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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 through ERK1/2 and Akt activation in SBOT3.1 cells.A, SBOT3.1 cells were treated for 48 hr with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF. E-cadherin and N-cadherin mRNA (left panel) and protein (right panel) levels were analyzed by RT-qPCR and western blot, respectively. B, SBOT3.1 cells were treated for with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF and Snail, and the Slug, Twist and ZEB1 mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) or LY294002 (20 µM). D, MPSC1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) SB203580 (10 µM) or LY294002 (20 µM). After treatment, cells were seeded into un-coated (migration) and Matrigel-coated (invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper side of the filter and the nuclei of invading cells were stained with Hoechst 33258. Results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with Ctrl. #p<0.05 compared with EGF.
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pone-0034071-g006: EGF induces cadherin switch through ERK1/2 and Akt activation in SBOT3.1 cells.A, SBOT3.1 cells were treated for 48 hr with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF. E-cadherin and N-cadherin mRNA (left panel) and protein (right panel) levels were analyzed by RT-qPCR and western blot, respectively. B, SBOT3.1 cells were treated for with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF and Snail, and the Slug, Twist and ZEB1 mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) or LY294002 (20 µM). D, MPSC1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) SB203580 (10 µM) or LY294002 (20 µM). After treatment, cells were seeded into un-coated (migration) and Matrigel-coated (invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper side of the filter and the nuclei of invading cells were stained with Hoechst 33258. Results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with Ctrl. #p<0.05 compared with EGF.

Mentions: It has been shown that the ERK1/2, p38 MAPK and PI3K/Akt pathways are involved in EGF-induced EMT [9], [22]. However, it is unknown whether these signaling pathways are also involved in EGF-induced EMT in SBOT cells. As shown in Figure 5, treatment with EGF induced the activation of ERK1/2 and Akt with the maximal effect observed at 5 min followed by a decrease after 180 min treatment. Interestingly, treatment with EGF did not activate p38 MAPK in SBOT3.1 cells. In contrast, EGF induced ERK1/2, p38 MAPK and Akt activation in MPSC1 cells. In SBOT3.1 cells, the EGF-induced down-regulation of E-cadherin and the up-regulation of N-cadherin mRNA and protein levels were diminished by treatment with the MEK1 inhibitor PD98059. Interestingly, treatment with the PI3K inhibitor LY294002 only diminished the EGF-induced down-regulation of E-cadherin but did not affect the EGF-induced up-regulation of N-cadherin (Figure 6A). In addition, treatment with PD98059 and LY294002 diminished EGF-induced up-regulation of Snail and Slug mRNA levels. However, the EGF-induced up-regulation of ZEB1 mRNA levels was only blocked by treatment with PD98059 and not with LY294002 (Figure 6B). Furthermore, EGF-induced cell migration and invasion were blocked by PD98059 and LY294002 treatments, although the inhibitory effect of LY294002 was less than that of PD98059 (Figure 6C). In MPSC1 cell, inhibition of ERK1/2, p38 MPAK and PI3K/Akt by PD98059, SB203580 and LY294002 attenuated EGF-induced cell migration and invasion (Figure 6D). Taken together, these results indicated that the ERK1/2 and PI3K/Akt pathways are involved in EGF-induced EMT and cell migration and invasion in SBOT cells. In addition, although EGF did not induce EMT in MPSC1 cells, our results indicate that ERK1/2, p38 MPAK and PI3K/Akt signaling pathways are involved in EGF-induced MPSC1 cell migration and invasion.


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 through ERK1/2 and Akt activation in SBOT3.1 cells.A, SBOT3.1 cells were treated for 48 hr with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF. E-cadherin and N-cadherin mRNA (left panel) and protein (right panel) levels were analyzed by RT-qPCR and western blot, respectively. B, SBOT3.1 cells were treated for with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF and Snail, and the Slug, Twist and ZEB1 mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) or LY294002 (20 µM). D, MPSC1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) SB203580 (10 µM) or LY294002 (20 µM). After treatment, cells were seeded into un-coated (migration) and Matrigel-coated (invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper side of the filter and the nuclei of invading cells were stained with Hoechst 33258. Results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with Ctrl. #p<0.05 compared with EGF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316602&req=5

pone-0034071-g006: EGF induces cadherin switch through ERK1/2 and Akt activation in SBOT3.1 cells.A, SBOT3.1 cells were treated for 48 hr with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF. E-cadherin and N-cadherin mRNA (left panel) and protein (right panel) levels were analyzed by RT-qPCR and western blot, respectively. B, SBOT3.1 cells were treated for with PD98059 (20 µM) or LY294002 (20 µM) in the presence or absence of 50 ng/ml EGF and Snail, and the Slug, Twist and ZEB1 mRNA levels were analyzed by RT-qPCR. C, SBOT3.1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) or LY294002 (20 µM). D, MPSC1 cells were treated with 50 ng/ml EGF in combination with PD98059 (20 µM) SB203580 (10 µM) or LY294002 (20 µM). After treatment, cells were seeded into un-coated (migration) and Matrigel-coated (invasion) transwell inserts. After 24 hr (migration) and 48 hr (invasion) incubation, non-invading cells were wiped from the upper side of the filter and the nuclei of invading cells were stained with Hoechst 33258. Results are expressed as the mean ± SEM of at least three independent experiments. *p<0.05 compared with Ctrl. #p<0.05 compared with EGF.
Mentions: It has been shown that the ERK1/2, p38 MAPK and PI3K/Akt pathways are involved in EGF-induced EMT [9], [22]. However, it is unknown whether these signaling pathways are also involved in EGF-induced EMT in SBOT cells. As shown in Figure 5, treatment with EGF induced the activation of ERK1/2 and Akt with the maximal effect observed at 5 min followed by a decrease after 180 min treatment. Interestingly, treatment with EGF did not activate p38 MAPK in SBOT3.1 cells. In contrast, EGF induced ERK1/2, p38 MAPK and Akt activation in MPSC1 cells. In SBOT3.1 cells, the EGF-induced down-regulation of E-cadherin and the up-regulation of N-cadherin mRNA and protein levels were diminished by treatment with the MEK1 inhibitor PD98059. Interestingly, treatment with the PI3K inhibitor LY294002 only diminished the EGF-induced down-regulation of E-cadherin but did not affect the EGF-induced up-regulation of N-cadherin (Figure 6A). In addition, treatment with PD98059 and LY294002 diminished EGF-induced up-regulation of Snail and Slug mRNA levels. However, the EGF-induced up-regulation of ZEB1 mRNA levels was only blocked by treatment with PD98059 and not with LY294002 (Figure 6B). Furthermore, EGF-induced cell migration and invasion were blocked by PD98059 and LY294002 treatments, although the inhibitory effect of LY294002 was less than that of PD98059 (Figure 6C). In MPSC1 cell, inhibition of ERK1/2, p38 MPAK and PI3K/Akt by PD98059, SB203580 and LY294002 attenuated EGF-induced cell migration and invasion (Figure 6D). Taken together, these results indicated that the ERK1/2 and PI3K/Akt pathways are involved in EGF-induced EMT and cell migration and invasion in SBOT cells. In addition, although EGF did not induce EMT in MPSC1 cells, our results indicate that ERK1/2, p38 MPAK and PI3K/Akt signaling pathways are involved in EGF-induced MPSC1 cell migration and invasion.

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