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Breast Cancer Research and Treatment

, Volume 146, Issue 2, pp 273–285 | Cite as

Tamoxifen through GPER upregulates aromatase expression: a novel mechanism sustaining tamoxifen-resistant breast cancer cell growth

  • Stefania Catalano
  • Cinzia Giordano
  • Salvatore Panza
  • Francesca Chemi
  • Daniela Bonofiglio
  • Marilena Lanzino
  • Pietro Rizza
  • Francesco Romeo
  • Suzanne A. W. Fuqua
  • Marcello Maggiolini
  • Sebastiano AndòEmail author
  • Ines BaroneEmail author
Preclinical study

Abstract

Tamoxifen resistance is a major clinical challenge in breast cancer treatment. Aromatase inhibitors are effective in women who progressed or recurred on tamoxifen, suggesting a role of local estrogen production by aromatase in driving tamoxifen-resistant phenotype. However, the link between aromatase activity and tamoxifen resistance has not yet been reported. We investigated whether long-term tamoxifen exposure may affect aromatase activity and/or expression, which may then sustain tamoxifen-resistant breast cancer cell growth. We employed MCF-7 breast cancer cells, tamoxifen-resistant MCF-7 cells (MCF-7 TR1 and TR2), SKBR-3 breast cancer cells, cancer-associated fibroblasts (CAFs1 and CAFs2). We used tritiated-water release assay, realtime-RT-PCR, and immunoblotting analysis for evaluating aromatase activity and expression; anchorage-independent assays for growth; reporter-gene, electrophoretic-mobility-shift, and chromatin-immunoprecipitation assays for promoter activity studies. We demonstrated an increased aromatase activity and expression, which supports proliferation in tamoxifen-resistant breast cancer cells. This is mediated by the G-protein-coupled receptor GPR30/GPER, since knocking-down GPER expression or treatment with a GPER antagonist reversed the enhanced aromatase levels induced by long-term tamoxifen exposure. The molecular mechanism was investigated in ER-negative, GPER/aromatase-positive SKBR3 cells, in which tamoxifen acts as a GPER agonist. Tamoxifen treatment increased aromatase promoter activity through an enhanced recruitment of c-fos/c-jun complex to AP-1 responsive elements located within the promoter region. As tamoxifen via GPER induced aromatase expression also in CAFs, this pathway may be involved in promoting aggressive behavior of breast tumors in response to tamoxifen treatment. Blocking estrogen production and/or GPER signaling activation may represent a valid option to overcome tamoxifen-resistance in breast cancers.

Keywords

Aromatase GPER Tamoxifen resistance Breast cancer 

Notes

Acknowledgments

This work was supported by Associazione Italiana Ricerca sul Cancro (AIRC) grant IG11595. Futuro in Ricerca 2012 RBFR12FI27 to IB. European Commission/FSE/Regione Calabria to FC and SP. MM was supported by Associazione Italiana per la Ricerca sul Cancro (project no. 12849/2012), AIRC project Calabria 2011 (http://www.airc.it/) and Fondazione Cassa di Risparmio di Calabria e Lucania.

Conflict of interest

The authors declared no conflict of interest and no financial relationship with the organization that sponsored the research.

Supplementary material

10549_2014_3017_MOESM1_ESM.ppt (154 kb)
Supplementary material 1 (PPT 151 kb)
10549_2014_3017_MOESM2_ESM.ppt (216 kb)
Supplementary material 2 (PPT 216 kb)

References

  1. 1.
    Harada N (1997) Aberrant expression of aromatase in breast cancer tissues. J Steroid Biochem Mol Biol 61:175–184PubMedCrossRefGoogle Scholar
  2. 2.
    James VH, McNeill JM, Lai LC et al (1987) Aromatase activity in normal breast and breast tumor tissues: in vivo and in vitro studies. Steroids 50:269–279PubMedCrossRefGoogle Scholar
  3. 3.
    Esteban JM, Warsi Z, Haniu M et al (1992) Detection of intratumoral aromatase in breast carcinomas. An immunohistochemical study with clinicopathologic correlation. Am J Pathol 140:337–343PubMedCentralPubMedGoogle Scholar
  4. 4.
    Chen S, Masri S, Wang X et al (2006) What do we know about the mechanisms of aromatase inhibitor resistance? J Steroid Biochem Mol Biol 102:232–240PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Simpson ER, Michael MD, Agarwal VR et al (1997) Cytochromes P450 11: expression of the CYP19 (aromatase) gene: an unusual case of alternative promoter usage. FASEB J 11:29–36PubMedGoogle Scholar
  6. 6.
    Bulun SE, Sebastian S, Takayama K et al (2003) The human CYP19 (aromatase P450) gene: update on physiologic roles and genomic organization of promoters. J Steroid Biochem Mol Biol 86:219–224PubMedCrossRefGoogle Scholar
  7. 7.
    Barone I, Giordano C, Malivindi R et al (2012) Estrogens and PTP1B function in a novel pathway to regulate aromatase enzymatic activity in breast cancer cells. Endocrinology 153:5157–5166PubMedCrossRefGoogle Scholar
  8. 8.
    Catalano S, Barone I, Giordano C et al (2009) Rapid estradiol/ERalpha signaling enhances aromatase enzymatic activity in breast cancer cells. Mol Endocrinol 23:1634–1645PubMedCrossRefGoogle Scholar
  9. 9.
    Maggiolini M, Carpino A, Bonofiglio D et al (2001) The direct proliferative stimulus of dehydroepiandrosterone on MCF7 breast cancer cells is potentiated by overexpression of aromatase. Mol Cell Endocrinol 184:163–171PubMedCrossRefGoogle Scholar
  10. 10.
    Sun XZ, Zhou D, Chen S (1997) Autocrine and paracrine actions of breast tumor aromatase. A three-dimensional cell culture study involving aromatase transfected MCF-7 and T-47D cells. J Steroid Biochem Mol Biol 63:29–36PubMedCrossRefGoogle Scholar
  11. 11.
    Yue W, Zhou D, Chen S et al (1994) A new nude mouse model for postmenopausal breast cancer using MCF-7 cells transfected with the human aromatase gene. Cancer Res 54:5092–5095PubMedGoogle Scholar
  12. 12.
    Tekmal RR, Ramachandra N, Gubba S et al (1996) Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities. Cancer Res 56:3180–3185PubMedGoogle Scholar
  13. 13.
    Prossnitz ER, Maggiolini M (2009) Mechanisms of estrogen signaling and gene expression via GPR30. Mol Cell Endocrinol 308:32–38PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Revankar CM, Cimino DF, Sklar LA et al (2005) A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science 307:1625–1630PubMedCrossRefGoogle Scholar
  15. 15.
    Filardo EJ (2002) Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer. J Steroid Biochem Mol Biol 80:231–238PubMedCrossRefGoogle Scholar
  16. 16.
    Prossnitz ER, Oprea TI, Sklar LA et al (2008) The ins and outs of GPR30: a transmembrane estrogen receptor. J Steroid Biochem Mol Biol 109:350–353PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Filardo EJ, Quinn JA, Bland KI et al (2000) Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF. Mol Endocrinol 14:1649–1660PubMedCrossRefGoogle Scholar
  18. 18.
    Pandey DP, Lappano R, Albanito L et al (2009) Estrogenic GPR30 signalling induces proliferation and migration of breast cancer cells through CTGF. EMBO J 28:523–532PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Thomas P, Pang Y, Filardo EJ et al (2005) Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology 146:624–632PubMedCrossRefGoogle Scholar
  20. 20.
    Vivacqua A, Bonofiglio D, Recchia AG et al (2006) The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells. Mol Endocrinol 20:631–646PubMedCrossRefGoogle Scholar
  21. 21.
    Vivacqua A, Bonofiglio D, Albanito L et al (2006) 17beta-estradiol, genistein, and 4-hydroxytamoxifen induce the proliferation of thyroid cancer cells through the g protein-coupled receptor GPR30. Mol Pharmacol 70:1414–1423PubMedCrossRefGoogle Scholar
  22. 22.
    Encarnacion CA, Ciocca DR, McGuire WL et al (1993) Measurement of steroid hormone receptors in breast cancer patients on tamoxifen. Breast Cancer Res Treat 26:237–246PubMedCrossRefGoogle Scholar
  23. 23.
    Giordano C, Cui Y, Barone I et al (2009) Growth factor-induced resistance to tamoxifen is associated with a mutation of estrogen receptor alpha and its phosphorylation at serine 305. Breast Cancer Res Treat 119:71–85PubMedCrossRefGoogle Scholar
  24. 24.
    Barone I, Brusco L, Fuqua SA (2010) Estrogen receptor mutations and changes in downstream gene expression and signaling. Clin Cancer Res 16:2702–2708PubMedCrossRefGoogle Scholar
  25. 25.
    Barone I, Iacopetta D, Covington KR et al (2010) Phosphorylation of the mutant K303R estrogen receptor alpha at serine 305 affects aromatase inhibitor sensitivity. Oncogene 29:2404–2414PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Barone I, Cui Y, Herynk MH et al (2009) Expression of the K303R estrogen receptor-alpha breast cancer mutation induces resistance to an aromatase inhibitor via addiction to the PI3K/Akt kinase pathway. Cancer Res 69:4724–4732PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Blume-Jensen P, Hunter T (2001) Oncogenic kinase signalling. Nature 411:355–365PubMedCrossRefGoogle Scholar
  28. 28.
    Martin LA, Farmer I, Johnston SR et al (2003) Enhanced estrogen receptor (ER) alpha, ERBB2, and MAPK signal transduction pathways operate during the adaptation of MCF-7 cells to long term estrogen deprivation. J Biol Chem 278:30458–30468PubMedCrossRefGoogle Scholar
  29. 29.
    Schiff R, Massarweh SA, Shou J et al (2004) Cross-talk between estrogen receptor and growth factor pathways as a molecular target for overcoming endocrine resistance. Clin Cancer Res 10:331S–336SPubMedCrossRefGoogle Scholar
  30. 30.
    Sabnis GJ, Jelovac D, Long B et al (2005) The role of growth factor receptor pathways in human breast cancer cells adapted to long-term estrogen deprivation. Cancer Res 65:3903–3910PubMedCrossRefGoogle Scholar
  31. 31.
    Staka CM, Nicholson RI, Gee JM (2005) Acquired resistance to oestrogen deprivation: role for growth factor signalling kinases/oestrogen receptor cross-talk revealed in new MCF-7X model. Endocr Relat Cancer 12(Suppl 1):S85–S97PubMedCrossRefGoogle Scholar
  32. 32.
    Evan GI, Brown L, Whyte M et al (1995) Apoptosis and the cell cycle. Curr Opin Cell Biol 7:825–834PubMedCrossRefGoogle Scholar
  33. 33.
    Ignatov A, Ignatov T, Roessner A et al (2010) Role of GPR30 in the mechanisms of tamoxifen resistance in breast cancer MCF-7 cells. Breast Cancer Res Treat 123:87–96PubMedCrossRefGoogle Scholar
  34. 34.
    Ignatov A, Ignatov T, Weissenborn C et al (2011) G-protein-coupled estrogen receptor GPR30 and tamoxifen resistance in breast cancer. Breast Cancer Res Treat 128:457–466PubMedCrossRefGoogle Scholar
  35. 35.
    Vivacqua A, Lappano R, De Marco P et al (2009) G protein-coupled receptor 30 expression is up-regulated by EGF and TGF alpha in estrogen receptor alpha-positive cancer cells. Mol Endocrinol 23:1815–1826PubMedCrossRefGoogle Scholar
  36. 36.
    Lin BC, Suzawa M, Blind RD et al (2009) Stimulating the GPR30 estrogen receptor with a novel tamoxifen analogue activates SF-1 and promotes endometrial cell proliferation. Cancer Res 69:5415–5423PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Barone I, Brusco L, Gu G et al (2011) Loss of Rho GDIalpha and resistance to tamoxifen via effects on estrogen receptor alpha. J Natl Cancer Inst 103:538–552PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Barone I, Catalano S, Gelsomino L et al (2012) Leptin mediates tumor-stromal interactions that promote the invasive growth of breast cancer cells. Cancer Res 72:1416–1427PubMedCrossRefGoogle Scholar
  39. 39.
    Catalano S, Marsico S, Giordano C et al (2003) Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line. J Biol Chem 278:28668–28676PubMedCrossRefGoogle Scholar
  40. 40.
    Albanito L, Sisci D, Aquila S et al (2008) Epidermal growth factor induces G protein-coupled receptor 30 expression in estrogen receptor-negative breast cancer cells. Endocrinology 149:3799–3808PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Catalano S, Malivindi R, Giordano C et al (2010) Farnesoid X receptor, through the binding with steroidogenic factor 1-responsive element, inhibits aromatase expression in tumor Leydig cells. J Biol Chem 285:5581–5593PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Plastina P, Bonofiglio D, Vizza D et al (2012) Identification of bioactive constituents of Ziziphus jujube fruit extracts exerting antiproliferative and apoptotic effects in human breast cancer cells. J Ethnopharmacol 140:325–332PubMedCrossRefGoogle Scholar
  43. 43.
    Catalano S, Panza S, Malivindi R et al (2013) Inhibition of Leydig tumor growth by farnesoid X receptor activation: the in vitro and in vivo basis for a novel therapeutic strategy. Int J Cancer 132:2237–2247PubMedCrossRefGoogle Scholar
  44. 44.
    Andrews NC, Faller DV (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res 19:2499PubMedCentralPubMedCrossRefGoogle Scholar
  45. 45.
    Gu G, Barone I, Gelsomino L et al (2012) Oldenlandia diffusa extracts exert antiproliferative and apoptotic effects on human breast cancer cells through ERalpha/Sp1-mediated p53 activation. J Cell Physiol 227:3363–3372PubMedCrossRefGoogle Scholar
  46. 46.
    Giordano C, Catalano S, Panza S et al (2011) Farnesoid X receptor inhibits tamoxifen-resistant MCF-7 breast cancer cell growth through downregulation of HER2 expression. Oncogene 30:4129–4140PubMedCrossRefGoogle Scholar
  47. 47.
    Macedo LF, Guo Z, Tilghman SL et al (2006) Role of androgens on MCF-7 breast cancer cell growth and on the inhibitory effect of letrozole. Cancer Res 66:7775–7782PubMedCrossRefGoogle Scholar
  48. 48.
    Lanzino M, Maris P, Sirianni R et al (2013) DAX-1, as an androgen-target gene, inhibits aromatase expression: a novel mechanism blocking estrogen-dependent breast cancer cell proliferation. Cell Death Dis 4:e724PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Maggiolini M, Vivacqua A, Fasanella G et al (2004) The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells. J Biol Chem 279:27008–27016PubMedCrossRefGoogle Scholar
  50. 50.
    Carmeci C, Thompson DA, Ring HZ et al (1997) Identification of a gene (GPR30) with homology to the G-protein-coupled receptor superfamily associated with estrogen receptor expression in breast cancer. Genomics 45:607–617PubMedCrossRefGoogle Scholar
  51. 51.
    Vivacqua A, Romeo E, De Marco P et al (2012) GPER mediates the Egr-1 expression induced by 17beta-estradiol and 4-hydroxitamoxifen in breast and endometrial cancer cells. Breast Cancer Res Treat 133:1025–1035PubMedCrossRefGoogle Scholar
  52. 52.
    Albanito L, Madeo A, Lappano R et al (2007) G protein-coupled receptor 30 (GPR30) mediates gene expression changes and growth response to 17beta-estradiol and selective GPR30 ligand G-1 in ovarian cancer cells. Cancer Res 67:1859–1866PubMedCrossRefGoogle Scholar
  53. 53.
    Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100:57–70PubMedCrossRefGoogle Scholar
  54. 54.
    Albanito L, Lappano R, Madeo A et al (2008) G-protein-coupled receptor 30 and estrogen receptor-alpha are involved in the proliferative effects induced by atrazine in ovarian cancer cells. Environ Health Perspect 116:1648–1655PubMedCrossRefGoogle Scholar
  55. 55.
    Filardo EJ, Graeber CT, Quinn JA et al (2006) Distribution of GPR30, a seven membrane-spanning estrogen receptor, in primary breast cancer and its association with clinicopathologic determinants of tumor progression. Clin Cancer Res 12:6359–6366PubMedCrossRefGoogle Scholar
  56. 56.
    Sirianni R, Chimento A, Ruggiero C et al (2008) The novel estrogen receptor, G protein-coupled receptor 30, mediates the proliferative effects induced by 17beta-estradiol on mouse spermatogonial GC-1 cell line. Endocrinology 149:5043–5051PubMedCrossRefGoogle Scholar
  57. 57.
    Kuo WH, Chang LY, Liu DL et al (2007) The interactions between GPR30 and the major biomarkers in infiltrating ductal carcinoma of the breast in an Asian population. Taiwan J Obstet Gynecol 46:135–145PubMedCrossRefGoogle Scholar
  58. 58.
    Angel P, Karin M (1991) The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta 1072:129–157PubMedGoogle Scholar
  59. 59.
    Morgan L, Gee J, Pumford S et al (2009) Elevated Src kinase activity attenuates tamoxifen response in vitro and is associated with poor prognosis clinically. Cancer Biol Ther 8:1550–1558PubMedCrossRefGoogle Scholar
  60. 60.
    Fan P, Wang J, Santen RJ et al (2007) 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 67:1352–1360PubMedCrossRefGoogle Scholar
  61. 61.
    Fidler IJ (2003) The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat Rev Cancer 3:453–458PubMedCrossRefGoogle Scholar
  62. 62.
    Witz IP (2008) Yin-Yang activities and vicious cycles in the tumor microenvironment. Cancer Res 68:9–13PubMedCrossRefGoogle Scholar
  63. 63.
    Martinez-Outschoorn UE, Goldberg A, Lin Z et al (2011) Anti-estrogen resistance in breast cancer is induced by the tumor microenvironment and can be overcome by inhibiting mitochondrial function in epithelial cancer cells. Cancer Biol Ther 12:924–938PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Madeo A, Maggiolini M (2010) Nuclear alternate estrogen receptor GPR30 mediates 17beta-estradiol-induced gene expression and migration in breast cancer-associated fibroblasts. Cancer Res 70:6036–6046PubMedCrossRefGoogle Scholar
  65. 65.
    Normanno N, Di Maio M, De Maio E et al (2005) Mechanisms of endocrine resistance and novel therapeutic strategies in breast cancer. Endocr Relat Cancer 12:721–747PubMedCrossRefGoogle Scholar
  66. 66.
    Mo Z, Liu M, Yang F et al (2013) GPR30 as an initiator of tamoxifen resistance in hormone-dependent breast cancer. Breast Cancer Res 15:R114PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Stefania Catalano
    • 1
  • Cinzia Giordano
    • 2
  • Salvatore Panza
    • 1
  • Francesca Chemi
    • 1
  • Daniela Bonofiglio
    • 1
  • Marilena Lanzino
    • 1
  • Pietro Rizza
    • 1
  • Francesco Romeo
    • 3
  • Suzanne A. W. Fuqua
    • 4
  • Marcello Maggiolini
    • 1
  • Sebastiano Andò
    • 1
    Email author
  • Ines Barone
    • 1
    Email author
  1. 1.Department of Pharmacy, Health and Nutritional SciencesUniversity of CalabriaArcavacata di RendeItaly
  2. 2.Centro SanitarioUniversity of CalabriaArcavacata di RendeItaly
  3. 3.Division of Anatomo-PathologyAnnunziata HospitalCosenzaItaly
  4. 4.Breast CenterBaylor College of MedicineHoustonUSA

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