Poorly Differentiated Colon Carcinoma Cell Lines Deficient in α-Catenin Expression Express High Levels of Surface E-cadherin but Lack Ca2+-Dependent Cell-Cell Adhesion

Studies on several different types of carcinomas, with the notable exception of colon carcinoma, have shown that poorly differentiated tumors are frequently deficient in E-cadherin dependent cell-cell adhesion. In this study, we examined Ca²⁺-dependent cell-cell adhesion in colon carcinoma cell lines. Five poorly differentiated (Clone A, MIP 101, RKO, CCL 222, CCL 228) and four moderately-well differentiated (CX-1, CCL 235, DLD-2, CCL 187) colon carcinoma cell lines were assayed for their ability to form cell-cell aggregates and for their levels of E-cadherin expression. All of the poorly differentiated cell lines exhibited low levels of Ca²⁺-dependent cell-cell aggregation, in contrast to the moderately-well differentiated cell lines. Contrary to most previous studies, however, we observed that three of the five poorly differentiated cell lines examined expressed E-cadherin by FACS analysis and immunoprecipitation using an E-cadherin mAb. In fact, two of these cell lines expressed a 3- to 4-fold higher level of E-cadherin than that found in the moderately-well differentiated cell lines. mRNA levels for E-cadherin, as evaluated by both RT-PCR and Northern hybridization, corresponded to the levels of protein expression in each of the cell lines. Immunoprecipitation with an E-cadherin mAb, which is known to co-precipitate the catenins, demonstrated that the three poorly differentiated cell lines expressing E-cadherin did not co-precipitate α-catenin, although all of the moderately-well differentiated cell lines expressed both α- and β-catenin. RT-PCR confirmed the absence of the α-catenin mRNA from two of these cell lines. Stable expression of an α-catenin cDNA in one of the poorly differentiated cell lines lacking α-catenin expression resulted in a 5-fold increase in its level of Ca²⁺-dependent cell-cell aggregation, providing evidence that α-catenin is directly responsible for the loss of cell-cell adhesion in some cell lines. The α-catenin transfectants also exhibited a marked reduction in migration on collagen I. These data indicate that loss of α-catenin expression, as well as E-cadherin expression, can lead to a phenotype associated with poorly differentiated colon carcinomas.     

Adherens junctions in the Drosophila embryo: The role of E-cadherin in their establishment and morphogenetic function

The integrity of epithelia depends largely on specialised adhesive structures, the adherens junctions. Several of the components required for building these structures are highly conserved between vertebrates and insects (e.g. E-cadherin and α- and β-catenin), while others have so far been found only in invertebrates (e.g. crumbs). Two recent papers^(1,2) show that the Drosophila E-cadherin is encoded by the gene shotgun. Phenotypic analyses of shotgun as well as armadillo (β-catenin) and crumbs mutants provide new insights into the mechanisms by which adherens junctions are built and, further, show that the requirement for E-cadherin largely depends on the morphogenetic activity of an epithelium.     

Unglycosylation at Asn-633 made extracellular domain of E-cadherin folded incorrectly and arrested in endoplasmic reticulum, then sequentially degraded by ERAD

The human E-cadherin is a single transmembrane domain protein involved in Ca²⁺-dependent cell–cell adhesion. In a previous study, we demonstrated that all of four potential N-glycosylation sites in E-cadherin are occupied by N-glycans in human breast carcinoma cells in vivo and the elimination of N-glycan at Asn-633 dramatically affected E-cadherin expression and made it degraded. In this study we investigated the molecular mechanism of E-cadherin, which lacks N-glycosylation at Asn-633 (M4), degradation and the role of the N-glycan at Asn-633 in E-cadherin folding. We treated cells stably expressed M4 E-cadherin with MG123, DMM, respectively. Either MG132 or DMM could efficiently block degradation of M4 E-cadherin. M4 E-cadherin was recognized as the substrate of ERAD and was retro-translocated from ER lumen to cytoplasm by p97. It was observed that the ration of M4 E-cadherin binding to calnexin was significantly increased compared with that of other variants, suggesting that it was a misfolded protein, though cytoplasmic domain of M4 E-cadherin could associate with β-catenin. Furthermore, we found that N-glycans of M4 E-cadherin were modified in immature high mannose type, suggesting that it could not depart to Golgi apparatus. In conclusion, this study revealed that N-glycosylation at Asn-633 is essential for E-cadherin expression, folding and trafficking.     

The Expression of the Zonula Adhaerens Protein PLEKHA7 Is Strongly Decreased in High Grade Ductal and Lobular Breast Carcinomas

PLEKHA7 is a junctional protein, which participates in a complex that stabilizes E-cadherin at the zonula adhaerens. Since E-cadherin is involved in epithelial morphogenesis, signaling, and tumor progression, we explored PLEKHA7 expression in cancer. PLEKHA7 expression was assessed in invasive ductal and lobular carcinomas of the breast by immunohistochemistry, immunofluorescence and quantitative RT-PCR. PLEKHA7 was detected at epithelial junctions of normal mammary ducts and lobules, and of tubular and micropapillary structures within G1 and G2 ductal carcinomas. At these junctions, the localization of PLEKHA7 was along the circumferential belt (zonula adhaerens), and only partially overlapping with that of E-cadherin, p120ctn and ZO-1, as shown previously in rodent tissues. PLEKHA7 immunolabeling was strongly decreased in G3 ductal carcinomas and undetectable in lobular carcinomas. PLEKHA7 mRNA was detected in both ductal and lobular carcinomas, with no observed correlation between mRNA levels and tumor type or grade. In summary, PLEKHA7 is a junctional marker of epithelial cells within tubular structures both in normal breast tissue and ductal carcinomas, and since PLEKHA7 protein but not mRNA expression is strongly decreased or lost in high grade ductal carcinomas and in lobular carcinomas, loss of PLEKHA7 is a newly characterized feature of these carcinomas.     

E-cadherin and adherens-junctions stability in gastric carcinoma: Functional implications of glycosyltransferases involving N-glycan branching biosynthesis,N-acetylglucosaminyltransferases III and V

Background

E-cadherin is a cell–cell adhesion molecule and the dysfunction of which is a common feature of more than 70% of all invasive carcinomas, including gastric cancer. Mechanisms behind the loss of E-cadherin function in gastric carcinomas include mutations and silencing at either the DNA or RNA level. Nevertheless, in a high percentage of gastric carcinoma cases displaying E-cadherin dysfunction, the mechanism responsible for E-cadherin dysregulation is unknown. We have previously demonstrated the existence of a bi-directional cross-talk between E-cadherin and two major N-glycan processing enzymes, N-acetylglucosaminyltransferase-III or -V (GnT-III or GnT-V).

Methods

In the present study, we have characterized the functional implications of the N-glycans catalyzed by GnT-III and GnT-V on the regulation of E-cadherin biological functions and in the molecular assembly and stability of adherens-junctions in a gastric cancer model. The results were validated in human gastric carcinoma samples.

Results

We demonstrated that GnT-III induced a stabilizing effect on E-cadherin at the cell membrane by inducing a delay in the turnover rate of the protein, contributing for the formation of stable and functional adherens-junctions, and further preventing clathrin-dependent E-cadherin endocytosis. Conversely, GnT-V promotes the destabilization of E-cadherin, leading to its mislocalization and unstable adherens-junctions with impairment of cell–cell adhesion.

Conclusions

This supports the role of GnT-III on E-cadherin-mediated tumor suppression, and GnT-V on E-cadherin-mediated tumor invasion.

General significance

These results contribute to fill the gap of knowledge of those human carcinoma cases harboring E-cadherin dysfunction, opening new insights into the molecular mechanisms underlying E-cadherin regulation in gastric cancer with potential translational clinical applications.     

Concomitant Loss of p120-Catenin and β-Catenin Membrane Expression and Oral Carcinoma Progression with E-Cadherin Reduction

The binding of p120-catenin and β-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and β-catenin in oral carcinomas (n = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, P < 0.05; β-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; β-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; β-catenin, P < 0.01). Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-β, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-β with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of β-catenin fine-tune the carcinoma progression.     

The promoting effect of retinoic acid on proliferation of chicken primordial germ cells by increased expression of cadherin and catenins

Proliferation and cellular aggregation are both crucial features for survival and self-renewal of primordial germ cells (PGCs). Adhesive proteins play pivotal roles in cell–cell adhesion and signal exchanges under the influence of cytokines, growth factors and bioactive metabolites such as retinoic acid (RA). In this study, proliferation-promoting effect of RA on chicken PGCs was investigated by revealing changes in adhesive proteins E-cadherin and α/β catenins. PGCs were isolated from the genital ridge of 4-day-old chicken embryos and cultured on embryonic fibroblast feeder. RA (10⁻⁷–10⁻⁵ M) increased PGCs aggregation and mRNA expression of E-cadherin and α/β-catenins. Furthermore, E-cadherin and β-catenin protein expression levels were increased by RA treatment. However, RA-elicited effect was significantly attenuated by a PKC inhibitor H₇. In addition, the number and area of PGC colonies were increased by RA treatment at 10⁻⁷–10⁻⁵ M. Again, this increase was reduced by combined treatment of H₇. The proliferating effect of RA on PGCs was further confirmed by increased mRNA expression of cyclins, CCND1 and CCNE1, and cyclin-dependent kinases 6 and 2, which are critical for G1–S progression in cell cycle. Moreover, flow cytometry analysis confirmed that RA-treated PGC populations displayed a significant increase in the proportion of S and G2 phase cells. Likewise, this stimulating action was hindered by combined H₇ treatment. These results indicate that RA, as a bioactive metabolite of vitamin A, may promote PGC proliferation and increase intercellular aggregation of PGCs via E-cadherin and α/β-catenins expression through the PKC signaling pathway.     

Membrane microdomains from early gastrula embryos of medaka, <Emphasis Type="Italic">Oryzias latipes</Emphasis>, are a platform of E-cadherin- and carbohydrate-mediated cell–cell interactions during epiboly

Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848–853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (β-catenin), transducer proteins (PLCγ, cSrc), and a cytoskeleton protein (β-actin) were enriched in the membrane microdomain. Le^X-containing glycolipids and glycoproteins (Le^X-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca²⁺. This membrane microdomain binding was achieved through the E-cadherin homophilic and the Le^X-glycan-mediated interactions. E-cadherin and Le^X-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and Le^X-glycan-mediated cell adhesion and signal transduction.

Allosteric Regulation of E-Cadherin Adhesion

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120^ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120^ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120^ctn dephosphorylation.     

Estradiol induces E-cadherin degradation in mouse uterine epithelium during the estrous cycle and early pregnancy

Mouse uterine epithelium is a tissue that undergoes cyclic endocrine-regulated cell dissociation and regeneration. It shows a dramatic cell loss following normal estrus. If pregnancy ensues, cell loss is averted during the first 2.5–3.5 days. However, this is followed by a precipitous loss of basal-lateral cell adhesion and apoptosis in preparation for blastocyst invasion. By comparing epithelia isolated by protease treatment, we show that a reduction of lateral cell adhesion is a primary event in these instances of normal tissue loss. It was readily induced in ovariectomized adult and immature mice by injections of estradiol (E₂), and to some extent also by progesterone (P₄). The reduction of lateral adhesion induced by including ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in the isolation medium mimicked and was additive to the effect of E₂ injection. However, the E₂ effect was different in not being prevented by adding Ca²⁺. The E₂ effect also was mimicked by the action on isolated epithelium of monoclonal antibody against the calcium-dependent cell adhesion molecule, E-cadherin, suggesting that inactivation of E-cadherin was induced by E₂. In detergent extracts of estrous and metestrous epithelium there was an increase in 80-kDa extracellular domain of E-cadherin relative to the intact 120-kDa molecule. The loss of adhesion between 3.5 and 4.5 days of pregnancy was associated with a loss of both intact membrane-associated 120-kDa E-cadherin and cleavage products. Cleavage of 80-kDa E-cadherin was uniquely induced by E₂ in ovariectomized adult and immature mice; P₄ was without effect. The cleavage of E-cadherin correlated with increased basal accumulation of E-cadherin antigen in estrous and E₂-injected mice and a loss of both basal and lateral antigen at 4.5 days of pregnancy. Only the E-cadherin antigen within junctional complexes appeared unaffected. The data are consistent with the hypothesis that the cyclic and pregnancy-dependent disruption of uterine epithelial integrity are promoted by E₂-dependent modification of E-cadherin, including its extracellular cleavage. © 1996 Wiley-Liss, Inc.     

1H, 15N and 13C resonance assignments and monomeric structure of the amino-terminal extracellular domain of epithelial cadherin

Summary E-cadherin is a transmembrane protein that provides Ca²⁺-dependent cell adhesion to epithelial cells. The large majority of the ¹H, ¹⁵N, ¹³C and ¹³CO resonances of a 146-amino acid polypeptide from epithelial (E-) cadherin have been assigned using multidimensional NMR spectroscopy. The structure of the amino-terminal 100 amino acids, corresponding to the first extracellular repeat of E-cadherin [Overduin et al. (1995) Science, 267, 386–389], has been refined. The monomeric state of this isolated domain is demonstrated by light scattering and sedimentation analysis. Seven -strands and two short helices were identified by patterns of NOE cross-peaks, vicinal coupling constants and chemical shift indices. A novel structural motif termed a quasi--helix found in the crystal structure of a neural (N-) cadherin domain [Shapiro et al. (1995) Nature, 374, 327–337] is characterized in detail for the first time by NMR. Slowly exchanging amides were concentrated in the -sheet region and quasi--helix. The -barrel fold of the cadherin domain is topologically similar to the immunoglobulin fold. Comparison of this solution structure to the crystallized dimers of the N-terminal pair of E-cadherin domains [Nagar et al. (1996) Nature, 380, 360–364] and of the homologous single domain of N-cadherin reveals a conserved cadherin fold with minor structural differences, which can be accounted for by differences in metal ligation and oligomeric state.Abbreviations cad extracellular cadherin repeat - CAM cell adhesion molecule - CSI chemical shift index - DTT dithiothreitol - E-cadherin epithelial cadherin - N-cadherin neural cadherin - NOE nuclear Overhauser enhancement - PFG pulsed field gradient - rmsd root-mean-square deviation     

E-Cadherin repression increases amount of cancer stem cells in human A549 lung adenocarcinoma and stimulates tumor growth

Here we show that cancer stem cells amount in human lung adenocarcinoma cell line A549 depends on E-cadherin expression. In fact, downregulation of E-cadherin expression enhanced expression of pluripotent genes (c-MYC, NESTIN, OCT3/4 and SOX2) and enriched cell population with the cells possessing the properties of so-called ‘cancer stem cells’ via activation of Wnt/β-catenin signaling. Repression of E-cadherin also stimulated cell proliferation and migration in vitro, decreased cell amount essential for xenografts formation in nude mice, increased tumors vascularization and growth. On the other hand, E-cadherin upregulation caused opposite effects i.e. diminished the number of cancer stem cells, decreased xenograft vascularization and decelerated tumor growth. Therefore, agents restoring E-cadherin expression may be useful in anticancer therapy.     

CA125/MUC16 interacts with Src family kinases,and over-expression of its C-terminal fragment in human epithelial cancer cells reduces cell–cell adhesion

MUC16/CA125 is over-expressed in human epithelial tumors including ovarian, breast and some other carcinomas. The purpose of this study is to investigate how cell surface MUC16 is functionally involved in tumor progression, with a special focus on the role of its cytoplasmic tail. Forced expression of C-terminal MUC16 fragment (MUC16C) in epithelial cancer cells increased cell migration. We found that MUC16C directly interacted with Src family kinases (SFKs). Notably, localizations of E-cadherin and β-catenin at the cell–cell contacts were more diffuse in MUC16C transfectants compared with mock transfectants. Furthermore, MUC16C transfectants showed reduced Ca²⁺-dependent cell–cell adhesion, but the treatment of cells with PP2, a SFKs inhibitor, restored this. Because cell surface MUC16 is also associated with the E-cadherin/β-catenin complex, the over-expression of MUC16 and its interaction with SFKs may enhance SFKs-induced deregulation of E-cadherin. Thus, our results suggest a role for cell surface MUC16 in cell–cell adhesion of epithelial cancer cells.     

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.     

Binding of cadmium (Cd2+) to E-CAD1, a calcium-binding polypeptide analog of E-cadherin

Recent studies have shown that Cd²⁺ can damage the Ca²⁺-dependent junctions between renal epithelial cells in culture, and preliminary evidence suggests that this effect may involve the interaction of Cd²⁺ with E-cadherin, a Ca²⁺-dependent cell adhesion molecule that is localized at the adhering junctions of epithelial cells. To determine whether or not Cd²⁺ might bind directly to the E-cadherin molecule, we studied the binding of Cd²⁺ to E-CAD1, a recombinant, 145-residue polypeptide that corresponds to one of the extracellular Ca²⁺-binding regions of mouse E-cadherin. By using an equilibrium microdialysis technique, we were able to show that Cd²⁺ could, in fact, bind to E-CAD1. The binding was saturable, with a maximum of one Cd²⁺ binding site per E-CAD1 molecule. The apparent dissociation constant (K_D) for the binding was about 20 μM, a concentration similar to that which has been shown to disrupt the junctions between epithelial cells. Other results showed that the binding of Cd²⁺ was greatly reduced when excess Ca²⁺ was included in the dialysis solution. These results suggest that Cd²⁺ can interact with the Ca²⁺ binding regions on the E-CAD1 molecule, and they provide additional support for the hypothesis that E-cadherin might be a molecular target for Cd²⁺ toxicity.     

Intestinal adenomagenesis involves core molecular signatures of the epithelial–mesenchymal transition

The epithelial–mesenchymal transition (EMT) occurs commonly during carcinoma invasion and metastasis, but not during early tumorigenesis. Microarray data demonstrated elevation of vimentin, a mesenchymal marker, in intestinal adenomas from Apc ^Min/+ (Min) mice. We have tested the involvement of EMT in early tumorigenesis in mammalian intestines by following EMT-associated markers. Elevated vimentin RNA expression and protein production were detected within neoplastic cells in murine intestinal adenomas. Similarly, vimentin protein was detected in both adenomas and invasive adenocarcinomas of the human colon, but not in the normal colonic epithelium or in hyperplastic polyps. Expression of E-cadherin varied inversely with vimentin. In addition, the expression of fibronectin was elevated while that of E-cadherin decreased. Canonical E-cadherin suppressors, such as Snail, were not elevated in the same tumor. Elevated vimentin expression in the adenoma was not correlated with persistent Ras signaling, but was strongly correlated with reduced proliferation indices, active Wnt signaling, and TGF-β signaling, as demonstrated by its dependence on Smad3. We designate our observations of expression of only some of the canonical features of EMT as “truncated EMT”. These unexpected observations are interpreted as reflecting the involvement of a core of the EMT system during the tissue remodeling of early tumorigenesis.     

Transforming Growth Factor β1Induces Squamous Carcinoma Cell Variants with Increased Metastatic Abilities and a Disorganized Cytoskeleton

Previous studies indicated that mouse transformed keratinocytes undergo an epithelial–fibroblastic conversion when cultured in the presence of TGF-β₁. This conversion is associatedin vivowith a squamous-spindle carcinoma transition. We derived epithelioid (A6, FPA6) and spindle (B5) clonal cell variants from a squamous carcinoma cell line (PDV) after treatment with TGF-β₁. FPA6 cells were isolated from the ascites fluid of an A6-tumor-bearing mouse. FPA6 and A6 cell lines produced in nude mice mixed carcinomas with a squamous and poorly differentiated component. Both cell lines coexpressed keratins and vimentin and synthesized E-cadherin protein, although FPA6 cells cultured at early passages (FPA6-ep) had reduced levels of E-cadherin mRNA and increased synthesis of keratin K8, a marker of malignant progression. Immunofluorescence analysis revealed that FPA6-ep cells exhibited a disorganized cytoskeleton with keratins forming focal juxtanuclear aggregates and loss of F-actin stress fibers and cortical bundles, and E-cadherin was localized in the cytoplasm out of cell–cell contact areas. Sporadic cells in A6 and PDV cultures also presented those anomalous keratin structures, suggesting that FPA6 cells originated from a subpopulation of A6 tumor cells that metastasized into the peritoneal cavity. The analysis of the spontaneous and experimental metastatic potentials of the cell lines showed that epithelioid and fibroblastic cell variants had acquired metastatic abilities compared to PDV which was nonmetastatic. The FPA6-ep cell line exhibited a highly aggressive behavior, killing the animals at about 17 days after intravenous injection of the cells into athymic mice. The phenotype of FPA6-ep cells was unstable and reverted at later passages in which the normal organization of keratin and F-actin in filaments and the localization of E-cadherin at cell–cell contacts were restored. This phenotypic reversion occurred concomitantly with a reduction of the experimental metastatic potential of FPA6 cells.