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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 blocked EGF-stimulated EGFR phosphorylation in breast cancer cells. a EBP50 overexpression retarded EGF-stimulated EGFR phosphorylation in MDA-MB-231 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-231 cells was significantly lower than that in its control cells. b EBP50 knockdown promoted EGF-stimulated EGFR phosphorylation in MCF-7 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-RNAi/MCF-7 cells was significantly higher than that in its control cells. Total expression levels of EGFR were unaffected by the different expression levels of EBP50 during EGF stimulation. The data presented are representative of a minimum of three independent experiments
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Fig4: EBP50 expression blocked EGF-stimulated EGFR phosphorylation in breast cancer cells. a EBP50 overexpression retarded EGF-stimulated EGFR phosphorylation in MDA-MB-231 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-231 cells was significantly lower than that in its control cells. b EBP50 knockdown promoted EGF-stimulated EGFR phosphorylation in MCF-7 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-RNAi/MCF-7 cells was significantly higher than that in its control cells. Total expression levels of EGFR were unaffected by the different expression levels of EBP50 during EGF stimulation. The data presented are representative of a minimum of three independent experiments

Mentions: EGF-stimulated ERK1/2 and AKT phosphorylation is mediated by triggering EGFR activation (Hsu et al. 2011). EBP50 was reported to interact with EGFR via its carboxyl terminal regulatory domain, which is adjacent to the autophosphorylation sites of the receptor. Therefore, it is possible that the association of EBP50 and EGFR masks the phosphorylation site of EGFR, thereby preventing EGFR activation and EGF-induced cell proliferation. To test this hypothesis, we detected the effect of EBP50 expression on EGF-stimulated EGFR phosphorylation. The overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation (Fig. 4a). After 5 min of EGF stimulation, EGFR phosphorylation was more than 9.3-fold over basal levels in parental cells, but only less than 2.5-fold over basal level in EBP-231 cells.Fig. 4


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 blocked EGF-stimulated EGFR phosphorylation in breast cancer cells. a EBP50 overexpression retarded EGF-stimulated EGFR phosphorylation in MDA-MB-231 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-231 cells was significantly lower than that in its control cells. b EBP50 knockdown promoted EGF-stimulated EGFR phosphorylation in MCF-7 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-RNAi/MCF-7 cells was significantly higher than that in its control cells. Total expression levels of EGFR were unaffected by the different expression levels of EBP50 during EGF stimulation. The data presented are representative of a minimum of three independent experiments
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472071&req=5

Fig4: EBP50 expression blocked EGF-stimulated EGFR phosphorylation in breast cancer cells. a EBP50 overexpression retarded EGF-stimulated EGFR phosphorylation in MDA-MB-231 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-231 cells was significantly lower than that in its control cells. b EBP50 knockdown promoted EGF-stimulated EGFR phosphorylation in MCF-7 cells. EGF (100 ng/ml for 5 or 30 min)-stimulated EGFR phosphorylation in EBP-RNAi/MCF-7 cells was significantly higher than that in its control cells. Total expression levels of EGFR were unaffected by the different expression levels of EBP50 during EGF stimulation. The data presented are representative of a minimum of three independent experiments
Mentions: EGF-stimulated ERK1/2 and AKT phosphorylation is mediated by triggering EGFR activation (Hsu et al. 2011). EBP50 was reported to interact with EGFR via its carboxyl terminal regulatory domain, which is adjacent to the autophosphorylation sites of the receptor. Therefore, it is possible that the association of EBP50 and EGFR masks the phosphorylation site of EGFR, thereby preventing EGFR activation and EGF-induced cell proliferation. To test this hypothesis, we detected the effect of EBP50 expression on EGF-stimulated EGFR phosphorylation. The overexpression of EBP50 in MDA-MB-231 cells inhibited EGF-stimulated EGFR phosphorylation (Fig. 4a). After 5 min of EGF stimulation, EGFR phosphorylation was more than 9.3-fold over basal levels in parental cells, but only less than 2.5-fold over basal level in EBP-231 cells.Fig. 4

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