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Cell Biology International (2003) 27, 477481 (Printed in Great Britain)
Erb-B2 homodimerization inhibits MUC1 transcription in cultured human mammary epithelial cells
Department of General Surgery, Faculty of Medicine, Cumhuriyet University, Sivas 58140, Turkey
MUC1 mucin is a heavily O-glycosylated transmembrane protein that is aberrantly expressed in many carcinomas, including breast cancer. In the present study, the effect of signaling generated from the Erb-B2 homodimer as a result of transcription of the MUC1 gene was investigated in human mammary epithelial cell lines (MTSV1-7 and Hb2) stably transfected with a pBAT/trk-neu construct in which the extracellular domain of Erb-B2 was replaced with the corresponding domain from the nerve growth factor (NGF) receptor. In this system, NGF stimulated homodimerization of Erb-B2 and phosphorylation of its intracellular domain. MTSV1-7/trk-neu and Hb2/trk-neu cells were transiently transfected with a construct in which the MUC1 promoter caused expression of a CAT reporter gene, and were then treated with NGF. These studies showed that MUC1 expression was inhibited by NGF treatment in both cell lines, suggesting that its expression can be regulated by signals resulting from the homodimerization of Erb-B2.
Keywords: MUC1, Gene expression regulation, Receptor, Erb-B2, Breast, Mammary epithelial cells, Transfection.
*Corresponding author. Tel.: +90-346-219-1300x2323; fax: +90-346-219-1284.
Fourteen MUC genes (Fowler et al., 2001) have been described that encode mucin-like glycoproteins expressed in epithelia. One member of this family, the transmembrane mucin MUC1 (also known as PEM, DF3, CD 227, episialin, EMA, CA 15-3), is expressed abundantly in the lactating mammary gland, in addition to being over-expressed in over 90% of human breast carcinomas and metastases (Zotter et al., 1988). In the normal mammary gland, MUC1 is expressed mainly on the apical surface of glandular epithelium and is believed to play a role in anti-adhesion and immune protection (Wesseling et al., 1996). In breast cancer, however, it is underglycosylated and apical localization is lost (Hilkens et al., 1995). The over-expression of MUC1 seen in breast cancers is paralleled by an increase in expression of the corresponding mRNA, which probably reflects an increase in gene transcription (Abe and Kufe, 1990).
Several proto-oncogenes have been characterized as active participants in signal transduction pathways that have downstream effects on gene transcription. The Erb-B2 (p185c-erbB2) proto-oncogene was identified through its homology to the gene coding for the Epidermal Growth Factor Receptor (EGF-R) (Semba et al., 1985), a transmembrane tyrosine kinase that undergoes autophosphorylation in over-expressing cells (Segatto et al., 1990).
To date there is no known ligand that binds and induces homodimerization of Erb-B2 receptors. Two mammary epithelial cell lines (MTSV1-7/trk-neu and Hb2/trk-neu) are able to induce ligand (NGF) binding to, and homodimerization of, the Erb-B2 receptors. Therefore, any possible effects of signalling generated by Erb-B2 homodimerization on MUC1 gene transcription were examined using the cell lines in this study.
2 Materials and Methods
2.1 Cell culture
The human mammary epithelial cell lines MTSV1-7 and Hb2 (Bartek et al. 1991; Berdichevsky et al., 1994) were maintained in DMEM supplemented with 10% FCS and 0.3 μg/ml glutamine, 5 μg/ml hydrocortisone and 10 μg/ml insulin. These cell lines were stably transfected with the pBAT/trk-neu construct, in which the extracellular region of Erb-B2 is replaced with the corresponding domain from the NGF receptor(Baeckström et al., 2000a).
2.2 Plasmid vectors
The −1400/+33 fragment of the human MUC1 promoter was ligated into the promoterless pGCAT-A reporter plasmid (Frebourg and Brison, 1988) using standard procedures (Kovarik et al., 1993). The pGL3-control vector was purchased from Promega.
2.3 Transient transfections
Transient transfections were performed by the calcium phosphate co-precipitation method, as described previously (Graham and Van Der Eb, 1973), using cells that had been plated 4–6 h previously to give 40% confluence. The DNA precipitate was left on the cells in the presence or absence of 100 ng/ml NGF for 14 h and then removed. Cells were then washed three times with serum-free medium to remove any remaining precipitate. 1–10 μg of the MUC1-CAT plasmid together with 0.5 μg of the luciferase expression plasmid pJ3Ω luc were used for each 60 mm dish, and duplicate dishes were analysed for each sample in individual experiments. PGCAT-A was kindly provided by Drs Frebourg and O. Brison (Frebourg and Brison, 1988). The cells were harvested after 48 h and lysed immediately with buffer containing 0.65% Nonidet P-40.
2.4 CAT activity assay
CAT activity was assayed by acetylation of chloromphenicol and solvent extraction of the radiolabelled product. Cell lysates were incubated at 68 °C for 5 min. The reaction was carried out at 37 °C for 2 h with 0.1 μCi of 14C-labelled acetyl coenzyme A (specific activity 2.046 gBq/mmol, Amersham) in a 100 μl reaction mixture consisting of 20 μl unlabeled acetyl-CoA, 20 μl of 8 mM chloromphenicol, and 30 μl of 0.25 M Tris HCl, pH.7.8. Luciferase activity was measured in each aliquot with the Wallac-Tri-lux system. In order to compensate for variations in transfection efficiency, CAT activity was expressed as the ratio of CAT activity (count/min/2 h):luciferase activity (arbitrary units of chemiluminescence). For each construct, four separate experiments were performed using duplicate plates.
2.5 Statistical analysis
The Mann–Whitney U test was performed using SPSS 9.0.1 for windows (SPSS Inc., Chicago, IL, USA).
3.1 Erb-B2 homodimerization inhibits MUC1 expression at the transcriptional level in MTSV1-7 and Hb2 cells
The MTSV1-7 cell line is derived from luminal epithelial cells cultured from milk (Bartek et al., 1991). The Hb2 cell line is a subclone of MTSV1-7 (Berdichevsky et al. 1994). They were both stably transfected with a trk-neu construct in which the extracellular domain of Erb-B2 receptor was replaced with the corresponding domain of the NGF receptor, so that the NGF ligand would induce homodimerization of the chimeric receptor (Baeckström et al., 2000a,b). The resulting cells are known as MTSV1-7/trk-neu and Hb2/trk-neu.
In order to analyse the effect of Erb-B2 homodimerization on transcription of the MUC-1 gene, MTSV1-7/trk-neu and Hb2/trk-neu cell lines weretransiently transfected with the −1400/+33 MUC-1 promoter sequence fused to a CAT reporter gene in the presence or absence of NGF. Cells were cotransfected with a construct expressing the luciferase reporter gene to control for differences in transfection efficiency. Following treatment with 100 ng/ml NGF, MTSV1-7/trk-neu and Hb2/trk-neu cells showed a strikingdecrease in CAT activity when Erb-B2 homodimerization was induced by NGF (Fig. 1), and differences were found to be significant in both trk-neu cell lines with NGF, compared to no NGF (P=0.08). When MTSV1-7/trk-neu cells transiently transfected with the MUC1-CAT construct were treated with different concentrations of NGF, transcription was reduced by 76% at the highest concentration used, and by 5% at the lowest concentration used (Fig. 2). In the Hb2/trk-neu cells, transcription of the MUC1-CAT construct was also reduced by increasing amounts of NGF (Fig. 2). In these cells, MUC1-CAT activity was reduced by 80% at the highest concentration and 7% at the lowest (Fig. 2). These results were found to be statistically significant (P=0.08).
Suppression of the MUC1 promoter by Erb-B2 homodimerization in a transfection assay. Five μg MUC1 promoter-CAT plasmid was transfected without NGF (white bars) or with 100 ng/ml NGF (grey bars) into Hb2, MTSV1-7, Hb2/trk-neu and MTSV1-7/trk-neu cells. CAT activity was analysed following transfection and normalized to the luciferase activity (LUC) of a cotransfected pGL3 control vector. The first set of columns represents the promoterless-Cat plasmid activity, called ‘PGCAT’. The results are expressed as a ratio of CAT:LUC. Each bar represents the mean±SE of four individual transfections. Differences were found to be statistically significant in both trk-neu transfected cell lines with NGF, compared to without NGF (P=0.08).
Percentage inhibition of the MUC1 promoter controlling CAT reporter activity with increasing amounts of NGF (1–200 ng/ml) in Hb2. Hb2/trk-neu and MTSV1-7/trk-neu cells were transfected with the MUC1 promoter-CAT hybrid plasmid vector and treated with 1–200 ng/ml NGF to induce homodimerization of Erb-B2. The results are shown as percentage inhibition of transcription. Each point represents mean±SE of four experiments. The results were found to be statistically significant (P=0.08).
MUC1 is normally embedded in the apical membrane of many secretory organs, such as salivary glands, breast and lung. It has a conserved 69 amino acid cytoplasmic domain that is believed to interact with the actin cytoskeleton and to have putative signalling functions (Li et al., 1998; Pandey et al., 1995; Patton et al., 1995). The extracellular domain consists of a variable number of tandem repeats of a 20 amino acid sequence (Patton et al., 1995). The role of MUC1 in breast carcinoma is seen primarily as reducing intercellular interactions between adjacent tumour cells and between tumour and immune effector cells (Agrawal et al., 1998a,b; Chan et al., 1999; Wesseling et al., 1995, 1996).
Further evidence for an anti-adhesive role for MUC1 is provided by the in vitro evidence that its cytoplasmic domain can compete for and bind to the β-catenin molecule, so inhibiting E-cadherin-mediated intracellular adhesion (Li et al., 1998). In addition to this, mucins are also tumour antigens, as they are present in greatly increased amounts in cancer. Because of the high cell surface expression of these mucins, they provide useful targets for antibodies, NK cells and T cells (Apostolopoulos and McKenzie, 1994), making the MUC1 gene product an important target for cancer therapies based on antibodies or immunotherapy (Taylor-Papadimitriou et al., 1993). Therefore, an analysis of the effects of oncogenes on the expression of MUC1 is of considerable clinical importance.
Erb-B2 belongs to the family of tyrosine kinase receptors that includes Erb-B1 (EGF-R), Erb-B3 and Erb-B4 (Kraus et al., 1989; Ullrich and Schlessinger, 1990). EGF and heregulin ligands that bind to Erb-B1 and Erb-B3 or Erb-B4, respectively, induce heterodimerization of these receptors with Erb-B2 and its cross phosphorylation. Erb-B receptors are expressed during mammary gland development (Schroeder and Lee, 1998), and are implicated in breast cancer initiation and progression, both in humans and rodents (Olayioye et al., 2000; Schroeder and Lee, 1997). It has previously been reported that the Erb-B2 gene is expressed at low levels in most tissues, but shows a dramatic increase in expression in 20–30% of breast cancers (McGuire et al., 1987). Over-expression of either the receptors or ligands of this family generally occurs in advanced and metastatic breast cancer, resulting in a poor prognosis (Olayioye et al., 2000). Erb-B2 is over-expressed by those in situ breast tumours that are more likely to become invasive (Iglehart et al., 1990), and is thus associated with a poor prognosis.
The exact mechanism by which over-expressed Erb-B2 contributes to the more invasive types of breast carcinoma is not known. There is also no known ligand that induces homodimerization of Erb-B2. MTSV1-7 cells and the Hb2 subclone have been transfected with the hybrid trk-neu receptor, consisting of the extracellular domain of the trkA NGF receptor and the transmembrane and cytoplasmic domains of Erb-B2 (Baeckström et al., 2000a,b). In cells expressing this construct, Erb-B2 homodimerization could be induced by the addition of NGF. In trk-neu transfectants of Hb2 cells, the effects of NGF addition on cell proliferation, apoptosis and α2β1 integrin expression have been extensively studied (Baeckström et al., 2000a,b). Therefore, these trk-neu cell lines enabled to investigate intracellular events such as downstream signaling resulting from Erb-B2 homodimerization.
Down regulation of α
Transcriptional regulatory factors that are involved in this regulation, such as cis and trans elements, as well as downstream signalling, need to be clarified by further studies. More recently, it has been shown that Erb-B2 effects MUC1 expression via the Ras pathway (Scibetta et al., 2001). In this study, there is a marked increase in the reporter activity of both cell lines transfected with trk-neu, but not stimulated with NGF, compared with control cells. Scibetta et al. (2001), in contrast, found that over-expression of c-erbB2 inhibited this activity compared with control cells. This finding suggests that the extracellular domain of erb-B2 has a particularly important role in the regulation of MUC1 transcriptional activity. Also, when erb-B2 homodimerization was induced with NGF, transcriptional activation returned to levels similar to the control cell line. This result also suggests that over-expression of trk-neuincreases activation of the MUC1 promoter, and that addition of NGF induces erb-B2 homodimerization and abrogates any increase caused by the transcriptional activation of the MUC1 promoter.
It has also been reported that EGFR signalling and MAP kinase activation may be linked to MUC1-associated tumorigenesis (Schroeder et al., 2001). As mentioned previously, MUC1's anti-adhesive role may be related to inhibition of E-cadherin-mediated intracellular adhesion, or to down regulation by Erb-B2 homodimerization. Therefore, the possibility of Erb-B2 homodimerization being involved in E-cadherin regulation, as well as MUC1 regulation by E-cadherin, should be considered. With regards to Erb-B2 over-expression, novel therapeutic approaches involving the use of antibodies against over-expressed mucin in tumours should be re-evaluated. Such an analysis would provide information, not only for understanding how expression of the gene is regulated by oncogenes, but also for the design of new MUC1-based promoters for targeting genes in carcinomas, and for the development of novel therapeutic approaches.
This work was carried out at the ICRF Breast Cancer Biology Group Laboratory, Thomas Guy House, Guy's Hospital, London, UK, with a grant from the Imperial Cancer Research Fund. The author is grateful to Dr Michael Norman for his critical reading the manuscript, to Prof. Dr Joyce Taylor-Papadimitriou for her generous support, and to Dr Moira Shearer for help with the Wallac-Trilux luminometer.
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Received 26 March 2002/9 November 2002; accepted 8 February 2003doi:10.1016/S1065-6995(03)00039-8