Breast Cancer Research and Treatment

, Volume 130, Issue 3, pp 791–807 | Cite as

Differential expression of arrestins is a predictor of breast cancer progression and survival

  • Allison M. Michal
  • Amy R. Peck
  • Thai H. Tran
  • Chengbao Liu
  • David L. Rimm
  • Hallgeir Rui
  • Jeffrey L. BenovicEmail author
Preclinical study


Emerging evidence has implicated G protein-coupled receptors, such as CXCR4 and PAR2, in breast cancer progression and the development of metastatic breast cancer. However, the role of proteins that regulate the function of these receptors, such as arrestins, in breast cancer has yet to be determined. Examination of the expression of the two nonvisual arrestins, arrestin2 and 3, in various breast cancer cell lines revealed comparable expression of arrestin3 in basal and luminal lines while arrestin2 expression was much higher in the luminal lines compared to the more aggressive basal lines. Analysis of normal human breast tissue revealed that arrestin2 and 3 were expressed in both luminal and myoepithelial cells of mammary epithelia with arrestin2 highest in myoepithelial cells and arrestin3 comparable in both cell types. Quantitative immunofluorescence-based examination of primary breast tumors revealed that arrestin2 expression significantly decreased with cancer progression from ductal carcinoma in situ to invasive carcinoma and further to lymph node metastasis (P < 0.001). Moreover, decreased arrestin2 expression was associated with decreased survival (P = 0.0007) as well as positive lymph node status and increased tumor size and nuclear grade. In contrast, arrestin3 expression significantly increased during breast cancer progression (P < 0.001) and increased expression was associated with decreased survival (P = 0.014). Arrestin3 was also an independent prognostic marker of breast cancer with a hazard ratio of 1.65. Overall, these studies demonstrate that arrestin2 levels decrease while arrestin3 levels increase during breast cancer progression and these changes correlate with a poor clinical outcome.


Arrestin Breast carcinoma Immunohistochemistry Metastasis Survival 



Automated quantitative analysis


Blocking peptide


Cutting edge matrix assembly






Ductal carcinoma in situ


Estrogen receptor


Extracellular signal-regulated kinase 2


Fetal bovine serum


G protein-coupled receptor


Glutathione S-Transferase


Human epidermal growth factor receptor 2


Hazard ratio


Horseradish peroxidase


Invasive ductal carcinoma


Insulin-like growth factor 1 receptor




Progesterone receptor


Tris buffered saline



We would like to thank Dr. Catherine Moore for helping to initiate some of the studies in the breast cancer cell lines and Shashi Rattan for excellent technical assistance. This work was partially supported by National Institutes of Health grants R01 CA129626 and R01 GM047417. The authors declare that there are no conflicts-of-interest.


  1. 1.
    American Cancer Society (2009) Breast cancer facts & figures 2009–2010. American Cancer Society Inc., Atlanta, GAGoogle Scholar
  2. 2.
    Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen H, Pesich R, Geisler S, Demeter J, Perou CM, Lonning PE, Brown PO, Borresen-Dale AL, Botstein D (2003) Repeated observation of breast tumor subtypes in independent gene expression data sets. Proc Natl Acad Sci USA 100:8418–8423. doi: 10.1073/pnas.0932692100 PubMedCrossRefGoogle Scholar
  3. 3.
    Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J, Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC (2006) Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA 295:2492–2502. doi: 10.1001/jama.295.21.2492 PubMedCrossRefGoogle Scholar
  4. 4.
    Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D (2000) Molecular portraits of human breast tumours. Nature 406:747–752. doi: 10.1038/35021093 PubMedCrossRefGoogle Scholar
  5. 5.
    Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10:515–527. doi: 10.1016/j.ccr.2006.10.008 PubMedCrossRefGoogle Scholar
  6. 6.
    Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA 98:10869–10874. doi: 10.1073/pnas.191367098 PubMedCrossRefGoogle Scholar
  7. 7.
    Luttrell LM (2006) Transmembrane signaling by G protein-coupled receptors. Methods Mol Biol 332:3–49PubMedGoogle Scholar
  8. 8.
    Dorsam RT, Gutkind JS (2007) G-protein-coupled receptors and cancer. Nat Rev Cancer 7:79–94PubMedCrossRefGoogle Scholar
  9. 9.
    Lefkowitz RJ (2004) Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol Sci 25:413–422PubMedCrossRefGoogle Scholar
  10. 10.
    Krupnick JG, Benovic JL (1998) The role of receptor kinases and arrestins in G protein-coupled receptor regulation. Annu Rev Pharmacol Toxicol 38:289–319. doi: 10.1146/annurev.pharmtox.38.1.289 PubMedCrossRefGoogle Scholar
  11. 11.
    Moore CA, Milano SK, Benovic JL (2007) Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 69:451–482. doi: 10.1146/annurev.physiol.69.022405.154712 PubMedCrossRefGoogle Scholar
  12. 12.
    DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK (2007) Beta-arrestins and cell signaling. Annu Rev Physiol 69:483–510. doi: 10.1146/ PubMedCrossRefGoogle Scholar
  13. 13.
    Kang J, Shi Y, Xiang B, Qu B, Su W, Zhu M, Zhang M, Bao G, Wang F, Zhang X, Yang R, Fan F, Chen X, Pei G, Ma L (2005) A nuclear function of beta-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription. Cell 123:833–847. doi: 10.1016/j.cell.2005.09.011 PubMedCrossRefGoogle Scholar
  14. 14.
    Ge L, Ly Y, Hollenberg M, DeFea K (2003) A beta-arrestin-dependent scaffold is associated with prolonged MAPK activation in pseudopodia during protease-activated receptor-2-induced chemotaxis. J Biol Chem 278:34418–34426. doi: 10.1074/jbc.M300573200 PubMedCrossRefGoogle Scholar
  15. 15.
    Sun Y, Cheng Z, Ma L, Pei G (2002) Beta-arrestin2 is critically involved in CXCR4-mediated chemotaxis, and this is mediated by its enhancement of p38 MAPK activation. J Biol Chem 277:49212–49219PubMedCrossRefGoogle Scholar
  16. 16.
    Zoudilova M, Kumar P, Ge L, Wang P, Bokoch GM, DeFea KA (2007) Beta-arrestin-dependent regulation of the cofilin pathway downstream of protease-activated receptor-2. J Biol Chem 282:20634–20646. doi: 10.1074/jbc.M701391200 PubMedCrossRefGoogle Scholar
  17. 17.
    Zoudilova M, Min J, Richards HL, Carter D, Huang T, DeFea KA (2010) beta-Arrestins scaffold cofilin with chronophin to direct localized actin filament severing and membrane protrusions downstream of protease-activated receptor-2. J Biol Chem 285:14318–14329. doi: 10.1074/jbc.M109.055806 PubMedCrossRefGoogle Scholar
  18. 18.
    Wang P, DeFea KA (2006) Protease-activated receptor-2 simultaneously directs beta-arrestin-1-dependent inhibition and Galphaq-dependent activation of phosphatidylinositol 3-kinase. Biochemistry 45:9374–9385PubMedCrossRefGoogle Scholar
  19. 19.
    Buchanan FG, Gorden DL, Matta P, Shi Q, Matrisian LM, DuBois RN (2006) Role of beta-arrestin 1 in the metastatic progression of colorectal cancer. Proc Natl Acad Sci USA 103:1492–1497PubMedCrossRefGoogle Scholar
  20. 20.
    Lakshmikanthan V, Zou L, Kim JI, Michal A, Nie Z, Messias NC, Benovic JL, Daaka Y (2009) Identification of betaArrestin2 as a corepressor of androgen receptor signaling in prostate cancer. Proc Natl Acad Sci USA 106:9379–9384. doi: 10.1073/pnas.0900258106 PubMedCrossRefGoogle Scholar
  21. 21.
    Shankar H, Michal A, Kern RC, Kang DS, Gurevich VV, Benovic JL (2010) Non-visual arrestins are constitutively associated with the centrosome and regulate centrosome function. J Biol Chem 285:8316–8329. doi: 10.1074/jbc.M109.062521 PubMedCrossRefGoogle Scholar
  22. 22.
    Raghuwanshi SK, Nasser MW, Chen X, Strieter RM, Richardson RM (2008) Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer. J Immunol 180:5699–5706PubMedGoogle Scholar
  23. 23.
    Zou L, Yang R, Chai J, Pei G (2008) Rapid xenograft tumor progression in beta-arrestin1 transgenic mice due to enhanced tumor angiogenesis. FASEB J 22:355–364. doi: 10.1096/fj.07-9046com PubMedCrossRefGoogle Scholar
  24. 24.
    LeBaron MJ, Crismon HR, Utama FE, Neilson LM, Sultan AS, Johnson KJ, Andersson EC, Rui H (2005) Ultrahigh density microarrays of solid samples. Nat Methods 2:511–513PubMedCrossRefGoogle Scholar
  25. 25.
    Tran TH, Utama FE, Lin J, Yang N, Sjolund AB, Ryder A, Johnson KJ, Neilson LM, Liu C, Brill KL, Rosenberg AL, Witkiewicz AK, Rui H (2010) Prolactin inhibits BCL6 expression in breast cancer through a Stat5a-dependent mechanism. Cancer Res 15:1711–1721CrossRefGoogle Scholar
  26. 26.
    Dolled-Filhart M, Ryden L, Cregger M, Jirstrom K, Harigopal M, Camp RL, Rimm DL (2006) Classification of breast cancer using genetic algorithms and tissue microarrays. Clin Cancer Res 12:6459–6468. doi: 10.1158/1078-0432.CCR-06-1383 PubMedCrossRefGoogle Scholar
  27. 27.
    Camp RL, Chung GG, Rimm DL (2002) Automated subcellular localization and quantification of protein expression in tissue microarrays. Nat Med 8:1323–1327. doi: 10.1038/nm791 PubMedCrossRefGoogle Scholar
  28. 28.
    Camp RL, Dolled-Filhart M, Rimm DL (2004) X-tile: a new bio-informatics tool for biomarker assessment and outcome-based cut-point optimization. Clin Cancer Res 10:7252–7259. doi: 10.1158/1078-0432.CCR-04-0713 PubMedCrossRefGoogle Scholar
  29. 29.
    McShane LM, Altman DG, Sauerbrei W, Taube SE, Gion M, Clark GM (2005) Reporting recommendations for tumor marker prognostic studies. J Clin Oncol 23:9067–9072PubMedCrossRefGoogle Scholar
  30. 30.
    Kenny PA, Lee GY, Myers CA, Neve RM, Semeiks JR, Spellman PT, Lorenz K, Lee EH, Barcellos-Hoff MH, Petersen OW, Gray JW, Bissell MJ (2007) The morphologies of breast cancer cell lines in three-dimensional assays correlate with their profiles of gene expression. Mol Oncol 1:84–96. doi: 10.1016/j.molonc.2007.02.004 PubMedCrossRefGoogle Scholar
  31. 31.
    Gudjonsson T, Adriance MC, Sternlicht MD, Petersen OW, Bissell MJ (2005) Myoepithelial cells: their origin and function in breast morphogenesis and neoplasia. J Mammary Gland Biol Neoplasia 10:261–272. doi: 10.1007/s10911-005-9586-4 PubMedCrossRefGoogle Scholar
  32. 32.
    Gudjonsson T, Ronnov-Jessen L, Villadsen R, Rank F, Bissell MJ, Petersen OW (2002) Normal and tumor-derived myoepithelial cells differ in their ability to interact with luminal breast epithelial cells for polarity and basement membrane deposition. J Cell Sci 115:39–50PubMedGoogle Scholar
  33. 33.
    Sainsbury JR, Anderson TJ, Morgan DA (2000) ABC of breast diseases: breast cancer. BMJ 321:745–750PubMedCrossRefGoogle Scholar
  34. 34.
    Gimpl G, Fahrenholz F (2001) The oxytocin receptor system: structure, function, and regulation. Physiol Rev 81:629–683PubMedGoogle Scholar
  35. 35.
    Sapino A, Macri L, Tonda L, Bussolati G (1993) Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland. Endocrinology 133:838–842PubMedCrossRefGoogle Scholar
  36. 36.
    Nishimori K, Young LJ, Guo Q, Wang Z, Insel TR, Matzuk MM (1996) Oxytocin is required for nursing but is not essential for parturition or reproductive behavior. Proc Natl Acad Sci USA 93:11699–11704PubMedCrossRefGoogle Scholar
  37. 37.
    Wagner KU, Young WS 3rd, Liu X, Ginns EI, Li M, Furth PA, Hennighausen L (1997) Oxytocin and milk removal are required for post-partum mammary-gland development. Genes Funct 1:233–244PubMedCrossRefGoogle Scholar
  38. 38.
    Ahn S, Wei H, Garrison TR, Lefkowitz RJ (2004) Reciprocal regulation of angiotensin receptor-activated extracellular signal-regulated kinases by beta-arrestins 1 and 2. J Biol Chem 279:7807–7811. doi: 10.1074/jbc.C300443200 PubMedCrossRefGoogle Scholar
  39. 39.
    Ren XR, Reiter E, Ahn S, Kim J, Chen W, Lefkowitz RJ (2005) Different G protein-coupled receptor kinases govern G protein and beta-arrestin-mediated signaling of V2 vasopressin receptor. Proc Natl Acad Sci USA 102:1448–1453. doi: 10.1073/pnas.0409534102 PubMedCrossRefGoogle Scholar
  40. 40.
    Ge L, Shenoy SK, Lefkowitz RJ, DeFea K (2004) Constitutive protease-activated receptor-2-mediated migration of MDA MB-231 breast cancer cells requires both beta-arrestin-1 and -2. J Biol Chem 279:55419–55424PubMedCrossRefGoogle Scholar
  41. 41.
    Defea KA (2007) Stop that cell! beta-arrestin-dependent chemotaxis: a tale of localized actin assembly and receptor desensitization. Annu Rev Physiol 69:535–560PubMedCrossRefGoogle Scholar
  42. 42.
    Balkwill F (2004) The significance of cancer cell expression of the chemokine receptor CXCR4. Semin Cancer Biol 14:171–179PubMedCrossRefGoogle Scholar
  43. 43.
    Bhandari D, Trejo J, Benovic JL, Marchese A (2007) Arrestin-2 interacts with the ubiquitin-protein isopeptide ligase atrophin-interacting protein 4 and mediates endosomal sorting of the chemokine receptor CXCR4. J Biol Chem 282:36971–36979. doi: 10.1074/jbc.M705085200 PubMedCrossRefGoogle Scholar
  44. 44.
    Fong AM, Premont RT, Richardson RM, Yu YR, Lefkowitz RJ, Patel DD (2002) Defective lymphocyte chemotaxis in beta-arrestin2- and GRK6-deficient mice. Proc Natl Acad Sci USA 99:7478–7483PubMedCrossRefGoogle Scholar
  45. 45.
    Kumar P, Lau CS, Mathur M, Wang P, DeFea KA (2007) Differential effects of beta-arrestins on the internalization, desensitization and ERK1/2 activation downstream of protease activated receptor-2. Am J Physiol Cell Physiol 293:C346–C357. doi: 10.1152/ajpcell.00010.2007 PubMedCrossRefGoogle Scholar
  46. 46.
    Saunders W (2005) Centrosomal amplification and spindle multipolarity in cancer cells. Semin Cancer Biol 15:25–32. doi: 10.1016/j.semcancer.2004.09.003 PubMedCrossRefGoogle Scholar
  47. 47.
    Doxsey S, Zimmerman W, Mikule K (2005) Centrosome control of the cell cycle. Trends Cell Biol 15:303–311. doi: 10.1016/j.tcb.2005.04.008 PubMedCrossRefGoogle Scholar
  48. 48.
    Salisbury JL, D’Assoro AB, Lingle WL (2004) Centrosome amplification and the origin of chromosomal instability in breast cancer. J Mammary Gland Biol Neoplasia 9:275–283. doi: 10.1023/B:JOMG.0000048774.27697.30 PubMedCrossRefGoogle Scholar
  49. 49.
    Lingle WL, Lukasiewicz K, Salisbury JL (2005) Deregulation of the centrosome cycle and the origin of chromosomal instability in cancer. Adv Exp Med Biol 570:393–421PubMedCrossRefGoogle Scholar
  50. 50.
    D’Assoro AB, Barrett SL, Folk C, Negron VC, Boeneman K, Busby R, Whitehead C, Stivala F, Lingle WL, Salisbury JL (2002) Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness. Breast Cancer Res Treat 75:25–34PubMedCrossRefGoogle Scholar
  51. 51.
    Lingle WL, Barrett SL, Negron VC, D’Assoro AB, Boeneman K, Liu W, Whitehead CM, Reynolds C, Salisbury JL (2002) Centrosome amplification drives chromosomal instability in breast tumor development. Proc Natl Acad Sci USA 99:1978–1983. doi: 10.1073/pnas.032479999 PubMedCrossRefGoogle Scholar
  52. 52.
    Molla-Herman A, Boularan C, Ghossoub R, Scott MG, Burtey A, Zarka M, Saunier S, Concordet JP, Marullo S, Benmerah A (2008) Targeting of beta-arrestin2 to the centrosome and primary cilium: role in cell proliferation control. PLoS ONE 3:e3728. doi: 10.1371/journal.pone.0003728 PubMedCrossRefGoogle Scholar
  53. 53.
    Strous GJ, Schantl JA (2001) Beta-arrestin and Mdm2, unsuspected partners in signaling from the cell surface. Sci STKE 2001(110):pe41. DOI: 10.1126/stke.2001.110.pe41
  54. 54.
    Zilfou JT, Lowe SW (2009) Tumor suppressive functions of p53. Cold Spring Harbor Perspect Biol 1:a001883. doi: 10.1101/cshperspect.a001883 CrossRefGoogle Scholar
  55. 55.
    Jones SN, Hancock AR, Vogel H, Donehower LA, Bradley A (1998) Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis. Proc Natl Acad Sci USA 95:15608–15612PubMedCrossRefGoogle Scholar
  56. 56.
    Turbin DA, Cheang MC, Bajdik CD, Gelmon KA, Yorida E, De Luca A, Nielsen TO, Huntsman DG, Gilks CB (2006) MDM2 protein expression is a negative prognostic marker in breast carcinoma. Mod Pathol 19:69–74. doi: 10.1038/modpathol.3800484 PubMedCrossRefGoogle Scholar
  57. 57.
    Girnita L, Shenoy SK, Sehat B, Vasilcanu R, Girnita A, Lefkowitz RJ, Larsson O (2005) β-Arrestin is crucial for ubiquitination and down-regulation of the insulin-like growth factor-1 receptor by acting as adaptor for the MDM2 E3 ligase. J Biol Chem 280:24412–24419PubMedCrossRefGoogle Scholar
  58. 58.
    Werner H, Bruchim I (2009) The insulin-like growth factor-I receptor as an oncogene. Arch Physiol Biochem 115:58–71. doi: 10.1080/13813450902783106 PubMedCrossRefGoogle Scholar
  59. 59.
    Dunn SE, Ehrlich M, Sharp NJ, Reiss K, Solomon G, Hawkins R, Baserga R, Barrett JC (1998) A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer. Cancer Res 58:3353–3361PubMedGoogle Scholar
  60. 60.
    Bonneterre J, Peyrat JP, Beuscart R, Demaille A (1990) Prognostic significance of insulin-like growth factor 1 receptors in human breast cancer. Cancer Res 50:6931–6935PubMedGoogle Scholar
  61. 61.
    Ellis MJ, Jenkins S, Hanfelt J, Redington ME, Taylor M, Leek R, Siddle K, Harris A (1998) Insulin-like growth factors in human breast cancer. Breast Cancer Res Treat 52:175–184PubMedCrossRefGoogle Scholar
  62. 62.
    Resnik JL, Reichart DB, Huey K, Webster NJ, Seely BL (1998) Elevated insulin-like growth factor I receptor autophosphorylation and kinase activity in human breast cancer. Cancer Res 58:1159–1164PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2011

Authors and Affiliations

  • Allison M. Michal
    • 1
  • Amy R. Peck
    • 2
  • Thai H. Tran
    • 2
  • Chengbao Liu
    • 2
  • David L. Rimm
    • 3
  • Hallgeir Rui
    • 2
  • Jeffrey L. Benovic
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyThomas Jefferson UniversityPhiladelphiaUSA
  2. 2.Department of Cancer Biology, Kimmel Cancer CenterThomas Jefferson UniversityPhiladelphiaUSA
  3. 3.Department of PathologyYale University School of MedicineNew HavenUSA

Personalised recommendations