E-Cadherin Suppresses Cellular Transformation by Inhibiting (cid:2) -Catenin Signaling in an Adhesion-independent Manner

E-cadherin is a tumor suppressor protein with a well-established role in cell–cell adhesion. Adhesion could contribute to tumor suppression either by physically joining cells or by facilitating other juxtacrine signaling events. Alternatively, E-cadherin tumor suppressor activity could result from binding and antago-nizing the nuclear signaling function of (cid:2) -catenin, a known proto-oncogene. To distinguish between an adhesion- versus a (cid:2) -catenin signaling–dependent mechanism, chimeric cadherin constructs were expressed in the SW480 colorectal tumor cell line. Expression of wild-type E-cadherin signiﬁcantly inhibits the growth of this cell line. Growth inhibitory activity is retained by all constructs that have the (cid:2) -catenin binding region of the cytoplasmic domain but not by E-cadherin constructs that exhibit adhesive activity, but lack the (cid:2) -catenin binding region. This growth suppression correlates with a reduction in (cid:2) -catenin/T cell factor (TCF) reporter gene activity. Importantly, direct inhibition of (cid:2) -catenin/TCF signaling inhibits the growth of SW480 cells, and the growth inhibitory activity of E-cadherin is rescued by constitutively activated forms of TCF. Thus, the growth suppressor activity of E-cadherin is adhesion independent and results from an inhibition of the (cid:2) -catenin/TCF signaling pathway, suggesting that loss of E-cadherin expression can contribute to upregulation of this pathway in human cancers. E-cadherin–mediated growth suppression was not accompanied by overall depletion of (cid:2) -catenin from the cytosol and nucleus. This appears to be due to the existence of a large pool of cytosolic (cid:2) -cate-nin in SW480 cells that is refractory to both cadherin binding and TCF binding. Thus, a small pool of (cid:2) -cate-nin that can bind TCF (i.e., the transcriptionally active pool) can be selectively depleted by E-cadherin expression. The existence of functionally distinct pools of cytosolic (cid:2) -catenin suggests that there are mechanisms to regulate (cid:2) -catenin signaling in addition to controlling its level of accumulation. with Lipofectamine reagent (GIBCO BRL), and colonies were selected in G418 (800 (cid:7) g/ml). Stable clones were first isolated with cloning cylinders, expanded, and screened for protein expression by Western analysis. Positives were then subjected to one round of subcloning by limiting dilution and then examined by immunofluorescence to ensure homogeneous clonal expression. Three inde- pendent clones were selected per cadherin construct based solely on clonal expression characteristics. 3 weeks, and the resultant colonies were fixed and stained with crystal violet. Transfections were done in trip- licate for each combination of plasmids. For colony formation of drug-selected stables, to confirm that colony formation reflects the anchorage- independent and -dependent growth properties of the stable cell lines characterized in Fig. 2, 500 cells from each cadherin construct–expressing stable cell line were seeded onto a 10-cm dish in DME supplemented with 10% FBS. After (cid:2) 14 d, cells were fixed, stained, and quantified.