Telomeres and metabolism : Part 1. Functional interplay between the telomere maintenance and mTOR pathways. Part 2. RAP1 role in chemically-induced hepatocellular carcinoma

Programa de Doctorado en Biociencias Moleculares

Telomeres and metabolism:

Part 1. Functional interplay between the telomere maintenance and mTOR pathways

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma

Doctoral thesis Iole Ferrara Romeo

Madrid, 2019

Facultad de Ciencias

Departamento de Biología Molecular

Telomeres and metabolism:

Part 1. Functional interplay between telomere maintenance and mTOR pathways

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma

Doctoral thesis Iole Ferrara Romeo

BSc,MSc

The entirety of the work presented in this thesis has been carried out at the Telomeres and Telomerase Group in the Spanish National Cancer Research Centre (CNIO, Madrid)

under the direction and supervision of

Dr. Maria Blasco Marhuenda and Dr. Paula Martínez Rodríguez

Madrid, 2019

Summary/Resumen

1. English

Telomeres are specialized nucleoprotein structures at the end of chromosomes that protect them from degradation and repair activities. Dysfunctional telomeres activate a DNA damage response, which eventually results in cell growth arrest, senescence and/or apoptosis, leading to a reduction of the regenerative potential of the tissues. Telomeres shorten within each cell division throughout the organismal life and telomere shortening is defined as a primary cause of aging-associated diseases. The development of metabolic disorders, in its turn, is also a phenotype of advancing age. Although some works have reported about the link between telomeres and metabolism, it still remains a fairly unexplored field to date.

Telomeres are elongated by the telomerase, a specialized enzyme whose expression is very restricted in the adulthood. Telomerase deficiency in mice and humans leads to short telomeres, premature appearance of age-related diseases and shorter lifespan. Inhibition of the nutrient sensing pathway regulated by the mechanistic target of rapamycin (mTOR) increases lifespan in numerous species from yeast to mice and is considered a major target to delay aging and age-related pathologies. In light of the beneficial effects of the mTOR pathway inhibition in extending longevity, we have addressed whether rapamycin treatment could ameliorate the premature aging phenotypes and the decreased longevity of telomerase-deficient mice with short telomeres. This is of potential relevance as mTOR inhibitors could represent new potential treatments for human patients suffering from “telomere syndromes”. To address the role of mTOR in the survival of telomerase-deficient mice with short telomeres (second generation, G2, Terc^-

/- mice), we treated them with rapamycin, an inhibitor of mTOR. We found that chronic rapamycin treatment decreased the survival of G2 Terc^-/- mice, in marked contrast to significant lifespan extension in the case of similarly treated wild-type controls. Telomerase-deficient mice with short telomeres have a hyper-activated mTOR pathway. By using mouse genetics, we further confirm that abrogation of the mTOR downstream target S6 kinase 1 in S6K1^-/-/Terc^-/- double mutant mice also decreases mouse longevity compared to single Terc^-/- controls, in contrast to lifespan extension in the case of single S6K1^-/- female mice. Together, these findings demonstrate that the mTOR pathway is an essential survival pathway in the context of telomerase deficiency and presence of short telomeres and its inhibition in this setting is deleterious.

The protein component of the telomere is a six-protein complex named shelterin. RAP1 is a component of shelterin known to have both telomeric and non-telomeric functions and is involved in the regulation of metabolic programs. RAP1-deficient mice develop obesity and hepatic steatosis, being these phenotypes more severe in females than in males. As hepatic steatosis and obesity have been related to increased liver cancer in mice and humans, we set out to address whether RAP1 deficiency results in increased liver cancer upon chemical liver carcinogenesis. We found that Rap1^-/- females were more susceptible to DEN-induced liver damage and hepatocellular carcinoma (HCC). DEN-treated Rap1^-/- female livers showed an earlier onset of both premalignant and malignant liver lesions that metastasize more rapidly to the lungs. These findings highlight an important role for RAP1 in protection from liver damage and liver cancer.

2. Español

Los telómeros son estructuras nucleoproteicas especializadas que se encuentran en el extremo de los cromosomas y los protegen de la degradación y de actividades de reparación. Cuando los telómeros se vuelven disfuncionales activan una respuesta frente al daño del ADN, que resulta en una progresiva pérdida de la capacidad regenerativa de los tejidos. Los telómeros se acortan en cada ciclo celular durante toda la vida del organismo y este acortamiento telomérico es considerado como causa primaria de la aparición de enfermedades asociadas con el envejecimiento. Las enfermedades metabólicas son también uno de los fenotipos asociados a la edad avanzada y aunque algunos trabajos han descrito cierta correlación entre telómeros y metabolismo, este campo de investigación es todavía poco conocido.

La telomerasa es una enzima especializada en alargar los telómeros. La deficiencia en telomerasa lleva, tanto en ratón como en humanos, a la acumulación de telómeros muy cortos, a la aparición prematura de enfermedades asociadas al envejecimiento y a una disminución de la longevidad. Por otro lado, la inhibición de la vía de señalización mTOR aumenta la longevidad en numerosas especies y además, en ratones, mejora varios fenotipos asociados con el envejecimiento. A la luz de los efectos beneficiosos de la inhibición de la ruta mTOR, nos propusimos estudiar si el tratamiento con rapamicina podría ameliorar el envejecimiento prematuro y la reducida longevidad de los ratones deficientes en telomerasa con telómeros cortos. Siendo así, los inhibidores de mTOR podrían constituir tratamientos potenciales en pacientes que sufren “síndromes teloméricos”. Para ello, tratamos ratones silvestres y ratones deficientes en telomerasa de segunda generación (G2 Terc^-/-) con el inhibidor de mTOR rapamicina. El tratamiento crónico con rapamicina disminuye la supervivencia de los ratones G2 Terc^-/-, en marcado contraste con la extensión significativa de la vida de los ratones silvestres tratados de manera similar. Por medio de análisis moleculares, hemos demostrado también que los ratones G2 Terc^-/- muestran una híper-activación de la vía mTOR. Además, utilizando medios genéticos, confirmamos que la falta de la proteína S6K1, un efector de mTORC1, en combinación con la deficiencia en telomerasa resulta en un acortamiento de la longevidad, en contraste con el alargamiento de la supervivencia en ratones hembras carentes de S6K1 que sí expresan telomerasa. Nuestros hallazgos demuestran que la ruta mTOR es una vía de supervivencia esencial en el contexto de deficiencia de telomerasa y presencia de telómeros cortos, y que su inhibición en este contexto es deletérea.

La proteína telomérica RAP1 tiene funciones tanto teloméricas como no teloméricas y está involucrada en la regulación del metabolismo. Ratones deficientes en RAP1 desarrollan obesidad y esteatosis hepática, siendo estos fenotipos más graves en las hembras que en los machos. Dado que la esteatosis hepática y la obesidad se asocian con una mayor incidencia en cáncer de hígado tanto en ratones como en humanos, nos propusimos analizar si la deficiencia de RAP1 producía un aumento del cáncer de hígado inducido químicamente. Tras tratar ratones Rap1^+/+ y Rap1^-/- con DEN, observamos que las hembras Rap1^-/- son más susceptibles al daño hepático y al carcinoma hepatocelular. Estos datos demuestran que RAP1 juega un papel importante en la protección contra el daño hepático y el cáncer de hígado.

Index

Summary/Resumen

1. English

2. Español

Index

Abbreviations

Introduction

1.1 Discovery and primary structure 21 1.1.1 The telomeric DNA 22

1.1.2 Shelterin: the telomeric complex 22 1.2 T-loop: the secondary structure of telomeres 23

1.3 Telomere shortening and re-elongation mechanisms 24

1.3.1 The end replication problem 24

1.3.2 Telomerase, the telomeric enzyme 25

1.3.3 Alternative Lengthening of Telomeres (ALT) 26

1.4 Telomeres and DNA Damage Response 26 1.5 Telomeres and telomerase in aging and cancer 27

1.5.1 Telomere syndromes 29

1.6 Telomerase mouse models 29

1.7 Telomeres and metabolism 31

2. The mTOR signaling pathway 33 2.1 mTOR field: a historical perspective 33

2.2 The two mTOR complexes, mTORC1 and mTORC2 34

2.3 The mTORC1 signaling 35

2.3.1 Routes downstream of mTORC1 35

2.3.2 Cellular processes upstream of mTORC1 37

2.4 The mTORC2 signaling 39

2.5 mTOR in cancer 40

2.6 mTOR in aging 41

2.6.1 Prolonged mTOR inhibition extends lifespan in mice 43

2.6.1.1 Chronic rapamycin treatment 43

2.6.1.2 Genetic depletion of the mTOR downstream target S6K1 44 3. Telomere biology and hepatocellular carcinoma 44

Objectives/Objetivos

Part 1. Functional interplay between the telomere maintenance

and mTOR pathways 51

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma 51 Parte 1. Interacción funcional entre las vías de mantenimiento telomérico

y la vía de señalización de mTOR 52

Parte 2. Papel de la proteína telomérica RAP1 en el carcinoma

hepatocelular inducido químicamente 52

Methods

1. In vivo experimentation 57

1.1 Mice generation 57

1.2 Mice maintenance 57

1.3 Mice genotyping 58

1.4 Rapamycin experimentation 59

1.4.1 Diet preparation 59

1.4.2 Rapamycin treatment 59

1.5 Chemical induction of hepatocellular carcinomas 60

1.6 Immunohistochemistry analysis 60

1.6.1 Pathological examination 61

1.7 Immunofluorescence analysis on tissue sections 62

1.8 ATP measurement 62

1.9 Gene expression analysis 62

1.10 Blood plasma analysis 63

1.11 Ultrasound imaging and tumors quantification 63

2. In vitro experimentation 63

2.1 Extraction and culture of Mouse Embryonic Fibroblasts 63

2.2 Rapamycin and torin 1 treatment 64

3. Western-blotting 64

4. In situ hybridization 65

4.1

In situ Hybridization (Q-FISH) in tissue sections 65

4.2 High-throughput Q-FISH 65

4.3 FISH analysis on metaphase spreads 65

5. Statistical analysis 66

Results

Part 1. Functional interplay between the telomere maintenance

and mTOR pathways 71

1.1 Rapamycin-mediated lifespan extension in wild-type mice is abrogated by telomerase deficiency

71

1.2 Rapamycin treated telomerase-deficient mice do not show increased

cancer susceptibility nor exacerbation of degenerative pathologies 74 1.3 Rapamycin treatment extends lifespan of wild-type mice in a telomere

length-independent manner and does not affect telomere shortening rates 76 1.4 Lifelong rapamycin treatment does not impact the intestinal defects driven by

telomerase deficiency 77

1.5 Persistent S6 phosphorylation in telomerase-deficient mice subjected to

chronic rapamycin treatment 82

1.6 Acute Rapamycin treatment inhibits mTORC1 in G2 Terc^-/- mice 83 1.7 Chronic rapamycin treatment does not inhibit mTORC1 in young healthy

G2 Terc^-/- mice 85

1.8 Intestine characterization of young mice fed rapamycin recapitulates what

observed at the humanitarian endpoint 87

1.9 Telomerase-deficient mice show a hyperactivation of the mTOR and other

survival pathways 88

1.10 Rapamycin treatment of Mouse Embryonic Fibroblasts 92 1.11 Reduced survival in late generation Terc^-/- S6K1^-/- compound mouse model 96

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma 98 2.1 DEN-induced liver damage hampers body weight gain as a consequence

of RAP1 deficiency 98

2.2 Rap1-deficient females are more susceptible to DEN-induced HCC

than wild-type mice 101

2.3 Role of RAP1 in HCC telomere dynamics 104

Discussion

Part 1. Functional interplay between the telomere maintenance

and mTOR pathways

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma 117

Conclusions/Conclusiones

Part 1. Functional interplay between the telomere maintenance

and mTOR pathways

Part 2. RAP1 role in chemically-induced hepatocellular carcinoma 125 Parte 1. Interacción funcional entre las vías de mantenimiento telomérico

y la vía de señalización de mTOR 126

Parte 2. Papel de la proteína telomérica RAP1 en el carcinoma

hepatocelular inducido químicamente 126

Bibliography

Acknowledgements

Annex I

Table S1 157

Table S2 163

Table S3 168

Table S4 174

Annex II

Abbreviations

3’-OH 3’-hydroxy group 4EBP1 eIF4E binding protein 1 53BP1 p53 binding protein 1

γH2AX Gamma-phosphorylated histone 2 variant A.X a.u.f./a.u. Arbitrary units (of fluorescence)

AC3 Active caspase 3 AFP a-fetoprotein

AKT AKT serine/threonine kinase ALT Alternative lengthening of telomeres ALT Alanine aminotransferase

AMP Adenosine 5'-monophosphate AMPK 5'-AMP-activated protein kinase APBs ALT-associated-PML nuclear bodies AST Aspartate aminotransferase

ATM Ataxia telangiectasia mutated ATP Adenosine 5'-triphosphate

ATR Ataxia telangiectasia and RAD3-related protein

bp Base pairs

BSA Bovine serum albumin

C57BL/6 C57 black 6 genetic background of inbred mice cDNA Complementary DNA

CHK1 Checkpoint kinase 1 CHK2 Checkpoint kinase 2 DC Dyskeratosis congenita

DDR DNA damage response DEN Diethylnitrosamine

DEPTOR DEP domain containing mTOR interacting protein DKC1 Dyskerin 1

DMEM Dulbecco’s modified Eagle’s medium DMSO Dimethyl sulfoxide

DR Dietary restriction

DSBs DNA double-strand breaks dUTP Deoxyuridine triphosphate

eIF3 eukaryotic translation initiation factor 3

eIF4A, B, E, F eukaryotic translation initiation factor 4A, B, E, F ERK Extra-cellular signal regulated kinase

EtOH Ethanol

FAH Foci of altered hepatocytes FBS Fetal bovine serum

FDR False discovery rate FKBP12 FK506-binding protein 12 FRB FKBP-rapamycin-binding G1, 2, 3, 4 Generation 1, 2, 3, 4 GAP GTPase-activating protein GDP Guanosine 5′-diphosphate

GRB10 GRB10-growth factor receptor-bound protein 10 GSEA gene-set enrichment analysis

GSK-3b Glycogen synthase kinase 3-b

GTP Guanosine-5'-triphosphate H&E Hematoxylin and eosin HCA Hepatocellular adenoma HCC Hepatocellular carcinoma HEP Humanitarian endpoint Hep Ag Hepatocyte antigen HIF-1 Hypoxia-inducible factor 1 HR Homologous recombination HT-qFISH High-throughput Q-FISH hTERC human Terc

hTERT human TERT

IGF1 Insulin like growth factor 1

IKK IκB kinase

i.p. intraperitoneal injection IRS1 Insulin receptor substrate 1 Kb Kilo base pairs

KEGG Kyoto encyclopedia of genes and genomes Ki67 Proliferation marker protein Ki-67

LIPIN1 phosphatidate phosphatase LPIN1 Mbp Mega base pairs

MEFs Mouse embryonic fibroblasts

mLST8 mammalian lethal with Sec13 protein 8

MRN Complex formed by MRE11, RAD50 and NBS1 mRNA messenger RNA

mSIN1 mammalian stress-activated protein kinase-interacting protein 1 mTOR Mechanistic target of rapamycin

mTORC1 mTOR Complex 1 mTORC2 mTOR Complex 2 MTS Multitelomeric signals

NAFDL Non-alcoholic fatty liver disease NASH Non-alcoholic steatohepatitis nCoR1 nuclear receptor corepressor 1 NFKB Nuclear factor kappa B

NHEJ Non-homologous end joining NK Natural killer

p19 19 kDa protein

p21 Cyclin-dependent kinase inhibitor 1 p53 Cellular tumor antigen p53

p65 Transcription factor p65

PBS Phosphate-buffered saline solution PCR Polymerase chain reaction

PDCD4 Programmed cell death protein 4

PDK1 3-phosphoinositide-dependent protein kinase 1 PEG 400 Polyethylene glycol 400

PGC1a Peroxisome proliferator-activated receptor gamma coactivator 1 alpha PGC1b Peroxisome proliferator-activated receptor gamma coactivator 1 beta PI3K Phosphoinositide 3-kinase

PIP3 Phosphatidylinositol 3,4,5-trisphosphate

PKA cAMP-dependent protein kinase 1

PKCa, g, d, e, z Protein kinase C alpha, gamma, delta, epsilon, zeta PKG cGMP-dependent protein kinase

PML Promyelocytic leukaemia POT1 Protection of telomeres 1

PPARa Peroxisome-proliferator activated receptor alpha PPARg Peroxisome-proliferator activated receptor gamma PPP Pentose phosphate pathway

PRAS40 Prolin-rich AKT substrates of 40kDa PROTOR 1/2 Protein observed with Rictor 1/2 pRPA phosphorylated replication protein A pS6 phosphorylated ribosomal protein S6

pS6K1 phosphorylated ribosomal protein p70 S6 Kinase beta 1 Q-FISH Quantitative fluorescence in Situ hybridization

RAGs Ras-related GTPases

RAGULATOR Ragulator complex protein LAMTOR4 RAP1 Repressor/activator protein 1

RAPTOR Regulatory protein associated with mTOR

REDD1 Regulated in development and DNA damage responses 1 RHEB Ras homolog, mTORC1 binding

RICTOR Raptor-indipendent companion of mTOR RPA Replication protein A

RSK Ribosomal protein S6 kinase alpha S6 or rpS6 Ribosomal protein S6

S6K1 Ribosomal protein p70 S6 kinase beta 1 S6K2 Ribosomal protein p70 S6 kinase beta 2 SGK1 Serine/threonine-protein kinase Sgk1 SKAR Polymerase delta-interacting protein 3

SMC1 Structural maintenance of chromosomes protein 1 SREBPs Sterol responsive element binding proteins SSBs DNA single strand breaks

T-SCE Telomeric sister-chromatid exchanges TBC1D7 TBC1 domain family member 7 TBS Tris-buffered saline

Terc Telomerase RNA component TERT Telomerase reverse transcriptase TIFs Telomere dysfunction induced foci TIN2 TRF1-interacting factor 2

TOR1/2 Target of rapamycin 1/2 TOS TOR signaling motif

TPP1 POT1-TIN2 organizing protein 1 TRF1 Telomere repeat factor 1

TRF2 Telomere repeat factor 2

TSC1, 2 Tuberous sclerosis complex 1, 2 U/L Units per liter

UM-HET3 Heterogeneous genetic background of mice population WNT Protein WNT

WT Wild-type

Introduction

Aging is defined as a multifactorial process that coincide with the progressive functional decline at both cellular and tissue levels, so resulting in decreased organismal fitness and increased risk of death (López-Otín et al. 2013; Martínez and Blasco 2018). Nine biological processes have been defined as the hallmarks of aging, i.e. the molecular and cellular causes of the aging phenotypes (López-Otín et al. 2013).

These processes cover shorten telomeres, genomic instability, cellular senescence, stem cell exhaustion, epigenetic alterations, mitochondrial dysfunction, deregulated nutrient sensing, loss of proteolysis and altered cellular communication (López-Otín et al. 2013). Notably, of these processes, shortened telomeres are considered a primary cause of aging-associated diseases as they can induce many of the other hallmarks of aging (López-Otín et al. 2013; Martínez and Blasco 2018). Short unprotected telomeres, indeed, lead to chromosome end-to-end fusions and genomic instability (Blasco et al. 1997a; Lee et al.

1998; Martínez and Blasco 2011), induce replicative senescence (Zou 2004; Fumagalli et al. 2012) thus leading to stem cell depletion and the consequent impairment of tissue-renewal (Harley et al. 1990; Collado et al. 2007; Sharpless and DePinho 2007; Flores et al. 2008; Muñoz-Espín and Serrano 2014), and trigger epigenetic changes at both telomeric and subtelomeric regions (Benetti et al. 2007). Furthermore, short telomeres have been causally associated with mitochondrial dysfunction (Sahin et al. 2011) as well as defective insulin secretion and consequent glucose intolerance (Kuhlow et al. 2010). The relationship between telomere biology and metabolism is however a rather unexplored field to date and further studies are needed to elucidate their connections.

1. Telomeres

1.1 Discovery and primary structure

The term telomere derives from the Greek words telos (end) and meros (part) and refers to a specialized nucleoprotein structure that forms the end of the linear eukaryotic chromosome (McClintock 1941; Muller 1938). Telomeres were first described by Hermann Muller and Barbara McClintock by studying the effects of X-rays on Drosophila Melanogaster and on Zea mays chromosomes. Based on these studies, they argued that the natural ends of chromosomes were discrete structures, absolutely essential for chromosome stability (McClintock 1941; Muller 1938).

Subsequently, Elisabeth Blackburn and Joe Gall deciphered the sequence of chromosome ends from the ciliated protozoan Tetrahymena thermophila. They discovered that these consisted of a variable number of tandem repeats of the hexameric sequence -TTGGGG- and argued that all eukaryotic telomeres are formed of tandem repeated oligonucleotide sequences (Blackburn and Gall 1978).

Since then, telomeric sequences of several organisms have been characterized and over time it became clear that telomeres are highly-conserved structures, consisting mostly of tandemly repeated short sequences rich in G nucleotides (Blackburn 1994).

1.1.1 The telomeric DNA

As mentioned above, telomeres are specialized nucleoprotein structures at the ends of linear eukaryotic chromosomes, they are formed by a tandem repeated sequence rich in G nucleotides and do not contain protein-coding genes (Wellinger and Sen 1997). In all vertebrates the repeated sequence is TTAGGG (Meyne et al. 1989) and at the end of the duplex repetitive sequence, there is a guanine-rich, single stranded 3’ overhang of approximately 150 to 200 bp, also known as G-strand overhang (Fig.1), that is essential for the protective function of telomeres (Klobutcher et al. 1981; Griffith et al. 1999).

Telomere length varies across species and within the same species, depending on the developmental stage and the cell type (Marion et al. 2009; Flores et al. 2008). Human telomeres span in average 5 to 15 kb (de Lange et al. 1990), whereas the average murine telomere spans between 15 and 40 kb depending on the genetic background (Hemann and Greider 2000; Zijlmans et al. 1997). These variations do not alter the function of the telomeres as long as their size does not decrease beyond a critical length.

However, when this occurs, telomeres become recognized as DNA double-strand breaks (DSBs) and lose their functionality, culminating in genomic instability (Blasco et al. 1997a; Lee et al. 1998).

1.1.2 Shelterin: the telomeric protein complex

In mammals, telomeric DNA is bound by a six-protein complex named shelterin (De Lange 2005;

Palm and de Lange 2008). It is formed by the telomere repeat factors 1 and 2 (TRF1 and TRF2), the TRF1- interacting factor 2 (TIN2), the protection of telomeres 1 (POT1), the POT1-TIN2 organizing protein 1 TPP1

Figure 1: Structure of telomeres. Top: mouse metaphase chromosome treated with a fluorescent-labeled probe that hybridizes to telomere repeats. Bottom: schematic depiction of telomeric DNA.

(also known as TINT1, PTOP or PIP1) and the repressor/activator protein 1 (RAP1) (De Lange 2002; Liu et al. 2004a; De Lange 2005). TRF1, TRF2 and POT1 are the only components that bind directly to the telomeric DNA repeats, the formers binding to the double-stranded DNA and the latter to the 3’-single stranded G-overhang (Fig.2). TRF1 and TRF2 present a high degree of homology in their primary structure and bind to the telomeric DNA as homodimers (Bianchi et al. 1997; Broccoli et al. 1997). Besides binding to the 3’ overhang, POT1 is connected to the shelterin complex through direct association to TPP1 (Fig.2) (Liu et al. 2004b). While humans contain only one Pot1 gene, mouse Pot1 has two paralogs, Pot1a and Pot1b, which carry out different functions (Hockemeyer et al. 2006). The linchpin of the entire complex is represented by TIN2, which anchors TPP1/POT1 to TRF1 and TRF2 (Chen et al. 2008; Kim et al. 2004; Ye et al. 2004). Finally, RAP1 is recruited to telomeres by its association to TRF2 (Fig.2) (Celli and de Lange 2005; Li 2003). Complete abrogation of each of these proteins in mouse models leads to early embryonic lethality, with the exception of RAP1 and POT1B (Karlseder et al., 2003; Chiang et al., 2004; Celli and de Lange, 2005; He et al., 2006; Hockemeyer et al., 2006b; Wu et al., 2006; Kibe et al., 2010; Martinez et al., 2010; Sfeir et al., 2010) (reviewed in (Martinez and Blasco 2010). Several shelterin transgenic mouse models as well astissue-specific conditional knockout mouse models have been generated, allowing to more clearly understand the molecular mechanisms of shelterin-induced telomere dysfunction.

Shelterin is essential for the generation of the so-called t-loop, the characteristic structure of telomere terminus (see below). It also prevents the telomeres from being recognized as DBSs and the subsequent activation of the DNA damage response. Moreover, shelterin is also implicated in the regulation of telomere length and aids at telomeres DNA replication (De Lange 2005).

In addition to the six core proteins that form the shelterin complex, several complementary proteins are transiently associated with telomeres, the majority of which being recruited to telomeres through their interaction with the shelterin components (mostly TRF1 and TRF2) (Chen et al. 2008) and playing important roles in maintaining the protective function of telomeres.

1.2 T-loop: the secondary structure of telomeres

As mentioned above, the 3’-single stranded G-overhang is essential for the protective function of telomeres (Griffith et al. 1999; Klobutcher et al. 1981). In order to protect the chromosome ends from being recognized as DSBs, the telomeric DNA forms a higher order conformation, called t-loop (telomere loop) (Fig.3), that stabilizes the telomere structure (Goytisolo et al. 2000; Griffith et al. 1999), being some shelterin

Figure 2: Shelterin complex.

Schematic depiction of the nucleoprotein structure of a telomere with bound the shelterin proteins.

(Adapted from: Martinez & Blasco, 2011).

components essential for its generation (Doksani et al. 2013; De Lange 2005). The G-rich overhang folds back and invades the double-stranded telomeric DNA, so giving rise to the t-loop and the associated structure D-loop or displacement loop (Fig.3) (Greider 1999; Griffith et al. 1999; Goytisolo et al. 2000). This higher order conformational structure protects telomeres from degradation and repair activities.

1.3 Telomere shortening and re-elongation mechanisms 1.3.1 The end replication problem

During each cell division cycle, telomeres shorten as a result of the incomplete replication of linear DNA molecules by conventional DNA-polymerases (Watson 1972; Olovnikov 1973). The reason for this shortening lies in the antiparallel nature of the DNA molecule and in the typical semiconservative and bidirectional mechanism of replication. DNA-polymerases can only add bases to the 3’ end of a newly synthesized DNA strand. Therefore, in replication forks, the daughter DNA strands are divided into a leading and a lagging strand. On the leading strand the DNA-polymerase can continuously operate, along with the progression of the replication fork machinery in a 5’-3’ direction. On the lagging strand, however, the polymerase moves in the opposite direction to the replication fork and DNA synthesis must be accomplished in a discontinuous manner. Therefore, in the open DNA of the replication fork, specific RNA-polymerases named DNA-primases, introduce short pieces of complementary RNA to the parental strand, and these are used until all template strand has been replicated. These RNAs function as primers giving the necessary 3’- hydroxy group (3’-OH) substrate to the DNA-polymerase for the synthesis of the lagging strand. The resulting discontinuous DNA fragments, the Okazaki fragments, appear interspersed by RNA primers (Okazaki et al.

2006). These RNA-primers are then degraded and a DNA-polymerase fills the gap with the corresponding DNA-bases. However, at the very end of the chromosome, when the most distal RNA priming sequence is degraded, the polymerase is not able to replicate the DNA, because of lack of the 3’OH group. This results

Figure 3: The t-loop structure. (A) Electron microscopy visualization of the mammalian t-loop in HeLa cells (Griffith et al., 1999).

(B) Schematic depiction of the t-loop and D-loop structures of a telomere with shelterin complexes bound to it (adapted from Martinez and Blasco, 2011).

in a shorter daughter strand at each cell division (Hastie et al. 1990; Ohki et al. 2002; Harley et al. 1990).

On the other hand, as the leading strand is synthesized on a complete manner, the resulting DNA molecule is blunt ended. Here, two nucleases (the EXO1 and Apollo/SNM1B) digest DNA from the 5’ end to form the G-strand overhang. This results in a shorter 5’ DNA strand and a shorter telomere (Huffman et al. 2000;

Longhese et al. 2010; Wu et al. 2012). In summary, telomeres shorten due to the discontinous replication in the lagging strand, and due to nuclease digestion in the leading strand.

This phenomenon is known as the “end replication problem” and leads to a loss of 50 to 200bp from telomeres in every cell cycle, resulting in a replication- or age-related telomere shortening (Ohki et al. 2002).

1.3.2 Telomerase, the telomere-elongating enzyme

The loss in telomere length occurring at every cell cycle can be compensated through de novo addition of telomeric repeats by the enzyme telomerase, a reverse transcriptase composed by a constitutive catalytic subunit (TERT) and an inducible RNA component (Terc) that functions as template (whose sequence is CCCUAA) (Fig.4) (Greider and Blackburn 1985). Telomerase was first described in the ciliate Tetrahymena termophila by Carol Greider and Elizabeth Blackburn in 1985 and acts by adding telomeric repeats to the 3’ end of the G-strand overhang. The complex recognizes the 3’-OH group of the G-strand overhang where it acts as a reverse transcriptase, adding telomeric repeats (Blackburn et al. 1989; Greider and Blackburn 1985). Additional components are required to stabilize the complex, among them the dyskerin 1 or DKC1 protein (Cohen et al. 2007; Mitchell et al. 1999) (Fig.4).

The telomerase reaction cycle can be divided into three main steps: 1) recognition of the 3’-hydroxy group of the G-strand overhang and binding of the enzyme; 2) synthesis of the first telomeric repeat in the 5’→3’ direction; 3) translocation of the enzyme and re-annealing of the template with new 3’-OH end to begin the next round of synthesis (Autexier and Lue 2006). Telomerase is able to catalyze multiple rounds of telomere synthesis, thus having a certain degree of processivity (Wyatt et al. 2010).

The accessibility for the telomerase complex to the telomeric DNA sequences depends on the telomere conformation. A short telomere, with low amounts of shelterins bound to it, is more accessible to telomerase due to its loose and open conformation. Consequently, in the closed form (the t-loop) the 3’ end

Figure 4: Telomerase structure and telomere elongation.

Schematic representation of the telomerase subunits TERT and Terc and the additional protein DKC1. Telomerase recognizes the 3’ end of the G-rich telomere strand and adds telomeric repeats de novo (adapted from: Blasco, 2007).

is less approachable, impeding access to telomerase. This has been proposed as a mechanism by which telomeres autoregulate their own length (Marcand et al. 1997). Also, telomere elongation is regulated by the cell cycle, occurring in the late S phase during DNA replication (Marcand et al. 2000).

Telomerase activity compensates for telomere shortening in those cells where it is expressed, such as embryonic stem cells, germ cells, adult stem cells, as well as the majority of advanced-stage human cancers (Blasco 2005; Deng and Chang 2007; Hiyama and Hiyama 2007; Shay and Bacchetti 1997; Shay and Wright 2010). In adult stem cells however, telomerase activity is not sufficient to maintain telomere length over successive cell divisions and differentiation, thus leading to telomere shortening, one of the molecular mechanisms underlying organismal aging (Harley et al. 1990; Flores et al. 2008; López-Otín et al. 2013).

1.3.3 Alternative Lengthening of Telomeres (ALT)

In the absence of functional telomerase, minor alternative mechanisms exist to elongate the telomeres, collectively known as ALT (Alternative Lengthening of Telomeres), and described for the first time in tumors (Bryan et al. 1997). These mechanisms are based on homologous recombination (see next paragraph for description) between telomeres and subtelomeres, the chromosomal regions immediately adjacent to them that expand around 3 Mbp from the telomeric sequences and also contain repetitive stretches of DNA. However, the molecular mechanisms initiating ALT and driving the recombination are not fully understood and several models exist (Cesare and Reddel 2010; Cho et al. 2014). The ALT gives rise to highly heterogeneous telomere length, with the simultaneous presence of long and short telomeres in the same nucleus (Dunham et al. 2000), as well as telomere association with the PML (promyelocytic leukaemia) protein, thus forming the ALT-Hayflick associated-PML nuclear bodies or APBs (Bryan et al. 1995; Muntoni and Reddel 2005; Bryan et al. 1997; Dunham et al. 2000). Around 10-15% of tumors show ALT, as well as several immortalized cell lines (Henson et al. 2002; Bryan et al. 1995, 1997) and cells from the early embryo cleavage stage (Liu et al. 2007).

1.4 Telomeres and DNA Damage Response

When DNA lesions occur, mammalian cells respond by activating a DNA damage response (DDR):

a network of signaling cascades that sense the damage, induce cell cycle arrest, repair or degrade the damaged fragment and if irreparable induce cell death. DNA breaks can activate two signaling pathways:

ataxia telangiectasia mutated (ATM) kinase pathway, primarily activated by DSBs, and ATM-RAD3-related (ATR) kinase pathway, activated by single-strand DNA breaks (SSBs). Once any of these two kinases are activated, they phosphorylate the histone 2 variant A.X on Serine 139 (γH2AX) which accumulates at the damaged site, giving rise to detectable DNA damage foci. ATM and/or ATR activation induces the checkpoint proteins CHK1 and CHK2 and ultimately leads to the activation of the tumor suppressors p53 (and p21,

triggering cell cycle arrest, senescence and/or apoptosis. In parallel, recognition of DNA breaks also drives the repair of these DNA fragments by two different pathways, the homologous recombination (HR) and the non-homologous end-joining (NHEJ) pathways (Denchi 2009).

Telomeres are potential sites of genome instability, thus requiring inhibition of DDR signals to be preserved. Dysfunctional telomeres that are critically short or unprotected due to shelterin defects are unable to maintain the t-loop and are recognized as DSBs by the DDR machinery (Martinez and Blasco 2010).

Upon telomere damage, the ATM and ATR kinases are among the earliest transducers to initiate the DDR signaling cascade. When the t-loop is disrupted, the MRN complex formed by MRE11, RAD50 and NBS1 (De Jager et al. 2001) recognizes the telomere as a DSB and activates ATM (Lee and Paull 2007). In contrast, unprotected SSBs are bound by the single-stranded DNA-binding protein RPA (replication protein A), which in turn recruits ATR (Zou and Elledge 2003). This leads to the accumulation of some DDR-related proteins at the dysfunctional telomeres, giving rise to the so-called telomere dysfunction induced foci (TIF) (such as foci of γH2AX) and successive chromosomal aberrations (De Lange 2009; Martínez et al. 2009;

Tejera et al. 2010; Denchi 2009), ultimately resulting in cell cycle arrest, senescence and/or apoptosis. The non-proper inhibition of NHEJ pathway can lead to end-to-end fusions between sister chromatids or fusions that involve different chromosomes. The activation of HR would lead to recombination between telomeres (telomeric sister-chromatid exchanges, T-SCE) or telomeres and other sequences in the genome. The NHEJ is the most frequent mechanism to repair dysfunctional telomeres in mammals during the G1 phase of cell cycle (Smogorzewska et al. 2002). Globally, it is an error-prone repair mechanism and generates chromosome fusions. The HR pathway is an error-free repair mechanism based on the recombination of homologous repeated sequences (Szostak et al. 1983). The activation of HR pathway in the presence of DSBs occurs in a lesser extent compared to the NHEJ, due to the fact that it requires sister-chromatid sequences as the template for the homologous recombination and hence it only takes place at S or G2 phases of the cell cycle (San Filippo et al. 2008).

1.5 Telomeres and telomerase in aging and cancer

A possible link between telomeres and aging was first suggested in 1961 by Hayflick and Moorhead, when they observed that upon serial passages in vitro, human fibroblasts entered a state of irreversible growth arrest, called replicative senescence, whereas cancer cells did not (Hayflick and Moorhead 1961).

They hypothesized the existence of cellular factors, the so-called “Hayflick factors”, whose loss through continuous cell divisions would impede normal cells to proliferate indefinitely. Later studies identified telomere shortening as the main cause of this limited duplication capacity (Harley et al. 1990). Indeed, it was observed that the ectopic re-expression of telomerase was able to prevent replicative senescence in many cell types (Bodnar et al. 1998; Vaziri and Benchimol 1998; Yang et al. 1999), whereas telomerase inhibition in immortalized cells induces a proliferation arrest (Ohmura et al. 1995).

Adult stem cells are responsible for maintaining the regenerative potential of the tissues by replacing the senescent or dead cells, being such maintenance especially important for the high renewal tissues, like the intestinal epithelium or the hematopoietic system (Rando 2006). The progressive loss of regenerative capacity of the tissues ultimately impairs the organs function, thus resulting in organism aging and finally death (Collado et al. 2007). As mentioned before, telomerase is active in the germline and most adult stem cell compartments (Fig.5) (Allsopp et al. 2006; Kim et al. 1994; Flores et al. 2008; Hiyama and Hiyama 2007). Nevertheless, telomerase activity is not sufficient to counteract telomere attrition associated with each cell division and therefore telomere shortening takes place with age in all the somatic cells of the tissues, both in differentiated and adult stem cells (Blasco 2007; Chiu et al. 1996; Homayoun et al. 1994;

Flores et al. 2008; Harley et al. 1990; Hiyama and Hiyama 2007). This progressive telomere shortening is one of the molecular pathways or “Hayflick factors” underlying organismal aging (Fig.5) (Rudolph et al. 1999;

Blasco 2007; López-Otín et al. 2013).

Cancer and aging are both intimately associated with the accumulation of DNA damage. Genomic instability is a prominent characteristic of most, if not all, cancer types and has an essential role in tumorigenesis, as it accelerates the accumulation of genetic changes that are responsible for cancer cell evolution, i.e. acquisition of malignant traits through the alteration of key genes (Halazonetis et al. 2008;

Tsantoulis et al. 2008). Telomere maintenance above a minimum length is essential to impede cells entering into crisis, which renders the telomere maintenance mechanisms indispensable for cancer cells. Describing a conceivable model, in the cancer cell scenario, upon an oncogenic stress, cells accelerate their proliferative rate, being telomere length a limiting factor to their cell division capacity. Usually telomeres are in fact shorter in tumor cells compared to the healthy surrounding tissue (Martinez and Blasco 2010). This telomere shortening provokes activation of the DDR, which in turn results in both end-to-end fusions and chromosome aberrations or polyploidization, in both cases leading to genomic instability (Blasco et al., 1997) (reviewed in: (Martínez and Blasco 2011). The subsequent reactivation of telomerase or activation of alternative mechanisms of telomere elongation would then provide the mutated precancerous cell with the capacity to divide indefinitely (Fig.5), thus probably leading to its malignant transformation (Martinez and Blasco 2010). In line with this, more than 90% of human tumors aberrantly over-express telomerase (Joseph et al. 2010; Kim et al. 1994; Shay and Bacchetti 1997), while the remaining telomerase-negative tumors activate ALT (Barthel et al. 2017; Bryan et al. 1997). In light of the described, telomeres are currently widely studied as potential anti-cancer targets. In fact, telomerase targeted inhibition has been proposed as an anticancer therapy by provoking telomere shortening to a critical length and loss of cell viability in the tumor (Blasco 2002).

1.5.1 Telomere syndromes

In accordance with the fact that telomere shortening is one of the mechanisms underlying aging, germline mutations in both telomerase components or some of the shelterins (e.g. TIN2) have been linked to a group of rare and degenerative genetic diseases in humans, collectively known as telomere syndromes or telomeropathies (Armanios and Blackburn 2012; Martínez and Blasco 2017). These diseases are characterized by the presence of short or dysfunctional telomeres and the consequent defects in tissues regenerative capacity, which ultimately result in premature aging and death (Fig.5) (Armanios and Blackburn 2012; Martínez and Blasco 2017). Among them, mutations in any of the components of telomerase Terc, TERT or DKC1, are linked to the Dyskeratosis Congenita (DC), a severe form of bone marrow deficiency, characterized by mucosal leukoplakia, nail dystrophy and abnormal skin pigmentation (Dokal 2000). Other examples of telomere syndromes are aplastic anemia, characterized by a hypocellular bone marrow, idiopathic pulmonary fibrosis and liver fibrosis (Armanios et al. 2007; Calado et al. 2009; Tsakiri et al. 2007;

Yamaguchi et al. 2005).

1.6 Telomerase mouse models

The generation of telomerase-deficient and telomerase-overexpressing mouse models during the last decades has been decisive to demonstrate the crucial role of the telomerase in tissue renewal and its implications in both aging and cancer.

Transgenic mice overexpressing Tert in stratified epithelia (K5-Tert mice) showed improved regenerative capacity of the skin. This phenotype was accompanied by higher incidence of preneoplastic and neoplastic lesions in various tissue types (González-Suárez et al. 2001). Conversely, concomitant

Figure 5: Graphic depiction of the causal role of telomere length in aging and cancer.

overexpression of Tert in mice genetically engineered to be cancer resistant by overexpression of the p53, p16 and p19ARF tumor suppressors (Sp53/ Sp16/ SpArf/TgTert), led to a significant extension of median and maximum lifespan, retarded onset of degenerative lesions and a general attenuation of the symptoms of aging, longer telomeres and decreased DNA damage burden than wild-type mice (Tomás-Loba et al.

2008).

In contrast to the constitutive deletion of most of the shelterins, full-body telomerase-deficient mouse models for either Terc or TERT are viable and survive to adulthood (Blasco et al., 1997; Yuan et al., 1999).

Terc-deficient mice show accelerated telomere attrition as well as decreased lifespan, as the consequence of premature development of aging-associated pathologies (Blasco et al., 1997; Lee et al., 1998; Herrera et al., 1999; Rudolph et al., 1999). In particular, longevity is progressively shortened with increasing generations of telomerase-deficient mice and this is paralleled by the anticipated onset of the aging pathologies, owing to inheritance of progressively shorter telomeres with each mouse generation. The phenotypes associated to telomere dysfunction in these mice include among others, small size, infertility, severe intestinal atrophy, alopecia and hair graying, heart and kidney dysfunction, proliferative defects of neural stem cells and defective bone marrow (Ferron 2004; García-Cao et al. 2006; Samper et al. 2002;

Blasco 2005; Herrera et al. 1999; Lee et al. 1998). Interestingly, owing to the fact that telomere maintenance by telomerase is essential for tumor growth, telomerase-deficient mice with short telomeres are cancer resistant (Gonzalez-Suarez et al. 2000). The first Terc^-/- mouse model was generated in a mixed genetic background (C57BL6/129Sv), that is characterized by unusually long telomeres, and the mice were bred for six generations (Lee et al. 1998). Afterwards, Terc^-/- mice were generated in a C57BL6 genetic background (Herrera 1999) which showed shorter telomeres than the original mixed C57BL6/129Sv background. The authors found that these mice could only be bred for four generations and the pathological phenotypes appeared as early as at the second generation, being already dramatic at generation fourth (Herrera 1999).

Similar phenotypes were described in Tert knockout mice and the phenotypic analysis showed a loss of tissue renewal capacity with progressive breeding of mice, that was indistinguishable from that of Terc^-/- mice (Liu et al. 2000; Erdmann et al. 2004; Strong et al. 2011; Yuan et al. 1999).

Furthermore, recent studies showed that the energy demand increases in telomere dysfunctional cells, resulting, at the organismal level, in enhanced glucose metabolism for the maintenance of energy homeostasis (Missios et al. 2014). G3 Terc-deficient mice, that are characterized by dramatically short telomeres, exhibit increased energy consumption and this results in compensatory increases in ATP production, oxygen consumption rates and glucose metabolism via both glycolysis and tricarboxylic acids (TCA) cycle (Missios et al. 2014). For aged G3 Terc-deficient mice, normal diet provides insufficient levels of glucose, thus resulting in impaired energy homeostasis, suppression of mitochondrial biogenesis and increased rates of catabolic metabolism, which in turn lead to weight loss, tissue atrophy and premature death. An increase in diet glucose content reverts these defects and leads to extension of the mice lifespan by stimulating glycolysis, mitochondrial biogenesis and oxidative glucose metabolism (Missios et al. 2014).

In line with these findings, gene expression profile analyses in keratinocytes from mice with dramatically short telomeres (telomerase-null and overexpressing TRF2 in stratified epithelia, K5TRF2/Terc^-

/- mice) revealed that telomere shortening is paralleled by deregulation of several genes, driving, among other changes, the up-regulation of the mTOR and AKT pathways (Schoeftner et al., 2009). Thus, in the context of dramatically short telomeres the mTOR/AKT pathways upregulation acts as a survival response (Schoeftner et al., 2009).

As described above, p53 is one of the main downstream mediators of the cellular response to DDR triggered by short telomeres. In line with this, concomitant p53 deficiency in Terc^-/- mice abrogates the proliferative defects of the stem cells compartments as well as the tumor suppressor effect of short telomeres (Artandl et al. 2000; Chin et al. 1999; Flores and Blasco 2009). On the other hand, doubly deficient Terc^-/- p21^-/- mice showed extended survival and lesser proliferative defects than the Terc single deficient mice, without displaying increased cancer incidence (Choudhury et al. 2007).

1.7 Telomeres and metabolism

Dysfunctional telomeres and defective metabolism have been linked to the pathogenesis of several age-related pathologies. The incidence of metabolic disorders is on the rise with advancing age and several observations suggest a link between telomere biology and metabolism. An association between shortened telomeres and type 2 as well as possibly type 1 diabetes mellitus has been described in humans (Adaikalakoteswari et al. 2005; Sampson et al. 2006; Tentolouris et al. 2007; Uziel et al. 2007; Salpea et al.

2010; Zee et al. 2010). By studying several metabolic parameters of late generation (G4) Terc-deficient mice, it was found that mice with short telomeres develop glucose intolerance and impaired insulin secretion due to the progressive failure of pancreatic beta cells regeneration (Kuhlow et al. 2010). The lack of telomerase and consequent presence of short telomeres causes the beta-cells loss, which is a hallmark of type 2 diabetes (Porte and Kahn E 2001; Dor et al. 2004; Georgia and Bhushan 2004; Rhodes 2005). In support of that, telomerase overexpression in old wild-type mice results in improved fasting insulin levels and glucose uptake (Bernardes de Jesus et al. 2012). In addition, a direct link between telomere dysfunction and defective mitochondrial metabolism has been described (Sahin et al. 2011). From studies in mice null for either Tert or Terc genes emerged that telomere dysfunction is associated with impaired mitochondrial biogenesis and function, decreased gluconeogenesis, cardiomyopathy, and increased reactive oxygen species (Sahin et al. 2011). Telomere dysfunction causes activation of p53 which in turn transcriptionally represses Pgc1a and Pgc1b (peroxisome proliferator-activated receptor gamma coactivator 1 a and b) expression and their downstream gene network, which controls many aspects of mitochondrial biology and cellular metabolism (Lin et al. 2005; Sahin et al. 2011). Indeed, enforced Tert or Pgc1a expression or p53 depletion in telomere-defective contexts restores the PGC network expression as well as mitochondrial respiration, gluconeogenesis and cardiac function (Sahin et al. 2011). This mitochondrial decline also occurs during physiological aging in wild-type mice and can be partially reversed by telomerase activation (Bernardes de Jesus et al. 2012). Consistently with these findings, it was shown that G3 Terc-deficient mice present increased energy consumption that results in compensatory increases in glucose metabolism

(Missios et al. 2014). Indeed, under standard feeding conditions, aged G3 Terc-deficient mice show impaired energy homeostasis, suppression of mitochondrial biogenesis and increased rates of catabolic metabolism, which in turn lead to weight loss, tissue atrophy and premature death (Missios et al. 2014). Accordingly, a glucose supplement in the diet reverts these defects by stimulating glycolysis, mitochondrial biogenesis and oxidative glucose metabolism that lead to an extended lifespan (Missios et al. 2014).

In line with these studies, genetically engineered mice characterized by telomeres longer than normal in the whole organism (hyper-long telomere mice) are significantly leaner than control mice and show clear metabolic improvements, such as improved glucose and insulin tolerance, throughout life (Muñoz- Lorente et al., 2019, submitted).

In addition to the involvement of telomerase in metabolism regulation, the telomeric protein RAP1 on metabolism has also been shown to play a crucial role in metabolism (Martínez et al. 2013; Martinez et al. 2010). Although being involved in repressing the HR pathway at telomeres (Chen et al. 2011; Sfeir et al.

2010), deletion of Rap1 in vivo does not result in lethal telomere dysfunction. Indeed, mice lacking telomeric RAP1 are born at the expected Mendelian ratios and are fertile (Yeung et al. 2013; Sfeir et al. 2010; Martínez et al. 2013) and no difference in telomere length was observed in liver and brown fat between Rap1-deficient and wild-type mice (Martínez et al. 2013). In spite of that, RAP1 is important for telomere length maintenance in the context of cellular stress conditions such as telomerase deficiency (Martínez et al. 2016).

It has been shown that mammalian RAP1 is involved in modulating metabolism by directly binding to and regulating transcription of the Ppara (peroxisome proliferator-activated receptor a) and Pgc1a genes (Martínez et al. 2013; Yeung et al. 2013). Hence, RAP1 deficiency leads to decreased expression of PPARa and PGC1a, causing deregulation of their downstream pathways, which are involved in energy homeostasis (Martínez et al. 2013; Yeung et al. 2013). Rap1-deficient mice develop liver and adipose tissue dysfunctions, exhibiting hepatic steatosis, visceral fat accumulation and progressive development of obesity, glucose intolerance and insulin resistance (Martínez et al. 2013; Yeung et al. 2013). Furthermore, in vitro Rap1- deficient MEFs showed upregulation of some genes involved in type 2 diabetes and downregulation of genes related to insulin secretion and genes involved in the peroxisome-proliferator-activated receptor metabolic signaling (Martinez et al. 2010).

The mechanistic target of rapamycin (mTOR) signaling pathway is an evolutionarily conserved nutrient-sensing protein kinase that regulates cell growth and metabolism (Kapahi et al. 2010). Although so far there is no evidence of in vivo studies documenting a connection between mTOR pathway and telomere biology, some in vitro studies reported about an association between these two biological processes. Thus, rapamycin at high concentrations (100-1000 nM) and long treatment conditions (48-72h) has been shown to inhibit both TERT messenger RNA (mRNA) levels and telomerase activity (Zhou et al. 2003; Zhao et al.

2008). Also, some immunoprecipitation experiments in transformed NK (natural killer) cells showed that TERT, mTOR, AKT, p70 S6 kinase 1 (Kawauchi et al. 2014), co-immunoprecipitate, suggesting that these proteins form a physical complex (Kawauchi et al. 2014). The authors determined that this complex was necessary for telomerase activity proposing that it is necessary for cancer cell survival (Sundin and Hentosh

2012; Kawauchi et al. 2014). Despite the intriguing potential of this idea, further studies and the generation of in vivo models are needed to confirm this hypothesis.

2. The mTOR signaling pathway

2.1 mTOR field: a historical perspective

The mTOR field started decades before the identification of the TOR gene, with the discovery of rapamycin (Sabatini 2017). From soil samples hailing from the Easter island (or Rapa Nui, as named by locals), Sehgal and colleagues identified the bacteria Streptomyces hygroscopicus and isolated one product, named rapamycin after the native name of the island (being later clinically referred to as sirolimus) (Vézina et al. 1975). Although originally identified as a new antifungal antibiotic agent (BAKER et al. 2012; Vézina et al. 1975), the antiproliferative and immunosuppressive properties of rapamycin became soon clear, having been used extensively in the clinic, during the next decades, to avoid rejection after organ transplantation as well as to treat autoimmune disorders (Martel et al. 1977; ENG et al. 1984; Garber 2001;

Lorberg and Hall 2004). It was soon discovered that rapamycin is able to suppress incorporation of amino acids into cellular proteins, thus inhibiting protein translation (Singh et al. 1979). Several further studies revealed that rapamycin forms a gain-of-function complex with the peptidyl-prolyl-isomerase FKBP12 (FK506-binding protein 12) to inhibit signal transduction pathways that are essential for cell growth and proliferation (Chung et al. 1992). From studies in yeast, two separate genes were identified as rapamycin target, TOR1/2 (target of rapamycin 1/2), as their loss of function mimicked rapamycin treatment and their mutations conferred resistance to the compound (Heitman et al. 1991; Stan et al. 1994; Zheng et al. 1995).

Shortly after its discovery in yeast, the mammalian TOR was identified as the direct target of the rapamycin- FKB12 complex in mammals, being referred to as mTOR (mechanistic target of rapamycin, being formerly

“mammalian”), given its sequence homology with the yeast TOR genes (Brown et al. 1994; Sabatini et al.

1994; Sabers et al. 1995). Differently from yeast, higher eukaryotes, including mammals, have only one MTOR gene, whose product forms two different complexes: mTORC1 and mTORC2 (mTOR complex 1 and 2).

Extensive studies have been performed since the initial discovery of rapamycin, leading to identify TOR as a highly evolutionarily conserved protein, that acts as a master regulator in coordinating cell growth and metabolism with environmental conditions.

2.2 The two mTOR complexes, mTORC1 and mTORC2

mTOR is a serin/threonine protein kinase that belongs to the PI3K (phosphoinositide 3-kinase)- related protein kinase (PIKK) family (Abraham and Eng 2008; Marone et al. 2008).As mentioned above, it can associate with different proteins to form two distinct protein complexes, mTORC1 and mTORC2, being mTOR the catalytic subunit of both complexes. mTORC1 is defined by three core components: mTOR, RAPTOR (regulatory protein associated with mTOR) and mLST8 (mammalian lethal with Sec13 protein8), also known as GbL (Fig.6A) (Kim et al. 2002, 2003b). RAPTOR facilitates substrate recruitment to mTORC1 by binding the TOR signaling motif (TOS) found on several mTORC1 substrates and is also required for subcellular localization of mTORC1 (Nojima et al. 2003; Schalm et al. 2003). mLST8 is a common core component of both mTORC1 and mTORC2 that associates with their catalytic domains (Fig.6A,B) (Yang et al. 2006). Besides the core components, other two proteins take part to the mTORC1 complex, both acting as inhibitory subunits: DEPTOR (DEP domain containing mTOR interacting protein) and PRAS40 (prolin-rich AKT substrates of 40 kDa) (Fig.6A) (Sancak et al. 2007; Haar et al. 2007; Wang et al. 2007). Structural studies showed that the rapamycin-FKBP12 complex binds to the FRB (FKBP- rapamycin-binding) domain of mTOR to partially occlude the catalytic cavity, thus preventing the access to the substrates (Fig.6A) (Yang et al. 2013). Like mTORC1, mTORC2 also includes mTOR, mLST8 and DEPTOR, however, instead of RAPTOR, it contains the so-called Raptor-independent companion of mTOR (RICTOR), in addition to the regulatory subunits mSIN1 (mammalian stress-activated protein kinase- interacting protein 1) and PROTOR1/2 (Protein observed with Rictor 1/2) (Fig.6B) (Dos et al. 2004; Jacinto et al. 2004, 2006; Frias et al. 2006; Pearce et al. 2007; Thedieck et al. 2007; Woo et al. 2007; Yang et al.

2006). While the rapamycin-FKBP12 complex directly binds to and inhibits mTORC1, it does not directly inhibit mTORC2. Recent studies demonstrated that this is due to the steric hindrance generated by RICTOR and mSIN1 that block the rapamycin-FKB12 access to mTOR (see Fig.6A,B) (Chen et al. 2018).

Nevertheless, it has been described that, although the rapamycin-FKBP12 complex does not directly bind and inhibit mTORC2, prolonged rapamycin treatment also abrogates the mTORC2 signaling, being this probably due to the inability of the mTOR molecules that are bound to rapamycin to incorporate into new mTORC2 complexes (Lamming et al. 2012; Sarbassov et al. 2006).

A B

mTOR mTOR

Figure 6: mTORC1 and mTORC2 complexes. Schematic depiction of mTORC1 and mTORC2 subunits (modified from: Kim &

Guan, 2019).

2.3 The mTORC1 signaling

Environmental changes in availability of nutrients and energy require metabolic adaptations in order to maintain the energy homeostasis of the cell. Under starvation, the levels of nutrients and growth factors drop, inducing the cell to suppress biosynthetic programs and increase the recycling of “old” proteins and organelles, as well as slow proliferation, in order to avoid energy imbalance and eventually death. On the contrary, under conditions of nutrient availability, the production of new cellular components, including proteins, nucleic acids and lipids, must be increased to allow the cell to grow and proliferate. mTORC1 plays a central role in regulating the balance between catabolic and anabolic processes in response to the environmental changes. In particular, in response to favorable environmental conditions, activated mTORC1 promotes the synthesis of new proteins, lipids and nucleic acids and inhibits the protein turnover by blocking autophagy.

2.3.1 Routes downstream of mTORC1

Of the processes controlled by mTORC1, protein synthesis is so far the best characterized.

mTORC1 directly phosphorylates p70S6 kinase 1 (S6K1) and eIF4E binding protein 1 (4EBP1) which in turn associate with messenger RNAs (mRNAs) and enhance both mRNA translation initiation and progression (Brown et al. 1995; von Manteuffel et al. 2002; Hara et al. 1997; Saxton and Sabatini 2017). Two S6Ks genes have been identified in mammals, S6K1 and S6K2, both being key effectors of mTORC1 (Gout et al.

1998; Lee-Fruman et al. 1999; Saitoh et al. 1998; Shima 2002). When inactive, S6K1 associates with the eukaryotic initiation factor 3 (eIF3), a multiprotein complex involved in the initiation phase of eukaryotic translation. Activated mTORC1 binds to the eIF3 complex and directly phosphorylates S6K1 at its hydrophobic motif site Thr389 (Holz et al. 2005). This results in S6K1 dissociation, activation, and subsequent phosphorylation of its translational targets, i.e. eIF4B, ribosomal protein S6, PDCD4 (programmed cell death protein 4) and SKAR (polymerase delta-interacting protein 3) (Fig.7) (Jeno et al.

2006; Shahbazian et al. 2006; Ma et al. 2008; Holz et al. 2005). eIF4B (eukaryotic initiation factor 4B) is a positive regulator of eIF4F (eukaryotic initiation factor 4F) (Holz et al. 2005), a heterotrimeric protein complex which includes the RNA helicase eIF4A (eukaryotic initiation factor 4A), the cap-binding protein eIF4E (eukaryotic initiation factor 4E), and the large "scaffold" protein eIF4G (eukaryotic initiation factor 4G), that binds the 5' cap of mRNAs complex to promote eukaryotic translation initiation (Aitken and Lorsch 2012;

Merrick 2015). The ribosomal protein S6 (rpS6 or S6) is a component of the 40S ribosome that represents the most extensively studied S6K1 substrate. However, although the phosphorylation of S6 has been described for the first time about four decades ago, the molecular mechanism(s) underlying the diverse effects of S6 phosphorylation on cellular and organismal physiology are still poorly understood (Meyuhas 2008, 2015). S6K1 also phosphorylates and promotes the degradation of PDCD4, an inhibitor of the RNA helicase eIF4A that catalyzes the unwinding of secondary structure at the mRNAs 5' untranslated region