РЕОРГАНИЗАЦИЯ МЕЖКЛЕТОЧНЫХ АДГЕЗИОННЫХ КОНТАКТОВ И ПОЯВЛЕНИЕ МИГРАЦИОННОЙ АКТИВНОСТИ У КЛЕТОК MCF-7-SNAI1 ПРИ ИНДУКЦИИ ЭПИТЕЛИАЛЬНО-МЕЗЕНХИМАЛЬНОГО ПЕРЕХОДА

Using DIC and confocal microscopy, changes in morphology, migratory characteristics and adherence junctions (AJs) were analyzed in the mammary carcinoma cell line MCF-7-SNAI1  after activation of the EMT transcription factor SNAI1. Western Blot analysis showed that  after removal of tetracycline from the cell culture medium expression of SNAI1 reached its  peak in 24 hours and then plateaued for 7 days. During the 7 days the cells continued to  express E-cadherin; however, tangential AJs typical for cells with stable cell-cell adhesion,  changed into radial AJs. The radial AJs continued to accumulate E-cadherin during 24‑72  hours after tetracycline removal. As a result of SNAI1 activation, the cells underwent  epithelial-mesenchymal transition (EMT) and became migratory. On a two-dimensional  substrate, cells exhibited both individual and collective migration. As the tetracycline  washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial  monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal  phenotype and accumulation of E-cadherin in AJs during early stages of EMT do not impede  disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity.С помощью DIC-микроскопии и конфокальной микроскопии были проанализированы изменения морфологии,  миграционных характеристик и межклеточных адгезионных контактов в культуре клеток рака молочной железы MCF-7-SNAI1 при активации экспрессии транскрипционного фактора ЭМП – SNAI1. Как показал Вестерн-блот  анализ, экспрессия SNAI1 достигала максимальных значений через 24 часа после переноса клеток в среду без  тетрациклина и поддерживалась на этом уровне в течение семи дней. В клетках в течение семи дней  сохранялась экспрессия Е-кадхерина, при этом тангенциальные межклеточные адгезионные контакты,  характерные для клеток со стабильной межклеточной адгезией, замещались радиальными контактами. В  радиальных контактах в течение 24–72 часов отмывки от тетрациклина продолжалась аккумуляция Е- кадхерина. В результате активации SNAI1 клетки вступали в ЭМП и приобретали миграционную активность. На  двумерном субстрате клетки мигрировали как индивидуально, так и коллективно. С увеличением  продолжительности отмывки от тетрациклина повышался процент клеток, мигрировавших через поры в  миграционных камерах, способность клеток инвазировать эпителиальный монослой, напротив, снижалась.  Полученные данные свидетельствуют о том, что сохранение гибридного эпителиально-мезенхимального  фенотипа и аккумуляция Е-кадхерина в межклеточных адгезионных контактах на ранних этапах ЭМП не  препятствуют разрушению стабильной межклеточной адгезии и приобретению клетками миграционной активности

Роль Е-кадхерина в неопластической эволюции эпителиальных клеток

Interaction of the extracellular domains of the transmembrane proteins cadherins provides cell-cell adhesion. For many years epithelial E- cadherin was regarded as a tumor suppressor and was used as a prognostic marker in cancer. Suppression of E-cadherin expression was observed in many carcinomas. During recent years, the tumor suppressor function of E-cadherin is being reconsidered. It has been shown that ductal breast carcinomas, colorectal carcinomas, oral cavity carcinomas, and squamous cell carcinomas of the head and neck can retain E-cadherin expression. Immunohistochemical staining with a panel of monoclonal antibodies revealed membrane localization of E-cadherin in many tumors. It was shown that transformed epithelial cells in vitro form dynamic adherens junctions that are essential for the effective collective migration of these cells.Трансмембранные белки кадхерины обеспечивают межклеточную адгезию через взаимодействие внеклеточных доменов. На протяжении многих лет эпителиальный Е-кадхерин считался опухолевым супрессором и рассматривался в качестве прогностического маркера у онкологических больных. Угнетение экспрессии Е-кадхерина наблюдали во многих карциномах. В последние несколько лет пересматриваются представления о супрессирующей оли Е-кадхерина. Показано, что протоковые карциномы молочной железы, карциномы толстой кишки, карциномы полости рта, плоскоклеточные карциномы головы и шеи могут сохранять экспрессию Е-кадхерина. При иммуногистохимическом окрашивании с использованием панели моноклональных антител во многих опухолях была выявлена мембранная локализация Е-кадхерина. В трансформированных эпителиоцитах in vitro были обнаружены динамичные межклеточные адгезионные контакты, образованные Е-кадхерином, которые важны для эффективной коллективной миграции клеток

A Novel Pathway of TEF Regulation Mediated by MicroRNA-125b Contributes to the Control of Actin Distribution and Cell Shape in Fibroblasts

BACKGROUND: Thyrotroph embryonic factor (TEF), a member of the PAR bZIP family of transcriptional regulators, has been involved in neurotransmitter homeostasis, amino acid metabolism, and regulation of apoptotic proteins. In spite of its relevance, nothing is known about the regulation of TEF. PRINCIPAL FINDINGS: p53-dependent genotoxic agents have been shown to be much more harmful for PAR bZIP-deficient mice as compared to wild type animals. Here we demonstrate that TEF expression is controlled by p53 through upregulation of microRNA-125b, as determined by both regulating the activity of p53 and transfecting cells with microRNA-125b precursors. We also describe a novel role for TEF in controlling actin distribution and cell shape in mouse fibroblasts. Lack of TEF is accompanied by dramatic increase of cell area and decrease of elongation (bipolarity) and dispersion (multipolarity). Staining of actin cytoskeleton also showed that TEF (-/-) cells are characterized by appearance of circumferential actin bundles and disappearance of straight fibers. Interestingly, transfection of TEF (-/-) fibroblasts with TEF induced a wild type-like phenotype. Consistent with our previous findings, transfection of wild type fibroblasts with miR-125b promoted a TEF (-/-)-like phenotype, and a similar but weaker effect was observed following exogenous expression of p53. CONCLUSIONS/SIGNIFICANCE: These findings provide the first evidence of TEF regulation, through a miR-125b-mediated pathway, and describes a novel role of TEF in the maintenance of cell shape in fibroblasts

Mechanisms and in vivo functions of contact inhibition of locomotion

Contact inhibition of locomotion (CIL) is a process whereby a cell ceases motility or changes its trajectory upon collision with another cell. CIL was initially characterized more than half a century ago and became a widely studied model system to understand how cells migrate and dynamically interact. Although CIL fell from interest for several decades, the scientific community has recently rediscovered this process. We are now beginning to understand the precise steps of this complex behaviour and to elucidate its regulatory components, including receptors, polarity proteins and cytoskeletal elements. Furthermore, this process is no longer just in vitro phenomenology; we now know from several different in vivo models that CIL is essential for embryogenesis and in governing behaviours such as cell dispersion, boundary formation and collective cell migration. In addition, changes in CIL responses have been associated with other physiological processes, such as cancer cell dissemination during metastasis

INDUCTION OF EPITHELIAL-TO-MESENCHYMAL TRANSITION IN MCF-7-SNAI1 CELLS LEADS TO REORGANIZATION OF ADHERENS JUNCTIONS AND ACQUISITION OF MIGRATORY ACTIVITY

Using DIC and confocal microscopy, changes in morphology, migratory characteristics and adherence junctions (AJs) were analyzed in the mammary carcinoma cell line MCF-7-SNAI1  after activation of the EMT transcription factor SNAI1. Western Blot analysis showed that  after removal of tetracycline from the cell culture medium expression of SNAI1 reached its  peak in 24 hours and then plateaued for 7 days. During the 7 days the cells continued to  express E-cadherin; however, tangential AJs typical for cells with stable cell-cell adhesion,  changed into radial AJs. The radial AJs continued to accumulate E-cadherin during 24‑72  hours after tetracycline removal. As a result of SNAI1 activation, the cells underwent  epithelial-mesenchymal transition (EMT) and became migratory. On a two-dimensional  substrate, cells exhibited both individual and collective migration. As the tetracycline  washout period progressed, the fraction of the cells capable of migrating through migration chamber membranes increased; on the contrary, cells’ ability to invade an epithelial  monolayer decreased. These results demonstrate that retaining a hybrid epithelial/mesenchymal  phenotype and accumulation of E-cadherin in AJs during early stages of EMT do not impede  disruption of stable cell-cell adhesion and cells’ acquisition of migratory activity

Phenotypic Plasticity of Cancer Cells Based on Remodeling of the Actin Cytoskeleton and Adhesive Structures

There is ample evidence that, instead of a binary switch, epithelial-mesenchymal transition (EMT) in cancer results in a flexible array of phenotypes, each one uniquely suited to a stage in the invasion-metastasis cascade. The phenotypic plasticity of epithelium-derived cancer cells gives them an edge in surviving and thriving in alien environments. This review describes in detail the actin cytoskeleton and E-cadherin-based adherens junction rearrangements that cancer cells need to implement in order to achieve the advantageous epithelial/mesenchymal phenotype and plasticity of migratory phenotypes that can arise from partial EMT

Normal and transformed IAR epithelial cells.

<p>(A) Single IAR-2 epithelial cells are discoid, they form islands in sparse culture and a confluent monolayer in dense culture. Scale bar 10 μm. (B) E-cadherin and N-cadherin expression in normal and transformed IAR cells. (C) IAR-2 cells were stained for E-cadherin (green) and actin (red). Top row (1.25 μm above the substrate) shows AJs organized as adhesion belts encircling each cell and co-localizing with circumferential actin bundles in the apical parts of cells. Bottom row (substrate level) shows parts of adhesion belts and irregular distributions of actin. Scale bar 10 μm. (D) Transformed IAR-6-1 cells were stained for E-cadherin (green) and actin (red). In these cells, radial AJs were associated with thin actin bundles. Scale bar 10 μm.</p

Transformed IAR-6-1 cells form E-cadherin-based AJs with underlying normal IAR-2 cells.

<p>GFP-E-cadherin-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A-B) Immunofluorescent staining for GFP. (A) E-cadherin accumulates in dot-like adhesions at the leading edge and in prominent AJs encircling the IAR-6-1 cell. Left—green channel. Boxed region is enlarged. Arrowhead indicates dot-like adhesions. Right—green and red channels. Dotted line indicates the position of the Y-projection. Scale bar 10 μm. (B) Y-projection. Arrows mark lateral AJs between IAR-6-1 and IAR-2 cells. (C) Selected confocal slices from time lapse Z-stacks (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133578#pone.0133578.s007" target="_blank">S4 Video</a>). The green channel is a “Z- projection” of all three slices in a confocal Z-stack, the red channel is the top slice. Asterisks indicate lateral AJs. Scale bar 10 μm. (D) A close-up view of the boxed region from (A). “Z-projection” of the green channel of the same video. At the leading edge of the IAR-6-1 cell, transient E-cadherin-based AJs are formed and quickly disassembled. Arrowheads mark spots where diffuse E-cadherin accumulates into dot-like adhesions, asterisks mark persisting E-cadherin dots, and arrows indicate disappearance of the dots. Scale bar 5 μm.</p

Transformed IAR-6-1 epithelial cells migrate over the monolayer of normal IAR-2 epithelial cells.

<p>EGFP-expressing IAR-6-1 cells were seeded onto the confluent monolayer of mKate2-expressing IAR-2 cells. (A) A scheme of experimental design used in the present study: a glass bottom culture dish with a confluent IAR-2 monolayer (red) and transformed IAR cells (green) seeded sparsely onto the monolayer. (B) Selected frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133578#pone.0133578.s005" target="_blank">S2 Video</a> with combined DIC and green channels. Asterisks indicate migration of an elongated fibroblast-like cell. Scale bar 40 μm. (C) Selected frames from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133578#pone.0133578.s006" target="_blank">S3 Video</a> with combined red and green channels of the top confocal slices out of time lapse Z-stacks. A corresponding 450-min track (1 point/15 min) of the migrating IAR-6-1 cell is shown on Frame 6. Scale bar 20 μm.</p

A Novel Role of E-Cadherin-Based Adherens Junctions in Neoplastic Cell Dissemination

<div><p>Using confocal microscopy, we analyzed the behavior of IAR-6-1, IAR1170, and IAR1162 transformed epithelial cells seeded onto the confluent monolayer of normal IAR-2 epithelial cells. Live-cell imaging of neoplastic cells stably expressing EGFP and of normal epithelial cells stably expressing mKate2 showed that transformed cells retaining expression of E-cadherin were able to migrate over the IAR-2 epithelial monolayer and invade the monolayer. Transformed IAR cells invaded the IAR-2 monolayer at the boundaries between normal cells. Studying interactions of IAR-6-1 transformed cells stably expressing GFP-E-cadherin with the IAR-2 epithelial monolayer, we found that IAR-6-1 cells established E-cadherin-based adhesions with normal epithelial cells: dot-like dynamic E-cadherin-based adhesions in protrusions and large adherens junctions at the cell sides and rear. A comparative study of a panel of transformed IAR cells that differ by their ability to form E-cadherin-based AJs, either through loss of E-cadherin expression or through expression of a dominant negative E-cadherin mutant, demonstrated that E-cadherin-based AJs are key mediators of the interactions between neoplastic and normal epithelial cells. IAR-6-1DNE cells expressing a dominant-negative mutant form of E-cadherin with the mutation in the first extracellular domain practically lost the ability to adhere to IAR-2 cells and invade the IAR-2 epithelial monolayer. The ability of cancer cells to form E-cadherin-based AJs with the surrounding normal epithelial cells may play an important role in driving cancer cell dissemination in the body.</p></div