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Downregulation of cell surface CA125/MUC16 induces epithelial-to-mesenchymal transition and restores EGFR signalling in NIH:OVCAR3 ovarian carcinoma cells.

Comamala M, Pinard M, Thériault C, Matte I, Albert A, Boivin M, Beaudin J, Piché A, Rancourt C - Br. J. Cancer (2011)

Bottom Line: CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin).Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities.Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and β-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

View Article: PubMed Central - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) cells are prone to metastasise throughout the peritoneal cavity. The epithelial-to-mesenchymal transition (EMT) is a necessary step towards metastatic tumour progression. CA125/MUC16 mucin is a high-molecular-weight glycoprotein overexpressed in the majority of serous carcinomas, suggesting a possible role in the pathogenesis of these cancers.

Methods: The role of CA125/MUC16 in EMT was investigated using single-chain antibody-mediated knockdown of cell surface CA125/MUC16 in overexpressing EOC NIH:OVCAR3 cells.

Results: CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin). Co-immunoprecipitation experiments revealed that CA125/MUC16 binds to E-cadherin and β-catenin complexes. The in vitro studies showed disruption of cell-cell junctions, enhanced motility, migration and invasiveness in CA125/MUC16 knockdown cells. Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities. Epidermal growth factor receptor inhibition strongly inhibited the motility of CA125/MUC16 knockdown cells.

Conclusions: Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and β-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

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Related in: MedlinePlus

CA125/MUC16 knockdown activates EGFR and its inhibition reduces the motility of these cells. (A) Western blot analysis showed activation of EGFR and its downstream targets Akt and ERK1/2 in CA125/MUC16 knockdown cells as compared with NIH:OVCAR3 and Ctrl scFv cells. (B) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of serum (20% FBS). The migration of CA125/MUC16 knockdown cells was dependent on the presence of serum ( × 100 magnification). (C) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells treated with serum, EGF (10 ng ml−1) and EGF inhibitors AG1478 (4 μ) or PD153035 (5 μ). EGF had an effect similar to serum on the motility of CA125/MUC16 cells, but serum failed to stimulate migration in Ctrl scFv cells. The presence of EGF inhibitor strongly decreased serum- or EGF-induced motility in knockdown cells ( × 100 magnification). (D) Western blot analysis of EGFR activation in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of AG1478. AG1478 completely abrogated serum-mediated EGFR activation in knockdown cells.
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fig7: CA125/MUC16 knockdown activates EGFR and its inhibition reduces the motility of these cells. (A) Western blot analysis showed activation of EGFR and its downstream targets Akt and ERK1/2 in CA125/MUC16 knockdown cells as compared with NIH:OVCAR3 and Ctrl scFv cells. (B) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of serum (20% FBS). The migration of CA125/MUC16 knockdown cells was dependent on the presence of serum ( × 100 magnification). (C) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells treated with serum, EGF (10 ng ml−1) and EGF inhibitors AG1478 (4 μ) or PD153035 (5 μ). EGF had an effect similar to serum on the motility of CA125/MUC16 cells, but serum failed to stimulate migration in Ctrl scFv cells. The presence of EGF inhibitor strongly decreased serum- or EGF-induced motility in knockdown cells ( × 100 magnification). (D) Western blot analysis of EGFR activation in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of AG1478. AG1478 completely abrogated serum-mediated EGFR activation in knockdown cells.

Mentions: We analysed the mechanism of CA135/MUC16-induced EMT in NIH:OVCAR3 cells. We observed an increase in the phosphorylation of EGFR (Y1068) in the CA125/MUC16 knockdown cells, although the levels of total EGFR remained unchanged (Figure 7A). We also determined the expression and phosphorylation of Akt and ERK1/2, which are downstream signalling molecules induced upon EGFR activation. CA125/MUC16 knockdown-induced activation of EGFR led to the activation of Akt and ERK1/2 as shown in Figure 7A. These data suggest that the knock down of CA125/MUC16 modulates the phosphorylation of EGFR and activation of its downstream targets to promote EMT.


Downregulation of cell surface CA125/MUC16 induces epithelial-to-mesenchymal transition and restores EGFR signalling in NIH:OVCAR3 ovarian carcinoma cells.

Comamala M, Pinard M, Thériault C, Matte I, Albert A, Boivin M, Beaudin J, Piché A, Rancourt C - Br. J. Cancer (2011)

CA125/MUC16 knockdown activates EGFR and its inhibition reduces the motility of these cells. (A) Western blot analysis showed activation of EGFR and its downstream targets Akt and ERK1/2 in CA125/MUC16 knockdown cells as compared with NIH:OVCAR3 and Ctrl scFv cells. (B) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of serum (20% FBS). The migration of CA125/MUC16 knockdown cells was dependent on the presence of serum ( × 100 magnification). (C) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells treated with serum, EGF (10 ng ml−1) and EGF inhibitors AG1478 (4 μ) or PD153035 (5 μ). EGF had an effect similar to serum on the motility of CA125/MUC16 cells, but serum failed to stimulate migration in Ctrl scFv cells. The presence of EGF inhibitor strongly decreased serum- or EGF-induced motility in knockdown cells ( × 100 magnification). (D) Western blot analysis of EGFR activation in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of AG1478. AG1478 completely abrogated serum-mediated EGFR activation in knockdown cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3065274&req=5

fig7: CA125/MUC16 knockdown activates EGFR and its inhibition reduces the motility of these cells. (A) Western blot analysis showed activation of EGFR and its downstream targets Akt and ERK1/2 in CA125/MUC16 knockdown cells as compared with NIH:OVCAR3 and Ctrl scFv cells. (B) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of serum (20% FBS). The migration of CA125/MUC16 knockdown cells was dependent on the presence of serum ( × 100 magnification). (C) Wound healing assay in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells treated with serum, EGF (10 ng ml−1) and EGF inhibitors AG1478 (4 μ) or PD153035 (5 μ). EGF had an effect similar to serum on the motility of CA125/MUC16 cells, but serum failed to stimulate migration in Ctrl scFv cells. The presence of EGF inhibitor strongly decreased serum- or EGF-induced motility in knockdown cells ( × 100 magnification). (D) Western blot analysis of EGFR activation in Ctrl scFv and CA125/MUC16 knockdown 1 : 9#9 cells in the presence or absence of AG1478. AG1478 completely abrogated serum-mediated EGFR activation in knockdown cells.
Mentions: We analysed the mechanism of CA135/MUC16-induced EMT in NIH:OVCAR3 cells. We observed an increase in the phosphorylation of EGFR (Y1068) in the CA125/MUC16 knockdown cells, although the levels of total EGFR remained unchanged (Figure 7A). We also determined the expression and phosphorylation of Akt and ERK1/2, which are downstream signalling molecules induced upon EGFR activation. CA125/MUC16 knockdown-induced activation of EGFR led to the activation of Akt and ERK1/2 as shown in Figure 7A. These data suggest that the knock down of CA125/MUC16 modulates the phosphorylation of EGFR and activation of its downstream targets to promote EMT.

Bottom Line: CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin).Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities.Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and β-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

View Article: PubMed Central - PubMed

Affiliation: Département de Microbiologie et Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, 3001, 12ième Avenue Nord, Sherbrooke, Quebec, Canada J1H 5N4.

ABSTRACT

Background: Epithelial ovarian cancer (EOC) cells are prone to metastasise throughout the peritoneal cavity. The epithelial-to-mesenchymal transition (EMT) is a necessary step towards metastatic tumour progression. CA125/MUC16 mucin is a high-molecular-weight glycoprotein overexpressed in the majority of serous carcinomas, suggesting a possible role in the pathogenesis of these cancers.

Methods: The role of CA125/MUC16 in EMT was investigated using single-chain antibody-mediated knockdown of cell surface CA125/MUC16 in overexpressing EOC NIH:OVCAR3 cells.

Results: CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin). Co-immunoprecipitation experiments revealed that CA125/MUC16 binds to E-cadherin and β-catenin complexes. The in vitro studies showed disruption of cell-cell junctions, enhanced motility, migration and invasiveness in CA125/MUC16 knockdown cells. Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities. Epidermal growth factor receptor inhibition strongly inhibited the motility of CA125/MUC16 knockdown cells.

Conclusions: Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and β-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

Show MeSH
Related in: MedlinePlus