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Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer

Nature Cell Biology volume 15, pages 967–977 (2013)Cite this article

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A Corrigendum to this article was published on 01 October 2013

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Abstract

The DNA damage response (DDR) pathway and ARF function as barriers to cancer development. Although commonly regarded as operating independently of each other, some studies proposed that ARF is positively regulated by the DDR. Contrary to either scenario, we found that in human oncogene-transformed and cancer cells, ATM suppressed ARF protein levels and activity in a transcription-independent manner. Mechanistically, ATM activated protein phosphatase 1, which antagonized Nek2-dependent phosphorylation of nucleophosmin (NPM), thereby liberating ARF from NPM and rendering it susceptible to degradation by the ULF E3-ubiquitin ligase. In human clinical samples, loss of ATM expression correlated with increased ARF levels and in xenograft and tissue culture models, inhibition of ATM stimulated the tumour-suppressive effects of ARF. These results provide insights into the functional interplay between the DDR and ARF anti-cancer barriers, with implications for tumorigenesis and treatment of advanced tumours.

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Figure 1: ATM regulates p14ARF protein levels in vitro.
Figure 2: ATM regulates p14ARF protein stability.
Figure 3: p14ARF protein stability is regulated by the ATM–PP1–Nek2-mediated pathway.
Figure 4: Phosphorylation of NPM/B23 at Ser 70 and Ser 88 enhances p14ARF protein stability.
Figure 5: ATM-dependent p14ARF regulation affects ribosomal biogenesis.
Figure 6: ATM-dependent p14ARF regulation affects cell growth.
Figure 7: ATM abrogation is inversely related to ARF expression in lung cancer.
Figure 8: ATM abrogation stalls growth in p53-null xenografts in an ARF-dependent manner.

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Change history

  • 28 August 2013

    In the version of this Article originally published, there was an error in Fig. 8e. The arrow beside ARF in green should have been pointing up and the arrow beside ARF in red should have been pointing down. This has been corrected in the HTML and PDF versions of the Article.

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Acknowledgements

We would like to thank G. Peters (Cancer Research UK London Research Institute, UK), D. Ginsberg (Bar Ilan University, Israel), P. G. Pelicci (European Institute of Oncology, Italy; University of Milan, Italy), M. Serrano (Spanish National Cancer Research Centre, Spain), Y. Shiloh (Tel Aviv University, Israel) and K. Vousden (Cancer Research-UK, Beatson Institute, UK) for providing reagents for this work. Special thanks for expert technical support from A. Damalas on the BJ cell treatments with serum starvation and T. Liloglou for manipulations with the HBECs and their derivatives. Financial support was from the European Commission FP7 (projects GENICA, INFLA-CARE, BioMedReg, DDResponse and INsPiRE), the Danish Cancer Society, and the Danish National Research Foundation. G.V. is a recipient of a Hellenic Association for Molecular Cancer Research scholarship. Dr A. Kotsinas is a recipient of an Empeirikeion Foundation fellowship. This work is dedicated to the memory of G. V. Gorgoulis.

Author information

Author notes

  1. Georgia Velimezi and Michalis Liontos: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Histology and Embryology, Molecular Carcinogenesis Group, School of Medicine, University of Athens, 75 Mikras Asias Str, Athens, GR-11527, Greece

    Georgia Velimezi, Michalis Liontos, Maria Sideridou, Ioannis S. Pateras, Kostas Evangelou, Athanassios Kotsinas & Vassilis G. Gorgoulis

  2. Biomedical Research Foundation of the Academy of Athens, 4 Soranou Ephessiou St., Athens, GR-11527, Greece

    Konstantinos Vougas, Theodoros Roumeliotis, George Papafotiou, Apostolos Klinakis & Vassilis G. Gorgoulis

  3. Centre for Proteomic Research, Institute for Life Sciences, University of Southampton, University Road, Highfield, Southampton, SO17 1BJ, UK

    Theodoros Roumeliotis

  4. Danish Cancer Society Research Center, Strandboulevarden 49, Copenhagen, DK-2100, Denmark

    Jirina Bartkova, Maciej Kocylowski & Jiri Bartek

  5. Department of Molecular Biology, University of Geneva, 30 quai Ernest-Ansermet, Geneva, CH-1211, Switzerland

    Ayguel Dereli-Oz & Thanos D. Halazonetis

  6. Department of Molecular Medicine and Biotechnology, School of Medicine, University of Rijeka, Brace Branchetta 20, Rijeka, 51000, Croatia

    Ines Orsolic, Sladana Bursac, Maja Cokaric-Brdovcak & Sinisa Volarevic

  7. Unit of Biomedical Applications, Institute of Biology, Medical Chemistry and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Ave., Athens, GR-11635, Greece

    Vassilis Zoumpourlis

  8. Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Centre for Scientific Research ‘Demokritos’, Agia Paraskevi Attikis, Athens, GR-15310, PO Box 60228, Greece

    Dimitris Kletsas

  9. and Department of Pathology and Cell Biology College of Physicians & Surgeons, Institute for Cancer Genetics, Columbia University, 1130 St. Nicholas Ave., New York, New York 10032, USA

    Wei Gu

  10. Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Hněvotı´nská 5, Olomouc, 779 00, Czech Republic

    Jiri Bartek

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  1. Georgia Velimezi
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Contributions

G.V. and M.L.: cell culture and manipulations, siRNA/plasmid/viral transfections/transductions/infections, ubiquitination assay, western blots, cell growth analyses and soft agar assays.

K.V., T.R. and V.G.G.: proteomic analysis design and experimentation.

E.K., J. Bartkova, I.S.P. and J. Bartek: human and mouse pathological evaluations, immunohistochemical analyses and evaluations, and tumour immunofluorescence analyses.

A.K.: real-time (RT-)PCR analyses.

M.S.: ChIP assay.

M.S. and G.V.: rRNA pulse-chase analysis, immunoprecipitation analyses, plasmid and lentiviral production.

A.D-O., M.K. and T.D.H.: I-2 mutagenesis analyses and corresponding immunofluorescence and western blot analyses.

I.O., S.B., M.C-B. and S.V.: nuclear fractionation, nucleolar immunofluorescence and western blot analyses, NPM site-directed mutagenesis and 47S pre-rRNA real-time RT–PCR analysis.

V.Z., D.K., G.P., A. Klinakis and V.G.G.: xenografts and mice manipulations.

K.V., M.L. and A. Kotsinas: Bioinformatic and statistical analyses.

W.G.: data analysis and production of reagents.

J. Bartek and T.D.H.: data analysis and interpretation, and assistance in manuscript preparation.

VG.: experimental design, guidance, manuscript preparation and writing.

Corresponding authors

Correspondence to Jiri Bartek or Vassilis G. Gorgoulis.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 HeLa and H1299 cells exhibit signs of DDR activation and detectable levels of ARF expression, but Chk2 silencing does not affect p14ARF protein levels.

a. Immunoblot (IB) analysis shows decreased phospho-Chk2 levels upon ATM inhibition (ATMi) in H1299 and HeLa cells, demonstrating the effectiveness of the inhibitor. b. IBs depicting signs of DDR activity, assessed by gH2AX (see also Fig. 1a), and detectable levels of p14ARF expression in HeLa and H1299 cells. c. IBs demonstrating that Chk2 silencing in HeLa and H1299 cells has no effect on p14ARF levels. p14ARF mRNA levels are induced by oncogenic stimuli (Cdk4, Ras and E2F1), but are unaltered after inhibition or silencing of ATM. Bars represent quantification of p14ARF mRNA levels as assessed by semi-quantitative real time RT–PCR in: (d) Human Bronchial Epithelial Cells (HBECS) (KT: hTERT, Cdk4 and KT transfected with activated K-RasV12), NARF cells (with or without IPTG induction) and Saos2-E2F1-ER (non-induced, TAM-induced and TAM-induced + ATMi), and in: (e) H1299, HeLa, HBECs cells transfected with control siRNA, siATM, DMSO (control) and Ku55933 (ATMi), induced NARF2 treated with Doxorubicin and Doxorubicin +Ku55933, respectively. (p<0.005, t-test, error bars indicate SDs, n = 4 real time RT–PCR runs). Silencing of ATM enhances oncogene-induced p14ARF expression in BJ cells and HBECs. f. IB analysis complementing Fig. 1c results showing the status of DDR activation and p14ARF levels in immortalized HBECs (hTERT, Cdk4) and immortalized HBECs with K-RasV12. Genetically manipulated HBECs expressing various oncogenes16,17,76 were employed as normal cells do not demonstrate signs of DDR activation47 and ARF levels are negligible1. g. IB analysis results showing that β-catenin transfected diploid BJ human fibroblasts exhibit DDR activation, while upregulation of p14ARF levels require additionally low serum conditions, as previously reported20. h. IB analyses, complementing panel g, demonstrating that the increased p14ARF protein levels in BJ primary human fibroblasts due to β-catenin transfection and serum depletion was further enhanced after silencing of ATM. ATM regulates p14ARF protein stability. i Silencing of ATM (shATM H1299 cells) protects ARF from DNA damage mediated downregulation. Oncogenic stimuli compete with active ATM in regulating ARF expression. j. IB analysis of p14ARF in H1299 cells infected with pBabe (control) or pBabe-Ha-RasV12 and in the presence or absence of ATM inhibitor Ku55933, showing that the oncogenic challenge of H1299 cell (that already harbour mutant and activated K-Ras) decreases the endogenous levels of ARF that were re-established when ATM was inhibited. Apparently, the oncogene-ARF pathway, in this setting, has reached an activation plateau and any additional oncogenic stimulus activates ATM leading to ARF suppression. b. Two signalling routes lead to ARF induction, oncogenic challenge1 and ATM suppression. Given that oncogenes activate ATM, as well4,7,47,50,60, oncogenic insults trigger two pathways with opposing effects on ARF expression. The outcome of this antagonism will depend on whether the rate of ARF production by oncogenes exceeds or not the rate of ARF destruction by the oncogene induced ATM pathway. Actin serves as loading control. ATMi = Ku55933 addition, PBGD = Porphobilinogen deaminase (house-keeping gene), Dox = Doxorubicin, ATMi = Ku55933 addition, ctrsi = control siRNA, TAM = 4-OH-Tamoxifen.

Supplementary Figure 2 Activation of ATM promotes the degradation of ARF by disrupting the ARF-NPM/B23 complex.

a. TRIP12/ULF protein levels remain unchanged after ATMi or Doxorubicin treatment in H1299 cells. Actin serves as loading control. ATMi = Ku55933 addition. b. IB analysis in siNPM/B23 H1299 cells showing downregulation of p14ARF denoting the significance of NPM/B23 in p14ARF stabilization. c. Nucleolar changes after exposure to ATM inhibition and Doxorubicine. H1299 cells were treated with doxorubicine (2uM) and the ATM inhibitor Ku55933 (10uM) for 24h. Untreated and treated cells were fixed and subjected to immunofluorescence staining with antibodies against the indicated nucleolar markers. Fibrillarin and UBF nucleolar cap structures are indicated by arrowheads. Fluorescence signals were analyzed by CLSM. Dox = Doxorubicin, ATMi = Ku55933 addition.

Supplementary Figure 3 ATM regulates PP1 activity through I-2.

a. IB analysis of I-2 in H1299 cells treated with DMSO, Ku55933 or Doxorubicin. pI-2 designates the slower migrating phosphorylated form of I-2. b. The phosphomimetic S43D mutation of I-2 abrogates its inhibitory effect over PP1. Immunoblot analysis for H3Ser10 in H1299 cells transfected with empty vector, wtI-2 or I-2S43D, which served as a positive control of increased PP1 activity. Histogram depicting quantification of H3Ser10 protein levels in H1299 cells transfected with the phosphomimetic S43D (p = NS and p<0.001 (S43D) respectively, t-test, error bars indicate SDs, n = 3 blots). c. Immunofluorescence analysis demonstrating absence of p14ARF staining in nuclei transfected with the phosphomimetic I-2S44D-GFP compared to I-2-GFP and I-2S44A-GFP mutant form in H1299 cells. Overexpression of wtI-2 or I-2S43A and I-2S43D mutants does not affect nucleolar integrity or NPM/B23 localization, as shown by immunofluorescence analysis for the nucleolar marker fibrillarin (d) and NPM/B23. (e) The efficiency of the transfection was examined with GFP. Nuclei were stained with DAPI. The efficiency of the transfection was examined with GFP. Dotted lines define nuclei. Actin and H3 serve as loading control. NS = non-significant, Dox = Doxorubicin, ATMi = Ku55933 addition, A.U. = arbitrary optical density units.

Supplementary Figure 4 Representative immunoblot showing the specificity of the sip14ARF as it does not affect p16INK4A expression.

Supplementary Figure 5 ATM-dependent p14ARF regulation affects ribosomal biogenesis.

a. Schematic presentation of the findings described in panels b and c. b. IBs depicting the localization over time of NPM/B23 and p14ARF in ATM compromised H1299 cells in total lysates, nucleoplasmic and nucleolar fractions. c. Modulation of p14ARF levels by ATMi affects TTF-I nucleoplasmic/nucleolar localization. IBs depicting the status of TTF-I and p14ARF in H1299 total cell lysates, nucleoplasmic and nucleolar fractions, respectively upon DMSO, ATMi and ATMi/siARF treatments.

Supplementary Figure 6 Gene Set enrichment analyses revealed the depicted ‘translation pathway’ to be among the most significantly affected ones (p<10−4) by ATM inhibition (ATMi). ATMi = Ku55933 addition.

Supplementary Figure 7 ATM abrogation is inversely related with ARF expression in human cancer.

a. Cumulative data (histograms) depicting the distribution of the lung cancer cases examined in the current work, according to the status of ATM, p-ATM, Chk2, p14ARF and p16INK4A66,67,68,69. b. Meta-analysis regarding alterations in the ATM gene and its protein product levels. Histograms represent the percentage of ATM alterations in the corresponding neoplasias. The red bar is the result of the current study. d. Phosphorylated I-2 PP1 regulatory subunit expression corroborates with ATM status in human lung carcinomas. Cases with normal ATM demonstrate by IB analysis increased expression of the phosphorylated I-2 PP1 regulatory subunit (pI-2). The reduced intensity of the slower migrating pI-2 band upon calf intestinal phosphatase (CIP) treatment of the blots denotes the specificity of the antibody used for the detection of the phosphorylated form of the I-2 subunit. In contrast, cases with low ATM levels do not show any levels of the pI-2 subunit14,30,33. Actin serves as loading control.

Supplementary Figure 8 ATM abrogation is inversely related with ARF expression in tumours generated from grafted H1299 cells in immuno-compromised mice.

a. Validation of lenti-shATM silencing sequences (see Suppl. Tables S4) in H1299 cells showing down-regulation of ATM followed by increased expression of ARF. Actin serves as loading control. b Successful transduction of H1299 cells with lenti-GFP. Scale bar: 25 μm. c. Successful delivery and expression of GFP after injecting H1299-mock xenografts with lenti-GFP. Nuclei were stained with DAPI. Scale bar: 50 μm. d. Timetable of injections containing either pLKO.1 lenti-shATM or pLKO.1 lenti-non-target shRNA to H1299-mock and H1299-shARF xenografts. e. Tumours generated from grafted H1299 cells in immuno-compromised mice exhibited significantly reduced size after suppressing ATM expression for a period of 4 weeks (see also panels f-h) by injecting the tumours with lenti-shATM. The tumours remained unaffected when shATM was administrated in H1299-shARF xenografts. f. Tumours generated from grafted H1299 cells in immuno-compromised mice exhibited significantly reduced size after inhibition of ATM activity (ATMi), but remained unaffected in size when ATM inhibitor (ATMi) was administrated in H1299-shARF xenografts. Hematoxylin-eosin sections from the developed tumours (yellow arrow depicts invasion of abdominal wall muscle layer). Scale bar: 200 μm. IB analysis depicting the p14ARF protein levels in the control (EV), ATMi/EV and ATMi/shARF tumours. g. There was no significant difference in mass between tumours developed from H1299-shARF injected cells, with or without ATMi administration. h. Histogram presents the average mass between the groups of generated tumours (p = 0.007 and p = NS respectively, t-test, error bars indicate SDs, from 3 groups of 4 animals/group). Dependence of ARF expression upon ATM status in MEFs and mouse testis. i IBs showing that ATM null mouse embryo fibroblasts (MEFs) demonstrate higher levels of ARF compared to ATM+/+ MEFs, which remain unaffected upon irradiation, while in irradiated ATM+/+ MEFs ARF expression is lost. j. IHC staining demonstrating loss of ARF expression in mouse seminiferous tubules after irradiation (Scale bar: 50 μm). Histogram depicts p19ARF mRNA levels in mouse testis before and after irradiation (p = NS, t-test, error bars indicate SDs, from 3 animals/group). EV = empty vector, ATMi = caffeine addition, IR = irradiation, NS = non-significant.

Supplementary Figure 9 Full scans of blots.

Supplementary Table 1 Table 3: Studies examining genetic and epigenetic alterations in ATM and its mRNA and protein expression status in several sporadic malignancies.
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Supplementary Table 2 Table 4: Lentiviral vectors (MISSION) and inserts used from Sigma-Aldrich.
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Supplementary Table 3 Table 5: List of antibodies employed in immunohistochemistry, immunofluorescence, immunoblotting, immunoprecipitation and chromatin immunoprecipitation analyses.
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Supplementary Table 4 Table 6: Primers for mutagenesis and sequencing.
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Supplementary Table 5 Table 7: Datasets accession numbers for proteomic results deposited in PRIDE.
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Supplementary information

Supplementary Information

Supplementary Information (PDF 2154 kb)

Supplementary Table 1

Analytical presentation of all peptides and their modification recognized by the LC-MS/MS analysis from NPM-immunoprecipitated H1299. (see full table organized in accompanying excel file). (XLSX 235 kb)

Supplementary Table 2

Peptides (a), proteins (b) and filtered proteins (c) presenting high degree of reproducibility. Section (c) also contains p-values indicative of differential expression with confidence being set to 95%. (see full table organized in accompanying excel file). (XLSX 4173 kb)

Supplementary Table 3

Studies examining genetic and epigenetic alterations in ATM and its mRNA and protein expression status in several sporadic malignancies. (XLSX 16 kb)

Supplementary Table 4

Lentiviral vectors (MISSION®) and inserts used from Sigma-Aldrich. (XLSX 11 kb)

Supplementary Table 5

List of antibodies employed in immunohistochemistry, immunofluorescence, immunoblotting, immunoprecipitation and chromatin immunoprecipitation analyses. (XLSX 14 kb)

Supplementary Table 6

Primers for mutagenesis and sequencing. (XLSX 11 kb)

Supplementary Table 7

Datasets accession numbers for proteomic results deposited in PRIDE. (XLSX 11 kb)

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Velimezi, G., Liontos, M., Vougas, K. et al. Functional interplay between the DNA-damage-response kinase ATM and ARF tumour suppressor protein in human cancer. Nat Cell Biol 15, 967–977 (2013). https://doi.org/10.1038/ncb2795

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