E-Cadherin Growth Regulation Figure 5. Loss of b-catenin by shRNA-mediated knock-down mimics E-cadherin engagement. (A) NHU cells expressing b-catenin shRNA (NHU-b-cat-KD) and their isogenic controls expressing scramble shRNA control (NHU-Con) were established by retrovirus transduction and the efficiency of the shRNA to down-regulate b-catenin was assessed by immunoblotting. Lysates from NHU-Con and NHU-b-cat-KD cells maintained in low calcium were probed for expression of active b-catenin (b-cat), E-cadherin (E-cad), phospho-AKT at S437 (p-AKT) and b-actin using mouse antibodies (b-cat, E-cad and b-actin) and rabbit antiserum (p-AKT), followed by goat anti-mouse antibody conjugated with Alexa Fluor 680 or goat anti-rabbit antibody conjugated with IRDye 800 as appropriate. Densitometry was performed to determine differences in protein expression with respect to b-actin and fold differences following normalisation are indicated. (B) NHU-Con and NHU-b-cat-KD cell lines were seeded into 96-well plates and cultured for a period of 6 days in medium containing either 0.09 mM (Low Ca) or 2.0 mM (Phys Ca) calcium concentration and in the absence (Control) or presence of 5 mM of PI3-K inhibitor LY294002. Proliferation was determined on the basis of cell biomass using the MTT assay and results are presented in upper and lower left panels. Data points represent mean absorbance values for 6 replicate wells (6S.E.M.). To allow easier comparison between inhibitor-treated (+ LY) and non-treated cells, graphs for each cell line are also presented (upper and lower middle panels). In addition, results for all data points are shown in the form of bar graphs (lower panel) for the purpose of statistical analysis. ns, non-significant; *, P,0.05; **, P,0.01; ***, P,0.001. doi:10.1371/journal.pone.0013621.g005 provides insight into the downstream sequelae of E-cadherin loss in cancer. The well-recognised property of E-cadherin as a metastasis suppressor is generally attributed to its function in mediating adherens junctions in the maintenance of tissue integrity. Although it is also accepted that E-cadherin has a modulatory role in cell proliferation and migration, its specific contribution has remained more equivocal. One problem of interpretation is that reintro- duction of E-cadherin expression into a malignant cell line tends to reveal idiospecific effects, as it is unlikely to restore all normal functions of E-cadherin against a background of multiple genetic/ epigenetic alterations. Pece and co-workers demonstrated E- cadherin-dependent, ligand-independent activation of EGFR and downstream MAPK pathways in the HaCaT cell line [7]. This was consistent with E-cadherin providing a proliferation signal in keratinocytes [8,26], or rescue from anoikis in squamous cell PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13621