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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 alters cell morphology and spheroid formation of NIH:OVCAR3 cells. (A) The morphology of control, CA125/MUC16 knockdown in NIH:OVCAR3 cells and OSE cells by phase-contrast microscopy ( × 200 magnification). (B) Immunofluorescence analysis of control, CA125/MUC16 knockdown cells and OSE cells ( × 1000 magnification). The panels show phalloidin–rhodamine staining of actin-cytoskeleton changes induced by CA125/MUC16 knockdown and DAPI for nuclear staining. (C) Control and knockdown cells were seeded in suspension for this aggregation assay. Control cells showed bigger and tighter cell aggregates in contrast to knockdown cells ( × 40 magnification).
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fig1: CA125/MUC16 knockdown alters cell morphology and spheroid formation of NIH:OVCAR3 cells. (A) The morphology of control, CA125/MUC16 knockdown in NIH:OVCAR3 cells and OSE cells by phase-contrast microscopy ( × 200 magnification). (B) Immunofluorescence analysis of control, CA125/MUC16 knockdown cells and OSE cells ( × 1000 magnification). The panels show phalloidin–rhodamine staining of actin-cytoskeleton changes induced by CA125/MUC16 knockdown and DAPI for nuclear staining. (C) Control and knockdown cells were seeded in suspension for this aggregation assay. Control cells showed bigger and tighter cell aggregates in contrast to knockdown cells ( × 40 magnification).

Mentions: We have chosen the widely used NIH:OVCAR3 epithelial ovarian carcinoma cell line for our studies because, like most serous ovarian carcinomas, these cells overexpress CA125/MUC16 at the their surface and they show a complete epithelial phenotype (Hamilton et al, 1983; Yin and Lloyd, 2001; Rump et al, 2004). In addition, NIH:OVCAR3 cells are non-invasive in Boyden chambers, poorly anchorage-independent, non-motile on plastic and do not invade in collagen gels (Hamilton et al, 1987). These properties make NIH:OVCAR3 cells ideally suitable to study EMT. To overcome the difficulties associated with silencing the large size of CA125/MUC16 RNA, we used a system where the cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells was downregulated by the expression of an endoplasmic reticulum-localised CA125/MUC16 scFv to study its role in EMT (Gubbels et al, 2006; Boivin et al, 2009). The expression of cell surface CA125/MUC16 was reduced by 90% in two independent NIH:OVCAR3 CA125/MUC16 scFv-expressing subclones (1 : 9#7 and 1 : 9#9 scFvs), whereas control NIH:OVCAR3 expressing a control (Ctrl) scFv did not show alteration of CA125/MUC16 expression compared with the parental NIH:OVCAR3 (Boivin et al, 2009). We noticed that CA125/MUC16 knockdown in NIH:OVCAR3 cells display significant changes in morphological features compared with controls. Control cells (parental NIH:OVCAR3 and Ctrl scFv cells) formed cobblestone-like monolayer, round boundary and cell–cell junctions and adhesion between neighbouring cells (Figure 1A). In contrast, knockdown cells have a longer and fibroblast-like shape more characteristic of mesenchymal cells and display scattering of the cells. These changes were compared with OSE cells that are CA125/MUC16 negative and display mesenchymal features as indicated by their fibroblast-like shape (Figure 1A). The distribution of filamentous actin was analysed by phalloidin staining and clearly revealed alterations in the actin cytoskeleton from the predominance of cortical actin (control cells) into actin stress fibres throughout the cells for CA125/MUC16 knockdown and OSE cells (Figure 1B). Re-organisation of filamentous actin is characteristic of a cell-spreading response (Cunningham et al, 1992). These observations suggest that the CA125/MUC16 knockdown induces a phenotypic switch in carcinoma cells that resemble 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 cell morphology and spheroid formation of NIH:OVCAR3 cells. (A) The morphology of control, CA125/MUC16 knockdown in NIH:OVCAR3 cells and OSE cells by phase-contrast microscopy ( × 200 magnification). (B) Immunofluorescence analysis of control, CA125/MUC16 knockdown cells and OSE cells ( × 1000 magnification). The panels show phalloidin–rhodamine staining of actin-cytoskeleton changes induced by CA125/MUC16 knockdown and DAPI for nuclear staining. (C) Control and knockdown cells were seeded in suspension for this aggregation assay. Control cells showed bigger and tighter cell aggregates in contrast to knockdown cells ( × 40 magnification).
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Related In: Results  -  Collection

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fig1: CA125/MUC16 knockdown alters cell morphology and spheroid formation of NIH:OVCAR3 cells. (A) The morphology of control, CA125/MUC16 knockdown in NIH:OVCAR3 cells and OSE cells by phase-contrast microscopy ( × 200 magnification). (B) Immunofluorescence analysis of control, CA125/MUC16 knockdown cells and OSE cells ( × 1000 magnification). The panels show phalloidin–rhodamine staining of actin-cytoskeleton changes induced by CA125/MUC16 knockdown and DAPI for nuclear staining. (C) Control and knockdown cells were seeded in suspension for this aggregation assay. Control cells showed bigger and tighter cell aggregates in contrast to knockdown cells ( × 40 magnification).
Mentions: We have chosen the widely used NIH:OVCAR3 epithelial ovarian carcinoma cell line for our studies because, like most serous ovarian carcinomas, these cells overexpress CA125/MUC16 at the their surface and they show a complete epithelial phenotype (Hamilton et al, 1983; Yin and Lloyd, 2001; Rump et al, 2004). In addition, NIH:OVCAR3 cells are non-invasive in Boyden chambers, poorly anchorage-independent, non-motile on plastic and do not invade in collagen gels (Hamilton et al, 1987). These properties make NIH:OVCAR3 cells ideally suitable to study EMT. To overcome the difficulties associated with silencing the large size of CA125/MUC16 RNA, we used a system where the cell surface expression of CA125/MUC16 in NIH:OVCAR3 cells was downregulated by the expression of an endoplasmic reticulum-localised CA125/MUC16 scFv to study its role in EMT (Gubbels et al, 2006; Boivin et al, 2009). The expression of cell surface CA125/MUC16 was reduced by 90% in two independent NIH:OVCAR3 CA125/MUC16 scFv-expressing subclones (1 : 9#7 and 1 : 9#9 scFvs), whereas control NIH:OVCAR3 expressing a control (Ctrl) scFv did not show alteration of CA125/MUC16 expression compared with the parental NIH:OVCAR3 (Boivin et al, 2009). We noticed that CA125/MUC16 knockdown in NIH:OVCAR3 cells display significant changes in morphological features compared with controls. Control cells (parental NIH:OVCAR3 and Ctrl scFv cells) formed cobblestone-like monolayer, round boundary and cell–cell junctions and adhesion between neighbouring cells (Figure 1A). In contrast, knockdown cells have a longer and fibroblast-like shape more characteristic of mesenchymal cells and display scattering of the cells. These changes were compared with OSE cells that are CA125/MUC16 negative and display mesenchymal features as indicated by their fibroblast-like shape (Figure 1A). The distribution of filamentous actin was analysed by phalloidin staining and clearly revealed alterations in the actin cytoskeleton from the predominance of cortical actin (control cells) into actin stress fibres throughout the cells for CA125/MUC16 knockdown and OSE cells (Figure 1B). Re-organisation of filamentous actin is characteristic of a cell-spreading response (Cunningham et al, 1992). These observations suggest that the CA125/MUC16 knockdown induces a phenotypic switch in carcinoma cells that resemble 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