Inbal Simcha

Autoregulation of E-cadherin expression by cadherin–cadherin interactions: the roles of β-catenin signaling, Slug, and MAPK

Transcriptional repression of E-cadherin, characteristic of epithelial to mesenchymal transition, is often found also during tumor cell invasion. At metastases, migratory fibroblasts sometimes revert to an epithelial phenotype, by a process involving regulation of the E-cadherin–β-catenin complex. We investigated the molecular basis of this regulation, using human colon cancer cells with aberrantly activated β-catenin signaling. Sparse cultures mimicked invasive tumor cells, displaying low levels of E-cadherin due to transcriptional repression of E-cadherin by Slug. Slug was induced by β-catenin signaling and, independently, by ERK. Dense cultures resembled a differentiated epithelium with high levels of E-cadherin and β-catenin in adherens junctions. In such cells, β-catenin signaling, ErbB-1/2 levels, and ERK activation were reduced and Slug was undetectable. Disruption of E-cadherin–mediated contacts resulted in nuclear localization and signaling by β-catenin, induction of Slug and inhibition of E-cadherin transcription, without changes in ErbB-1/2 and ERK activation. This autoregulation of E-cadherin by cell–cell adhesion involving Slug, β-catenin and ERK could be important in tumorigenesis.

Excess beta-catenin promotes accumulation of transcriptionally active p53.

β‐catenin is a multifunctional protein, acting both as a structural component of the cell adhesion machinery and as a transducer of extracellular signals. Deregulated β‐catenin protein expression, due to mutations in the β‐catenin gene itself or in its upstream regulator, the adenomatous polyposis coli (APC) gene, is prevalent in colorectal cancer and in several other tumor types, and attests to the potential oncogenic activity of this protein. Increased expression of β‐catenin is an early event in colorectal carcinogenesis, and is usually followed by a later mutational inactivation of the p53 tumor suppressor. To examine whether these two key steps in carcinogenesis are interrelated, we studied the effect of excess β‐catenin on p53. We report here that overexpression of β‐catenin results in accumulation of p53, apparently through interference with its proteolytic degradation. This effect involves both Mdm2‐dependent and ‐independent p53 degradation pathways, and is accompanied by augmented transcriptional activity of p53 in the affected cells. Increased p53 activity may provide a safeguard against oncogenic deregulation of β‐catenin, and thus impose a pressure for mutational inactivation of p53 during the later stages of tumor progression.

Prototypical Type I E-cadherin and Type II Cadherin-7 Mediate Very Distinct Adhesiveness through Their Extracellular Domains

Using a dual pipette assay that measures the force required to separate adherent cell doublets, we have quantitatively compared intercellular adhesiveness mediated by Type I (E- or N-cadherin) or Type II (cadherin-7 or -11) cadherins. At similar cadherin expression levels, cells expressing Type I cadherins adhered much more rapidly and strongly than cells expressing Type II cadherins. Using chimeric cadherins, we found that the extracellular domain exerts by far the dominant effect on cell adhesivity, that of E-cadherin conferring high adhesivity, and that of cadherin-7 conferring low adhesivity. Type I cadherins were incorporated to a greater extent into detergent-insoluble cytoskeletal complexes, and their cytoplasmic tails were much more effective in disrupting strong adherent junctions, suggesting that Type II cadherins form less stable complexes with β-catenin. The present study demonstrates compellingly, for the first time, that cadherins are dramatically different in their ability to promote intercellular adhesiveness, a finding that has profound implications for the regulation of tissue morphogenesis.

Differential interaction of plakoglobin and β-catenin with the ubiquitin-proteasome system

β-Catenin and plakoglobin are closely related armadillo family proteins with shared and distinct properties; Both are associated with cadherins in actin-containing adherens junctions. Plakoglobin is also found in desmosomes where it anchors intermediate filaments to the desmosomal plaques. β-Catenin, on the other hand, is a component of the Wnt signaling pathway, which is involved in embryonic morphogenesis and tumorigenesis. A key step in the regulation of this pathway involves modulation of β-catenin stability. A multiprotein complex, regulated by Wnt, directs the phosphorylation of β-catenin and its degradation by the ubiquitin-proteasome system. Plakoglobin can also associate with members of this complex, but inhibition of proteasomal degradation has little effect on its levels while dramatically increasing the levels of β-catenin. β-TrCP, an F-box protein of the SCF E3 ubiquitin ligase complex, was recently shown to play a role in the turnover of β-catenin. To elucidate the basis for the apparent differences in the turnover of β-catenin and plakoglobin we compared the handling of these two proteins by the ubiquitin-proteasome system. We show here that a deletion mutant of β-TrCP, lacking the F-box, can stabilize the endogenous β-catenin leading to its nuclear translocation and induction of β-catenin/LEF-1-directed transcription, without affecting the levels of plakoglobin. However, when plakoglobin was overexpressed, it readily associated with β-TrCP, efficiently competed with β-catenin for binding to β-TrCP and became polyubiquitinated. Fractionation studies revealed that about 85% of plakoglobin in 293 cells, is Triton X-100-insoluble compared to 50% of β-catenin. These results suggest that while both plakoglobin and β-catenin can comparably interact with β-TrCP and the ubiquitination system, the sequestration of plakoglobin by the membrane-cytoskeleton system renders it inaccessible to the proteolytic machinery and stabilizes it.