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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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CA125/MUC16 knockdown disrupts cell–cell junctions. (A) Assessment of cell–cell junctions in Ctrl scFv and CA125/MUC16 knockdown cells and OSE cells by electronic microscopy. Arrows indicate the presence of three desmosome spots in Ctrl scFv cells and the lack of such desmosomes in knockdown cells (scale bar – 100 nm). (B) Immunofluorescence analysis of claudin-7 expression, a protein involved in tight junctions, in Ctrl scFv and knockdown cells showing the disruption of the protein at the cell surface of knockdown cells ( × 1000 magnification).
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fig5: CA125/MUC16 knockdown disrupts cell–cell junctions. (A) Assessment of cell–cell junctions in Ctrl scFv and CA125/MUC16 knockdown cells and OSE cells by electronic microscopy. Arrows indicate the presence of three desmosome spots in Ctrl scFv cells and the lack of such desmosomes in knockdown cells (scale bar – 100 nm). (B) Immunofluorescence analysis of claudin-7 expression, a protein involved in tight junctions, in Ctrl scFv and knockdown cells showing the disruption of the protein at the cell surface of knockdown cells ( × 1000 magnification).

Mentions: Epithelial-to-mesenchymal transition usually involves the disruption of tight junctions, adherens junctions and desmosomes, which contribute to the separation into individual cells (Sundfeldt, 2003). Because of the morphological changes and the absence of spheroid formation in suspension observed in CA125/MUC16 knockdown cells (Figure 1), and their enhanced potential to migrate (Figure 4), we evaluated cell junctions by electronic microscopy in controls and knockdown NIH:OVCAR3 cells. Figure 5A shows a representative electron micrograph of three spot desmosomes between two Ctrl scFv-expressing NIH:OVCAR3 cells forming adhering junctions. In contrast, OSE and CA125/MUC16 knockdown cells lack such adhering junctions, resulting in larger intercellular space. Consistent with these results, the staining pattern for claudin-7 proteins, which are involved in tight junctions, were altered in CA125/MUC16 knockdown cells as compared with control cells (Figure 5B). Altogether, these data show that CA125/MUC16 knockdown in NIH:OVCAR3 cells disrupts cell–cell junctions and promotes cell migration.


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 disrupts cell–cell junctions. (A) Assessment of cell–cell junctions in Ctrl scFv and CA125/MUC16 knockdown cells and OSE cells by electronic microscopy. Arrows indicate the presence of three desmosome spots in Ctrl scFv cells and the lack of such desmosomes in knockdown cells (scale bar – 100 nm). (B) Immunofluorescence analysis of claudin-7 expression, a protein involved in tight junctions, in Ctrl scFv and knockdown cells showing the disruption of the protein at the cell surface of knockdown cells ( × 1000 magnification).
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Related In: Results  -  Collection

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fig5: CA125/MUC16 knockdown disrupts cell–cell junctions. (A) Assessment of cell–cell junctions in Ctrl scFv and CA125/MUC16 knockdown cells and OSE cells by electronic microscopy. Arrows indicate the presence of three desmosome spots in Ctrl scFv cells and the lack of such desmosomes in knockdown cells (scale bar – 100 nm). (B) Immunofluorescence analysis of claudin-7 expression, a protein involved in tight junctions, in Ctrl scFv and knockdown cells showing the disruption of the protein at the cell surface of knockdown cells ( × 1000 magnification).
Mentions: Epithelial-to-mesenchymal transition usually involves the disruption of tight junctions, adherens junctions and desmosomes, which contribute to the separation into individual cells (Sundfeldt, 2003). Because of the morphological changes and the absence of spheroid formation in suspension observed in CA125/MUC16 knockdown cells (Figure 1), and their enhanced potential to migrate (Figure 4), we evaluated cell junctions by electronic microscopy in controls and knockdown NIH:OVCAR3 cells. Figure 5A shows a representative electron micrograph of three spot desmosomes between two Ctrl scFv-expressing NIH:OVCAR3 cells forming adhering junctions. In contrast, OSE and CA125/MUC16 knockdown cells lack such adhering junctions, resulting in larger intercellular space. Consistent with these results, the staining pattern for claudin-7 proteins, which are involved in tight junctions, were altered in CA125/MUC16 knockdown cells as compared with control cells (Figure 5B). Altogether, these data show that CA125/MUC16 knockdown in NIH:OVCAR3 cells disrupts cell–cell junctions and promotes cell migration.

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