TRF2 inhibits a cell-extrinsic pathway through which ...

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TRF2 inhibits a cell-extrinsic pathway through whichnatural killer cells eliminate cancer cellsAnnamaria Biroccio1,2,18,20, Julien Cherfils-Vicini3,18, Adeline Augereau1,3,18, Sébastien Pinte1,19, Serge Bauwens1,Jing Ye1,3,4, Thomas Simonet1, Béatrice Horard1, Karine Jamet3, Ludovic Cervera3, Aaron Mendez-Bermudez3,Delphine Poncet1,19, Renée Grataroli1, Claire T’kint de Rodenbeeke1, Erica Salvati2, Angela Rizzo2,Pasquale Zizza2, Michelle Ricoul5, Céline Cognet6,7, Thomas Kuilman8, Helene Duret9, Florian Lépinasse10,11,Jacqueline Marvel12, Els Verhoeyen13, François-Loïc Cosset13, Daniel Peeper8, Mark J. Smyth9,14,Arturo Londoño-Vallejo15, Laure Sabatier5, Vincent Picco3, Gilles Pages3, Jean-Yves Scoazec10,11,Antonella Stoppacciaro16, Carlo Leonetti2, Eric Vivier6,7 and Eric Gilson1,3,17,20

Dysfunctional telomeres suppress tumour progression by activating cell-intrinsic programs that lead to growth arrest. Increasedlevels of TRF2, a key factor in telomere protection, are observed in various human malignancies and contribute to oncogenesis. Wedemonstrate here that a high level of TRF2 in tumour cells decreased their ability to recruit and activate natural killer (NK) cells.Conversely, a reduced dose of TRF2 enabled tumour cells to be more easily eliminated by NK cells. Consistent with these results,a progressive upregulation of TRF2 correlated with decreased NK cell density during the early development of human colon cancer.By screening for TRF2-bound genes, we found that HS3ST4—a gene encoding for the heparan sulphate (glucosamine)3-O -sulphotransferase 4—was regulated by TRF2 and inhibited the recruitment of NK cells in an epistatic relationship with TRF2.Overall, these results reveal a TRF2-dependent pathway that is tumour-cell extrinsic and regulates NK cell immunity.

Telomeres are key features of chromosome termini that preservegenome integrity1 and emerge as cellular integrators of variousstresses2. Consequently, changes in their structure profoundly affectthe ability of cells to proliferate and to adapt to a new environment.Cells respond intrinsically to the perception of telomere dysfunctionresulting from their excessive shortening by initiating the DNA damageresponse (DDR) that leads to senescence or apoptosis. For instance,critically short telomeres are recognized as damaged DNA and signalgrowth arrest3. Consequently, telomere dysfunction prevents cancerprogression through DDR activation4–9.

1Laboratory of Molecular Biology of the Cell, CNRS UMR5239, IFR128, Ecole Normale Supérieure de Lyon, Lyon 69364, France. 2Regina Elena National CancerInstitute, Rome 00158, Italy. 3Institute for Research on Cancer and Aging, Nice (IRCAN), Nice University, CNRS UMR7284/INSERM U1081, Faculty of Medicine,Nice 06107, France. 4Shanghai Jiaotong University, Shanghai Ruijin Hospital, Shanghai 200025, China. 5Commissariat à l’Energie Atomique, DSV-Radiobiology andOncology Unit, BP6 92265 Fontenay-aux-Roses, France. 6Centre d’Immunologie de Marseille-Luminy, Aix-Marseille Université,UM2 INSERM UMR1104, CNRSUMR7280, Marseille 13288, France. 7Hôpital de la Conception, Assistance Publique–Hôpitaux de Marseille, Marseille 13005, France. 8Division of MolecularGenetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 9Cancer Immunology Program, Peter MacCallum Cancer Centre, St Andrews Place,East Melbourne, 3002 Victoria, Australia. 10Hospices Civils de Lyon, Hôpital Edouard Herriot, Service d’Anatomie Pathologique, 69437 Lyon cedex 03, France.11INSERM, UMR1052, Faculté Laennec, 69372 Lyon cedex 08, France. 12Université Lyon 1, IFR128, Institut National de la Santé et de la Recherche Médicale,U851, Lyon 69366, France. 13INSERM U758. ENS de Lyon, 46 Allee d’Italie, 69364 Lyon Cedex 07, France. 14Immunology in Cancer and Infection, QueenslandInstitute of Medical Research, 300 Herston Road, Herston, 4006 Queensland, Australia. 15UMR3244, Institut Curie-UPMC-CNRS 26, rue d’Ulm, 75248 Paris,France. 16Experimental Medicine and Pathology Department II Faculty, S. Andrea, Rome 00189, Italy. 17Department of Medical Genetics, Archet 2 Hospital, CHU ofNice, BP 3079, 06202 Nice cedex 3, France. 18These authors contributed equally to this work. 19Present addresses: Biology Institute of Lille, CNRS UMR8161,Pasteur Institute of Lille, BP 447, 59021, LILLE cedex, France (S.P.); EA 3738, Laboratoire de Radiobiologie Cellulaire et Moléculaire, Faculté de Médecine Lyon-sud,69 921 Oullins cedex, Lyon, France (D.P.).20Correspondence should be addressed to A.B or E.G. (e-mail: [email protected] or [email protected])

Received 25 February 2013; accepted 1 May 2013; published online 23 June 2013; DOI: 10.1038/ncb2774

The integrity of telomeres depends on at least two types ofmechanism. The first relies on telomerase, which can compensatefor replicative erosion1,10. The second relies on a particular chromatinorganization protecting chromosome ends from aberrant signalizationand repair11. The chromatin-mediated pathway depends on thebinding of several telomeric DNA-binding proteins, including theshelterin complex12.The shelterin protein TRF2 (telomere repeats binding factor 2)

is at the heart of the molecular events that maintain telomereintegrity in mammals13,14 and is essential for embryogenesis15. At

818 NATURE CELL BIOLOGY VOLUME 15 | NUMBER 7 | JULY 2013

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Figure 1 TRF2 dosage modulates the tumorigenicity of mouse and humantumour cells. (a–d) Immunocompetent C57/Bl6J mice were injectedsubcutaneously (s.c.) with B16F10 cells (1×106 cells per mouse)overexpressing or not mouse TRF2 (mTRF2) or expressing scramble shRNAversus mTERF2 shRNA1 or shRNA2 (see also Supplementary Fig. S1).The tumour volume (mean± s.d.) was determined every 2–3 days (a) andthe tumour volume on day 11 is shown in b by minimum to maximumbox-and-whiskers graph. P values were determined using the Mann–Whitneytest (∗P <0.05; ∗∗P <0.005; ∗∗∗P <0.001). (c) Tumour take was assessedon the basis of palpability; the results are expressed as the percentageof tumour-free mice at the indicated time points after cell injection.P values were determined using the log-rank Mantel–Cox test (∗P <0.05;∗∗∗P <0.001). (d) The overall survival of TRF2-compromised-tumour-bearingC57/Bl6J mice was followed over 35 days, and a log-rank Mantel–Coxtest was performed (∗P < 0.05;∗∗P < 0.005). (a–d) n = 7 mice percondition. One of two independent experiments is shown. Data for both

experiments are presented in Supplementary Table S5 (Statistics SourceData). (e) Immunodeficient nude mice were injected intramuscularly (i.m.)with infected BJ-HELTRas cells (1×106 cells per mouse) or cells expressingscramble shRNA or TERF2 shRNA1 (5×105 cells per mouse). Tumourtake was assessed on the basis of palpability; the results are expressed asthe percentage of tumour-free mice at the indicated time points after cellinjection. n =12 mice per condition. One of two independent experimentsis shown. Data for both experiments are presented in SupplementaryTable S5 (Statistics Source Data). P values were determined using thelog-rank Mantel–Cox test (∗∗∗P <0.001). (f) Nude mice were injected s.c.with infected BJ-HELTRas at 1×106 cells per mouse and tumour volume(mean±s.d.) on day 18 is shown by minimum to maximum box-and-whiskersgraph. n =7 mice per condition. One of two independent experiments isshown. Data for both experiments are presented in Supplementary Table S5(Statistics Source Data). P values were calculated using the Mann–Whitneytest (∗∗P <0.005).

the cellular level, suppression of TRF2 activates an ATM-dependentDDR pathway16–18. Moreover, TRF2 helps to fold the telomeric DNAinto a t-loop, which might contribute to telomere protection bymasking the 3′ overhang19,20.It has previously been shown that an increased dosage of TRF2 is

observed in human malignancies21–30, is required for tumorigenicity31,contributes to carcinogenesis in mice32 and is regulated by the Wnt/β-catenin pathway33. However, themechanism bywhich TRF2 controlscancer formation is unknown.In this study, we address the question of the role of TRF2 overexpres-

sion in cancer development.We demonstrated in immunodeficient andimmunocompetent mice that a reduced dosage of TRF2 can impairtumorigenicity in the absence of overt activation of DDR, withoutpromoting an intrinsic anti-proliferative program and in a natural

killer (NK)-cell-dependent manner. In fact, TRF2 inhibition in tumourcells modulates HS3ST4 gene expression that triggers a cell-extrinsicprogram leading to NK cell activation. These findings raise the intrigu-ing possibility that overexpression of TRF2 is a critical step in humanoncogenesis by contributing to bypass innate immune surveillance.

RESULTSTRF2 level modulates tumorigenicity in a DDR-independentmannerConsistent with its oncogenic role in human cancers, an increaseddosage of TRF2 in a variety of human andmouse tumour cells enhancedtheir tumorigenicity and aggressiveness, whereas TRF2 depletion, byRNA interference or the expression of a dominant-negative form(TRF21B1M, referred to here as TRF2dn; ref. 17), noticeably reduced

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S5 (Statistics Source Data). (d) To evaluate the ability to form colonies,increasing numbers of indicated cells from each condition were plated andcolonies were quantified at 10 days. Data represent the mean±s.d. of n=3independent experiments. (e) Transduced BJ-HELTRas cells were subjectedto an SA-β-galactosidase assay to visualize senescence. Senescent WI38cells were used as a positive control. Images representative of 3 independentexperiments are shown. Scale bar, 10 µm. (f) A flow cytometry analysis of theDNA content (upper panel) and Annexin V staining (lower panel) performedin BJHELT-Ras cells infected with a control lentiviral vector (pWPIR) orlentivirus expressing the wild-type (TRF2) or dominant-negative (TRF2dn)form of TRF2. The numbers reported in the Annexin V+ region of eachhistogram represent the percentage of apoptotic cells. Data are from oneexperiment performed in triplicate.

tumour growth (Fig. 1 and Supplementary Fig. S1 and Table S1).The effect of TRF2 modulation on the tumorigenic potential ofseveral cancer lines cannot be merely explained by variation in growthrate, viability, clonogenicity, senescence or apoptosis during their

in vitro expansion and it appeared also distinct from the effectsof the TRF2-binding protein Rap1 on NF-κB (ref. 34; Fig. 2 andSupplementary Fig. S2a–d). Similarly, the anti-tumour effects of TRF2inhibition cannot be attributed to DDR activation because the level of



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Figure 3 Partial TRF2 inhibition in various tumour cell lines does notuncap telomeres. (a) Immunoblotting analysis of transduced Myc–TRF2and DDR proteins were performed with the indicated antibodies. All of theinformation about antibodies (clones, dilutions and references) is reportedin Supplementary Table S4. (b) Immunoblotting for endogenous TRF2 inBJ-HELT and BJ-HELTRas cells infected with a lentivirus expressing ascrambled shRNA and an shRNA directed against TERF2 mRNA (TERF2shRNA). (c) Representative images of confocal sections used in the detectionof TRF1 (green) and 53BP1 (red) co-localization at telomere damage-inducedfoci (TIF) in the indicated situations using BJ-HELT and BJ-HELTRas cells.All of the information about antibodies is reported in Supplementary TableS4. Scale bar, 5 µm. (d) Quantification of the mean number of TIF pernucleus in the indicated cell lines 5 days post infection, as estimated

by scoring the number of TIF counted in all of the confocal sections ofa nucleus. TIF assay was achieved through a double immunofluorescencemethod using anti-TRF1 and anti-53BP1 antibodies. For the mouse B16F10cells, TIF assay was achieved through a FISH-IF procedure labelling telomericDNA with a PNA probe and 53BP1. For each condition, n = 30 nucleiwere assessed over 3 independent experiments. Data represent means±s.d.Results from each experiment are reported in Supplementary Table S5(Statistics source data). P values were calculated using Student’s t -test,ANOVA or the Kruskal–Wallis test (∗∗∗P <0.001). (e) PNA-FISH analysis ofmetaphase spreads analysed three weeks after infection does not show anincrease in telomere aberration in cells that express TRF2dn. Data representthe mean± s.d. of n = 3 independent experiments. Uncropped images ofblots are shown in Supplementary Fig. S9.

TRF2 inhibition, exhibiting an oncosuppressive effect in several of thecancer cells used in this study, neither activated DDR pathways noruncapped nor shortened telomeres nor increased the rate of telomerefusions and chromosome rearrangements (Fig. 3 and SupplementaryFig. S3a–e). These results are consistent with a recent publicationreporting that telomeres of certain cancer lines can be resistant toa partial loss of TRF2 (ref. 35).In line with these in vitro results, the up- or downregulation

of TRF2 in BJ-HELTRas and MDA-MB231 tumour xenografted inmice does not change the DDR or stress response and the reduced

proliferation of TRF2-compromised cells in vivo did not stem froman increased number of senescent cells (Supplementary Fig. S2e,f andTable S2). We conclude that an up- or downregulation of TRF2 intumour cells of different histotypes strongly affects tumorigenicitywithout activating DDR.It is worth noting that, differently from H-RasV12 transformed BJ

fibroblasts, TRF2 inhibition in the parental cell line BJ-HELT (notexpressing H-RasV12) or HeLa cells triggered telomere uncappingand DDR activation (Fig. 3a–d and Supplementary Fig. S3a). Ofnote, both BJ-HELT and BJ-HELTRas cells efficiently responded to



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Figure 4 IL-6 is necessary and sufficient to prevent telomere uncappingon TRF2 partial inhibition. (a) The indicated cells were analysed forIL6 transcript levels by qRT-PCR. Levels are represented relative tothose found in control-infected BJ-HELT cells, as mean± s.d. from 3independent experiments. (b) Quantification of the mean number oftelomere damage-induced foci (TIF) per nucleus in the indicated cell lines5 days post infection, estimated by scoring the number of TIF counted inall of the confocal sections of a nucleus. n =30 nuclei were analysed foreach condition assessed over 3 independent experiments and the data showthe mean±s.d., ∗∗P <0.005; ∗∗∗P <0.001 with Student’s t -test, ANOVAor the Kruskal–Wallis test. (c) Representative images of confocal sectionsfrom BJ-HELTRas cells used to detect TRF1 and 53BP1 co-localization inthe indicated situations. Scale bar, 2 µm. (d) In the indicated situations of

transduced BJ-HELTRas cells, the clonogenic ability is determined as thenumber of viable colonies issued from the indicated number of plated cells.Data are from 1 experiment performed in triplicate. All values are plotted onthe graph. (e) TIF analysis of BJ-HELT cells infected with a control emptylentivirus (vE) or with a lentivirus expressing full-length IL-6 (vIL-6) at theindicated MOI (multiplicity of infection), in the presence or not of TRF2dn.n=30 nuclei were analysed for each condition assessed over 2 independentexperiments; the data represent the mean±s.d. (f) Samples from e wereanalysed for IL-6 mRNA levels by qRT-PCR in a single experiment. Levelsare represented relative to those found in cells infected with the emptycontrol (vE=1) as mean±s.d. (g) Clonogenicity determined as in d withvarious situations of transduced BJ-HELT cells. Data from one experimentperformed in triplicate. All values are plotted on the graph.

γ-ray irradiation (Supplementary Fig. S3f–h), excluding a generalimpairment of DDR pathways. As expected36–38, the ectopic expressionof H-RasV12 in BJ-HELT cells triggered a marked increase in theexpression of IL-6 (Fig. 4a). A high level of IL-6 expression is alsoobserved in MDA-MB231 cells, expressing a mutated K-Ras isoform39,but not in HeLa cells, revealing a striking correlation between TRF2resistance and a high level of IL-6 expression (Fig. 4a).The link between IL6 expression and telomere resistance to TRF2

dysfunction was directly assessed by showing that, on one hand, areduced IL-6 expression by two different short hairpin RNAs (shRNAs)restored the sensitivity of BJ-HELTRas and MDA-MB231 cells toTRF2 dysfunction (Fig. 4b–d and Supplementary Fig. S4a) and, onthe other hand, an ectopic expression of IL-6 in BJ-HELT cells wassufficient to render the cells resistant to TRF2 dysfunction (Fig. 4e–gand Supplementary Fig. S4b). We conclude that a high level of IL-6expression in transformed cells can protect telomeres from DDRactivation in response to a partial loss of TRF2 function.

TRF2 level in tumour cells modulates NK cell mobilizationThe above results show that the oncogenic properties of TRF2 can beuncoupled from a cell-autonomous pathway of telomere protectionand growth control, raising the interesting possibility that TRF2triggers cell non-autonomous functions. In agreement with thisview, TRF2-compromised cells exerted an oncosuppressive paracrineeffect on RasV12-transformed fibroblasts when both types of cell wereco-injected into mice (Supplementary Table S1).Therefore, we explored whether TRF2 could modify the tumour

microenvironment by examining immune cell infiltration intoMatrigelplugs after the subcutaneous injection of B16F10 cells. Neither theup- nor the downregulation of mouse TRF2 (mTRF2) modulated theglobal infiltration of CD45+ cells or CD45+CD3+ T cells (Fig. 5a,b andSupplementary Fig. S5a,b). In contrast, the modulation of mTRF2markedly affected NK cell infiltration, as revealed by the presenceof NKp46+CD3− cells (Fig. 5c,d), and activation, as determined byscoring NK cells positive for the activation markers CD107a and



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pWPIR pWPIR-mTRF2

Scramble shRNA mTERF2 shRNA1 mTERF2 shRNA2

Figure 5 The modulation of tumorigenicity by TRF2 dosage correlates withNK cell recruitment and activation. (a–d) Immune recruitment inducedby mTRF2 in B16F10 cells was assayed using Matrigel plugs. SevenC57/Bl6J mice were injected s.c. with 1×106 mTRF2-overexpressingB16F10 cells or mTERF2 -shRNA-expressing B16F10 cells in Matrigel.After 5 days of incubation, the immune infiltrate was examined by flowcytometry. Total immune cell infiltration (CD45+ cells) is representedin a, T lymphocyte infiltration (CD45+ CD3+ cells) is represented inb, and NK cell infiltration (CD45+CD3−NKp46+ cells) is representedin c,d as minimum to maximum box-and-whiskers graphs. n = 5 miceper condition. One of two independent experiments is shown. Data for

both experiments are presented in Supplementary Table S5 (StatisticsSource Data), and P values were determined using the Mann–Whitneytest (∗P < 0.05; ∗∗P < 0.005). (e,f) The activation of NK cells towardsTRF2-compromised B16F10 cells was examined using a Matrigel assay.The percentage of CD107a-positive and CD69-positive NK cells infiltratingthe Matrigel plugs was determined by flow cytometry 5 days after injectioninto C57/Bl6J mice (n =6 mice per group). Data from 1 experiment arerepresented with a minimum to maximum box-and-whiskers graph andare presented in Supplementary Table S5 (Statistics Source Data). Foreach panel, P values were determined using the Mann–Whitney test(∗P <0.05).

CD69 (Fig. 5e,f). The effects of TRF2 dosage on NK cell immunitywere not dependent on cell type or mouse background because anidentical correlation was observed with human TRF2 modulationin BJ-HELTRas cells injected into nude mice (Fig. 6a). That TRF2dn

tumour cells can activate NK cells was directly assayed by co-culturingmouse splenocytes with tumour cells expressing various amounts ofTRF2, showing that the production of IFN-γ by NK cells increased 24 hafter incubation with BJ-HELTRas TRF2dn cells (Fig. 6b).Consistent with these observations, as soon as 2 days after

cell injection, histological analysis revealed an inverse correlationbetween the density of NKp46+ cells and TRF2 dosage (Fig. 6c andSupplementary Fig. S5c,d and Table S2). Moreover, the high densityof NK cells triggered by TRF2 inhibition correlated with a strongincrease of IFN-γ in infiltrating cells (Fig. 6c and Supplementary TableS2), with a slight increase in apoptosis (Supplementary Table S2),

which may have resulted from the cytotoxic effects of NK cells, andfinally with less neovascularization, as revealed by a decreased numberof CD31+ cells (Fig. 6c and Supplementary Table S2). Confirmingthe specificity of the effect of TRF2 on NK cell infiltration observedin B16F10 cells, we found that the sites of the injection of TRF2-compromised BJ-HELTRas cells did not contain larger numbers ofinfiltrating macrophages, neutrophils or DEC-205+ dendritic cells(Supplementary Fig. S5d and Table S2), even though we cannot excludethe possibility that other types of dendritic cell or immunoregulatorycell may have been recruited.

The reduced tumorigenicity of TRF2-compromised cellsdepends on NK cellsThe above results, together with the observation that BJ-HELTRasTRF2-compromised cells did not form tumours in SCID or Rag1−/−



ART I C L E S

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NA

TERF2

shRNA1

0

2

4

6

8

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pWPIR-hTRF2dn

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12 16 200

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pWPIR-hTRF2dn

(NK1.1-treatedmice)pWPIR-hTRF2dna b c

Figure 6 The reduced tumorigenicity of TRF2-compromised cells isdependent on NK cells. (a) Nude mice were injected s.c. with 1×106 TRF2or TRF2dn-overexpressing BJ-HELTRas cells or TERF2 -shRNA-expressingBJ-HELTRas cells in Matrigel. After 5 days, the immune infiltrate wasexamined by flow cytometry. NK cell infiltration (CD45+ CD3– NKp46+cells) is represented with a minimum to maximum box-and-whiskersgraph. n = 5 mice per condition. One of two independent experimentsis shown. Data for both experiments are presented in SupplementaryTable S5 (Statistics Source Data). P values were determined using theMann–Whitney test (∗P < 0.05; ∗∗P < 0.005). (b) C57BL/6J splenocyteswere incubated for 24h at 37 ◦C with the indicated TRF2-compromisedBJ-HELTRas cells. The percentage of IFN-γ-producing NK cells was thendetermined by flow cytometry. Results are mean±s.d. of n=3 independentexperiments. P values were determined using Student’s t -test (∗P <0.05).(c) Biopsies from BJ-HELTRas tumour-bearing mice were analysed byimmunohistochemistry. Representative images (NKp46, Ki67, IFN-γ andCD31) are shown. See Supplementary Table S2 for quantification. All

information about antibodies is reported in Supplementary Table S4. Scalebar, 10 µm. (d,e) Mice were injected with BJ-HELTRas cells at 5×105 cellsper mouse on day 1. The mice were treated with anti-NK1.1 antibodies at100 µg per mouse. Tumour formation was assessed on the basis of palpability;the results are expressed as the percentage of tumour-free mice at theindicated time points after cell injection following NK cell depletion. n =7mice per condition. One of two independent experiments is shown. Datafor both experiments are presented in Supplementary Table S5 (StatisticsSource Data). (f) Nude mice were injected i.m. with TRF2-compromisedBJ-HELTRas cells and treated or not with the ATM inhibitor KU-55933.After 5 days, microtumours were analysed by immunohistochemistry. Imagequantification shown in Supplementary Fig. S5h is represented. Positivecells scored in 7 high-powered fields for each condition were counted. n=7mice per condition. Mean±s.d. from one of two independent experiments isshown. Data for both experiments are presented in Supplementary Table S5(Statistics Source Data). P values were determined using a Mann–Whitneytest (∗P <0.05).

mice (Supplementary Table S1), which excludes the possibility that Tor B cells play a role, suggested that the anti-tumour effects of TRF2inhibition result, at least in part, from NK cell activation. Indeed,TRF2dn cells readily formed tumours in NK-cell-immunodepletedRag1−/− mice (Fig. 6d,e). These tumours exhibited a similar histolog-ical phenotype to those formed by TRF2-expressing cells: a high Ki67index, high density of CD31+ cells, and no expression of IFN-γ (Fig. 6cand Supplementary Table S2). In addition, they still expressed TRF2dn,excluding the possibility of selection against cells expressing the trans-gene (Supplementary Fig. S5e,f). Notably, IFN-γ neutralizationwas alsoshown to allow the formation of tumours from TRF2-compromisedcells (Supplementary Fig. S5g). Thus, the induction of IFN-γ by NKcells is a critical factor in the inability of TRF2-compromised cellsto form tumours. The fact that the immunodepletion of NK cells

restored the vascularization of TRF2dn tumours (Fig. 6c) suggests thatIFN-γ contributes to the anti-angiogenic effects of TRF2 dysfunction40.In agreement with this hypothesis, the IFN-γ-induced angiostaticmolecule MIG/CXCL9 was overexpressed by lymphoid cells presentwithin the TRF2dn microtumours ofNK-cell-competentmice but not ofNK-cell-immunodepletedmice (Supplementary Fig. S5d andTable S2).Overall, these results demonstrate that NK cells and IFN-γ are

required for the anti-tumour effects of TRF2 dysfunction and reveal theexistence of a cell-extrinsic pathway controlled by TRF2 that regulatesinnate immune surveillance. This seems to be the main mechanism bywhich TRF2dn reduces tumorigenicity, although one cannot exclude aminor contribution from cell-intrinsic effects as suggested by a slightreduction of clonogenic ability in soft agar conferred by TRF2dn (Fig. 2and Supplementary Fig. S2).



ART I C L E S

HS3ST4 (25,610,847–26,056,510)

Chr.16

50 kb

5'–CTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAACCCTAAC–3'

Interstitial telomeric sequence

0

pWPIR

Scram

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Scram

ble shR

NA

Unt

reat

ed

Bleom

ycin

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mg

ml–1

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shR

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hHS3S

T4 shR

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-hTR

F2 W

T

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1.0

1.5

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∗

∗∗

∗

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3ST4

mR

NA

level

pW

PIR

pW

PIR

-hT

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le s

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shR

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shR

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hR

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b c

d

a

Figure 7 TRF2 dosage influences NK cell infiltration in a HS3ST4-dependent manner. (a) Schematic diagram of the HS3ST4 gene andrepresentation of the interstitial localization of telomeric sequences withinthe introns of HS3ST4. (b) Analysis of HS3ST4 mRNA expression relativeto GAPDH mRNA by RT-qPCR in BJ-HELTRas either overexpressing ordownregulating TRF2 or treated with the genotoxic drug bleomycin. Thedata represent the mean±s.d. of n=5 independent experiments performedin triplicate. P values were calculated using the Mann–Whitney test(∗P <0.05; ∗∗P <0.005). (c) Western blot analysis of the phosphorylationof ATM and CHK1 in BJ-HELTRas cells treated with bleomycin. Allinformation about antibodies is reported in Supplementary Table S4.(d) Analysis of the HS3ST4 protein level by western blotting usingproteins from BJ-HELTRas cells either overexpressing or downregulatingTRF2. (e) Quantitative PCR analysis of chromatin immunoprecipitates

obtained from empty or TRF2 WT-overexpressing BJ-HELTRas cells probedfor the HS3ST4 ITS are reported as mean± s.d. of n = 3 independentChIP experiments. (f,g) Scramble-shRNA- or mTERF2 -shRNA-transducedBJ-HELTRas cells were infected with lentiviruses expressing scrambleshRNA or HS3ST4 shRNA. The different cell batches were assessedfor NK cell recruitment in a Matrigel assay as described previously (f),and the activity of the infiltrating NK cells was determined on the basisof the percentage of CD107a-positive NK cells in the Matrigel plug (g).Data are represented by minimum to maximum box-and-whiskers graphs.n = 6 mice per condition. One experiment is shown. Data are presentedin Supplementary Table S5 (Statistics Source Data); P values weredetermined using the Mann–Whitney test (∗P < 0.05; ∗∗P < 0.005; NS,not significant). Uncropped images of blots are shown in SupplementaryFig. S9.

TRF2 level regulates the expression of HS3ST4, a gene involvedin heparan sulphate biosynthesisThe key question remained the molecular mechanism by which TRF2regulates NK cells.We first excluded the possibility that TRF2-mediatedNK cell activation was a result of its role in telomere protectionand DNA damage control. Indeed, KU-55933 (2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one), a specific and very potent small-molecule inhibitor of ATM (ref. 41), the main kinase mediatingtelomere dysfunction triggered by TRF2 inhibition42, as well asknockdown of ATM and ATR by RNA interference, did not affect theability of TRF2dn to activate NK cells, to trigger IFN-γ production andto impair tumorigenicity (Fig. 6f and Supplementary Fig. S5h and TableS1). We conclude that TRF2 can control NK cells and tumorigenicityindependently of its role in telomere protection andDDR.Next, we examined the possibility that TRF2 modulates the

expression of NK cell receptor ligands, but we failed to detect anychanges in the expression of a vast panel of NK cell receptor ligands43–45

(Supplementary Fig. S6a). Next, by examining whether TRF2 could

modify the secretion of other molecules involved in NK cell immunity,we found that, as expected from RasV12 expression, BJ-HELTRas cellssecreted large quantities of factors included in the senescence-associatedsecretory profile37 (SASP) or senescence-messaging secretome46 (SMS;Supplementary Fig. S6b). However, we failed to detect any notableSASP/SMS differences between control fibroblasts and cells expressingTRF2 and TRF2dn, suggesting that modification of the SASP/SMS is notresponsible for the anti-tumour properties of TRF2-compromised cells.Nevertheless, among the SASP/SMS factors, high levels of IL-6 and -12were triggered by the injection of TRF2-compromised cells comparedwith control cells (Supplementary Table S2), even if the depletion ofIL-6 in tumour cells or the neutralization of IL-6 and -12 in mice didnot affect the ability of TRF2-compromised cells to activate NK cellsand did not restore the tumorigenicity (Supplementary Tables S1 andS2). Therefore, the NK cell stimulation and oncosuppression caused byTRF2 inhibition in BJ-HELTRas cells can be explained neither by thehigh expression level of IL-6 in these cells nor by the increased level ofIL-6 and IL-12 at the site of injection.



ART I C L E S

Normal colon

mucosa

Low-grade

adenoma

High-grade

adenoma

TRF2

Focal intramucous

adenocarcinoma

∗ ∗ ∗ ∗ ∗ ∗N

um

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n = 60

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adenoma

n = 30

High-grade

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Strong and heterogeneous expression

Strong and homogeneous expression

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n' = 5

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Figure 8 TRF2 expression is negatively correlated with NK cell densityduring the early stages of colon carcinogenesis. (a) Representativeimages showing TRF2 expression in normal, preneoplastic and neoplasticcolonic mucosa. Original magnifications: normal mucosa, ×320; low-and high-grade adenomas, ×270; adenocarcinoma, ×260. (b) Scoringof TRF2 expression. The apparent levels of expression were gradedaccording to the colour code shown in the panel. (c) Representativeimages of CD56 and hNKp46 expression. Original magnifications: normalmucosa, ×400; low- and high-grade adenomas, ×420; adenocarcinoma,×430. (d) Mean±s.e.m. of CD56+ (black bars; n = 60 normal colon

mucosa; n=30 low-grade adenoma; n=30 high-grade adenoma; n=15focal intramucous adenoma) and hNKp46+ (open bars; n = 6 normalcolon mucosa; n=5 low-grade adenoma; n=5 high-grade adenoma andn=5 focal intramucous adenoma) cell densities. Significant differencesfrom the control (normal mucosa) are indicated by asterisks. P valueswere determined using the Mann–Whitney test (∗P < 0.05). Data arepresented in Supplementary Table S5. (e) Three-parameter graphrepresenting the relative TRF2 expression score; NK cell infiltration persquare millimetre; and relative clinical score, which is proportional to thespherical diameter.

As we could explain the effect of TRF2 on NK cells neither byits canonical role in telomere protection against the DDR, nor bythe identification of known modulators of NK cells regulated byTRF2, we reasoned that TRF2 could directly regulate the expressionof NK cell modulator genes. Therefore, we used TRF2 chromatinimmunoprecipitation sequencing (ChIP-seq) data, previously ob-tained using BJ-HELTRas cells47, to search for TRF2 direct targetgenes involved in NK cell function. Among the genes containing ahigh-affinity DNA-binding site for TRF2 either within an intron or<10 kb from the transcription start site47, HS3ST4 was the only onethat encoded a protein involved in extracellular functions (sulphationof heparan sulphate proteoglycans) and whose expression (at boththe messenger RNA and protein levels) was positively regulated byTRF2 (Fig. 7a–d). In agreement with a DDR-independent role of TRF2

in this regulation, the TRF2-compromised cells used for assayingHS3ST4 expression did not show DDR activation (SupplementaryFig. S7) and an activation of the DDR by bleomycin treatment did notalter HS4ST4 expression (Fig. 7b,c). In line with the ChIP-seq data,TRF2 bound the interstitial telomeric sequence48 (ITS) located withinthe HS3ST4 intron, as revealed by the quantitative PCR analysis ofchromatin immunoprecipitates, and this binding was dependent onTRF2 expression (Fig. 7e).Reduced HS3ST4 expression triggered by two different shRNAs

increased the recruitment of NK cells in Matrigel co-injected withBJ-HELTRas cells to a level similar to that seen with the knockdownof TERF2 (Fig. 7d,f), and the double knockdown of HS3ST4 andTERF2 did not increase NK cell recruitment further (Fig. 7f), indicatingthat these genes act in the same pathway. It is worth noting that



ART I C L E S

HS3ST4 does not seem to be involved in NK cell activation, as revealedby the quantity of NK cells within the plugs expressing CD107amarkers (Fig. 7g), suggesting that other genes involved in NK cellimmunity are regulated by TRF2. We conclude that the level of TRF2in tumour cells shapes the NK cell response independently of aneffect on telomere protection by directly targeting the expression ofHS3ST4. This provides an explanation for the DDR-independent effectof TRF2 on tumorigenicity.

Inverse correlation between TRF2 level and NK cell densityduring early stages of human colon carcinogenesisFinally, we addressed the relevance of these findings to humanoncogenesis by analysing samples of human colon for TRF2 expressionand NK cell density. Although TRF2 was typically undetectable innormal colonic mucosa, most epithelial cells in neoplastic lesionsexhibited faint to strong nuclear TRF2 expression (Fig. 8a). Weidentified an increasing number of TRF2-positive cells in low- tohigh-grade adenomas and intramucosal adenocarcinomas (Fig. 8a,b).These data further support the notion that TRF2 is frequentlyoverexpressed during the early stages of human oncogenesis21,28. Whatis remarkable is that the density of NK cells in the same samples, assayedusing antibodies raised against both CD56 andNKp46, was significantlyhigher in normal colonic mucosa than in early neoplastic lesions andtended to decrease with neoplastic progression (Fig. 8c,d).We observedno apparent change in the density of CD3+ cells, which account formost of the resident lymphocytes in the studied samples or in CD8+or CD20+ cells, which were more infrequent (Supplementary Fig. S8).A three-parameter graph in which values of NK cell density within thetumour microenvironment were plotted against the relative expressionof TRF2 in tumour cells and the severity of disease (Fig. 8e) revealedan inverse association between TRF2 expression, clinical grade andNK cell infiltration. Taken together, these data reveal a specific declinein NK cell number with a concomitant, progressive increase in TRF2expression during the early stages ofmalignant transformation.

DISCUSSIONMany unknowns remain in the description and understanding of thecell non-autonomous response to genome damage. Our work bringsthe notion that NK cells can be called by TRF2 changes in tumour cellsthat do not lead to DDR and growth arrest. These findings suggest asophisticated mechanism of co-evolution between the telomere andthe immune systems to keep tissue integrity in check.The fact that the expression of H-RasV12 protects telomeres against

a partial TRF2 dysfunction by increasing IL-6 expression offered usthe unique opportunity to separate the in vivo growth defects ofTRF2 loss in cancer cells from telomere deprotection, DDR activationand cell-intrinsic inhibition of cell growth. The finding that IL-6 isrequired for the growth of TRF2-compromised H-RasV12 cells seemsat odds with its role in oncogene-induced senescence, in which itmaintains the senescent state38. Overall, these findings suggest that IL-6has antagonist effects on proliferation, being anti-mitogenic in DNAdamage checkpoint-proficient cells and pro-mitogenic in transformedcells such as the BJ-HELTRas cells used in this study.Importantly, we identified one mechanism by which TRF2 controls

NK cell biology in a DDR-independent manner. The level of TRF2positively regulates the expression of HS3ST4, a gene encoding

for the heparan sulphate (glucosamine) 3-O-sulphotransferase 4,involved in NK cell modulation (this study) and containing theintronic ITS (ref. 47). This finding sheds light on the mechanismsof NK cell recognition by tumour cells through the regulation of thesulphation of heparan sulphate molecules. It is tempting to speculatethat the sulphation of cell surface heparan sulphate proteoglycan byHS3ST4 affects the binding of cytokines and chemokines around thetumour cells49,50, thereby modifying their capacity to be eliminatedby NK cells. The role of carbohydrate recognition by NK cells isa new avenue of research that remains to be explored in depth51.In addition, the identification of the ITS-associated HS3ST4 geneas a TRF2 target provides a paradigm for studying ITS function.As TRF2 binds a large number of non-telomeric sites throughoutthe genome47,52,53, we propose that, in addition to HS3ST4, TRF2regulates the expression of a network of genes involved in tissuehomeostasis. Whether these extratelomeric roles of TRF2 are coupledto telomere chromatin structure, for example by releasing TRF2molecules from telomeres on shortening, is an interesting possibilitythat would be reminiscent of the budding yeast situation where changesin telomere structure can influence the expression of genes locatedat the interior of chromosomes by triggering the delocalization ofspecific telomeric factors54,55.In summary, we propose a model in which a high TRF2 level in

tumour cells contributes to early oncogenesis, not only by delayingreplicative senescence but also by a non-cell autonomous mechanismpreventing innate immune surveillance. �

METHODSMethods and any associated references are available in the onlineversion of the paper.

Note: Supplementary Information is available in the online version of the paper

ACKNOWLEDGEMENTSThe work done in the laboratory of E.G. was supported by La Ligue NationaleContre Le Cancer (Équipe Labellisée), Institut National du Cancer (TELOFUNand TELOCHROM programme), ANR (INNATELO programme) and theEuropean Union (FP7-Telomarker, Health-F2-2007-200950). The E.V. laboratoryis supported by ANR (programme INNATELO) and the European Union (ERCadvanced grant THINK). We thank L. Zitvogel (Institut Gustave Roussy, France)for providing the XMG1.2 clone, R. Weinberg (Whitehead Institute for BiomedicalResearch, Cambridge, Massachusetts, USA) for providing the BJ-HELT cells, C.Delprat (University of Lyon, France) for Luminex analyses, J. Lingner (EcolePolytechnique Fdrale de Lausanne, Switzerland) for providing the ATR shRNAand ATM shRNA plasmids, and V. Leopold (IRCAN, France) for providing thesubcloning vectors.We are also grateful toC.D’Angelo andM. Scarsella for technicalsupport. This work was performed using the microscopy (PICMI), cytometry(CYTOMED) and animal house facilities of IRCAN. The work done by the A.B.group was supported by grants from the Italian Association for Cancer Research(#11567 and #9979). A.B. was supported by the Short-Term Fellowship Programmeof the EMBO. M.J.S. was supported by a National Health and Medical ResearchCouncil Australia Fellowship. J.C-V. was supported by a postdoctoral fellowshipfrom La Ligue Nationale Contre Le Cancer.

AUTHOR CONTRIBUTIONSA.B. designed and interpreted most of the experiments and wrote the manuscript;J.C-V. designed, performed and interpreted syngenic mouse experiments, NK cellexperiments and HS3ST4 experiments, and wrote the manuscript; A.A. designed,performed and interpreted the IL-6 experiments, contributed to several cell biologyexperiments and wrote the manuscript; S.P. performed and interpreted cell biologyexperiments; S.B. performed and interpreted TIF analyses and lentivirus production;J.Y. designed and performed NK cell experiments, and contributed to TIF analysesand lentivirus production; T.S., B.H. and A.M-B. performed and interpreted ChIPexperiments; K.J. performed bioinformatic analyses; L.C. performed cytometry






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analyses; C.T.d.R. and D. Poncet performed gene expression analysis; E.S., A.R., P.Z.and R.G. performed cell biology and mouse experiments; L.S. and M.R. performedand analysed metaphase experiments; C.C. performed and interpreted NK cellexperiments; T.K. and D. Peeper provided IL-6 tools and help in analysing thedata; H.D. produced IL-12 antibodies; F.L. performed the pathological analyses oncolon samples; J.M. provided mouse cell lines; E. Verhoeyen and F-L.C. contributedto lentiviral production; M.J.S. designed experiments, provided 1L-12 antibodiesand edited the manuscript; A.L.V. provided cell lines and contributed to telomereanalyses; V.P. and G.P. designed, performed and analysed the A375 experiments;J-Y.S. designed and interpreted the colon sample experiments, and wrote themanuscript; A.S. designed, performed and interpreted the pathological experimentswith mouse tumours; C.L. designed, performed and interpreted most of thexenograft experiments; E. Vivier designed and interpreted the NK cell experiments,and wrote the manuscript; E.G. designed and coordinated all of the experiments,interpreted the results and wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

Published online at www.nature.com/doifinder/10.1038/ncb2774Reprints and permissions information is available online at www.nature.com/reprints

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DOI: 10.1038/ncb2774 METHODS

METHODSCell lines and reagents. The details of the DNA lentiviral constructs containingvarious forms of Myc–TRF2 will be furnished on request. The sequences of theshRNAs used in this study are given in Supplementary Table S3. The list ofantibodies used for western blotting, immunofluorescence and cytometry is givenin Supplementary Table S4.

We used human fibroblasts (BJ) and kidney epithelial (HEK) cells renderedtumorigenic by successive retroviral transductions of telomerase (hTERT), simianvirus 40 early region (SV40 ER) and oncogenic Ras (H-Ras[G12V] or RasV12)56

as well as MDA-MB231 human mammary carcinoma cells and A375 melanomacells. From the immortalized but not tumorigenic fibroblasts expressing hTERT andSV40 ER genes (termed BJ-HELT) we derived both a polyclonal and monoclonalpopulation of RasV12-transduced cells named, respectively, BJ-HELTRaspc andBJ-HELTRas. To obtain HEK-HELTRas, we introduced RasV12 into ER-SV40-HA1+hTERT cells57 and selected a clone overexpressing the protein. Mousemelanoma B16F10 cells were obtained from the American Type Culture Collection(Manassas). Cells were grown at 37 ◦C in DMEM with 10% fetal calf serum (FCS)and penicillin–streptomycin.

To create the lentiviruses, we transfected 10-cm dishes of 293T cells with8.6 µg pCMV1R8.91, 2.8 µg phCMV-G and 8.6 µg pWPIR-GFP plasmid58

containing different forms of the TRF2-coding sequence or pLKO plasmid bycalcium phosphate precipitation. Viral supernatants were collected 24 h aftertransfection. The infection efficiencywas investigated by determining the percentageof GFP-positive cells using FACS analysis 3 days after infection or the number ofclones after 1 week of selection with ampicillin.

A375 cells, which express the Tet repressor, were used for transfection withpcDNA4/TO, pcDNA4/TO-TRF2wt or pcDNA4/TO-TRF2dbdc (pTrex-A; LifeTechnologies). Zeocine-resistant clones (5 µgml−1) for each constructwere screenedby western blotting using anti-TRF2 antibodies (4A794; Imgenex) and anti-c-Mycantibodies (9E10; Santa Cruz Biotechnology) after tetracycline induction.

Immunofluorescence detection of telomere dysfunction-induced foci. Slideswere fixed with methanol at −20 ◦C or 4% formaldehyde at room temperaturefor 15min, and then incubated for 1 h with blocking buffer (0.8× PBS, 50mMNaCl, 0.5% Triton X-100 and 3% milk), followed by incubation overnight at 4 ◦Cwith anti-TRF1 (ab10579; Abcam) and -53BP1 (NB100-305; Novus Biologicals)antibodies. The cells were thenwashedwith 0.8×PBS, 50mMNaCl and 0.1%TritonX-100 and incubated with anti-mouse Alexa488 (A21202; Life Technologies) andanti-rabbit Alexa555 (A-31572; Life Technologies) antibodies. After washing with0.8× PBS, 50mM NaCl, and 0.1% Triton X-100, the nucleus was labelled withDAPI and/or TOTO-3. B16F10 cells were unmasked for 40minwith Target Retrievalsolution (Dako #S1699) at 95 ◦C and then for 20min at room temperature, followedby PBS rinses and successive ethanol dehydrations (3× 3min, 50%, 75%, 100%),then PNA probe was hybridized for 3min at 80 ◦C and for 2 h at room temperaturein a moist chamber in the dark (0.3 ng µl−1 dissolved in 70% formamide, 10mMTris at pH 7.2, 1% blocking reagent (from Roche Biochemicals)), washes were asfollows: 2×15min at room temperature in 70% formamide, 10mM Tris at pH 7.2and 3×5min in 50mMTris at pH 7.5, 150mMNaCl, 0.05% Tween-20; then 53BP1was stained as mentioned below. The list of antibodies is given in SupplementaryTable S4.

Confocal images were produced using a Zeiss LSM-510 confocal scanning lasermicroscope (Jena). Optical sections were recorded at an interval of×200 nm, with aPlan-Apochromat (×63, NA 1.4, oil-immersion lens). The excitation wavelengthswere 488, 543 and 633 nm for the FITC-labelled PNA and DNA probes, for theAlexa555-labelled secondary antibodies, and for TOTO-3-stainedDNA, respectively.Co-localizations were scored in each optical section by scrolling through the z stack.The appearance of the images was improved by whole-image operations withPhotoshop (Adobe Systems).

Mice. All procedures involving mice and their care were performed as describedpreviously59 following the guidelines set forth in European Directive 86/609/EEC,which are in compliance with national and international laws.

Tumorigenicity. For the intramuscular (i.m.) and intravenous (i.v.) injections,CD-1 male nude (nu/nu) mice, 6–8 weeks old and weighing 22–24 g each, werepurchased from Charles River Laboratories. To assess tumorigenicity, we injecteddifferent numbers of cells (from 5× 105 to 2× 106 per mouse) i.m. Mice (12 foreach group) were observed daily to establish tumour take, the time of tumourappearance, and tumour weight59. For the subcutaneous (s.c.) injections, male Swissnu/nu mice (Charles River Laboratories), 6–7 weeks old and weighing 22–24 g each,were housed according to the guidelines of the French Agriculture Ministry. Mice(three for each group) were injected s.c. in both flanks (n= 6) with 1× 106 cells

in 0.1ml Matrigel (Becton, Dickinson, Franklin Lakes) diluted (v/v) in PBS. Forthe A375-control, A375-TRF2 and A375-TRF2dn cells were induced overnight withtetracycline (1 µgml−1) and 106 cells were injected s.c. into the flanks of 6-week-oldnude (nu/nu) mice (Harlan Laboratories). Doxycycline (750 µgml−1) was added tothe drinking water beginning immediately after injection. B16F10 cells (1×106 cellsin 0.1ml PBS) were injected s.c. into the backs of 8-week-old C57BL/6Jmice (JanvierSAS), and mice were surveyed daily. Tumour volumes were calculated as follows:V =L × l2×0.5, where L and l represent the largest and smallest tumour diameters,respectively, measured at the indicated times. Rag1tm1Bal (ref. 60) mice deficientin Rag1 backcrossed in a C57BL/10JCrl background were bred at the Plateau deBiologie Expérimentale de la Souris (IFR128).

Tissue analysis of the mouse tumours. Fresh tissue samples were embedded inOCT compound (Miles Laboratories), snap-frozen in liquid nitrogen, stored at−80 ◦C, and used after sectioning for use with a TUNEL Apoptosis Detection Kitand β-galactosidase staining following themanufacturer’s instructions. Immunohis-tochemical analysis was performed on 100% acetone-fixed sections after quenchingendogenous peroxidase activity with 1% H2O2 in 100% methanol and inhibitingbiotin. The sections were incubated with optimal dilutions of primary antibodies(listed below) after pre-incubation to inhibit nonspecific protein binding. Thereactions were revealed using an UltraTek HRP Anti-Polivalent Lab Pack (ScytekLaboratories) according to the manufacturer’s instructions and counterstained withhaematoxylin.

For NKp46 immunofluorescence microscopy, frozen mouse muscle sections(8mm)were fixedwith acetone and stainedwith the following antibodies: polyclonalgoat anti-NKp46 (R&DSystems) or biotin-conjugated anti-CD3 (145-2C11; Becton,Dickinson). These antibodies were visualized, using Alexa488-donkey anti-goatIgG and Alexa-546-conjugated streptavidin (Life Technologies), respectively. Nucleiwere stainedwith Topro-3 (Life Technologies). Confocalmicroscopywas performedusing a Zeiss LSM510microscope. Image processing was performed using Zeiss LSMsoftware and Photoshop (Adobe Systems).

In vivoMatrigel assay. Each group of cells (1×106 BJ-HELTRas or B16F10 cellsin 100 µl PBS) was mixed with 400 µl Matrigel (BD Biosciences) and injected s.c.Groups of eight mice were used for each experiment. The cells were replaced by100 µl PBS in the negative control and by 100 µl PBS supplemented with TNF-α,VEGF, SDF-1 and S1P in the positive control. Five days after injection, the Matrigelplugs were dissociated mechanically in serum-free medium containing DNase I(Roche), collagenase A (Roche) and dispase (BD Biosciences) and then incubatedfor 30min at 37 ◦Cwith agitation. Next, the cells were filtered, washed and saturatedwith anti-CD16/32 antibodies (clone 2.4G2; BD Biosciences) in PBS containing 2%FCS and 0.5mM EDTA. Finally, the cells were stained for 30min at 4 ◦C with FITC-conjugated anti-CD3, APC-conjugated anti-NKp46 or APC-Cy7-conjugated anti-CD45 antibodies (all from BDBiosciences). After washing and fixation, staining wasdetected by flow cytometry using a CANTO cytometer andDIVA6 software (BDBio-sciences). All samples are defined positive when the fluorescence intensity is higherthan that of the isotypic control. However, we noticed that the mean fluorescenceintensity of the NK cell population is low owing to the autofluorescence of Matrigel.

NK cell analysis. C57BL/6J splenocytes were used for the NK cell effectorfunction assay. The splenocytes were incubated for 24 h at 37 ◦C in the presenceof transformed human fibroblasts or rmIL-12 (20 ngml−1; R&D Systems) andrmIL-18 (5 ngml−1; R&D Systems). The effector/target ratio was 2.5:1. Six hoursbefore the end of stimulation, Golgi Stop (1/1500; Becton, Dickinson) was added.The cells were then stained for 30min at 4 ◦C with PerCP-conjugated anti-CD3(145- 2C11; Becton, Dickinson) or APC-conjugated anti-NK1.1 (PK136; Becton,Dickinson) antibodies. After fixation and permeabilization, the expression of IFN-γwas detected using PE-conjugated anti-IFN-γ antibodies (Becton, Dickinson) for 30min at 4 ◦C.

Tissue analysis of human colon samples. The study group consisted of a totalof 90 cases of early neoplastic lesions of the colon selected from the Departmentof Pathology, Hôpital Edouard Herriot, Hospices Civils. All cases were reviewedby an experienced gastrointestinal pathologist and classified according to therecommendations of the Vienna classification61.

In 75 cases, only archival formalin-fixed, paraffin-embedded tissue samples wereavailable. These were obtained from 75 different patients (40 males and 35 females,aged 41–75 years (median, 61± 8.9 years)) who underwent routine colonoscopy fordiagnostic, screening or follow-up purposes. Five patients had familial adenomatouspolyposis. Of the lesions examined, 30 were from the right colon, 20 from thetransverse colon and 25 from the left colon. A total of 30 lesions were classified aslow-grade adenomas, 30 were classified as high-grade adenomas and 15 contained

NATURE CELL BIOLOGY


METHODS DOI: 10.1038/ncb2774

at least a focus of well-differentiated, intramucosal adenocarcinoma developed withan adenoma. A total of 60 samples of normal colonic mucosa were studied inparallel.

In 15 cases, both formalin-fixed and frozen tissue samples were available. Thesecases represented 15 different patients who underwent routine colonoscopy (10males and 5 females, aged 22–56 years median, 44± 7.6 years)): 5 patients hadfamilial adenomatous polyposis and 1 had HNPCC syndrome. Of the lesionsexamined, 5 were classified as low- grade adenomas, 5 as high-grade adenomas and5 as intramucosal adenocarcinomas. Six samples of normal colonic mucosa werestudied in parallel.

For immunohistochemical analysis, anti-TRF2 (clone 4A794; Abcam), -CD56(clone C5.9; Dako, Glostrup), -CD3 (clone UCHT1; Dako) and -CD8 antibodies(clone C8/144B; Dako) were applied to deparaffinized serial sections (4 µm inthickness) of formalin-fixed tissue. Anti-hNKp46 antibodies (clone 195314; R&DSystems) were applied to acetone-fixed, air-dried, frozen tissue sections (6 µm inthickness). In all cases, after incubation with the primary antibody, an indirectimmunoperoxidase assay was performed according to the streptavidin–biotintechnique with DAB as the chromogen.

The apparent level of expression of TRF2 was evaluated against internal controlsformed by the proliferative lymphoid cells of germinal centres present in the sametissue sections in the adjacent colonic mucosa. The level of expression of TRF2 innormal or neoplastic epithelial cells was defined as faint when it was weaker than thatexhibited by the internal controls and as strong when it was at least equivalent to thatof the internal controls. A semiquantitative scoring system was used. The density of

CD56+ and hNKp46+ cells was evaluated in at least 10 consecutive surfaces of 1mm2

or across the entire lesion for lesions measuring<10mm2.

Statistics. All graphing and statistical analyses were done using GraphPad Prismsoftware. All results represent the mean± s.d. or with minimum to maximumbox-and-whiskers plots. The significance of differences between the means wasdetermined using the Mann–Whitney two-tailed test. To determine overall survival,we used a log-rank (Mantel–Cox) test. For each test, P < 0.05 was consideredstatistically significant.

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NATURE CELL BIOLOGY


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S U P P L E M E N TA RY I N F O R M AT I O N

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DOI: 10.1038/ncb2774

Figure S1 TRF2 dosage modulates the tumorigenicity of mouse and human tumor cells. (a) Left: immunoblotting with antibodies directed against the Myc tag of the transduced TRF2 forms after the lentiviral infection of BJ-HELT cells. Right: immunoblotting of endogenous TRF2 in cells infected with a lentivirus expressing scramble shRNA (shCtrl) and an shRNA directed against TERF2 (shTERF2). Results of one representative experiment are shown. (b) Modulation of TRF2 expression in B16F10 cells. After the lentiviral transduction of B16F10 with lentivirus expressing TRF2 WT, the level of expression of the GFP tag was analyzed by flow cytometry and (c) the level of expression of mTERF2 after transduction of B16F10 cells with mTRF2-expressing or mTERF2 shRNA-expressing lentivirus was determined by RT-qPCR and expressed relative to GAPDH. Results of one representative experiment are shown. (d) Nude mice were injected intravenously with infected BJ-EHLT Rasmc at 1x105 cells/mouse.

The mean (SE) number of lung nodules on day 50 is indicated. Arrows indicate metastatic nodules. Data from one experiment with n=6 mice per condition are shown and source data are presented in Table S5 (Statistics Source Data). p-values were determined using the Mann-Whitney test. (e) Derivatives of the A375 melanoma cell line injection into nude mice for tumor take. Tumor take was assessed based on palpability; the results are expressed as the percentage of tumor-free mice at the indicated time points after cell injection. n=7 mice per condition, results of one representative experiment are shown and data for two independent experiments are presented in Table S5 (Statistics Source Data). (f) The tumor volume (mean +/- s.d.) was determined and representative images of the tumors are presented in (g). Scale bar represent 1 cm; p-values were determined using the Mann-Whitney test (*p < 0.05; **p < 0.005; ***p < 0.001).


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Figure S2 Partial TRF2 inhibition in BJ-HELTRas cells does not trigger NF-κB, DNA damage and stress response. (a) Anchorage independent growth assay performed 2 weeks pos-infection. Five thousand cells were seeded and the colonies counted three weeks subsequently. Data represent the mean +/- s.d. of n=3 independent experiments and source data are provided in Table S5 (Statistics Source Data). *p<0.05 p-values were determined using the Mann-Whitney test. (b) An increasing number of cells from each sample were plated and colonies were numerated after 10 days. Data represent the mean +/- s.d. of n=3 independent experiments. (c) Growth properties of the indicated cell lines, shown as plots of cumulative population doubling versus the number of days postinfection. Data represent the mean of n=3 independent experiments and source data are provided in Table S5 (Statistics Source Data). (d) NF-κB gel shift analyses using nuclear extracts of the indicated cells. The probe used was a consensus sequence for the transcription factor NF-κB. A nuclear

extract of Jurkat cells was used as a positive control. These experiments were performed using a Gelshift NFκB/Rel Family Kit (#37319; Active Motif, Carlsbad, CA). (e) Histology of BJ-HELTRas cells transduced with the indicated lentiviral vector analyzed 7 days after their subcutaneous injection. Biopsies from at least four different mice were analyzed, and at least four sections from each tumor were evaluated. The boxes in the SA-β-galactosidase images represent positive staining in hair follicle and sebaceous gland. See Supplementary Table 2 for quantification. The white bar represent 10 µm. (f) Staining of mouse skin before and after γ-ray irradiation showing the activation of CHK2 and ATM. Immunoblotting of proteins from two TRF2dn BJ-HELTRas tumors formed in nude mice with anti-CHK1 and -phospho-CHK1 antibodies showing an absence of detectable DDR activation in contrast to cultured BJ-HELTRas cells treated with the DNA damaging agent campthotecin. The white bar represents 10 µm. All the information about antibodies is reported in Table S4.




Figure S3 Partial TRF2 inhibition in various tumor cell lines does not uncap telomeres. (a) Chromatin immunoprecipitation experiments performed with BJ-HELT and RasV12 derivatives cell lines five days after infection with the indicated lentiviral vector using γ-H2AX and Myc antibodies and analyzed by slot-blotting hybridized with an Telo or an Alu probe. It is worth noting that we observed a significant binding of the TRF2dn protein at telomeres, although less pronounced than the one of an equivalent amount of overexpressed myc-TRF2. This was unanticipated since TRF2dn does not contain a DNA binding domain and is believed to work by displacing the endogenous TRF2 molecules. The telomeric binding of TRF2dn might be explained by an interaction with other shelterin components, data fromone experiment performed in triplicate, all values are plotted on the graph. (b,c) Multiplex fluorescence in situ hybridization analyses (M-FISH) establish that the lines transduced with pWPIR-TRF2dn does not show an increased number of chromosome rearrangement, both clonal, i.e. shared by at least two cells, or non-clonal. M-FISH was performed using multi-FISH probes

(MetaSystems, GmbH) according to the manufacturers’ recommendations. Images of hybridized metaphases were captured with a CCD camera (Zeisss) coupled to a Zeiss Axioplan microscope and were processed with ISIS software (MetaSystems, GmbH). Data represent the mean +/- S.D. of n=30 metaphases from a single experiment are shown. (d) Telomere length measurement by a Southern procedure. Genomic DNA was digested with HinfI and RsaI and hybridized with a radiolabeled TTAGGG probe, data from 1 experiment are shown. (e) Immunoblotting analysis of shelterin components with the indicated antibodies. (f) Immunoblotting analysis of DNA damage, with the indicated antibodies after exposure to 5 Gy of ionizing radiation. All the information about antibodies is reported in Table S4. (g) Analysis of the phospho-(Ser/Thr) ATM/ATR substrates. (h) Quantification of the total number of 53BP1 foci per nucleus in the indicated cell lines 5 days post-infection or 5 hours after exposure to 5 Gy of ionizing radiation. Data represents mean+/-S.D. of n=30 nuclei assessed over 2 independent experiments, ***p<0.001. p-values were determined using ANOVA test.




Figure S4 IL-6 is necessary and sufficient to prevent growth inhibition upon TRF2 partial inhibition. (a,b) In the indicated situations, the clonogenic ability is determined as the number of viable colonies issued from the indicated number

of plated cells. MOI = Multiplicity of Infection. vIL-6 : lentiviral vector expressing full length IL-6; vE : control lentiviral vector not expressing IL-6. Data from 1 experiment performed in triplicate, all values are plotted on the graph.




Figure S5 The modulation of tumorigenicity by TRF2 dosage correlates with Natural Killer (NK) cell recruitment and activation. (a) Positive controls from the Matrigel assays. PBS alone or containing TNF-α, VEGF-α, SDF-1, and sphingosine-1-phosphate (S1P) was mixed with Matrigel prior to its injection into 5 mice. Five days later, the immune infiltration of the Matrigel plug was assessed using flow cytometry. The min to max box-and-whiskers graph represents the NK cell infiltration of n=5 mice from1 experiment. Data are also presented in Table S5 (Statistics Source Data). p-values were calculated using the Mann-Whitney test (*p < 0.05). (b) Representative FACS dot plots showing CD45+ and CD45+ CD3+ infiltration into the Matrigel plugs quantified in Fig. 5. The data are representative of n=2 independent experiments conducted with 5 mice each. (c) Biopsies from BJ-HELTRas tumor-bearing mice were analyzed by immunohistochemistry. Images showing immunofluorescence staining for NKp46 are shown. The white bar represents 20 µm. (d) Histology of BJ-HELTRas cells transduced with the indicated lentiviral vector and analyzed 7 days after their subcutaneous injection. See Supplementary Table 2 for quantification. All the information about antibodies is reported in Table S4. The white bar represents 10 µm. (e) The expression of TRF2dn was assessed in tumors formed in NK1.1-treated mice by immunoblotting with

anti-Myc antibodies, which recognized Myc-tagged TRF2. (f) The percentage of tumor cells expressing TRF2dn was assayed by tissue analysis with anti-Myc antibodies. The values represent the mean percentage of Myc-positive cells from four tumors. “Untreated” refer to samples of TRF2dn cells two days after their injection in mice that have not been treated with NK1.1 antibodies. The white bar represents 10 µm. (g) Mice were injected with BJ-HELTRas cells at 5 × 105 cells/mouse on day 1. The mice were treated with anti-IFN-γ monoclonal antibodies (clone XMG 1.2) at 0.5 mg/mouse on days 0, 4, and 7. Tumor formation was assessed based on palpability; the results are expressed as the percentage of tumor-free mice at the indicated time points after cell injection following NK cell depletion. n=3 mice per condition. One of two independent experiments is shown. Data for both experiments are presented in Table S5 (Statistics Source Data). (h) Nude mice were injected with i.m. with BJ-HELTRas cells or TRF2dn overexpressing BJ-HELTRas cells and treated or not with the ATM inhibitor KU-55933. After 5 days, microtumors were analyzed by immunohistochemistry for the phosphorylation of ATM, NKp46 infiltration and IFN-γ expression. The white bar represents 10 µm. Data for n=2 independent experiments are presented in Table S5 (Statistics Source Data). Quantifications are presented in Figure 6f.




Figure S6 TRF2 dysfunction in BJ-HELTRas cells does not alter the expression of NKG2D and DNAM-1 ligands and senescence-associated secretory proteins. (a) Analysis of the expression of NKG2D and DNAM-1 ligands on transformed human fibroblasts by flow cytometry. TRF2- or TRF2dn- BJ-HELTRas cells were incubated at 4°C for 30 min with purified antibodies: anti-MIC-A, MIC-B, ULBP-1, ULBP-2, ULBP-3, CD112 and CD155. All the informations about antibodies are reported in Table S4. Representative FACS histograms

of 3 independent experiments. (b) The concentration of 24 cytokines was determined in the indicated CM by Luminex Technology using the Milliplex Human Cytokine/Chemokine panel (Millipore Corp., Billerica, MA). The experiment, including sample dilution, beads, antibodies, and cytokine standard preparation, was performed according to the specifications of the manufacturer and run on a Bio-Rad Bio-Plex System (Hercules, CA). Data represent the mean +/- standard deviation of n=3 independent experiments.




Figure S7 Cells used for assaying HS3ST4 expression did not show DDR activation. Western blot analysis of the phosphorylation of ATM and CHK1 in the samples of BJ-HELTRas cells either overexpressing or down-regulating TRF2 used for the HS3ST4 experiments of Figure 7. All the information about antibodies is reported in Table S4.




Figure S8 Quantification of CD3+, CD8+ and CD20+ cells in early stages of colon carcinogenesis. Patients assessed for NKp46 and TRF2 staining (Figure 8) where also scored for CD3+ (n=30 low grade adenoma; n=30 high grade adenoma and n=15 focal intramucous adenoma) (a), CD8+ (n=30 low grade adenoma; n=30 high grade

adenoma and n=15 focal intramucous adenoma) (b) and CD20+ (n=8 low grade adenoma; n=8 high grade adenoma and n=8 focal intramucous adenoma) (c) infiltration. Data represents mean +/- s.d. of cell densities per mm2. Data are presented in Table S5 (Statistics Source Data) for the Figure 8.




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Figure S9 Uncropped Western blots. The white boxes refer to the part of the blot presented in the main figure.


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