Breast Cancer Research and Treatment

, Volume 120, Issue 2, pp 317–326 | Cite as

SOX9 mediates the retinoic acid-induced HES-1 gene expression in human breast cancer cells

  • Patrick MüllerEmail author
  • Justin D. Crofts
  • Ben S. Newman
  • Laura C. Bridgewater
  • Chin-Yo Lin
  • Jan-Åke Gustafsson
  • Anders Ström
Preclinical study


We have previously shown that the anti-proliferative effect of retinoic acid in human breast cancer cell line MCF-7 is dependent on HES-1 expression. Here we show that retinoic acid induces HES-1 expression via upregulation of transcription factor SOX9. By expressing a dominant negative form of SOX9, disrupting endogenous SOX9 activity, the retinoic acid-induced HES-1 mRNA expression was inhibited. We found an enhancer regulating HES-1 expression: two SOX9 binding sites upstream of the HES-1 gene that were capable of binding SOX9 in vitro. By performing chromatin immunoprecipitation, we showed that SOX9 binding to the HES-1 enhancer was induced by retinoic acid in vivo. In reporter assays, transfection of a SOX9 expression plasmid increased the activity of the HES-1 enhancer. The enhancer responded to retinoic acid; furthermore, the expression of a dominant negative SOX9 abolished this response. Taken together, we present here a novel transcriptional mechanism in regulating hormone-dependent cancer cell proliferation.


atRA HES-1 SOX9 Proliferation 



We thank Véronique Lefebvre and Gerd Scherer for kindly providing us SOX9 and truncated SOX9 plasmids. This work was supported by Magnus Bergwall’s foundation and by the Swedish Cancer Fund.


  1. 1.
    Ross SA, McCaffery PJ, Drager UC, De Luca LM (2000) Retinoids in embryonal development. Physiol Rev 80(3):1021–1054PubMedGoogle Scholar
  2. 2.
    Altucci L, Leibowitz MD, Ogilvie KM, de Lera AR, Gronemeyer H (2007) RAR and RXR modulation in cancer and metabolic disease. Natl Rev 6(10):793–810CrossRefGoogle Scholar
  3. 3.
    Niles RM (2000) Vitamin A and cancer. Nutrition 16(7–8):573–576CrossRefPubMedGoogle Scholar
  4. 4.
    Clarke N, Germain P, Altucci L, Gronemeyer H (2004) Retinoids: potential in cancer prevention and therapy. Expert Rev Mol Med 6(25):1–23. doi: 10.1017/S1462399404008488 CrossRefPubMedGoogle Scholar
  5. 5.
    Chambon P (1996) A decade of molecular biology of retinoic acid receptors. FASEB J 10(9):940–954PubMedGoogle Scholar
  6. 6.
    Glass CK, Rosenfeld MG (2000) The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14(2):121–141PubMedGoogle Scholar
  7. 7.
    Dilworth FJ, Chambon P (2001) Nuclear receptors coordinate the activities of chromatin remodeling complexes and coactivators to facilitate initiation of transcription. Oncogene 20(24):3047–3054. doi: 10.1038/sj.onc.1204329 CrossRefPubMedGoogle Scholar
  8. 8.
    Niles RM (2000) Recent advances in the use of vitamin A (retinoids) in the prevention and treatment of cancer. Nutrition 16(11–12):1084–1089CrossRefPubMedGoogle Scholar
  9. 9.
    Preisler HD, Gopal V, Banavali SD, Finke D, Bokari SA (1992) Multiparameter assessment of the cell cycle effects of bioactive and cytotoxic agents. Cancer Res 52(15):4090–4095PubMedGoogle Scholar
  10. 10.
    Ueda H, Takenawa T, Millan JC, Gesell MS, Brandes D (1980) The effects of retinoids on proliferative capacities and macromolecular synthesis in human breast cancer MCF-7 cells. Cancer 46(10):2203–2209. doi: 10.1002/1097-0142(19801115)46:10<2203::AID-CNCR2820461017>3.0.CO;2-A CrossRefPubMedGoogle Scholar
  11. 11.
    Fontana JA, Mezu AB, Cooper BN, Miranda D (1990) Retinoid modulation of estradiol-stimulated growth and of protein synthesis and secretion in human breast carcinoma cells. Cancer Res 50(7):1997–2002PubMedGoogle Scholar
  12. 12.
    Fitzgerald P, Teng M, Chandraratna RA, Heyman RA, Allegretto EA (1997) Retinoic acid receptor alpha expression correlates with retinoid-induced growth inhibition of human breast cancer cells regardless of estrogen receptor status. Cancer Res 57(13):2642–2650PubMedGoogle Scholar
  13. 13.
    Sheikh MS, Shao ZM, Chen JC, Hussain A, Jetten AM, Fontana JA (1993) Estrogen receptor-negative breast cancer cells transfected with the estrogen receptor exhibit increased RAR alpha gene expression and sensitivity to growth inhibition by retinoic acid. J Cell Biochem 53(4):394–404. doi: 10.1002/jcb.240530417 CrossRefPubMedGoogle Scholar
  14. 14.
    Zhu WY, Jones CS, Kiss A, Matsukuma K, Amin S, De Luca LM (1997) Retinoic acid inhibition of cell cycle progression in MCF-7 human breast cancer cells. Exp Cell Res 234(2):293–299. doi: 10.1006/excr.1997.3589 CrossRefPubMedGoogle Scholar
  15. 15.
    Yang L, Ostrowski J, Reczek P, Brown P (2001) The retinoic acid receptor antagonist, BMS453, inhibits normal breast cell growth by inducing active TGFbeta and causing cell cycle arrest. Oncogene 20(55):8025–8035. doi: 10.1038/sj.onc.1204911 CrossRefPubMedGoogle Scholar
  16. 16.
    Strom A, Arai N, Leers J, Gustafsson JA (2000) The Hairy and Enhancer of Split homologue-1 (HES-1) mediates the proliferative effect of 17beta-estradiol on breast cancer cell lines. Oncogene 19(51):5951–5953. doi: 10.1038/sj.onc.1203990 CrossRefPubMedGoogle Scholar
  17. 17.
    Hartman J, Muller P, Foster JS, Wimalasena J, Gustafsson JA, Strom A (2004) HES-1 inhibits 17beta-estradiol and heregulin-beta1-mediated upregulation of E2F–1. Oncogene 23(54):8826–8833. doi: 10.1038/sj.onc.1208139 CrossRefPubMedGoogle Scholar
  18. 18.
    Muller P, Kietz S, Gustafsson JA, Strom A (2002) The anti-estrogenic effect of all-trans-retinoic acid on the breast cancer cell line MCF-7 is dependent on HES-1 expression. J Biol Chem 277(32):28376–28379. doi: 10.1074/jbc.C200340200 CrossRefPubMedGoogle Scholar
  19. 19.
    Castella P, Sawai S, Nakao K, Wagner JA, Caudy M (2000) HES-1 repression of differentiation and proliferation in PC12 cells: role for the helix 3-helix 4 domain in transcription repression. Mol Cell Biol 20(16):6170–6183. doi: 10.1128/MCB.20.16.6170-6183.2000 CrossRefPubMedGoogle Scholar
  20. 20.
    Kunnimalaiyaan M, Yan S, Wong F, Zhang YW, Chen H (2005) Hairy Enhancer of Split-1 (HES-1), a Notch1 effector, inhibits the growth of carcinoid tumor cells. Surgery 138(6):1137–1142. doi: 10.1016/j.surg.2005.05.027 discussion 1142CrossRefPubMedGoogle Scholar
  21. 21.
    Ishibashi M, Moriyoshi K, Sasai Y, Shiota K, Nakanishi S, Kageyama R (1994) Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J 13(8):1799–1805PubMedGoogle Scholar
  22. 22.
    Sasai Y, Kageyama R, Tagawa Y, Shigemoto R, Nakanishi S (1992) Two mammalian helix-loop-helix factors structurally related to Drosophila hairy and enhancer of split. Genes Dev 6(12B):2620–2634. doi: 10.1101/gad.6.12b.2620 CrossRefPubMedGoogle Scholar
  23. 23.
    Kopan R, Nye JS, Weintraub H (1994) The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix-loop-helix region of MyoD. Development 120(9):2385–2396PubMedGoogle Scholar
  24. 24.
    Tomita K, Ishibashi M, Nakahara K, Ang SL, Nakanishi S, Guillemot F, Kageyama R (1996) Mammalian hairy and enhancer of split homolog 1 regulates differentiation of retinal neurons and is essential for eye morphogenesis. Neuron 16(4):723–734. doi: 10.1016/S0896-6273(00)80093-8 CrossRefPubMedGoogle Scholar
  25. 25.
    Tomita K, Hattori M, Nakamura E, Nakanishi S, Minato N, Kageyama R (1999) The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev 13(9):1203–1210. doi: 10.1101/gad.13.9.1203 CrossRefPubMedGoogle Scholar
  26. 26.
    Wagner T, Wirth J, Meyer J, Zabel B, Held M, Zimmer J, Pasantes J, Bricarelli FD, Keutel J, Hustert E et al (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79(6):1111–1120. doi: 10.1016/0092-8674(94)90041-8 CrossRefPubMedGoogle Scholar
  27. 27.
    Vidal VP, Chaboissier MC, de Rooij DG, Schedl A (2001) Sox9 induces testis development in XX transgenic mice. Nat Genet 28(3):216–217. doi: 10.1038/90046 CrossRefPubMedGoogle Scholar
  28. 28.
    Drivdahl R, Haugk KH, Sprenger CC, Nelson PS, Tennant MK, Plymate SR (2004) Suppression of growth and tumorigenicity in the prostate tumor cell line M12 by overexpression of the transcription factor SOX9. Oncogene 23(26):4584–4593. doi: 10.1038/sj.onc.1207603 CrossRefPubMedGoogle Scholar
  29. 29.
    Panda DK, Miao D, Lefebvre V, Hendy GN, Goltzman D (2001) The transcription factor SOX9 regulates cell cycle and differentiation genes in chondrocytic CFK2 cells. J Biol Chem 276(44):41229–41236. doi: 10.1074/jbc.M104231200 CrossRefPubMedGoogle Scholar
  30. 30.
    Afonja O, Raaka BM, Huang A, Das S, Zhao X, Helmer E, Juste D, Samuels HH (2002) RAR agonists stimulate SOX9 gene expression in breast cancer cell lines: evidence for a role in retinoid-mediated growth inhibition. Oncogene 21(51):7850–7860. doi: 10.1038/sj.onc.1205985 CrossRefPubMedGoogle Scholar
  31. 31.
    Lefebvre V, Huang W, Harley VR, Goodfellow PN, de Crombrugghe B (1997) SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alpha1(II) collagen gene. Mol Cell Biol 17(4):2336–2346PubMedGoogle Scholar
  32. 32.
    Hwang CK, Wu X, Wang G, Kim CS, Loh HH (2003) Mouse mu opioid receptor distal promoter transcriptional regulation by SOX proteins. J Biol Chem 278(6):3742–3750. doi: 10.1074/jbc.M208780200 CrossRefPubMedGoogle Scholar
  33. 33.
    Schreiber E, Matthias P, Muller MM, Schaffner W (1989) Rapid detection of octamer binding proteins with ‘mini-extracts’, prepared from a small number of cells. Nucleic Acids Res 17(15):6419. doi: 10.1093/nar/17.15.6419 CrossRefPubMedGoogle Scholar
  34. 34.
    Frasor J, Danes JM, Komm B, Chang KC, Lyttle CR, Katzenellenbogen BS (2003) Profiling of estrogen up- and down-regulated gene expression in human breast cancer cells: insights into gene networks and pathways underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144(10):4562–4574. doi: 10.1210/en.2003-0567 CrossRefPubMedGoogle Scholar
  35. 35.
    Hirata H, Yoshiura S, Ohtsuka T, Bessho Y, Harada T, Yoshikawa K, Kageyama R (2002) Oscillatory expression of the bHLH factor Hes1 regulated by a negative feedback loop. Science 298(5594):840–843CrossRefPubMedGoogle Scholar
  36. 36.
    Yoshiura S, Ohtsuka T, Takenaka Y, Nagahara H, Yoshikawa K, Kageyama R (2007) Ultradian oscillations of Stat, Smad, and Hes1 expression in response to serum. Proc Natl Acad Sci USA 104(27):11292–11297. doi: 10.1073/pnas.0701837104 CrossRefPubMedGoogle Scholar
  37. 37.
    Bernard P, Tang P, Liu S, Dewing P, Harley VR, Vilain E (2003) Dimerization of SOX9 is required for chondrogenesis, but not for sex determination. Hum Mol Genet 12(14):1755–1765. doi: 10.1093/hmg/ddg182 CrossRefPubMedGoogle Scholar
  38. 38.
    Bridgewater LC, Walker MD, Miller GC, Ellison TA, Holsinger LD, Potter JL, Jackson TL, Chen RK, Winkel VL, Zhang Z et al (2003) Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements. Nucleic Acids Res 31(5):1541–1553. doi: 10.1093/nar/gkg230 CrossRefPubMedGoogle Scholar
  39. 39.
    Karlsson C, Brantsing C, Svensson T, Brisby H, Asp J, Tallheden T, Lindahl A (2007) Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors. J Orthop Res 25(2):152–163. doi: 10.1002/jor.20287 CrossRefPubMedGoogle Scholar
  40. 40.
    Oldershaw RA, Tew SR, Russell AM, Meade K, Hawkins R, McKay TR, Brennan KR, Hardingham TE (2008) Notch signaling through Jagged-1 is necessary to initiate chondrogenesis in human bone marrow stromal cells, but must be switched off to complete chondrogenesis. Stem Cells 26(3):666–674CrossRefPubMedGoogle Scholar
  41. 41.
    Weston AD, Chandraratna RA, Torchia J, Underhill TM (2002) Requirement for RAR-mediated gene repression in skeletal progenitor differentiation. J Cell Biol 158(1):39–51. doi: 10.1083/jcb.200112029 CrossRefPubMedGoogle Scholar
  42. 42.
    Seymour PA, Freude KK, Tran MN, Mayes EE, Jensen J, Kist R, Scherer G, Sander M (2007) SOX9 is required for maintenance of the pancreatic progenitor cell pool. Proc Natl Acad Sci USA 104(6):1865–1870. doi: 10.1073/pnas.0609217104 CrossRefPubMedGoogle Scholar
  43. 43.
    Georgia S, Soliz R, Li M, Zhang P, Bhushan A (2006) p57 and Hes1 coordinate cell cycle exit with self-renewal of pancreatic progenitors. Dev Biol 298(1):22–31. doi: 10.1016/j.ydbio.2006.05.036 CrossRefPubMedGoogle Scholar
  44. 44.
    Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi M, Kageyama R, Guillemot F, Serup P, Madsen OD (2000) Control of endodermal endocrine development by Hes-1. Nat Genet 24(1):36–44. doi: 10.1038/71657 CrossRefPubMedGoogle Scholar
  45. 45.
    Kayahara T, Sawada M, Takaishi S, Fukui H, Seno H, Fukuzawa H, Suzuki K, Hiai H, Kageyama R, Okano H et al (2003) Candidate markers for stem and early progenitor cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine. FEBS Lett 535(1–3):131–135. doi: 10.1016/S0014-5793(02)03896-6 CrossRefPubMedGoogle Scholar
  46. 46.
    Yang Q, Bermingham NA, Finegold MJ, Zoghbi HY (2001) Requirement of Math1 for secretory cell lineage commitment in the mouse intestine. Science 294(5549):2155–2158CrossRefPubMedGoogle Scholar
  47. 47.
    Fre S, Huyghe M, Mourikis P, Robine S, Louvard D, Artavanis-Tsakonas S (2005) Notch signals control the fate of immature progenitor cells in the intestine. Nature 435(7044):964–968. doi: 10.1038/nature03589 CrossRefPubMedGoogle Scholar
  48. 48.
    van Es JH, van Gijn ME, Riccio O, van den Born M, Vooijs M, Begthel H, Cozijnsen M, Robine S, Winton DJ, Radtke F et al (2005) Notch/gamma-secretase inhibition turns proliferative cells in intestinal crypts and adenomas into goblet cells. Nature 435(7044):959–963. doi: 10.1038/nature03659 CrossRefPubMedGoogle Scholar
  49. 49.
    Mori-Akiyama Y, van den Born M, van Es JH, Hamilton SR, Adams HP, Zhang J, Clevers H, de Crombrugghe B (2007) SOX9 is required for the differentiation of paneth cells in the intestinal epithelium. Gastroenterology 133(2):539–546. doi: 10.1053/j.gastro.2007.05.020 CrossRefPubMedGoogle Scholar
  50. 50.
    Bastide P, Darido C, Pannequin J, Kist R, Robine S, Marty-Double C, Bibeau F, Scherer G, Joubert D, Hollande F et al (2007) Sox9 regulates cell proliferation and is required for Paneth cell differentiation in the intestinal epithelium. J Cell Biol 178(4):635–648. doi: 10.1083/jcb.200704152 CrossRefPubMedGoogle Scholar
  51. 51.
    Suzuki K, Fukui H, Kayahara T, Sawada M, Seno H, Hiai H, Kageyama R, Okano H, Chiba T (2005) Hes1-deficient mice show precocious differentiation of Paneth cells in the small intestine. Biochem Biophys Res Commun 328(1):348–352. doi: 10.1016/j.bbrc.2004.12.174 CrossRefPubMedGoogle Scholar
  52. 52.
    Curry CL, Reed LL, Nickoloff BJ, Miele L, Foreman KE (2006) Notch-independent regulation of Hes-1 expression by c-Jun N-terminal kinase signaling in human endothelial cells. Lab invest; J tech methods pathol 86(8):842–852Google Scholar
  53. 53.
    Murata K, Hattori M, Hirai N, Shinozuka Y, Hirata H, Kageyama R, Sakai T, Minato N (2005) Hes1 directly controls cell proliferation through the transcriptional repression of p27Kip1. Mol Cell Biol 25(10):4262–4271. doi: 10.1128/MCB.25.10.4262-4271.2005 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2009

Authors and Affiliations

  • Patrick Müller
    • 1
    Email author
  • Justin D. Crofts
    • 2
  • Ben S. Newman
    • 2
  • Laura C. Bridgewater
    • 2
  • Chin-Yo Lin
    • 2
  • Jan-Åke Gustafsson
    • 1
    • 3
  • Anders Ström
    • 3
  1. 1.Department of Biosciences and NutritionKarolinska InstitutetHuddingeSweden
  2. 2.Department of Microbiology and Molecular BiologyBrigham Young UniversityProvoUSA
  3. 3.Center for Nuclear Receptors and Cell Signaling, Department of Cell Biology and BiochemistryUniversity of HoustonHoustonUSA

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