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EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells?

Cheng JC, Auersperg N, Leung PC - PLoS ONE (2012)

Bottom Line: In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment.Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt.The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.

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EGF induces cadherin switch in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells.A, Cells were treated with 50 ng/ml EGF for 24 hr. E-cadherin and N-cadherin mRNA levels were analyzed by RT-qPCR. B, Cells were treated with 50 ng/ml EGF for 48 hr. E-cadherin and N-cadherin protein levels were analyzed by western blot. C and D, SBOT3.1 cells were treated with the EGFR inhibitor, AG1478 (10 µM), in the presence or absence of 50 ng/ml EGF, and the levels of E-cadherin and N-cadherin mRNA (24 hr EGF treatment) and protein (48 hr EGF treatment) were analyzed. E, SBOT3.1 cells were transfected with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF for 48 hr. The protein levels of E-cadherin and N-cadherin were analyzed by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with time-matched Ctrl. #p<0.05 compared with EGF or EGF in si-Ctrl.
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pone-0034071-g003: EGF induces cadherin switch in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells.A, Cells were treated with 50 ng/ml EGF for 24 hr. E-cadherin and N-cadherin mRNA levels were analyzed by RT-qPCR. B, Cells were treated with 50 ng/ml EGF for 48 hr. E-cadherin and N-cadherin protein levels were analyzed by western blot. C and D, SBOT3.1 cells were treated with the EGFR inhibitor, AG1478 (10 µM), in the presence or absence of 50 ng/ml EGF, and the levels of E-cadherin and N-cadherin mRNA (24 hr EGF treatment) and protein (48 hr EGF treatment) were analyzed. E, SBOT3.1 cells were transfected with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF for 48 hr. The protein levels of E-cadherin and N-cadherin were analyzed by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with time-matched Ctrl. #p<0.05 compared with EGF or EGF in si-Ctrl.

Mentions: A characteristic of EMT is a switch from E-cadherin to N-cadherin expression. In SBOT3.1 and SBOT4-LT cells, RT-qPCR analysis showed that EGF treatment down-regulated E-cadherin mRNA levels. Concurrently, N-cadherin mRNA levels increased with EGF treatment. Unexpectedly, EGF treatment did not alter the mRNA levels of E-cadherin or N-cadherin in MPSC1 and ILGC cells (Figure 3A). Similarly, western blot analysis performed on total cell lysates collected from cells treated with EGF for 48 hr showed that EGF down-regulated E-cadherin and up-regulated N-cadherin total protein levels in SBOT3.1 cells, but not in MPSC1 cells (Figure 3B). In addition, the effects of EGF on the mRNA and protein levels of E- and N-cadherin in SBOT3.1 cells were eliminated by treatment with the EGFR inhibitor, AG1478 (Figures 3C and D). Moreover, EGFR siRNA abolished the EGF-induced switch from E-cadherin to N-cadherin (Figure 3E). It has been shown that the binding of EGF to EGFR rapidly induces clustering and internalization of the ligand-receptor complexes, ultimately resulting in lysosomal degradation of both EGF and its receptor [21]. This process was supported by the data in Figure 3E, which shows that EGFR was down-regulated in SBOT3.1 cells in response to EGF treatment.


EGF-induced EMT and invasiveness in serous borderline ovarian tumor cells: a possible step in the transition to low-grade serous carcinoma cells?

Cheng JC, Auersperg N, Leung PC - PLoS ONE (2012)

EGF induces cadherin switch in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells.A, Cells were treated with 50 ng/ml EGF for 24 hr. E-cadherin and N-cadherin mRNA levels were analyzed by RT-qPCR. B, Cells were treated with 50 ng/ml EGF for 48 hr. E-cadherin and N-cadherin protein levels were analyzed by western blot. C and D, SBOT3.1 cells were treated with the EGFR inhibitor, AG1478 (10 µM), in the presence or absence of 50 ng/ml EGF, and the levels of E-cadherin and N-cadherin mRNA (24 hr EGF treatment) and protein (48 hr EGF treatment) were analyzed. E, SBOT3.1 cells were transfected with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF for 48 hr. The protein levels of E-cadherin and N-cadherin were analyzed by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with time-matched Ctrl. #p<0.05 compared with EGF or EGF in si-Ctrl.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316602&req=5

pone-0034071-g003: EGF induces cadherin switch in SBOT3.1 and SBOT4-LT cells, but not in MPSC1 and ILGC cells.A, Cells were treated with 50 ng/ml EGF for 24 hr. E-cadherin and N-cadherin mRNA levels were analyzed by RT-qPCR. B, Cells were treated with 50 ng/ml EGF for 48 hr. E-cadherin and N-cadherin protein levels were analyzed by western blot. C and D, SBOT3.1 cells were treated with the EGFR inhibitor, AG1478 (10 µM), in the presence or absence of 50 ng/ml EGF, and the levels of E-cadherin and N-cadherin mRNA (24 hr EGF treatment) and protein (48 hr EGF treatment) were analyzed. E, SBOT3.1 cells were transfected with control siRNA (si-Ctrl) or EGFR siRNA (si-EGFR) for 48 hr and then treated with 50 ng/ml EGF for 48 hr. The protein levels of E-cadherin and N-cadherin were analyzed by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with time-matched Ctrl. #p<0.05 compared with EGF or EGF in si-Ctrl.
Mentions: A characteristic of EMT is a switch from E-cadherin to N-cadherin expression. In SBOT3.1 and SBOT4-LT cells, RT-qPCR analysis showed that EGF treatment down-regulated E-cadherin mRNA levels. Concurrently, N-cadherin mRNA levels increased with EGF treatment. Unexpectedly, EGF treatment did not alter the mRNA levels of E-cadherin or N-cadherin in MPSC1 and ILGC cells (Figure 3A). Similarly, western blot analysis performed on total cell lysates collected from cells treated with EGF for 48 hr showed that EGF down-regulated E-cadherin and up-regulated N-cadherin total protein levels in SBOT3.1 cells, but not in MPSC1 cells (Figure 3B). In addition, the effects of EGF on the mRNA and protein levels of E- and N-cadherin in SBOT3.1 cells were eliminated by treatment with the EGFR inhibitor, AG1478 (Figures 3C and D). Moreover, EGFR siRNA abolished the EGF-induced switch from E-cadherin to N-cadherin (Figure 3E). It has been shown that the binding of EGF to EGFR rapidly induces clustering and internalization of the ligand-receptor complexes, ultimately resulting in lysosomal degradation of both EGF and its receptor [21]. This process was supported by the data in Figure 3E, which shows that EGFR was down-regulated in SBOT3.1 cells in response to EGF treatment.

Bottom Line: In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment.Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt.The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Child and Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada.

ABSTRACT
In high-grade ovarian cancer cultures, it has been shown that epidermal growth factor (EGF) induces cell invasion by activating an epithelial-mesenchymal transition (EMT). However, the effect of EGF on serous borderline ovarian tumors (SBOT) and low-grade serous carcinomas (LGC) cell invasion remains unknown. Here, we show that EGF receptor (EGFR) was expressed, that EGF treatment increased cell migration and invasion in two cultured SBOT cell lines, SBOT3.1 and SV40 large T antigen-infected SBOT cells (SBOT4-LT), and in two cultured LGC cell lines, MPSC1 and SV40 LT/ST-immortalized LGC cells (ILGC). However, EGF induced down-regulation of E-cadherin and concurrent up-regulation of N-cadherin in SBOT cells but not in LGC cells. In SBOT cells, the expression of the transcriptional repressors of E-cadherin, Snail, Slug and ZEB1 were increased by EGF treatment. Treatment with EGF led to the activation of the downstream ERK1/2 and PI3K/Akt. The MEK1 inhibitor PD98059 diminished the EGF-induced cadherin switch and the up-regulation of Snail, Slug and ZEB1 and the EGF-mediated increase in SBOT cell migration and invasion. The PI3K inhibitor LY294002 had similar effects, but it could not block the EGF-induced up-regulation of N-cadherin and ZEB1. This study demonstrates that EGF induces SBOT cell migration and invasion by activating EMT, which involves the activation of the ERK1/2 and PI3K/Akt pathways and, subsequently, Snail, Slug and ZEB1 expression. Moreover, our results suggest that there are EMT-independent mechanisms that mediate the EGF-induced LGC cell migration and invasion.

Show MeSH
Related in: MedlinePlus