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The Glue Signals Too
The initial observations were made with endogenous E-cadherin and RTKs in the epithelial Madin-Darby canine kidney (MDCK) cell line, which is widely used to explore epithelial cell physiology. When the cells were confluent, the density-dependent inhibition of their growth was correlated with a strong inhibition of ligand-dependent RTK activation. The RTK inhibition was found to have resulted from a severe reduction in the binding affinity of RTK ligands, as determined by Scatchard analysis, without a concomitant reduction in the number of binding sites or a reduction in the surface accessibility of the RTKs. The inhibition of ligand binding and of RTK activation was E-cadherin dependent, as the activity was specifically abrogated by a neutralizing E-cadherin antibody. Consistent with the inhibition being attributable primarily to impaired ligand-receptor interaction, E-cadherin did not regulate a mutationally activated neu oncoprotein whose constitutive activity is ligand independent. The negative regulation did not extend to G-protein–coupled receptors (GPCR), since the ligand-dependent activation of two classes of GPCR—lysophosphatidic acid (LPA) and muscarinic receptors—was not regulated by E-cadherin. The findings in MDCK cells were extended to two human tumor cell lines—a melanoma line and a breast cancer line—that did not express endogenous E-cadherin (Figure 1). When E-cadherin was transfected into these lines, it inhibited their anchorage-independent growth and conferred ligand-dependent RTK regulation similar to that observed in confluent MDCK cells, whereas ligand-dependent GPCR activation was not regulated by the E-cadherin. RTK regulation by E-cadherin was associated with the colocalization of both classes of protein to the basolateral region of polarized cells. Furthermore, E-cadherin was found to form a complex with both EGFR and IGF-1R in MDCK cells, as determined by immunoprecipitation and Western blotting. The cytoplasmic portion of E-cadherin possesses two well-characterized sites for noncovalent binding to heterologous proteins: a C-terminal domain that binds beta-catenin and a more proximal domain that binds p120-catenin. When E-cadherin mutants deficient for binding either beta-catenin or p120-catenin were transfected into human embryo kidney 293 (HEK293) cells that stably express EGFR, E-cadherin-EGFR complexes were formed with the mutants as efficiently as with wild-type E-cadherin, and both mutants retained some adhesion activity and the ability to negatively regulate RTKs. By contrast, an E-cadherin mutant lacking most of the extracellular domain did not form complexes with EGFR, did not have adhesive activity, and did not regulate RTKs. Complex formation between EGFR and E-cadherin was specific, in that complexes were not detected between EGFR and N-cadherin, or between E-cadherin and a GPCR muscarinic receptor. Together, these findings indicate that the interaction between E-cadherin and EGFR is specific and requires the extracellular domain of E-cadherin. The interaction between E-cadherin and EGFR is not, however, mediated by the interaction between E-cadherin and its two well-recognized binding partners, beta-catenin and p120-catenin. Furthermore, it is independent of the sites in E-cadherin to which these proteins bind. We can therefore infer that the interaction between E-cadherin and EGFR involves a site in E-cadherin not previously identified as mediating interactions with heterologous proteins. We conclude that negative regulation of RTKs by E-cadherin is a physiologic adhesion-dependent activity that results from a reduction in the efficiency of RTK ligand binding. The activity is abrogated in tumors when E-cadherin is downregulated. It can be restored when E-cadherin is reexpressed. The pleiotropic inhibitory effects of E-cadherin imply that restoration of its expression in cancers in which it has been silenced may represent a potent means for reversing the malignant phenotype. |
-cadherin
is a tumor suppressor whose expression is frequently silenced in
human cancers (Hajra KM et al. Genes Chromosomes Cancer 34:
255–68, 2002). In normal cells, E-cadherin maintains the integrity
of virtually all epithelial tissues through its ability to form
cell-cell adhesions via interactions between E-cadherin molecules
on the surface of adjacent cells.
E-cadherin may also regulate signaling, but no adhesion-dependent
signaling activity had previously been clearly identified: We have
now made the unexpected observation that E-cadherin can negatively
regulate the activation of several classes of receptor tyrosine
kinases (RTK), including epidermal growth factor receptor (EGFR)/neu,
insulin-like growth factor-1 receptor (IGF-1R), and hepatocyte growth
factor (HGF) receptor
(c-Met). This adhesion-dependent activity represents a new function
for E-cadherin. As the activities of many RTKs have been implicated
in the pathogenesis of a wide range of cancers, loss of this form
of negative RTK regulation may account,
at least in part, for the frequent selection of silenced E-cadherin
expression in cancer.
