Loss of p27Kip¹ promotes metaplasia in the pancreas via the regulation of Sox9 expression

Abstract

p27Kip1 (p27) is a negative regulator of proliferation and a tumor suppressor via the inhibition of cyclin-CDK activity in the nucleus. p27 is also involved in the regulation of other cellular processes, including transcription by acting as a transcriptional co-repressor. Loss of p27 expression is frequently observed in pancreatic adenocarcinomas in human and is associated with decreased patient survival. Similarly, in a mouse model of K-Ras-driven pancreatic cancer, loss of p27 accelerates tumor development and shortens survival, suggesting an important role for p27 in pancreatic tumorigenesis. Here, we sought to determine how p27 might contribute to early events leading to tumor development in the pancreas. We found that K-Ras activation in the pancreas causes p27 mislocalization at pre-neoplastic stages. Moreover, loss of p27 or expression of a mutant p27 that does not bind cyclin-CDKs causes the mislocalization of several acinar polarity markers associated with metaplasia and induces the nuclear expression of Sox9 and Pdx1 two transcription factors involved in acinar-to-ductal metaplasia. Finally, we found that p27 directly represses transcription of Sox9, but not that of Pdx1. Thus, our results suggest that K-Ras activation, the earliest known event in pancreatic carcinogenesis, may cause loss of nuclear p27 expression which results in derepression of Sox9, triggering reprogramming of acinar cells and metaplasia.

Keywords: CDK; cell cycle; p27Kip1; pancreas.

Figure 1. K-Ras activation causes loss of nuclear p27 localization

Sections of paraffin embedded pancreas were stained for p27 or H&E. A.-B. Localization of p27 in normal pancreas of p27−/− (n = 3), p27+/+ (n = 3) and ElasK-Ras^G12V (n = 6) mice. Asterisks indicate β-islets. C.-E. H&E and p27 staining of consecutive sections in areas of ElasK-Ras^G12V pancreas showing either metaplasia C., PanIN-1 D. or adenocarcinoma E. Scale bars in all images are 50 μm except in E. were the scale bars are 100 μm in the two top images.

Figure 2. Mislocalization of integrinβ1 and Munc18 in acinar cells of p27^CK− and p27−/− pancreas

A.-B. Sections of paraffin-embedded pancreas from p27+/+, p27−/−, and p27^CK−/CK− mice were stained for the basal markers laminin α2 and Munc18 A. or laminin α2 and Integrinβ1 B. DNA was stained with Hoechst. All images were acquired using a 60x objective. In A. the graph displays the percentage of p27+/+ (n = 12), p27^CK−/CK− (n = 13) and p27−/− (n = 12) mice with milocalized Munc18. In B., the graph displays the percentage of p27+/+ (n = 11), p27CK−/CK− (n=12) and p27−/− (n = 17) mice with mislocalized Integrinβ1. Mice were considered positive for Integrinβ1or Munc18 mislocalization when several areas with non-basal staining were observed.

Figure 3. Expression of Sox9 and Pdx1 in acinar cells of p27^CK− and p27−/− pancreas

A.-B. Sections of paraffin-embedded pancreas from p27+/+, p27−/−, and p27^CK−/CK− mice were stained for E-Cadherin and Sox9 A. or E-Cadherin and Pdx1 B. DNA was stained with Hoechst. All images were acquired using a 60x objective. The graphs display the percentage of p27+/+ (n = 15), p27^CK−/CK− (n = 19) and p27−/− (n = 19) pancreas with Sox9 expression A. and Pdx1 expression B. Mice were considered positive for Sox9 when more than a half of the pancreas expressed Sox9. Mice were considered positive for Pdx1 when more than a quarter of the pancreas expressed Pdx1.

Figure 4. p27 represses transcription of Sox9 in a CDK dependent manner

A. Schematic of the reporter construct used in transcription reporter assays. The Sox9 promoter is cloned upstream of destabilized GFP (half-life of approximately 1 h). B.-C. 293 cells were co-transfected with the Sox9 reporter construct or a control reporter construct in which the GAPDH promoter is cloned upstream of GFP and the indicated amount of p27 B. or p27CK- C. The amount of DNA transfected was normalized using a plasmid encoding β-Gal. Fluorescence levels were monitored and quantified using an Incucyte FLR on 25 images in each well. The graphs in B and C represent the mean fluorescence intensity of the Sox9 promoter normalized to that of the GAPDH promoter in the same condition from six (p27) and five (p27^CK−) independent experiments. Data were compared by ANOVA followed by Neuman-Keuls multiple comparison test, ** = p < 0.01. D. A chromatin immunoprecipitation (ChIP) analysis was performed to determine if p27 could bind the Sox9 promoter in vivo in various cell lines. PCR products using primers specific for the Sox9 promoter were separated on an agarose gel. For each cell line, PCRs were performed on a fraction of the input and DNA from ChIPs with anti-p27 or isotype control antibodies.