Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance

Oncogene volume 32, pages 3543–3551 (2013)Cite this article

Abstract

Protein kinase A (PKA)-induced estrogen receptor alpha (ERα) phosphorylation at serine residue 305 (ERαS305-P) can induce tamoxifen (TAM) resistance in breast cancer. How this phospho-modification affects ERα specificity and translates into TAM resistance is unclear. Here, we show that S305-P modification of ERα reprograms the receptor, redirecting it to new transcriptional start sites, thus modulating the transcriptome. By altering the chromatin-binding pattern, Ser305 phosphorylation of ERα translates into a 26-gene expression classifier that identifies breast cancer patients with a poor disease outcome after TAM treatment. MYC-target genes and networks were significantly enriched in this gene classifier that includes a number of selective targets for ERαS305-P. The enhanced expression of MYC increased cell proliferation in the presence of TAM. We demonstrate that activation of the PKA signaling pathway alters the transcriptome by redirecting ERα to new transcriptional start sites, resulting in altered transcription and TAM resistance.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM . Cancer incidence and mortality worldwide. IARC CancerBase No. 10 2010.

  2. Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H et al. Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 2001; 98: 10869–10874.

    Article  CAS  Google Scholar 

  3. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998; 95: 927–937.

    Article  CAS  Google Scholar 

  4. Robertson JF . Selective oestrogen receptor modulators/new antioestrogens: a clinical perspective. Cancer Treat Rev 2004; 30: 695–706.

    Article  CAS  Google Scholar 

  5. Holm C, Kok M, Michalides R, Fles R, Koornstra RH, Wesseling J et al. Phosphorylation of the oestrogen receptor alpha at serine 305 and prediction of tamoxifen resistance in breast cancer. J Pathol 2009; 217: 372–379.

    Article  CAS  Google Scholar 

  6. Kok M, Zwart W, Holm C, Fles R, Hauptmann M, Van't Veer LJ et al. PKA-induced phosphorylation of ERalpha at serine 305 and high PAK1 levels is associated with sensitivity to tamoxifen in ER-positive breast cancer. Breast Cancer Res Treat 2011; 125: 1–12.

    Article  CAS  Google Scholar 

  7. Bostner J, Skoog L, Fornander T, Nordenskjold B, Stal O . Estrogen receptor-alpha phosphorylation at serine 305, nuclear p21-activated kinase 1 expression, and response to tamoxifen in postmenopausal breast cancer. Clin Cancer Res 2010; 16: 1624–1633.

    Article  CAS  Google Scholar 

  8. Massarweh S, Osborne CK, Creighton CJ, Qin L, Tsimelzon A, Huang S et al. Tamoxifen resistance in breast tumors is driven by growth factor receptor signaling with repression of classic estrogen receptor genomic function. Cancer Res 2008; 68: 826–833.

    Article  CAS  Google Scholar 

  9. Fan P, Wang J, Santen RJ, Yue W . Long-term treatment with tamoxifen facilitates translocation of estrogen receptor alpha out of the nucleus and enhances its interaction with EGFR in MCF-7 breast cancer cells. Cancer Res 2007; 67: 1352–1360.

    Article  CAS  Google Scholar 

  10. Shou J, Massarweh S, Osborne CK, Wakeling AE, Ali S, Weiss H et al. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer. J Natl Cancer Inst 2004; 96: 926–935.

    Article  CAS  Google Scholar 

  11. Riggins RB, Schrecengost RS, Guerrero MS, Bouton AH . Pathways to tamoxifen resistance. Cancer Lett 2007; 256: 1–24.

    Article  CAS  Google Scholar 

  12. Kirkegaard T, Witton CJ, McGlynn LM, Tovey SM, Dunne B, Lyon A et al. AKT activation predicts outcome in breast cancer patients treated with tamoxifen. J Pathol 2005; 207: 139–146.

    Article  CAS  Google Scholar 

  13. Gee JM, Robertson JF, Ellis IO, Nicholson RI . Phosphorylation of ERK1/2 mitogen-activated protein kinase is associated with poor response to anti-hormonal therapy and decreased patient survival in clinical breast cancer. Int J Cancer 2001; 95: 247–254.

    Article  CAS  Google Scholar 

  14. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995; 270: 1491–1494.

    Article  CAS  Google Scholar 

  15. Rayala SK, Molli PR, Kumar R . Nuclear p21-activated kinase 1 in breast cancer packs off tamoxifen sensitivity. Cancer Res 2006; 66: 5985–5988.

    Article  CAS  Google Scholar 

  16. Rayala SK, Talukder AH, Balasenthil S, Tharakan R, Barnes CJ, Wang RA et al. P21-activated kinase 1 regulation of estrogen receptor-alpha activation involves serine 305 activation linked with serine 118 phosphorylation. Cancer Res 2006; 66: 1694–1701.

    Article  CAS  Google Scholar 

  17. Michalides R, Griekspoor A, Balkenende A, Verwoerd D, Janssen L, Jalink K et al. Tamoxifen resistance by a conformational arrest of the estrogen receptor alpha after PKA activation in breast cancer. Cancer Cell 2004; 5: 597–605.

    Article  CAS  Google Scholar 

  18. de Leeuw R, Neefjes J, Michalides RA . Role for Estrogen Receptor Phosphorylation in the Resistance to Tamoxifen. Int J Breast Cancer 2011; 2011: 232435.

    Article  Google Scholar 

  19. Skliris GP, Rowan BG, Al Dhaheri M, Williams C, Troup S, Begic S et al. Immunohistochemical validation of multiple phospho-specific epitopes for estrogen receptor alpha (ERalpha) in tissue microarrays of ERalpha positive human breast carcinomas. Breast Cancer Res Treat 2009; 118: 443–453.

    Article  CAS  Google Scholar 

  20. Skliris GP, Nugent ZJ, Rowan BG, Penner CR, Watson PH, Murphy LC . A phosphorylation code for oestrogen receptor-alpha predicts clinical outcome to endocrine therapy in breast cancer. Endocr Relat Cancer 2010; 17: 589–597.

    Article  CAS  Google Scholar 

  21. Zwart W, Griekspoor A, Berno V, Lakeman K, Jalink K, Mancini M et al. PKA-induced resistance to tamoxifen is associated with an altered orientation of ERalpha towards co-activator SRC-1. EMBO J 2007; 26: 3534–3544.

    Article  CAS  Google Scholar 

  22. Dudek P, Picard D . Genomics of signaling crosstalk of estrogen receptor alpha in breast cancer cells. PLoS One 2008; 3: e1859.

    Article  Google Scholar 

  23. Miller WR . Regulatory subunits of PKA and breast cancer. Ann NY Acad Sci 2002; 968: 37–48.

    Article  CAS  Google Scholar 

  24. Wu RC, Qin J, Yi P, Wong J, Tsai SY, Tsai MJ et al. Selective phosphorylations of the SRC-3/AIB1 coactivator integrate genomic reponses to multiple cellular signaling pathways. Mol Cell 2004; 15: 937–949.

    Article  CAS  Google Scholar 

  25. Yi P, Feng Q, Amazit L, Lonard DM, Tsai SY, Tsai M et al. Atypical protein kinase C regulates dual pathways for degradation of the oncogenic coactivator SRC-3/AIB1. Mol Cell 2008; 29: 465–476.

    Article  CAS  Google Scholar 

  26. Carascossa S, Dudek P, Cenni B, Briand PA, Picard D . CARM1 mediates the ligand-independent and tamoxifen-resistant activation of the estrogen receptor alpha by cAMP. Genes Dev 2010; 24: 708–719.

    Article  CAS  Google Scholar 

  27. Lupien M, Eeckhoute J, Meyer CA, Krum SA, Rhodes DR, Liu XS et al. Coactivator function defines the active estrogen receptor alpha cistrome. Mol Cell Biol 2009; 29: 3413–3423.

    Article  CAS  Google Scholar 

  28. Lupien M, Meyer CA, Bailey ST, Eeckhoute J, Cook J, Westerling T et al. Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance. Genes Dev 2010; 24: 2219–2227.

    Article  CAS  Google Scholar 

  29. Reiner GC, Katzenellenbogen BS . Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells. Cancer Res 1986; 46: 1124–1131.

    CAS  PubMed  Google Scholar 

  30. Al Dhaheri MH, Rowan BG . Protein kinase A exhibits selective modulation of estradiol-dependent transcription in breast cancer cells that Is associated with decreased ligand binding, altered estrogen receptor {alpha} promoter interaction, and changes in receptor phosphorylation. Mol Endocrinol 2007; 21: 439–456.

    Article  CAS  Google Scholar 

  31. Bossis I, Stratakis CA . Minireview: PRKAR1A: normal and abnormal functions. Endocrinology 2004; 145: 5452–5458.

    Article  CAS  Google Scholar 

  32. Loi S, Haibe-Kains B, Desmedt C, Lallemand F, Tutt AM, Gillet C et al. Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol 2007; 25: 1239–1246.

    Article  CAS  Google Scholar 

  33. Buffa FM, Camps C, Winchester L, Snell CE, Gee HE, Sheldon H et al. microRNA-associated progression pathways and potential therapeutic targets identified by integrated mRNA and microRNA expression profiling in breast cancer. Cancer Res 2011; 71: 5635–5645.

    Article  CAS  Google Scholar 

  34. Chen D, Pace PE, Coombes RC, Ali S . Phosphorylation of human estrogen receptor alpha by protein kinase A regulates dimerization. Mol Cell Biol 1999; 19: 1002–1015.

    Article  CAS  Google Scholar 

  35. Schmidt D, Wilson MD, Spyrou C, Brown GD, Hadfield J, Odom DT . ChIP-seq: using high-throughput sequencing to discover protein-DNA interactions. Methods 2009; 48: 240–248.

    Article  CAS  Google Scholar 

  36. Robinson JL, Macarthur S, Ross-Innes CS, Tilley WD, Neal DE, Mills IG et al. Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1. EMBO J 2011; 30: 3019–3027.

    Article  CAS  Google Scholar 

  37. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J et al. Genome-wide analysis of estrogen receptor binding sites. Nat Genet 2006; 38: 1289–1297.

    Article  CAS  Google Scholar 

  38. Madak-Erdogan Z, Lupien M, Stossi F, Brown M, Katzenellenbogen BS . Genomic collaboration of estrogen receptor alpha and extracellular signal-regulated kinase 2 in regulating gene and proliferation programs. Mol Cell Biol 2011; 31: 226–236.

    Article  CAS  Google Scholar 

  39. Miller TW, Balko JM, Ghazoui Z, Dunbier A, Anderson H, Dowsett M et al. A gene expression signature from human breast cancer cells with acquired hormone independence identifies MYC as a mediator of antiestrogen resistance. Clin Cancer Res 2011; 17: 2024–2034.

    Article  CAS  Google Scholar 

  40. Musgrove EA, Sergio CM, Loi S, Inman CK, Anderson LR, Alles MC et al. Identification of functional networks of estrogen- and c-Myc-responsive genes and their relationship to response to tamoxifen therapy in breast cancer. PLoS One 2008; 3: e2987.

    Article  Google Scholar 

  41. Louie MC, McClellan A, Siewit C, Kawabata L . Estrogen receptor regulates E2F1 expression to mediate tamoxifen resistance. Mol Cancer Res 2010; 8: 343–352.

    Article  CAS  Google Scholar 

  42. Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB et al. An oestrogen-receptor-alpha-bound human chromatin interactome. Nature 2009; 462: 58–64.

    Article  CAS  Google Scholar 

  43. Wang C, Mayer JA, Mazumdar A, Fertuck K, Kim H, Brown M et al. Estrogen induces c-myc gene expression via an upstream enhancer activated by the estrogen receptor and the AP-1 transcription factor. Mol Endocrinol 2011; 25: 1527–1538.

    Article  CAS  Google Scholar 

  44. Inoue A, Omoto Y, Yamaguchi Y, Kiyama R, Hayashi SI . Transcription factor EGR3 is involved in estrogen-signaling pathway in breast cancer cells. J Mol Endocrinol 2004; 32: 649–661.

    Article  CAS  Google Scholar 

  45. Suzuki T, Inoue A, Miki Y, Moriya T, Akahira J, Ishida T et al. Early growth responsive gene 3 in human breast carcinoma: a regulator of estrogen-meditated invasion and a potent prognostic factor. Encocr Relat Cancer 2007; 14: 279–292.

    Article  CAS  Google Scholar 

  46. Frietze S, Lupien M, Silver PA, Brown M . CARM1 regulates estrogen-stimulated breast cancer growth through up-regulation of E2F1. Cancer Res 2008; 68: 301–306.

    Article  CAS  Google Scholar 

  47. Callero MA, Loaiza-Pérez AI . The role of aryl hydrocarbon receptor and crosstalk with estrogen receptor in response of breast cancer cells to the novel antitumor agents benzothiazoles and aminoflavone. Int J Breast Cancer 2011; 2011: 923250.

    Article  Google Scholar 

  48. Madak-Erdogan Z, Katzenellenbogen BS . Aryl hydrocarbon receptor modulation of estroge receptor a-mediated gene regulation by a multimeric chromatin complex involving the two receptors and the coregulator RIP140. Toxicol Sci 2012; 125: 401–411.

    Article  CAS  Google Scholar 

  49. Chandra V, Huang P, Hamuro Y, Raghuram S, Wang Y, Burris TP et al. Structure of the intact PPAR-gamma-RXR-alpha nuclear receptor complex on DNA. Nature 2008; 456: 350–356.

    Article  Google Scholar 

  50. Krum SA, Miranda-Carboni GA, Lupien M, Eeckhoute J, Carroll JS, Brown M . Unique ERalpha cistromes control cell type-specific gene regulation. Mol Endocrinol 2008; 22: 2393–2406.

    Article  CAS  Google Scholar 

  51. Welboren WJ, van Driel MA, Janssen-Megens EM, van Heeringen SJ, Sweep FC, Span PN et al. ChIP-Seq of ERalpha and RNA polymerase II defines genes differentially responding to ligands. EMBO J 2009; 28: 1418–1428.

    Article  CAS  Google Scholar 

  52. Hurtado A, Holmes KA, Ross-Innes CS, Schmidt D, Carroll JS . FOXA1 is a key determinant of estrogen receptor function and endocrine response. Nat Genet 2009; 43: 27–33.

    Article  Google Scholar 

  53. Carroll JS, Liu XS, Brodsky AS, Li W, Meyer CA, Szary AJ et al. Chromosome-wide mapping of estrogen receptor binding reveals long-range regulation requiring the forkhead protein FoxA1. Cell 2005; 15: 33–43.

    Article  Google Scholar 

  54. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol 2008; 9: R137.

    Article  Google Scholar 

  55. Zang C, Schones DE, Zeng C, Cui K, Zhao K, Peng W . A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics 2009; 25: 1952–1958.

    Article  CAS  Google Scholar 

  56. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci USA 1995; 92: 7297–7301.

    Article  CAS  Google Scholar 

  57. Ji X, Li W, Song J, Wei L, Liu XS . CEAS: cis-regulatory element annotation system. Nucleic Acids Res 2006; 34: W551–W554.

    Article  CAS  Google Scholar 

  58. He HH, Meyer CA, Shin H, Bailey ST, Wei G, Wang Q et al. Nucleosome dynamics define transcriptional enhancers. Nat Genet 2010; 42: 343–347.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Central Microarray Facility of the Netherlands Cancer Institute for processing the microarray samples. RL is in part supported by Top Institute Pharma. WZ is supported by a KWF Dutch Cancer Society Fellowship. Author contributions: RL, JN, RM and WZ designed all the experiments. RL and KF conducted all the experiments, with help from CBT and XA. RL and SC conducted all bioinformatics analyses. The paper was written by RL, JN and WZ, with help from all other authors.

Author information

Authors and Affiliations

  1. Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    R de Leeuw, C Bentin Toaldo, J Neefjes & R Michalides

  2. Department of Molecular Pathology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    K Flach, X Alexi, S Canisius & W Zwart

  3. Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    S Canisius

Authors
  1. R de Leeuw
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K Flach
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C Bentin Toaldo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. X Alexi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S Canisius
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. J Neefjes
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R Michalides
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. W Zwart
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to W Zwart.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

de Leeuw, R., Flach, K., Bentin Toaldo, C. et al. PKA phosphorylation redirects ERα to promoters of a unique gene set to induce tamoxifen resistance. Oncogene 32, 3543–3551 (2013). https://doi.org/10.1038/onc.2012.361

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2012.361

Keywords

This article is cited by

Search

Advanced search

Quick links