Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions Dmitry V. Ayollo., Irina Y. Zhitnyak . , Jury M. Vasiliev, Natalya A. Gloushankova* Institute of Carcinogenesis, N.N. Blokhin Cancer Research Center of the Russian Academy of Medical Sciences, Moscow, Russia 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. Citation: Ayollo DV, Zhitnyak IY, Vasiliev JM, Gloushankova (2009) Rearrangements of the Actin Cytoskeleton and E-Cadherin–Based Adherens Junctions Caused by Neoplasic Transformation Change Cell–Cell Interactions. PLoS ONE 4(11): e8027. doi:10.1371/journal.pone.0008027 Editor: Dominik Hartl, LMU University of Munich, Germany Received August 5, 2009; Accepted November 2, 2009; Published November 30, 2009 Copyright: 2009 Ayollo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded from the Russian Foundation for Basic Research (Grant 08-04-00408a). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: natglu@hotmail.com . These authors contributed equally to this work. Introduction 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’ [1] for a phenomenon that has recently been described for neural crest cell migration in vivo [2]. 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 ectodo- mains. 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 [3]. Cell-cell adhesion is often modified during cancer progression [4]. 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 [5]. Many tumors, however, continue to express E- cadherin [6], 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. It has been shown that Rho family small GTPases, which regulate cytoskeletal dynamics [7], are involved in cadherin- dependent adhesion. In epithelial cells, inhibition of endogenous Rho or Rac decreased accumulation of E-cadherin at cell-cell contacts [8,9]. Rac1, which regulates Arp2/3-mediated actin polymerization, is associated with the newly formed cell-cell contacts [10]. Early AJs incorporate exogenous G-actin and PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e8027