Breast Cancer Research and Treatment

, Volume 86, Issue 1, pp 55–73

Growth and EGFR Regulation in Breast Cancer Cells by Vitamin D and Retinoid Compounds

  • Kenneth R. McGaffin
  • Lisa E. Acktinson
  • Susan A. Chrysogelos


The effect of 1,25-dihydroxyvitamin D3, analog C (1,25-(OH)2-16-en-23-yn-26,27-F6-vitamin D3), 9-cis retinoic acid, and all-trans retinoic acid on the growth and expression of EGFR in MCF7, T47D, BT474, and BT549 breast cancer cells was examined. Significant growth inhibition was noted in MCF7, T47D, and BT474 cells by 8 days of treatment, while BT549 cells showed none. MCF7, T47D, and BT549 cells treated with 1,25-dihydroxyvitamin D3 demonstrated a 50% decrease in EGFR mRNA within 2 h which was sustained to 72 h, while BT474 cells demonstrated a 200–500% increase. EGFR protein levels correlated with these mRNA changes in BT474 and BT549 cells. Measurement of mRNA stability in vitamin D treated BT474 cells indicated that there was no change in EGFR mRNA half-life. Transfection of an EGFR promoter containing reporter plasmid demonstrated vitamin D induced changes in reporter gene activity that paralleled the changes observed in EGFR mRNA and protein. Electrophoretic mobility shift assays using a putative vitamin D response element within this region of the EGFR promoter demonstrated specific VDR binding. These results indicate that the vitamin D effect on EGFR expression in breast cancer cells has a transcriptional component likely mediated through a vitamin D responsive promoter sequence. They also suggest that growth inhibition and EGFR down-regulation by vitamin D and retinoids may be related events in some breast cancer cells, but not in all.

breast cancer EGFR retinoids transcription vitamin D 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rajkumar T, Gullick WJ: The type I growth factor receptors in human breast cancer. Breast Cancer Res Treat 29: 3–9, 1994CrossRefPubMedGoogle Scholar
  2. 2.
    Buras RR, Schumaker LM, Davoodi F, Brenner RV, Shabahang M, Nauta RJ, Evans SR: Vitamin D receptors in breast cancer cells. Breast Cancer Res Treat 31: 191–202, 1994CrossRefPubMedGoogle Scholar
  3. 3.
    Looi LM, Cheah PL: C-erbB-2 oncoprotein amplification in infiltrating ductal carcinoma of the breast relates to high histological grade and loss of estrogen receptor protein. Malays J Pathol 20: 19–23, 1998PubMedGoogle Scholar
  4. 4.
    Harris AL: What is the biological, prognostic, and therapeutic role of the EGF receptor in human breast cancer? Breast Cancer Res Treat 29: 1–2, 1994CrossRefPubMedGoogle Scholar
  5. 5.
    Sainsbury J, Farndon J, Needham G, Malcolm A, Harris A: Epidermal growth factor receptor status as a predictor of early recurrence of and death from breast cancer. Lancet I: 1398–1402, 1987Google Scholar
  6. 6.
    Chrysogelos SA, Dickson RB: EGF receptor expression, regulation, and function in breast cancer. Breast Cancer Res Treat 29: 29–40, 1994CrossRefPubMedGoogle Scholar
  7. 7.
    Ulrich A, Schlessinger J: Signal transduction by receptors with tyrosine kinase activity. Cell 61: 203–212, 1990CrossRefPubMedGoogle Scholar
  8. 8.
    Jardines L, Weiss M, Fowble B, Green M: Neu(c-erbB-2/HER2) and the epidermal growth factor receptor (EGFR) in breast cancer. Pathobiology 61: 268–282, 1993PubMedGoogle Scholar
  9. 9.
    Davidson NE, Gelmann EP, Lipmann ME, Dickson RB: Epidermal growth factor receptor gene expression in estrogen receptor-positive and negative human breast cancer cell lines. Mol Endocrinol 1: 216–223, 1987PubMedGoogle Scholar
  10. 10.
    DiFiore P, Pierce J, Fleming T, Hazon R, Ulrich A, King CR, Schlessinger J, Aaronson S: Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH3T3 cells. Cell 51: 1063–1070, 1987CrossRefPubMedGoogle Scholar
  11. 11.
    Liu YE, Lupu R, Kern FG: Characterization of MCF7 breast carcinoma cells singly or doubly transfected with c-erbB2 and EGFR. Proc AARC 33: 368, 1992Google Scholar
  12. 12.
    Agthoven TV, Agthoven TLAV, Portengen H, Foekers JA, Dorsse LCJ: Ectopic expression of epidermal growth factor receptors induces hormone independence in ZR-75-1 human breast cancer cells. Cancer Res 52: 5082–5088, 1992PubMedGoogle Scholar
  13. 13.
    Baselga J, Medelsohn J: The epidermal growth factor receptor as a target for therapy in breast carcinoma. Breast Cancer Res Treat 29: 157–138, 1994CrossRefGoogle Scholar
  14. 14.
    Clark AJL, Ishii S, Richert N, Merlino GT, Pastan I: Epidermal growth factor regulates the expression of its own receptor. Proc Natl Acad Sci USA 82: 8374–8378, 1985PubMedGoogle Scholar
  15. 15.
    Desprez PY, Poujol D, Falette N, Lefebvre NF, Saez S: 1,25-dihydroxyvitamin D3 increases epidermal growth factor gene expression in BT-20 breast carcinoma cells. Biochem Biophys Res Comm 176: 1–6, 1991CrossRefPubMedGoogle Scholar
  16. 16.
    Prasad KN, Edwards-Prasad J, Kumar S, Meyers A: Vitamins regulate gene expression and induce differentiation and growth inhibition in cancer cells. Their relevance in cancer prevention. Arch Otolaryngol Head Neck Surg 119: 1133–1140, 1993PubMedGoogle Scholar
  17. 17.
    Welsh J, VanWeelden K, Flanagan L, Byrne I, Nolan E, Narvaez CJ: The role of vitamin D3 and antiestrogens in modulating apoptosis of breast cancer cells and tumors. Subcell Biochem 30: 245–270, 1998PubMedGoogle Scholar
  18. 18.
    Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfiled AE: 1 alpha,25-dihydroxyvitamin D3 inhibits angiogenesis in vitro and in vivo. Circ Res 87: 214–220, 2000PubMedGoogle Scholar
  19. 19.
    Hansen CM, Frandsen TL, Brunner N, Binderup L: 1 alpha,25-dihydroxyvitamin D3 inhibits the invasive potential of human breast cancer cells in vitro. Clin Exp Metastasis 12: 195–202, 1994CrossRefPubMedGoogle Scholar
  20. 20.
    Chouvet C, Vicard E, Devonec M, Saez S: 1,25-dihydroxyvitamin D3 inhibitory effect on the growth of two human breast cancer cell lines (MCF7, BT-20). J Steroid Biochem 24: 373–376, 1986CrossRefPubMedGoogle Scholar
  21. 21.
    Koga M, Sutherland RL: Retinoic acid acts synergistically with 1,25-dihydroxyvitamin D3 or antioestrogen to inhibit T-47D human breast cancer cell proliferation. J Steroid Biochem Mol Biol 39: 455–460, 1991CrossRefPubMedGoogle Scholar
  22. 22.
    Zheng ZS, Polakowska R, Johnson A, Goldsmith LA: Transcriptional control of epidermal growth factor receptor by retinoic acid. Cell Grow Diff 3: 255–232, 1992Google Scholar
  23. 23.
    Chomczynski P, Sacchi N: Single step method of RNA isolation by acid guanidinium thiocyanate-pheno-chloroform extraction. Anal Biochem 162: 156–159, 1987CrossRefPubMedGoogle Scholar
  24. 24.
    Laborda J: 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucl Acids Res 19: 3998, 1991PubMedGoogle Scholar
  25. 25.
    Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254, 1976CrossRefPubMedGoogle Scholar
  26. 26.
    Salinovich O, Montelaro RC: Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analytical Biochem 156: 341–347, 1986CrossRefGoogle Scholar
  27. 27.
    Dignam JD, Lebovitz RM, Roeder RG: Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucl Acids Res 11: 1475–1489, 1983PubMedGoogle Scholar
  28. 28.
    Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, Evans RM: The nuclear receptor superfamily: the second decade. Cell 83: 835–839, 1995CrossRefPubMedGoogle Scholar
  29. 29.
    Roman SD, Clarke CL, Hall RE, Alexander IE, Sutherland RL: Expression and regulation of retinoic acid receptors in human breast cancer cells. Cancer Res 52: 2236–2242, 1992PubMedGoogle Scholar
  30. 30.
    Shao ZM, Sheikh MS, Chen JC, Kute T, Aisner S, Schnaper L, Fontana JA: Expression of the retinoic acid nuclear receptors (RARs) and retinoid X receptor genes in estrogen receptor positive and negative breast cancer. Int J Oncol 4: 859–8563, 1994Google Scholar
  31. 31.
    Zhou JY, Norman AW, Akashi M, Chen DL, Uskokovic MR, Aurrecoehea JM, Dauben WG, Okamura WH, Koeffler HP: Development of a novel 1,25(OH)2-vitamin D3 analog with potent ability to induce HL-60 cell differentiation without modulating calcium metabolism. Blood 78: 75–82, 1991PubMedGoogle Scholar
  32. 32.
    MacDonald PN, Dowd DR, Nakajima S, Galligan MA, Reeder MC, Haussler CA, Ozato K, Haussler MR: Retinoid X receptors stimulate and 9-cis retinoic acid inhibits 1,25-dihydroxyvitamin D3-activated expression of the rat osteocalcin gene. Mol Cell Biol 13: 5907–5917, 1993PubMedGoogle Scholar
  33. 33.
    DeLuca HF, Zierold C: Mechanisms and functions of vitamin D. Nutr Rev 56: S54–S75, 1998Google Scholar
  34. 34.
    Fridovich-Keil JL, Gudas JM, Bryan IB, Pardee AB: Improved expression vectors for eukaryotic promoter/enhancer studies. BioTechniques 11: 572–579, 1991PubMedGoogle Scholar
  35. 35.
    Umesono K, Murakami KK, Thompson CC, Evans RM: Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65: 1255–1266, 1991CrossRefPubMedGoogle Scholar
  36. 36.
    Ozono K, Liao J, Kerner SA, Scott RA, Pike JW: The vitamin D-responsive element in the human osteocalcin gene. J Biol Chem 265: 21881–21888, 1990PubMedGoogle Scholar
  37. 37.
    Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM: DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87: 369–373, 1990PubMedGoogle Scholar
  38. 38.
    Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT: Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phospoprotein 1 (Spp-1 or osteopontin.). Proc Natl Acad Sci USA 87: 9995–9999, 1990PubMedGoogle Scholar
  39. 39.
    Lippman SM, Kessler JF, Meyskens FL: Retinoids as preventive and therapeutic anticancer agents (part 1). Cancer Treat Rep 71: 391–405, 1987PubMedGoogle Scholar
  40. 40.
    Hunter DJ, Manson JE, Colditz GA, Stampfer MJ. Rosner B, Hennekens CH, Speizer FE, Wilett WC: A prospective study of the intake of vitamins C, E, and A and the risk of breast cancer. N Engl J Med 329: 234–240, 1993CrossRefPubMedGoogle Scholar
  41. 41.
    Blot WJ, Fraumeni JF, Stone BJ: Geographic patterns of breast cancer in the United States. JNCI 59: 1407–1411, 1977PubMedGoogle Scholar
  42. 42.
    Gorham ED, Garland CF, Garland FC: Acid haze air pollution in breast and colon cancer mortality in 20 Canadian cities. Can J Public Health 80: 96–100, 1989PubMedGoogle Scholar
  43. 43.
    Garland FC, Garland CF, Gorham ED, Young JF: Geographic variation in breast cancer mortality in the United States: a hypothesis involving exposure to solar radiation. Prev Med 19: 614–622, 1990CrossRefPubMedGoogle Scholar
  44. 44.
    Gorham ED, Garland FC, Garland CF: Sunlight and breast cancer incidence in the USSR. Int J Epidemiol 19: 820–824, 1990PubMedGoogle Scholar
  45. 45.
    Garland FC, Garland CF, Gorham ED: Calcium and vitamin D: their potential roles in colon and breast cancer prevention. Ann NY Acad Sci 889: 107–119, 1999CrossRefPubMedGoogle Scholar
  46. 46.
    Haddad JG, Hahn TJ: Natural and synthetic sources of circulating 25-hydroxyvitamin D in man. Nature 244: 515–517, 1973CrossRefPubMedGoogle Scholar
  47. 47.
    Janowsky EC, Lester GE, Weinberg CR, Millikan RC, Schildkraut JM, Garrett PA, Hulka BS: Association between low levels of 1,25-dihydroxyvitamin D3 and breast cancer risk. Public Health Nutr 2: 283–291, 1999PubMedGoogle Scholar
  48. 48.
    Bower M, Colston KW, Stein RC, Hedley A, Gazet J-C, Ford HT, Coombes RC: Topical calcipotriol treatment in advanced breast cancer. Lancet 337: 701–702, 1991CrossRefPubMedGoogle Scholar
  49. 49.
    Colston KW, Berger U, Coombes RC: Possible role for vitamin D in controlling breast cancer cell proliferation. Lancet 1(8631): 188–191, 1989CrossRefPubMedGoogle Scholar
  50. 50.
    Sheikh MS, Shao Z-M, Chen J-C, Hussain A, Jetten AM, Fontana JA: Estrogen receptor-negative breast cancer cells transfected with the estrogen receptor exhibit increased RARa gene expression and sensitivity to growth inhibition by retinoic acid. J Cell Biochem 53: 394–404, 1993CrossRefPubMedGoogle Scholar
  51. 51.
    Welsh J, Wietzke J, Zinser G, Smyczek S, Romu S, Tribble E, Welsh J, Byrne B, Narvaez C: Impact of the vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer. J Steroid Biochem Mol Biol 83: 85–92, 2003CrossRefGoogle Scholar
  52. 52.
    Bortman P, Folgueira M, Katayama M, Snitcovsky I, Brentani M: Antiproliferative effects of 1,25-dihydroxyvitamin D3 on breast cells-a mini review. Braz J Med Biol Res 35: 1–9, 2002CrossRefPubMedGoogle Scholar
  53. 53.
    Farach-Carson MC, Sergeev I, Norman AW: Nongenomic actions of 1,25-dihydroxyvitamin D3 in rat osteosarcoma cells: structure-function studies using ligand analogs. Endocrinology 129: 1876–1884, 1991PubMedGoogle Scholar
  54. 54.
    Nemere I, Norman AW: Steroid hormone actions at the plasma membrane: induced calcium uptake and exocytotic events. Mol Cell Endocrinol 80: C165–C169, 1991CrossRefPubMedGoogle Scholar
  55. 55.
    Sorensen AM, Bowman D, Baran DT: 1α,25-dihydroxyvitamin D3 rapidly increases nuclear calcium levels in rat osteosarcoma cells. J Cell Biochem 52: 237–242, 1993CrossRefPubMedGoogle Scholar
  56. 56.
    Lieberherr M: Effects of vitamin D3 metabolites on cytosolic free calcium in confluent mouse osteoblasts. J Biol Chem 262: 13168–13173, 1987PubMedGoogle Scholar
  57. 57.
    Grosse B, Bourdeau A, Lieberherr M: Oscillation in inositiol 1,4,5-triphosphate and diacylglycerol induced by vitamin D metabolites in confluent mouse osteoblasts. Bone Min Res 8: 1059–1069, 1993Google Scholar
  58. 58.
    Jeni LG, Lian JB, Stein GS, Baran DT: 1?,25-dihydroxyvitamin D3-induced changes in intracellular pH in osteoblast-like cells modulate gene expression. J Cell Biochem 53: 234–239, 1993CrossRefPubMedGoogle Scholar
  59. 59.
    Laird AD, Shalloway D: Oncoprotein signalling and mitosis. Cell Signal 9: 249–255, 1997CrossRefPubMedGoogle Scholar
  60. 60.
    Codero JB, Cozzolino M, Lu Y, Vidal M, Slatopolsky E, Stahl P, Barbieri MA, Dusso A: 1,25-dihydroxyvitamin D downregulates cell membrane growth and nuclear-growth promoting signals by the epidermal growth factor receptor. J Biol Chem 277: 38965–38971, 2002CrossRefPubMedGoogle Scholar
  61. 61.
    Koga M, Eisman JA, Sutherland RL: Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells. Cancer Res 48: 2734–2739, 1988PubMedGoogle Scholar
  62. 62.
    Falette N, Frappart L, Lefebvre MF, Saez S: Increased epidermal growth factor receptor level in breast cancer cells treated by 1,25-dihydroxyvitamin D3. Mol Cell Endo 63: 189–198, 1989CrossRefGoogle Scholar
  63. 63.
    Yarden RI, Wilson MA, Barth M, Chrysogelos SA: The role of estrogen in the regulation of EGFR expression. In: Lichtner RB, Harkins RN (eds) Ernest Schering Research Foundation Workshop 19: EGF Receptor in Tumor Growth and Progression. Springer Scientific Publishers, Berlin, 1996, pp 129–154Google Scholar
  64. 64.
    Pike JW: Vitamin D3 receptors: structure and function in transcription. Annu Rev Nutr 11: 189–216, 1991CrossRefPubMedGoogle Scholar
  65. 65.
    Gonzales EA, Disthabanchong S, Kowalewski R, Martin K: Mechanisms of regulation of egf receptor gene expression by calcitriol and parathyroid hormone in UMR 106-01 cells. Kidney Int 61: 1627–1634, 2002CrossRefPubMedGoogle Scholar
  66. 66.
    Haley J, Whittle N, Bennet P, Kinchington D, Ulrich A, Waterfield M: The human egf receptor gene: structure of the 110 kb locus and identification of sequences regulating its transcription. Oncogene Res 1: 375–396, 1987PubMedGoogle Scholar
  67. 67.
    Johnson AC, Ishii S, Jinno Y, Pastan I, Merlino GT: Epidermal growth factor gene promoter. Deletion analysis and identification of nuclear protein binding sites. J Biol Chem 263: 5693–5699, 1988PubMedGoogle Scholar
  68. 68.
    Kageyama R, Pastan I: Molecular cloning and characterization of a human DNA binding factor that represses transcription. Cell 59: 815–825, 1989CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Kenneth R. McGaffin
    • 1
  • Lisa E. Acktinson
    • 2
  • Susan A. Chrysogelos
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyLombardi Cancer CenterUSA
  2. 2.School of MedicineGeorgetown UniversityUSA
  3. 3.University of Pittsburgh Medical CenterPittsburghUSA

Personalised recommendations