Short Communication

Oncogene (2009) 28, 1162–1167; doi:10.1038/onc.2008.458; published online 12 January 2009

FEN1 contributes to telomere stability in ALT-positive tumor cells

A Saharia1 and S A Stewart1,2

  1. 1Department of Cell Biology and Physiology, Washington University School of Medicine, Saint Louis, MO, USA
  2. 2Department of Medicine, Washington University School of Medicine, Saint Louis, MO, USA

Correspondence: Dr SA Stewart, Department of Cell Biology and Physiology and Medicine, Washington University School of Medicine, Campus Box 8228, 660 South Euclid Avenue, St Louis, MO 63110, USA. E-mail: sheila.stewart@cellbiology.wustl.edu

Received 23 June 2008; Revised 16 October 2008; Accepted 7 November 2008; Published online 12 January 2009.

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Abstract

Abrogation of telomere stability through loss-of-function mutations in telomere binding proteins contributes to genomic instability and cancer progression. Recently, Flap endonuclease 1 (FEN1) was shown to contribute to telomere stability in human cells that had not yet activated a telomere maintenance mechanism, suggesting that abrogation of FEN1 function influences the transformation process by compromising telomere stability and driving genomic instability. Here, we analyse the telomeres in human cancer cells following FEN1 depletion. We show that FEN1 is required for telomere stability in cells that rely on the alternative lengthening of telomere (ALT) mechanism. Indeed, FEN1 depletion resulted in telomere dysfunction, characterized by formation of telomere dysfunction-induced foci (TIFs) and end-to-end fusions in ALT-positive cells. In contrast, no telomere phenotype was observed in telomerase-positive cells on FEN1 depletion, suggesting that ongoing telomerase activity protected telomeres. In consonance with this, we found that expression of the catalytic component of telomerase (hTERT) but not an inactive allele rescued telomere dysfunction on FEN1 depletion in ALT cells. Our data suggest that mutations that arise in FEN1 affect telomere stability and genome fidelity by promoting telomere fusions and anaphase–bridge–breakage cycles, which further drive genome instability and thereby contribute to the transformation process.

Keywords:

telomere, FEN1, genomic stability, ALT

Loss-of-function mutations in genes involved in detection, signaling and repair of DNA damage correlate with increased genomic instability and cancer incidence. Proper maintenance of telomere function is critical to genomic stability. As a functional DNA–protein complex, the telomere distinguishes the end of a chromosome from a bona fide double-strand break. Destabilization of telomere structure compromises its function and renders it susceptible to the actions of the DNA repair machinery, often leading to chromosome end-to-end fusions (de Lange, 2005). Telomeric fusions result in anaphase–bridge–breakage cycles, which contribute to genomic instability and drives the transformation process (Artandi and DePinho, 2000).

The telomere consists of repetitive double- and single-stranded DNA (TTAGGG) and six core proteins, referred to as Shelterin (or Telosome) (Liu et al., 2004; de Lange, 2005), which together shield the telomere from the DNA repair machinery. In addition to the Shelterin components, a growing list of accessory proteins localize to the telomere and play essential roles in telomere maintenance (Blasco, 2005). For example, ATM, WRN and Ku influence telomere stability where mutation and/or depletion of these proteins result in cancer syndromes (Blasco, 2005). Together, these data underscore the importance of these DNA replication and repair proteins in telomere maintenance and high-fidelity maintenance of the genome.

Recently, we showed that Flap endonuclease 1 (FEN1) is a telomere-binding protein that plays an important role in maintaining telomere stability in human cells (Saharia et al., 2008). RNA interference-directed depletion of FEN1 led to sister telomere loss, which was restricted to telomeres replicated by lagging strand DNA synthesis (Saharia et al., 2008). Flap endonuclease 1 is a multifunctional nuclease that participates in replication (Li et al., 1995), long-patch base excision repair (Prasad et al., 2000), homologous recombination (Kikuchi et al., 2005), re-initiation of stalled replication forks and DNA degradation in apoptotic cells (Zheng et al., 2005, 2007). Work in yeast showed that disruption of the FEN1 homolog, Rad27, results in a DNA mutator phenotype and telomere dysfunction (Tishkoff et al., 1997; Parenteau and Wellinger, 1999, 2002). Similarly, mice heterozygotic for FEN1 show a mutator phenotype and are predisposed to develop neoplasias (Kucherlapati et al., 2007). Given that the initiation and development of cancer results in part from accumulation of genetic instability, and that telomere dysfunction can contribute to this instability, abrogation and/or mutation of genes such as FEN1 may contribute to this process. Indeed, such a role for FEN1 was suggested by a recent report that showed that knock-in of a FEN1 mutant gene identified in human cancers resulted in cancer predisposition in a murine model (Zheng et al., 2007). Specifically, when a FEN1 mutant that abrogates a repair function known as the gap endonuclease activity was knocked into the analogous murine locus, animals developed several pathologies, including lung tumors (Zheng et al., 2007). This observation, together with our earlier findings, raises the possibility that FEN1 depletion (and/or mutation) affects genomic stability by abrogating telomere stability, and in this way contributes to the transformation process.

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Flap endonuclease 1 is required for telomere stability in alternative lengthening of telomere cells

In earlier work we found that FEN1 depletion in somatic cells that have not activated a telomere maintenance mechanism led to telomere dysfunction, which was compensated for by ectopic expression of the catalytic component of telomerase (hTERT) (Saharia et al., 2008). This observation raised the possibility that FEN1 depletion or mutation might affect telomere stability in transformed cells that utilized the alternative lengthening of telomere (ALT) mechanism of telomere maintenance. Thus, we investigated whether FEN1 depletion affected telomere stability in a human osteosarcoma cell line (U2OS) that is telomerase negative and maintains its telomeres through the recombination-dependent ALT mechanism (Bryan et al., 1997). To control for possible off-target effects associated with RNA interference, we utilized two independent lentiviral constructs expressing short hairpin RNAs targeted to FEN1's coding region and 3′ untranslated region (shFEN and shFEN3, respectively). In addition, a short hairpin consisting of a scrambled sequence (shSCR) was also introduced into these cells and functioned as a negative control.

Following transduction, FEN1 protein expression was determined by western blot analysis. Expression of the two hairpins (shFEN and shFEN3) led to a significant reduction in FEN1 protein levels (Figure 1a). To determine the effect of FEN1 depletion on telomere stability, metaphase spreads were prepared and analysed for telomere dysfunction. Metaphases were labeled using fluorescent in situ hybridization (FISH) with telomere (red) and centromere (green) probes and analysed (Figure 1b). Analysis of metaphase spreads showed that FEN1 depletion led to telomere dysfunction characterized by chromosomal end-to-end fusions that retained telomeric sequences at the fusion points (Figure 1b). U2OS cells expressing shSCR displayed 0.1 telomere fusion events per cell. In contrast, FEN1 depletion resulted in a significant increase in the number of telomere fusions observed in U2OS cells to 0.33 (P<0.001) and 0.38 (P<0.01) events in the shFEN- and shFEN3-expressing cells, respectively (Figure 1c). Moreover, the percentage of cells having one or more fusion events increased from 6.7% in the shSCR-expressing cells to 27 and 33%, respectively, in the shFEN- and shFEN3-expressing cells.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Flap endonuclease 1 (FEN1) depletion leads to telomere dysfunction in alternative lengthening of telomere (ALT) cells. (a) Two independent shRNAs targeting FEN1 (shFEN and shFEN3) and one consisting of a scrambled sequence (shSCR) were introduced by lentiviral infection into GM847 and U2OS cells and FEN1 expression was determined by Western blot analysis. (b) Representative metaphases from GM847 and U2OS cells following indicated shRNA expression. FISH analysis was conducted using Cy3-labeled TTAGGG probes (in red) and FITC-labeled centromere probes (in green). DNA was stained using 4′,6-diamidino-2-phenylindole (DAPI; in blue). The lower panel shows a higher magnification image of the metaphase chromosomes. (c) Quantification of telomere fusion events observed after indicated treatments of GM847 (purple bars) and U2OS (white bars) cells. A minimum of 60 metaphases were analysed per treatment in a blinded fashion. Statistical analysis was conducted using the Wilcoxon two-sample test (*P<0.001; #P<0.01). (d) FEN1 depletion increases telomere dysfunction-induced foci (TIF) formation in GM847 cells. Immunofluorescence was conducted using anti-TRF2 (green: Santa Cruz, CA, USA; H-300), anti-γH2AX (red: Upstate, NY, USA; 05-636) and DAPI (blue). Confocal images were acquired on a Zeiss Axiovert 200 microscope. (e) TIF quantification in GM847 cells. A minimum of 100 cells were counted for each condition and the average for two experiments is presented. Cell culture, western blot analysis, viral constructs and production as well as metaphase preparation and statistical analyses were as described earlier (Stewart et al., 2002, 2003; Saharia et al., 2008).

Full figure and legend (235K)

The telomeric impact of FEN1 depletion was not unique to U2OS cells. Indeed, depletion of FEN1 in a second ALT cell line, GM847 (Figure 1a), also resulted in a significant increase in telomere dysfunction (Figures 1b–e). GM847 cells infected with a control virus displayed 0.07 telomeric fusions. In contrast, expression of shFEN and shFEN3 led to 0.8 and 0.57 telomeric fusions, respectively, with 50–60% of the metaphases analysed showing one or more telomeric fusion (P<0.0001; Figure 1c).

Several groups have shown that DNA damage foci, referred to as telomere dysfunction-induced foci (TIFs), are readily detectable when telomere stability is compromised (Takai et al., 2003; d’Adda di Fagagna et al., 2003). Therefore, to confirm the presence of telomere dysfunction on FEN1 depletion, we examined cells for the presence of γH2AX foci at telomeres. As expected, we found that FEN1 depletion led to an increase in TIFs (Figures 1d and e). In GM847 cells infected with a control hairpin, we noted that 27.9% of the cells hadgreater than or equal to5 TIFs per cell, whereas upon infection with the shFEN 3 expressing virsus, the number of cells with greater than or equal to5 TIFs increased to 78.2% (Figure 1e). Together, these data show that FEN1 contributes to telomere stability in immortal cells, and that its depletion leads to telomere dysfunction in cells that maintain their telomeres through the ALT mechanism.

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Flap endonuclease 1 depletion in telomerase-positive cells does not impact telomere stability

Human cancer cells maintain stable telomere lengths through activation of either ALT or the telomerase enzyme. We showed above that FEN1 depletion leads to telomeric fusions in ALT cells. In addition, in a previous report we showed that FEN1 depletion in mortal human fibroblasts led to sister telomere loss, which were rescued by expression of catalytically active telomerase. Together, these data argue that tumor cells that have activated telomerase would be insensitive to FEN1 depletion. To test this hypothesis directly, we examined how FEN1 depletion affected telomere stability in telomerase-positive cells. HeLa cells, a cervical cancer cell line that utilizes telomerase for telomere maintenance, were transduced with viral vectors expressing shSCR, shFEN or shFEN3. Upon FEN1 depletion (Figure 2a), cells were analysed for telomeric fusions as described above. As expected, depletion of FEN1 did not result in telomeric fusions (Figures 2b and c). Similar results were observed in a second telomerase-positive ovarian cancer cell line, 36M (Figure 2). These results indicate that cells that utilize endogenous telomerase for telomere maintenance are insensitive to FEN1 depletion at the telomere.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Flap endonuclease 1 (FEN1) is not essential for telomere stability in telomerase-positive cells. (a) Western blot analysis shows that introduction of two different short hairpin RNAs (shRNAs) targeted to FEN1 leads to reduction in FEN1 protein levels in HeLa and 36M cells. (b) Representative metaphases from HeLa and 36M cells following shRNA expression. (c) Quantification of telomere fusion events observed following shRNA expression in HeLa (grey bars) and 36M (white bars) cells. A minimum of 60 metaphases were analysed per treatment in a blinded fashion. Statistical analysis was conducted using the Wilcoxon two-sample test.

Full figure and legend (146K)

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Catalytically active telomerase rescues Flap endonuclease 1 depletion at the telomeres

Depletion of FEN1 in cells that maintain stable telomeres through the ALT mechanism resulted in telomere dysfunction. In contrast, telomere stability was unperturbed in telomerase-positive cells after FEN1 depletion. These results were reminiscent of our earlier findings that telomerase rescued sister telomere loss in cell lines that had not yet activated a telomere maintenance program (Saharia et al., 2008). To determine whether the catalytic activity of telomerase was required to protect telomeres in cells that utilized the ALT mechanism, we expressed the catalytic component of telomerase (hTERT) in GM847 cells (GM847-hTERT) (Figure 3a) (Hahn et al., 1999). Expression of hTERT reconstitutes telomerase activity in these cells, leading to lengthening of the shortest telomeres (Grobelny et al., 2001; Hemann et al., 2001; Perrem et al., 2001; Teixeira et al., 2004).

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Catalytically active telomerase rescues telomere instability on Flap endonuclease 1 (FEN1) depletion. (a) Reverse transcriptase (RT) PCR showing exogenous expression of the catalytic component of telomerase (hTERT), dominant-negative hTERT (DN-hTERT) or uninfected (CTRL). RNA isolation, PCR and primers were as described earlier (Hahn et al., 1999). (b) Western blot analysis shows that introduction of two different shRNAs targeted to FEN1 leads to reduction in FEN1 protein levels in hTERT and DN-hTERT cells. (c) Quantification of telomere fusion events following shRNA expression in hTERT (grey bars) and DN-hTERT (white bars) cells. A minimum of 60 metaphases were analysed per treatment in a blinded fashion. Statistical analysis was conducted using the Wilcoxon two-sample test (*P<0.01).

Full figure and legend (86K)

Introduction of short hairpin RNA constructs targeting FEN1 into GM847-hTERT cells resulted in a significant reduction in protein expression (Figure 3b). Analysis of metaphase spreads from cells expressing the FEN1 hairpins compared with those expressing a control hairpin did not show an increase in telomere dysfunction (Figure 3c). To determine whether it was the telomere extension activity of telomerase that compensated for FEN1 depletion at the telomeres as suggested by our earlier work, we utilized a catalytically inactive, dominant-negative allele of hTERT (DN-hTERT) (Figure 3a). This allele was chosen because earlier work showed that it had no impact on telomere stability in GM847 cells (Stewart et al., 2002). In contrast to that observed in GM847-hTERT cells, FEN1 depletion in GM847-DN-hTERT cells resulted in increased telomeric fusions (Figures 3b and c). Flap endonuclease 1 depletion increased the number of telomere fusion events per cell from 0.15 events in control cells, to 0.55 and 0.53 in cells expressing the two hairpins against FEN1 (P<0.01; Figure 3c). There was also a large increase in the percentage of metaphases possessing one or more fusions (46% versus 13% in the control cells). The inability of DN-hTERT to rescue FEN1 depletion at the telomere indicates that the catalytic activity of telomerase is important for this rescue, and suggests that telomeric extension by telomerase is important in the absence of FEN1.

Flap endonuclease 1 is a structure-specific endonuclease that acts in DNA replication and repair. Here, we assessed the role of FEN1 in the context of telomere stability. We found that depletion of FEN1 in cancer cells that maintain their telomeres through the ALT mechanism results in telomere dysfunction characterized by increases in the number of TIFs and telomeric fusions. In contrast, FEN1 depletion did not lead to telomere dysfunction in telomerase-positive cells. Telomere fusions observed in ALT cells were rescued by the expression of catalytically active telomerase, but not a catalytically dead enzyme. Given our earlier work showing that single telomeres were lost in pre-crisis human cells upon FEN1 depletion (Saharia et al., 2008), this result suggests that the ability of telomerase to elongate the shortest telomeres was protective. Together, these results suggest that abrogation of FEN1 function in telomerase-negative tumor cells results in increased genomic instability by compromising telomere stability that may contribute to tumor progression.

DNA replication is a challenging cellular event that is prone to errors that can result in loss of genomic fidelity. Sequences that offer significant challenges to the DNA replication machinery are repetitive DNA sequences, particularly those containing triplicate repeats (Fouche et al., 2006), which hinder replication fork progression. Therefore, it is not surprising that the repetitive G-rich nature of the telomere presents a challenging template for the replication machinery (Gilson and Geli, 2007). Indeed, this has been underscored by in vitro DNA replication systems that have shown that replication of telomeric sequences is less efficient than randomized sequences due to a significant increase in stalled DNA replication forks within telomeric sequences (Ohki and Ishikawa, 2004). For these reasons, the impact of loss-of-function mutations in genes that facilitate replication fork progression and restart would be expected to have a profound impact at the telomere. Indeed, this has been observed with the Werner protein (Crabbe et al., 2004) as well as FEN1 (this study). Flap endonuclease 1 functions with the Werner protein to process branch-migrating structures that resemble stalled replication forks (Sharma et al., 2004). Therefore, given that unresolved stalled replication forks lead to DNA double-strand breaks (Branzei and Foiani, 2005), loss of Werner or FEN1 activity would be expected to result in telomere loss and subsequent end-to-end fusions.

Our earlier work showed that FEN1 depletion led to sister telomere loss, but no significant telomeric fusions were observed (Saharia et al., 2008). Why then do we observe telomeric fusions in cells that utilize the ALT mechanism? Telomeres within ALT cells are in a constant state of flux, where they undergo rapid elongation and shortening (Londono-Vallejo et al., 2004). This dynamic fluctuation results in chromosome ends with extremely short telomeres that are unlikely to adequately protect telomere ends from recognition by DNA damage surveillance mechanisms. As a result, telomeres within ALT cells are recognized as DNA damage, as evidenced by the presence of TIFs or γH2AX foci at many telomeres (Figures 1d and e, shSCR) (Nabetani et al., 2004). Depletion of FEN1 appears to exacerbate telomere dysfunction by producing signal-free ends in fibroblasts (Saharia et al., 2008) and increasing the number of TIFs in ALT cells (Figures 1d and e), thus leading to additional substrates capable of participating in end-to-end fusions. As telomerase acts on the shortest telomeres (Marcand et al., 1999; Forstemann et al., 2000; Ouellette et al., 2000), it would be recruited to those chromosome ends that experienced a catastrophic loss because of a stalled and unresolved replication fork or failure to cap the telomere. Telomerase could then extend the short telomeres, stabilizing them and rescuing telomere dysfunction. These studies suggest that FEN1 mutation contributes to the transformation process by increasing genomic instability through telomere loss and subsequent end-to-end fusions. Further, abrogation of FEN1 function in tumor cells that do not utilize telomerase may result in additional genomic instability leading to progression of the neoplastic state.

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Acknowledgements

This work was supported by the Sidney Kimmel Foundation for Cancer Research and the Edward Mallinckrodt, Jr Foundation. AS was supported by the Lucille P Markey Program. SAS is a Sidney Kimmel Scholar. We are grateful to Yu Tao for help with statistical analyses and members of the Stewart Laboratory for valuable discussions.

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