Differential regulation of cell proliferation and protease secretion by epidermal growth factor and amphiregulin in tumoral versus normal breast epithelial cells

Abstract

Summary 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 dosedependent increase of AR mRNA level in both normal and tumoral cells. Thus, 10 Ϫ8 M 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. Whereas the expression of AR in a variety of both nontransformed and tumoral cells appears to be stimulated in the presence of EGF (Normanno et al, 1994b;Sehgal et al, 1994), the potential regulation of AR by growth factors in normal breast epithelial cells has never been examined. Because previous studies suggest that dysregulated expression of AR may be a component of mammary tumorigenesis we proposed to analyse and to compare the regulation of amphiregulin gene expression and protein secretion by EGF in normal and tumoral breast epithelial cells. Moreover, our aim was to examine potential role of AR in the regulation of uPA and PAi-1 protein secretion that could account for breast cancer progression. MATERIAL AND METHODS MaterialsAnti-smooth muscle α-actin, -actin, -vimentin and -cytokeratin 14, 18 and 19 antibodies were provided by Sigma (St Quentin Fallavier, France); Anti-vimentin antibody was from DAKO (Denmark). The AR cDNA probe was obtained from Dr. Plowman (Bristol-Myers Squibb, Seattle, WA). Matrigel was provided by Becton-Dickinson (France). Anti-uPA receptor neutralizing antibody, recombinant human AR and AR enzyme-immunoassay kit were from R & D Systems (Abingdon, UK). Antibodies used in ELISA determination of AR recognize various forms of...

Mammary tumorigenesis induced by fibroblast growth factor receptor 1 requires activation of the epidermal growth factor receptor

“…Both AREG and EREG have been implicated in breast cancer (Bieche et al, 2004;Eltarhouny et al, 2008;Gupta et al, 2007;Kenney et al, 1996b;Kondapaka et al, 1997;Ma et al, 1999;Silvy et al, 2001). High levels of AREG expression have been identified in cancerous lesions but not in neighboring noncancerous tissue (LeJeune et al, 1993;Panico et al, 1996;Qi et al, 1994).…”

“…Furthermore, both ligands are known activators of the EGF receptor (EGFR); AREG exclusively binds to and signals through EGFR (Shoyab et al, 1989), whereas EREG can also bind to and signal through ERBB4 Shelly et al, 1998). AREG is essential for normal ductal morphogenesis in the mammary gland (Ciarloni et al, 2007;Luetteke et al, 1999) and has also been linked to breast cancer progression (Kenney et al, 1996a;Kondapaka et al, 1997;Ma et al, 1999;Silvy et al, 2001). Studies examining AREG expression in human breast cancers have found AREG expression to significantly correlate with regional lymph node metastases, large tumor size and high-grade tumors (LeJeune et al, 1993;Ma et al, 2001;Qi et al, 1994).…”

Epidermal Growth Factor Receptor Pathway Analysis Identifies Amphiregulin as a Key Factor for Cisplatin Resistance of Human Breast Cancer Cells

“…A comparison between the biological effects of EGF and amphiregulin reveals distinct differences (43). Amphiregulin increases invasion capabilities of MCF-7 breast cancer cells, and transcriptional profiling experiments revealed that amphiregulin and EGF promote dramatically distinct patterns of gene expression (43,44). Several genes involved in cell motility and invasion were up-regulated when nontumorigenic breast epithelial cells were cultivated in the presence of amphiregulin (43).…”

Autocrine and Juxtacrine Effects of Amphiregulin on the Proliferative, Invasive, and Migratory Properties of Normal and Neoplastic Human Mammary Epithelial Cells

“…AR expression was also found to be strongly correlated with inflammatory breast cancer, and a putative AR/EGFR autocrine loop is suggested to contribute to breast cancer progression (30). Indeed, AR may play a specific role in cancer progression, since it has been shown that AR activation of EGFR contributes to the synthesis, secretion, and activation of some proteins involved in invasion and metastasis, such as urokinase-type plasminogen activator, matrix metalloproteinase 9, and extracellular matrix metalloproteinase inducer (31)(32)(33).…”