Abstract
Amphiregulin (AR) is a heparin-binding epidermal growth factor (EGF)-related peptide that seems to play an important role in mammary epithelial cell growth regulation. We have investigated the regulation of AR-gene expression and -protein secretion by EGF in normal breast epithelial cells (HMECs), as well as in the tumoral breast epithelial cell lines MCF-7 and MDA-MB231. EGF induced a dose-dependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10−8M EGF stimulated AR expression in HMECs to 140–300% of control. A similar EGF concentration increased AR mRNA level to 550% and 980% of control in MCF-7 and MDA-MB231 cells, respectively. This was accompanied by an accumulation of AR into conditioned culture media. However, HMECs secreted in response to EGF, 5–10 fold more AR than tumour cells. Furthermore, the potential participation of AR in the regulation of the plasminogen activator (PA)/plasmin system was investigated. Whereas HMEC-proliferation was stimulated by AR, the levels of secreted urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAi-1) remained unaffected. Conversely, AR failed to regulate the proliferation of tumoral cell lines but induced an accumulation of uPA and PAi-1 into culture media. This was accompanied by an increase of the number of tumoral cells that invaded matrigel in vitro. Moreover, the presence of a neutralizing anti-uPA receptor antibody reversed the increased invasiveness of MDA-MB231 cells induced by AR. These data reveal differential behaviour of normal versus tumoral breast epithelial cells in regard to the action of AR and demonstrate that, in a number of cases, AR might play a significant role in tumour progression through the regulation of the PA/plasmin system. © 2001 Cancer Research Campaign
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References
Andreasen PA, Kjoller L, Christensen L and Duffy MJ (1997) The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer 72: 1–22
Bouchet C, Spyratos F, Martin PM, Hacene K, Gentile A and Oglobine J (1994) Prognosis value of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitors PAI-1 and PAI-2 in breast carcinomas. Br J Cancer 69: 398–405
Ciardiello F, Kim N, Liscia DS, Bianco C, Lidereau R, Merlo G, Callahan R, Brattain M, Greiner J, Szpak C, Kidwell W, Derynck R, Schlom J and Salomon DS (1989) Transforming growth factor α (TGFα) mRNA expression in human breast carcinomas and TGFα activity in the effusions of breast cancer patients. J Natl Cancer Inst 81: 1165–1171
Feinberg AP and Vogelstein BA (1983) Technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132: 6–13
Foeckens JA, Schmitt M, van Putten WL, Peters HA, Bontenbal M, Janicke F and Klijn GJ (1992) Prognostic value of urokinase-type plasminogen activator in 671 primary breast cancer patients. Cancer Res 52: 6101–6105
Fontanini G, De Laurentiis M, Vignati S, Chine S, Lucchi M, Silvestri V, Mussi A, De Placido S, Tortora G, Bianco AR, Gullick W, Angeletti CA, Bevilacqua G and Ciardiello F (1998) Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognosis indicators of survival. Clinical Cancer Res 4: 241–249
Fry CJ and Farnham PJ (1999) Context-dependent transcriptional regulation. J Biol Chem 274: 29583–29586
Higashiyama S, Abraham JA, Miller J, Fiddes JC and Klagsbrun M (1991) A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science 251: 936–9391
Johnson GR, Saeki T, Gordon A, Shoyab M, Salomon DS and Stromberg K (1992) Autocrine action of amphiregulin in a colon carcinoma cell line and immunocytochemical localization of amphiregulin in human colon. J Cell Biol 118: 741–751
Johnson GR, Kannan B, Shoyab M and Stromberg K (1993a) Amphiregulin induces tyrosine phosphorylation of the epidermal growth factor receptor and p185erbB2. Evidence that amphiregulin acts exclusively through the epidermal growth factor receptor at the surface of human epithelial cells. J Biol Chem 268: 2924–2931
Johnson GR, Prigent SA, Gullick WJ and Stromberg K (1993b) Characterization of high and low molecular forms of amphiregulin that differ in glycosylation and peptide core length. J Biol Chem 268: 18835–18843
Kenney N, Johnson G, Selvam MP, Kim N, Cheng-Feng Q, Saeki T, Brandt R, Wallace-Jones B, Ciardiello F, Shoyab M, Plowman G, Day A, Salomon DS and Normanno N (1993) Transforming growth factor α (TGFα) and amphiregulin (AR) as autocrine growth factors in nontransformed, immortalized 184A1N4 human mammary epithelial cells. Mol Cell Diff 1: 163–184
Kenney NJ, Huang RP, Johnson GR, Wu JX, Okamura D, Matheny W, Kordon E, Gullick WJ and Plowman G (1995) Detection and location of amphiregulin and Cripto-1 expression in the developing postnatal mouse mammary gland. Molecular Reproduction and Development 41: 277–286
Kenney NJ, Smith GH, Rosenberg K, Cutler ML and Dickson RB (1996) Induction of ductal morphogenesis and lobular hyperplasia by amphiregulin in the mouse mammary gland. Cell Growth Diff 7: 1769–1781
Kondapaka SB, Fridman R and Reddy KB (1997) Epidermal growth factor an amphiregulin up-regulate matrix metalloproteinase-9 (MMP-9) in human breast cancer cells. Int J Cancer 70: 722–726
LeJeune S, Leek R, Horak E, Plowman G, Greenall M and Harris AL (1993) Amphiregulin, epidermal growth factor receptor, and estrogen receptor expression in human primary breast cancer. Cancer Res 53: 3597–3602
Li S, Plowman GD, Buckley SD and Shipley GD (1992) Heparin inhibition of autonomous growth implicates amphiregulin as an autocrine growth factor for normal human mammary epithelial cells. J Cell Physiol 153: 103–111
Ma L, Gauvillé C, Berthois Y, Johnson GR and Calvo F (1999) Antisense expression for amphiregulin suppresses tumorigenicity of a transformed human breast epithelial cell line. Oncogene 18: 6513–6520
Martinez-Lacaci I, Saceda M, Plowman GD, Johnson GR, Normanno N, Salomon DS and Dickson RB (1995) Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell lines. Endocrinology 136: 3983–3992
Normanno N, Ciardiello F, Brandt R and Salomon DS (1994a) Epidermal growth factor-related peptides in the pathogenesis of human breast cancer. Breast Cancer Res Treat 29: 11–27
Normanno N, Selvam MP, Qi CF, Saeki T, Johnson G, Kim N, Ciardiello F, Shoyab M, Plowman G, Brandt R, Todaro G and Salomon DS (1994b) Amphiregulin as an autocrine growth factor for c-Ha-ras and c-erbB-2-transformed human mammary epithelial cells. Proc Natl Acad Sci USA 91: 2790–2794
Normanno N, Selvam MP, Bianco C, Damiano V, De Angelis E, Grassi M, Magliulo G, Tortora G, Salomon DS and Ciardiello F (1995) Amphiregulin anti-sense oligodeoxynucleotides inhibit growth and transformation of a human colon carcinoma cell line. Int J Cancer 62: 762–766
Panico L, D'Antonio A, Salvatore G, Mezza E, Tortora G, De Laurentiis M, De Placido S, Giordano T, Merino M, Salomon DS, Gullick WJ, Pettinato G, Schnitt SJ, Bianco AR and Ciardiello F (1996) Differential immunohistochemical detection of transforming growth factor α, amphiregulin and cripto in human normal and malignant breast tissues. Int J Cancer 65: 51–56
Qi CF, Liscia DS, Normanno N, Merlo G, Johnson GR, Gullick WJ, Ciardiello F, Saeki T, Brandt R, Kim N, Kenney N and Salomon DS (1994) Expression of transforming growth factor α, amphiregulin and cripto-1 in human breast carcinomas. Br J Cancer 69: 903–910
Riese DJ, Kim ED, Elenius K, Buckley S, Klagsbrun M, Plowman GD and Stern DF (1996) The epidermal growth factor receptor couples transforming growth factor-α, heparin-binding epidermal growth factor-like factor, and amphiregulin to Neu, ErbB-3, and ErbB-4. J Biol Chem 271: 20047–20052
Rosenthal EL, Johnson TM, Allen ED, Apel IJ, Punturieri A and Weiss SJ (1998) Role of the plasminogen activator and matrix metalloproteinase systems in epidermal growth factor- and scatter factor-stimulated invasion of carcinoma cells. Cancer Res 58: 5221–5230
Salomon DS, Normanno N, Ciardiello F, Brandt R, Shoyab M and Todaro GJ (1995) The role of amphiregulin in breast cancer. Breast Cancer Res Treat 33: 103–114
Sehgal I, Bailey J, Hitzemann K, Pittelkow MR and Maihle NJ (1994) Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells. Mol Biol Cell 5: 339–347
Shoyab M, McDonald VL, Bradley JG and Todaro GJ (1988) Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc Natl Acad Sci USA 85: 6528–6532
Silvy M, Martin PM, Chajry N and Berthois Y (1998) Differential dose-dependent effects of epidermal growth factor on gene expression in A431 cells: evidence for a signal transduction pathway that can bypass Raf-1 activation. Endocrinology 139: 2382–2391
Souttou B, Hamelin R and Crepin M (1994) FGF2 as an autocrine growth factor for immortal human breast epithelial cells. Cell Growth & Differentiation 5: 615–623
Sundareshan P, Nagle RB and Bowden GT (1999) EGF induces the expression of matrilysin in the human prostate adenocarcinoma cell line, LNCaP. Prostate 40: 159–166
Visscher DW, Sarkar FH, Kasunic TC and Reddy KB (1997) Clinicopathological analysis of amphiregulin and heregulin immunostaining in breast neoplasia. Breast Cancer Res Treat 45: 75–80
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Silvy, M., Giusti, C., Martin, PM. et al. Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells. Br J Cancer 84, 936–945 (2001). https://doi.org/10.1054/bjoc.2000.1678
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DOI: https://doi.org/10.1054/bjoc.2000.1678
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