Commentaries PinX1 the tail on the F. Brad Johnson

Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

The PinX1 protein inhibits , an enzyme that lengthens — promoting factors that may abet cancers the structures that protect the ends of . Loss of PinX1 leads to derived from neighboring cells (7). Thus, increased length along with defects in chromosome dynamics. In telomere shortening can have both positive this issue of the JCI, Zhou et al. present novel evidence from human tumors and negative effects on carcinogenesis. and mouse models indicating that PinX1 is a clinically significant tumor A cytogenetic characteristic of epithelial suppressor. Importantly, the genome-destabilizing effects of PinX1 loss cancers (i.e., ) is frequent and appear to depend on telomerase activity, raising new models and questions complex nonreciprocal translocations. for how telomeres and telomerase contribute to the development of cancer. This contrasts with the comparatively stable genomes of nonepithelial cancers. Telomeres and telomerase ing proteins TRF1 and TRF2 (1). Although the basis of cytogenetic instabil- Telomeres cap the ends of chromosomes, proteins recruit a host of other factors to ity in is not fully understood, concealing them from the checkpoint the telomere including PinX1. TRF1 binds a leading candidate is chromosome end- responses that recognize broken DNA PinX1, which directly interacts with and fusion caused by uncapped telomeres (8). ends and protecting them from inappro- inhibits telomerase action (6). Telomere dysfunction may thus drive the priate exonucleolytic attack and recombi- development of carcinomas more than it nation (1). Telomeres are thus essential to Telomere biology and cancer drives nonepithelial cancers. Consistent chromosome stability. However, for sev- Telomeres and telomerase influence can- with this idea, telomere shortening in p53+/– eral reasons, telomeres shorten with each cer in several ways (3). It is thought that mTerc–/– mice shifts the spectrum of tumors round of DNA replication and can even- the shortening of telomeres in human cells observed in p53+/– mice from lymphoma tually become uncapped by this attrition, provides a brake to the unlimited cell divi- and sarcoma toward carcinoma (9). leading to their recognition as DNA breaks sion characteristic of cancers. Telomere and thus to permanent cell-cycle arrest (cell shortening and eventual uncapping can PinX1 is a haploinsufficient tumor senescence) or . thus be considered a type of clock, whose suppressor Telomere shortening can be slowed or alarm is set off when cells have undergone PinX1 is encoded within the chromosome reversed by the enzyme telomerase, which more divisions than would be expected 8p23 region, which is frequently affected at its core contains a catalytic subunit, within a natural human lifespan — an by loss of heterozygosity in human carci- telomerase reverse transcriptase (TERT), indication that the proliferative controls nomas (e.g., ref. 10). In this issue of the JCI, and an associated RNA template, telom- of such cells have gone awry and that divi- Zhou et al. report on their investigation of erase RNA component (TERC), which sion should be stopped. There is abundant the possibility that PinX1 may be a tumor codes for the telomere repeat. However, evidence supporting this idea. For example, suppressor (11). Consistent with this idea, in most somatic cells, telomerase is pres- the key tumor suppressors p53 and Rb they found that PinX1 protein levels are ent at levels that are insufficient to pre- respond to uncapped telomeres by limiting generally reduced in breast cancer tissues vent telomere shortening with cell divi- cell proliferation or survival, and premalig- and cell lines, lending support to earlier sion and advancing age. In addition to its nant lesions contain cells with very short reports of lower levels in some other carci- role at telomeres, evidence has mounted telomeres and that have become senescent. nomas including liver and gastric tumors. that telomerase may also have “extracur- However, although telomere length–based Next they generated mice lacking one copy ricular” activities, including functions barriers likely provide net inhibition of of PinX1, which yielded reduced levels of in mitochondria, DNA repair, stem cell cancer progression, the rare cells that PinX1 protein, two-fold increased telom- maintenance, and modulation of Wnt sig- evade these barriers (e.g., via compromised erase activity, and an approximately 50% naling (2–5). The last activity (and possi- p53 and Rb pathways) will incur the con- increase in telomere lengths in embryonic bly the others) apparently requires TERT sequences of dysfunctional telomeres. For fibroblasts (MEFs) and adult tissues. Strik- but not TERC and is thus independent of example, these can include chromosome ingly, the heterozygous mice developed classical telomerase activity. end-to-end fusions, leading to dicentric increased rates of cancer, with a shift in In addition to telomerase, a large num- chromosomes and yielding aneuploidy or tumor spectrum away from the lymphomas ber of factors help maintain telomere chromosome rearrangements. The even- and sarcomas typical of mice and toward integrity. In mammals, chief among these tual activation of a telomere maintenance epithelial cancers more frequently seen in are the members of the shelterin complex, mechanism, typically taking the form of humans, including lung, liver, mammary, including the duplex telomere repeat bind- elevated expression of telomerase, in these and gastrointestinal carcinomas. This shift incipient cancer cells can then support also occurred when one or both copies of further cell division. In addition, whereas p53 were inactivated. Furthermore, the Conflict of interest: The author has declared that no conflict of interest exists. senescence caused by telomere uncapping tumors retained the intact copy of PinX1. Citation for this article: J Clin Invest. 2011; limits carcinogenesis in a cell-autonomous These data indicate that PinX1 is a haplo- 121(4):1242–1244. doi:10.1172/JCI57024. fashion, senescent cells produce tumor- insufficient tumor suppressor.

1242 The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 4 April 2011 commentaries

spectrum of the K5-mTert mice; and carci- nomas develop in PinX1+/– mice regardless of p53 status, whereas mTerc–/– mice with intact p53 or homozygous p53 inactiva- tion do not develop the striking carcino- mas observed in mTerc–/–p53+/– mice. Thus carcinomas may arise via distinct mecha- nisms in these different models. Also of note, the dual requirement for TERT and TERC for the chromosome instability of PinX1+/– cells suggests TERC-independent extracurricular TERT activities are unlikely to be involved. How else might PinX1 deficiency lead to Figure 1 genome instability? Just as a blindfolded Consequences of insufficient PinX1 that may contribute to chromosome instability and cancer. The chromosome instability observed in cultured PinX1+/– MEFs required both TERT and TERC com- child playing the party game of pin the ponents of telomerase, and so the processes that are known or suspected to require TERT and tail on the donkey sometimes misses the TERC are indicated. The chromosome instability, and presumably cancer, in PinX1+/– mice is due mark, telomerase can mistakenly append to a single process, or some combination of processes, that depend on telomerase and are sensi- telomere repeats onto the termini of a tive to PinX1 dosage. However, the key processes are not known, and processes not yet defined broken chromosome, an event known as could be involved. Speculative or uncertain aspects of the figure are indicated by question marks. chromosome healing (18). These events are rare in normal cells but might be disinhib- ited by PinX1 deficiency, similar to healing The mechanisms underlying cancer and one can imagine that excessive tracts of broken chromosomes in yeast lacking in PinX1+/– mice may involve increased of telomere repeats might destabilize chro- the Pif1 protein, which normally inhibits genome instability, because with passage mosomes, for example, by being especially telomerase action at telomeres and DNA in culture PinX1+/– MEFs developed ana- prone to replication or recombination breaks (19). Although healing can stabi- phase bridges, aneuploidy, and chromo- abnormalities, by outstripping the capac- lize the broken end (at least temporarily), some rearrangements, including nonre- ity of natural levels of shelterin proteins it interferes with other repair mechanisms ciprocal translocations. Similar instability to protect them, or possibly by soaking that would otherwise restore the chromo- was observed in the tumors from these up factors that normally function at other some. The acentric fragment would be mice. Importantly, genome instability in genomic loci. On the other hand, TIF assays lost and the new terminus bearing short MEFs required both TERT and TERC, did not reveal uncapped telomeres in the telomere repeats might recombine with suggesting an important role for telom- PinX1+/– cells, and so if there are defects in interstitial telomere repeats to generate erase activity in this phenotype. Also of telomere capping, they must be relatively translocations. It would be interesting to note, telomere dysfunction–induced foci subtle or infrequent. Furthermore, previous examine the translocation breakpoints in (TIF) assays indicated no apparent defects studies have not provided any clear indica- PinX1+/– cells to test this speculative idea. in telomere capping. tions that long telomeres per se contribute Another possibility is that telomerase plays directly to genome instability. For example, an indirect permissive role that cooperates How does diminished PinX1 forced expression of telomerase in cultured with defects caused by reduced PinX1 levels function drive chromosome normal human fibroblasts, although it to allow survival of cells with chromosome instability and cancer? lengthens telomeres, has only a beneficial instability. These PinX1-related defects Beyond suggesting that PinX1 is an impor- effect on chromosome stability (15). In could be at nontelomeric sites, e.g., at tant tumor suppressor, the new findings yeast, artificial lengthening of a telomere causing mitotic segregation may have uncovered a novel dimension of can lead to elevated levels of recombina- defects or at the telomere. TRF1 has been the interplay of telomere biology and can- tion-based telomere shortening, but this found to facilitate telomere replication cer. A key question is how PinX1 deficiency occurs only when a subset of the telomeres (20), and it is conceivable PinX1 cooperates leads to chromosome instability (Figure 1). is lengthened, indicating it is not an intrin- with TRF1 in this capacity. In these scenar- In addition to regulating telomerase, PinX1 sic property of long telomeres (16). Further- ios, the permissive role of telomerase could localizes to kinetochores (12) and to nucle- more, Zhou et al. suggest the mechanisms be at telomeres or possibly at other targets oli (13), but whereas defects at these sites underlying the preferential increases in car- of telomerase such as mitochondria. In this certainly might have an impact on genome cinomas observed in PinX1+/– mice might later case, the longer telomeres of PinX1+/– stability and nucleolar PinX1 may regulate be related to those driving similar tumor cells would merely correlate with, rather telomerase biogenesis (14), it is not clear spectra following telomerase overexpres- than contribute to, the instability. how telomerase would act directly at these sion and telomere lengthening in K5-mTert sites to drive genome instability. Zhou et (where K5 indicates Keratin 5) transgenic Additional questions al. instead suggest that long telomeres may mice or following telomere shortening in The new findings raise additional ques- themselves induce instability, an idea con- mTerc–/–p53+/– mice (9, 17). However, the tions. For example, at what stages of car- sistent with the requirement for telomerase K5 promoter is expressed principally in cinogenesis are PinX1 levels decreased, activity. This is a fascinating suggestion, epithelia, which might explain the tumor and how does this timing correlate with

The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 4 April 2011 1243 commentaries the onset of chromosome instability? How School of Medicine, 422 Curie Boulevard, loci on chromosome 8p in hepatocellular carcino- ma, colorectal cancer, and lung cancer. Cancer Res. might the status of PinX1 in tumors guide Philadelphia, Pennsylvania 19104, USA. 1992;52(19):5368–5372. therapy? The authors suggest that telom- Phone: 215.573.5057; Fax: 215.573.6317; 11. Zhou XZ, et al. The telomerase inhibitor PinX1 is erase inhibitors may be useful for treat- E-mail: [email protected]. a major haploinsufficient tumor suppressor essen- tial for chromosome stability in mice. J Clin Invest. ing cancers lacking PinX1 function. This 2011;121(4):1266–1282. is an intriguing idea, provided the benefit 1. Palm W, de Lange T. How shelterin protects mam- 12. Yuan K, et al. PinX1 is a novel microtubule-binding to cancer cells of PinX1 loss persists in malian telomeres. Annu Rev Genet. 2008;42:301–334. protein essential for accurate chromosome segrega- 2. Kovalenko OA, et al. A mutant telomerase defective tion. J Biol Chem. 2009;284(34):23072–23082. established tumors (e.g., via promoting in nuclear-cytoplasmic shuttling fails to immortal- 13. Yoo JE, Oh BK, Park YN. Human PinX1 mediates telomere lengthening); however, if the key ize cells and is associated with mitochondrial dys- TRF1 accumulation in nucleolus and enhances benefit occurs early, via enhanced genome function. Aging Cell. 2010;9(2):203–219. TRF1 binding to telomeres. J Mol Biol. 2009; 3. Artandi SE, DePinho RA. Telomeres and telomer- instability, PinX1 status may not affect the 388(5):928–940. ase in cancer. Carcinogenesis. 2010;31(1):9–18. 14. Lin J, Blackburn EH. Nucleolar protein PinX1p sensitivity of mature tumors to telomerase 4. Park JI, et al. Telomerase modulates Wnt signalling regulates telomerase by sequestering its protein inhibition. Fortunately, existing tools can by association with target chromatin. Nature. catalytic subunit in an inactive complex lacking 2009;460(7251):66–72. be combined to address whether cancer in telomerase RNA. Dev. 2004;18(4):387–396. 5. Flores I, Benetti R, Blasco MA. Telomerase regula- 15. Morales CP, et al. Absence of cancer-associated +/– PinX1 mice or the growth of PinX1-defi- tion and stem cell behaviour. Curr Opin Cell Biol. changes in human fibroblasts immortalized with cient tumor cell lines is particularly sus- 2006;18(3):254–260. telomerase. Nat Genet. 1999;21(1):115–118. 6. Soohoo CY, Shi R, Lee TH, Huang P, Lu KP, Zhou ceptible to telomerase inhibition, and so 16. Li B, Lustig AJ. A novel mechanism for telomere XZ. Telomerase inhibitor PinX1 provides a link size control in Saccharomyces cerevisiae. Genes Dev. answers should soon be forthcoming. between TRF1 and telomerase to prevent telomere 1996;10(11):1310–1326. elongation. J Biol Chem. 2011;286(5):3894–3906. 17. Gonzalez-Suarez E, Flores JM, Blasco MA. Coop- Acknowledgments 7. Davalos AR, Coppe JP, Campisi J, Desprez PY. eration between p53 mutation and high telomerase Senescent cells as a source of inflammatory fac- transgenic expression in spontaneous cancer devel- The author thanks P. Lieberman, Y. Tzfati, tors for tumor progression. Cancer Metastasis Rev. opment. Mol Cell Biol. 2002;22(20):7291–7301. R. Greenberg, and members of the Johnson 2010;29(2):273–283. 18. Gao Q, et al. Telomerase-dependent and -indepen- lab for helpful discussions. This work was 8. Thompson SL, Compton DA. Chromosomes dent chromosome healing in mouse embryonic stem and cancer cells [published online ahead of print cells. DNA Repair (Amst). 2008;7(8):1233–1249. supported by NIH grant AG021521. December 29, 2010]. Chromosome Res. doi:10.1007/ 19. Boule JB, Zakian VA. Roles of Pif1-like helicases in s10577-010-9179-y. the maintenance of genomic stability. Nucleic Acids Address correspondence to: F. Brad John- 9. Artandi SE, et al. Telomere dysfunction promotes Res. 2006;34(15):4147–4153. non-reciprocal translocations and epithelial can- 20. Sfeir A, et al. Mammalian telomeres resemble frag- son, Department of Pathology and Labora- cers in mice. Nature. 2000;406(6796):641–645. ile sites and require TRF1 for efficient replication. tory Medicine, University of Pennsylvania 10. Emi M, et al. Frequent loss of heterozygosity for Cell. 2009;138(1):90–103. The adaptive stroma joining the antiangiogenic resistance front Oriol Casanovas

Tumor Angiogenesis Group, Translational Research Laboratory, Catalan Institute of Oncology — IDIBELL, L’Hospitalet de Llobregat, Spain.

Resistance to antiangiogenic therapies in cancer involves both tumor cells theless, clinical and experimental evidence and stromal components, but their relative contributions differ in each can- has been mounting that resistance to anti- cer subtype. In this issue of the JCI, Cascone et al. describe a stromal adapta- angiogenic therapy does indeed occur (3). tion to antiangiogenic therapy in non–small cell lung carcinoma (NSCLC) Among the tumor responses to therapy, it models that include EGFR-driven vascular remodeling promoting resistance is critical to distinguish between refractori- to VEGF inhibition. Their results suggest that the added benefit of dual ness, sometimes called intrinsic resistance VEGF/R and EGFR targeting in these models could be clinically relevant to (4), and acquired resistance. Tumors have fight resistance in NSCLC patients. long been shown to have remarkable plas- ticity and adaptability to classical chemo- The growth of a tumor depends on vascular the main target of the currently approved therapy and radiation, and this plasticity remodeling to ensure a continuous supply antiangiogenic drugs. Nevertheless, clinical contributes to evasion from antiangiogenic of nutrients and oxygen, and blockade of results demonstrate only moderate gains therapy (5–7). However, the specific mecha- the formation of new blood vessels with in time to progression and scarce benefits nisms of acquired resistance to antiangio- antiangiogenic drugs is currently used to in overall survival, despite long-term treat- genic therapies are unique, and may reverse treat certain types of cancer. For its central ment (1). Why are there such modest and after antiangiogenic therapy has been role in promoting angiogenesis, VEGF is short-lasting benefits of antiangiogenic stopped (M. Pàez-Ribes and O. Casanovas, therapies in the clinic? The initial hypoth- unpublished observations). This suggests esis was that antiangiogenic therapy would that these forms of resistance may reflect Conflict of interest: The author has declared that no conflict of interest exists. not induce resistance (i.e., “resistant to resis- adaptations to therapy rather than the Citation for this article: J Clin Invest. 2011; tance”) because it targeted endothelial cells mutations or gene amplifications that char- 121(4):1244–1247. doi:10.1172/JCI46430. instead of the tumor cell itself (2). Never- acterize acquired resistance to other thera-

1244 The Journal of Clinical Investigation http://www.jci.org Volume 121 Number 4 April 2011