Breast Cancer Research and Treatment

, Volume 134, Issue 1, pp 41–51 | Cite as

Integration of BRCA1-mediated miRNA and mRNA profiles reveals microRNA regulation of TRAF2 and NFκB pathway

  • Miljana Tanic
  • Magdalena Zajac
  • Gonzalo Gómez-López
  • Javier Benítez
  • Beatriz Martínez-DelgadoEmail author
Preclinical study


Microarray-based techniques are being useful to obtain miRNA and gene expression signatures associated with different tumors. BRCA1 deregulation is a frequent event in the pathogenesis of breast as well as other cancers. In addition to DNA repair functions of BRCA1, it is involved in a wide range of cellular processes such as cell cycle, chromatin remodeling or transcription. However, the molecular events underlying BRCA1-associated tumorigenesis are still largely unknown. In order to deepen our understanding of BRCA1-associated tumorigenesis, we integrated data from mRNA and miRNA microarray experiments on HCC1937 breast cancer cell line, and the isogenic HCC1937 stably expressing BRCA1, to obtain significant miRNA–mRNA relationships associated with the presence of BRCA1 gene. By using bioinformatic integration of gene and miRNA expression data, we found significant miRNA–gene relationships underlying the array signatures. We additionally evaluated the role of these statistically significant pairs at the biological pathways level and identified MAPK and NF-κB pathways associated with these expression changes. Furthermore, we experimentally validated miRNAs induced by BRCA1 that commonly regulate TRAF2, a key regulator of NF-κB and MAPK pathways. We demonstrate that miR-146a, miR-99b and miR-205, induced in HCC1937 BRCA1-expressing cells, bind and regulate TRAF2 gene. In addition, re-expression of miR-146a, miR-99b or miR-205 in HCC1937 BRCA1-null cells was sufficient to modulate NF-κB activity. Our results demonstrate that integration of mRNA and miRNA associated with BRCA1 expression was useful to discover new miRNA–gene interactions as molecular events underlying BRCA1-mediated tumorigenesis.


BRCA1 Hereditary breast cancer microRNA mRNA expression Data integration TRAF2 NF-KappaB 



We thank Alicia Barroso, Fernando Fernandez and Victoria Fernandez for excellent technical assistance. This study was supported by grants from the Asociación Española contra el cancer (AECC). MT has financial support by Fundacion La Caixa. The CIBER de Enfermedades Raras is an initiative of the ISCIII.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10549_2011_1905_MOESM1_ESM.xls (899 kb)
Supplementary material 1 (XLS 899 kb)
10549_2011_1905_MOESM2_ESM.doc (40 kb)
Supplementary material 2 (DOC 40 kb)
10549_2011_1905_MOESM3_ESM.doc (44 kb)
Supplementary material 3 (DOC 43 kb)


  1. 1.
    Bertwistle D, Ashworth A (1998) Functions of the BRCA1 and BRCA2 genes. Curr Opin Genet Dev 8(1):14–20PubMedCrossRefGoogle Scholar
  2. 2.
    Rahman N, Stratton MR (1998) The genetics of breast cancer susceptibility. Annu Rev Genet 32:95–121PubMedCrossRefGoogle Scholar
  3. 3.
    Esteller M, Silva JM, Dominguez G, Bonilla F, Matias-Guiu X, Lerma E, Bussaglia E, Prat J, Harkes IC, Repasky EA, Gabrielson E, Schutte M, Baylin SB, Herman JG (2000) Promoter hypermethylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J Natl Cancer Inst 92(7):564–569PubMedCrossRefGoogle Scholar
  4. 4.
    Taylor J, Lymboura M, Pace PE, A’Hern RP, Desai AJ, Shousha S, Coombes RC, Ali S (1998) An important role for BRCA1 in breast cancer progression is indicated by its loss in a large proportion of non-familial breast cancers. Int J Cancer 79(4):334–342PubMedCrossRefGoogle Scholar
  5. 5.
    Turner NC, Reis-Filho JS, Russell AM, Springall RJ, Ryder K, Steele D, Savage K, Gillett CE, Schmitt FC, Ashworth A, Tutt AN (2007) BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene 26(14):2126–2132PubMedCrossRefGoogle Scholar
  6. 6.
    Wei M, Xu J, Dignam J, Nanda R, Sveen L, Fackenthal J, Grushko TA, Olopade OI (2008) Estrogen receptor alpha, BRCA1, and FANCF promoter methylation occur in distinct subsets of sporadic breast cancers. Breast Cancer Res Treat 111(1):113–120PubMedCrossRefGoogle Scholar
  7. 7.
    Bertwistle D, Ashworth A (1999) The pathology of familial breast cancer: How do the functions of BRCA1 and BRCA2 relate to breast tumour pathology? Breast Cancer Res 1(1):41–47PubMedCrossRefGoogle Scholar
  8. 8.
    Starita LM, Parvin JD (2003) The multiple nuclear functions of BRCA1: transcription, ubiquitination and DNA repair. Curr Opin Cell Biol 15(3):345–350PubMedCrossRefGoogle Scholar
  9. 9.
    Deng CX (2006) BRCA1: cell cycle checkpoint, genetic instability, DNA damage response and cancer evolution. Nucleic Acids Res 34(5):1416–1426PubMedCrossRefGoogle Scholar
  10. 10.
    Huen MS, Sy SM, Chen J (2010) BRCA1 and its toolbox for the maintenance of genome integrity. Natl Rev Mol Cell Biol 11(2):138–148CrossRefGoogle Scholar
  11. 11.
    Cable PL, Wilson CA, Calzone FJ, Rauscher FJ 3rd, Scully R, Livingston DM, Li L, Blackwell CB, Futreal PA, Afshari CA (2003) Novel consensus DNA-binding sequence for BRCA1 protein complexes. Mol Carcinog 38(2):85–96PubMedCrossRefGoogle Scholar
  12. 12.
    Chapman MS, Verma IM (1996) Transcriptional activation by BRCA1. Nature 382(6593):678–679PubMedCrossRefGoogle Scholar
  13. 13.
    Croce CM (2009) Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet 10(10):704–714PubMedCrossRefGoogle Scholar
  14. 14.
    Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR (2005) MicroRNA expression profiles classify human cancers. Nature 435(7043):834–838PubMedCrossRefGoogle Scholar
  15. 15.
    Thomson JM, Newman M, Parker JS, Morin-Kensicki EM, Wright T, Hammond SM (2006) Extensive post-transcriptional regulation of microRNAs and its implications for cancer. Genes Dev 20(16):2202–2207PubMedCrossRefGoogle Scholar
  16. 16.
    Lima RT, Busacca S, Almeida GM, Gaudino G, Fennell DA, Vasconcelos MH (2011) MicroRNA regulation of core apoptosis pathways in cancer. Eur J Cancer 47(2):163–174PubMedCrossRefGoogle Scholar
  17. 17.
    Schmittgen TD (2008) Regulation of microRNA processing in development, differentiation and cancer. J Cell Mol Med 12(5B):1811–1819PubMedCrossRefGoogle Scholar
  18. 18.
    Blenkiron C, Goldstein LD, Thorne NP, Spiteri I, Chin SF, Dunning MJ, Barbosa-Morais NL, Teschendorff AE, Green AR, Ellis IO, Tavare S, Caldas C, Miska EA (2007) MicroRNA expression profiling of human breast cancer identifies new markers of tumor subtype. Genome Biol 8(10):R214PubMedCrossRefGoogle Scholar
  19. 19.
    Cheng C, Fu X, Alves P, Gerstein M (2009) mRNA expression profiles show differential regulatory effects of microRNAs between estrogen receptor-positive and estrogen receptor-negative breast cancer. Genome Biol 10(9):R90PubMedCrossRefGoogle Scholar
  20. 20.
    Enerly E, Steinfeld I, Kleivi K, Leivonen SK, Aure MR, Russnes HG, Ronneberg JA, Johnsen H, Navon R, Rodland E, Makela R, Naume B, Perala M, Kallioniemi O, Kristensen VN, Yakhini Z, Borresen-Dale AL (2011) miRNA–mRNA integrated analysis reveals roles for miRNAs in primary breast tumors. PLoS ONE 6(2):e16915PubMedCrossRefGoogle Scholar
  21. 21.
    Andrews HN, Mullan PB, McWilliams S, Sebelova S, Quinn JE, Gilmore PM, McCabe N, Pace A, Koller B, Johnston PG, Haber DA, Harkin DP (2002) BRCA1 regulates the interferon gamma-mediated apoptotic response. J Biol Chem 277(29):26225–26232PubMedCrossRefGoogle Scholar
  22. 22.
    Furuta S, Wang JM, Wei S, Jeng YM, Jiang X, Gu B, Chen PL, Lee EY, Lee WH (2006) Removal of BRCA1/CtIP/ZBRK1 repressor complex on ANG1 promoter leads to accelerated mammary tumor growth contributed by prominent vasculature. Cancer Cell 10(1):13–24PubMedCrossRefGoogle Scholar
  23. 23.
    Harkin DP, Bean JM, Miklos D, Song YH, Truong VB, Englert C, Christians FC, Ellisen LW, Maheswaran S, Oliner JD, Haber DA (1999) Induction of GADD45 and JNK/SAPK-dependent apoptosis following inducible expression of BRCA1. Cell 97(5):575–586PubMedCrossRefGoogle Scholar
  24. 24.
    Hartman AR, Ford JM (2002) BRCA1 induces DNA damage recognition factors and enhances nucleotide excision repair. Nat Genet 32(1):180–184PubMedCrossRefGoogle Scholar
  25. 25.
    Lamber EP, Horwitz AA, Parvin JD (2010) BRCA1 represses amphiregulin gene expression. Cancer Res 70(3):996–1005PubMedCrossRefGoogle Scholar
  26. 26.
    MacLachlan TK, Somasundaram K, Sgagias M, Shifman Y, Muschel RJ, Cowan KH, El-Deiry WS (2000) BRCA1 effects on the cell cycle and the DNA damage response are linked to altered gene expression. J Biol Chem 275(4):2777–2785PubMedCrossRefGoogle Scholar
  27. 27.
    MacLachlan TK, Takimoto R, El-Deiry WS (2002) BRCA1 directs a selective p53-dependent transcriptional response towards growth arrest and DNA repair targets. Mol Cell Biol 22(12):4280–4292PubMedCrossRefGoogle Scholar
  28. 28.
    Di Lisio L, Gomez-Lopez G, Sanchez-Beato M, Gomez-Abad C, Rodriguez ME, Villuendas R, Ferreira BI, Carro A, Rico D, Mollejo M, Martinez MA, Menarguez J, Diaz-Alderete A, Gil J, Cigudosa JC, Pisano DG, Piris MA, Martinez N (2010) Mantle cell lymphoma: transcriptional regulation by microRNAs. Leukemia 24(7):1335–1342PubMedCrossRefGoogle Scholar
  29. 29.
    Carpentier I, Declercq W, Malinin NL, Wallach D, Fiers W, Beyaert R (1998) TRAF2 plays a dual role in NF-kappaB-dependent gene activation by mediating the TNF-induced activation of p38 MAPK and IkappaB kinase pathways. FEBS Lett 425(2):195–198PubMedCrossRefGoogle Scholar
  30. 30.
    Horwitz AA, Affar el B, Heine GF, Shi Y, Parvin JD (2007) A mechanism for transcriptional repression dependent on the BRCA1 E3 ubiquitin ligase. Proc Natl Acad Sci USA 104(16):6614–6619PubMedCrossRefGoogle Scholar
  31. 31.
    Horwitz AA, Sankaran S, Parvin JD (2006) Direct stimulation of transcription initiation by BRCA1 requires both its amino and carboxyl termini. J Biol Chem 281(13):8317–8320PubMedCrossRefGoogle Scholar
  32. 32.
    Shen J, Ambrosone CB, DiCioccio RA, Odunsi K, Lele SB, Zhao H (2008) A functional polymorphism in the miR-146a gene and age of familial breast/ovarian cancer diagnosis. Carcinogenesis 29(10):1963–1966PubMedCrossRefGoogle Scholar
  33. 33.
    Alexiou P, Maragkakis M, Papadopoulos GL, Reczko M, Hatzigeorgiou AG (2009) Lost in translation: an assessment and perspective for computational microRNA target identification. Bioinformatics 25(23):3049–3055PubMedCrossRefGoogle Scholar
  34. 34.
    Maziere P, Enright AJ (2007) Prediction of microRNA targets. Drug Discov Today 12(11–12):452–458PubMedCrossRefGoogle Scholar
  35. 35.
    Hori M, Inagawa S, Shimazaki J, Itabashi M (2000) Overexpression of mitogen-activated protein kinase superfamily proteins unrelated to Ras and AF-1 of estrogen receptor alpha mutation in advanced stage human breast cancer. Pathol Res Pract 196(12):817–826PubMedCrossRefGoogle Scholar
  36. 36.
    Whyte J, Bergin O, Bianchi A, McNally S, Martin F (2009) Key signalling nodes in mammary gland development and cancer. Mitogen-activated protein kinase signalling in experimental models of breast cancer progression and in mammary gland development. Breast Cancer Res 11(5):209PubMedCrossRefGoogle Scholar
  37. 37.
    Baldwin AS Jr (1996) The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu Rev Immunol 14:649–683PubMedCrossRefGoogle Scholar
  38. 38.
    Beg AA, Baltimore D (1996) An essential role for NF-kappaB in preventing TNF-alpha-induced cell death. Science 274(5288):782–784PubMedCrossRefGoogle Scholar
  39. 39.
    Van Antwerp DJ, Martin SJ, Kafri T, Green DR, Verma IM (1996) Suppression of TNF-alpha-induced apoptosis by NF-kappaB. Science 274(5288):787–789PubMedCrossRefGoogle Scholar
  40. 40.
    Wang CY, Mayo MW, Baldwin AS Jr (1996) TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-kappaB. Science 274(5288):784–787PubMedCrossRefGoogle Scholar
  41. 41.
    Madhusoodhanan R, Natarajan M, Veeraraghavan J, Herman TS, Aravindan N (2009) NFkappaB activity and transcriptional responses in human breast adenocarcinoma cells after single and fractionated irradiation. Cancer Biol Ther 8(9):765–773PubMedCrossRefGoogle Scholar
  42. 42.
    Zhou Y, Eppenberger-Castori S, Marx C, Yau C, Scott GK, Eppenberger U, Benz CC (2005) Activation of nuclear factor-kappaB (NFkappaB) identifies a high-risk subset of hormone-dependent breast cancers. Int J Biochem Cell Biol 37(5):1130–1144PubMedCrossRefGoogle Scholar
  43. 43.
    Zhou J, Zhang H, Gu P, Bai J, Margolick JB, Zhang Y (2008) NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res Treat 111(3):419–427PubMedCrossRefGoogle Scholar
  44. 44.
    Benezra M, Chevallier N, Morrison DJ, MacLachlan TK, El-Deiry WS, Licht JD (2003) BRCA1 augments transcription by the NF-kappaB transcription factor by binding to the Rel domain of the p65/RelA subunit. J Biol Chem 278(29):26333–26341PubMedCrossRefGoogle Scholar
  45. 45.
    Bhaumik D, Scott GK, Schokrpur S, Patil CK, Campisi J, Benz CC (2008) Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27(42):5643–5647PubMedCrossRefGoogle Scholar
  46. 46.
    Taganov KD, Boldin MP, Chang KJ, Baltimore D (2006) NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA 103(33):12481–12486PubMedCrossRefGoogle Scholar
  47. 47.
    Fernandez-Ramires R, Sole X, De Cecco L, Llort G, Cazorla A, Bonifaci N, Garcia MJ, Caldes T, Blanco I, Gariboldi M, Pierotti MA, Pujana MA, Benitez J, Osorio A (2009) Gene expression profiling integrated into network modelling reveals heterogeneity in the mechanisms of BRCA1 tumorigenesis. Br J Cancer 101(8):1469–1480PubMedCrossRefGoogle Scholar
  48. 48.
    Arch RH, Thompson CB (1998) 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptor-associated factors and activate nuclear factor kappaB. Mol Cell Biol 18(1):558–565PubMedGoogle Scholar
  49. 49.
    Duckett CS, Gedrich RW, Gilfillan MC, Thompson CB (1997) Induction of nuclear factor kappaB by the CD30 receptor is mediated by TRAF1 and TRAF2. Mol Cell Biol 17(3):1535–1542PubMedGoogle Scholar
  50. 50.
    Hsu H, Shu HB, Pan MG, Goeddel DV (1996) TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84(2):299–308PubMedCrossRefGoogle Scholar
  51. 51.
    Reinhard C, Shamoon B, Shyamala V, Williams LT (1997) Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2. EMBO J 16(5):1080–1092PubMedCrossRefGoogle Scholar
  52. 52.
    Rothe M, Sarma V, Dixit VM, Goeddel DV (1995) TRAF2-mediated activation of NF-kappa B by TNF receptor 2 and CD40. Science 269(5229):1424–1427PubMedCrossRefGoogle Scholar
  53. 53.
    Takeuchi M, Rothe M, Goeddel DV (1996) Anatomy of TRAF2. Distinct domains for nuclear factor-kappaB activation and association with tumor necrosis factor signaling proteins. J Biol Chem 271(33):19935–19942PubMedCrossRefGoogle Scholar
  54. 54.
    Liu ZG, Hsu H, Goeddel DV, Karin M (1996) Dissection of TNF receptor 1 effector functions: JNK activation is not linked to apoptosis while NF-kappaB activation prevents cell death. Cell 87(3):565–576PubMedCrossRefGoogle Scholar
  55. 55.
    Natoli G, Costanzo A, Ianni A, Templeton DJ, Woodgett JR, Balsano C, Levrero M (1997) Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway. Science 275(5297):200–203PubMedCrossRefGoogle Scholar
  56. 56.
    Cao Z, Henzel WJ, Gao X (1996) IRAK: a kinase associated with the interleukin-1 receptor. Science 271(5252):1128–1131PubMedCrossRefGoogle Scholar
  57. 57.
    Song HY, Regnier CH, Kirschning CJ, Goeddel DV, Rothe M (1997) Tumor necrosis factor (TNF)-mediated kinase cascades: bifurcation of nuclear factor-kappaB and c-jun N-terminal kinase (JNK/SAPK) pathways at TNF receptor-associated factor 2. Proc Natl Acad Sci USA 94(18):9792–9796PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Miljana Tanic
    • 1
  • Magdalena Zajac
    • 1
  • Gonzalo Gómez-López
    • 3
  • Javier Benítez
    • 1
    • 2
  • Beatriz Martínez-Delgado
    • 1
    • 2
    Email author
  1. 1.Human Genetics Group, Spanish National Cancer Research Centre (CNIO)MadridSpain
  2. 2.Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), (CNIO)MadridSpain
  3. 3.Bioinformatics Unit, Spanish National Cancer Research Centre (CNIO)MadridSpain

Personalised recommendations