Lack of telomerase reduces cancer incidence and increases lifespan of zebrafish tp53M214K mutants

Telomerase activity is restricted in humans and telomere attrition occurs in several tissues accompanying natural aging. Critically short telomeres trigger DNA damage responses and activate p53 which leads to apoptosis or replicative senescence. These processes reduce cell proliferation and disrupt tissue homeostasis, thus contributing to systemic aging. Similarly, zebrafish have restricted telomerase expression, and telomeres shorten to critical length during their lifespan. Telomerase-deficient zebrafish (tert −/−) is a premature model of aging that anticipates aging phenotypes due to early telomere shortening. tert −/− zebrafish have impaired cell proliferation, accumulation of DNA damage markers and p53 response. These cellular defects lead to disruption of tissue homeostasis, resulting in premature infertility, gastrointestinal atrophy, sarcopenia and kyphosis. Such consequences contribute to its premature death. Here we reveal a genetic interdependence between tp53 and telomerase function. Mutation of tp53 abrogates premature aging of tert −/− zebrafish, prolonging male fertility and lifespan. However, it does not fully rescue healthspan. tp53mut tert −/− zebrafish retain high levels of inflammation and increased spontaneous cancer incidence. Conversely, loss of telomerase prolongs the lifespan of tp53mut single mutants. Lack of telomerase reduces two-fold the cancer incidence in double mutants and increases lifetime survival. Thus, we observe a reciprocal rescue of tp53mut and tert −/− that ameliorates lifespan but not spontaneous cancer incidence of tp53mut, likely due to higher levels of inflammation.

www.nature.com/scientificreports/dysfunction in humans due to its naturally shorter telomeres 21 .First generation telomerase Cast/Ei KO mice exhibit defects that better resemble human TBDs 21 .
TP53 is a tumor suppressor gene that plays a crucial role as the "guardian" of genome.Mutations in TP53 are found in more than half of all human tumors.TP53 regulates cell cycle progression and genome stability by activating DNA repair, apoptosis and senescence.Zebrafish tp53 M214K mutants (equivalent to human M246 in the DNA binding domain and mutated in several cancers) lack transcription of p21, Puma and Bax and G1 checkpoint activation 28 .Similar to mice and humans, tp53 M214K zebrafish mutants develop soft-tissue spontaneous tumors by the age 9 months 28 .
Several phenotypes associated to deficiency of telomerase require p53 function.Deletion of p53 is sufficient to prevent germ-cell apoptosis and infertility in late-generation Tert −/− mice 29 .However, this comes at the high cost of increased genome instability and cancer 29,30 .In tert −/− zebrafish, although tp53 deficiency rescues cell-proliferation defects and premature death 12 , its effect at the organism level and tumor incidence remained unknown.Conversely, it was not investigated if lack of telomerase would have an impact on tp53 mutant zebrafish.Here we show that tp53 mutation rescues male fertility, attenuates aging phenotypes, and increases lifespan of tert −/− zebrafish.However, similar to tert −/− fish, double mutants retain higher inflammation and early cancer incidence when compared to wild type.

tp53 mutation rescues premature male infertility of tert −/− zebrafish
Loss of male fertility is one of the early phenotypes of zebrafish aging in both wild type and tert hu3430/hu3430 mutants (referred as tert −/− in this study) 23 .tp53 M214K/M214K homozygous mutants (tp53mut in this study) rescues cell proliferation decline of testis and gut of tert −/− zebrafish, resulting in partial restoration of tissue integrity 22 .To test whether tp53 mutation had a functional effect on tert −/− zebrafish fertility, we have conducted fertility assays over their lifespan.We selected 6-month-old fish as the earliest time point, outcrossed mutant males with wild type females and evaluated the percentage of fertilized eggs.Consistent with our previous reports, by the age of 6 months, tert −/− males were almost completely infertile (Fig. 1A, WT vs tert −/− p < 0.0001, tp53mut vs tert −/− p < 0.0001, tert −/− vs tert −/− ; tp53mut p < 0.0001).In the tert −/− ; tp53mut background, fertility was slightly lower than that of wild type males (Fig. 1A, WT vs tert −/− ;tp53mut p = 0.014).However, approximately 60% of the eggs laid by wild-type females were fertilized, indicating a clear functional rescue of tert −/− male infertility (Fig. 1A, tert −/− vs tert −/− ;tp53mut p < 0.0001).

tp53 mutation reduces tert −/− premature aging phenotypes
The rescue of male fertility prompted us to investigate whether tp53 mutation improved other aging phenotypes, such as kyphosis (abnormal curvature of the spine), caused by increased weakness of the spinal bones, and cachexia (excessive muscle wasting), caused by muscle tissue atrophy.The incidence of these phenotypes was monitored from 12 months of age until the time of death.At 12 months of age, 52% of tert −/− zebrafish developed cachexia and/or kyphosis (Fig. 2A and B, WT vs tert −/− p < 0.001, tert −/− vs tert −/− ;tp53mut p < 0.001).This increase in incidence of aging phenotypes was accompanied by weight loss of tert −/− zebrafish by a mean weight of about 0.4 g compared to 0.55 g in wild type and tp53mut siblings (Fig. 2C, WT vs tert −/− p = 0.004, tp53mut vs tert −/− p = 0.016).In the tert −/− ;tp53mut background, cachexia and kyphosis incidence was about 13% and significantly lower than tert −/− siblings (Fig. 2B, tert −/− vs tert −/− ;tp53mut p < 0.001).

tp53 mutation does not reduce tert −/− inflammation
Low-grade sterile inflammation is a recognized feature of aging and is a preeminent phenotype of tert −/− zebrafish.We have recently shown that senescence-associated inflammatory phenotype (SASP) and inflammation are key features of telomerase mutants that are initiated by specific organs during aging 26 .To test whether tp53 mutation could attenuate the effects of increased cell senescence of tert −/− zebrafish, we examined the expression of genes related to type I interferon inflammation (Interferon-stimulated gene 15, isg15 and Type I interferon, ifn-i) 31 and SASP (matrix metallopeptidase 15a, mmp15a) 32,33 in 12-month-old zebrafish.The head kidney was chosen because it hosts the adult hematopoietic system and produces blood cells that regulate communication between organs.In tert −/− zebrafish, inflammation significantly higher than wild type and tp53mut siblings (isg15, Fig. 3A, WT vs tert −/− p = 0.006, tp53mut vs tert −/− p = 0.012) and (ifn-i, Fig. 3B, WT vs tert −/− p = 0.049, tp53mut vs tert −/− p = 0.031).Similarly, expression levels of the matrix metalloproteinase mmp15a associated with inflammation and SASP were elevated in tert −/− zebrafish (Fig. 3C).However, inflammation observed in tert −/− zebrafish was not reduced in tert −/− ;tp53mut (Fig. 3A-C), denoting an incomplete rescue of double mutants likely due to ongoing DNA damage triggered by critically short telomeres.

tert −/− reduces tumor incidence of tp53mut zebrafish
During our observation of zebrafish aging, we noticed that tp53mut zebrafish started to develop macroscopically visible tumors from the age of 11 months on.By the time they reached 18 months of age, about 65% of tp53mut fish had developed visible tumors (Fig. 4A, WT vs tp53mut p < 0.001).At this time, incidence of cancer was 30% in tert −/− ;tp53mut (Fig. 4A, WT vs tert −/−; tp53mut p < 0.001) and 15% in tert −/− zebrafish (Fig. 4A, WT vs tert −/− ns p = 0.072).However, a more detailed histological analysis of 12 months old fish revealed that macroscopic assessment of tumors led to underestimation of true tumor incidence and onset.We were able to detect both carcinomas and sarcomas in these fish (Fig. 4B).Nevertheless, all genotypes exhibited a similar trend concerning susceptibility to tumor incidence.Approximately 10% of tert −/− zebrafish developed tumors, exclusively carcinomas (Fig. 4C).In tert −/−; tp53mut zebrafish, tumor incidence was about 20%, and most tumors were sarcomas.Consistent with the macroscopic assessment, tp53mut had a high tumor incidence, of  www.nature.com/scientificreports/approximately 30%, all sarcomas.In contrast to 18 months of age, tumor incidence at 12 months did not reach statistical significance between mutant genotypes (Fig. 4C).

Discussion
Decline in cell proliferation constitutes one of the most prominent physiological changes that occurs during aging, which ultimately leads to tissue dysfunction 30 .Telomere attrition is a shared characteristic of both normal aging and premature aging syndromes.Zebrafish tert −/− mutants exhibit premature short telomeres resulting in increased levels of DNA damage markers, 53BP1 and P-γH2AX 22 .Importantly, DNA damage markers colocalize with telomeres in tert −/− zebrafish 23 .DNA damage response was corroborated by increase of p53 levels and transcription of its target genes: puma, cyclinG1 and cdkn1a 22 .Proliferative tissues showed a marked decline of cell proliferation (PCNA) with an initial increase in apoptosis (TUNEL) and, later, cell senescence (SA-B-Gal, p15/16 and p21), accompanied by increased mitochondria dysfunction (disrupted mitochondria membranes, low ATP and increased ROS) and appreciated tissue damage 22,23,25 .Our current study extends the understanding of the tp53-dependent effects of telomere shortening at the organism level and how p53 stabilization accelerates aging phenotypes.Loss of tp53 function in telomerase mutants extends the fertile period of males and, at the organism level, ameliorates aging phenotypes and longevity.These findings provide further support to the hypothesis that aging is a consequence of loss of tissue homeostasis partly due to reduced cell proliferation.Inhibition of p53 increases longevity of tert −/− zebrafish, but the underlying mechanism is not unidirectional.We previously reported that tert −/− zebrafish are more susceptible to tumorigenesis and develop spontaneous tumors, mostly carcinomas, at an earlier age 23 .Loss of p53 activity is also strongly associated with increased cancer incidence 34 .Approximately 28% of tp53mut zebrafish develop sarcomas by 9 months of age 28 .Consistent with these findings, we observed an acceleration in spontaneous tumor formation upon loss of tp53.Interestingly, tert −/− developed spontaneous tumors later and less frequently than tp53mut zebrafish, suggesting that while telomerase deficiency drives tumorigenesis, it can also restrain the onset of tp53mut tumors.Lack of telomerase also modifies the types of tumors of tp53mut, as double mutants now exhibited not only sarcomas but a majority of carcinomas.
While scoring for tumor incidence, we observed that tp53mut zebrafish died shortly after developing tumors.Survival analysis revealed that among the four genotypes, tp53mut zebrafish had the shortest lifespan.Importantly, the shortened lifespan observed in both tp53mut and tert −/− was rescued when both genes were mutated.These findings are in agreement with previous studies that used p53 morpholinos and pharmacology-based methods to inhibit p53 in G2 tert −/− larvae 24,25 .However, these studies did not explore the effect of p53 mutations www.nature.com/scientificreports/ on lifespan, suggesting a unidirectional rescue, whereas we now show a mutual rescue between tp53mut and tert −/− zebrafish.
In addition to proliferative arrest, chronic inflammation is another consequence of telomere shortening that contributes to the fragility of tert −/− zebrafish and accelerated tumor incidence.The source and mechanism of action of short telomere-driven inflammation is not fully identified yet.Dead, damaged, or stressed tissues can trigger chronic inflammation 35 .In our previous studies, we showed that tissue integrity was improved in the tert −/− ;tp53mut zebrafish.However, these improvements did not rescue the inflammation signature in the kidney marrow, the zebrafish hematopoietic organ.Our work reveals that restoring cell proliferation and improving tissue integrity rescues several premature aging phenotypes of tert −/− zebrafish, such as premature infertility, body wasting and reduced life span.However, like in our previous studies 27 , chronic inflammation is not rescued and allowing cell proliferation in presence of persistent DNA damage have pro-tumorigenic effects, ultimately impairing the healthspan of the organism.

Ethics statement
All zebrafish studies and methods were performed in accordance with the guidelines and regulations of the IRCAN Animal Care Committee, in addition experimental protocols were approved by the regional (CIEPAL Cote d' Azur #784) and national authorities (French Ministry of Research #27673-2020092817202619).This study was conducted as recommended by the ARRIVE guidelines.

Zebrafish lines and maintenance
Zebrafish were maintained in accordance with Institutional and National animal care protocols.The stock line was preserved as tert hu340/+ tp53 M214K/+ double heterozygote and maintained strictly by outcrossing it to AB WT fish to avoid the effects of haploinsufficiency in the progeny.Experimental fish were obtained by in-crossing the stock line.Overall characterization of these four genotypes was performed in F1 sibling animals at 9 months of age.Due to male sex bias in our crosses, that affected mostly tert −/− progeny, we were unable to obtain significant numbers of females for analysis and so all data are restricted to males, except the survival analysis, scoring of aging phenotypes and macroscopic tumor formation.

Fertility assays
In order to assess male fertility, single 9-month-old males from the four different genotypes were separately housed overnight in external breeding tanks with a single young (3-6 months old) wild type female.Breeding pairs were left to cross and lay eggs in the following morning and embryos were collected approximately 4 h post fertilization (hpf) and allowed to develop at 28 °C.Assessment of fertilized eggs and embryo viability was conducted between 4 and 6 hpf.At least 12 independent crosses were conducted for each genotype to evaluate male fertility.Only successful breeding trials, defined as events in which a clutch of eggs was laid by the female, were scored.

Real-time quantitative PCR
Twelve-months-old zebrafish were sacrificed in 1 g/L of MS-222 (Sigma Aldrich) and the head kidney was collected and immediately snap frozen in liquid nitrogen.RNA extraction was performed in TRIzol (Invitrogen) by mashing tissues with a motorized pestle in a 1.5 mL eppendorf tube.After incubation at room temperature (RT) for 10 min TRIzol, chloroform extractions were performed.Quality of RNA samples was assessed through BioAnalyzer (Agilent 2100).Retro-transcription into cDNA was performed using QuantiTect Reverse Transcription kit (Qiagen).
Quantitative PCR (qPCR) using primers described in Table 1 was performed using FastStart Universal SYBR Green Master mix (Roche) and an StepOne plus Real time PCR Detection System (Applied Biosystems).qPCRs were carried out in duplicate for each cDNA sample.Relative mRNA expression was normalized against the housekeeping gene Ribosomal protein S11 (rps11) mRNA expression using the 2 −ddCT method compared to control condition.

Scoring of aging phenotypes
Aging fish were observed macroscopically for signs of body wasting (cachexia) by evaluating the dorsal-ventral width at the dorsal fin level and comparing with their length, as performed in our previous study 22 .To further evaluate body wasting, fish were weight at the same age.Spinal curvature was assessed macroscopically as any age-associated deformation of the spine in adult fish.