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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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Association of CA125/MUC16 with E-cadherin and β-catenin. Lysate from adherent NIH:OVCAR3 cells were immunoprecipitated with anti-CA125/MUC16 (M11) antibody (A), anti-E-cadherin antibody (B) or anti-β-catenin antibody (C) in the presence or absence of EGF. Lysates were also immunoprecipitated with a control IgG (c). The immunoprecipitates were analysed for reactivity with CA125/MUC16, E-cadherin and β-catenin. Lysates were directly analysed by immunoblotting as controls (input).
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fig3: Association of CA125/MUC16 with E-cadherin and β-catenin. Lysate from adherent NIH:OVCAR3 cells were immunoprecipitated with anti-CA125/MUC16 (M11) antibody (A), anti-E-cadherin antibody (B) or anti-β-catenin antibody (C) in the presence or absence of EGF. Lysates were also immunoprecipitated with a control IgG (c). The immunoprecipitates were analysed for reactivity with CA125/MUC16, E-cadherin and β-catenin. Lysates were directly analysed by immunoblotting as controls (input).

Mentions: As MUC1 has been previously shown to bind β-catenin (Yamamoto et al, 1997), and as E-cadherin and β-catenin expressions were altered by the knock down of CA125/MUC16, we assessed whether these proteins associate with CA125/MUC16 in the presence or absence of EGF because EGF may affect E-cadherin expression (Lu et al, 2003). Co-immunoprecipitation experiments confirmed the association of CA125/MUC16 with E-cadherin complexes in NIH:OVCAR3 cells. As shown in Figure 3A, antibodies to CA125/MUC16 immunoprecipitated CA125/MUC16 from the lysates of NIH:OVCAR3 cells. More importantly, E-cadherin was co-immunoprecipitated with CA125/MUC16 in these cells with or without EGF treatment. In reciprocal experiments, antibodies to E-cadherin immunoprecipitated E-cadherin and co-immunoprecipitated CA125/MUC16 from the lysate of NIH:OVCAR3 cells, suggesting that CA125/MUC16 forms complexes with E-cadherin (Figure 3B). In a similar set of experiments, we found that antibodies to β-catenin co-immunoprecipitated CA125/MUC16 (Figure 3C). These findings indicate that CA125/MUC16 associates with E-cadherin and β-catenin complexes in NIH:OVCAR3 cells.


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)

Association of CA125/MUC16 with E-cadherin and β-catenin. Lysate from adherent NIH:OVCAR3 cells were immunoprecipitated with anti-CA125/MUC16 (M11) antibody (A), anti-E-cadherin antibody (B) or anti-β-catenin antibody (C) in the presence or absence of EGF. Lysates were also immunoprecipitated with a control IgG (c). The immunoprecipitates were analysed for reactivity with CA125/MUC16, E-cadherin and β-catenin. Lysates were directly analysed by immunoblotting as controls (input).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Association of CA125/MUC16 with E-cadherin and β-catenin. Lysate from adherent NIH:OVCAR3 cells were immunoprecipitated with anti-CA125/MUC16 (M11) antibody (A), anti-E-cadherin antibody (B) or anti-β-catenin antibody (C) in the presence or absence of EGF. Lysates were also immunoprecipitated with a control IgG (c). The immunoprecipitates were analysed for reactivity with CA125/MUC16, E-cadherin and β-catenin. Lysates were directly analysed by immunoblotting as controls (input).
Mentions: As MUC1 has been previously shown to bind β-catenin (Yamamoto et al, 1997), and as E-cadherin and β-catenin expressions were altered by the knock down of CA125/MUC16, we assessed whether these proteins associate with CA125/MUC16 in the presence or absence of EGF because EGF may affect E-cadherin expression (Lu et al, 2003). Co-immunoprecipitation experiments confirmed the association of CA125/MUC16 with E-cadherin complexes in NIH:OVCAR3 cells. As shown in Figure 3A, antibodies to CA125/MUC16 immunoprecipitated CA125/MUC16 from the lysates of NIH:OVCAR3 cells. More importantly, E-cadherin was co-immunoprecipitated with CA125/MUC16 in these cells with or without EGF treatment. In reciprocal experiments, antibodies to E-cadherin immunoprecipitated E-cadherin and co-immunoprecipitated CA125/MUC16 from the lysate of NIH:OVCAR3 cells, suggesting that CA125/MUC16 forms complexes with E-cadherin (Figure 3B). In a similar set of experiments, we found that antibodies to β-catenin co-immunoprecipitated CA125/MUC16 (Figure 3C). These findings indicate that CA125/MUC16 associates with E-cadherin and β-catenin complexes in NIH:OVCAR3 cells.

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