Expression of N-cadherin by human squamous carcinoma cells induces a scattered fibroblastic phenotype with disrupted cell-cell adhesion

E-cadherin is a transmembrane glycoprotein that mediates calcium- dependent, homotypic cell-cell adhesion and plays an important role in maintaining the normal phenotype of epithelial cells. Disruption of E- cadherin activity in epithelial cells correlates with formation of metastatic tumors. Decreased adhesive function may be implemented in a number of ways including: (a) decreased expression of E-cadherin; (b) mutations in the gene encoding E-cadherin; or (c) mutations in the genes that encode the catenins, proteins that link the cadherins to the cytoskeleton and are essential for cadherin mediated cell-cell adhesion. In this study, we explored the possibility that inappropriate expression of a nonepithelial cadherin by an epithelial cell might also result in disruption of cell-cell adhesion. We showed that a squamous cell carcinoma-derived cell line expressed N-cadherin and displayed a scattered fibroblastic phenotype along with decreased expression of E- and P-cadherin. Transfection of this cell line with antisense N- cadherin resulted in reversion to a normal-appearing squamous epithelial cell with increased E- and P-cadherin expression. In addition, transfection of a normal-appearing squamous epithelial cell line with N-cadherin resulted in downregulation of both E- and P- cadherin and a scattered fibroblastic phenotype. In all cases, the levels of expression of N-cadherin and E-cadherin were inversely related to one another. In addition, we showed that some squamous cell carcinomas expressed N-cadherin in situ and those tumors expressing N- cadherin were invasive. These studies led us to propose a novel mechanism for tumorigenesis in squamous epithelial cells; i.e., inadvertent expression of a nonepithelial cadherin.     

N-cadherin expression is involved in malignant behavior of head and neck cancer in relation to epithelial-mesenchymal transition

The loss of E-cadherin and the gain of N-cadherin expression are known as "cadherin switching". Cadherin switching is a major hallmark of epithelial-mesenchymal transition (EMT). EMT is a crucial process in cancer progression, providing cancer cells with the ability to escape from the primary focus, to invade stromal tissues and to migrate to distant regions. Although down-regulation of E-cadherin is well known in various cancers, there are a few studies on N-cadherin expression in cancer. Here, therefore, we investigated whether N-cadherin expression was associated with the progression of head and neck squamous cell carcinoma (HNSCC). First, we examined the expression of N-cadherin by immunohistochemistry and its correlation with clinico-pathological findings. High expression of N-cadherin was observed in 52 of 80 HNSCC cases and was significantly correlated with malignant behaviors. Next, we examined the correlation between N-cadherin and E-cadherin. Cadherin switching (high expression of N-cadherin and low expression of E-cadherin) was found in 30 of 80 HNSCC cases and was well correlated with histological differentiation, pattern of invasion and lymph node metastasis in HNSCC cases. Moreover, we examined the expression of N-cadherin and E-cadherin by RT-PCR in 16 HNSCC cell lines to confirm the immunohistochemical findings. N-cadherin expression was observed in 7 of 16 HNSCC cells, and cadherin switching was observed in 2 HNSCC cells. Interestingly, HNSCC cells with cadherin switching have EMT features. In conclusion, we suggest that i) N-cadherin may play an important role in malignant behaviors of HNSCC, and ii) cadherin switching might be considered as a discrete critical event in EMT and metastatic potential of HNSCC.     

Promoter methylation regulates cadherin switching in squamous cell carcinoma

Cadherins are cell adhesion molecules that modulate the epithelial phenotype and regulate tumor invasion. To identify the role of promoter methylation in regulating E-cadherin expression and in the "switching" of cadherins in oral squamous cell carcinoma (SCC), we studied 14 cell lines for cadherin expression. Immunoblotting revealed that only two (HOC-313 and HA-376) showed strong up-regulation of N-cadherin, and neither expressed E-cadherin. These results were confirmed by PCR. Furthermore, analysis of genomic DNA showed that the lack of E-cadherin expression in the two cell lines was not due to gene deletion. In both cell lines, methylation-specific PCR indicated extensive methylation of the 5' CpG island in the E-cadherin promoter. After treatment with a DNA methylation inhibitor (5-Aza-2-deoxycytidine), both immunoblotting and immunofluorescence staining showed that HA-376 cells newly expressed E-cadherin with a parallel decrease in their N-cadherin expression. Multiplex RT-PCR demonstrated that the down-regulation of N-cadherin mRNA was coordinately regulated with E-cadherin expression. Thus, methylation of the 5' CpG island in the E-cadherin promoter induces reciprocal expression of E- and N-cadherins in oral SCC by an unknown mechanism that appears to be mediated at the level of N-cadherin gene expression. These events may play an important role in the regulation of tumor cell mobility and invasion.     

Ectopic expression of cadherin 8 is sufficient to cause cyst formation in a novel 3D collagen matrix renal tubule culture

While a variety of genetic mutations have been shown to be associated with renal cyst formation, mechanisms of renal cyst formation are largely unknown. In prior communications we described alterations in E-cadherin assembly in cultured cystic epithelial cells (Charron AJ, Nakamura S, Bacallao R, Wandinger-Ness A. J Cell Biol 149: 111-124, 2000). Using the same cell line we assayed cadherin expression by RT-PCR using primer pairs that anneal to highly conserved sequences of cadherin genes but flank informative regions of cadherins. Using this approach we found that autosomal dominant polycystic kidney disease (ADPKD) cells express cadherin 8, a neuronal cadherin with limited expression in the kidney. Immunohistochemistry confirmed cadherin 8 expression in cystic epithelia. To test the functional significance of cadherin 8 expression in renal epithelial cells, we adapted a three-dimensional collagen culture method in which HK-2 cells form tubule structures and microinjected adenovirus into the matrix space surrounding tubule structures. Adenovirus expressing cadherin 8 under the control of a tet promoter caused cyst structures to grow out of the tubules when coinjected with adenovirus expressing a tet transactivator. Microinjection of single adenovirus expressing either tet transactivator or cadherin 8 failed to cause cyst formation. When doxycycline was added to the culture, following coinjection of adenovirus, there was a dose-response reduction in cadherin 8 expression and cyst formation. Similarly, HK-2 cells transfected with Flag-tagged cadherin 8 form cysts in addition to tubular structures. HK-2 cells transfected with Flag-tagged N-cadherin do not form cysts. These data suggest that ectopic expression of cadherin 8 in renal epithelial cells is sufficient to cause the morphogenic pattern of cyst formation.     

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.     

Insulin cells of pancreas extend neurites but do not arise from the neuroectoderm.

It is generally believed that during mammalian embryogenesis neurons arise only from the ectodermal germ layer, while the other two germ layers, mesoderm and endoderm, give rise to connective tissue and gut, respectively. Pancreatic islet cells, however, may be an exception to this classical cell lineage derivation. These cells, of endodermal origin, can express several neuronal antigens in addition to the peptide hormones which regulate carbohydrate metabolism. This study sought to determine whether islet cells of adult mice, in addition to displaying biochemical homology to neurons, are also capable of extending neurites, the cytoplasmic elongations that are recognized as a hallmark of the neuronal phenotype. It was found that dissociated pancreatic islet cells can extend neurite-like processes when maintained in vitro and that these processes contain neurofilament, the intermediate filament protein specific to neurons. Islet cells maintained in vitro as explants, however, did not form neurites thereby indicating that normal histotypical contacts inhibit process formation. This observation may account for the absence of process elaboration by intact islets in vivo. These results demonstrate that cells derived from the endoderm share the ability to display a characteristic neuronal phenotype with neuroectodermal cells and, furthermore, that the expression of these traits is regulated by epigenetic cues.     

Cadherin expression in ovarian carcinoma and malignant mesothelioma cell effusions

OBJECTIVE: To analyze potential differences in cadherin expression between ovarian carcinoma/primary peritoneal carcinoma (OC/PPC) and malignant mesothelioma (MM) at this anatomic site. STUDY DESIGN: MM (N=24) and OC/PPC (N= 53) effusions were analyzed for E-cadherin, N-cadherin and P-cadherin protein expression using immunocytochemistry. RESULTS: Both MM and OC/PPC cells showed frequent expression of all 3 cadherins. OC/PPC specimens expressed E-cadherin and N-cadherin in 52 of 53 cases and P-cadherin in 51 of 53 cases. MM effusions expressed E-cadherin, N-cadherin and P-cadherin in 22 of 24, 21 of24 and 23 of24 cases, respectively. The differences in the percentage of cadherin-positive cells was weakly significant for P-cadherin (higher expression in MM, p = 0.04), but E-cadherin and N-cadherin expression was comparable (p > 0.05). CONCLUSION: MM and OC/PPC coexpress different cadherin family members. P-cadherin, E-cadherin and N-cadherin are not useful for differentiation between OC/PPC and MM in effusions.     

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.     

E-cadherin is functionally involved in the maturation of the erythroid lineage

Differentiation and proliferation of hematopoietic progenitors take place in the bone marrow and is a tightly controlled process. Cell adhesion molecules of the integrin and immunoglobulin families have been shown to be involved in these processes, but almost nothing was known about the involvement of the cadherin family in the hematopoietic system. A PCR screening of RNA of human bone marrow mononuclear cells with specific primers for classical cadherins revealed that E-cadherin, which is mainly expressed by cells of epithelial origin, is also expressed by bone marrow cells. Western blot analysis and immunofluorescence staining of bone marrow sections confirmed this unexpected finding. A more detailed analysis using immunoaffinity columns and dual color flow cytometry showed that the expression of E- cadherin is restricted to defined maturation stages of the erythropoietic lineage. Erythroblasts and normoblasts express E- cadherin, mature erythrocytes do not. A functional role of E-cadherin in the differentiation process of the erythroid lineage was indicated by antibody-inhibition studies. The addition of anti-E-cadherin antibody to bone marrow mononuclear cultures containing exogeneous erythropoietin drastically diminished the formation of erythropoietic cells. These data suggest a non-anticipated expression and function of E-cadherin in one defined hematopoietic cell lineage.     

Expression of cell type-specific markers during pancreatic development in the mouse: implications for pancreatic cell lineages

Summary The islet cells of the mammalian pancreas are comprised of four different endocrine cell types, each containing a specific hormone. Islet cells also contain two enzymes of the catecholamine biosynthetic pathway: tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC). The cell lineage relationships of these different cell types have not been examined and it is not known whether, during development, they originate from the same or from different precursor populations. In this study we used immunocytochemical procedures to determine whether developing pancreatic cells express markers common to endocrine and exocrine cell types. We found that acinar cell precursors express AADC prior to the appearance of an exocrine marker and that the expression of AADC in acinar cells persists throughout embryogenesis to the first month of postnatal life. At this time, acinar cells do not contain AADC. We also found that exocrine cells containing AADC never express other islet-cell markers. These findings suggest that while acinar and islet cells both arise from precursor cells containing AADC, these progenitor cells do not express a combined endocrine-exocrine phenotype.     

The Novel Catenin p120Binds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts

p120^cas(CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and β-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutantsin vivoindicated that the CAS–N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS–cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the stucturally similar β-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus β-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes.     

Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.     

Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.     

Expression of the neural cell adhesion molecule NCAM in endocrine cells

We examined the expression of the neural cell adhesion molecule NCAM in a number of endocrine tissues of adult rat and in an endocrine tumor cell line. NCAM was found by immunoelectron microscopy to be present on the surface of all endocrine cells in the three lobes of the hypophysis, although staining was relatively less intense in the intermediate lobe, and in pancreatic islets. Pituicytes, hypophyseal glial cells, were also labeled for NCAM. A rat insulinoma cell line (RIN A2) also expressed NCAM as judged by immunocytochemistry. Analysis of NCAM antigenic determinants (Mr 180, 140, and 120 KD) revealed large variations in the relative proportions of NCAM polypeptides present in the different tissues. Although all tissues and cell lines expressed NCAM-140, NCAM-180 was not detected in the adenohypophysis, pancreas, or adrenal medulla, and NCAM-120 was found in none of the endocrine tissues or cell lines except at low levels in the neurohypophysis. The tumor cell line expressed significant levels of NCAM-180, which was most abundant in the neurohypophysis. These results show that NCAM expression appears to be a general property of endocrine cells, although the antigenic composition differs markedly from that in brain tissue. These data are discussed with regard to the embryological origins of the different endocrine tissues, and possible functional implications are suggested.     

Expression of beta1-integrins and N-cadherin in bladder cancer and melanoma cell lines

Changes in the expression of integrins and cadherins might contribute to the progression, invasion and metastasis of transitional cell cancer of the bladder and of melanomas. The expression of alpha5 (P < 0.001), alpha2 and beta1 (P < 0.05 - P < 0.001) integrin subunits in melanoma cells from noncutaneous metastatic sites (WM9, A375) were significantly increased as compared to cutaneous primary tumor (WM35) and metastatic (WM239) cell lines. These differences might be ascribed to the invasive character of melanoma cells and their metastasis to the noncutaneous locations. The significantly heterogeneous expression of beta1 integrin subunit in two malignant bladder cancer cell lines (T24 and Hu456) and nonsignificant differences in the expression of alpha2, alpha3, and alpha5 subunits between malignant and non-malignant human bladder cell lines do not allow an unanimous conclusion on the role of these intergrin subunits in the progression of transitional cancer of bladder. The adhesion molecule, expressed in all studied melanoma and bladder cell lines, that reacted with anti-Pan cadherin monoclonal antibodies was identified as N-cadherin except in the HCV29 non-malignant ureter cell line. However, neither this nor any other bladder or melanoma cell line expressed E-cadherin. The obtained results imply that the replacement of E-cadherin by N-cadherin accompanied by a simultaneous increase in expression of alpha2, alpha3 and alpha5 integrin subunits clearly indicates an increase of invasiveness of melanoma and, to a lesser extent, of transitional cell cancer of bladder. High expression of N-cadherin and alpha5 integrin subunit seems to be associated with the most invasive melanoma phenotype.     

Identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization.

The function of cadherin cell adhesion molecules is thought to be regulated by a group of cytoplasmic proteins, including alpha-catenin. We identified a subtype of alpha-catenin, termed alpha N-catenin, which is associated with N-cadherin and expressed mainly in the nervous system. cDNA transfection experiments showed that alpha N-catenin can also bind with E-cadherin. To investigate the role of alpha N-catenin, we transfected lung carcinoma PC9 cells, which express E-cadherin and beta-catenin but neither alpha- nor alpha N-catenin, with alpha N-catenin cDNA. While parental PC9 grew as isolated cells, the transfectant lines formed aggregates in which cells were tightly adhered to each other, showing epithelial arrangements, and they occasionally gave rise to cystic spheres. These results suggest that alpha N-catenin is crucial not only for cadherin function but also for organization of multicellular structures.     

Endogenous N-cadherin in a subpopulation of MDCK cells: distribution and catenin complex composition

Epithelial (E)-cadherin plays a critical role in developing a normal epithelial phenotype but neural (N)-cadherin can disrupt epithelial shape, at least in carcinoma-derived cells. Here the normal epithelial cell line MDCK was used to select for a trypsin-sensitive (TS-MDCK) subpopulation that expresses low levels of endogenous N-cadherin. Similar amounts of E-cadherin and all catenins are found in both TS-MDCK and trypsin-resistant cells (TR-MDCK), but TS-MDCK are less phenotypically epithelioid and more motile, and junctional proteins are more detergent soluble. In TS-MDCK, N-cadherin is largely nonjunctional; a similar N-cadherin distribution and mesenchymal phenotype are found in TR-MDCK transfected to express low levels of exogenous N-cadherin. Little N-cadherin was attracted to junctions between TS-MDCK and hTERT-RPE1 cells, a retinal pigment epithelium-derived line that expresses dominantly N-cadherin. No differences were seen in E-cadherin-catenin complexes in TS- and TR-MDCK, but N-cadherin-catenin complexes in TS-MDCK have more abundant p120 catenin. Overall, the results indicate that E- and N-cadherin assemble stoichiometrically different complexes with p120 in the same cells. Further, N-cadherin does not participate with E-cadherin in a zonular epithelial junction in normal MDCK epithelial cells. Rather, even low levels of endogenous N-cadherin contribute to a disrupted epithelial phenotype, resembling the effect of N-cadherin on carcinoma-derived epithelial cells.