Abstract P6-08-07: Claudin-20 Promotes an Aggressive Phenotype in Human Breast Cancer Cells
Authors: Martin TA , Mansel RE , Jiang WG. 
Published: April 2011

Abstract
Background: Claudin-20 is a member of the claudin family of transmembrane proteins located in the tight junction (TJ) of cells of epithelial origin. Claudin-20 was originally described as being distributed in the TJ of intestinal epithelial cells. Due to the increasing evidence supporting the role of TJ proteins in preventing tumour cell metastatic behaviour, this study sought to evaluate the effect of claudin-20 overexpression in human breast cancer cells.
Methods: Breast cancer primary tumours (n= 114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR to obtain survival curves. 42 human cancer cell lines were screened for expression of claudin-20. Invasive and non-invasive breast cancer cell lines, MDA-MB-231 (MDAwt) and MCF-7 (MCF-7wt) respectively, which exhibited negligible expression were chosen for over-expression of the claudin-20 gene (MDAexp & MCF-7exp). The gene, after amplification from normal breast cDNA was cloned into a GFP-Fusion TOPO plasmid before electroporation. Expression of the gene product was confirmed by RT-PCR and fluorescent microscopy. Functional testing was carried out using TER (trans-epithelial resistance), paracellular permeability (PCP), cell invasion, cell adhesion and cell growth assays. In addition, in vivo growth was evaluated using an athymic nu/nu mice model.
Results: Q-PCR data showed that high claudin-20 expression was correlated with poor survival of patients with breast cancer (p=0.022). We therefore decided to investigate the effect of claudin-20 over-expression in human breast cancer cell behaviour. Following transformation of the breast cancer cell lines MDA-MB-231 & MCF-7 with a claudin-20 expression construct, the expression of the gene was demonstrated by the presence of mRNA expression and by the protein signal clearly observable at the cell-cell membrane. Claudin-20 transformed cells showed significantly increased invasion compared to WT cells (MDAexp 0.332±0.02 RU, MDAwt 0.304±0.001; p=0.05: MCF-7exp0 0.07±0.002 RU, MCF-7wt 0.05±0.0001; p=0.0014). Both MDAexp and MCF-7exp cells were significantly less adhesive than WT cells, (number of cells adhering to matrix: MDAexp 0.2±0.02 RU versus MDAwt 0.27±0.004, p=0.007; MCF-7exp 0.043±0.0005 RU versus MCF-7wt 0.052±0.0.005, p=0.05). There was no effect on growth (either in vitro or in vivo) for either cell line. Over-expression of Claudin-20 resulted in reduced TER (induced by the motogen HGF at 25 ng/ml: MDAexp change in TER at 2h -74+/−1.5 vs MDAwt at 2h -40+/−4.4, p=0.0007). Interestingly, this was not mirrored by PCP, as over-expression of claudin-20 caused a reduction in permeability (MDAexp change in PCP at 2h 182+/−5 vs MDAwt at 2h 260+/−10). Conclusion: The introduction of claudin-20 into human breast cancer cells resulted in breast cancer cells with an aggressive phenotype and reduced trans-epithelial resistance. There was no corresponding decrease in paracellular permeability, indicating that this claudin has a differential function in epithelial TJ. This provides further insight into the importance of correctly functioning TJ in preventing the progression of human breast cancer.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-07.