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. 2012 Oct 21:11:79.
doi: 10.1186/1476-4598-11-79.

Epidermal growth factor (EGF) and interleukin (IL)-1β synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion

Liqiang Ma  1 , Fenghua Lan, Zhiyong Zheng, Feilai Xie, Lie Wang, Wei Liu, Junyong Han, Feng Zheng, Yanchuan Xie, Qiaojia Huang
Affiliations

Epidermal growth factor (EGF) and interleukin (IL)-1β synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion

Liqiang Ma et al. Mol Cancer. .

Abstract

Background: Patients with invasive breast ductal carcinoma (IBDC) with metastasis have a very poor prognosis. Little is known about the synergistic action of growth and inflammatory factors in IBDC metastases.

Methods: The expression of activated extracellular signal-regulated kinase1/2 (phosphorylated or p-ERK1/2) was analyzed by immunohistochemistry in IBDC tissue samples from 80 cases. BT474 IBDC cell migration and invasion were quantified using the Transwell assay. Matrix metalloproteinase (MMP)-9 expression and activity were analyzed by RT-PCR, Western blotting and zymography. Activator protein (AP)-1 activity was measured with a luciferase reporter gene assay. The Wilcoxon signed-rank test, Chi-square test, the partition of Chi-square test, independent t-test, and Spearman's method were used for the statistical analysis.

Results: Phosphorylated ERK1/2 was detected in 58/80 (72.5%) IBDC tissues, and was associated with higher TNM stage and lymph node metastasis, but not patient age or tumor size. Individually, epidermal growth factor (EGF), and interleukin (IL)-1β activated ERK1/2, increased cell migration and invasion, MMP-9 expression and activity, AP-1 activation in vitro and the expression of p-ERK1/2 was positively correlated with EGF expression levels, as well as IL-1β, MMP-9 and c-fos in IBDC tissue samples. Co-stimulation with EGF and IL-1β synergistically increased ERK1/2 and AP-1 activation, cell migration and invasion, and MMP-9 expression and activity. Inhibition of ERK1/2 using U0126 or siRNA abolished EGF and/or IL-1β-induced cell migration and invasion in a dose-dependent manner.

Conclusion: Activated ERK1/2 was associated with higher TNM stage and lymph node metastasis in IBDC. Both in vitro and in vivo studies indicated that ERK-1/2 activation may increase the metastatic ability of IBDC cells. Growth and inflammatory factors synergistically induced IBDC cell migration and invasion via ERK1/2 signaling, AP-1 activation and MMP-9 upregulation.

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Figures

Figure 1
Figure 1
Detection of p-ERK1/2 expression in IBDC and non-neoplastic tissues using immunohistochemistry. P-ERK1/2 was not detected or very weakly expressed in non-neoplastic breast ductal cells (A), but was frequently expressed in both the cytoplasm and nucleus of IBDC cells (B to D). B to D represented different intensities of p-ERK1/2 positive staining in IBDC. Images in the left panel were ×100 magnification; and expanded on the right at ×200.
Figure 2
Figure 2
EGF and IL-1β synergistically promote ERK1/2-mediated IBDC cell migration and invasion.A: Increased expression of activated ERK1/2 was detected in BT474 cells after stimulation with EGF. Western blotting demonstrating that pre-treatment with U0126 significantly inhibited EGF-induced ERK1/2 activation in BT474 cells. B, Increased expression of activated ERK1/2 was detected after the stimulation of BT474 cells with IL-1β. Western blotting demonstrating that pre-treatment with U0126 significantly inhibited IL-1β-induced ERK1/2 activation in BT474 cells. C, Western blots confirmed that the transfection of ERK1/2 siRNA dose-dependently reduced ERK1/2 expression in BT474 cells. D, Stronger activation of ERK1/2 was detected in EGF plus IL-1β-stimulated BT474 cells (an approximate 2- to 3-fold increase) compared to EGF or IL-1β alone. ERK1/2 siRNA and U0126 inhibited both EGF and IL-1β-induced ERK1/2 activation. E and F, Increased numbers of migrating and invading BT474 cells were observed after stimulation with EGF plus IL-1β, leading to a 2-fold increase compared to either EGF or IL-1β alone. ERK1/2 siRNA and U0126 inhibited EGF plus IL-1β-induced cell migration and invasion. G, Representative light microscopy images of BT474 cell migration and invasion in the Transwell assay. ** and ▼▼, P < 0.05 vs. siRNA negative control-transfected cells stimulated with EGF plus IL-1β; ΔΔ and ◇◇, P < 0.05 vs. untransfected cells stimulated with EGF plus IL-1β. Bars are the mean ± SD of migrating and invading cells. Cells were counted as the number of cells per field of view.
Figure 3
Figure 3
EGF plus IL-1β synergistically upregulate MMP-9 in IBDC cells via the ERK1/2 pathway.A, RT-PCR showing that MMP-9 mRNA was expressed in BT474 cells and increased after EGF treatment. Knockdown of ERK1/2 using siRNA or pre-treatment with U0126 dose-dependently attenuated EGF-induced MMP-9 mRNA upregulation in the cells. B, Bars indicate the relative expression levels of MMP-9 mRNA normalized to GAPDH mRNA. ** P < 0.05 vs. control siRNA-transfected cells stimulated with EGF; ΔΔP < 0.05 vs. untransfected cells stimulated with EGF. C, IL-1β also increased MMP-9 mRNA expression in BT474 cells; EGF plus IL-1β synergistically increased MMP-9 expression compared to either EGF or IL-1β stimulation alone; this effect could be inhibited by ERK1/2 siRNA or pre-treatment with U0126. D, Bars indicate the relative expression levels of MMP-9 mRNA normalized to GAPDH mRNA. ** and ▼▼, P < 0.05 vs. siRNA negative control-transfected cells stimulated with EGF plus IL-1β; ΔΔ and ◇◇, P < 0.05 vs. untransfected cells stimulated with EGF plus IL-1β. E, EGF plus IL-1β synergistically increased MMP-9 expression compared to either EGF or IL-1β stimulation alone as analyzed by Western blotting. F to H, MMP-9 activity was analyzed using zymography. F, EGF increased MMP-9 activity; this effect could be attenuated by both ERK1/2 siRNA and U0126 in the cells. G, IL-1β also increased MMP-9 activity in BT474 cells; H, EGF plus IL-1β synergistically increased MMP-9 activity compared to either EGF or IL-1β stimulation alone.
Figure 4
Figure 4
Knockdown of ERK1/2 inhibits the synergistic activation of AP-1 by EGF and IL-1β in IBDC cells. EGF and IL-1β stimulation increased the activity of an AP-1 luciferase reporter gene in BT474 cells; however, EGF plus IL-1β synergistically increased AP-1 luciferase activity. Transfection of BT474 cells with ERK1/2 siRNA decreased EGF or IL-1β, or EGF plus IL-1β-induced AP-1 activation in a dose-dependent manner. ** or ◇◇, P < 0.05 vs. control siRNA and AP-1 luc-transfected cells stimulated with EGF or IL-1β alone; ▼▼ and ΔΔ, P < 0.05 vs. control siRNA and AP-1 luc-transfected cells stimulated with EGF plus IL-1β; relative luciferase activity was normalized against B-gal.
Figure 5
Figure 5
The expression of p-ERK1/2 was closely related to EGF with IL-1β, MMP-9 and AP-1 in IBDC tissue samples.A, Strong expression levels of EGF, IL-1β, and EGF plus IL-1β, stronger expression of p-ERK1/2, MMP-9 and c-fos in IBDC tissue sample. B, Weaker expression levels of EGF, IL-1β, and EGF plus IL-1β, weaker expression levels of p-ERK1/2, MMP-9 and c-fos in IBDC tissue sample.
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