Breast Cancer Research and Treatment

, Volume 147, Issue 2, pp 295–309 | Cite as

Phosphorylation of activating transcription factor-2 (ATF-2) within the activation domain is a key determinant of sensitivity to tamoxifen in breast cancer

  • Bharath Rudraraju
  • Marjolein Droog
  • Tarek M. A. Abdel-Fatah
  • Wilbert Zwart
  • Athina Giannoudis
  • Mohammed I. Malki
  • David Moore
  • Hetal Patel
  • Jacqui Shaw
  • Ian O. Ellis
  • Steve Chan
  • Greg N. Brooke
  • Ekaterina Nevedomskaya
  • Christiana Lo Nigro
  • Jason Carroll
  • R. Charles Coombes
  • Charlotte Bevan
  • Simak Ali
  • Carlo Palmieri
Preclinical study


Activating transcription factor-2 (ATF-2) has been implicated as a tumour suppressor in breast cancer (BC). c-JUN N-terminal kinase (JNK) and p38 MAPK phosphorylate ATF-2 within the activation domain (AD), which is required for its transcriptional activity. To date, the role of ATF-2 in determining response to endocrine therapy has not been explored. Effects of ATF-2 loss in the oestrogen receptor (ER)-positive luminal BC cell line MCF7 were explored, as well as its role in response to tamoxifen treatment. Genome-wide chromatin binding patterns of ATF-2 when phosphorylated within the AD in MCF-7 cells were determined using ChIP-seq. The expression of ATF-2 and phosphorylated ATF-2 (pATF-2-Thr71) was determined in a series of 1,650 BC patients and correlated with clinico-pathological features and clinical outcome. Loss of ATF-2 diminished the growth-inhibitory effects of tamoxifen, while tamoxifen treatment induced ATF-2 phosphorylation within the AD, to regulate the expression of a set of 227 genes for proximal phospho-ATF-2 binding, involved in cell development, assembly and survival. Low expression of both ATF-2 and pATF-2-Thr71 was significantly associated with aggressive pathological features. Furthermore, pATF-2 was associated with both p-p38 and pJNK1/2 (< 0.0001). While expression of ATF-2 is not associated with outcome, pATF-2 is associated with longer disease-free (p = 0.002) and BC-specific survival in patients exposed to tamoxifen (p = 0.01). Furthermore, multivariate analysis confirmed pATF-2-Thr71 as an independent prognostic factor. ATF-2 is important for modulating the effect of tamoxifen and phosphorylation of ATF-2 within the AD at Thr71 predicts for improved outcome for ER-positive BC receiving tamoxifen.


Breast cancer Activating transcription factor-2 Phosphorylation Tamoxifen 



Carlo Palmieri was supported by a clinician scientist fellowship from Cancer Research UK, Wilbert Zwart by a KWF Dutch Cancer Society Fellowship and a VENI scholarship from the Dutch Organisation for Scientific Research NWO, and Jason Carroll by an ERC starting grant and an EMBO Young investigator award. We thank Angie Gillies (University of Leicester) for technical help with immunohistochemistry. We would also like to acknowledge the support of Cancer Research UK Cambridge Research Institute, The Netherlands Cancer Institute and A Sisters Hope. The Department of Molecular and Clinical Cancer Medicine forms part of the North West Cancer Centre-University of Liverpool which is funded by North West Cancer Research. Research support is also received from The Clatterbridge Cancer Charity.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary material

10549_2014_3098_MOESM1_ESM.pdf (982 kb)
Supplementary material 1 (PDF 981 kb)
10549_2014_3098_MOESM2_ESM.pdf (330 kb)
Supplementary material 2 (PDF 330 kb)
10549_2014_3098_MOESM3_ESM.docx (13 kb)
Supplementary material 3 (DOCX 13 kb)


  1. 1.
    Goldhirsch A, Glick JH, Gelber RD, Coates AS, Thürlimann B, Senn HJ, Panel members (2005) Meeting highlights: international expert consensus on the primary therapy of early breast cancer. Ann Oncol 16:1569–1583PubMedCrossRefGoogle Scholar
  2. 2.
    Early Breast Cancer Trialists’ Collaborative Group (2011) Relevance of breast cancer hormone receptors and other factors to the efficacy of adjuvant tamoxifen: patient-level meta-analysis of randomised trials. Lancet 378:771–784CrossRefGoogle Scholar
  3. 3.
    Ali S, Coombes RC (2002) Endocrine-responsive breast cancer and strategies for combating resistance. Nat Rev Cancer 2:101–112PubMedCrossRefGoogle Scholar
  4. 4.
    Palmieri C, Patten DK, Januszewski A, Zucchini G, Howell SJ (2014) Breast Cancer: current and future endocrine therapies. Mol Cell Endo 382:695–723CrossRefGoogle Scholar
  5. 5.
    Hai T, Hartman MG (2001) The molecular biology and nomenclature of the activating transcription factor/cAMP responsive element binding family of transcription factors: activating transcription factor proteins and homeostasis. Gene 273:1–11PubMedCrossRefGoogle Scholar
  6. 6.
    Hai TW, Liu F, Coukos WJ, Green MR (1989) Transcription factor ATF cDNA clones: an extensive family of leucine zipper proteins able to selectively form DNA-binding heterodimers. Genes Dev 3:2083–2090PubMedCrossRefGoogle Scholar
  7. 7.
    Hai T, Curran T (1991) Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity. Proc Natl Acad Sci USA 88:3720–3724PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Matsuda S, Maekawa T, Ishii S (1991) Identification of the functional domains of the transcriptional regulator CRE-BP1. J Biol Chem 266:18188–18193PubMedGoogle Scholar
  9. 9.
    van Dam H, Duyndam M, Rottier R, Bosch A, de Vries-Smits L, Herrlich P, Zantema A, Angel P, van der Eb AJ (1993) Heterodimer formation of c-Jun and ATF-2 is responsible for induction of c-jun by the 243 amino acid adenovirus E1A protein. EMBO J 12:479–487PubMedCentralPubMedGoogle Scholar
  10. 10.
    Kim HS, Choi ES, Shin JA, Jang YK, Park SD (2004) Regulation of Swi6/HP1-dependent heterochromatin assembly by cooperation of components of the mitogen-activated protein kinase pathway and a histone deacetylase Clr6. J Biol Chem 279:42850–42859PubMedCrossRefGoogle Scholar
  11. 11.
    Agelopoulos M, Thanos D (2006) Epigenetic determination of a cell-specific gene expression program by ATF-2 and the histone variant macroH2A. EMBO J 25:4843–4853PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Bruhat A, Cherasse Y, Maurin AC, Breitwieser W, Parry L, Deval C, Jones N, Jousse C, Fafournoux P (2007) ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation. Nucleic Acids Res 35:1312–1321PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Li XY, Green MR (1996) Intramolecular inhibition of activating transcription factor-2 function by its DNA binding domain. Genes Dev 10:517–527PubMedCrossRefGoogle Scholar
  14. 14.
    Gupta S, Campbell D, Derijard B, Davis RJ (1995) Transcription factor ATF2 regulation by the JNK signal transduction pathway. Science 267:389–393PubMedCrossRefGoogle Scholar
  15. 15.
    Livingstone C, Patel G, Jones N (1995) ATF-2 contains a phosphorylation-dependent transcriptional activation domain. EMBO J 14:1785–1797PubMedCentralPubMedGoogle Scholar
  16. 16.
    Raingeaud J, Whitmarsh AJ, Barrett T, Dérijard B, Davis RJ (1996) MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol 16:1247–1255PubMedCentralPubMedGoogle Scholar
  17. 17.
    Ouwens DM, de Ruiter ND, van der Zon GC, Carter AP, Schouten J, van der Burgt C, Kooistra K, Bos JL, Maassen JA, van Dam H (2002) Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38. EMBO J 21:3782–3793PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    van Dam H, Wilhelm D, Herr I, Steffen A, Herrlich P, Angel P (1995) ATF-2 is preferentially activated by stress-activated protein kinases to mediate c-jun induction in response to genotoxic agents. EMBO J 14:1798–1811PubMedCentralPubMedGoogle Scholar
  19. 19.
    Tsay YG, Wang YH, Chiu CM, Shen BJ, Lee SC (2000) A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry. Anal Biochem 287:55–64PubMedCrossRefGoogle Scholar
  20. 20.
    Sakurai A, Maekawa T, Sudo T, Ishii S, Kishimoto A (1991) Phosphorylation of cAMP response element-binding protein, CRE-BP1, by cAMP-dependent protein kinase and protein kinase C. Biochem Biophys Res Commun 181:629–635PubMedCrossRefGoogle Scholar
  21. 21.
    Yamasaki T, Takahashi A, Pan J, Yamaguchi N, Yokoyama KK (2009) Phosphorylation of activation transcription factor-2 at serine 121 by protein kinase C controls c-Jun-mediated activation of transcription. J Biol Chem 284:8567–8581PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Bhoumik A, Takahashi S, Breitweiser W, Shiloh Y, Jones N, Ronai Z (2005) ATM-dependent phosphorylation of ATF2 is required for the DNA damage response. Mol Cell 18:577–587PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Lewis JS, Vijayanathan V, Thomas TJ, Pestell RG, Albanese C, Gallo MA, Thomas T (2005) Activation of cyclin D1 by estradiol and spermine in MCF-7 breast cancer cells: a mechanism involving the p38 MAP kinase and phosphorylation of ATF-2. Oncol Res 15:113–128PubMedGoogle Scholar
  24. 24.
    Song H, Ki SH, Kim SG, Moon A (2006) Activating transcription factor 2 mediates matrix metalloproteinase-2 transcriptional activation induced by p38 in breast epithelial cells. Cancer Res 66:10487–10496PubMedCrossRefGoogle Scholar
  25. 25.
    Lewis JS, Thomas TJ, Pestell RG, Albanese C, Gallo MA, Thomas T (2005) Differential effects of 16α-hydroxyestrone and 2-methoxyestradiol on cyclin D1 involving the transcription factor ATF-2 in MCF-7 breast cancer cells. J Mol Endo 34:91–105CrossRefGoogle Scholar
  26. 26.
    Hayakawa J, Depatie C, Ohmichi M, Mercola D (2003) The activation of c-Jun NH2-terminal kinase (JNK) by DNA-damaging agents serves to promote drug resistance via activating transcription factor 2 (ATF2)-dependent enhanced DNA repair. J Biol Chem 278:20582–20592PubMedCrossRefGoogle Scholar
  27. 27.
    Hayakawa J, Mittal S, Wang Y, Korkmaz KS, Adamson E, English C, Ohmichi M, McClelland M, Mercola D (2004) Identification of promoters bound by c-Jun/ATF2 during rapid large-scale gene activation following genotoxic stress. Mol Cell 16:521–535PubMedCrossRefGoogle Scholar
  28. 28.
    Maekawa T, Shinagawa T, Sano Y, Sakuma T, Nomura S, Nagasaki K, Miki Y, Saito-Ohara F, Inazawa J, Kohno T, Yokota J, Ishii S (2007) Reduced levels of ATF-2 predispose mice to mammary tumourtumours. Mol Cell Biol 27:1730–1744PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Liu Y, Wang Y, Li W, Zheng P (2009) Activating transcription factor 2 and c-Jun-mediated induction of FoxP3 for experimental therapy of mammary tumour in the mouse. Cancer Res 69:5954–5960PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Knippen S, Löning T, Müller V, Schröder C, Jänicke F, Milde-Langosch K (2009) Expression and prognostic value of activating transcription factor 2 (ATF2) and its phosphorylated form in mammary carcinomas. Anticancer Res 29:183–189PubMedGoogle Scholar
  31. 31.
    Lopez-Garcia J, Periyasamy M, Thomas RS, Christian M, Leao M, Jat P, Kindle KB, Heery DM, Parker MG, Buluwela L, Kamalati T, Ali S (2006) ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases. Nucleic Acids Res 34:6126–6136PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    He HH, Meyer CA, Shin H, Bailey ST, Wei G, Wang Q, Zhang Y, Xu K, Ni M, Lupien M, Mieczkowski P, Lieb JD, Zhao K, Brown M, Liu XS (2010) Nucleosome dynamics define transcriptional enhancers. Nat Genet 42:343–347PubMedCentralPubMedCrossRefGoogle Scholar
  33. 33.
    Loi S, Haibe-Kains B, Desmedt C, Lallemand F, Tutt AM, Gillet C, Ellis P, Harris A, Bergh J, Foekens JA, Klijn JG, Larsimont D, Buyse M, Bontempi G, Delorenzi M, Piccart MJ, Sotiriou C (2007) Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol 25:1239–1246PubMedCrossRefGoogle Scholar
  34. 34.
    Wang Y, Klijn JG, Zhang Y, Sieuwerts AM, Look MP, Yang F, Talantov D, Timmermans M, Meijer-van Gelder ME, Yu J, Jatkoe T, Berns EM, Atkins D, Foekens JA (2005) Gene-expression profiles to predict distant metastasis of lymph-node-negative primary breast cancer. Lancet 365:671–679PubMedCrossRefGoogle Scholar
  35. 35.
    Ellis IO, Galea M, Broughton N, Locker A, Blamey RW, Elston CW (1992) Pathological prognostic factors in breast cancer. II. Histological type. Relationship with survival in a large study with long-term follow-up. Histopathology 20:479–489PubMedCrossRefGoogle Scholar
  36. 36.
    Abdel-Fatah TM, Perry C, Dickinson P, Ball G, Moseley P, Madhusudan S, Ellis IO, Chan SY (2013) Bcl2 is an independent prognostic marker of triple negative breast cancer (TNBC) and predicts response to anthracycline combination (ATC) chemotherapy (CT) in adjuvant and neoadjuvant settings. Ann Oncol 11:2801–2807CrossRefGoogle Scholar
  37. 37.
    Sultana R, Abdel-Fatah T, Abbotts R, Hawkes C, Albarakati N, Seedhouse C, Ball G, Chan S, Rakha EA, Ellis IO, Madhusudan S (2013) Targeting XRCC1 deficiency in breast cancer for personalized therapy. Cancer Res 73:1621–1634PubMedCrossRefGoogle Scholar
  38. 38.
    Berger AJ, Kluger HM, Li N, Kielhorn E, Halaban R, Ronai Z, Rimm DL (2003) Subcellular localization of activating transcription factor 2 in melanoma specimens predicts patient survival. Cancer Res 63:8103–8107PubMedGoogle Scholar
  39. 39.
    Bhoumik A, Fichtman B, Derossi C, Breitwieser W, Kluger HM, Davis Subtil A, Meltzer P, Krajewski S, Jones N, Ronai Z (2008) Suppressor role of activating transcription factor 2 (ATF2) in skin cancer. Proc Natl Acad Sci USA 105:1674–1679PubMedCentralPubMedCrossRefGoogle Scholar
  40. 40.
    Choi JH, Cho HK, Choi YH, Cheong J (2009) Activating transcription factor 2 increases transactivation and protein stability of hypoxia-inducible factor 1alpha in hepatocytes. Biochem J 42:4285–4296Google Scholar
  41. 41.
    Reimold AM, Grusby MJ, Kosaras B, Fries JW, Mori R, Maniwa S, Clauss IM, Collins T, Sidman RL, Glimcher MJ, Glimcher LH (1996) Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice. Nature 379:262–265PubMedCrossRefGoogle Scholar
  42. 42.
    Lau E, Ronai ZA (2012) ATF2: at the crossroad of nuclear and cytosolic functions. J Cell Sci 125:2815–2824PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Lindaman LL, Yeh DM, Xie C, Breen KM, Coss D (2013) Phosphorylation of ATF2 and interaction with NFY induces c-Jun in the gonadotrope. Mol Cell Endocrinol 365:316–326PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Nogueira EF, Rainey WE (2010) Regulation of aldosterone synthase by activator transcription factor/cAMP response element-binding protein family members. Endocrinology 151:1060–1070PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Mynard V, Latchoumanin O, Guignat L, Devin-Leclerc J, Bertagna X, Barré B, Fagart J, Coqueret O, Catelli MG (2004) Synergistic signaling by corticotropin-releasing hormone and leukemia inhibitory factor bridged by phosphorylated 3′,5′-cyclic adenosine monophosphate response element binding protein at the Nur response element (NurRE)-signal transducers and activators of transcription (STAT) element of the proopiomelanocortin promoter. Mol Endocrinol 18:2997–3010PubMedCrossRefGoogle Scholar
  46. 46.
    Kodama S, Moore R, Yamamoto Y, Negishi M (2007) Human nuclear pregnane X receptor cross-talk with CREB to repress cAMP activation of the glucose-6-phosphatase gene. Biochem J 407:373–381PubMedCentralPubMedCrossRefGoogle Scholar
  47. 47.
    Mandlekar S, Yu R, Tan TH, Kong AN (2000) Activation of caspase-3 and c-Jun NH2-terminal kinase-1 signaling pathways in tamoxifen-induced apoptosis of human breast cancer cells. Cancer Res 60:5995–6000PubMedGoogle Scholar
  48. 48.
    Zhang CC, Shapiro DJ (2000) Activation of the p38 mitogen-activated protein kinase pathway by estrogen or by 4-hydroxytamoxifen is coupled to estrogen receptor-induced apoptosis. J Biol Chem 275:479–486PubMedCrossRefGoogle Scholar
  49. 49.
    Buck MB, Pfizenmaier K, Knabbe C (2004) Antiestrogens induce growth inhibition by sequential activation of p38 mitogen-activated protein kinase and transforming growth factor-beta pathways in human breast cancer cells. Mol Endocrinol 18:1643–1657PubMedCrossRefGoogle Scholar
  50. 50.
    Ellis MJ, Perou CM (2013) The genomic landscape of breast cancer as a therapeutic roadmap. Cancer Dis 3:27–34CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Bharath Rudraraju
    • 1
  • Marjolein Droog
    • 2
  • Tarek M. A. Abdel-Fatah
    • 3
  • Wilbert Zwart
    • 2
  • Athina Giannoudis
    • 1
  • Mohammed I. Malki
    • 1
  • David Moore
    • 4
  • Hetal Patel
    • 5
  • Jacqui Shaw
    • 4
  • Ian O. Ellis
    • 6
  • Steve Chan
    • 3
  • Greg N. Brooke
    • 7
  • Ekaterina Nevedomskaya
    • 2
    • 8
  • Christiana Lo Nigro
    • 9
  • Jason Carroll
    • 10
  • R. Charles Coombes
    • 5
  • Charlotte Bevan
    • 5
  • Simak Ali
    • 5
  • Carlo Palmieri
    • 1
    • 11
    • 12
  1. 1.Department of Molecular and Clinical Cancer Medicine, Institute of Translational MedicineUniversity of LiverpoolLiverpoolUK
  2. 2.Division of Molecular PathologyThe Netherlands Cancer InstituteAmsterdamThe Netherlands
  3. 3.Division of Pathology, School of Molecular Medical SciencesNottingham University Hospitals and University of NottinghamNottinghamUK
  4. 4.Department of Cancer Studies and Molecular MedicineUniversity of LeicesterLeicesterUK
  5. 5.Cancer Research UK Laboratories, Division of CancerImperial College LondonLondonUK
  6. 6.Division of Pathology, School of Molecular Medical SciencesUniversity of NottinghamNottinghamUK
  7. 7.School of Biological SciencesUniversity of EssexEssexUK
  8. 8.Department of Molecular CarcinogenesisThe Netherlands Cancer InstituteAmsterdamThe Netherlands
  9. 9.Laboratory of Cancer Research and Translational Oncology, Oncology DepartmentS. Croce General HospitalCuneoItaly
  10. 10.Cancer Research UK, Cambridge Research InstituteCambridgeUK
  11. 11.Liverpool & Merseyside Academic Breast UnitRoyal Liverpool University HospitalLiverpoolUK
  12. 12.Academic Department of Medical OncologyClatterbridge Cancer Centre NHS Foundation TrustWirralUK

Personalised recommendations