The BRCA1 and BRCA2 proteins are involved in the maintenance of genome stability and germ-line loss-of-function mutations in either BRCA1 or BRCA2 strongly predispose carriers to cancers of the breast and other organs. It has been demonstrated previously that inhibiting elements of the cellular DNA maintenance pathways represents a novel therapeutic approach to treating tumors in these individuals. Here, we show that inhibition of the telomere-associated protein, Tankyrase 1, is also selectively lethal with BRCA deficiency. We also demonstrate that the selectivity caused by inhibition of Tankyrase 1 is associated with an exacerbation of the centrosome amplification phenotype associated with BRCA deficiency. We propose that inhibition of Tankyrase 1 could be therapeutically exploited in BRCA-associated cancers.
Germ-line mutations in either BRCA1 or BRCA2 strongly predispose individuals to cancers of the breast and also to malignancies of the ovaries, pancreas and prostate gland. Tumors arising in women carrying a single germ-line mutant BRCA allele exhibit loss of heterozygosity at the BRCA locus, losing the wild-type allele and retaining the mutant copy of the gene, suggesting that BRCA1 and BRCA2 act as tumor suppressors (Tutt et al., 2005, and references therein). BRCA1- or BRCA2-deficient cells have defects in the repair of DNA double-strand breaks (DSBs) by the conservative, error-free pathway of homologous recombination (HR), leading to cellular sensitivity to specific DNA-damaging agents (Tutt et al., 2005). This has been previously exploited in the design of novel therapies to treat BRCA-associated cancers, including the demonstration that inhibition of the DNA repair enzyme poly(ADP)-ribose polymerase (PARP1) is particularly selective for BRCA-deficient cells (Bryant et al., 2005; Farmer et al., 2005). The profound sensitivity of BRCA-deficient cells to PARP inhibition exemplifies the concept of synthetic lethality. Two genes or proteins exhibit a synthetic lethal interaction when loss of either gene is not overtly deleterious but loss of both is lethal (Kaelin, 2005). In scenarios such as BRCA-associated cancer, where recapitulation of a wild-type tumor suppressor is impractical, synthetic lethality presents an attractive approach to the identification of therapeutic targets (Iorns et al., 2007). Despite the potential of PARP inhibition in BRCA-associated cancers, it is possible that some patients will either be inherently refractory to this approach or develop drug resistance. Therefore, it is vital to identify additional therapeutic targets for BRCA-associated cancers.
Our previous observations on the efficacy of PARP inhibition led us to an assessment of the other members of the PARP superfamily, which includes at least 17 members divided into three subfamilies based on their enzymatic characteristics (Ame et al., 2004; Kleine et al., 2008). The first subfamily includes members with PARP activity (PARP1–5), the second includes proteins with mono(ADP)ribosyltransferase activity (PARP6, 8, 10–12 and 14–16) and the third constitutes catalytically inactive members of the superfamily (PARP9, 13) (Kleine et al., 2008). In particular, we have focused on one member, Tankyrase 1 (PARP5a). Tankyrase 1 was identified by its ability to bind TRF1, a positive regulator of telomere length (Smith et al., 1998; Seimiya et al., 2004). Tankyrase 1 binding to TRF1 results in poly(ADP-ribosyl)ation of TRF1 and leads to TRF1 release from the telomeres and its proteolytic degradation (Chang et al., 2003). Consistent with these observations, overexpression of Tankyrase 1 results in telomere elongation (Seimiya et al., 2004). The interaction between Tankyrase 1 and TRF1 requires the C-terminal region of Tankyrase 1 (Seimiya et al., 2004). This region contains a domain of 24 ankyrin (ANK) repeats, which are organized in five highly conserved subdomains (ARC1-V, or ANK repeat clusters) (Seimiya and Smith, 2002; Seimiya et al., 2004). Although TRF1 binds to all five ARC subdomains, only the ARCV domain is required for its poly(ADP-ribosyl)ation and release from the telomeres (Seimiya et al., 2004). In agreement with an involvement of Tankyrase 1 in telomere maintenance, a recent report showed that inhibition of Tankyrase 1 accentuates the ability of a telomerase inhibitor, MST-312 to induce telomere shortening (Seimiya et al., 2005), indicating that Tankyrase 1 may be a potential therapeutic target (Seimiya, 2006). Besides its telomere function, Tankyrase 1 has also been localized to the Golgi and to the mitotic spindle poles (Smith and de Lange, 1999; Chi and Lodish, 2000). A reduction in Tankyrase 1 expression has also been shown to cause cells to accumulate in M phase (Dynek and Smith, 2004) and have abnormal spindle structures (Chang et al., 2005), suggesting involvement of Tankyrase 1 in spindle structure and function.
It has previously been demonstrated that targeting DNA repair pathways in BRCA-deficient cells can illuminate synthetic lethal interactions and lead to the development of novel therapeutic approaches (Farmer et al., 2005). Therefore, we investigated the possibility of synthetic lethal interactions between Tankyrase 1 and BRCA1 or BRCA2. We demonstrate that targeting Tankyrase 1 is selectively lethal in the context of BRCA deficiency.
Results and discussion
To assess the possibility that targeting Tankyrase 1 may have utility in the treatment of tumors associated with loss of BRCA1 or BRCA2 function, we developed a human colon cancer cell line, HCT116, with stable reduction of Tankyrase 1 expression (Figure 1a). This was achieved by infection with a retroviral vector expressing either a short-hairpin RNA (shRNA) targeting Tankyrase 1 (catalogue no. RHS1764-9688056) or a control nontargeting shRNA (catalogue no. RHS1703; Open Biosystems, Huntsville, AL, USA). Tankyrase-silenced cells (HCT116-Tankyrase 1 shRNA) and control cells (HCT116-Control shRNA) were subsequently transfected with pSUPER shRNA constructs targeting either BRCA1 or BRCA2 (Figure 1b; Tutt et al., 2005; McCabe et al., 2006) and clonogenic survival assays were performed. Our results showed that inhibition of Tankyrase 1 was selectively lethal in cells with a reduction in either BRCA1 or BRCA2 expression, but had no effect in control cells (Figure 1c).
To validate these observations, we used HTC75 human fibrosarcoma cell lines expressing three truncated forms of Tankyrase 1 (ARCV, ARC1 and ΔANK). We silenced BRCA1 or BRCA2 expression in these cells and compared their survival to cell lines that stably express full-length Tankyrase 1 (TANK-1) or the empty vector (MOCK) (Figure 2a; Seimiya et al., 2004). Loss of the ARCV domain of Tankyrase 1 has previously been shown to reduce significantly the ability of Tankyrase 1 to poly(ADP-ribosyl)ate TRF1 and release TRF1 from telomeres, thereby preventing telomere elongation (Seimiya and Smith, 2002; Seimiya et al., 2004). Silencing of BRCA1 or BRCA2 significantly increased lethality in the ARCV-expressing cells compared to the isogenic control cell lines MOCK and TANK-1 (Figure 2b). Interestingly, cells stably expressing other truncated forms of Tankyrase 1, ARC1 and ΔANK, did not show any selective lethality when transfected with pSUPER-BRCA1 or pSUPER-BRCA2 compared to the control cells. As the ARC1 and ΔANK cell lines do not show the same TRF1 telomeric release defects present in the ARCV cell line (Seimiya et al., 2004), this result suggests that the mechanism of lethality might be dependent on modifying TRF1 function. TRF1 is a member of the shelterin complex and functions predominantly at the telomeres (de Lange, 2005). Therefore, we analysed whether the synthetic lethality between Tankyrase 1 and BRCA was due to a cumulative negative impact on telomere maintenance. Loss of functional telomeres results in the appearance of DNA damage foci (known as TIFs or telomere-dysfunction-induced foci) and eventually end-to-end fusions (d'Adda di Fagagna et al., 2003). We monitored the presence of TIFs by immunofluorescence and the frequency of chromosome fusions by fluorescent in situ hybridization in ARCV and MOCK cells transfected with either BRCA-silencing plasmids or the control plasmid. Our results showed that the combined loss of function of Tankyrase 1 and BRCA did not cooperate in inducing telomere dysfunction (data not shown), indicating that the main mechanism of synthetic lethality between Tankyrase 1 and BRCA is unlikely to be dependent on their effects at the telomeres.
BRCA mutations are predominantly found in breast and ovarian cancers. To validate our findings we used two human tumor cell lines with known BRCA1 mutations and loss of function, the breast cancer cell line HCC1937 and the ovarian cancer cell line UWB1.289 (DelloRusso et al., 2007). We silenced Tankyrase 1 expression in these cells by retroviral infection with a vector expressing either a nontargeting shRNA (sh-ctrl) or two shRNA targeting Tankyrase (sh-TNK5 and sh-TNK6; see Supplementary Figure 1S) and performed clonogenic survival assays in the presence of puromycin selection. As shown in Figure 3, inhibition of Tankyrase expression in both BRCA1 mutant lines resulted in loss of viability, confirming our previous results.
To further validate this effect, we transfected the breast cancer cell line, CAL51, with combinations of BRCA1 and Tankyrase siRNA (Supplementary Figure 2S) and performed clonogenic survival assays. As shown in Supplementary Figure 2S, knockdown of both BRCA1 and Tankyrase resulted in reduction of cell viability compared to control cells as well as cells transfected with either siRNA alone.
Besides its telomere function, Tankyrase 1 has also been localized to the mitotic spindle poles (Smith and de Lange, 1999). BRCA1- and BRCA2-deficient cells have also been previously shown to display centrosome amplification and furthermore BRCA1 has been shown to interact with the centrosome in M-phase cells (Tutt et al., 1999; Xu et al., 1999). Therefore, we investigated whether the synthetic lethality between BRCA and Tankyrase 1 was because of the exacerbation of known mitotic phenotypes associated with BRCA deficiency. HTC75 cells expressing the truncated forms of Tankyrase 1 and the isogenic control cell lines, MOCK and TANK-1, were transfected with BRCA1- or BRCA2-silencing plasmids and the number of centrosomes analysed by immunofluorescence. We observed a significant increase in the number of cells with centrosome amplification after silencing of BRCA1 or BRCA2 alone compared to controls, consistent with previous studies (Figures 4a and b; Tutt et al., 1999; Xu et al., 1999). However, this phenotype was considerably exacerbated in cells with silenced BRCA1 or BRCA2 in the presence of the ARCV Tankyrase 1 mutant (Figures 4a and b). This difference was statistically significant (P<0.05) when compared to control cells.
A similar phenotype was also observed with HCT116 derivatives. A higher percentage of HTC116-Tankyrase 1 shRNA cells displayed centrosome amplification when transfected with pSUPER-BRCA1 or pSUPER-BRCA2 compared to cells transfected with the control plasmid (Figures 4a and c). These results suggest that the increased lethality associated with combined loss of BRCA1 or BRCA2 and Tankyrase 1 is characterized by excessive centrosome amplification. As multiple centrosomes are known to result in severe chromosomal missegregation at cell division (Nigg, 2002), we hypothesize that loss of Tankyrase 1 function in combination with BRCA deficiency leads to excessive centrosome amplification and high levels of chromosomal missegregation that is incompatible with cell viability. Although the mechanisms that control centrosome amplification are unclear, it has previously been proposed that genetic instability and centrosome amplification aberrations enhance each other (Nigg, 2002). Given the well-documented roles of BRCA1 and BRCA2 in the maintenance of genome stability (Tutt et al., 2005), it is likely that BRCA deficiency contributes to centrosome amplification by this route. Tankyrase 1 dysfunction had not previously been directly associated with centrosome amplification. However, Tankyrase-1-silenced cells have been demonstrated to accumulate in M phase (Dynek and Smith, 2004) and have abnormal spindle structures (Chang et al., 2005). Therefore, it is possible that a combination of genomic instability (caused by BRCA deficiency) and spindle dysfunction (caused by Tankyrase 1 deficiency) causes centrosome amplification and, ultimately, cell death. Regardless of the exact mechanism, the demonstration of synthetic lethality suggests that a therapeutic approach that is based on interfering with Tankyrase 1 function in patients with BRCA-associated tumors is worthy of further investigation. Given that specific, high potency, small-molecule inhibitors of PARP1 and PARP2 proteins have already been developed, it seems likely that similar inhibitors could be developed for Tankyrase 1. Furthermore, it is conceivable that such therapeutics might be exploited to treat not only BRCA-associated cancers but also sporadic cancers displaying properties of ‘BRCAness’ or those with deficiencies in the HR pathway (Turner et al., 2004).
Ame JC, Spenlehauer C, de Murcia G . (2004). The PARP superfamily. Bioessays 26: 882–893.
Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D, Lopez E et al. (2005). Specific killing of BRCA2-deficient tumours with inhibitors of poly(ADP-ribose) polymerase. Nature 434: 913–917.
Chang W, Dynek JN, Smith S . (2003). TRF1 is degraded by ubiquitin-mediated proteolysis after release from telomeres. Genes Dev 17: 1328–1333.
Chang W, Dynek JN, Smith S . (2005). NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis. Biochem J 391: 177–184.
Chi NW, Lodish HF . (2000). Tankyrase is a Golgi-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J Biol Chem 275: 38437–38444.
d'Adda di Fagagna F, Reaper PM, Clay-Farrace L, Fiegler H, Carr P, Von Zglinicki T et al. (2003). A DNA damage checkpoint response in telomere-initiated senescence. Nature 426: 194–198.
de Lange T . (2005). Shelterin: the protein complex that shapes and safeguards human telomeres. Genes Dev 19: 2100–2110.
DelloRusso C, Welcsh PL, Wang W, Garcia RL, King MC, Swisher EM . (2007). Functional characterization of a novel BRCA1-null ovarian cancer cell line in response to ionizing radiation. Mol Cancer Res 5: 35–45.
Dynek JN, Smith S . (2004). Resolution of sister telomere association is required for progression through mitosis. Science 304: 97–100.
Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB et al. (2005). Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434: 917–921.
Iorns E, Lord CJ, Turner N, Ashworth A . (2007). Utilizing RNA interference to enhance cancer drug discovery. Nat Rev Drug Discov 6: 556–568.
Kaelin Jr WG . (2005). The concept of synthetic lethality in the context of anticancer therapy. Nat Rev Cancer 5: 689–698.
Kleine H, Poreba E, Lesniewicz K, Hassa PO, Hottiger MO, Litchfield DW et al. (2008). Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation. Mol Cell 32: 57–69.
McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S et al. (2006). Deficiency in the repair of DNA damage by homologous recombination and sensitivity to poly(ADP-ribose) polymerase inhibition. Cancer Res 66: 8109–8115.
Nigg EA . (2002). Centrosome aberrations: cause or consequence of cancer progression? Nat Rev Cancer 2: 815–825.
Seimiya H . (2006). The telomeric PARP, tankyrases, as targets for cancer therapy. Br J Cancer 94: 341–345.
Seimiya H, Muramatsu Y, Ohishi T, Tsuruo T . (2005). Tankyrase 1 as a target for telomere-directed molecular cancer therapeutics. Cancer Cell 7: 25–37.
Seimiya H, Muramatsu Y, Smith S, Tsuruo T . (2004). Functional subdomain in the ankyrin domain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation. Mol Cell Biol 24: 1944–1955.
Seimiya H, Smith S . (2002). The telomeric poly(ADP-ribose) polymerase, tankyrase 1, contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor, 182-kDa tankyrase-binding protein (TAB182). J Biol Chem 277: 14116–14126.
Silva JM, Li MZ, Chang K, Ge W, Golding MC, Rickles RJ et al. (2005). Second-generation shRNA libraries covering the mouse and human genomes. Nat Genet 37: 1281–1288.
Smith S, de Lange T . (1999). Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes. J Cell Sci 112 (Part 21): 3649–3656.
Smith S, Giriat I, Schmitt A, de Lange T . (1998). Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282: 1484–1487.
Turner N, Tutt A, Ashworth A . (2004). Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer 4: 814–819.
Tutt A, Gabriel A, Bertwistle D, Connor F, Paterson H, Peacock J et al. (1999). Absence of Brca2 causes genome instability by chromosome breakage and loss associated with centrosome amplification. Curr Biol 9: 1107–1110.
Tutt AN, Lord CJ, McCabe N, Farmer H, Turner N, Martin NM et al. (2005). Exploiting the DNA repair defect in BRCA mutant cells in the design of new therapeutic strategies for cancer. Cold Spring Harb Symp Quant Biol 70: 139–148.
Xu X, Weaver Z, Linke SP, Li C, Gotay J, Wang XW et al. (1999). Centrosome amplification and a defective G2–M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol Cell 3: 389–395.
This work was funded by Breakthrough Breast Cancer and Cancer Research UK. We thank Jill Williamson and Dave Robertson for assistance with karyotyping and microscopy, respectively.
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McCabe, N., Cerone, M., Ohishi, T. et al. Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer. Oncogene 28, 1465–1470 (2009). https://doi.org/10.1038/onc.2008.483
- Tankyrase 1
- synthetic lethality
- anticancer therapy
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