Breast Cancer Research and Treatment

, Volume 134, Issue 2, pp 511–517 | Cite as

Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of olaparib in breast cancer cells: a proof of concept study for synthetic lethal therapeutic option

  • Ziyan Yuan Pessetto
  • Ying Yan
  • Tadayoshi Bessho
  • Amarnath NatarajanEmail author
Preclinical Study


Synthetic lethal therapeutic strategy using poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor olaparib in carriers of BRCA1 or BRCA2 mutation has shown promise in clinical settings. Since <5 % of patients are BRCA1 or BRCA2 mutation carriers, small molecules that functionally mimic BRCA1 or BRCA2 mutations will extend the synthetic lethal therapeutic option for non-mutation carriers. Here we provide proof of principle for this strategy using a BRCA1 inhibitor peptide 2 that targets the BRCT(BRCA1)-phosphoprotein interaction and mimics the M177R/K BRCA1 mutation. Reciprocal immunoprecipitation and immunoblotting of BRCA1 and Abraxas was used to demonstrate inhibitor 2 targets BRCT(BRCA1)-Abraxas interface. Immunostaining of γH2AX, cell cycle analysis and homologous recombination (HR) assays were conducted to confirm that inhibitor 2 functionally mimics a chemosensitizing BRCA1 mutation. The concept of synthetic lethal therapeutic strategy with the BRCA1 inhibitor 2 and the PARP inhibitor Olaparib was explored in HeLa, MDA-MB-231, and HCC1937 cell lines. The results show that inhibition of BRCA1 by 2 sensitizes HeLa and MDA-MB-231 cells but not HCC1937 to Olaparib mediated growth inhibition and apoptosis. These results provide the basis for developing high affinity BRCT(BRCA1) inhibitors as adjuvants to treat sporadic breast and ovarian cancers.


Synthetic lethal therapeutic strategy BRCA1 inhibitor Abraxas Chemosensitization Olaparib and IR 



Poly(adenosine diphosphate [ADP]-ribose) polymerase






Homologous recombination


Ionizing Radiation



This study was supported by NIH R01CA127239. We thank the UNMC flow cytometry and confocal laser scanning microscopy core facilities.

Conflict of interest

The authors have no conflict of interest.

Supplementary material

10549_2012_2079_MOESM1_ESM.docx (25 kb)
Supplementary material 1 (DOCX 25 kb)


  1. 1.
    Fong PC, Boss DS, Yap TA, Tutt A, Wu P, Mergui-Roelvink M, Mortimer P, Swaisland H, Lau A, O’Connor MJ, Ashworth A, Carmichael J, Kaye SB, Schellens JH, de Bono JS (2009) Inhibition of poly(ADP-ribose) polymerase in tumors from BRCA mutation carriers. N Engl J Med 361(2):123–134PubMedCrossRefGoogle Scholar
  2. 2.
    Lokesh GL, Muralidhara BK, Negi SS, Natarajan A (2007) Thermodynamics of phosphopeptide tethering to BRCT: the structural minima for inhibitor design. J Am Chem Soc 129(35):10658–10659PubMedCrossRefGoogle Scholar
  3. 3.
    Yuan Z, Kumar EA, Kizhake S, Natarajan A (2011) Structure–activity relationship studies to probe the phosphoprotein binding site on the carboxy terminal domains of the breast cancer susceptibility gene 1. J Med Chem 54(12):4264–4268PubMedCrossRefGoogle Scholar
  4. 4.
    Campbell SJ, Edwards RA, Glover JN (2010) Comparison of the structures and peptide binding specificities of the BRCT domains of MDC1 and BRCA1. Structure 18(2):167–176PubMedCrossRefGoogle Scholar
  5. 5.
    Joseph PR, Yuan Z, Kumar EA, Lokesh GL, Kizhake S, Rajarathnam K, Natarajan A (2010) Structural characterization of BRCT-tetrapeptide binding interactions. Biochem Biophys Res Commun 393(2):207–210PubMedCrossRefGoogle Scholar
  6. 6.
    Nakanishi K, Yang YG, Pierce AJ, Taniguchi T, Digweed M, D’Andrea AD, Wang ZQ, Jasin M (2005) Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proc Natl Acad Sci USA 102(4):1110–1115PubMedCrossRefGoogle Scholar
  7. 7.
    Richardson C, Elliott B, Jasin M (1999) Chromosomal double-strand breaks introduced in mammalian cells by expression of I-Sce I endonuclease. Methods Mol Biol 113:453–463PubMedCrossRefGoogle Scholar
  8. 8.
    Manke IA, Lowery DM, Nguyen A, Yaffe MB (2003) BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science 302(5645):636–639PubMedCrossRefGoogle Scholar
  9. 9.
    Yu X, Chini CC, He M, Mer G, Chen J (2003) The BRCT domain is a phospho-protein binding domain. Science 302(5645):639–642PubMedCrossRefGoogle Scholar
  10. 10.
    Wender PA, Mitchell DJ, Pattabiraman K, Pelkey ET, Steinman L, Rothbard JB (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci USA 97(24):13003–13008PubMedCrossRefGoogle Scholar
  11. 11.
    Lokesh GL, Rachamallu A, Kumar GD, Natarajan A (2006) High-throughput fluorescence polarization assay to identify small molecule inhibitors of BRCT domains of breast cancer gene 1. Anal Biochem 352(1):135–141PubMedCrossRefGoogle Scholar
  12. 12.
    Simeonov A, Yasgar A, Jadhav A, Lokesh GL, Klumpp C, Michael S, Austin CP, Natarajan A, Inglese J (2008) Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction. Anal Biochem 375(1):60–70PubMedCrossRefGoogle Scholar
  13. 13.
    Pierce AJ, Hu P, Han M, Ellis N, Jasin M (2001) Ku DNA end-binding protein modulates homologous repair of double-strand breaks in mammalian cells. Genes Dev 15(24):3237–3242PubMedCrossRefGoogle Scholar
  14. 14.
    Hu Y, Scully R, Sobhian B, Xie A, Shestakova E, Livingston DM (2011) RAP80-directed tuning of BRCA1 homologous recombination function at ionizing radiation-induced nuclear foci. Genes Dev 25(7):685–700PubMedCrossRefGoogle Scholar
  15. 15.
    Yu X, Chen J (2004) DNA damage-induced cell cycle checkpoint control requires CtIP, a phosphorylation-dependent binding partner of BRCA1 C-terminal domains. Mol Cell Biol 24(21):9478–9486PubMedCrossRefGoogle Scholar
  16. 16.
    Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau LA, Xia B, Livingston DM, Greenberg RA (2007) RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites. Science 316(5828):1198–1202PubMedCrossRefGoogle Scholar
  17. 17.
    Wang B, Matsuoka S, Ballif BA, Zhang D, Smogorzewska A, Gygi SP, Elledge SJ (2007) Abraxas and RAP80 form a BRCA1 protein complex required for the DNA damage response. Science 316(5828):1194–1198PubMedCrossRefGoogle Scholar
  18. 18.
    Kim H, Chen J, Yu X (2007) Ubiquitin-binding protein RAP80 mediates BRCA1-dependent DNA damage response. Science 316(5828):1202–1205PubMedCrossRefGoogle Scholar
  19. 19.
    Xu B, Kim ST, Lim DS, Kastan MB (2002) Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol 22(4):1049–1059PubMedCrossRefGoogle Scholar
  20. 20.
    Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA, Richardson TB, Santarosa M, Dillon KJ, Hickson I, Knights C, Martin NM, Jackson SP, Smith GC, Ashworth A (2005) Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy. Nature 434(7035):917–921PubMedCrossRefGoogle Scholar
  21. 21.
    Underhill C, Toulmonde M, Bonnefoi H (2011) A review of PARP inhibitors: from bench to bedside. Ann Oncol 22(2):268–279PubMedCrossRefGoogle Scholar
  22. 22.
    Narod SA (2010) BRCA mutations in the management of breast cancer: the state of the art. Nat Rev Clin Oncol 7(12):702–707PubMedCrossRefGoogle Scholar
  23. 23.
    Yuan Z, Kumar EA, Campbell SJ, Palermo NY, Kizhake S, Glover JNM, Natarajan A (2011) Exploiting the P-1 pocket of BRCT domains towards a structure guided inhibitor design. ACS Med Chem Lett 2(10):764–767PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Ziyan Yuan Pessetto
    • 1
  • Ying Yan
    • 1
  • Tadayoshi Bessho
    • 1
  • Amarnath Natarajan
    • 1
    • 2
    • 3
    Email author
  1. 1.Eppley Institute for Cancer ResearchUniversity of Nebraska Medical CenterOmahaUSA
  2. 2.Department of Pharmaceutical SciencesUniversity of Nebraska Medical CenterOmahaUSA
  3. 3.Department of Genetics Cell Biology and AnatomyUniversity of Nebraska Medical CenterOmahaUSA

Personalised recommendations