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Tumour growth and resistance to gemcitabine of pancreatic cancer cells are decreased by AP-2alpha overexpression.

Jonckheere N, Fauquette V, Stechly L, Saint-Laurent N, Aubert S, Susini C, Huet G, Porchet N, Van Seuningen I, Pigny P - Br. J. Cancer (2009)

Bottom Line: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells.Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity.Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U837, Université de Lille 2, Centre de Recherche Jean-Pierre Aubert, Place de Verdun, 59045 Lille cedex, France.

ABSTRACT

Background: Activator protein-2alpha (AP-2alpha) is a transcription factor that belongs to the family of AP-2 proteins that have essential roles in tumorigenesis. Indeed, AP-2alpha is considered as a tumour-suppressor gene in different tissues such as colonic, prostatic or breast epithelial cells. Moreover, AP-2alpha also participates in the control of colon and breast cancer cells sensitivity towards chemotherapeutic drugs. Despite its potential interest, very few data are available regarding the roles of AP-2alpha in pancreatic cancer.

Methods: We have developed a stable pancreatic CAPAN-1 cell line overexpressing AP-2alpha. Consequences of overexpression were studied in terms of in vivo cell growth, gene expression, migration capacity and chemosensitivity.

Results: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells. An altered expression pattern of cell cycle-controlling factors (CDK-4, CDK-6, cyclin-G1, p27(kip1) and p57(kip2)) was observed in AP-2alpha-overexpressing clones by microarrays and western blot analysis. Promoter activity and ChIP analysis indicated that AP-2alpha induces p27(kip1) protein levels by direct binding to and transactivation of its promoter. Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity.

Conclusion: Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

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Expression pattern of MUC1 membrane-bound mucin and ErbB-2 receptor in the CAPAN-1 clones stably transfected with an AP-2α expression vector (α27 to α45). (A) Western blot analysis of the expression of ErbB-2 and MUC1. One or two upper arrow(s) correspond to a low or high levels of expression of AP-2α, respectively. Bands were quantified by densitometry and a ratio (specific protein to β-actin) was calculated to evaluate differences between mock cells and the clones. Ratio values are indicated under each panel. Total cellular extracts were used. Ca1, parental cell line; C5, mock cells. (B) Topography of MUC1 and MUC4 expression was analysed by confocal microscopy.
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fig1: Expression pattern of MUC1 membrane-bound mucin and ErbB-2 receptor in the CAPAN-1 clones stably transfected with an AP-2α expression vector (α27 to α45). (A) Western blot analysis of the expression of ErbB-2 and MUC1. One or two upper arrow(s) correspond to a low or high levels of expression of AP-2α, respectively. Bands were quantified by densitometry and a ratio (specific protein to β-actin) was calculated to evaluate differences between mock cells and the clones. Ratio values are indicated under each panel. Total cellular extracts were used. Ca1, parental cell line; C5, mock cells. (B) Topography of MUC1 and MUC4 expression was analysed by confocal microscopy.

Mentions: We previously produced stable clones deriving from CAPAN-1 cell line that overexpressed moderate (clones α42 and α45, one arrow on Figure 1A) or high levels (α27 and α38, two arrows on Figure 1A) of AP-2α, and consequently exhibited a partial to complete inhibition of MUC4 expression, respectively (Fauquette et al, 2007). To further characterise the pattern of expression of these clones, levels of expression of ErbB-2 (the putative MUC4 receptor) and of the membrane-bound mucin MUC1 were determined by western blotting. As previously shown with MUC4 (Fauquette et al, 2007), AP-2α overexpression is accompanied by a decrease of the expression levels of total ErbB-2 and MUC1 (Figure 1A). A nearly complete extinction of MUC1 was observed in clones expressing high levels of AP-2α. We further examined whether AP-2α overexpression could affect the localisation of MUC4 and MUC1 using confocal microscopy analysis. In parental CAPAN-1 cells and in C5 control cells (mock cells), MUC4 was mostly localised at the apical surface, whereas MUC1 showed both apical and basolateral staining (Figure 1B). In α42 and α45 clones that still expressed membrane-bound mucins, MUC4 apical staining was strongly decreased, whereas MUC1 showed a slight decrease of the apical staining together with an enhancement of the basolateral staining.


Tumour growth and resistance to gemcitabine of pancreatic cancer cells are decreased by AP-2alpha overexpression.

Jonckheere N, Fauquette V, Stechly L, Saint-Laurent N, Aubert S, Susini C, Huet G, Porchet N, Van Seuningen I, Pigny P - Br. J. Cancer (2009)

Expression pattern of MUC1 membrane-bound mucin and ErbB-2 receptor in the CAPAN-1 clones stably transfected with an AP-2α expression vector (α27 to α45). (A) Western blot analysis of the expression of ErbB-2 and MUC1. One or two upper arrow(s) correspond to a low or high levels of expression of AP-2α, respectively. Bands were quantified by densitometry and a ratio (specific protein to β-actin) was calculated to evaluate differences between mock cells and the clones. Ratio values are indicated under each panel. Total cellular extracts were used. Ca1, parental cell line; C5, mock cells. (B) Topography of MUC1 and MUC4 expression was analysed by confocal microscopy.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736821&req=5

fig1: Expression pattern of MUC1 membrane-bound mucin and ErbB-2 receptor in the CAPAN-1 clones stably transfected with an AP-2α expression vector (α27 to α45). (A) Western blot analysis of the expression of ErbB-2 and MUC1. One or two upper arrow(s) correspond to a low or high levels of expression of AP-2α, respectively. Bands were quantified by densitometry and a ratio (specific protein to β-actin) was calculated to evaluate differences between mock cells and the clones. Ratio values are indicated under each panel. Total cellular extracts were used. Ca1, parental cell line; C5, mock cells. (B) Topography of MUC1 and MUC4 expression was analysed by confocal microscopy.
Mentions: We previously produced stable clones deriving from CAPAN-1 cell line that overexpressed moderate (clones α42 and α45, one arrow on Figure 1A) or high levels (α27 and α38, two arrows on Figure 1A) of AP-2α, and consequently exhibited a partial to complete inhibition of MUC4 expression, respectively (Fauquette et al, 2007). To further characterise the pattern of expression of these clones, levels of expression of ErbB-2 (the putative MUC4 receptor) and of the membrane-bound mucin MUC1 were determined by western blotting. As previously shown with MUC4 (Fauquette et al, 2007), AP-2α overexpression is accompanied by a decrease of the expression levels of total ErbB-2 and MUC1 (Figure 1A). A nearly complete extinction of MUC1 was observed in clones expressing high levels of AP-2α. We further examined whether AP-2α overexpression could affect the localisation of MUC4 and MUC1 using confocal microscopy analysis. In parental CAPAN-1 cells and in C5 control cells (mock cells), MUC4 was mostly localised at the apical surface, whereas MUC1 showed both apical and basolateral staining (Figure 1B). In α42 and α45 clones that still expressed membrane-bound mucins, MUC4 apical staining was strongly decreased, whereas MUC1 showed a slight decrease of the apical staining together with an enhancement of the basolateral staining.

Bottom Line: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells.Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity.Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U837, Université de Lille 2, Centre de Recherche Jean-Pierre Aubert, Place de Verdun, 59045 Lille cedex, France.

ABSTRACT

Background: Activator protein-2alpha (AP-2alpha) is a transcription factor that belongs to the family of AP-2 proteins that have essential roles in tumorigenesis. Indeed, AP-2alpha is considered as a tumour-suppressor gene in different tissues such as colonic, prostatic or breast epithelial cells. Moreover, AP-2alpha also participates in the control of colon and breast cancer cells sensitivity towards chemotherapeutic drugs. Despite its potential interest, very few data are available regarding the roles of AP-2alpha in pancreatic cancer.

Methods: We have developed a stable pancreatic CAPAN-1 cell line overexpressing AP-2alpha. Consequences of overexpression were studied in terms of in vivo cell growth, gene expression, migration capacity and chemosensitivity.

Results: In vivo tumour growth of CAPAN-1 cells overexpressing AP-2alpha was significantly decreased by comparison to control cells. An altered expression pattern of cell cycle-controlling factors (CDK-4, CDK-6, cyclin-G1, p27(kip1) and p57(kip2)) was observed in AP-2alpha-overexpressing clones by microarrays and western blot analysis. Promoter activity and ChIP analysis indicated that AP-2alpha induces p27(kip1) protein levels by direct binding to and transactivation of its promoter. Moreover, AP-2alpha overexpression increased the chemosensitivity of CAPAN-1 cells to low doses of gemcitabine and reduced their in vitro migration capacity.

Conclusion: Our data suggested that AP-2alpha overexpression could be exploited to decrease in vivo tumour growth of pancreatic cancer cells and to increase their sensitivity to gemcitabine.

Show MeSH
Related in: MedlinePlus