Activation of Wnt signalling in stroma from pancreatic cancer identified by gene expression profiling

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an abundant desmoplastic stroma. Interactions between cancer and stromal cells play a critical role in tumour invasion, metastasis and chemoresistance. Therefore, we hypothesized that gene expression profile of the stromal components of pancreatic carcinoma is different from chronic pancreatitis and reflects the interaction with the tumour. We investigated the gene expression of eleven stromal tissues from PDAC, nine from chronic pancreatitis and cell lines of stromal origin using the Affymetrix U133 GeneChip set. The tissue samples were microdissected, the RNA was extracted, amplified and labelled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified and validated using quantitative RT-PCR and immuno-histochemistry. We found 255 genes to be overexpressed and 61 genes to be underexpressed within the stroma of pancreatic carcinoma compared to the stroma of chronic pancreatitis. Analysis of the involved signal transduction pathways revealed a number of genes associated with the Wnt pathway of which the differential expression of SFRP1 and WNT5a was confirmed using immunohistochemistry. Moreover, we could demonstrate that WNT5a expression was induced in fibroblasts during cocultivation with a pancreatic carcinoma cell line. The identified differences in the expression profile of stroma cells derived from tumour compared to cells of inflammatory origin suggest a specific response of the tissue surrounding malignant cells. The overexpression of WNT5a, a gene involved in the non canonical Wnt signalling and chondrocyte development might contribute to the strong desmoplastic reaction seen in pancreatic cancer.

Fig. 1

(A) Hierarchical clustering of the stromal tissue samples using the identified differentially expressed genes. (B) Hierarchical clustering of pancreatic tissues and cell lines using the 205 probe sets identified asdifferentially expressed between tumour stroma and all other primary tissues from the 331 probe set. Probe set within the black lines are highly expressed in tumour stroma and the genes of this cluster are depicted on the right. (C) Heatmap of the expression of the 331 probe sets in the data set. Probe sets within the black line are highly expressed in cell lines and are depicted on the right. The cell line PT45 is the last column to the right in Fig. 1B and C.

Fig. 2

(A) Association of the differentially expressed genes with KEGG signal transduction pathways. Green: genes underexpressed; red: genes overex-pressed in pancreatic ductal adenocarcinoma (PDAC) stroma compared to stroma from chronic pancreatitis. (B) Genes identified in the KEGG pathways, TS, tumour stroma; CP, stroma of chronic pancreatitis.

Fig. 3

Boxplots of the distribution of expression of SFRP1, WNT5a and CXCL14 in stroma of PDAC and chronic pancreatitis. (A–C) Arbitrary intensity units of the probe sets from the Affymetrix U133 GeneChips; (D–F): Δ-cT values from the quantitative PCR, data were calculated as Δ-cT values using β-actin values for normalization, therefore lower values indicate higher expression of a given gene in the samples; (G) Relative expression of WNT5a in KIF5 stromal cells cocultured with Panc89 cells. WNT5a is induced in fibroblasts by a median of 2.82-fold if WNT5a expression is normalized to G6PDH expression and referred to the expression of WNT5a in KIF5 cells grown in monolayers; dotted lines: median; FC, fold change; TS, tumour stroma; CP, stroma of chronic pan-creatitis. P-values were calculated using the t-test.

Fig. 4

Immunohistochemistry of Wnt pathway associated genes. (A) Strong expression of WNT5a in PDAC stroma cell and (B) lack of expression of WNT5a in stromal tissue adjacent to normal ducts in two different samples. The surrounding acinar cells of the pancreas show staining for WNT5a, whereas the normal duct epithelia show only a faint stain and staining is absent within the stromal cells; (C) CXCL14 expression in PDAC epithelia and adjacent stromal cells. (D) Lack of expression of CXCL14 in a stroma of chronic pancreatitis. (E) Expression of SFRP1 in the stroma of chronic pancreatitis and blood vessels; (F) Loss of SFRP1 expression in the stroma of a well-differentiated PDAC. (A–F) 200×.