Functional characterization of E- and P-cadherin in invasive breast cancer cells

Abstract

Background: Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood.

Methods: To compare the functional role of E-cadherin and P-cadherin in invasive breast cancer, we stably transfected these molecules into the MDA-MB-231 cell line, and investigated their effects on motility, invasion and gene expression regulation.

Results: Expression of either E- and P-cadherin significantly increased cell aggregation and induced a switch from fibroblastic to epithelial morphology. Although expression of these cadherins did not completely reverse the mesenchymal phenotype of MDA-MB-231 cells, both E- and P-cadherin decreased fibroblast-like migration and invasion through extracellular matrix in a similar way. Moreover, microarray gene expression analysis of MDA-MB-231 cells after expression of E- and P-cadherins revealed that these molecules can activate signaling pathways leading to significant changes in gene expression. Although the expression patterns induced by E- and P-cadherin showed more similarities than differences, 40 genes were differentially modified by the expression of either cadherin type.

Conclusion: E- and P-cadherin have similar functional consequences on the phenotype and invasive behavior of MDA-MB-231 cells. Moreover, we demonstrate for the first time that these cadherins can induce both common and specific gene expression programs on invasive breast cancer cells. Importantly, these identified genes are potential targets for future studies on the functional consequences of altered cadherin expression in human breast cancer.

Figure 1

Exogenous expression of E- and P-cadherin promotes formation of functional adherens junctions and morphological changes in 231 cells. A: Western blot showing E- and P-cadherin expression before (-) and after (+) addition of Dexamethasone in 231 clones. MCF7 and BT549 cells were used as controls for endogenous expression of E- and P-cadherin, respectively. B: E- or P-cadherin expression promotes epitheloid morphology (first column, bright field) and recruits the catenins (p120 and beta-catenin) to the adherens junctions (immunofluorescence, columns two to four). C: E- or P-cadherin expression enhances cell aggregation. Left pictures show representative examples of cellular aggregation in each condition ("-", before and "+" after addition of Dex). E-cadherin positive MCF7 cells were used as control. Right: Quantification of mean particle size. Each bar represents mean ± SEM size (measured in pixels) of cell aggregates (n = 25, for each condition). Mean differences were compared by Student's t test (* indicates differences statistically significant p < 0.05; NS, not significant).

Figure 2

Expression of E- or P-cadherin does not completely reverse the mesenchymal phenotype of 231 cells. First row: Double immunofluorescence of E- or P-cadherin (Alexa-594, red) and actin cytoskeleton (Phalloidin-Alexa-488, green). Second row: Focal adhesions (arrows) co-stained with Phalloidin (green) and anti-paxillin (red). Note that no evident changes in the number or organization of focal adhesions are seen among the different conditions. Third and fourth rows: expression of the mesenchymal marker vimentin and the epithelial marker cytokeratin.

Figure 3

E- and P-cadherin modify the migratory and invasive behaviour of 231 cells. A: Study of cell migration by wound healing assay. Left pictures show that control cells migrate as single cells (arrow) whereas 231_E-cadh and 231_P-cadh cells migrate as clusters. Right: Cell migration was quantified by measuring the wounded area (in pixels) that was covered by the cells in the indicated time points. Each bar represents mean ± SEM of four independent experiments. B: Invasion ability through extracellular matrix (Matrigel) assessed by Transwell assays. Left: Representative images showing the number of invaded cells (nuclei stained by DAPI) after 24 hours. Right: Quantification of invasion. Each bar represents mean number ± SEM of invaded cells for each condition (four independent experiments). Mean differences were compared by Student's t test (* indicates differences statistically significant p < 0.05; NS, not significant).

Figure 4

E- and P-cadherin modulate transcriptional changes. A: Unsupervised hierarchical clustering of the gene expression patterns induced by E- or P-cadherin expression. Columns represent microarray experiments, and rows gene expression of the 149 genes modified at least two-fold with respect to control cells. Intensity of color is a function of the gene expression level as depicted in the scale bar. B: Identification of genes differentially expressed (with a statistical significance of FDR < 0.15) between 231_E-cadh and 231_P-cadh cells. C: Validation of microarray data of selected genes by qRT-PCR. Bars represent mean gene expression ± SEM (mRNA levels relative to control B2M transcript) from four different experiments.ARHGDIB is down-regulated and VEGFC is over-expressed in 231_E-Cadh and 231_P-cadh cells compared to control cells. MMP14 is down-regulated only in 231_P-cadh clones.