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MCF-7 breast cancer cells overexpressing transfectedc-erbB-2 have anin vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivityin vivo

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Summary

Ac-erbB-2 expression vector was transfected into the estrogen receptor positive (ER+) MCF-7 human breast cancer cell line to determine if overexpression of this transmembrane tyrosine kinase could increase the malignant phenotype of this cell line. Loss of transfectedc-erbB-2 expression was observed when cells were carried in medium containing estrogen. Homogeneous populations stably overexpressing levels of the 185 kDac-erbB-2 observed in the SKBR-3 a breast cancer cell line which overexpressesc-erbB-2 as a result of gene amplification could be obtained by continually maintaining the transfected cell lines in estrogen-free conditions. Levels of constitutively activatedc-erbB-2 varied among clonal isolates. Whereas some over-expressing lines did acquire the ability to form transient tumor nodules in ovariectomized nude mice without estrogen supplementation, as well as in mice that received the antiestrogen tamoxifen, one cell line that exhibited the highest levels of constitutively activatedc-erbB-2 was able to form static tumors of a larger size under both conditions. This same cell line formed progressively growing tumors in estrogen-supplemented mice that were much larger than observed in mice injected with control cell lines, and also showed reduced sensitivity to antiestrogensin vitro, but it continued to have a low metastatic phenotype. These results suggest that signal transduction mediated by thec-erbB-2 tyrosine kinase can partially overcome the estrogen dependence of ER+ breast cancer cells for growth and thatc-erbB-2 overexpression confers a selective advantage to such cells in the absence of estrogen.

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References

  1. Kraus MH, Fedi P, Starks V, Muraro R, Aaronson SA: Demonstration of ligand-dependent signaling by theerb-B3 tyrosine kinase and its constitutive activation in human breast tumor cells. Proc Natl Acad Sci USA 90: 2900–2904, 1993

    Google Scholar 

  2. Plowman GD, Culouscou J-M, Whitney GS, Green JM, Carolton GW, Foy L, Neubauer MG, Shoyab M: Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc Natl Acad Sci USA 90: 1746–1750, 1993

    Google Scholar 

  3. Bargmann CL, Hung M-C, Weinberg RA: The neu oncogene encodes an epidermal growth factor receptor-related protein. Nature 319: 226–230, 1986

    Google Scholar 

  4. Coussens L, Yang-Feng T, Liao Y-C, Chen L, Gray A, McGrath J, Seeburg PH, Lieberman TA, Schlessinger J, Francke U, Levinson A, Ullrich A: Tyrosine kinase receptor with extensive homology to EGF receptor shares chromosomal location with neu oncogene. Science 230: 1132–1139, 1985

    Google Scholar 

  5. Plowman GD, Whitney GS, Neubauer MG, Green JM, McDonald VL, Todaro GJ, Shoyab M: Molecular cloning and expression of an additional epidermal growth factor receptor-related gene. Proc Natl Acad Sci USA 87:4905–4909, 1990

    Google Scholar 

  6. Lonardo F, Di Marco E, King CR, Pierce JH, Segatto O, Aaronson SA, Di Fiore PP: The normal erbB-2 product is an atypical receptor-like tyrosine kinase with constitutive activity in the absence of ligand. New Biol 2: 992–1003, 1990

    Google Scholar 

  7. DiFiore P, Pierce J, Fleming T, Hazan R, Ullrich A, King CR, Schlessinger J, Aaronson S: Overexpression of the human EGF receptor confers an EGF-dependent transformed phenotype to NIH 3T3 cells. Cell 51: 1063–1070, 1987

    Google Scholar 

  8. Hudziak RM, Schlessinger J, Ullrich A: Increased expression of the putative growth factor receptor p185HER2 causes transformation and tumorigenesis of NIH3T3 cells. Proc Natl Acad Sci USA 84: 7159–7163, 1987

    Google Scholar 

  9. DiFiore PP, Pierce J, Kraus M, Segatto O, King CR, Aaronson SA: erb-B2 is a potent oncogene when overexpressed in NIH/3T3 cells. Science 237: 178–182, 1987

    Google Scholar 

  10. Gusterson BA: Identification and interpretation of epidermal growth factor and c-erbB-2 overexpression. Eur J Cancer 28: 263–267, 1992

    Google Scholar 

  11. Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Stuart SG, Udove J, Ullrich Aet al.: Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707–712, 1989

    Google Scholar 

  12. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuire WL: Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235: 177–182, 1987

    Google Scholar 

  13. Perren TJ: cv-erbB-2 oncogene as a prognostic marker in breast cancer [editorial]. Br J Cancer 63: 328–332, 1991

    Google Scholar 

  14. Gullick WJ, Love SB, Wright C, Barnes DM, Gusterson B, Harris AL, Altman DG: c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes. Br J Cancer 63: 434–438, 1991

    Google Scholar 

  15. Lodato RF, Maguire HC Jr, Greene MI, Weiner DB, LiVolsi VA: Immunohistochemical evaluation of c-erbB-2 oncogene expression in ductal carcinomain situ and atypical ductal hyperplasia of the breast. Mod Pathol 3: 449–454, 1990

    Google Scholar 

  16. Allred DC, Clark GM, Molina R, Tandon AK, Schnitt SJ, Gilchrist KW, Osborne CK, Tormey DC, McGuire WL: Overexpression of HER-2/neu and its relationship with other prognostic factors change during the progression ofin situ to invasive breast cancer. Hum Pathol 23: 974–979, 1992

    Google Scholar 

  17. van de Vijver MJ, Peterse JL, Mooi WJ, Wisman P, Lomans J, Dalesio O, Nusse R: Neu-protein overexpression in breast cancer. Association with comedo-type ductal carcinomain situ and limited prognostic value in stage II breast cancer. N Engl J Med 319: 1239–1245, 1988

    Google Scholar 

  18. Shih C, Padhy LC, Murray M, Weinberg RA: Transforming genes of carcinomas and neuroblastomas introduced into mouse fibroblasts. Nature 290: 261–264, 1981

    Google Scholar 

  19. Bargmann CJ, Hung M-C, Weinberg RA: Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain. Cell 45: 649–657, 1986

    Google Scholar 

  20. Lemoine NR, Staddon S, Dickson C, Barnes DM, Gullick WJ: Absence of activating transmembrane mutations in the c-erbB-2 proto-oncogene in human breast cancer. Oncogene 5: 237–239, 1990

    Google Scholar 

  21. Guy CT, Webster MA, Schaller M, Parsons TJ, Cardiff RD, Muller WJ: Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc Natl Acad Sci USA 89: 10578–10582, 1992

    Google Scholar 

  22. Pierce JH, Arnstein P, DiMarco E, Artrip J, Kraus MH, Lonardo F, Di Fiore PP, Aaronson SA: Oncogenic potential of erbB-2 in human mammary epithelial cells. Oncogene 6: 1189–1194, 1991

    Google Scholar 

  23. Ciardiello F, McGeady ML, Kim N, Basolo F, Hynes N, Langton BC, Yokozaki H, Saeki T, Elliott JW, Masui H: Transforming growth factor-alpha expression is enhanced in human mammary epithelial cells transformed by an activated c-Ha-ras protooncogene but not by the c-neu protooncogene, and overexpression of the transforming growth factor-alpha complementary DNA leads to transformation. Cell Growth Differ 1: 407–420, 1990

    Google Scholar 

  24. D'Souza B, Berdichevsky F, Kyprianou N, Taylor-Papadimitriou J: Collagen-induced morphogenesis and expression of the alpha 2-integrin subunit is inhibited in c-erbB2-transfected human mammary epithelial cells. Oncogene 8: 1797–1806, 1993

    Google Scholar 

  25. Berns EM, Klijn JG, van Staveren IL, Portengen H, Noordegraaf E, Foekens JA: Prevalence of amplification of the oncogenes c-myc, HER2/neu, and int-2 in one thousand human breast tumours: correlation with steroid receptors. Eur J Cancer 28: 697–700, 1992

    Google Scholar 

  26. Wright C, Nicholson S, Angus B, Sainsbury JR, Farndon J, Cairns J, Harris AL, Horne CH: Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Br J Cancer 65: 118–121, 1992

    Google Scholar 

  27. Lippman ME, Dickson RB: Mechanisms of normal and malignant breast epithelial growth regulation. J Steroid Biochem 34: 107–121, 1989

    Google Scholar 

  28. Dickson R, Lippman ME: Estrogenic regulation of growth and polypeptide growth factor secretion in human breast carcinoma. Endocr Rev 8: 29–43, 1987

    Google Scholar 

  29. Lippman ME, Dickson RB: Mechanisms of normal and malignant breast epithelial growth regulation. J Steroid Biochem 34: 107–121, 1989

    Google Scholar 

  30. Miller DL, El-Ashry D, Cheville AL, Liu Y, McLeskey SW, Kern FG: Emergence of MCF-7 cells overexpressing a transfected epidermal growth factor receptor under estrogen-depleted conditions: evidence for a role of EGFR in breast cancer growth and progression. Cell Growth Differ, submitted: 1994

  31. Chen C, Okayama H: High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7: 2745–2752, 1987

    Google Scholar 

  32. Estes PA, Suba EJ, Lawler-Heavner J, Elashry-Stowers D, Wei LL, Toft DO, Sullivan WP, Horwitz KB, Edwards DP: Immunological analysis of progesterone receptors in human breast cancer. I. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies. Biochemistry 26: 6250–6262, 1987

    Google Scholar 

  33. Bradford M: A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976

    Google Scholar 

  34. Edwards DP, McGuire WL: 17-beta estradiol is a biologically active estrogen in human breast cancer cells in tissue culture. Endocrinology 107: 884–891, 1980

    Google Scholar 

  35. Kraus MH, Popescu NC, Amsbaugh SC, King CR: Over-expression of the EGF receptor-related proto-oncogene erbB-2 in human mammary tumor cell lines by different molecular mechanisms. EMBO J 6: 605–610, 1990

    Google Scholar 

  36. Sarup JC, Johnson RM, King KL, Fendly BM, Lipari MT, Napier MA, Ullrich A, Shepard HM: Characterization of an anti-p185HER2 monoclonal antibody that stimulates receptor function and inhibits tumor cell growth. Growth Regul 1: 72–82, 1991

    Google Scholar 

  37. Benz CC, Scott GK, Sarup JC, Johnson RM, Tripathy D, Coronado E, Shepard HM, Osborne CK: Estrogen-dependent, tamoxifen-resistant tumorigenic growth of MCF-7 cells transfected with HER2/neu. Breast Cancer Res Treat 24: 85–95, 1993

    Google Scholar 

  38. Read LD, Keith D Jr, Slamon DJ, Katzenellenbogen BS: Hormonal modulation of HER-2/neu protooncogene messenger ribonucleic acid and p185 protein expression in human breast cancer cell lines. Cancer Res 50: 3947–3951, 1990

    Google Scholar 

  39. Dati C, Antoniotti S, Taverna D, Perroteau I, De Bortoli M: Inhibition of c-erbB-2 oncogene expression by estrogens in human breast cancer cells. Oncogene 5: 1001–1006, 1990

    Google Scholar 

  40. King CR, Borrello I, Porter L, Comoglio P, Schlessinger J: Ligand-independent tyrosine phosphorylation of EGF receptor and the erbB-2/neu proto-oncogene product is induced by hyperosmotic shock. Oncogene 4: 13–18, 1989

    Google Scholar 

  41. Holme TC: Cancer cell structure: actin changes in tumour cells - possible mechanisms for malignant tumour formation. Eur J Surg Oncol 16: 161–169, 1990

    Google Scholar 

  42. Welshons WV, Jordan VC: Adaption of estrogen-dependent MCF-7 cells to low estrogen (phenol red-free) culture. Eur J Cancer Clin Oncol 23: 1935–1939, 1987

    Google Scholar 

  43. Ciardiello F, Gottardis M, Basolo F, Pepe S, Normanno N, Dickson RB, Bianco AR, Salomon DS: Additive effects of c-erbB-2, c-Ha-ras, and transforming growth factor-alpha genes onin vitro transformation of human mammary epithelial cells. Mol Carcinog 6: 43–52, 1992

    Google Scholar 

  44. Clarke R, Brünner N, Katzenellenbogen BS, Thompson EW, Norman MJ, Koppi C, Paik S, Lippman ME, Dickson RB: Progression of human breast cancer cells from hormone-dependent to hormone-independent growth bothin vitro andin vivo. Proc Natl Acad Sci USA 86: 3649–3653, 1989

    Google Scholar 

  45. Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y: Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens. Cancer Res 47: 4355–4360, 1987

    Google Scholar 

  46. Zhang L, Ding IYF, Kharbanda S, McLeskey SW, Honig S, Chen D, Kern FG: MCF-7 breast carcinoma cells overexpressing recombinant FGF-1 cDNAs with or without an appended signal peptide form vascularized, metastatic tumors in ovariectomized nude mice. Cancer Res, submitted: 1994

  47. Antoniotti S, Maggiora P, Dati C, De Bortoli M: Tamoxifen up-regulates c-erbB-2 expression in oestrogen-responsive breast cancer cellsin vitro. Eur J Cancer 28: 318–321, 1992

    Google Scholar 

  48. Sainsbury JRC, Farndon JR, Sherbet GV, Harris AL: Epidermal growth factor receptors and oestrogen receptors in human breast cancer. Lancet 1: 364–366, 1985

    Google Scholar 

  49. Dickson RB, Huff KK, Spencer EM, Lippman ME: Induction of epidermal growth factor-related polypeptides by 17 beta-estradiol in MCF-7 human breast cancer cells. Endocrinology 118: 138–142, 1986

    Google Scholar 

  50. Barnes DW: Epidermal growth factor inhibits the growth of A431 epidermoid carcinoma in serum-free cell culture. J Cell Biol 93: 1–4, 1982

    Google Scholar 

  51. Kan M, Huang J, Mansson P-E, Yasumitsu H, Carr B, McKeehan WL: Heparin-binding growth factor type 1 (acidic fibroblast growth factor): A potential biphasic autocrine and paracrine regulator of hepatocyte regeneration. Proc Natl Acad Sci USA 86: 7432–7436, 1989

    Google Scholar 

  52. Lupu L, Colomer R, Zugmaier G, Sarup J, Shepard M, Slamon D, Lippman ME: Direct interaction of a ligand for the erbB-2 oncogene product with the EGF receptor and p185erbB-2. Science 249: 1552–1555, 1990

    Google Scholar 

  53. Peles E, Bacus SS, Koski RA, Lu HS, Wen D, Ogden SG, Levy RB, Yarden Y: Isolation of the neu/HER-2 stimulatory ligand: a 44 kd glycoprotein that induces differentiation of mammary tumor cells. Cell 69: 205–216, 1992

    Google Scholar 

  54. Bacus SS, Huberman E, Chin D, Kiguchi K, Simpson S, Lippman M, Lupu R: A ligand for the erbB-2 oncogene product (gp30) induces differentiation of human breast cancer cells. Cell Growth Differ 3: 401–411, 1992

    Google Scholar 

  55. Holmes WE, Sliwkowski MX, Akita RW, Henzel WJ, Lee J, Park JW, Yansura D, Abadi N, Raab H, Lewis GD, Shepard HM, Kuang W-J, Wood WI, Goeddel DV, Vandlen RL: Identification of heregulin, a specific activator of p185erbB2 Science 256: 1205–1210, 1992

    Google Scholar 

  56. Plowman GD, Green JM, Culouscou JM, Carlton GW, Rothwell VM: Heregulin induces tyrosine phosphorylation of HER4/p180 (ErbB4). Nature 366: 473–475, 1993

    Google Scholar 

  57. King CR, Borrello I, Bellot F, Comoglio P, Schlessinger J: EGF binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3. EMBO J 7: 1647–1651, 1988

    Google Scholar 

  58. Stern DF, Kamps MP: EGF-stimulated tyrosine phosphorylation of p185neu: a potential model for receptor interactions. EMBO J 7: 995–1001, 1988

    Google Scholar 

  59. Peles E, Ben-Levy R, Tzahar E, Liu N, Wen D, Yarden Y: Cell-type specific interaction of Neu differentiation factor (NDF/heregulin) with Neu/HER-2 suggests complex ligand-receptor relationships. EMBO J 12: 961–971, 1993

    Google Scholar 

  60. Samanta A, LeVea CM, Dougall WC, Qian XL, Greene MI: Ligand and p185 (c-neu) density govern receptor interactions and tyrosine kinase activation. Proc Natl Acad Sci USA 91: 1711–1715, 1994

    Google Scholar 

  61. McLeskey SW, Kurebayashi J, Honig SF, Zwiebel J, Lippman ME, Dickson RB, Kern FG: Fibroblast growth factor 4 transfection of MCF-7 cells produces cell lines that are tumorigenic and metastatic in ovariectomized or tamoxifen-treated athymic nude mice. Cancer Res 53: 2168–2177, 1993

    Google Scholar 

  62. Lin YZ, Clinton GM: A soluble protein related to the HER-2 proto-oncogene product is released from human breast carcinoma cells. Oncogene 6: 639–643, 1991

    Google Scholar 

  63. Langton BC, Crenshaw MC, Chao LA, Stuart SG, Akita RW, Jackson JE: An antigen immunologically related to the external domain of gp185 is shed from nude mouse tumors overexpressing the c-erbB-2 (HER-2/neu) oncogene. Cancer Res 51: 2593–2598, 1991

    Google Scholar 

  64. Alper O, Yamaguchi K, Hitomi J, Honda S, Matsushima T, Abe K: The presence of c-erbB-2 gene product-related protein in culture medium conditioned by breast cancer cell line SK-BR-3. Cell Growth Differ 1: 591–599, 1990

    Google Scholar 

  65. Scott GK, Robles R, Park JW, Montgomery PA, Daniel J, Holmes WE, Lee J, Keller GA, Li WL, Fendly BM: A truncated intracellular HER2/neu receptor produced by alternative RNA processing affects growth of human carcinoma cells. Mol Cell Biol 13: 2247–2257, 1993

    Google Scholar 

  66. Basu A, Raghunath M, Bishayee S, Das M: Inhibition of tyrosine kinase activity of the epidermal growth factor (EGF) receptor by a truncated receptor form that binds to EGF: role for interreceptor interaction in kinase regulation. Mol Cell Biol 9: 671–677, 1989

    Google Scholar 

  67. Kashles O, Yarden Y, Fischer R, Ullrich A, Schlessinger J: A dominant negative mutation suppresses the function of normal epidermal growth factor receptors by heterodimerization. Mol Cell Biol 11: 1454–1463, 1991

    Google Scholar 

  68. Flickinger TW, Maihle NJ, Kung HJ: An alternatively processed mRNA from the avian c-erbB gene encodes a soluble, truncated form of the receptor that can block ligand-dependent transformation. Mol Cell Biol 12: 883–893, 1992

    Google Scholar 

  69. Zhai YF, Beittenmiller H, Wang B, Gould MN, Oakley C, Esselman WJ, Welsch CW: Increased expression of specific protein tyrosine phosphatases in human breast epithelial cells neoplastically transformed by the neu oncogene. Cancer Res 53: 2272–2278, 1993

    Google Scholar 

  70. Yu DH, Hung MC: Expression of activated rat neu oncogene is sufficient to induce experimental metastasis in 3T3 cells. Oncogene 6: 1991–1996, 1991

    Google Scholar 

  71. Yusa K, Sugimoto Y, Yamori T, Yamamoto T, Toyoshima K, Tsuruo T: Low metastatic potential of clone from murine colon adenocarcinoma 26 increased by transfection of activated c-erbB-2 gene. J Natl Cancer Inst 82: 1633–1636, 1990

    Google Scholar 

  72. Price JE, Polyzos A, Zhang RD, Daniels LM: Tumorigenicity and metastasis of human breast carcinoma cell lines in nude mice. Cancer Res 50: 717–721, 1990

    Google Scholar 

  73. Kurebayashi J, McLeskey SW, Johnson MD, Lippman ME, Dickson RB, Kern FG: Quantitative demonstration of spontaneous metastasis by MCF-7 human breast cancer cells cotransfected with fibroblast growth factor 4 and LacZ. Cancer Res 53: 2178–2187, 1993

    Google Scholar 

  74. Engel LW, Young NA: Human breast carcinoma cells in continuous culture: a review. Cancer Res 38: 4327–4339, 1978

    Google Scholar 

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  1. Lombardi Cancer Research Center, Room S107, Georgetown University Medical Center, 3800 Reservoir Road, NW, 20007, Washington, DC, USA

    Yiliang Liu, Dorraya El-Ashry, Denise Chen, Ivan Yi Fan Ding & Francis G. Kern

  2. Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3800 Reservoir Road, NW, 20007, Washington, DC, USA

    Dorraya El-Ashry & Francis G. Kern

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Liu, Y., El-Ashry, D., Chen, D. et al. MCF-7 breast cancer cells overexpressing transfectedc-erbB-2 have anin vitro growth advantage in estrogen-depleted conditions and reduced estrogen-dependence and tamoxifen-sensitivityin vivo . Breast Cancer Res Tr 34, 97–117 (1995). https://doi.org/10.1007/BF00665783

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