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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 alters expression of epithelial and mesenchymal markers. (A) Immunofluorescence analysis showing a decreased or absence of cell surface expression of E-cadherin and cytokeratin-18 and increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown and OSE cells ( × 1000 magnification). (B) Immunoblot analysis of N-cadherin and vimentin expression in control and knockdown cells. (C) Immunofluorescence analysis of β-catenin expression in control and CA125/MUC16 knockdown cells.
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fig2: CA125/MUC16 knockdown alters expression of epithelial and mesenchymal markers. (A) Immunofluorescence analysis showing a decreased or absence of cell surface expression of E-cadherin and cytokeratin-18 and increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown and OSE cells ( × 1000 magnification). (B) Immunoblot analysis of N-cadherin and vimentin expression in control and knockdown cells. (C) Immunofluorescence analysis of β-catenin expression in control and CA125/MUC16 knockdown cells.

Mentions: Epithelial-to-mesenchymal transition is a process characterised by the loss of epithelial markers such as E-cadherin and cytokeratin-18 and gain of mesenchymal markers such as N-cadherin and vimentin (Thiery, 2003; Ahmed et al, 2007; Vergara et al, 2010). During progression, carcinoma cells with increased invasiveness and metastatic potential usually acquire mesenchymal markers and show a reduction or absence of E-cadherin expression (Hugo et al, 2007). However, in contrast to most adenocarcinomas, advanced and poorly differentiated ovarian tumours continue to express E-cadherin (Ahmed et al, 2007). This contrasts with OSE cells, which do not express E-cadherin (Auersperg et al, 2001). Immunofluorescence microscopy analysis showed that E-cadherin was detected at the contacts between NIH:OVCAR3 control cells, whereas there was a loss of E-cadherin staining at the cell surface and an internalisation of E-cadherin in CA125/MUC16 knockdown cells (Figure 2A). As expected, OSE did not express E-cadherin. Knockdown cells behave very much like OSE cells; they displayed decreased expression of cytokeratin-18, whereas N-cadherin and vimentin expression were increased compared with NIH:OVCAR3- and Ctrl scFv-transfected cells (Figure 2A). Western blot analysis confirmed the increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown cells (Figure 2B). The cytoplasmic domain of E-cadherin has been shown to bind to β-catenin (Sasaki et al, 2000). β-Catenin staining was observed to localise at the contacts of cells in a manner similar to E-cadherin in NIH:OVCAR3- and Ctrl scFv-transfected cells, but partially re-localised in the cytoplasm in CA125/MUC16 knockdown cells (Figure 2C). Altogether, these data show that CA125/MUC16 knockdown induces a re-distribution of E-cadherin and β-catenin epithelial markers throughout the cytoplasm and increased expression of mesenchymal markers, such as N-cadherin and vimentin, consistent with an EMT. The data also show that OVCAR3 knockdown cells acquired a phenotype that was very similar to that of CA125-negative OSE 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)

CA125/MUC16 knockdown alters expression of epithelial and mesenchymal markers. (A) Immunofluorescence analysis showing a decreased or absence of cell surface expression of E-cadherin and cytokeratin-18 and increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown and OSE cells ( × 1000 magnification). (B) Immunoblot analysis of N-cadherin and vimentin expression in control and knockdown cells. (C) Immunofluorescence analysis of β-catenin expression in control and CA125/MUC16 knockdown cells.
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Related In: Results  -  Collection

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

fig2: CA125/MUC16 knockdown alters expression of epithelial and mesenchymal markers. (A) Immunofluorescence analysis showing a decreased or absence of cell surface expression of E-cadherin and cytokeratin-18 and increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown and OSE cells ( × 1000 magnification). (B) Immunoblot analysis of N-cadherin and vimentin expression in control and knockdown cells. (C) Immunofluorescence analysis of β-catenin expression in control and CA125/MUC16 knockdown cells.
Mentions: Epithelial-to-mesenchymal transition is a process characterised by the loss of epithelial markers such as E-cadherin and cytokeratin-18 and gain of mesenchymal markers such as N-cadherin and vimentin (Thiery, 2003; Ahmed et al, 2007; Vergara et al, 2010). During progression, carcinoma cells with increased invasiveness and metastatic potential usually acquire mesenchymal markers and show a reduction or absence of E-cadherin expression (Hugo et al, 2007). However, in contrast to most adenocarcinomas, advanced and poorly differentiated ovarian tumours continue to express E-cadherin (Ahmed et al, 2007). This contrasts with OSE cells, which do not express E-cadherin (Auersperg et al, 2001). Immunofluorescence microscopy analysis showed that E-cadherin was detected at the contacts between NIH:OVCAR3 control cells, whereas there was a loss of E-cadherin staining at the cell surface and an internalisation of E-cadherin in CA125/MUC16 knockdown cells (Figure 2A). As expected, OSE did not express E-cadherin. Knockdown cells behave very much like OSE cells; they displayed decreased expression of cytokeratin-18, whereas N-cadherin and vimentin expression were increased compared with NIH:OVCAR3- and Ctrl scFv-transfected cells (Figure 2A). Western blot analysis confirmed the increased expression of N-cadherin and vimentin in CA125/MUC16 knockdown cells (Figure 2B). The cytoplasmic domain of E-cadherin has been shown to bind to β-catenin (Sasaki et al, 2000). β-Catenin staining was observed to localise at the contacts of cells in a manner similar to E-cadherin in NIH:OVCAR3- and Ctrl scFv-transfected cells, but partially re-localised in the cytoplasm in CA125/MUC16 knockdown cells (Figure 2C). Altogether, these data show that CA125/MUC16 knockdown induces a re-distribution of E-cadherin and β-catenin epithelial markers throughout the cytoplasm and increased expression of mesenchymal markers, such as N-cadherin and vimentin, consistent with an EMT. The data also show that OVCAR3 knockdown cells acquired a phenotype that was very similar to that of CA125-negative OSE 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