In most cancers, transformation begins in a single cell in an epithelial cell sheet. However, it is not known what happens at the interface between non-transformed (normal) and transformed cells once the initial transformation has occurred. Using Madin-Darby canine kidney (MDCK) epithelial cells that express constitutively active, oncogenic Ras (Ras(V12)) in a tetracycline-inducible system, we investigated the cellular processes arising at the interface between normal and transformed cells. We show that two independent phenomena occur in a non-cell-autonomous manner: when surrounded by normal cells, Ras(V12) cells are either apically extruded from the monolayer, or form dynamic basal protrusions and invade the basal matrix. Neither apical extrusion nor basal protrusion formation is observed when Ras(V12) cells are surrounded by other Ras(V12) cells. We show that Cdc42 and ROCK (also known as Rho kinase) have vital roles in these processes. We also demonstrate that E-cadherin knockdown in normal cells surrounding Ras(V12) cells reduces the frequency of apical extrusion, while promoting basal protrusion formation and invasion. These results indicate that Ras(V12)-transformed cells are able to recognize differences between normal and transformed cells, and consequently leave epithelial sheets either apically or basally, in a cell-context-dependent manner.

SummaryAt the initial stage of carcinogenesis, transformation occurs in a single cell within an epithelial sheet. However, it remains unknown what happens at the boundary between normal and transformed cells. Using Madin-Darby canine kidney (MDCK) cells transformed with temperature-sensitive v-Src, we have examined the interface between normal and Src-transformed epithelial cells. We show that Src-transformed cells are apically extruded when surrounded by normal cells, but not when Src cells alone are cultured, suggesting that apical extrusion occurs in a cell-context-dependent manner. We also observe apical extrusion of Src-transformed cells in the enveloping layer of zebrafish gastrula embryos. When Src-transformed MDCK cells are surrounded by normal MDCK cells, myosin-II and focal adhesion kinase (FAK) are activated in Src cells, which further activate downstream mitogen-activated protein kinase (MAPK). Importantly, activation of these signalling pathways depends on the presence of surrounding normal cells and plays a crucial role in apical extrusion of Src cells. Collectively, these results indicate that interaction with surrounding normal epithelial cells influences the signalling pathways and behaviour of Src-transformed cells.

Recent studies have shown that certain types of transformed cells are extruded from an epithelial monolayer. However, it is not known whether and how neighbouring normal cells play an active role in this process. In this study, we demonstrate that filamin A and vimentin accumulate in normal cells specifically at the interface with Src-or RasV12-transformed cells. Knockdown of filamin A or vimentin in normal cells profoundly suppresses apical extrusion of the neighbouring transformed cells. In addition, we show in zebrafish embryos that filamin plays a positive role in the elimination of the transformed cells. Furthermore, the Rho/Rho kinase pathway regulates filamin accumulation and filamin acts upstream of vimentin in the apical extrusion. This is the first report demonstrating that normal epithelial cells recognize and actively eliminate neighbouring transformed cells and that filamin is a key mediator in the interaction between normal and transformed epithelial cells.

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Mahjong is a novel Lethal giant larvae-binding protein that plays a vital role in cell competition in both flies and mammals.

SummaryIn Drosophila, normal and transformed cells compete with each other for survival in a process called cell competition. However, it is not known whether comparable phenomena also occur in mammals. Scribble is a tumor suppressor protein in Drosophila and mammals. In this study we examine the interface between normal and Scribble-knockdown epithelial cells using Madin-Darby Canine Kidney (MDCK) cells expressing Scribble short hairpin RNA (shRNA) in a tetracycline-inducible manner. We observe that Scribbleknockdown cells undergo apoptosis and are apically extruded from the epithelium when surrounded by normal cells. Apoptosis does not occur when Scribble-knockdown cells are cultured alone, suggesting that the presence of surrounding normal cells induces the cell death. We also show that death of Scribble-knockdown cells occurs independently of apical extrusion. Finally, we demonstrate that apoptosis of Scribble-knockdown cells depends on activation of p38 mitogen-activated protein kinase (MAPK). This is the first demonstration that an oncogenic transformation within an epithelium induces cell competition in a mammalian cell culture system.

Cell-cell contacts play a vital role in intracellular signaling, although the molecular mechanisms of these signaling pathways are not fully understood. E-cadherin, an important mediator of cell-cell adhesions, has been shown to be cleaved by ␥-secretase. This cleavage releases a fragment of E-cadherin, E-cadherin C-terminal fragment 2 (E-cad/CTF2), into the cytosol. Here, we study the fate and function of this fragment. First, we show that coexpression of the cadherin-binding protein, p120 catenin (p120), enhances the nuclear translocation of E-cad/CTF2. By knocking down p120 with short interfering RNA, we also demonstrate that p120 is necessary for the nuclear localization of E-cad/CTF2. Furthermore, p120 enhances and is required for the specific binding of E-cad/CTF2 to DNA. Finally, we show that E-cad/CTF2 can regulate the p120-Kaiso-mediated signaling pathway in the nucleus. These data indicate a novel role for cleaved E-cadherin in the nucleus.In multicellular organisms, individual cells are often connected with each other via cell-cell adhesions to form threedimensionally structured tissues or organs. Formation of tight and compact cell-cell adhesions suppresses free cell movement and provides cells with a positional cue for the establishment of cell polarity. In addition to the structural roles, it has been long known that cell-cell contacts play an important role in various signal transduction pathways (1, 2). In other words, through cell-cell contacts, cells can exchange a variety of information with their neighbors to behave properly in their community.Cadherins are a family of transmembrane cell-cell adhesion proteins that can be subdivided into several groups including classical cadherins and protocadherins (3). Classical cadherins, of which E-cadherin is the best characterized, play a crucial role in mediating cell-cell contacts at adherens junctions. The extracellular domains of classical cadherins form homophilic ligations. The cytoplasmic domain of cadherin includes two cadherin homology (CH) 2 domains: CH2 domain (located at the membrane proximal region) and CH3 domain (located at the distal region). These domains are conserved between classical cadherins. The CH2 and CH3 domains of cadherins interact with p120-catenin (p120) and ␤-catenin, respectively (4, 5). Furthermore, the CH2 domain of E-cadherin also interacts with Hakai (6, 7).Cadherins, especially classical cadherins, have been shown to be involved in several signaling pathways regulating cell proliferation, differentiation, and survival (8 -11). However, the molecular mechanisms whereby cadherins regulate varied cellular processes are not fully understood. The cytoplasmic domain of cadherins does not possess any intrinsic enzymatic activity that could directly mediate signaling pathways in the cytosol. However, cadherin-binding proteins, especially ␤-catenin and p120, have been shown to localize in the nucleus and play a key role in signal transduction. The crucial role of ␤-catenin in the canonical Wnt signaling pathway has been well...

Immunoglobulin-like Necl-5/Tage4/poliovirus receptor (PVR)/CD155, originally identified as the PVR, has been shown to be up-regulated in cancer cells and to enhance growth factor–induced cell movement and proliferation. In addition, Necl-5 heterophilically trans-interacts with nectin-3, a cell–cell adhesion molecule known to form adherens junctions in cooperation with cadherin. We show here that Necl-5 was down-regulated from cell surface upon cell–cell contacts in NIH3T3 cells. This down-regulation of Necl-5 was initiated by its interaction with nectin-3 and was mainly mediated by clathrin-dependent endocytosis. Then, the down-regulation of Necl-5 induced in this way reduced movement and proliferation of NIH3T3 cells. These results indicate that the down-regulation of Necl-5 induced by its interaction with nectin-3 upon cell–cell contacts may be at least one mechanism underlying contact inhibition of cell movement and proliferation.