Stromal upregulation of lateral epithelial adhesions: gene expression analysis of signalling pathways in prostate epithelium

Abstract

Background: Stromal signalling increases the lateral cell adhesions of prostate epithelial cells grown in 3D culture. The aim of this study was to use microarray analysis to identify significant epithelial signalling pathways and genes in this process.

Methods: Microarray analysis was used to identify genes that were differentially expressed when epithelial cells were grown in 3D Matrigel culture with stromal co-culture compared to without stroma. Two culture models were employed: primary epithelial cells (ten samples) and an epithelial cell line (three experiments). A separate microarray analysis was performed on each model system and then compared to identify tissue-relevant genes in a cell line model.

Results: TGF beta signalling was significantly ranked for both model systems and in both models the TGF beta signalling gene SOX4 was significantly down regulated. Analysis of all differentially expressed genes to identify genes that were common to both models found several morphology related gene clusters; actin binding (DIAPH2, FHOD3, ABLIM1, TMOD4, MYH10), GTPase activator activity (BCR, MYH10), cytoskeleton (MAP2, MYH10, TMOD4, FHOD3), protein binding (ITGA6, CD44), proteinaceous extracellular matrix (NID2, CILP2), ion channel/ ion transporter activity (CACNA1C, CACNB2, KCNH2, SLC8A1, SLC39A9) and genes associated with developmental pathways (POFUT1, FZD2, HOXA5, IRX2, FGF11, SOX4, SMARCC1).

Conclusions: In 3D prostate cultures, stromal cells increase lateral epithelial cell adhesions. We show that this morphological effect is associated with gene expression changes to TGF beta signalling, cytoskeleton and anion activity.

Figure 1

Primary component analysis to show clustering of genes from acini cultured with stroma (blue) compared to acini cultured without (red).

Figure 2

Evidence of heterogeneity in gene expression between different primary culture samples. Primary epithelial cells were grown for 14 days in Matrigel with stromal co-cultures and compared to cultures grown without stroma. RNA was prepared from single acini. Data is shown for all ten matched primary epithelial cell cultures. A) the mean fold change in gene expression levels for FGFBP1 measured by Operon microarray (black bars) or QRT-PCR (open bars). B) the mean fold change in gene expression levels for DNMBP measured by Operon microarray. C) the mean fold change in gene expression levels for CLDN6 measured by Operon microarray. Changes in gene expression detected by Operon were from the 1.1 fold gene list (p < 0.05).* insufficient sample available for QRT-PCR

Figure 3

Genes identified in the TGF beta pathway. The Kegg pathway for TGF beta signalling illustrating the significant genes identified by pathway express. Genes circled in red indicate genes indicate genes from the primary cell microarray dataset (Operon) and those in blue from the BPH-1 cell microarray dataset (Affymetrix). Continuous lines are upregulated genes whereas broken lines are down-regulated.

Figure 4

Validation of human TGF beta/ BMP signalling pathway genes. Average changes in gene expression between 3D BPH-1 acini cultured with stroma compared to without stroma detected by Affymetrix (white bars) and QRT-PCR techniques (black bars). Changes in gene expression detected by Affymetrix were from the 1.1 fold gene list (p < 0.05).

Figure 5

Identification of common genes that were up regulated or down regulated in both cell line and primary epithelial models in response to stroma. Fold change lists were generated using Genesping (both used fold change lists of 1.1 fold and p < 0.05) for both the primary (blue) and cell line (red) arrays. The gene lists were compared using Entrez gene IDs to find common upregulated (A) and downregulated genes (B).

Figure 6

Confirmation of MAP2 expression. QRT-PCR was performed on mRNA isolated from 3D BPH-1 acini cultured for 7 days with (white) or without stroma (black) from three different patients (in duplicate). The average fold difference in gene expression was expressed relative to growth without stroma ± standard error ** P < 0.05.