Recovery of cellular E-cadherin precedes replenishment of estrogen receptor and estrogen-dependent proliferation of breast cancer cells rescued from a death stimulus

Loss of estrogen-responsiveness and impaired E-cadherin expression/function has been linked to increased metastatic potential of breast cancer cells. In this study, we report that proliferation of breast cancer cells can resume following removal of a toxic stimulus causing severe impairment of cell adhesion and estrogen responsiveness. This type of response was induced by okadaic acid (OA) in MCF-7 cells, and was accompanied by an almost complete block of DNA synthesis, loss of cell-cell contact and cell detachment from culture dishes, loss of estrogen receptor (ER), progesterone receptor (PR) and E-cadherin, whereas only a weak, if any, inhibition of protein synthesis could be observed. These responses were detected in MCF-7 cells after a 1-day treatment with 50 nM OA, and could be reversed if OA-treated cells were recovered in a culture medium devoid of the toxin, so that rescued cells resumed growth 8-12 days after replating. By pulse-chase experiments, we found that protein synthesis was not significantly affected in rescued cells, whose DNA synthesis, instead, was almost completely blocked during the first days of MCF-7 cell rescue from OA treatment. We also analyzed E-cadherin, mitogen activated protein kinase isoforms ERK1 and ERK2, Bcl-2 and BAX proteins during the rescue of MCF-7 cells from OA-induced cell death, and found that their expression followed temporally defined patterns. Cellular levels of E-cadherin returned to control levels within the first days of the rescue, followed by ER, ERK1, and ERK2, and finally by Bcl-2 and BAX proteins. Under our experimental conditions, restoration of cell adhesion did not require a functional ER system, but recovery of a normal ER pool accompanied resumption of estrogen-dependent proliferation of OA-treated MCF-7 cells.     

γ-Secretase-dependent cleavage of E-cadherin by staurosporine in breast cancer cells

E-cadherin is a transmembrane protein that serves as a cell adhesion molecule component of the adherens junction. We previously showed that cadmium induced γ-secretase-dependent E-cadherin cleavage via oxidative stress. In this study, we report that staurosporine (STS)-induced apoptosis induces caspase-2 and/or -8-dependent E-cadherin cleavage. STS increased γ-secretase-dependent cleavage of E-cadherin in breast cancer cells through caspase activation. The ability of the γ-secretase inhibitor DAPT and the caspase inhibitor zVAD-FMK to block E-cadherin cleavage provided support for these results. The cleavage of E-cadherin was blocked by caspase-2 and -8 inhibitors. Immunofluorescence analysis confirmed that, along with the disappearance of E-cadherin staining at the cell surface, the E-cadherin cytoplasmic domain accumulated in the cytosol. In the presence of an inhibitor of γ-secretase or caspase, the cleavage of E-cadherin was partially blocked. Our findings suggest that activation of caspase-2/-8 stimulated the disruption of cadherin-mediated cell-cell contacts in apoptotic cells via γ-secretase activation.     

E-cadherin regulates the function of the EphA2 receptor tyrosine kinase.

EphA2 is a member of the Eph family of receptor tyrosine kinases, which are increasingly understood to play critical roles in disease and development. We report here the regulation of EphA2 by E-cadherin. In nonneoplastic epithelia, EphA2 was tyrosine-phosphorylated and localized to sites of cell-cell contact. These properties required the proper expression and functioning of E-cadherin. In breast cancer cells that lack E-cadherin, the phosphotyrosine content of EphA2 was decreased, and EphA2 was redistributed into membrane ruffles. Expression of E-cadherin in metastatic cells restored a more normal pattern of EphA2 phosphorylation and localization. Activation of EphA2, either by E-cadherin expression or antibody-mediated aggregation, decreased cell-extracellular matrix adhesion and cell growth. Altogether, this demonstrates that EphA2 function is dependent on E-cadherin and suggests that loss of E-cadherin function may alter neoplastic cell growth and adhesion via effects on EphA2.     

Cell binding function of E-cadherin is regulated by the cytoplasmic domain.

Cadherins are a family of transmembrane glycoproteins responsible for Ca2+-dependent cell-cell adhesion. Their amino acid sequences are highly conserved in the cytoplasmic domain. To study the role of the cytoplasmic domain in the function of cadherins, we constructed expression vectors with cDNAs encoding the deletion mutants of E-cadherin polypeptides, in which the carboxy terminus was truncated at various lengths. These vectors were introduced into L cells by transfection, and cell lines expressing the mutant E-cadherin molecules were isolated. In all transfectants obtained, the extracellular domain of the mutant E-cadherins was exposed on the cell surface, and had normal Ca2+-sensitivity and molecular size. However, these cells did not show any Ca2+-dependent aggregation, indicating that the mutant molecules cannot mediate cell-cell binding. The mutant E-cadherin molecules could be released from cells by nonionic detergents, whereas a fraction of normal E-cadherin molecules could not be extracted with the detergent and appeared to be anchored to the cytoskeleton at cell-cell junctions. These results suggest that the cytoplasmic domain regulates the cell-cell binding function of the extracellular domain of E-cadherin, possibly through interaction with some cytoskeletal components.     

HGF/SF-induced spreading of MDCK cells correlates with disappearance of barmotin/7H6, a tight junction-associated protein, from the cell membrane

Changes in expression of the two tight junction-associated proteins, barmotin/7H6 and ZO-1, as well as the adherence junction-associated protein, E-cadherin, were followed during hepatocyte growth factor/scatter factor (HGF/SC)-induced migration process of MDCK cells. Modulation of the HGF/SF-induced migration process by staurosporine, an inhibitor of protein kinase C (PKC), was also examined. Cell migration induced by HGF/SF consisted of two distinct phases, initial cell spreading between 2 and 9 h after the start of treatment, and the scattering phase which started approximately 12 h after treatment. Both ZO-1 and E-cadherin were expressed at the cell-cell border of adherent cells in the scattering phase, whereas barmotin/7H6, a barrier function-related tight junction protein, was not seen during the early spreading phase. Confluent cultures of MDCK cells, which did not spread after HGF/SF treatment, were positive for barmotin/7H6 expression at cell-cell borders. Blocking PKC activation during HGF/SF treatment with staurosporine inhibited cell spreading, and the cells retained barmotin/7H6 expression until at 6 h after HGF/SF treatment. The results indicate that disappearance of the tight junction protein, barmotin/7H6, is closely associated with cell spreading, with both barmotin/7H6 expression and cell spreading seemingly being regulated by PKC-mediated signaling.     

Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E- cadherin

Gap junctional intercellular communication (GJIC) of cultured mouse epidermal cells is mediated by a gap junction protein, connexin 43, and is dependent on the calcium concentration in the medium, with higher GJIC in a high-calcium (1.2 mM) medium. In several mouse epidermal cell lines, we found a good correlation between the level of GJIC and that of immunohistochemical staining of E-cadherin, a calcium-dependent cell adhesion molecule, at cell-cell contact areas. The variant cell line P3/22 showed both low GJIC and E-cadherin protein expression in low- and high-Ca2+ media. P3/22 cells showed very low E-cadherin mRNA expression. To test directly whether E-cadherin is involved in the Ca(2+)-dependent regulation of GJIC, we transfected the E-cadherin expression vector into P3/22 cells and obtained several stable clones which expressed high levels of E-cadherin mRNA. All transfectants expressed E-cadherin molecules at cell-cell contact areas in a calcium-dependent manner. GJIC was also observed in these transfectants and was calcium dependent. These results suggest that Ca(2+)-dependent regulation of GJIC in mouse epidermal cells is directly controlled by a calcium-dependent cell adhesion molecule, E-cadherin. Furthermore, several lines of evidence suggest that GJIC control by E-cadherin involves posttranslational regulation (assembly and/or function) of the gap junction protein connexin 43.     

Inducible expression of p120Cas1B isoform corroborates the role for p120-catenin as a positive regulator of E-cadherin function in intestinal cancer cells

Over the past decade, the exact function of p120-catenin in regulation of E-cadherin/catenins complex has remained particularly controversial. We have previously reported that E-cadherin-mediated adhesion is tightly regulated by tyrosine phosphorylation of catenins. However, this effect is not observed in human colon carcinoma cell line Caco-2. Here, we have generated inducible Caco-2 clones that display p120Cas1B, a p120-catenin isoform poorly expressed by these cells. As a result, neither expression of the transgene nor tyrosine phosphorylation of catenins induces redistribution of E-cadherin to the cytosol and disassembly of adherens and tight junctions. In contrast, E-cadherin appears markedly increased reinforcing cell-cell adhesion. Interestingly, a substantial decrease in p120-catenin levels is found in MDCK cells expressing Snail, in which E-cadherin expression is strongly inhibited. Additionally, we show that the specific depletion of p120-catenin decreases cell-cell contacts, and increases cell motility and scattering of colonies established by HT-29 M6 cells. Together our results corroborate that p120-catenin plays an essential role in the maintenance of the required E-cadherin protein levels that prevent the loss of epithelial characteristics occurred during tumorigenesis.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation

Gene amplification and protein overexpression of MDM2, which is often found in certain types of cancers, indicate that MDM2 plays an important role in tumorigenesis. Interestingly, several clinical reports have demonstrated that amplification of the MDM2 gene correlates with the metastatic stage. Using an antibody array assay, we identified E-cadherin as an MDM2-binding protein and confirmed that E-cadherin is a substrate for the MDM2 E3 ubiquitin ligase. We demonstrate that MDM2 interacts in vivo with E-cadherin, resulting in its ubiquitination and degradation. This regulation appears to be clinically relevant, as we found a significant correlation between high MDM2 and low E-cadherin protein levels in resected tumor specimens recovered from breast cancer patients with lymph node metastases. Ectopic expression of MDM2 in breast cancer cells was found to disrupt cell-cell contacts and enhance cell motility and invasive potential. We found that E-cadherin and MDM2 colocalized on the plasma membrane and in the early endosome, where ubiquitin moieties were attached to E-cadherin. Blocking endocytosis with dominant-negative mutants of dynamin abolished the association of MDM2 with E-cadherin, prevented E-cadherin degradation, and attenuated cell motility as observed by fluorescence microscopy. Thus, we provide evidence to support a novel role for MDM2 in regulating cell adhesions by a mechanism that involves degrading and down-regulating the expression of E-cadherin via an endosome pathway. This novel MDM2-regulated pathway is likely to play a biologically relevant role in cancer metastasis.     

E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.     

Aberrant O-glycosylation inhibits stable expression of dysadherin,a carcinoma-associated antigen,and facilitates cell-cell adhesion

Recently, we identified dysadherin, a novel carcinoma-associated glycoprotein, and showed that overexpression of dysadherin in human hepatocarcinoma PLC/PRF/5 cells could suppress E-cadherin-mediated cell-cell adhesion and promote tumor metastasis. The present study shows evidence that dysadherin is actually O-glycosylated. This was based on a direct carbohydrate composition analysis of a chimera protein of an extracellular domain of dysadherin fused to an Fc fragment of immunoglobulin. To assess the importance of O-glycosylation in dysadherin function, dysadherin-transfected hepatocarcinoma cells were cultured in a medium containing benzyl-alpha-GalNAc, a modulator of O-glycosylation. This treatment facilitated homotypic cell adhesion among dysadherin transfectants accompanied with morphological changes, indicating that the anti-adhesive effect of dysadherin was weakened. Modification of O-glycan synthesis also resulted in down-regulation of dysadherin expression and up-regulation of E-cadherin expression in dysadherin transfectants but did not affect E-cadherin expression in mock transfectants. Structural analysis of O-glycans released from the dysadherin chimera proteins indicated that a series of O-glycans with core 1 and 2 structures are attached to dysadherin, and their sialylation is remarkably inhibited by benzyl-alpha-GalNAc treatment. However, sialidase treatment of the cells did not affect calcium-dependent cell aggregation, which excluded the possibility that sialic acid itself is directly involved in cell-cell adhesion. We suggest that aberrant O-glycosylation in carcinoma cells inhibits stable expression of dysadherin and leads to the up-regulation of E-cadherin expression by an unknown mechanism, resulting in increased cell-cell adhesion. The carbohydrate-directed approach to the regulation of dysadherin expression might be a new strategy for cancer therapy.     

Induction of E-cadherin endocytosis by loss of protein phosphatase 2A expression in human breast cancers

The cell-cell adhesion molecule E-cadherin is stabilized by linking intracellularly with the actin cytoskeleton through PP2A-mediated recruitment of IQGAP1 to Rac1-bound E-cadherin-catenins complex in nonmalignant HME cells. However, little is known about the dysfunction of E-cadherin by loss or reduced expression of PP2A in human breast cancer cells. We report here that both human breast cancer MDA-MB-231 and MCF-7 cells were deficient in expression of the PP2A-A protein and lost the IQGAP1 recruitment to Rac1-bound catenins. In MDA-MB-231 cells, E-cadherin was also deficient. Immunohistochemical analysis of the normal-carcinoma matched human breast tissue arrays revealed that PP2A-A was expressed in 96% of normal tissue specimens but not in 57% of carcinoma specimens. Expression of E-cadherin in MCF-7 cells was 1.5-fold higher than that in HME cells, however, 80% of E-cadherin was endocytosed and incompletely anchored to F-actin. Therefore, we propose that the dysfunction of E-cadherin due to its endocytosis may occur in some proportion of human breast carcinomas in which the PP2A-A protein is lost or significantly reduced.     

IL-6 promotes head and neck tumor metastasis by inducing epithelial-mesenchymal transition via the JAK-STAT3-SNAIL signaling pathway

Epithelial-mesenchymal transition (EMT) is a key process in tumor metastatic cascade that is characterized by the loss of cell-cell junctions and cell polarity, resulting in the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process in head and neck cancers are poorly understood. Increasing evidence suggests that tumor microenvironment plays an important role in promoting EMT in tumor cells. We have previously shown that head and neck tumors exhibit significantly higher Bcl-2 expression in tumor-associated endothelial cells and overexpression of Bcl-2 alone in tumor-associated endothelial cells was sufficient to enhance tumor metastasis of oral squamous cell carcinoma in a severe combined immunodeficient (SCID) mouse model. In this study, we show that endothelial cells expressing Bcl-2 (EC-Bcl-2), when cocultured with head and neck tumor cells (CAL27), significantly enhance EMT-related changes in tumor cells predominantly by the secretion of IL-6. Treatment with recombinant IL-6 or stable IL-6 overexpression in CAL27 cells or immortalized oral epithelial cells (IOE) significantly induced the expression of mesenchymal marker, vimentin, while repressing E-cadherin expression via the JAK/STAT3/Snail signaling pathway. These EMT-related changes were further associated with enhanced tumor and IOE cell scattering and motility. STAT3 knockdown significantly reversed IL-6-mediated tumor and IOE cell motility by inhibiting FAK activation. Furthermore, tumor cells overexpressing IL-6 showed marked increase in lymph node and lung metastasis in a SCID mouse xenograft model. Taken together, these results show a novel function for IL-6 in mediating EMT in head and neck tumor cells and increasing their metastatic potential.     

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.