Domain Of E-cadherin Research Articles

Evaluation of the physical stability of the EC5 domain of E-cadherin: Effects of pH, temperature, ionic strength, and disulfide bonds

The development of protein drugs has been hampered by difficulties in formulating them due to their inherent chemical and physical stability, which could generate problems during the late stages of development. Thus, a basic understanding of the effect of structural features on the physicochemical stability of proteins can provide fundamental solutions to the formation of proteins. In this work, the physical stability of the EC5 protein under variable pH, temperature, and ionic strength and the role of the disulfide bond on the physical stability of EC5 were evaluated. All spectroscopic measurements were integrated in empirical phase diagrams, and these diagrams showed the stable and unstable regions of EC5. The native EC5 is more stable at high than at low ionic strength in a wide pH range during temperature elevation to 70°C. The empirical phase diagrams also show that the reduced EC5 has lower stability than the parent EC5. The reduced EC5 has secondary structure only at pH 3 and 4 and is unfolded at other pH values. Finally, the reduced EC5 rapidly forms a precipitate at pH 4 and 5 upon heating. In conclusion, this study shows that ionic strength and the presence of the disulfide bonds are critical for the stability of EC5.

Interaction of KLRG1 with E‐cadherin: New functional and structural insights

The killer cell lectin-like receptor G1 (KLRG1) is an inhibitory receptor expressed by memory T cells and NK cells in man and mice. It is frequently used as a cell differentiation marker and members of the cadherin family are ligands for KLRG1. The present study provides new insights into the interaction of mouse KLRG1 with E-cadherin. Firstly, we demonstrate that co-engagement of KLRG1 and CD3/TCR in a spatially linked manner was required for inhibition arguing against the notion that KLRG1-ligation per se transmits inhibitory signals. Secondly, experiments with T cells carrying Y(7)F-mutant KLRG1 molecules with a replacement of the tyrosine residue to phenylalanine in the single ITIM indicated that the inhibitory activity of KLRG1 is counteracted to some degree by increased interaction of KLRG1(+) T cells with E-cadherin expressing target cells. Thirdly, we demonstrate that deletion of the first or the second external domain of E-cadherin abolished reactivity in KLRG1-reporter cell assays. Finally, we made the intriguing observation that KLRG1 formed multimeric protein complexes in T cells in addition to the previously described mono- and dimeric molecules.

The first “Slit” is the deepest: the secret to a hollow heart

Tubular organs are essential for life, but lumen formation in nonepithelial tissues such as the vascular system or heart is poorly understood. Two studies in this issue (Medioni, C., M. Astier, M. Zmojdzian, K. Jagla, and M. Sémériva. 2008. J. Cell Biol. 182:249–261; Santiago-Martínez, E., N.H. Soplop, R. Patel, and S.G. Kramer. 2008. J. Cell Biol. 182:241–248) reveal unexpected roles for the Slit–Robo signaling system during Drosophila melanogaster heart morphogenesis. In cardioblasts, Slit and Robo modulate the cell shape changes and domains of E-cadherin–based adhesion that drive lumen formation. Furthermore, in contrast to the well-known paracrine role of Slit and Robo in guiding cell migrations, here Slit and Robo may act by autocrine signaling. In addition, the two groups demonstrate that heart lumen formation is even more distinct from typical epithelial tubulogenesis mechanisms because the heart lumen is bounded by membrane surfaces that have basal rather than apical attributes. As the D. melanogaster cardioblasts are thought to have significant evolutionary similarity to vertebrate endothelial and cardiac lineages, these findings are likely to provide insights into mechanisms of vertebrate heart and vascular morphogenesis.

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 Ca(2+)-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 beta-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.

A Role for the Cleaved Cytoplasmic Domain of E-cadherin in the Nucleus

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 γ-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.

KLRG1, Cadherins and their Interactions

The killer cell lectin-like receptor G1 (KLRG1) is a unique inhibitory receptor expressed on a phenotypically mature subset of resting NK cells as well as effector memory CD8+ T cells. KLRG1 is a C-type lectin inhibitory receptor that contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. It has been demonstrated that engagement of the KLRG1 molecule inhibits NK cell functions. KLRG1 ligands have been recently identified and are members of the broadly expressed cadherin family, E, N, and R-cadherin. In this study, we attempt to elucidate the consequences of cadherin engagement by KLRG1 as well as KLRG1 engagement by cadherin. We find that KLRG1 engagement by E-cadherin induces a moderate but significant inhibition of TCR signaling. In addition, we showed that recombinant extra cellular domain of E-cadherin blocks KLRG1 tetramer binding to cadherin expressing cells. Interestingly, upon analysis of E-cadherin mutants, we found that E-cadherin cell surface expression is regulated by its cytoplasmic domain. Finally, we found that E-cadherin engagement by KLRG1 influences the dynamics of E-cadherin expression. Taken together, our data suggest that immune receptor interaction with cadherin may have an impact not only on immune cells such as NK cells but also on cells expressing cadherin such as epithelial cells. Supported by NIH Grant AI58181.

Frequent accumulation of nuclear E-cadherin and alterations in the Wnt signaling pathway in esophageal squamous cell carcinomas

Esophageal squamous cell carcinoma is frequently associated with poor prognosis, as a result of high levels of lymph node metastasis. So far, very few genetic abnormalities have been associated with this disease, and its molecular etiology remains largely unknown. To assess whether the Wnt pathway contributes to esophageal squamous cell carcinoma, we characterized the expression and subcellular localization of the key Wnt signaling components in all 30 cases of esophageal squamous cell carcinomas analyzed. We found abnormal expression and/or localization in glycogen synthase kinase-3 α/β (34%), Axin2 (48%), α-catenin (31%), MYC (73%) and cyclin D1 in 46% of cases. Only 13% of tumors showed nuclear accumulation of β-catenin. By contrast, 60% showed nuclear expression of E-cadherin using an antibody that recognizes the cytoplasmic domain of E-cadherin. When the same tumors were stained with antibody raised against the extracellular domain of E-cadherin, the expression was lost. A direct correlation was found between nuclear E-cadherin and the increased nuclear cyclin D1, one of the AP-1 target genes in these tumors. By transfection experiments, the cytoplasmic portion of E-cadherin was found to activate the AP-1 transcription factor pathway and induced cyclin D1 promoter activity, but β-catenin/Tcf transcription activity was unaffected. Nuclear expression of E-cadherin was also detected in tumors other than squamous cell carcinoma, including pancreatic and colon cancers, albeit at lower frequency. Nuclear accumulation of a portion of E-cadherin in esophageal squamous cell carcinoma and the other types of tumors indicates that, in addition to the previously implicated tumor suppressor activity of E-cadherin, modified forms of this glycoprotein might also play a role in growth promotion.

Enterotoxigenic Bacteroides fragilis

Bacteroides fragilis is a minor component of the microbial flora of the intestine but the most frequent disease--causing anaerobe. Virulence characteristics are its capsule, which induces abscess formation, and the production of fragilysin, a Zn-metalloprotease. This toxin's action is to hydrolyze the extracellular domain of E-cadherin, the effect of which is to disrupt intercellular adhesion and thus increase permeability of the epithelium, causing intracellular redistribution of actin with morphologic changes to the cells and release of beta-catenin, which translocates to the nucleus and ultimately increases cellular proliferation. Clinically, enterotoxigenic B. fragilis is linked to secretory diarrhea, particularly in children. Preliminary evidence suggests that enterotoxigenic B. fragilis may also be linked to inflammatory bowel disease and colon cancer.

Membrane loss and aberrant nuclear localization of E‐cadherin are consistent features of solid pseudopapillary tumour of the pancreas. An immunohistochemical study using two antibodies recognizing different domains of the E‐cadherin molecule

To examine the expression of E-cadherin in solid pseudopapillary tumours (SPT) of the pancreas using two monoclonal antibodies recognizing two different domains of the E-cadherin molecule. Twenty cases of SPT were collected and a tissue microarray (TMA) constructed. The TMA was stained with commercially available antibodies to E-cadherin and beta-catenin. All 20 cases displayed nuclear beta-catenin as well as aberrant E-cadherin expression. With the antibody that stains the cytoplasmic domain of E-cadherin (clone 36, BD Transduction Laboratories), all 20 cases demonstrated nuclear E-cadherin reactivity, whereas with use of the antibody that recognizes the extracellular domain (clone 36B5, Vector Laboratories), no reactivity was observed in any of the cases. This study shows that aberrant beta-catenin and E-cadherin protein expression occurs in 100% of SPT, is probably linked mechanistically to beta-catenin nuclear localization, and two distinct patterns of E-cadherin immunoreactivity are seen in SPT: nuclear (with the antibody against the cytoplasmic domain), or immunonegativity (complete loss) when stained with the antibody for the E-cadherin extracellular fragment.

Dendritic Cells Break Bonds to Tolerize

Typically, dendritic cells (DCs) induce peripheral tolerance under steady state but immunity during inflammation or infection. In this issue of Immunity, Jiang et al. (2007) identify disruption of E-cadherin interactions as a unique maturation pathway by which DCs are capable of mediating tolerance.

Ankyrin-G Is a Molecular Partner of E-cadherin in Epithelial Cells and Early Embryos

E-cadherin is a ubiquitous component of lateral membranes in epithelial tissues and is required to form the first lateral membrane domains in development. Here, we identify ankyrin-G as a molecular partner of E-cadherin and demonstrate that ankyrin-G and beta-2-spectrin are required for accumulation of E-cadherin at the lateral membrane in both epithelial cells and early embryos. Ankyrin-G binds to the cytoplasmic domain of E-cadherin at a conserved site distinct from that of beta-catenin. Ankyrin-G also recruits beta-2-spectrin to E-cadherin-beta-catenin complexes, thus providing a direct connection between E-cadherin and the spectrin/actin skeleton. In addition to restricting the membrane mobility of E-cadherin, ankyrin-G and beta-2-spectrin also are required for exit of E-cadherin from the trans-Golgi network in a microtubule-dependent pathway. Ankyrin-G and beta-2-spectrin co-localize with E-cadherin in preimplantation mouse embryos. Moreover, knockdown of either ankyrin-G or beta-2-spectrin in one cell of a two-cell embryo blocks accumulation of E-cadherin at sites of cell-cell contact. E-cadherin thus requires both ankyrin-G and beta-2-spectrin for its cellular localization in early embryos as well as cultured epithelial cells. We have recently reported that ankyrin-G and beta-2-spectrin collaborate in biogenesis of the lateral membrane ( Kizhatil, K., Yoon, W., Mohler, P. J., Davis, L. H., Hoffman, J. A., and Bennett, V. (2007) J. Biol. Chem. 282, 2029-2037 ). Together with the current findings, these data suggest a ankyrin/spectrin-based mechanism for coordinating membrane assembly with extracellular interactions of E-cadherin at sites of cell-cell contact.

The Crystal Structure of Human E-cadherin Domains 1 and 2, and Comparison with other Cadherins in the Context of Adhesion Mechanism

Cell adhesion mediated by type I cadherins involves homophilic “ trans” interactions that are thought to be brought about by a strand exchange mechanism involving the N-terminal extracellular domain. Here, we present the high-resolution crystal structure of the N-terminal two domains of human E-cadherin. Comparison of this structure with other type I cadherin structures reveals features that are likely to be critical to facilitate dimerization by strand exchange as well as dimer flexibility. We integrate this structural knowledge to provide a model for type I cadherin adhesive interactions. Intra-molecular docking of the conserved N-terminal “adhesion arm” into the acceptor pocket in monomeric E-cadherin appears largely identical to inter-molecular docking of the adhesion arm in adhesive trans dimers. A strained conformation of the adhesion arm in the monomer, however, may create an equilibrium between “open” and “closed” forms that primes the cadherin for formation of adhesive interactions, which are then stabilized by additional dimer-specific contacts. By contrast, in type II cadherins, strain in the adhesion arm appears absent and a much larger surface area is involved in trans adhesion, which may compensate the activation energy required to peel off the intra-molecularly docked arm. It seems that evolution has selected slightly different adhesion mechanisms for type I and type II cadherins.

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling

BackgroundClusterin (CLU) is an enigmatic molecule associated with various physiological processes and disease states. Different modes of cellular stress lead to increased CLU levels, and additionally numerous growth factors and cytokines affect the expression of the CLU gene. APC and c-MYC, both intimately linked to the Wnt signaling pathway have previously been shown to influence CLU levels, and we therefore investigated if changes in Wnt signaling activity in vitro could regulate the expression of one, or more, of several CLU mRNA and protein variants.ResultsOver-expression of the cytoplasmic domain of E-cadherin tagged with GFP was used to abrogate Wnt signaling activity in LS174T and HCT116 colon carcinoma cells. This fusion construct sequestered signaling competent β-catenin whereby Wnt signaling was abrogated, and consequently cytoplasmic CLU protein levels increased as demonstrated by immunofluorescence. To determine which branch of the Wnt pathway was mediating the CLU response, we over-expressed dominant negative (dn) TCF1 and TCF4 transcription factors in stably transfected LS174T cells. We observed both intra- and extracellular levels of CLU protein to be induced by dnTCF1 but not dnTCF4. Subsequent analysis of the expression levels of three CLU mRNA variants by real time RT-PCR revealed only one CLU mRNA variant to be responsive to dnTCF1 over-expression. 5'-end RACE indicated that this CLU mRNA variant was shorter at the 5'-end than previously reported, and accordingly the translated protein was predicted to be shorter at the N-terminus and destined to the secretory pathway which fit our observations. Examination of the immediate expression kinetics of CLU after dnTCF1 over-expression using real time RT-PCR indicated that CLU might be a secondary Wnt target.ConclusionIn conclusion, we have demonstrated that the Wnt signaling pathway specifically regulates one out of three CLU mRNA variants via TCF1. This CLU transcript is shorter at the 5' end than reported by the RefSeq database, and produces the intracellular 60 kDa CLU protein isoform which is secreted as a ~80 kDa protein after post-translational processing.

E-Cadherin Inhibits the Growth of Single Cells

E-cadherin is found at the junctions of epithelial cells. Besides mediating cell-cell adhesion through homophilic interactions (E-cadherin from one cell binding to E-cadherin on an adjacent cell), E-cadherin mediates contact inhibition of cell growth, and loss of E-cadherin is associated with tumorigenesis. As a result of the many molecular interactions that occur between cells at junctions, it has been unclear whether E-cadherin alone can inhibit cell growth in the absence of cell contact. Perrais et al . devised an experimental system designed to monitor the effects of ligation of E-cadherin on the growth of single cells. The authors exposed single epithelial cells, attached to fibronectin-coated coverslips, to microspheres coated with chimeric proteins of the extracellular domain of E-cadherin fused to the immunoglobulin Fc domain (Fc-hE). Exposure of single cells to Fc-hE-microspheres resulted in decreased BrdU incorporation (a marker of DNA replication), as measured by immunofluorescence microscopy, compared with that in cells exposed to control microspheres, which indicates that ligation of E-cadherin on these cells resulted in decreased cellular proliferation. Cadherins mediate cell-cell adhesion in part through interactions of their cytoplasmic domains with catenins. Through the use of mutated forms of E-cadherin in epithelial cell lines, the authors found that the β-catenin binding domain of E-cadherin was necessary for the inhibition of cellular proliferation. Knockdown of β-catenin by siRNA eliminated E-cadherin-mediated inhibition of cell growth, resulting in increased cellular proliferation. E-cadherin-mediated inhibition of cell growth, however, was not dependent on β-catenin-dependent transcriptional activation. Ligation of E-cadherin by Fc-hE-microspheres inhibited cell growth stimulated by epidermal growth factor (EGF) in epithelial cell lines. This effect of E-cadherin was also dependent on the presence of β-catenin. Whereas E-cadherin ligation did not inhibit either the autophosphorylation of the EGF receptor (EGFR) or EGFR-mediated activation of extracellular signal-regulated kinase (ERK), it did inhibit the transphosphorylation of the Tyr845 residue of EGFR and the subsequent activation of signal transducer and activator of transcription 5 (STAT5). Together these data suggest that homophilic ligation of E-cadherin can inhibit cell growth and EGFR signaling independently of cell-cell contact. M. Perrais, X. Chen, M. Perez-Moreno, B. M. Gumbiner, E-cadherin homophilic ligation inhibits cell growth and epidermal growth factor receptor signaling independently of other cell interactions. Mol. Biol. Cell 18 , 2013-2025 (2007). [Abstract] [Full Text]

Bacteroides fragilis toxin stimulates intestinal epithelial cell shedding and γ-secretase-dependent E-cadherin cleavage

Enterotoxigenic Bacteroides fragilis - organisms that live in the colon - secrete a metalloprotease toxin, B. fragilis toxin. This toxin binds to a specific intestinal epithelial cell receptor and stimulates cell proliferation, which is dependent, in part, on E-cadherin degradation and beta-catenin-T-cell-factor nuclear signaling. Gamma-secretase (or presenilin-1) is an intramembrane cleaving protease and is a positive regulator of E-cadherin cleavage and a negative regulator of beta-catenin signaling. Here we examine the mechanistic details of toxin-initiated E-cadherin cleavage. B. fragilis toxin stimulated shedding of cell membrane proteins, including the 80 kDa E-cadherin ectodomain. Shedding of this domain required biologically active toxin and was not mediated by MMP-7, ADAM10 or ADAM17. Inhibition of gamma-secretase blocked toxin-induced proteolysis of the 33 kDa intracellular E-cadherin domain causing cell membrane retention of a distinct beta-catenin pool without diminishing toxin-induced cell proliferation. Unexpectedly, gamma-secretase positively regulated basal cell proliferation dependent on the beta-catenin-T-cell-factor complex. We conclude that toxin induces step-wise cleavage of E-cadherin, which is dependent on toxin metalloprotease and gamma-secretase. Our results suggest that differentially regulated beta-catenin pools associate with the E-cadherin-gamma-secretase adherens junction complex; one pool regulated by gamma-secretase is important to intestinal epithelial cell homeostasis.

Casein Kinase 1 Is a Novel Negative Regulator of E-Cadherin-Based Cell-Cell Contacts

Cadherins are the most crucial membrane proteins for the formation of tight and compact cell-cell contacts. Cadherin-based cell-cell adhesions are dynamically established and/or disrupted during various physiological and pathological processes. However, the molecular mechanisms that regulate cell-cell contacts are not fully understood. In this paper, we report a novel functional role of casein kinase 1 (CK1) in the regulation of cell-cell contacts. Firstly, we observed that IC261, a specific inhibitor of CK1, stabilizes cadherin-based cell-cell contacts, whereas the overexpression of CK1 disrupts them. CK1 colocalizes with E-cadherin and phosphorylates the cytoplasmic domain of E-cadherin in vitro and in a cell culture system. We show that the major CK1 phosphorylation site of E-cadherin is serine 846, a highly conserved residue between classical cadherins. Constitutively phosphorylated E-cadherin (S846D) is unable to localize at cell-cell contacts and has decreased adhesive activity. Furthermore, phosphorylated E-cadherin (S846D) has weaker interactions with beta-catenin and is internalized more efficiently than wild-type E-cadherin. These data indicate that CK1 is a novel negative regulator of cadherin-based cell-cell contacts.

Phosphorylation of β-Catenin by AKT Promotes β-Catenin Transcriptional Activity

Increased transcriptional activity of beta-catenin resulting from Wnt/Wingless-dependent or -independent signaling has been detected in many types of human cancer, but the underlying mechanism of Wnt-independent regulation is poorly understood. We have demonstrated that AKT, which is activated downstream from epidermal growth factor receptor signaling, phosphorylates beta-catenin at Ser552 in vitro and in vivo. AKT-mediated phosphorylation of beta-catenin causes its disassociation from cell-cell contacts and accumulation in both the cytosol and the nucleus and enhances its interaction with 14-3-3zeta via a binding motif containing Ser552. Phosphorylation of beta-catenin by AKT increases its transcriptional activity and promotes tumor cell invasion, indicating that AKT-dependent regulation of beta-catenin plays a critical role in tumor invasion and development.

Evidence that the V832M E-Cadherin Germ-Line Missense Mutation does not Influence the Affinity of α -Catenin for the Cadherin/Catenin Complex

Mutations in E-cadherin are associated with a number of diseases, and have been shown to contribute to disease progression. In particular, 50% of hereditary diffuse gastric cancer cases have inactivating mutations in the E-cadherin gene. An interesting mutation near the beta-catenin-binding site on the cytoplasmic domain of E-cadherin (V832M) was recently reported that produces full-length protein, but exhibits decreased binding of alpha -catenin to the cadherin/catenin complex. The study was done by transfecting mutant E-cadherin into Chinese hamster ovary fibroblast cells. Here we show that the previously reported characteristics of this mutation do not apply to human epithelial cells expressing this mutant protein and suggest that the mechanism whereby the V832M mutation in human E-cadherin promotes gastric cancer is not yet understood.

Coordinated Functions of E-Cadherin and Transforming Growth Factor β Receptor II In vitro and In vivo

In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and loss of E-cadherin is a hallmark of tumor progression fostering cancer cell invasion and metastasis. To examine E-cadherin loss in squamous cell cancers, we used primary human esophageal epithelial cells (keratinocytes) as a platform and retrovirally transduced wild-type and dominant-negative forms of E-cadherin into these cells. We found decreased cell adhesion in the cells expressing dominant-negative E-cadherin, thereby resulting in enhanced migration and invasion. To analyze which molecular pathway(s) may modulate these changes, we conducted microarray analysis and found up-regulation of transforming growth factor beta receptor II (TbetaRII) in the wild-type E-cadherin-overexpressing cells, which was confirmed by real-time PCR and Western blot analyses. To investigate the in vivo relevance of this finding, we analyzed tissue microarrays of paired esophageal squamous cell carcinomas and adjacent normal esophagus, and we could show a coordinated loss of E-cadherin and TbetaRII in approximately 80% of tumors. To determine if there may be an E-cadherin-dependent regulation of TbetaRII, we show the physical interaction of E-cadherin with TbetaRII and that this is mediated through the extracellular domains of E-cadherin and TbetaRII, respectively. In addition, TbetaRI is recruited to this complex. When placed in the context of three-dimensional cell culture, which reflects the physiologic microenvironment, TbetaRII-mediated cell signaling is dependent upon intact E-cadherin function. Our results, which suggest that E-cadherin regulates TbetaRII function, have important implications for epithelial carcinogenesis characterized through the frequent occurrence of E-cadherin and TbetaRII loss.

E-Cadherin Regulates Human Nanos1, which Interacts with p120ctn and Induces Tumor Cell Migration and Invasion

Down-regulation of the epithelial cell-cell adhesion molecule E-cadherin is frequently associated with tumor formation and progression. Besides its role in physical cell-cell adhesion, E-cadherin is also thought to be involved in intracellular signaling in normal epithelial cells. In these cells, the Armadillo catenin p120ctn binds to the cytoplasmic domain of E-cadherin and stabilizes the adhesion complexes. On loss of E-cadherin, cytoplasmic p120ctn might accumulate and contribute to tumor malignancy. We used suppression subtractive hybridization to search for genes regulated by E-cadherin expression. We isolated human Nanos1 as a transcript of which levels decrease on E-cadherin reexpression in a human breast cancer cell line. The hNanos1 protein bears a COOH-terminal (CCHC)(2) zinc finger domain and belongs to an evolutionarily conserved protein family sharing functions in germ cell development in both vertebrates and invertebrates. We found an inverse correlation between E-cadherin and hNanos1 expression in various cell lines and under diverse conditions. Conditional expression of hNanos1 in human colorectal DLD1 cancer cells functionally abolished cell-cell adhesion. It induced cytoplasmic translocation of p120ctn, as well as strong migratory and invasive properties. We also found that the NH(2)-terminal domain of hNanos1, which is conserved only among mammals, interacts with p120ctn. hNanos1 counteracted the stimulatory effect of p120ctn on cell protrusion formation. Together, these findings describe a new function for hNanos1 as a downstream effector of E-cadherin loss contributing to tumor progression. Targeting hNanos1 might be a promising strategy in the treatment of E-cadherin-negative tumors in particular.