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Comparative Study
. 2004;6(3):R239-45.
doi: 10.1186/bcr780. Epub 2004 Mar 18.

Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells

Haiyan Pang  1 , Brian G Rowan, Mariam Al-Dhaheri, Lee E Faber
Affiliations
Comparative Study

Epidermal growth factor suppresses induction by progestin of the adhesion protein desmoplakin in T47D breast cancer cells

Haiyan Pang et al. Breast Cancer Res. 2004.

Abstract

Introduction: Although the effects of progesterone on cell cycle progression are well known, its role in spreading and adhesion of breast cancer cells has not attracted much attention until recently. Indeed, by controlling cell adhesion proteins, progesterone may play a direct role in breast cancer invasion and metastasis. Progesterone has also been shown to modulate epidermal growth factor (EGF) effects in neoplasia, although EGF effects on progesterone pathways and targets are less well understood. In the present study we identify an effect of EGF on a progesterone target, namely desmoplakin.

Methods: Initially flow cytometry was used to establish the growing conditions and demonstrate that the T47D breast cancer cell line was responding to progesterone and EGF in a classical manner. Differential display RT-PCR was employed to identify differentially expressed genes affected by progesterone and EGF. Western and Northern blotting were used to verify interactions between EGF and progesterone in three breast cancer cell lines: T47D, MCF-7, and ZR-75.

Results: We found the cell adhesion protein desmoplakin to be upregulated by progesterone - a process that was suppressed by EGF. This appears to be a general but not universal effect in breast cancer cell lines.

Conclusion: Our findings suggest that progesterone and EGF may play opposing roles in metastasis. They also suggest that desmoplakin may be a useful biomarker for mechanistic studies designed to analyze the crosstalk between EGF and progesterone dependent events. Our work may help to bridge the fields of metastasis and differentiation, and the mechanisms of steroid action.

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Figures

Figure 1
Figure 1
The effects of R5020 and epidermal growth factor (EGF) on S-phase of T47D cells. T47D cells were incubated for 48 hours with 100 nmol/l R5020, 10 nmol/l EGF, or 100 nmol/l R5020 plus 10 nmol/l EGF. Controls received vehicle and were allowed to grow for 48 hours. S-phase was measured by flow cytometry. Each bar is the mean of three individual experiments. Statistical significance was determined using t-test. *P < 0.05, **P < 0.01, versus control.
Figure 2
Figure 2
Differential display and Northern blot analysis of R5020 and epidermal growth factor (EGF) regulation of desmoplakin I (DPI) and II (DPII) in T47D cells. (a) Autoradiography of a representative differential display RT-PCR acrylamide gel. 33P-labeled DNA fragments generated by RT-PCR from RNAs of control T47D cells (lane 1), 10 nmol/l R5020 treatment (lane 2), 10 nmol/l EGF treatment (lane 3), 10 nmol/l R5020 plus 10 nmol/l EGF treatment (lane 4). Each lane contains 5 μL PCR mixture. (A) 1:20 dilution of RT-PCR cDNA. (B) 1:40 dilution of RT-PCR cDNA. The arrow indicates desmoplakin (DP). Molecular weight standards of 0.65, 0.42, and 0.41 kb are indicated in lane 5. (b) Photograph of a representative Northern blot analysis of DPI and DPII, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in T47D cells. Lane 1: control T47D cells were left untreated for 48 hours. Matched sister colonies of T47D cells received 10nmol/l R5020 (lane 2), 10 nmol/l EGF (lane 3), or 10 nmol/l R5020 plus 10 nmol/l EGF for 48 hours (lane 4). Ten micrograms of total RNA was subjected to Northern blot analysis as described in the Methods section. (c) The Northern blot films were scanned. The graphs indicate the fold difference of desmoplakin expression compared vehicle (set at a value of 1) following measurement of the band intensities. In each independent experiment, the desmoplakin band for each treatment condition was normalized to the GAPDH signal. The experiment was repeated three times and the mean values from these experiments ± SEM are reported. Statistical significance was determined using t-test. *P < 0.05, **P < 0.01, versus control.
Figure 3
Figure 3
Regulation of desmoplakin expression in breast cancer cell lines by R5020 and epidermal growth factor (EGF). (a) T47D cells were plated in RPMI media containing 5% stripped serum. (b) ZR-75 and (c) MCF-7 cells were plated in Dulbecco's modified Eagle medium (DMEM) containing 5% stripped serum. Cells were incubated with vehicle, R5020, EGF, or R5020 plus EGF for 48 hours before preparation of total cellular extract for Western blot analysis with antibody to desmoplakin or β-actin. The graphs indicate the fold difference of desmoplakin expression compared with vehicle (set at a value of 1) following measurement of Western blot signal intensity by Kodak Image Station 440CF and 1D Image Analysis software. In each independent experiment, duplicate samples for each treatment condition were normalized to the β-actin levels. The experiment was repeated two times and the mean values from these experiments ± SEM are reported. Representative Western blots for desmoplakin and β-actin levels in each cell line are shown below the graphs. *P < 0.05 versus control (by one-way analysis of variance).
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