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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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AP-2α activates CDKN1B by direct interactions with its promoter. (A) Schematic representation of human CDKN1B promoter. The main cis elements for Sp1, CAAT box-binding factor and AP-2 are shown. The location of the AP-2 cis elements that undergo site-directed mutagenesis is also shown. (B) The different CDKN1B promoter constructs were transiently cotransfected with 0.2 μg of pRSV-AP-2α or corresponding empty vector. Results are expressed as fold induction of luciferase activity relative to the empty expression vector (value was set at 1.0). Values are means±s.d. for two independent experiments in which cotransfections were run in triplicate (*, P<0.05). (C) ChIP assay. AP-2 represents the fraction precipitated with the anti-AP-2α antibody, IgG those precipitated with a normal rabbit IgG. PCR results obtained on the proximal region are shown.
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fig3: AP-2α activates CDKN1B by direct interactions with its promoter. (A) Schematic representation of human CDKN1B promoter. The main cis elements for Sp1, CAAT box-binding factor and AP-2 are shown. The location of the AP-2 cis elements that undergo site-directed mutagenesis is also shown. (B) The different CDKN1B promoter constructs were transiently cotransfected with 0.2 μg of pRSV-AP-2α or corresponding empty vector. Results are expressed as fold induction of luciferase activity relative to the empty expression vector (value was set at 1.0). Values are means±s.d. for two independent experiments in which cotransfections were run in triplicate (*, P<0.05). (C) ChIP assay. AP-2 represents the fraction precipitated with the anti-AP-2α antibody, IgG those precipitated with a normal rabbit IgG. PCR results obtained on the proximal region are shown.

Mentions: We previously showed that AP-2α overexpression led to an upregulation of p27kip1 protein levels in pancreatic cancer cells (Fauquette et al, 2007). As p27kip1 expression is regulated at transcriptional and post-transcriptional levels, we next wanted to determine whether AP-2α could act at the promoter level. To do so, CAPAN-1 cells were transiently cotransfected with a series of three deletion mutants of CDKN1B promoter (Figure 3A) along with an AP-2α expression vector. As shown in Figure 3B, loss of the −3568 to −774 nucleotides in the CDKN1B promoter did not affect AP-2α effect that remained weak. Further deletion up to −549 nucleotides allowed AP-2α to increase the luciferase activity of the CDKN1B promoter activity by 4.3-fold, suggesting the presence of inhibitory cis elements in the upstream region. Interestingly the −549/−1 region contains two putative binding sites for AP-2 located at −315 and −243, and we decided to check their implication in AP-2α effect by site-directed mutagenesis. Disruption of AP-2 site#1 located at −315 led to a strong reduction of AP-2α stimulatory effect (P<0.05), whereas disruption of AP-2 site #2 at −243 had no consequence. To decipher what happens in vivo, ChIP assay was carried out on two regions of CDKN1B promoter, that is, a distal part (−610/−479) that did not contain the two AP-2 cis elements mentioned earlier and a proximal part (−426/−20) that did contain these elements. As expected, a strong binding of AP-2α to the proximal part of CDKN1B promoter was observed in the α45 clone that overexpressed the p27 protein (Figure 3C).


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)

AP-2α activates CDKN1B by direct interactions with its promoter. (A) Schematic representation of human CDKN1B promoter. The main cis elements for Sp1, CAAT box-binding factor and AP-2 are shown. The location of the AP-2 cis elements that undergo site-directed mutagenesis is also shown. (B) The different CDKN1B promoter constructs were transiently cotransfected with 0.2 μg of pRSV-AP-2α or corresponding empty vector. Results are expressed as fold induction of luciferase activity relative to the empty expression vector (value was set at 1.0). Values are means±s.d. for two independent experiments in which cotransfections were run in triplicate (*, P<0.05). (C) ChIP assay. AP-2 represents the fraction precipitated with the anti-AP-2α antibody, IgG those precipitated with a normal rabbit IgG. PCR results obtained on the proximal region are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2736821&req=5

fig3: AP-2α activates CDKN1B by direct interactions with its promoter. (A) Schematic representation of human CDKN1B promoter. The main cis elements for Sp1, CAAT box-binding factor and AP-2 are shown. The location of the AP-2 cis elements that undergo site-directed mutagenesis is also shown. (B) The different CDKN1B promoter constructs were transiently cotransfected with 0.2 μg of pRSV-AP-2α or corresponding empty vector. Results are expressed as fold induction of luciferase activity relative to the empty expression vector (value was set at 1.0). Values are means±s.d. for two independent experiments in which cotransfections were run in triplicate (*, P<0.05). (C) ChIP assay. AP-2 represents the fraction precipitated with the anti-AP-2α antibody, IgG those precipitated with a normal rabbit IgG. PCR results obtained on the proximal region are shown.
Mentions: We previously showed that AP-2α overexpression led to an upregulation of p27kip1 protein levels in pancreatic cancer cells (Fauquette et al, 2007). As p27kip1 expression is regulated at transcriptional and post-transcriptional levels, we next wanted to determine whether AP-2α could act at the promoter level. To do so, CAPAN-1 cells were transiently cotransfected with a series of three deletion mutants of CDKN1B promoter (Figure 3A) along with an AP-2α expression vector. As shown in Figure 3B, loss of the −3568 to −774 nucleotides in the CDKN1B promoter did not affect AP-2α effect that remained weak. Further deletion up to −549 nucleotides allowed AP-2α to increase the luciferase activity of the CDKN1B promoter activity by 4.3-fold, suggesting the presence of inhibitory cis elements in the upstream region. Interestingly the −549/−1 region contains two putative binding sites for AP-2 located at −315 and −243, and we decided to check their implication in AP-2α effect by site-directed mutagenesis. Disruption of AP-2 site#1 located at −315 led to a strong reduction of AP-2α stimulatory effect (P<0.05), whereas disruption of AP-2 site #2 at −243 had no consequence. To decipher what happens in vivo, ChIP assay was carried out on two regions of CDKN1B promoter, that is, a distal part (−610/−479) that did not contain the two AP-2 cis elements mentioned earlier and a proximal part (−426/−20) that did contain these elements. As expected, a strong binding of AP-2α to the proximal part of CDKN1B promoter was observed in the α45 clone that overexpressed the p27 protein (Figure 3C).

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