Telomere length is an epigenetic trait - Implications for the use of telomerase-deficient organisms to model human disease.

Telomere length, unlike most genetic traits, is epigenetic, in the sense that it is not fully coded by the genome. Telomeres vary in length and randomly assort to the progeny leaving some individuals with longer and others with shorter telomeres. Telomerase activity counteracts this by extending telomeres in the germline and during embryogenesis but sizeable variances remain in telomere length. This effect is exacerbated by the absence of fully active telomerase. Telomerase heterozygous animals (tert+/-) have reduced telomerase activity and their telomeres fail to be elongated to wild-type average length, meaning that - with every generation - they decrease. After a given number of successive generations of telomerase-insufficient crosses, telomeres become critically short and cause organismal defects that, in humans, are known as telomere biology disorders. Importantly, these defects also occur in wild-type (tert+/+) animals derived from such tert+/- incrosses. Despite these tert+/+ animals being proficient for telomerase, they have shorter than average telomere length and, although milder, develop phenotypes that are similar to those of telomerase mutants. Here, we discuss the impact of this phenomenon on human pathologies associated with telomere length, provide a brief overview of telomere biology across species and propose specific measures for working with telomerase-deficient zebrafish.

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.

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast.

Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks.

Taurine deficiency as a driver of aging.

Aging is associated with changes in circulating levels of various molecules, some of which remain undefined. We find that concentrations of circulating taurine decline with aging in mice, monkeys, and humans. A reversal of this decline through taurine supplementation increased the health span (the period of healthy living) and life span in mice and health span in monkeys. Mechanistically, taurine reduced cellular senescence, protected against telomerase deficiency, suppressed mitochondrial dysfunction, decreased DNA damage, and attenuated inflammaging. In humans, lower taurine concentrations correlated with several age-related diseases and taurine concentrations increased after acute endurance exercise. Thus, taurine deficiency may be a driver of aging because its reversal increases health span in worms, rodents, and primates and life span in worms and rodents. Clinical trials in humans seem warranted to test whether taurine deficiency might drive aging in humans.

Gut-specific telomerase expression counteracts systemic aging in telomerase-deficient zebrafish.

Telomere shortening is a hallmark of aging and is counteracted by telomerase. As in humans, the zebrafish gut is one of the organs with the fastest rate of telomere decline, triggering early tissue dysfunction during normal zebrafish aging and in prematurely aged telomerase mutants. However, whether telomere-dependent aging of an individual organ, the gut, causes systemic aging is unknown. Here we show that tissue-specific telomerase expression in the gut can prevent telomere shortening and rescues premature aging of tert-/-. Induction of telomerase rescues gut senescence and low cell proliferation, while restoring tissue integrity, inflammation and age-dependent microbiota dysbiosis. Averting gut aging causes systemic beneficial impacts, rescuing aging of distant organs such as reproductive and hematopoietic systems. Conclusively, we show that gut-specific telomerase expression extends the lifespan of tert-/- by 40%, while ameliorating natural aging. Our work demonstrates that gut-specific rescue of telomerase expression leading to telomere elongation is sufficient to systemically counteract aging in zebrafish.

Whole-mount Senescence-Associated Beta-Galactosidase (SA-ß-GAL) Activity Detection Protocol for Adult Zebrafish.

Senescence-associated beta-galactosidase (SA-ß-GAL) is an enzyme that accumulates in the lysosomes of senescent cells, where it hydrolyses ß-galactosides. With p16, it represents a well-recognized biomarker used to assess senescence both in vivo and in cell culture. The use of a chromogenic substrate, such as 5-bromo-4-chloro-3-indoyl-ß-d-galactopyranoside (X-Gal), allows the detection of SA-ß-GAL activity at pH 6.0 by the release of a visible blue product. Senescence occurs during aging and is part of the aging process itself. We have shown that prematurely aged zebrafish accumulate senescent cells detectable by SA-ß-GAL staining in different tissues, including testis and gut. Here, we report a detailed protocol to perform an SA-ß-GAL assay to detect senescent cell accumulation across the entire adult zebrafish organism ( Danio rerio ). We also identify previously unreported organs that show increased cell senescence in telomerase mutants, including the liver and the spinal cord.

Predictive and Therapeutic Implications of a Novel PLCγ1/SHP2-Driven Mechanism of Cetuximab Resistance in Metastatic Colorectal Cancer.

PURPOSE: Cetuximab is an EGFR-targeted therapy approved for the treatment of RAS wild-type (WT) metastatic colorectal cancer (mCRC). However, about 60% of these patients show innate resistance to cetuximab. To increase cetuximab efficacy, it is crucial to successfully identify responder patients, as well as to develop new therapeutic approaches to overcome cetuximab resistance. EXPERIMENTAL DESIGN: We evaluated the value of EGFR effector phospholipase C gamma 1 (PLCγ1) in predicting cetuximab responses, by analyzing progression-free survival (PFS) of a multicentric retrospective cohort of 94 treated patients with mCRC (log-rank test and Cox regression model). Furthermore, we used in vitro and zebrafish xenotransplant models to identify and target the mechanism behind PLCγ1-mediated resistance to cetuximab. RESULTS: In this study, levels of PLCγ1 were found increased in RAS WT tumors and were able to predict cetuximab responses in clinical samples and in vitro and in vivo models. Mechanistically, PLCγ1 expression was found to bypass cetuximab-dependent EGFR inhibition by activating ERK and AKT pathways. This novel resistance mechanism involves a noncatalytic role of PLCγ1 SH2 tandem domains in the propagation of downstream signaling via SH2-containing protein tyrosine phosphatase 2 (SHP2). Accordingly, SHP2 inhibition sensitizes PLCγ1-resistant cells to cetuximab. CONCLUSIONS: Our discoveries reveal the potential of PLCγ1 as a predictive biomarker for cetuximab responses and suggest an alternative therapeutic approach to circumvent PLCγ1-mediated resistance to cetuximab in patients with RAS WT mCRC. In this way, this work contributes to the development of novel strategies in the medical management and treatment of patients with mCRC.

Lifespan and telomere length variation across populations of wild-derived African killifish.

Telomeres and telomerase prevent the continuous erosion of chromosome-ends caused by lifelong cell division. Shortened telomeres are associated with age-related pathologies. While short telomere length is positively correlated with increased lethality at the individual level, in comparisons across species short telomeres are associated with long (and not short) lifespans. Here, we tested this contradiction between individual and evolutionary patterns in telomere length using African annual killifish. We analysed lifespan and telomere length in a set of captive strains derived from well-defined wild populations of Nothobranchius furzeri and its sister species, N. kadleci, from sites along a strong gradient of aridity which ultimately determines maximum natural lifespan. Overall, males were shorter-lived than females, and also had shorter telomeres. Male lifespan (measured in controlled laboratory conditions) was positively associated with the amount of annual rainfall in the site of strain origin. However, fish from wetter climates had shorter telomeres. In addition, individual fish which grew largest over the juvenile period possessed shorter telomeres at the onset of adulthood. This demonstrates that individual condition and environmentally-driven selection indeed modulate the relationship between telomere length and lifespan in opposite directions, validating the existence of inverse trends within a single taxon. Intraindividual heterogeneity of telomere length (capable to detect very short telomeres) was not associated with mean telomere length, suggesting that the shortest telomeres are controlled by regulatory pathways other than those that determine mean telomere length. The substantial variation in telomere length between strains from different environments identifies killifish as a powerful system in understanding the adaptive value of telomere length.

Using Zebrafish Larvae as a Xenotransplantation Model to Study Ewing Sarcoma.

Tumor models allowing for the in vivo investigation of molecular mechanisms driving tumor progression and metastasis are important to develop novel strategies for cancer treatment. Unfortunately, for Ewing sarcoma no adequate genetic animal models are currently available. Mouse xenograft models are the state of the art to model Ewing sarcoma in vivo. Here, we describe an alternative Ewing sarcoma xenograft model in embryonic and larval zebrafish. This xenograft model offers live imaging and easy compound testing opportunities hereby complementing mouse xenograft models. In this chapter, we provide a detailed protocol how to xenograft Ewing sarcoma cells (shSK-E17T) into 2-day-old zebrafish and how xenografted zebrafish can be imaged and analyzed over consecutive days to study tumor proliferation.

Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status.

Poly (ADP-ribose) polymerase (PARP) inhibition in BRCA-mutated cells results in an incapacity to repair DNA damage, leading to cell death caused by synthetic lethality. Within the treatment options for advanced triple negative breast cancer, the PARP inhibitor olaparib is only given to patients with BRCA1/2 mutations. However, these patients may show resistance to this drug and BRCA1/2 wild-type tumors can show a striking sensitivity, making BRCA status a poor biomarker for treatment choice. Aiming to investigate if the zebrafish model can discriminate sensitivities to olaparib, we developed zebrafish xenografts with different BRCA status and measured tumor response to treatment, as well as its impact on angiogenesis and metastasis. When challenged with olaparib, xenografts revealed sensitivity phenotypes independent of BRCA. Moreover, its combination with ionizing radiation increased the cytotoxic effects, showing potential as a combinatorial regimen. In conclusion, we show that the zebrafish xenograft model may be used as a sensitivity profiling platform for olaparib in monotherapy or in combinatorial regimens. Hence, this model presents as a promising option for the future establishment of patient-derived xenografts for personalized medicine approaches beyond BRCA status.

Zebrafish xenografts as a fast screening platform for bevacizumab cancer therapy.

Despite promising preclinical results, average response rates to anti-VEGF therapies, such as bevacizumab, are reduced for most cancers, while incurring in remarkable costs and side effects. Currently, there are no biomarkers available to select patients that can benefit from this therapy. Depending on the individual tumor, anti-VEGF therapies can either block or promote metastasis. In this context, an assay able to predict individual responses prior to treatment, including the impact on metastasis would prove of great value to guide treatment options. Here we show that zebrafish xenografts are able to reveal different responses to bevacizumab in just 4 days, evaluating not only individual tumor responses but also the impact on angiogenesis and micrometastasis. Importantly, we perform proof-of-concept experiments where clinical responses in patients were compared with their matching zebrafish Patient-Derived Xenografts - zAvatars, opening the possibility of using the zebrafish model to screen bevacizumab therapy in a personalized manner.

Telomere shortening produces an inflammatory environment that increases tumor incidence in zebrafish.

Cancer incidence increases exponentially with age when human telomeres are shorter. Similarly, telomerase reverse transcriptase (tert) mutant zebrafish have premature short telomeres and anticipate cancer incidence to younger ages. However, because short telomeres constitute a road block to cell proliferation, telomere shortening is currently viewed as a tumor suppressor mechanism and should protect from cancer. This conundrum is not fully understood. In our current study, we report that telomere shortening promotes cancer in a noncell autonomous manner. Using zebrafish chimeras, we show increased incidence of invasive melanoma when wild-type (WT) tumors are generated in tert mutant zebrafish. Tissues adjacent to melanoma lesions (skin) and distant organs (intestine) in tert mutants exhibited higher levels of senescence and inflammation. In addition, we transferred second generation (G2) tert blastula cells into WT to produce embryo chimeras. Cells with very short telomeres induced increased tumor necrosis factor1-α (TNF1-α) expression and senescence in larval tissues in a noncell autonomous manner, creating an inflammatory environment. Considering that inflammation is protumorigenic, we transplanted melanoma-derived cells into G2 tert zebrafish embryos and observed that tissue environment with short telomeres leads to increased tumor development. To test if inflammation was necessary for this effect, we treated melanoma transplants with nonsteroid anti-inflammatory drugs and show that higher melanoma dissemination can be averted. Thus, apart from the cell autonomous role of short telomeres in contributing to genome instability, we propose that telomere shortening with age causes systemic chronic inflammation leading to increased tumor incidence.

Opposing p53 and mTOR/AKT promote an in vivo switch from apoptosis to senescence upon telomere shortening in zebrafish.

Progressive telomere shortening during lifespan is associated with restriction of cell proliferation, genome instability and aging. Apoptosis and senescence are the two major outcomes upon irreversible cellular damage. Here, we show a transition of these two cell fates during aging of telomerase deficient zebrafish. In young telomerase mutants, proliferative tissues exhibit DNA damage and p53-dependent apoptosis, but no senescence. However, these tissues in older animals display loss of cellularity and senescence becomes predominant. Tissue alterations are accompanied by a pro-proliferative stimulus mediated by AKT signaling. Upon AKT activation, FoxO transcription factors are phosphorylated and translocated out of the nucleus. This results in reduced SOD2 expression causing an increase of ROS and mitochondrial dysfunction. These alterations induce p15/16 growth arrest and senescence. We propose that, upon telomere shortening, early apoptosis leads to cell depletion and insufficient compensatory proliferation. Following tissue damage, the mTOR/AKT is activated causing mitochondrial dysfunction and p15/16-dependent senescence.

Platinum-Triggered Bond-Cleavage of Pentynoyl Amide and N-Propargyl Handles for Drug-Activation.

The ability to create ways to control drug activation at specific tissues while sparing healthy tissues remains a major challenge. The administration of exogenous target-specific triggers offers the potential for traceless release of active drugs on tumor sites from antibody-drug conjugates (ADCs) and caged prodrugs. We have developed a metal-mediated bond-cleavage reaction that uses platinum complexes [K2PtCl4 or Cisplatin (CisPt)] for drug activation. Key to the success of the reaction is a water-promoted activation process that triggers the reactivity of the platinum complexes. Under these conditions, the decaging of pentynoyl tertiary amides and N-propargyls occurs rapidly in aqueous systems. In cells, the protected analogues of cytotoxic drugs 5-fluorouracil (5-FU) and monomethyl auristatin E (MMAE) are partially activated by nontoxic amounts of platinum salts. Additionally, a noninternalizing ADC built with a pentynoyl traceless linker that features a tertiary amide protected MMAE was also decaged in the presence of platinum salts for extracellular drug release in cancer cells. Finally, CisPt-mediated prodrug activation of a propargyl derivative of 5-FU was shown in a colorectal zebrafish xenograft model that led to significant reductions in tumor size. Overall, our results reveal a new metal-based cleavable reaction that expands the application of platinum complexes beyond those in catalysis and cancer therapy.

Developments in zebrafish avatars as radiotherapy sensitivity reporters - towards personalized medicine.

BACKGROUND: Whereas the role of neoadjuvant radiotherapy in rectal cancer is well-established, the ability to discriminate between radioresistant and radiosensitive tumors before starting treatment is still a crucial unmet need. Here we aimed to develop an in vivo test to directly challenge living cancer cells to radiotherapy, using zebrafish xenografts. METHODS: We generated zebrafish xenografts using colorectal cancer cell lines and patient biopsies without in vitro passaging, and developed a fast radiotherapy protocol consisting of a single dose of 25 Gy. As readouts of the impact of radiotherapy we analyzed proliferation, apoptosis, tumor size and DNA damage. FINDINGS: By directly comparing isogenic cells that only differ in the KRASG13D allele, we show that it is possible to distinguish radiosensitive from radioresistant tumors in zebrafish xenografts, even in polyclonal tumors, in just 4 days. Most importantly, we performed proof-of-concept experiments using primary rectum biopsies, where clinical response to neoadjuvant chemoradiotherapy correlates with induction of apoptosis in their matching zebrafish Patient-Derived Xenografts-Avatars. INTERPRETATION: Our work opens the possibility to predict tumor responses to radiotherapy using the zebrafish Avatar model, sparing valuable therapeutic time and unnecessary toxicity.

Pericentrin-mediated SAS-6 recruitment promotes centriole assembly.

The centrosome is composed of two centrioles surrounded by a microtubule-nucleating pericentriolar material (PCM). Although centrioles are known to regulate PCM assembly, it is less known whether and how the PCM contributes to centriole assembly. Here we investigate the interaction between centriole components and the PCM by taking advantage of fission yeast, which has a centriole-free, PCM-containing centrosome, the SPB. Surprisingly, we observed that several ectopically-expressed animal centriole components such as SAS-6 are recruited to the SPB. We revealed that a conserved PCM component, Pcp1/pericentrin, interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly its elongation. We further explored how yeasts kept this interaction even after centriole loss and showed that the conserved calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction. Our work suggests that the PCM not only recruits and concentrates microtubule-nucleators, but also the centriole assembly machinery, promoting biogenesis close by.

Ssu72 phosphatase is a conserved telomere replication terminator.

Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3'-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication.

FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence.

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.

The Zebrafish as an Emerging Model to Study DNA Damage in Aging, Cancer and Other Diseases.

Cancer is a disease of the elderly, and old age is its largest risk factor. With age, DNA damage accumulates continuously, increasing the chance of malignant transformation. The zebrafish has emerged as an important vertebrate model to study these processes. Key mechanisms such as DNA damage responses and cellular senescence can be studied in zebrafish throughout its life course. In addition, the zebrafish is becoming an important resource to study telomere biology in aging, regeneration and cancer. Here we review some of the tools and resources that zebrafish researchers have developed and discuss their potential use in the study of DNA damage, cancer and aging related diseases.