorylate and thus enhance the activity of TERT); and (v) gain of copy number of *TERT* and/or *TER* [13]. Somatic mutations in the *TERT* gene promoter region are frequent events in cancers of the bladder, central nervous system, skin (melanoma) and thyroid (follicular cell-derived) [19]. Two mutually exclusive and highly recurrent *TERT* promoter mutations are C250T and C228T [20, 21]. Although both mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, they are functionally distinct in such a way that the the C250T *TERT* promoter but not the C228T *TERT* promoter additionally requires non-canonical NF-κB signalling in order to be transcriptionally driven [22]. Collec‐ tively, these findings highlight the contribution of *TERT* promoter mutations and noncanonical NF-κB signalling to tumourigenesis and decipher a fundamental mechanism for the reactivation of TERT in various tumours.
