Date Published: November 30, 2009
Publisher: Public Library of Science
Author(s): Dmitry V. Ayollo, Irina Y. Zhitnyak, Jury M. Vasiliev, Natalya A. Gloushankova, Dominik Hartl. http://doi.org/10.1371/journal.pone.0008027
Abstract: E-cadherin–mediated cell–cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell–cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin–based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell–cell interactions, preventing contact paralysis after the establishment of cell–cell contact and promoting dynamic cell–cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.
Partial Text: Analyses of cell-cell interactions and of the establishment of cell-cell adhesion are important directions in the studies of normal morphogenesis and its pathological alterations during tumor progression. Long ago, Abercrombie coined the term ‘contact inhibition of locomotion’  for a phenomenon that has recently been described for neural crest cell migration in vivo. The nature of this phenomenon and of changes of cell-cell interactions of neoplastic cells has still not been elucidated. Classical cadherins are transmembrane proteins that mediate cell-cell adhesion through Ca+2-dependent homophilic interactions of their ectodomains. The intracellular domain of the cadherin molecule interacts with proteins of cytoplasmic plaque (catenins, vinculun) that link it to actin filaments to maintain the stability of adherens junctions (AJs). The stable E-cadherin-based AJs play a pivotal role in the integrity of the epithelium and the maintenance of tissue homeostasis . Cell-cell adhesion is often modified during cancer progression . Epithelial-mesenchymal transition (EMT), which includes the downregulation of E-cadherin expression, the disruption of cell-cell contacts, and the acquisition of an elongated fibroblast-like phenotype by epithelial cells, has been observed in many carcinoma cell lines. It has been thought for a long time that a decrease in the E-cadherin level on the cell surface in the course of EMT is a key step of the progression from adenoma to carcinoma . Many tumors, however, continue to express E-cadherin , and thus there should be other mechanisms to regulate the E-cadherin adhesive function in carcinomas. The early stages of the morphological transformation of epithelial cells, including rearrangements of the actin cytoskeleton and cell-cell interactions, have not yet been characterized.
This study is the first to demonstrate that transformed epithelial cells can use the disappearance of the marginal actin bundle and the remodeling of E-cadherin-based AJs to weaken cell-cell adhesion and to acquire motile behavior and migratory activity. When we studied transformed IAR-6-1 and IAR1170 epithelial lines, we found that while cells of these lines retain E-cadherin expression, have a morphology slightly changed from the morphology of the parent nontransformed IAR-2 cells, and form monolayers in confluent culture that are similar to IAR-2 monolayer, immunofluorescence staining demonstrated dramatic rearrangements of the actin cytoskeleton and AJs. In transformed IAR-6-1 and IAR1170 cells, the adhesion belt is broken and replaced by E-cadherin-based puncta or strands resembling radial AJs of fibroblasts. AJs in transformed epithelial cells are associated with short straight actin bundles. Our data demonstrated that in contrast with stable tangential AJs in nontransformed epithelial cells, radial E-cadherin-based AJs in transformed cells are very dynamic.