**3. Cancer stemness**

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

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

component of human tumours.

188 Telomere - A Complex End of a Chromosome

**2. Telomerase and the cancer connection**

Cancer cells within a single tumour often exist in distinct phenotypic states which differ in functional attributes. This so-called intratumoural heterogeneity originates from a myriad of cell types recruited to the tumour as well as from genetic, epigenetic and metabolic differences amongst the cancer cells themselves and may result in variable or unpredictable responses to treatment [23]. Postulated to be the driving force behind tumour maintenance, hypermalignant stem-like cells called cancer stem cells (CSCs) represent a unique dimension of intratumoural heterogeneity. This often-small subpopulation of cancer cells is thought to play pivotal roles in tumour initiation and progression, spreading, therapy resistance, and recurrence, all of which lead to poor prognosis.

#### **3.1. Definitions and measurements**

CSCs have critical implications for nearly, if not quite, all types of cancers, including leukae‐ mias [24–26], lymphomas [27], melanomas [28, 29], sarcomas [30], and various carcinomas such as brain [31], skin [32], head and neck [33], lung [34, 35], liver [36], gastric [37], colorectal [38, 39], bladder [40], pancreatic [41, 42], prostate [43], breast [44] and ovarian [45] cancers. The functions assumed by CSCs in human cancers are diverse, ranging from sustaining tumour growth and dissemination to treatment failure and tumour relapse. The three clear-cut features that contribute to the aforementioned functions of CSCs, aka cancer stemness traits, are: (i) their unrestricted ability to self-renew; (ii) their aberrant ability to differentiate into mixed populations of tumour cells; and (iii) their high ability to transition from a proliferative to a quiescent state. In spite of the fact that these operational characteristics are, to a large extent, shared by both CSCs and physiological stem cells, CSCs are the ones that are known to be related to several malignant phenotypes, including induction of invasion and metastasis and resistance to apoptosis. In addition, CSCs are distinguished from bulk tumour cells by their capacity to form nonadherent spheres when cultured in stem cell media, their propensity to found fresh tumours when transplanted into severe combined immunodeficient mice and their expression of a selected repertoire of stem cell-surface markers [46]. Therefore, it is important here to realise that cancer stemness, no matter how it is measured, stresses the ways in which CSCs differ from bulk cancer cells as well as from physiological stem cells. The cancer stemness phenomenon is of considerable clinical importance and significance because it prognosticates that successful anticancer therapy must involve strategies that will eradicate CSCs, as these cells are able to dominate any residual tumour cells that survive conventional anticancer therapies.

#### **3.2. Determinants and signatures**

In the past, the cancer stemness model was widely seen as a static one which suggested a stable CSC population and a hierarchical organisation of cell division and differentiation. In recent times, however, the cancer stemness model has evolved into a dynamic one where CSCs are rather accepted as a functional subpopulation of cancer cells and can also be formed by the process of dedifferentiation from mature cancer cells under proper environmental conditions [47]. Accumulating data have revealed that cancer stemness is governed by genetic changes (such as oncogene activation and oncosuppressor gene inactivation), epigenetic changes (such as miRNA targeting and promoter DNA hypomethylation/hypermethylation) and metabolic changes (such as the shift to aerobic glycolysis) concomitant with changes in the tumour microenvironment, especially the CSC niche (Figure 1). These changes are a prerequisite for the oncogenic transformation and cellular (nuclear and metabolic) reprogramming of non-CSCs to CSCs and precipitate a spectrum of drastic cellular consequences, including overpro‐ duction of certain oncoproteins, disruption of certain oncosuppressor proteins, upregulation/ reactivation of telomerase, reactivation of the epithelial-to-mesenchymal transition (EMT) programme, modulation of energy metabolism, stimulation of a number of embryonic/ oncogenic signalling pathways, and differential expression of several microRNAs (miRNAs).

in tumour initiation and progression, spreading, therapy resistance, and recurrence, all of

CSCs have critical implications for nearly, if not quite, all types of cancers, including leukae‐ mias [24–26], lymphomas [27], melanomas [28, 29], sarcomas [30], and various carcinomas such as brain [31], skin [32], head and neck [33], lung [34, 35], liver [36], gastric [37], colorectal [38, 39], bladder [40], pancreatic [41, 42], prostate [43], breast [44] and ovarian [45] cancers. The functions assumed by CSCs in human cancers are diverse, ranging from sustaining tumour growth and dissemination to treatment failure and tumour relapse. The three clear-cut features that contribute to the aforementioned functions of CSCs, aka cancer stemness traits, are: (i) their unrestricted ability to self-renew; (ii) their aberrant ability to differentiate into mixed populations of tumour cells; and (iii) their high ability to transition from a proliferative to a quiescent state. In spite of the fact that these operational characteristics are, to a large extent, shared by both CSCs and physiological stem cells, CSCs are the ones that are known to be related to several malignant phenotypes, including induction of invasion and metastasis and resistance to apoptosis. In addition, CSCs are distinguished from bulk tumour cells by their capacity to form nonadherent spheres when cultured in stem cell media, their propensity to found fresh tumours when transplanted into severe combined immunodeficient mice and their expression of a selected repertoire of stem cell-surface markers [46]. Therefore, it is important here to realise that cancer stemness, no matter how it is measured, stresses the ways in which CSCs differ from bulk cancer cells as well as from physiological stem cells. The cancer stemness phenomenon is of considerable clinical importance and significance because it prognosticates that successful anticancer therapy must involve strategies that will eradicate CSCs, as these cells are able to dominate any residual tumour cells that survive conventional anticancer

In the past, the cancer stemness model was widely seen as a static one which suggested a stable CSC population and a hierarchical organisation of cell division and differentiation. In recent times, however, the cancer stemness model has evolved into a dynamic one where CSCs are rather accepted as a functional subpopulation of cancer cells and can also be formed by the process of dedifferentiation from mature cancer cells under proper environmental conditions [47]. Accumulating data have revealed that cancer stemness is governed by genetic changes (such as oncogene activation and oncosuppressor gene inactivation), epigenetic changes (such as miRNA targeting and promoter DNA hypomethylation/hypermethylation) and metabolic changes (such as the shift to aerobic glycolysis) concomitant with changes in the tumour microenvironment, especially the CSC niche (Figure 1). These changes are a prerequisite for the oncogenic transformation and cellular (nuclear and metabolic) reprogramming of non-CSCs to CSCs and precipitate a spectrum of drastic cellular consequences, including overpro‐ duction of certain oncoproteins, disruption of certain oncosuppressor proteins, upregulation/ reactivation of telomerase, reactivation of the epithelial-to-mesenchymal transition (EMT)

which lead to poor prognosis.

190 Telomere - A Complex End of a Chromosome

therapies.

**3.2. Determinants and signatures**

**3.1. Definitions and measurements**

**Figure 1. How do CSCs arise?** The joint impact of genetic, epigenetic, metabolic and microenvironmental factors is believed to determine the conversion of non-CSCs (which could be normal stem cells, mature cancer cells, or others) to CSCs. This process somehow is a dynamic one and CSCs are a functional and not merely territorial subpopulation of cancer cells.

Disruption of diverse oncosuppressor proteins with antiproliferative, prodifferentiative and/or proapoptotic effects accounts for an early molecular event accompanying the emer‐ gence of cancer stemness traits. p53, pRB, PTEN, and p16INK4A are by far among the most commonly inactivated oncosuppressor proteins in advanced human tumours [48]. Their inactivation permits premalignant cells to avoid replicative senescence, apoptosis, or both, thereby continuing to divide and accumulating further tumourigenic alterations like genomic (chromosomal) instability that follows telomere erosion [49]. The subsequent upregulation/ reactivation of telomerase compensates for telomere erosion (which would otherwise trigger entry of cells into a period of crisis with massive cell death), suppressing genomic (chromo‐ somal) instability and allowing premalignant cells to proliferate for a virtually infinite number of cell divisions. Additionally, and surprisingly, there is accumulating evidence that telomer‐ ase upregulation/reactivation provides susceptible cells with cancer stemness traits. The many functions of telomerase in the development and maintenance of cancer stemness will be addressed in more detail in the next sections of this chapter.

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.

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‐ lation, not by glycolysis [57, 58].

CSCs maintain their prolonged residence in the stemness state through diverting and co-opting elegant signalling pathways that are normally active during embryonic development. The embryonic/oncogenic signalling pathways operating in CSCs include Notch, Hedgehog (HH), Wnt/β-catenin, cytokine receptor-mediated JAK/STAT, TNF-α receptor-mediated NF-κB, growth factor receptor (receptor tyrosine kinase)-mediated PI3K/AKT/mTOR, TGF-β/BMP receptor-mediated SMAD, and Hippo-YAP/TAZ [59–61]. Sustained activation of and crosstalk between these cascades ultimately enhance the expression of cell-surface proteins (for example, CD133, CD44, integrins, and CXCR4), prosurvival proteins (for example, BCL-2, BCL-XL, MCL-1, survivin, and MIC-1), induced pluripotency-associated transcription factors (for example, BMI-1, OCT-3/4, SOX-2, MYC, and NANOG), EMT-associated proteins (for example, MMPs, vimentin, N-cadherin, SNAI, TWIST, and ZEB), glycolysis-associated proteins (for example, GLUTs and glycolytic enzymes), treatment resistance-associated proteins (for example, GSH, ALDH1, ABCB1, ABCC1, ABCG2, CHK-1, and CHK-2), proan‐ giogenic factors (for example, VEGF and COX-2), and proinflammatory cytokines (for example, IL-6 and TNF-α) [60, 62, 63].

Lastly, several miRNAs have been observed to support the emergence of cancer stemness traits through targeting signalling elements and gene groups implicated in CSC biology. miRNAs are a fast-growing class of short (19 to 22 nt), noncoding, regulatory RNA molecules that customarily bind to the 3′-untranslated region (3′-UTR) of their target transcripts to induce translational repression, degradation, or destabilisation. Although miRNAs generally help regulate the transitions between different stages of development, they are also linked with tumourigenesis. As such, CSC-specific miRNA expression profiles may be useful for prog‐ nostic purposes. Those miRNAs that are highly expressed in CSCs of a specific tumour are termed oncomiRs; those that are excluded from CSCs of the same tumour are known as tumour-suppressor miRs. Breast CSCs were the first CSCs in which differential expression of miRNAs was demonstrated [64].
