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EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation.

Yao W, Feng D, Bian W, Yang L, Li Y, Yang Z, Xiong Y, Zheng J, Zhai R, He J - Amino Acids (2012)

Bottom Line: Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression.Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation.These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, People's Republic of China.

ABSTRACT
Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

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EBP50 expression inhibited EGF-stimulated ERK1/2 and AKT phosphorylation in breast cancer cells. a EBP50 overexpression inhibited EGF-stimulated ERK1/2 phosphorylation in MDA-MB-231 cells. ERK1/2 activation in EBP-231 cells was significantly suppressed upon 5-min or 30-min EGF treatment. b EBP50 knockdown enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells. ERK1/2 activation in EBP-RNAi cells was significantly enhanced compared with that in its control cells upon EGF treatment. c EBP50 overexpression inhibited EGF-stimulated AKT phosphorylation in MDA-MB-231 cells. Phosphorylation of AKT in EBP-231 cells was significantly suppressed compared with that in its control cells upon 5-min or 30-min EGF treatment. d EBP50 knockdown enhanced EGF-stimulated AKT phosphorylation in MCF-7 cells. Phosphorylation of AKT in EBP-RNAi cells was significantly enhanced upon EGF treatment. e Knockdown of EBP50 in EBP-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression in EBP-231 cells was knocked down by EBP50 siRNA, the ERK1/2 activation was increased back to similar levels to that in HA-231 cells upon 5-min EGF treatment. f Restoration of EBP50 expression in EBP50-knockdown cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression level in EBP-RNAi/MCF-7 cells was restored by transfection of EBP50 constructs, the ERK1/2 activation in EBP-RNAi/MCF-7 cells was decreased back to similar levels to that in Luc-RNAi/MCF-7 cells upon 5-min EGF treatment. The data presented are representative of a minimum of three independent experiments
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Fig3: EBP50 expression inhibited EGF-stimulated ERK1/2 and AKT phosphorylation in breast cancer cells. a EBP50 overexpression inhibited EGF-stimulated ERK1/2 phosphorylation in MDA-MB-231 cells. ERK1/2 activation in EBP-231 cells was significantly suppressed upon 5-min or 30-min EGF treatment. b EBP50 knockdown enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells. ERK1/2 activation in EBP-RNAi cells was significantly enhanced compared with that in its control cells upon EGF treatment. c EBP50 overexpression inhibited EGF-stimulated AKT phosphorylation in MDA-MB-231 cells. Phosphorylation of AKT in EBP-231 cells was significantly suppressed compared with that in its control cells upon 5-min or 30-min EGF treatment. d EBP50 knockdown enhanced EGF-stimulated AKT phosphorylation in MCF-7 cells. Phosphorylation of AKT in EBP-RNAi cells was significantly enhanced upon EGF treatment. e Knockdown of EBP50 in EBP-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression in EBP-231 cells was knocked down by EBP50 siRNA, the ERK1/2 activation was increased back to similar levels to that in HA-231 cells upon 5-min EGF treatment. f Restoration of EBP50 expression in EBP50-knockdown cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression level in EBP-RNAi/MCF-7 cells was restored by transfection of EBP50 constructs, the ERK1/2 activation in EBP-RNAi/MCF-7 cells was decreased back to similar levels to that in Luc-RNAi/MCF-7 cells upon 5-min EGF treatment. The data presented are representative of a minimum of three independent experiments

Mentions: EGF-induced increase of cell proliferation was mediated through EGFR activation. ERK1/2 mitogen-activated protein kinase (MAPK) and AKT are signaling molecules on major downstream pathways initiated by the activation of EGFR and related with cell proliferation (Lim and Cha 2011; Kim and Lim 2011). Therefore, we first explored whether EBP50 expression could inhibit ERK1/2 phosphorylation in breast cancer cells. As shown in Fig. 3a, ERK1/2 phosphorylation was inhibited in EBP-231 cells. After 5 min of EGF stimulation, ERK1/2 phosphorylation increased 12.3-fold over basal levels in the parental cells, but only 5.3-fold in EBP-231 cells. After 30 min of EGF stimulation, ERK1/2 phosphorylation increased 22.8-fold over basal levels in its parental cells, but only 9.7-fold in EBP-231 cells. Conversely, EBP50 knockdown led to enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells (Fig. 3b). After 5 min of EGF stimulation, ERK1/2 phosphorylation increased 6.1-fold over basal levels in its parental cells, but more than 11.0-fold in EBP-RNAi cells. After 30 min of EGF stimulation, ERK1/2 phosphorylation increased 5.0-fold over basal levels in its parental cells, but more than 10.0-fold in EBP-RNAi cells. These results indicated that EBP50 could inhibit EGF-stimulated ERK1/2 phosphorylation in breast cancer cells.Fig. 3


EBP50 inhibits EGF-induced breast cancer cell proliferation by blocking EGFR phosphorylation.

Yao W, Feng D, Bian W, Yang L, Li Y, Yang Z, Xiong Y, Zheng J, Zhai R, He J - Amino Acids (2012)

EBP50 expression inhibited EGF-stimulated ERK1/2 and AKT phosphorylation in breast cancer cells. a EBP50 overexpression inhibited EGF-stimulated ERK1/2 phosphorylation in MDA-MB-231 cells. ERK1/2 activation in EBP-231 cells was significantly suppressed upon 5-min or 30-min EGF treatment. b EBP50 knockdown enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells. ERK1/2 activation in EBP-RNAi cells was significantly enhanced compared with that in its control cells upon EGF treatment. c EBP50 overexpression inhibited EGF-stimulated AKT phosphorylation in MDA-MB-231 cells. Phosphorylation of AKT in EBP-231 cells was significantly suppressed compared with that in its control cells upon 5-min or 30-min EGF treatment. d EBP50 knockdown enhanced EGF-stimulated AKT phosphorylation in MCF-7 cells. Phosphorylation of AKT in EBP-RNAi cells was significantly enhanced upon EGF treatment. e Knockdown of EBP50 in EBP-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression in EBP-231 cells was knocked down by EBP50 siRNA, the ERK1/2 activation was increased back to similar levels to that in HA-231 cells upon 5-min EGF treatment. f Restoration of EBP50 expression in EBP50-knockdown cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression level in EBP-RNAi/MCF-7 cells was restored by transfection of EBP50 constructs, the ERK1/2 activation in EBP-RNAi/MCF-7 cells was decreased back to similar levels to that in Luc-RNAi/MCF-7 cells upon 5-min EGF treatment. The data presented are representative of a minimum of three independent experiments
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Fig3: EBP50 expression inhibited EGF-stimulated ERK1/2 and AKT phosphorylation in breast cancer cells. a EBP50 overexpression inhibited EGF-stimulated ERK1/2 phosphorylation in MDA-MB-231 cells. ERK1/2 activation in EBP-231 cells was significantly suppressed upon 5-min or 30-min EGF treatment. b EBP50 knockdown enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells. ERK1/2 activation in EBP-RNAi cells was significantly enhanced compared with that in its control cells upon EGF treatment. c EBP50 overexpression inhibited EGF-stimulated AKT phosphorylation in MDA-MB-231 cells. Phosphorylation of AKT in EBP-231 cells was significantly suppressed compared with that in its control cells upon 5-min or 30-min EGF treatment. d EBP50 knockdown enhanced EGF-stimulated AKT phosphorylation in MCF-7 cells. Phosphorylation of AKT in EBP-RNAi cells was significantly enhanced upon EGF treatment. e Knockdown of EBP50 in EBP-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression in EBP-231 cells was knocked down by EBP50 siRNA, the ERK1/2 activation was increased back to similar levels to that in HA-231 cells upon 5-min EGF treatment. f Restoration of EBP50 expression in EBP50-knockdown cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation. When EBP50 expression level in EBP-RNAi/MCF-7 cells was restored by transfection of EBP50 constructs, the ERK1/2 activation in EBP-RNAi/MCF-7 cells was decreased back to similar levels to that in Luc-RNAi/MCF-7 cells upon 5-min EGF treatment. The data presented are representative of a minimum of three independent experiments
Mentions: EGF-induced increase of cell proliferation was mediated through EGFR activation. ERK1/2 mitogen-activated protein kinase (MAPK) and AKT are signaling molecules on major downstream pathways initiated by the activation of EGFR and related with cell proliferation (Lim and Cha 2011; Kim and Lim 2011). Therefore, we first explored whether EBP50 expression could inhibit ERK1/2 phosphorylation in breast cancer cells. As shown in Fig. 3a, ERK1/2 phosphorylation was inhibited in EBP-231 cells. After 5 min of EGF stimulation, ERK1/2 phosphorylation increased 12.3-fold over basal levels in the parental cells, but only 5.3-fold in EBP-231 cells. After 30 min of EGF stimulation, ERK1/2 phosphorylation increased 22.8-fold over basal levels in its parental cells, but only 9.7-fold in EBP-231 cells. Conversely, EBP50 knockdown led to enhanced EGF-stimulated ERK1/2 phosphorylation in MCF-7 cells (Fig. 3b). After 5 min of EGF stimulation, ERK1/2 phosphorylation increased 6.1-fold over basal levels in its parental cells, but more than 11.0-fold in EBP-RNAi cells. After 30 min of EGF stimulation, ERK1/2 phosphorylation increased 5.0-fold over basal levels in its parental cells, but more than 10.0-fold in EBP-RNAi cells. These results indicated that EBP50 could inhibit EGF-stimulated ERK1/2 phosphorylation in breast cancer cells.Fig. 3

Bottom Line: Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression.Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation.These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing 100069, People's Republic of China.

ABSTRACT
Ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) suppresses breast cancer cell proliferation, potentially through its regulatory effect on epidermal growth factor receptor (EGFR) signaling, although the mechanism by which this occurs remains unknown. Thus in our studies, we aimed to determine the effect of EBP50 expression on EGF-induced cell proliferation and activation of EGFR signaling in the breast cancer cell lines, MDA-MB-231 and MCF-7. In MDA-MB-231 cells, which express low levels of EBP50, EBP50 overexpression inhibited EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. In MCF-7 cells, which express high levels of EBP50, EBP50 knockdown promoted EGF-induced cell proliferation, ERK1/2 and AKT phosphorylation. Knockdown of EBP50 in EBP50-overexpressed MDA-MB-231 cells abrogated the inhibitory effect of EBP50 on EGF-stimulated ERK1/2 phosphorylation and restoration of EBP50 expression in EBP50-knockdown MCF-7 cells rescued the inhibition of EBP50 on EGF-stimulated ERK1/2 phosphorylation, further confirming that the activation of EGF-induced downstream molecules could be specifically inhibited by EBP50 expression. Since EGFR signaling was triggered by EGF ligands via EGFR phosphorylation, we further detected the phosphorylation status of EGFR in the presence or absence of EBP50 expression. Overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation, whereas knockdown of EBP50 in MCF-7 cells enhanced EGF-stimulated EGFR phosphorylation. Meanwhile, total expression levels of EGFR were unaffected during EGF stimulation. Taken together, our data shows that EBP50 can suppress EGF-induced proliferation of breast cancer cells by inhibiting EGFR phosphorylation and blocking EGFR downstream signaling in breast cancer cells. These results provide further insight into the molecular mechanism by which EBP50 regulates the development and progression of breast cancer.

Show MeSH
Related in: MedlinePlus