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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 suppressed EGF-induced breast cancer cell growth. a That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by CCK-8 method. Growth rate of EBP-231 cells was consistently slower than that of the control cells (P < 0.05). b That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by CCK-8 method. Growth rate of EBP-RNAi/MCF-7 cells was consistently faster than that of the control cells (P < 0.05). c That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-231 cells was consistently lower than that of its control cells (P < 0.05). d That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-RNAi/MCF-7 cells was consistently higher than that of its control cells (P < 0.01). All data shown are the mean ± SD of a representative experiment performed in quadruplicate (n = 4)
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Fig2: EBP50 expression suppressed EGF-induced breast cancer cell growth. a That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by CCK-8 method. Growth rate of EBP-231 cells was consistently slower than that of the control cells (P < 0.05). b That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by CCK-8 method. Growth rate of EBP-RNAi/MCF-7 cells was consistently faster than that of the control cells (P < 0.05). c That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-231 cells was consistently lower than that of its control cells (P < 0.05). d That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-RNAi/MCF-7 cells was consistently higher than that of its control cells (P < 0.01). All data shown are the mean ± SD of a representative experiment performed in quadruplicate (n = 4)

Mentions: First, we detected the effect of EBP50 overexpression on EGF-induced proliferation of MDA-MB-231 cells using a CCK-8 kit to measure the number of viable cells at different time points (Fig. 2a). The results showed that overexpression of EBP50 significantly inhibited EGF-induced cell proliferation (P < 0.05). Compared to its vector control cells, cell proliferation was suppressed by 26 % in EBP-231 cells at day 4. This suggested that restoring EBP50 expression in EBP50 deficient MDA-MB-231 cells inhibited EGF-induced breast cancer cell proliferation. We then determined the effect of EBP50 knockdown on EGF-induced cell proliferation in MCF-7 cells. We found that EBP50 knockdown enhanced EGF-induced MCF-7 cell proliferation compared to its control cells, promoting up to 40 % increase in cell proliferation at day 4 (P < 0.05) (Fig. 2b). The cell counting assay confirmed this result (data not shown). Taken together, these results suggested that EBP50 inhibited EGF-induced cell proliferation in breast cancer cells.Fig. 2


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 suppressed EGF-induced breast cancer cell growth. a That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by CCK-8 method. Growth rate of EBP-231 cells was consistently slower than that of the control cells (P < 0.05). b That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by CCK-8 method. Growth rate of EBP-RNAi/MCF-7 cells was consistently faster than that of the control cells (P < 0.05). c That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-231 cells was consistently lower than that of its control cells (P < 0.05). d That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-RNAi/MCF-7 cells was consistently higher than that of its control cells (P < 0.01). All data shown are the mean ± SD of a representative experiment performed in quadruplicate (n = 4)
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getmorefigures.php?uid=PMC3472071&req=5

Fig2: EBP50 expression suppressed EGF-induced breast cancer cell growth. a That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by CCK-8 method. Growth rate of EBP-231 cells was consistently slower than that of the control cells (P < 0.05). b That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by CCK-8 method. Growth rate of EBP-RNAi/MCF-7 cells was consistently faster than that of the control cells (P < 0.05). c That EBP50 overexpression inhibited the EGF-induced proliferative response of MDA-MB-231 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-231 cells was consistently lower than that of its control cells (P < 0.05). d That EBP50 knockdown enhanced EGF-induced cell proliferation of MCF-7 cells was assayed by BrdU incorporation method. DNA synthesis rate of EBP-RNAi/MCF-7 cells was consistently higher than that of its control cells (P < 0.01). All data shown are the mean ± SD of a representative experiment performed in quadruplicate (n = 4)
Mentions: First, we detected the effect of EBP50 overexpression on EGF-induced proliferation of MDA-MB-231 cells using a CCK-8 kit to measure the number of viable cells at different time points (Fig. 2a). The results showed that overexpression of EBP50 significantly inhibited EGF-induced cell proliferation (P < 0.05). Compared to its vector control cells, cell proliferation was suppressed by 26 % in EBP-231 cells at day 4. This suggested that restoring EBP50 expression in EBP50 deficient MDA-MB-231 cells inhibited EGF-induced breast cancer cell proliferation. We then determined the effect of EBP50 knockdown on EGF-induced cell proliferation in MCF-7 cells. We found that EBP50 knockdown enhanced EGF-induced MCF-7 cell proliferation compared to its control cells, promoting up to 40 % increase in cell proliferation at day 4 (P < 0.05) (Fig. 2b). The cell counting assay confirmed this result (data not shown). Taken together, these results suggested that EBP50 inhibited EGF-induced cell proliferation in breast cancer cells.Fig. 2

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