Analysis of HER2 genomic binding in breast cancer cells identifies a global role in direct gene regulation

Autoři: Aisling M. Redmond aff001;  Soleilmane Omarjee aff001;  Igor Chernukhin aff001;  Muriel Le Romancer aff002;  Jason S. Carroll aff001
Působiště autorů: University of Cambridge, Cancer Research UK Cambridge Institute, Cambridge, United Kingdom aff001;  Université Lyon 1, Lyon, France aff002;  Inserm U1052, Centre de Recherche en Cancérologie de Lyon, Lyon, France aff003;  CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon, Lyon, France aff004
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225180


HER2 is a transmembrane receptor tyrosine kinase, which plays a key role in breast cancer due to a common genomic amplification. It is used as a marker to stratify patients in the clinic and is targeted by a number of drugs including Trastuzumab and Lapatinib. HER2 has previously been shown to translocate to the nucleus. In this study, we have explored the properties of nuclear HER2 by analysing the binding of this protein to the chromatin in two breast cancer cell lines. We find genome-wide re-programming of HER2 binding after treatment with the growth factor EGF and have identified a de novo motif at HER2 binding sites. Over 2,000 HER2 binding sites are found in both breast cancer cell lines after EGF treatment, and according to pathway analysis, these binding sites were enriched near genes involved in protein kinase activity and signal transduction. HER2 was shown to co-localise at a small subset of regions demarcated by H3K4me1, a hallmark of functional enhancer elements and HER2/H3K4me1 co-bound regions were enriched near EGF regulated genes providing evidence for their functional role as regulatory elements. A chromatin bound role for HER2 was verified by independent methods, including Proximity Ligation Assay (PLA), which confirmed a close association between HER2 and H3K4me1. Mass spectrometry analysis of the chromatin bound HER2 complex identified EGFR and STAT3 as interacting partners in the nucleus. These findings reveal a global role for HER2 as a chromatin-associated factor that binds to enhancer elements to elicit direct gene expression events in breast cancer cells.

Klíčová slova:

Binding analysis – Breast cancer – Cell binding – Gene expression – Gene regulation – Chromatin – Sequence motif analysis – BT474 cells


1. Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ, et al. The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups. Nature. 2012;486(7403):346–52. Epub 2012/04/24. doi: 10.1038/nature10983 22522925

2. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235(4785):177–82. Epub 1987/01/09. doi: 10.1126/science.3798106 3798106.

3. Carter P, Presta L, Gorman CM, Ridgway JB, Henner D, Wong WL, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proceedings of the National Academy of Sciences of the United States of America. 1992;89(10):4285–9. Epub 1992/05/15. doi: 10.1073/pnas.89.10.4285 1350088

4. Ryan Q, Ibrahim A, Cohen MH, Johnson J, Ko CW, Sridhara R, et al. FDA drug approval summary: lapatinib in combination with capecitabine for previously treated metastatic breast cancer that overexpresses HER-2. The oncologist. 2008;13(10):1114–9. Epub 2008/10/14. doi: 10.1634/theoncologist.2008-0816 18849320.

5. Krop IE, Kim SB, Martin AG, LoRusso PM, Ferrero JM, Badovinac-Crnjevic T, et al. Trastuzumab emtansine versus treatment of physician’s choice in patients with previously treated HER2-positive metastatic breast cancer (TH3RESA): final overall survival results from a randomised open-label phase 3 trial. Lancet Oncol. 2017. doi: 10.1016/S1470-2045(17)30313-3 28526538.

6. Xie Y, Hung MC. Nuclear localization of p185neu tyrosine kinase and its association with transcriptional transactivation. Biochem Biophys Res Commun. 1994;203(3):1589–98. doi: 10.1006/bbrc.1994.2368 7945309.

7. Giri DK, Ali-Seyed M, Li LY, Lee DF, Ling P, Bartholomeusz G, et al. Endosomal transport of ErbB-2: mechanism for nuclear entry of the cell surface receptor. Molecular and cellular biology. 2005;25(24):11005–18. Epub 2005/11/30. doi: 10.1128/MCB.25.24.11005-11018.2005 16314522

8. Beguelin W, Diaz Flaque MC, Proietti CJ, Cayrol F, Rivas MA, Tkach M, et al. Progesterone receptor induces ErbB-2 nuclear translocation to promote breast cancer growth via a novel transcriptional effect: ErbB-2 function as a coactivator of Stat3. Molecular and cellular biology. 2010;30(23):5456–72. doi: 10.1128/MCB.00012-10 20876300

9. Diaz Flaque MC, Galigniana NM, Beguelin W, Vicario R, Proietti CJ, Russo R, et al. Progesterone receptor assembly of a transcriptional complex along with activator protein 1, signal transducer and activator of transcription 3 and ErbB-2 governs breast cancer growth and predicts response to endocrine therapy. Breast Cancer Res. 2013;15(6):R118. doi: 10.1186/bcr3587 24345432

10. Diaz Flaque MC, Vicario R, Proietti CJ, Izzo F, Schillaci R, Elizalde PV. Progestin drives breast cancer growth by inducing p21(CIP1) expression through the assembly of a transcriptional complex among Stat3, progesterone receptor and ErbB-2. Steroids. 2013;78(6):559–67. doi: 10.1016/j.steroids.2012.11.003 23178160.

11. Wang SC, Lien HC, Xia W, Chen IF, Lo HW, Wang Z, et al. Binding at and transactivation of the COX-2 promoter by nuclear tyrosine kinase receptor ErbB-2. Cancer cell. 2004;6(3):251–61. Epub 2004/09/24. doi: 10.1016/j.ccr.2004.07.012 15380516.

12. Venturutti L, Romero LV, Urtreger AJ, Chervo MF, Cordo Russo RI, Mercogliano MF, et al. Stat3 regulates ErbB-2 expression and co-opts ErbB-2 nuclear function to induce miR-21 expression, PDCD4 downregulation and breast cancer metastasis. Oncogene. 2016;35(17):2208–22. doi: 10.1038/onc.2015.281 26212010.

13. Li LY, Chen H, Hsieh YH, Wang YN, Chu HJ, Chen YH, et al. Nuclear ErbB2 enhances translation and cell growth by activating transcription of ribosomal RNA genes. Cancer Res. 2011;71(12):4269–79. doi: 10.1158/0008-5472.CAN-10-3504 21555369

14. Li X, Kuang J, Shen Y, Majer MM, Nelson CC, Parsawar K, et al. The atypical histone macroH2A1.2 interacts with HER-2 protein in cancer cells. The Journal of biological chemistry. 2012;287(27):23171–83. doi: 10.1074/jbc.M112.379412 22589551

15. Dillon MF, Stafford AT, Kelly G, Redmond AM, McIlroy M, Crotty TB, et al. Cyclooxygenase-2 predicts adverse effects of tamoxifen: a possible mechanism of role for nuclear HER2 in breast cancer patients. Endocrine-related cancer. 2008;15(3):745–53. Epub 2008/05/13. doi: 10.1677/ERC-08-0009 18469157.

16. Schillaci R, Guzman P, Cayrol F, Beguelin W, Diaz Flaque MC, Proietti CJ, et al. Clinical relevance of ErbB-2/HER2 nuclear expression in breast cancer. BMC cancer. 2012;12:74. Epub 2012/02/24. doi: 10.1186/1471-2407-12-74 22356700

17. Cordo Russo RI, Beguelin W, Diaz Flaque MC, Proietti CJ, Venturutti L, Galigniana N, et al. Targeting ErbB-2 nuclear localization and function inhibits breast cancer growth and overcomes trastuzumab resistance. Oncogene. 2015;34(26):3413–28. Epub 2014/09/02. doi: 10.1038/onc.2014.272 25174405.

18. Serandour AA, Brown GD, Cohen JD, Carroll JS. Development of an Illumina-based ChIP-exonuclease method provides insight into FoxA1-DNA binding properties. Genome biology. 2013;14(12):R147. Epub 2014/01/01. doi: 10.1186/gb-2013-14-12-r147 24373287.

19. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based analysis of ChIP-Seq (MACS). Genome biology. 2008;9(9):R137. Epub 2008/09/19. doi: 10.1186/gb-2008-9-9-r137 18798982

20. Heintzman ND, Stuart RK, Hon G, Fu Y, Ching CW, Hawkins RD, et al. Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome. Nat Genet. 2007;39(3):311–8. doi: 10.1038/ng1966 17277777.

21. Poulard C, Rambaud J, Le Romancer M, Corbo L. Proximity ligation assay to detect and localize the interactions of ERalpha with PI3-K and Src in breast cancer cells and tumor samples. Methods Mol Biol. 2014;1204:135–43. doi: 10.1007/978-1-4939-1346-6_12 25182767.

22. Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nature genetics. 2003;34(3):267–73. Epub 2003/06/17. doi: 10.1038/ng1180 12808457.

23. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102(43):15545–50. doi: 10.1073/pnas.0506580102 16199517

24. Mohammed H, D’Santos C, Serandour AA, Ali HR, Brown GD, Atkins A, et al. Endogenous purification reveals GREB1 as a key estrogen receptor regulatory factor. Cell Rep. 2013;3(2):342–9. doi: 10.1016/j.celrep.2013.01.010 23403292.

25. Lin SY, Makino K, Xia W, Matin A, Wen Y, Kwong KY, et al. Nuclear localization of EGF receptor and its potential new role as a transcription factor. Nature cell biology. 2001;3(9):802–8. Epub 2001/09/05. doi: 10.1038/ncb0901-802 11533659.

26. Andrique L, Fauvin D, El Maassarani M, Colasson H, Vannier B, Seite P. ErbB3(80 kDa), a nuclear variant of the ErbB3 receptor, binds to the Cyclin D1 promoter to activate cell proliferation but is negatively controlled by p14ARF. Cellular signalling. 2012;24(5):1074–85. Epub 2012/01/21. doi: 10.1016/j.cellsig.2012.01.002 22261253.

27. Wang YN, Lee HH, Lee HJ, Du Y, Yamaguchi H, Hung MC. Membrane-bound trafficking regulates nuclear transport of integral epidermal growth factor receptor (EGFR) and ErbB-2. The Journal of biological chemistry. 2012;287(20):16869–79. Epub 2012/03/28. doi: 10.1074/jbc.M111.314799 22451678

28. Dawson MA, Bannister AJ, Gottgens B, Foster SD, Bartke T, Green AR, et al. JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin. Nature. 2009;461(7265):819–22. doi: 10.1038/nature08448 19783980

29. Tiwari VK, Stadler MB, Wirbelauer C, Paro R, Schubeler D, Beisel C. A chromatin-modifying function of JNK during stem cell differentiation. Nat Genet. 2012;44(1):94–100. doi: 10.1038/ng.1036 22179133.

30. 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(3):240–8. Epub 2009/03/12. doi: 10.1016/j.ymeth.2009.03.001 19275939.

31. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9(4):357–9. doi: 10.1038/nmeth.1923 22388286

32. 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(15):1952–8. doi: 10.1093/bioinformatics/btp340 19505939

33. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57–74. doi: 10.1038/nature11247 22955616

34. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, et al. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 2009;37(Web Server issue):W202–8. doi: 10.1093/nar/gkp335 19458158

35. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009;25(9):1105–11. doi: 10.1093/bioinformatics/btp120 19289445

36. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome biology. 2014;15(12):550. doi: 10.1186/s13059-014-0550-8 25516281

Článek vyšel v časopise


2019 Číslo 11